new therapies

Transcription

new therapies

EUROPEAN COMMISSION
NEW THERAPIES
EU-supported Research in Genomics and Biotechnology for Health
Sixth Framework Programme (2002-2006)
Edited by
Charles Kessler
Directorate-General for Research
2007
NewTherapy_02.indd 3
Life Sciences, Genomics and Biotechnology for Health
EUR 22841
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Luxembourg: Office for Official Publications of the European Communities, 2007
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NEW THERAPIES
TABLE OF CONTENTS
INTRODUCTION
11
REGENERATIVE
MEDICINE
1
EuroStemCell
European consortium for stem cell research 17
GENOSTEM
Adult mesenchymal stem cells engineering for
connective tissue disorders. From the bench to
the bed side
22
OsteoCord
Bone from blood: optimised isolation, characterisation and osteogenic induction of mesenchymal stem cells from umbilical cord blood
27
TherCord
Development and preclinical testing of cord
blood-derived cell therapy products
30
EPISTEM
Role of p63 and related pathways in epithelial
stem cell proliferation and differentiation and in
rare EEC-related syndromes.
32
Ulcer Therapy
Gene transfer in skin equivalnts and stem cells:
novel strategies for chronic ulcer repair and tissue regeneration
37
Skintherapy
Gene therapy for Epidermolysis Bullosa: a model
system for treatment of inherited skin diseases.
41
THERAPEUSKIN
Ex vivo gene therapy for recessive dystrophic epidermlysis bullosa : preclinical and clinical studies
44
BetaCellTherapy
Beta cell programming for treatment of diabetes
47
EuroSTEC
Soft tissue engineering for congenital birth
defects in children: new treatment modalities
for spina biida, urogenital and abdominal wall
defects
2
SC&CR
Application and process optimisation of human
stem cells for myocardium reapair
7
STEMSTROKE
Towards a stem cell therapy for stroke
61
STEMS
Preclinical evaluation of stem cell therapy in
stroke
64
STROKEMAP
Multipotent Adult Progenitor Cells to treat Stroke
67
RESCUE
From stem cell technology to functional restoration after spinal cord injury
69
New Therapies – Table of contents
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TABLE OF CONTENTS
GENE THERAPY
83
STEM-HD
Embryonic stem cells for therapy and exploration
of mechanisms in Huntington Disease
72
CLINIGENE
European Network for the Advancement of Clinical Gene Transfer and Therapy
8
NEUROscreen
The discovery of future neuro-therapeutic
molecules
7
CONSERT
Concerted Safety & Eficiency Evaluation of Retroviral Transgenesis in Gene Therapy of Inherited
Diseases
92
myoamp
Ampliication of human myogenic stem cells in
clinical conditions
77
CRYSTAL
CRYo-banking of Stem cells for human Therapeutic AppLicationp
80
GIANT
Gene therapy: an Integrated Approach for Neoplastic Treatment
98
BACULOGENES
Baculovirus vectors for gene therapy
102
THOVLEN
Targeted Herpesvirus-derived Oncolytic Vectors
for Liver cancer European Network
104
THERADPOX
Optimised and novel oncolytic adenoviruses and
pox viruses in the treatment of cancer: Virotherapy combined with molecular chemotherapy107
RIGHT
RNA Interference Technology as Human Therapeutic Tool
110
ZNIP
Therapeutic in vivo DNA repair by site-speciic
double-strand breaks
11
SNIPER
Sequence Speciic Oligomers for in vivo DNA
Repair
118
Improved precision
Improved precision of nucleic acid based therapy
of cystic ibrosis
121
6
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NEW THERAPIES
INTHER
Development and application of transposons
and site-speciic integration technologies as
non-viral gene delivery methods for ex vivo genebased therapies
124
Epi-Vector
Episomal vectors as gene delivery systems for
therapeutic application
129
InVivoVectorTrain
European labcourse: towards clinical gene therapy: preclinical gene transfer assessment
147
IndustryVectorTrain
European labcourse: advanced methods for industrial production, puriication and characterisation of gene vectors
10
PolExGene
Biocompatible non-viral polymeric gene delivery
systems for the ex vivo treatment of ocular and
cardiovascular diseases with high unmet medical
need
133
Magselectofection
Combined isolation and stable nonviral transfection of hematopoietic cells ‘ a novel platform
technology for ex vivo hematopoietic stem cell
gene therapy
13
SyntheGeneDelivery
Ex vivo gene delivery for stem cells of clinical
interests using synthetic processes of cellular
and nuclear import and targeted chromosomal
integration
138
MOLEDA
Molecular optimisation of laser/electrotransfer
DNA administration into muscle and skin for
gene therapy
141
ANGIOSKIN
DNA electrotransfer of plasmids coding for antiangiogenic factors as a proof of principle of
non-viral gene therapy for the treatment of skin
disease
144
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TABLE OF CONTENTS
IMMUNOTHERAPY AND TRANSPLANTATION
Allostem
The development of immunotherapeutic strategies to treat haematological and neoplastic diseases on the basis of optimised allogeneic stem
cell transplantation
1
DC-THERA
Dendritic cells for novel immmunotherapies
19
DC-VACC
Dendritic cells as natural adjuvants for novel vaccine technologies
16
THERAVAC
Optimised delivery systems for vaccines targeted
to dendritic cells
169
DENDRITOPHAGES
Therapeutic cancer vaccines
173
Genomes To Vaccines
Translating genome and proteome information
into immune recognition
177
COMPUVAC
Ration design and standardised evaluation of
novel genetic vaccines.
181
HEPACIVAC
New preventative and therapeutic Hepatits C
vaccines: from preclinical to phase I
186
BacAbs
Assessment of structural requirements in complement-mediated bactericidal events: towards a
global approach to the selection of new vaccine
candidates
189
8
13
MimoVax
Alzheimer’s disease-treatment targeting truncated Aß40/42 by active immunisation
193
Pharma-Planta
Recombinant pharmaceuticals from plants for
human health
196
SAGE
SME-led antibody glyco-engineering
200
BMC
Bispeciic monoclonal antibody technology
concept
202
AUTOCURE
Curing autoimmune disease. A translational approach to autoimmune diseases in the post-genomic era using inlammatory arthritis and myositis as prototypes and learning examples 204
INNOCHEM
Innovative chemokine-based therapeutic strategies for autoimmunity and chronic inlammation
209
CELLAID
European symposia for the evaluation of potentials and perspectives of curative cell therapies
for autoimmune diseases
214
STEMDIAGNOSTICS
The development of new diagnostic tests, new
tools and non-invasive methods for the prevention, early diagnosis and monitoring for haematopoietic stem cell transplantation
217
RISET
Reprogramming the immune system for the establishment of tolerance
221
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NEW THERAPIES
XENOME
Engineering of the porcine genome for
xenotransplantation studies in primates: a step
towards clinical application
224
CLINT
Facilitating international prospective clinical
trials in stem cell transplantation
228
TRIE
Transplantation research integration across
Europe
230
INDEX OF PROJECTS
233
INDEX OF
COORDINATORS
234
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10
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NEW THERAPIES
INTRODUCTION
“N
ew therapies” as used in the title here
refers to treatments such as gene and
cell therapy, immunotherapy, tissue
engineering and regenerative medicine which
offer hope for therapy of diseases which are currently untreatable, where life is at stake, and for
regenerating diseased, damaged or defective
tissues and organs. Their main characteristic is
that they employ large biomolecules, genes,
cells and tissues as therapy rather than drugs or
pharmaceuticals. By addressing such conditions
as arthritis, diabetes, heart and neurodegenerative disease, new therapies address problems of
an ageing population, and as high-value, new
technologies, they represent an opportunity for
developing industrial competitiveness.
The purpose of this compilation of information
on EU-sponsored research in new therapies is
to demonstrate the range of activities undertaken in this ield during the course of the EU’s
Sixth Framework Programme for Research (20022006), notably under the heading “Applications
of knowledge and technologies in the ield of
genomics and biotechnology for health” in the
“Life sciences, genomics and biotechnology for
health” thematic priority.
The projects described have been ordered as
much as possible on the basis of similarity and
have been grouped into three chapters:
REGENERATIVE MEDICINE. This refers to innovative medical therapies enabling the body to repair, replace, restore and regenerate damaged
or diseased cells, tissues and organs. The ield includes research areas such as cell therapy, tissue
engineering, biomaterials engineering, growth
factors and transplantation science. Projects
described here focus on regeneration of tissues
such as bone and cartilage, skin, muscle, heart,
pancreas and nervous tissue, through the use of
haematopoietic, mesenchymal, cord blood, human embryonic and adult stem cells. In many cases projects take a comparative approach to identify the best sources of cells and to see how they
perform in different tissues. Such comparisons
can also reveal insights into underlying knowledge of the biological processes involved. Highlights of the research include using stem cells to
reverse muscular dystrophy in a dog model of
the disease, and developing a technique to grow
pure brain stem cells.
GENE THERAPY. Gene therapy is the insertion
of genes into an individual’s cells and tissues to
treat a disease; its success depends on isolation
of effective therapeutic genes and on inding
suitable vectors to deliver them into the patient’s
cells. The main approaches for gene delivery are
viral, which often displays a high rate of gene
transfer but which can be immunogenic and dificult to produce on a large scale, and nonviral,
which results in less effective gene transfer and
limited expression periods, but which has no
insert size limitation, is relatively non-immunogenic and easy to manufacture. Cell-based vectors for gene delivery use a patient’s own cells,
which are transfected ex vivo and injected back
into the same patient, where they are non-immunogenic and recognised as self. Reports here
focus on development of tools and technologies
to overcome the existing limitations and allow
clinical application of gene therapy.
New Therapies – Introduction
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INTRODUCTION
IMMUNOTHERAPY AND TRANSPLANTATION. Immunotherapy aims to modulate the immune system
to achieve a prophylactic and/or therapeutic goal by inducing, enhancing, or suppressing an immune
response. Projects in this sector include cell therapy using stem cell transplantation, dendritic cells,
vaccines and antibodies to speciically target cells, and research on autoimmune diseases. Transplantation projects are also grouped here since immune suppression and tolerance are major research
goals. Shortage of organs for transplantation is addressed through optimisation strategies, development of xenotransplants and the regenerative medicine approach.
While these three areas do possess their own identity, they also contain parts that are interrelated;
hence use of the term new therapies to cover them all. A summary of the main topics covered by the
activities is shown in schematic version in the igure below:
EU New Therapies Research (FP6)
REGENERATIVE MEDICINE
IMMUNOTHERAPY
hESC
Mesenchymal
SC
Adult
Dendritic Cells
Cancer
hematopoietic
SC
Cord Blood
Banking
Rheuma
heart
Soft tissue
Zn- finger
Transposon
Oncolytic
AAV
Viral/
Hep C
spine
Alzheimer
Minichr
romosome
Selection
Baculo
Engineered cells
Design
Clinic
Ab Production
Retro
Viral vectors
Lenti
Vaccinology
Bacteria
muscle
Electrotransfer
Plasmid
Autoimmune
Tissue repair skin
& regeneration
siRNA
Cytokines
Transplantation
brain
Oligomers
Episomal
Non viral vectors
Infectious diseases
Mechanisms
islets
GENE THERAPY
Safety
Standardisation
Main topics of new therapies research projects
described in this review
The aim of the projects described here was to
create technological platforms for the development of new therapeutic tools. To achieve this
projects were built around ive different funding
schemes:
12
Integratedprojects
(16 projects). These are the largest size
projects and integrate a range of different activities, such as multidisciplinary research, demonstration and training. They
also permit projects to take a translational
approach linking underlying biology and
therapy and enable scientists, clinicians
and other stakeholders to work together
to achieve their deliverables.
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NEW THERAPIES
SpeciicTargetedResearchProjects
(29 projects). These are smaller projects
which focus on speciic research issues.
They may be translational but are less
multi-disciplinary and wide-ranging than
the Integrated Projects.
SME-SpeciicTargetedResearchProjects
(7 projects). Targeted Research Projects
designed to encourage research and innovation efforts of Small and MediumSized Enterprises and where research-led
SMEs play a leading role.
SpeciicSupportActions (5 actions) for training,
conferences or prospective studies in support of the programme.
The number of project supported in each of the
three areas and the EC inancial contribution to
them is shown in the table below. It can be seen
that overall 59 projects were supported with an
EC contribution of almost €270 million; this was
distributed among about 700 research teams,
almost 100 of which are SMEs.
European research projects are encouraged
to be open towards the general public and to
engage with stakeholders and interest groups.
Accordingly many projects organise public
meetings and dialogue and set up websites
on the consortium, the research and results.
Website addresses are given in the details of
each project and provide more and more up-todate information than this publication.
Networks of Excellence (2 networks), whose
objective is to reduce fragmentation in EU
research, structure the way it is carried out
and strengthen its excellence.
Area
Number of projects
EC inancial contribution (million €)
Regenerative Medicine
19
80.6
Gene therapy
19
77.4
Immunotherapy
and Transplantation
21
107.8
Total
59
265.8
Numbers of projects supported and EC inancial contribution to new therapies research
It is clear from this compilation that new therapies is an extremely active area of research for European
scientists both from the public and private sectors. Since the projects described here take a platform
approach to tool and technology development applications are expected in a range of different practical settings. The recently launched Seventh Framework Programme and approval of the Regulation
on advanced therapy medicinal products will act as a further stimulus for the area and open the way
for much-needed clinical applications.
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14
New Therapies – Regenerative Medicine
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EUROSTEMCELL
EuropeanConsortiumforStemCellResearch
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LSHB-CT-2003-0300
IntegratedProject
e฀11900000
1February2004
48months
www.eurostemcell.org
Background and objectives:
The EuroStemCell consortium aims to harness
the intrinsic potential of embryonic, foetal and
adult stem cells for the continuous generation
of specialised cell types. The ability to produce
differentiated cell populations at scale will create
new opportunities for the assignment of gene
function, identiication and validation of drug
targets, pharmaceutical screening, toxicology
testing, and, in the longer perspective, for the
repair of diseased or damaged tissue by cell
transplantation.
EuroStemCell is an integrated trans-European
effort to develop well-characterised cellular resources of therapeutic potential, derived from
embryonic, neural, myogenic, haematopoietic
and epithelial stem cells. EuroStemCell applies
genomic and transgenic technologies in conjunction with state of the art in vitro and in vivo functional assays, to dissect the molecular machinery
that governs self-renewal versus differentiation.
The consortium undertakes comparative molecular and functional evaluations of stem cells
from embryonic, foetal and adult sources. Knowledge thus acquired is used to achieve scaleable
production of identiied and characterised cell
lineages for in vitro gene and compound screening. These cell line resources are also subjected
to preclinical evaluation of tissue integration and
repair potential in animal models.
Alongside basic and applied research, EuroStem-
Cell examines the ethical and societal impacts of
stem cell research and exploitation. The consortium develops outreach resources to engage patient groups, high school students and lay public
in the issues that surround stem cell research and
medicine.
Approach and methodology:
The fundamental research work of EuroStemCell is focused on stem cell identiication, isolation, and in vitro propagation and differentiation. This includes the generation and validation
of key tools, such as antibodies and genetically
engineered cell lines and mice. Allied to this, the
consortium is collating microarray expression
proiling data on different stem cells. This foundation work is accompanied by the evaluation of
candidate stem cell resources for in vitro up-scaling and drug screening potential. Longer-term
studies will evaluate the survival and functional
integration of transplanted cells in normal and
injured tissue.
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EUROSTEMCELL
es between partner laboratories funded, while
2 three-day meetings of the entire consortium
(more than 100 researchers) were held, to promote interaction and collaboration between
partner laboratories, and reine project planning.
The overall low of the project can be illustrated
schematically, as shown below:
A number of parallel activities have been designed to support, extend and maximise the impact of the project partners’ scientiic research. In
the irst 3 years, EuroStemCell has carried out the
following:
• it developed a prototype stem cell database to encourage data exchange and
dissemination;
• it encouraged junior group leaders to
take a higher proile in the project by according Associate Principal Investigator
status to 8 individuals;
• it organised specialist workshops focusing on disease areas, to bring stem
cell researchers and clinical practitioners
together and develop a ‘roadmap to the
clinic’;
• it held a series of workshops on the
ethical implications of stem cell research,
involving scientists, ethicists, legislators,
clinicians and patient representatives in
discussions, and making outcomes widely
available via the group’s website.
There were 25 training and workgroup exchang-
18
Moreover, EuroStemCell arranged a major European stem cell conference series, ‘Advances
in Stem Cell Research’. The irst conference took
place in Milan in 2005, with an attendance of over
300. The conference was successfully repeated in
Lausanne in 2006, and will continue in Stockholm
in 2007. The series attracts leading international
speakers and participants from around the world,
providing an important opportunity to highlight
European stem cell research. The consortium
also organised annual summer schools on “Stem
Cells and Regenerative Medicine” to develop and
train the next generation of European stem cell
scientists. Each school is attended by 54 pre-and
post-doctoral students, including clinicians and
regulators, and is open to non-members of EuroStemCell.
A ‘Glossary for Stem Cell Biology’, was prepared:
it was published in Nature and adopted by the
EMBO Science & Society Programme in its report
on stem cell research. Web-based information
about stem cell research was made accessible to
a range of audiences at www.eurostemcell.org.
EuroStemCell responded individually to hundreds of requests for information from media,
patient groups and individuals, and it also issued
13 press releases, and attracted worldwide media coverage for key publications.
The project partners successfully produced an
award-winning animated ilm, A Stem Cell Story,
about stem cell research.The ilm has been distributed online, via iTunes, through festival and public
screenings, and on DVD, and has been viewed by
audiences totalling more than 10 000. Three additional short ilms to provide in-depth resources on ethics, cell culture and cloning have been
developed, in addition to a careers workshop for
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schoolchildren aged 13 and over, and a hands-on
activity programme for younger children.
EuroStemCell initiated joint activities and research projects with the BetaCellTherapy Integrated project, supported by ‘Bridge’ funding
from the Juvenile Diabetes Research Foundation
International.
Expected outcome:
Over the four-year funding period, EuroStemCell
has been contributing to advances in technological platforms, cellular resources and scientiic
knowledge in stem cell biology. This will provide
the basics for translational research. EuroStemCell is accordingly beginning to establish criteria
for preclinical validation and future clinical trials
in the areas of neurological repair, muscle repair
and epithelial repair. A major outcome of the
project will be a ‘Roadmap to the clinic’ for cell
therapy.
EuroStemCell research will also facilitate biopharmaceutical exploitation of stem cells, in part
mediated via the SME partners. In this regard, EuroStemCell partners have, to date, iled 24 patent
applications, several of which have been licensed
for exploitation by SME partners. EuroStemCell is
committed to developing ethical guidelines and
promoting public engagement with stem cell research, with the aim of positively impacting public and political opinion.
Main findings:
The consortium has now published 56 scientiic
papers directly supported by EuroStemCell. Many
of these studies have appeared in the leading
peer-review journals including Nature, Science,
Cell, PLoS Biology, Developmental Cell, Genes &
Development, Development, Neuron and Journal of Neuroscience.
In the area of basic stem cell biology, EuroStemCell investigators have published fundamental
observations, challenging dogmas about the
origins and properties of myogenic, haematopoietic and hair follicle stem cells (Nature 431; Genes
Dev 19; Cell 121; Development 133; Nature Cell
Biology 8).
Partners in Edinburgh, Milan and Bonn, working
on stem cells for the nervous system have collaborated, achieving for the irst time expansion of
pure populations of neural stem (NS) cells, with
the potential to generate the three cell types of
the central nervous system (PLoS Biology 3; Cerebral Cortex 16).
Importantly, these neural stem cells can be obtained both from embryonic stem (ES) cells and
from in vivo tissue sources. The consortium has
also reported advances in directing both mouse
and human embryonic stem cells into the nervous system lineage by manipulation of deined
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EUROSTEMCELL
signalling pathways (PLoS Biology 4). Other partners are investigating the potential of resident
stem cells in the adult brain to be harnessed for
repair (Neuron 52).
In the muscle stem cell ield, members of the consortium have demonstrated the high capacity of
freshly isolated muscle progenitor cells to repair
damaged muscle (Science 309) and the potential
of expanded mesangioblast cells for tissue repair
in a large animal model of muscular dystrophy
(Nature 444).
Partners in Lausanne and London are advancing
in the development of a large animal model for
epidermal stem cell transplantation.
SME partners are coordinating projects to improve culture conditions for the expansion of
various stem cell types. Several reined media formulations have been commercialised, including
ESGRO complete media for the growth of murine
ES cells in serum free conditions, HEScGRO for the
growth of human ES cells in serum and animal
component free conditions, and N2B27 and RHBA media for the differentiation of human and
mouse stem cells into cells of the neural lineage.
The goal is to develop fully deined conditions
for stem cell propagation for future therapeutic
grade production.
The EuroStemCell ethics group has produced reports on stem cell registers and stem cell banking following a workshop arranged jointly with
the Commission, and on commercialisation of
stem cell research. These reports can be viewed
at the following websites:
http://www.eurostemcell.org/Ethics/ethics_wrkshp2005.htm
http://www.eurostemcell.org/Ethics/ethics_wrkshp2006.htm
Major publications
Tonlorenzi, R., Innocenzi, A., Mognol, P., Thibaud,
J.L., Galvez, B.G., Barthélémy, I., Perani, L., Mantero,
S., Guttinger, M., Pansarasa, O., Rinaldi, C., Cusella
De Angelis, M.G., Torrente, Y., Bordignon, C., Bottinelli R., Cossu, G., ‘Mesoangioblast stem cells
ameliorate muscle function in dystrophic dogs’,
Nature 444, 574 – 579.
Shinin, V., Gayraud-Morel, B., Gomès, D., Tajbakhsh, S., ‘Asymmetric division and cosegregation
of template DNA strands in adult muscle satellite
cells’, Nature Cell Biology 8, 677 – 682.
Lowell, S., Benchoua, A., Heavey, B., Smith, A.G.,
‘Notch Promotes Neural Lineage Entry By
Pluripotent Embryonic Stem Cells’, PLoS Biology
4(5): e121.
Gustafsson, M.V., Zheng, X., Pereira, T., Gradin, K.,
Jin S., Lundkvist, J., Ruas, J.L., Poellinger, L., Lendahl, U., Bondesson, M., ‘Hypoxia requires Notch
signaling to maintain the undifferentiated cell
state’, Developmental Cell 9: 617-628.
Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaffran, S., Cumano, A., Partridge, T., Buckingham, M.,
‘Direct Isolation of Satellite Cells for Skeletal Muscle Regeneration’, Science 309: 2064-2067
Conti, L., Pollard, S.M., Gorba, T., Reitano, E., Toselli,
M., Biella, G., Sun, Y., Sanzone, S., Ying, Q.L., Cattaneo, E., Smith, A., ‘Niche-independent symmetrical self-renewal of a mammalian tissue stem
cell’, PLoS Biology 3(9): e283
Adolfsson, J., Månsson, R., Buza-Vidas, N., Hultquist,
A., Liuba, K., Jensen, C.T., Bryder, D., Yang, L., Borge,
O.J., Thoren, L.A.M., Anderson, K., Sitnicka, E., Sasaki, Y., Sigvardsson, M., Jacobsen, S.E.W., ‘Identiication of Flt3+ Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised
Road Map for Adult Blood Lineage Commitment’,
Cell 121: 295-306.
Sampaolesi, M., Blot, S., D’Antona, G., Granger N.,
20
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Coordinator
AustinSmith
Wellcome Trust Centre for Stem Cell Research
University of Cambridge
Tennis Court Road
Cambridge CB2 1QR, UK
E-mail: [email protected]
Partners
TimAllsopp
Stem Cell Sciences UK
Cambridge, UK
ErnestArenas,JonasFrisen,UrbanLendhal
Karolinska Institute
Stockholm, Sweden
YannBarrandon
Swiss Federal Institute for Technology
Lausanne, Switzerland
AndersBjorklund,GoranHermeren,StenEirikJacobsen,
OlleLindvall,ZaalKokaia
Lund University
Lund, Sweden
ClareBlackburnAlexanderMedvinsky,SimonTomlinson,
ValWilson,BrianHendrich
University of Edinburgh
Institute for Stem Cell Research
Edinburgh, UK
OlivierBrustleandFrankEdenhofer
University of Bonn Medical Centre
Bonn, Germany
MargaretBuckingham,AnaCumano,Jean-François
Nicolas,ShahragimTajbakhsh,BenoîtRobert
Institut Pasteur
Paris, France
ElenaCattaneoandLucianoConti
Milano University
Milan, Italy
GiulioCossu
Milano Hospital SCRI
Milan, Italy
AndersHaegerstrand
NeuroNova AB
Stockholm, Sweden
MengLi
Imperial College
London, UK
JohnMcCafferty
Wellcome Trust Sanger Institute
Cambridge, UK
ClausNerlovandLilianaMinichiello
EMBL-Monterotondo
Rome, Italy
LarsWahlberg
NsGene A/S
Ballerup, Denmark
FionaWatt
Cancer Research UK
Cambridge, UK
ChristianDani
CNRS
Nice, France
TariqEnver
Weatherall Institute of Molecular Medicine
Oxford, UK
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GENOSTEM
Adult mesenchymal stem cells engineering for connective tissue disorders:
fromthebenchtothebedside
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Regeneration takes place in the body throughout life. However, bone, cartilage, tendons, vessels and cardiac muscle have a limited capacity
for self-repair, and after injury or disease the regenerative power of adult tissue is often not suficient, leading to non-functional scaring. When
organs or tissues are irreparably damaged, they
may be replaced by an artiicial device or donor
organ. However, the number of available donor
organs is critically limited.
Generation of tissue-engineered replacement
organs by means of ex vivo culture of autologous organ-speciic cells seeding a scaffold may
represent a suitable substitute for donor organ
implantation. It also prevents the risks of disease
transmission from donors, a common problem
in organ implantation. Tissue engineering using
stem cells takes full advantage of the high proliferative capacity and the multi-potentiality of
these immature cells. The use of embryonic stem
cells is limited because of ethical drawbacks, as
well as the allogeneic immune response, inducing rejection of the implants. The alternative is
the use of adult stem cells.
The goal of GENOSTEM is to establish a European
international scientiic leadership for stem cell
regenerative medicine in the ield of connective
tissue disorders. Autologous adult Mesenchymal
Stem Cells (MSCs) are optimal candidates to serve
as building blocks for the engineering of con-
22
LSHB-CT-2003-3161
IntegratedProject
e฀872000
1January2004
48months
www.genostem.org
nective tissues, since they are the multipotential
stem cells that give rise to skeletal cells (osteoblasts, chondrocytes and tenocytes), vascular cells
(endothelial cells, pericytes and vascular smooth
muscle cells), sarcomeric muscle (skeletal and
cardiac) and adipocytes.
GENOSTEM will purify and optimise cultures of
MSCs collected from bone marrow, skeletal muscles, fat pads, cord blood vasculature, placental
and adult peripheral blood of humans, as well as
rodents. By combining different cells sources, isolating different cell subsets, and providing better
accessibility of growth factors (using scaffolds,
matrices, microcarriers coated with cytokines,
etc.), the inal aim is to obtain cell populations
with different differentiation and proliferation
potentials:
• undifferentiated MSCs where the full
differentiation potential would be preserved;
• committed MSCs that would express,
in addition to mesenchymal factors, at
least one lineage-speciic marker;
• differentiated progeny blocked at a
certain differentiation stage;
• fully differentiated progeny.
The consortium will also identify novel factors
essential for MSC proliferation and differentiation using genomic and proteomic analysis, and
engineered cells in gain (transfer of genes) and
11/8/07 10:48:57 AM
loss (transfer of small interfering RNAs) of function
studies.
To engineer MSCs (or differentiated progeny)
with the appropriate scaffold to secure repair of
the target tissue and to deine optimal conditions
for transplantation of MSCs in normal and pathological tissue, is another approach undertaken by
GENOSTEM. Undifferentiated MSCs or MSC-differentiated progeny will be assessed in normal and
in pathological animal models, to investigate the
reparative potential of MSC strains. The project
strategy will include the use of sophisticated culture conditions, using biodegradable scaffolds,
matrices and microcarriers binding soluble growth
factors, inhibitors and adhesive glycoproteins and
vectors containing gene sequences adapted to
the pathological state under study. Gene transfer
will be carried out using original lentiviral vectors,
non-viral DNA delivery complexes (polymersomes)
or matrix-anchoring, DNA-binding peptides.
The project will deine the best strategy for translating GENOSTEM research into phase I clinical trials in bone defect, in partnership with SMEs and
regulatory bodies for the scaling-up of safe procedures. GENOSTEM will be taking advantage of the
experience already acquired by one of the partners, in clinical trials using cultured muscle cells.
Figure 1: Stem cells injected in merinos sheep differentiating to cartilage, visualised in vivo with confocal celviso
system (Photo Inserm)
oped scaffolds with a DNA delivery system, and a
speciic microsphere able to deliver differentiation factors.
Standard conditions for culturing adult stem cells
from umbilical cords, adipose tissue and bone
marrow have been developed. The cells were
cultured in vitro and infected with Adeno-Smad8
and BMP-2 at different ratios, or just with AdenoSmad8, and seeded on a Collagen sponge and
implanted intramuscularly in NOD/SCID mice.
Tendon-like tissue was generated with the combination of Smad8 and BMP-2 expression. The
factors involved in chondrogenesis and the engineering of the cells were successfully identiied.
Main findings:
GENOSTEM partners have compared in vivo the
different scaffolds with MSCs, including hydrogel, chitosan, polymer, PAMs and ceramics. The
project partners developed procedures that
could combine cell attachment eficiency with
local growth factor-releasing capacity. Furthermore, a large-scale in vivo model for cartilage
repair was developed: a chitosan-poly(butylene
succinate) (PBS)(50/50%wt) blend was produced
and processed into scaffolds, using melt spinning and ibre bonding. This scaffold was used in
the merino sheep model for cartilage repair with
a chondral defect. The partners have also devel-
Chitosan scaffolds combined with TGF-treated
ovine stem cells have been injected in merino
sheep and followed for 2 months (Fig 1). Results
obtained with this pertinent osteoarthritis model
have validated the protocol, and will allow clinical development of the procedure.
Moreover, the consortium has focused on bone
repair for the setting-up of clinical trials. For this,
animal models of femoral bone defect have
been initiated. The partners developed a cranial
critical size defect assay in nude mice for in vivo
implantation. A femur critical size defect model
using a novel external ixation device is avail-
23
11/8/07 10:48:58 AM
GENOSTEM
able in immunodeicient mice for studying bone
repair. The analysis of the osteogenic potential
of various hMSCs implanted with HA/TCP (with
various %) and different materials (hydrogel/ceramic scaffold, chitosan) has been performed in
this heterotopic assay in vivo. For bone induction,
the partners identiied IGF2 and IGF-BP2 through
the microarray analyses. Treatment with IGF2 or/
and IGF-BP2 at the appropriate dosage, slightly
increased ALP staining, suggesting osteoblastic
differentiation.
Large-scale GMP production of autologous MSC
for clinical purposes is available.
stem cells in bone bioengineering
• Hofmann A, Gross G. Tendon and ligament engineering: From cell biology to in
vivo application
• Mendez-Peruz M, Hughes C, Annenkov
A, Daly G, Chernajovsky Y. Engineering
stem cells for therapy
• Vilquin JT, Rosset P. Mesenchymal stem
cells in bone and cartilage repair: current
status.
Snedeker, J.G., Pelled, G., Zilberman, Y., Gerhard, F.,
Muller, R., Gazit, D., ‘Endoscopic cellular microscopy for in vivo biomechanical assessment of tendon function’, J Biomed Opt, 2006, 11(6):064010.
Major publications
Hoffmann, A., Pelled, G., Turgeman, G., Eberle, P.,
Zilberman, Y., Shinar, H., Keinan Adamsky, K., Winkel, A., Shahab, S., Navon, G., Gross, G., Gazit, D.,
‘Neotendon formation induced by manipulation
of the Smad8 signalling pathway in mesenchymal stem cells’, J. Clin. Invest, 2006, 116(4):940-52.
Review articles Special Focus: Mesenchymal Stem
Cells, Regenerative Medicine, 2006, 1 (4):517-604
• Jorgensen C. Tissue engineering
through mesenchymal stem cells: role of
the Genostem Consortium
• Delorme B, Chateauvieux S, Charbord
P. The concept of mesenchymal stem cells.
• Roche S, Provansal M, Tiers L, Jorgensen
C, Lehmann S. Proteomics of primary mesenchymal stem cells
• Srouji S, Kizhner T, Livne E. 3D scaffolds
for bone marrow stem cell support in
bone repair
• Djouad F, Mrugala D, Noël D, Jorgensen
C. Engineered mesenchymal stem cells for
cartilage repair
• Marie PJ, Fromigué O. Osteogenic differentiation of human marrow-derived
mesenchymal stem cells
• Kimelman N, Pelled G, Gazit Z, Gazit D.
Applications of gene therapy and adult
24
Chernayovsky, Y., ‘Gene therapy for arthritis
- where do we stand?’ Arthritis Res Ther, 2005,
7(6):227-9.
Arnulf, B., Lecourt, S., Soulier, J., Ternaux, B., Lacassagne, M.N., Crinquette, A., Dessoly, J., Sciaini,
A.K., Benbunan, M., Chomienne, C., Fermand, J.P.,
Marolleau, J.P., Larghero, J., ‘Phenotypic and functional characterization of bone marrow mesenchymal stem cells derived from patients with
multiple myeloma’, Leukemia, 2007, 21(1):158-63.
Epub 2006 Nov 9.
Kulbe, H., Thompson, R., Wilson, J.L., Robinson, S.,
Hagemann, T., Fatah, R., Gould, D., Ayhan, A., Balkwill, F., ‘The inlammatory cytokine tumor necrosis factor-alpha generates an autocrine tumorpromoting network in epithelial ovarian cancer
cells’, Cancer Res, 2007, 67(2):585-92.
Bianco, P., Kuznetsov, S.A., Riminucci, M., Gehron
Robey, P., ‘Postnatal skeletal stem cells’, Methods
Enzymol, 2007, 419:117-48.
Delorme, B., Charbord, P., ‘Culture and characterisation of human bone marrow mesenchymal
stem cells’, Methods in Molecular Medicine – Tissue
Engineering 2nd Edition. Edited by Hansjörg Hauser and Martin Fussenegger. (in press)
11/8/07 10:48:58 AM
Coordinator
ChristianJorgensen
INSERM
Unit 475
Hôpital Lapeyronie, Service Immuno-Rhumatologie
34000 Montpellier, France
E-mail : [email protected]
Partners
PierreMarie
INSERM - U349
Hôpital Lariboisiere
Paris, France
Jean-ThomasVilquin
INSERM – U582
Institut de MyologieGroupe Hospitalier Pitié-Salpêtrière
Paris, France
ClaudiaMontero-Menei
INSERM – U646
Laboratoire de Vectorisation Particulaire
Angers, France
PierreCharbord
Université François Rabelais
Laboratoire d’Hématopoïèse, Faculté de Médecine
Tours, France
YutiChernajovsky
University of London
Queen Mary and Westield College
Bone & Joint Research Unit, Barts and The London
London, UK
AntoinetteHatzfeld
Centre National de la Recherche Scientiique (CNRS)
UPR 9045 - Laboratoire de Biologie des Cellules Souches
Humaines
Villejuif, France
LouisCasteilla
CNRS- UMR 5018
Toulouse, France
RalphMüller
ETH - Swiss Federal Institute of Technology
Institute for Biomedical Engineering
Zürich, Switzerland
DanGazit
The Hebrew University of Jerusalem
Skeletal Biotechnology Lab
Jerusalem, Israel
GerhardGross
Helmholtz Zentrum für Infektionsforschung
Department of Gene Regulation and Differentiation
Braunschweig, Germany
PaoloBianco
Università di Roma La Sapienza
Institute for Cell Biology and Tissue Engineering
Rome, Italy
VirgilPaunescu
University of Medicine and Pharmacy Victor Babes
Department of Physiology and Immunology
Timisoara, Romania
RobertOostendorp
Technische Universität München
Labor für Stammzellphysiologie
Munich, Germany
UlrikeNuber
Lund University
Stem Cell Centre
Lund, Sweden
ErellaLivne
Technion - Israel Institute of Technology
Anatomy and Cell Biology Faculty of Medicine Technion
Haifa, Israel
2
11/8/07 10:48:58 AM
GENOSTEM
ThomasHäupl
Charité, Medical Faculty
Department of Rheumatology
Berlin, Germany
ChristianeDascher-Nadel
Inserm-Transfert SA
Department of European and International Affaires
Marseille, France
JerónimoBlanco
Centro de Investigación Cardiovascular
Barcelona, Spain
JeffreyHubble
EPFL - Swiss Federal Institute of Technology
Institute for Biological Engineering and Biotechnology
EleniPapadaki
University of Crete
Medical School
Heraklion, Crete, Greece
MariaKalmanti
University of Crete
Jean-PierrePujol
Université de Caen
Laboratoire de Biochimie du Tissu Conjonctif
Caen, France
NunoNeves
University de Minho
Department of Polymer Engineering
Braga, Portugal
Jean-PierreMouscadet
Abcys SA
Paris, France
JohnWatson
Kuros Biosurgery AG
NathalieRougier
Biopredic International
Rennes, France
AndresCrespo
GENOPOIETIC
Miribel, France
26
11/8/07 10:48:58 AM
OSTEOCORD
Bone from blood: Optimised isolation, characterisation and osteogenic
inductionofmesenchymalstemcellsfromumbilicalcordblood
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Stem cells are ‘blank’ cells that can replicate indeinitely, or given the right triggers, grow into
specialised cells for speciic areas of the body. In
adults, a few types of cell, such as blood, bone
marrow or nerves, are unable to replicate themselves by normal cell division. Within these parts
of the body, a small quantity of stem cells is found,
that is used to repair and replace damaged cells.
The intrinsic ability of stem cells to self-repair
on demand has generated a signiicant level of
interest in determining how scientists might be
able to exploit stem cells for the treatment of certain disease conditions.
There is an urgent clinical requirement for appropriate bone substitutes that are able to replace current grafting procedures for the repair
of diseased or damaged bone. Mesenchymal
stem cells (MSCs) are found predominantly in
the bone marrow and are able to differentiate
into osteogenic (bone), chondrogenic (cartilage),
adipogenic (fat) and tenogenic (tendon) tissue
types, thus offering considerable therapeutic
potential for tissue engineering applications in
orthopaedic surgery.
However, invasive extraction procedures and
poor cell yields have demanded the identiication of alternative tissue sources of MSCs. Growing evidence suggests that umbilical cord blood
(UCB) contains a population of rare MSCs that are
able to give rise to many different cell types in
LSHB-CT-200-018999
SpeciicTargetedResearchProject
e฀200000
1January2006
36months
www.bonefromblood.org
a manner similar to bone marrow-derived MSCs.
The aim of this project is to optimise the isolation
and expansion of MSCs from human UCB (CBMSCs). The differentiation capacity of CB-MSCs
will be examined, with a speciic focus on osteogenesis, and we will tissue engineer 3-D bone replacement structures.
OsteoCord is a nine-partner EC FP7 project with
representatives from academia and industry. The
CB-MSCs will be characterised using a range of
techniques, including examination of the gene
and protein expression proiles, before and after
osteogenic differentiation, compared to MSCs
isolated from human bone marrow, as well as
embryonic stem cells. The integrated datasets
will allow the consortium to identify speciic and/
or novel signalling pathways associated with CBMSCs, that will help them to understand CB-MSC
biology and may help identify new targets for
cell-based therapies.
Bioimpedance measurements of CB-MSCs in 2D and 3-D growth conigurations will be determined, using purpose-built microchips for highthroughput characterisation. The immune status
of CB-MSCs will also be determined. Allogeneic
transplantation of MSCs between different individuals may be possible, as MSCs appear to be
’immune-privileged’, in that they are not necessarily rejected when implanted into unmatched
recipients. It is important to point out that MSCs
27
11/8/07 10:48:58 AM
OSTEOCORD
display immuno-suppressive characteristics, and
are able to reduce an immune response and
promote the engraftment of different cell types,
such as skin cells and blood cells. Therefore, it is
necessary to determine how CB-MSCs react under different immune environments.
To deliver suficient cell numbers for viable therapies, key components of the project are focused
on expansion protocols. Comparative analyses
of growth rates and ageing characteristics will
identify the lifespan of CB-MSCs in culture. Novel
techniques will be combined with scale-up procedures and the generation of CB-MSC lines for
banking, following optimised cryopreservation
protocols. Biocompatibility assays using a range
of bespoke, biomimetic scaffolds will be used to
develop tissue engineering applications.
An independent ethical evaluation of the work
will determine how the work has contributed
to the ethics of stem cell research and other issues such as cord blood banking. OsteoCord
will also provide a social science assessment of
the different expectations of MSCs held across
the research community. The aim is to assist the
research community in better understanding
broader technical, regulatory, commercial and
clinical factors in the future shaping of MSCs, and
articulate, robust and empirically informed scenarios for the exploitation of MSCs.
Main findings:
Since its launch, the OsteoCord project and all of
its work packages have gained ground. The major achievements , are set out below:
• determined growth characteristics and
CD antigen expression proile of CB-MSCs,
following recovery from cryopreservation;
• determined electrochemical impedance measurements of BM-MSCs, using a
planar electrode chip system;
• developed BM-MSC spheroids for 3-D
electrode chip assays;
• optimisation of MSC alloreactivity assays in progress;
• analysed dynamic microcarrier-assisted bioreactors for CB-MSC growth;
• established osteogenic differentiation
protocols;
• identiied candidate osteogenic scaffolds and initiated biocompatibility studies;
• completed an extensive international
patents review, on the therapeutic applications of MSCs.
Expected outcome:
OsteoCord’s integrated approach using existing and complementary expertise will provide a
timely and thorough evaluation of CB-MSCs, and
deine appropriate routes for their therapeutic
implementation. The work ranges from fundamental biological understanding through to the
delivery of CB-MSC-based therapies, under appropriate regulatory conditions.
28
11/8/07 10:48:59 AM
Coordinator
PaulGenever
University of York
Biomedical Tissue Research
Department of Biology (Area 9)
York, YO10 5YW, UK
HelderCruz
ECBio
Cell biotechnologies
Oeiras, Portugal
RobinQuirk
RegenTec Ltd
Nottingham, UK
Partners
MoustaphaKassem
University Hospital of Odense
Department of Endocrinology
Odense, Denmark
JensAndersen
University of Southern Denmark
Department of Biochemistry and Molecular Biology
Odense, Denmark
HagenThielecke
Fraunhofer Institute for Biomedical Engineering
Biohybrid Systems Department
St Ingbert, Germany
LeeButtery
University of Nottingham
Tissue Engineering Group
School of Pharmacy
Nottingham, UK
KarenBieback
Ruprecht-Karls-Universität Heidelberg
Institute of Transfusion Medicine and Immunology
Stem Cell Laboratory
Mannheim, Germany
TrevorWalter
Angel Biotechnology Ltd
Northumberland, UK
29
11/8/07 10:48:59 AM
THERCORD
Development and preclinical testing of cord blood-derived cell therapy
products
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
As scientists continue to discover new applications for human stem cells, they are targeting
diseases with a signiicant need for more eficacious treatment options. Until recently, patients
affected by acute renal failure (ARF) were treated
by dialytic and pharmacological approaches
therapies, with very limited eficacy. Human cord
blood (CB) mesenchymal stem cell (MSCs) could
represent an interesting alternative source for
therapeutic purposes in renal repair.
The Thercord consortium isolated MSCs from
full-term umbilical CB to test their therapeutic
potential on renal tissue in vitro and in vivo. These
cells were extensively characterised by low cytometry, and the differentiation assays were
performed towards adipogenic, osteogenic and
chondrogenic lineages in order to conirm their
mesenchymal features. Moreover, it tested their
ability to migrate in the presence of in vitro damage, and to produce soluble factors to promote
renal repair. Their ability to promote renal repair
when transplanted into NOD-SCID mice with
acute renal failure was also assessed.
Main findings:
The group successfully isolated MSCs from CB
units at a rate of approximately 18%. What is clear
is that the greater the percentage of extracted
30
LHSB-CT-2006-018817
e฀1800000
1May2006
36months
www.thercord.eu
MSCs, the greater the chances of a successful
transplant and long-term engraftment. Testing of
the MSCs also conirmed certain characteristics
that help induce tissue repair, like the development of bone and cartilage.
CB MSCs were positive for CD44, CD105, CD90,
HLA class I and negative for CD31, CD45, CD34,
HLA class II. Moreover, within the CB MSCs, the
consortium identiied a high percentage of
CD146+/34-/45- cells, consistent with the perivascular/pericyte-like phenotype, a new stem cell
subpopulation. In the migration assay, they were
able to observe that these cells were capable of
migrating and integrating into the damaged renal cell line.
With the proteome assay, they analysed several
solubles, including Hepatocyte Growth Factor,
Vascular Endothelial Growth Factor, Fibroblast
Growth Factor — factors well known to be involved in renal protection and differentiation.
They tested the CB MSCs in immunocompromised mice with acute renal failure, showing that
these cells strongly protect the mice from renal
function impairment. Kidneys were assessed
functionally (blood urea nitrogen: BUN), as well
as by histology, immunohistochemistry and
Tunel assay, effectively showing lower levels of
BUN, reduced tubular changes and reduced apoptosis in animals treated with CB MSCs.
Interestingly, inlammatory cytokines such as ILalpha, IL-beta and TNF beta were statistically re-
11/8/07 10:48:59 AM
duced in mice receiving CB MSCs, in comparison
to the control group. In conclusion, they demonstrated that CB MSCs produce soluble factors
that play a critical role, providing a protective
and reparative effect in acute renal failure. And
in vivo CB MSCs exhibit reparative potential in
acute renal failure.
Coordinator
LorenzaLazzari
Fondazione Ospedale Maggiore Policlinico
Mangiagalli e Regina Elena
Milan, Italy
Partners
MaurizioPesce
Centro Cardiologico Monzino
Milan, Italy
DominiqueBonnet
Cancer Research UK
London, UK
AnnaMagdaleneWobus
Insitut fur Planzengenetik und Kulturplanzenforschung
Gatersleben, Germany
WillemFibbe
Leiden University Medical Centre
Leiden, Netherlands
JoanGarcia
Barcelona Cord Blood Bank
Barcelona, Spain
MarcelaContreras
National Blood Service
London, UK
RitaMaccario
IRCCS Policlinico San Matteo di Pavia
Pavia, Italy
31
11/8/07 10:48:59 AM
EPISTEM
Role of p63 and related pathways in epithelial stem cell proliferation and
differentiationandinrareEEC-relatedsyndromes
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The focus of EPISTEM is on generating new
knowledge and translating it into applications
that enhance human health. To this end, both
fundamental and applied research will be involved. EPISTEM integrates multidisciplinary and
coordinated efforts to understand the molecular
basis of factors involved in epidermal stem cell
generation, maintenance and differentiation, as
well as skin disease. Moreover, the core molecule
that will be studied in this Integrated Project (IP)
is p63 (and related pathways), a molecule genetically proven to be involved in the development
of rare skin diseases.
EPISTEM is collecting and culturing keratinocytes
from EDS patients with p63 mutations, as well as
carrying out an extensive analysis of phenotypegenotype correlation. These keratinocytes will
be used to uncover the role of p63 proteins and
pathways in normal and abnormal skin development.
Collectively, the prevalence of ectodermal dysplasia syndromes (EDS) is estimated at 7 cases in
10 000 births. Currently, there is no cure for these
patients. By creating the EPISTEM consortium,
the project partners will address from different
angles (genetics, gene proiling, molecular and
cellular biology, structural biology, drug design,
bioinformatics) the molecular pathways involved
in epidermal dysplasia syndromes, making use of
different technologies (mutation analysis, microarray, ChiP, transgenes, proteomics, in vitro skin
cultures, crystallography, etc.). The consortium
brings together leading European clinicians, geneticists, molecular and cellular biologists, structural biologists, a drug designer and bioinformatics specialists in the ield of p63 (and related
molecules) research.
32
LSHB-CT-200-019067
IntegratedProject
e฀8130000
1January2006
48months
www.epistem.eu
The consortium is building relevant in vitro and in
vivo skin disease models for studying the role of
p63 in EDS disease, and the genetic assessment
of novel pathways discovered during this project.
These models will be used for drug assessment
as well. It will also provide insight into the regulation and involvement of p63 and related pathways in skin differentiation, the maintenance of
the proliferative capacity of epithelial stem cells
and the transition of ectodermal cells to epidermal stem cells.
Screening for and design of novel therapeutic
drugs, based on three dimensional p63 models,
which will refold/reactivate or inhibit p63 mutants and induce biological responses in relevant
disease models, is also key.
Expected outcome:
First of all, the EPISTEM consortium will generate thorough insight into the molecular biology
of a rare disease such as EDS, for which no cure
is currently available. Second, characterising and
11/8/07 10:48:59 AM
understanding how epidermal stem cell maintenance is regulated by p63 could be beneicial
for the treatment of burn victims, since these
stem cells could be used for tissue regeneration.
Therefore, as currently estimated, the EPISTEM
research proposal may have a far broader impact
on clinical practice and for biomedical industry
in the long run. Third, this integrated project will
generate knowledge and technology that is applicable not only to p63 itself, but also to its family
members p53 and p73; p53 being an important
target that is mutated in most cancers and p73
being an important molecule for neurogenesis.
Fourth, from a technological point of view, the
EPISTEM proposal will invest in the development
of chIP (chromatin immunoprecipitation) on chip
technology.
Main findings:
The project partners have collected material
from 226 patients in 119 families, all with mutations in the p63 gene. Expression vectors suitable for transient expression in mammalian cells
are available for 6 of the p63 isoforms ( N and TA
combined with alpha, beta and gamma). Transactivation assays have been carried out for several p63 mutations found in patients. EPISTEM
has generated two transgenic mice lines overexpressing TAp63 and two lines overexpressing Np63 under the K5 promoter p63 wild-type
background. The consortium recently developed
a model of ectodermal/epidermal differentiation from mouse embryonic stem (ES) cells, and
showed that BMP4 leads to the apoptosis of ESderived neuroectodermal cells while inducting
epidermal fate.
Target genes for speciic p63 isoforms selectively
expressed in proliferating and differentiating
keratinocytes have been identiied. EPISTEM has
validated several p63 target genes identiied by
the gene array in vivo, using the transgenic mice
and the TAp63 and/or DNp63 genetically complemented mice. The partners have shown that
Figure 1: Various combinations of ectodermal dysplasia,
orofacial clefting and limb malformations are the hallmark of
p63-associated syndromes. EEC syndrome is the prototype
of these syndromes and together with LMS shows all three
hallmarks. ADULT syndrome patients never show orofacial
clefting, whereas AEC and RHS never show limb defects.
Non-syndromic limb defect condition (SHFM4) and non-syndromic cleft lip/palate (NSCL) are also caused by mutations
in the p63 gene.
p63 functions as a negative regulator of p21
expression in keratinocytes through an unexpected and complex mechanism that involves a
reciprocal negative cross-talk between the p63
and Notch signaling pathways.
In order to determine the role of caspase-14, the
consortium generated caspase-14 deicient mice.
They determined the NMR solution structure of
the SAM domain mutant (L518F) which causes
the Hay-Wells syndrome.
Initial experiments using Saos-2 cells expressing
exogenous temperature sensitive (ts) A167P mutant TAp63 revealed that treatment with PRIMA1 inhibited cell growth in a mutant p63-dependent manner, as shown by the WST-1 proliferation
assay. Two human p63 point mutants have been
engineered, TAp63 -R204W and TAp63 -R304W.
Both have been cloned in the pcDNAHA vector
and in the pTRE2pur vector, to allow regulation
by Tet (doxycyclin).
33
11/8/07 10:48:59 AM
EPISTEM
Figure 2: Pathogenic p63 mutations cause at least ive different syndromes and two non-syndromic conditions. Mutations
causing different diseeases are illustrated in different colours. Only mutations that are discussed in the text are indicated, an
overview of all mutations is given in Table 1. EEC hostpot mutations are clustered in DNA binding domain, and RHS and AEC
syndrome mutations in SAM and TI domains. Several mutations, such as R280, R313, I510, S541 and 1850 Del A, can have a
variable clinical outcome, probably due to genetic background effects. Tha black asterisks illustrate sites needed for upiquitination (K193, K194 and PY) and the white asterisk represents a sumoylation site (fKXD/E).40,42,58
Several data sets involving p53, p63 and p73 have
been collected and constructed. Furthermore, a
web tool was developed and is now available in
the private section of the website http://tools.
epistem.eu. This tool makes it possible to remain
informed of all the datasets and the tools that are
available.
transcriptional up-regulation of the IGF-I gene’, J
Biol Chem, 2006, 281, 30463-30470.
Ponassi, R., Terrinoni, A., Chikh, A., Ruini, A., Lena,
A.M., Sayan, B.S., Melino, G., Candi, E.,‘p63 and p73,
members of the p53 gene family, transactivate
PKCdelta’, Biochem Pharmacol, ,2006, 72, 14171422.
Major publications
Candi, E., Ruini, A., Terrinoni, A., Dinsdale, D.,
Ranalli, M., Paradisi, A., De Laurenzi, V., Spagnoli,
L.G., Catani, M.V., Ramadan, S., Knight, R.A., Melino,
G., ‘Differential roles of p63 isoforms in epidermal
development: selective genetic complementation in p63 null mice’, Cell Death Differ, 2006, 13,
1037-1047.
Devgan, V., Nguyen, B.C., Oh, H., Dotto, G.P.,
‘p21WAF1/Cip1 suppresses keratinocyte differentiation independently of the cell cycle through
34
Rinne, T., Hamel, B., van Bokhoven, H., Brunner,
H.G., ‘Pattern of p63 mutations and their phenotypes—update’, Am J Med Genet A, 2006, 140,
1396-1406.
Testoni, B., Borrelli, S., Tenedini, E., Alotto, D., Castagnoli, C., Piccolo, S., Tagliaico, E., Ferrari, S., Vigano, M.A., Mantovani, R., ‘Identiication of new p63
targets in human keratinocytes’, Cell Cycle, 2006,
5, 2805-2811.
Vigano, M.A., Lamartine, J., Testoni, B., Merico,
11/8/07 10:49:00 AM
D., Alotto, D., Castagnoli, C., Robert, A., Candi, E.,
Melino, G., Gidrol, X., Mantovani, R., ‘New p63 targets in keratinocytes identiied by a genomewide approach’, Embo J, 2006, 25, 5105-5116.
Coordinator
PeterVandenabeele
Flanders Interuniversity Institute for Biotechnology
VIB/UGent
Ghent, Belgium
or [email protected]
Partners
HansvanBokhoven,GijsSchaftenaar,
MartijnHuynen
Radboud University Nijmegen
Nijmegen, Netherlands
GerryMelinoandVincenzoDeLaurenzi
Università Degli Studi di Roma Tor Vergata
Rome, Italy
GianPaoloDotto
University of Lausanne
JohnMcGrath
King’s College London
London, UK
KlasWiman
Karolinska Institutet
Stockholm, Sweden
VolkerDoetsch
J.W. Goethe-Universitat Frankfurt am main
Frankfurt, Germany
RobertoMantovani
Università degli Studi di Milano
Milan, Italy
MassimoGulisano
Istituto Oncologico del Mediterraneo
Viagrande, Catania, Italy
3
11/8/07 10:49:01 AM
EPISTEM
DanielAberdam
INSERM
Nice, France
GeneSpinSrL
Milano, Italy
PiranitKantaputra
CMU
Chiang Mai, Thailand
JingdeZhu
SJTU
Shanghai, China
36
11/8/07 10:49:01 AM
ULCER THERAPY
Gene transfer in skin equivalents and stem cells: novel strategies for chronic
ulcerrepairandtissueregeneration
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Chronic skin ulceration is a frequent pathological
condition and represents a major health problem
for humans. In particular, cutaneous ulcers are a
common complication of diabetes (due to macrovascular and microvascular disease), and peripheral neuropathy, associated with skin more
vulnerable to traumatic injuries and growth factor expression reduction. Furthermore, chronic
ulcers are characterised by a tissue imbalance
between proteolytic enzymes and their inhibitors, often leading to enhanced growth factor
degradation.
Conventional therapeutic approaches (relief of
pressure at the wound site, surgical debridement,
control of infection, and in selected cases, arterial
reconstruction) are often not enough to guarantee adequate healing. Non-conventional tools for
the treatment of chronic ulcers include topical
application of recombinant growth factors — directly or mediated by viral vector gene delivery
— and transplantation of skin equivalents. However, the short half-life of exogenous growth factors, their degradation, or the ineficient delivery
of genes to target cells, are major limitations for
the treatment with recombinant factors. Analogously, the therapeutic effect of skin-equivalent
grafting is limited, mostly due to infections and
an impaired production of granulation tissue by
the host.
The goal of Ulcer Therapy is to assess the thera-
LSHB-CT-200-12102
e฀239193
1July200
36months
http://ulcertherapy.idi.it
peutic eficacy in wound healing, of a combination of the two previous approaches, based on the
generation and optimisation of skin equivalents,
engineered ex vivo to over-express therapeutic
proteins. In parallel, the therapeutic potential in
wound healing of endothelial progenitor cells
(EPCs) and mesenchymal stem cells (MSCs), genetically modiied ex vivo to express angiogenic
molecules or genes involved in stem cell survival
and proliferation, is under evaluation.
The project’s inal goal will be achieved through
the three main objectives set out below.
1. Understanding the role of growth factors,
proteases, and protease inhibitors in the pathophysiology of wound healing. To this end, a basic research approach is used to characterise the
role and mechanism of the action of potentially
therapeutic proteins. Transgenic and conditional
knockout mice are generated for studying the
involvement of novel growth factors in wound
healing. In parallel, the analysis of the proteolytic degradation of growth factors, the characterisation of the involved proteolytic enzymes,
and the examination of presence and activity of
tissue protease inhibitors is performed. The cDNA’s coding for the studied growth factors and
protease inhibitors are inserted into adenoviral
vectors that are used to deliver these proteins
into wounds practised on mice. For this activity,
animal models characterised by impaired wound
37
11/8/07 10:49:01 AM
ULCER THERAPY
Figure 1. Schematic representation of the in vivo model of
“skin-humanised mice”. Human skin equivalents are generated from cultured primary keratinocytes and ibroblasts
obtained from skin biopsies. Skin equivalents are then
orthotopically transplanted onto the back of nude mice.
healing are used. Most of the study is performed
on diabetic animals.
2. Evaluating the therapeutic potential of genetically modiied stem cells in impaired wound
healing. Circulating endothelial precursor cells
(EPCs) and mesenchymal stem cells (MSCs) secrete angiogenic growth factors, and can differentiate into endothelial cells, thus improving
angiogenesis. The capacity of isolated EPCs and
MSCs per se, or infected with adenoviral vectors
carrying angiogenic molecules or genes involved
in stem cell survival and proliferation, injected at
the wound margin in mouse model of impaired
wound healing (NOD/SCID or diabetic mice), is
therefore investigated. In parallel, a broad analysis is conducted to identify the most suitable adenoviral and adeno-associated viral vectors for
gene transfer into these stem cell types.
3. Assessing the safety and eficacy of genetically modiied skin equivalents as a delivery system for therapeutic proteins in wound healing.
To this end, quality controls, safety and eficacy
preclinical studies in vitro and on animal models
are performed. Fibrin is used as an optimal substrate to reconstitute genetically modiied composite skin grafts for the proper delivery of therapeutic factors. After a comparative analysis of the
38
Figure 2. EGFP-bioengineered “skin-humanised mice”.
Human cultured primary keratinocytes transfected with viral
vectors carrying EGFP are used to generate skin equivalents
that are grafted onto nude mice. Epiluorescence
microphotographs show the EGFP-positive epithelium,
corresponding to the human grafted skin, surrounded by
mouse skin.
gene transfer eficacy into keratinocytes and skin
equivalents of adeno and adeno-associated viral
vectors, the nerve growth factor (NGF) and the
best candidates from the genes analysed in the
course of this project, are employed for further
in vitro and in vivo preclinical studies using skin
grafts on immunodeicient mice.
Expected outcome:
Ulcer Therapy anticipates the following outcome:
• characterisation of the role and mechanism of the action of potentially therapeutic proteins in wound healing;
• characterisation of the proteolytic enzyme and the tissue protease inhibitor
function in the ulcer environment;
• identiication of the most suitable adenoviral and adeno-associated viral vectors
for gene transfer into keratinocytes and
stem cells (EPCs and MSCs);
• assessment of the quality, safety and
potential therapeutic effects of genetically-modiied skin equivalents;
11/8/07 10:49:03 AM
• assessment of the safety and eficacy
in ulcer therapy of stem cells per se or in
genetically modiied form;
• development of standard operating
procedures for the ex vivo genetic modiication of skin equivalents and their production and testing;
• design of a protocol for a phase I trial
of gene therapy in humans affected by
chronic diabetic ulcers.
Main findings:
The main results obtained during the irst period
of the Ulcer Therapy project are set out below:
• The analysis of transgenic and knockout mice indicated that increased expression of the angiogenic factor PlGF or
activin or the soluble IGF-I isoform at the
wound site, exerts a beneicial effect in
wound healing. PlGF activity has also been
investigated in diabetic mice and proved
to accelerate healing by promoting angiogenesis. Importantly, adeno-mediated
PlGF gene transfer to diabetic wounds
was able to signiicantly accelerate healing, approximating the wound closure
time of healthy controls. Different aspects
of the repair process were improved following PlGF gene transfer.
• The analysis of growth factor degradation, protease and protease inhibitors
in the ulcer environment revealed that in
chronic wounds PlGF and HGF, as already
described for VEGF, are targets of proteolytic cleavage that attenuates their activities. Site-directed mutagenesis protected
VEGF from proteolytic degradation and
enhanced its therapeutic activity in diabetic wound healing.
• Adipose tissue-derived MSCs (ATMSCs), which are easier to obtain and
propagate in culture compared to bone
marrow-derived ones (BM-MSCs), and
show capacity to differentiate towards
different lineages, were chosen for assessing the eficacy in wound healing of
MSCs. Preliminary data indicated a role
in promoting wound healing of AT-MSCs
topically administered into wounds of
diabetic mice.
• Of the different viral vectors tested,
irst generation Ad5 vectors proved to be
the most eficient in transfecting keratinocytes, while serotype-2 adeno-associated
vectors were able to eficiently transduce
genes into EPCs.
• Quality control, safety and eficacy
studies on cultured keratinocytes and skin
equivalents transfected with an adenoviral vector containing the NGF cDNA (Ad5NGF-EGF) indicated that high transgene
expression is achieved without any relevant negative effect. Moreover, the analysis of wound healing in skin equivalents
transduced with a viral vector containing the keratinocyte growth factor (KGF)
cDNA and grafted onto nude mice, indicated that KGF overexpression was able
to enhance closure. This data indicates
that humanised mice are a suitable model
for testing the effect of selected factors in
vivo.
Major publications
Cianfarani, L., Zambruno, G., Brogelli, L., Sera, F.,
Lacal, P.M., Pesce, M., Capogrossi, M.C., Failla, C.M.,
Napolitano, M., Odorisio, T., ‘Placenta growth factor in diabetic wound healing: altered expression
and therapeutic potential’, Am J Pathol, 2006, 169:
1167-1182.
Roth, D., Piekarek, M., Christ, H., Paulsson, M., Bloch,
W., Krieg, T., Davidson, J., Eming, S.A., ‘Plasmin
modulates VEGF-A mediated angiogenesis during wound repair’, Am J Pathol, 2006, 168:670-684.
Carretero, M., Escàmez, M.J., Prada, F., Mirones, I.,
Garcìa, M., Holguìn, A., Duarte, B., Podhajcer, O.,
39
11/8/07 10:49:03 AM
ULCER THERAPY
Jorcano, J.L., Larcher, F., Del Rìo, M., ‘Skin gene
therapy for acquired and inherited disorders’, Histol Histopathol, 2006, 21: 1233-1247.
Eming, S.A., Krieg, T., ‘Molecular mechanisms of
VEGF-A action during tissue repair’, J Investig Dermatol Symp Proc, 2006, 11: 79-86.
Eming, S.A., Smola-Hess, S., Kurschat, P., Hirche, D.,
Krieg, T., Smola, H., ‘A novel property of povidoniodine: inhibition of excessive protease levels in
chronic non-healing wounds’, J Invest Dermatol,
2006, 126: 2731-2733.
Perabo, L., Goldnau, D., White, K., Endell, J., Boucas,
J., Humme, S., Work, L.M., Janicki, H., Hallek, M.,
Baker, A.H., Buening, H., ‘Heparan sulfate proteoglycan binding properties of adeno-associated
virus retargeting mutants and consequences for
their in vivo tropism’, J Virol, 2006, 80: 7265-7269.
Eming, S.A., Werner, S., Bugnon, P., Wickenahuser,
C., Siewe, L., Utermöhlen, O., Davidson, J., Krieg, T.,
Roers, A., ‘Accelerated wound closure in mice deicient for interleukin-10’, Am J Pathol, 2007, 170:
188-202.
Coordinator
GiovannaZambruno
Istituto Dermopatico dell’Immacolata, IDI-IRCCS
Lab. Biologia Molecolare e Cellulare
Via dei Monti di Creta, 104
00167 Rome, Italy
Partners
MaurizioPesce
Milan, Italy
SabineWerner
Swiss Federal Institute of Technology
Zurich, Switzerland
FernandoLarcher
Centro Investigaciones Energeticas, Medioambientales y
Tecnologicas
Madrid, Spain
MichaelHallek
Clinic I of Internal Medicine
University of Cologne
Cologne, Germany
SabineEming
University of Cologne
Department of Dermatology
Cologne, Germany
NadiaRosenthal
European Molecular Biology Laboratory
Monterotondo, Rome, Italy
ElenaDellambra
IDI Farmaceutici S.p.A
Pomezia, Italy
40
11/8/07 10:49:03 AM
SKINTHERAPY
Gene therapy for edidermolysis bullosa: a model system for treatment of
inheritedskindiseases
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Epidermolysis Bullosa (EB) is a rare genetic skin
disease, affecting approximately 30 000 individuals in Europe, and approximately 400 000 to 500
000 people worldwide. The patients are predominantly children, and there is no treatment available yet.
EB is characterised by an extreme fragility of the
skin, resulting in unremitting blisters and erosions with unceasing wound healing. The skin
lesions stem from the poor adhesion of the epidermis to the underlying mesenchyme, which
makes the skin vulnerable to damage caused by
mild friction and trauma. The genetic bases of the
different clinical forms of EB have been elucidated, and a precise correlation has now been made
between genetic defects of the basement membrane components, and the types of EB which
have been classiied within the Simplex (EBS),
Junctional (JEB), and Dystrophic (DEB) forms.
In DEB, generalised blisters heal with scarring.
Extensive and progressive mutilating scarring results in increased disability and deformity of the
ingers, as well as in contractures of the joints. The
disease may also affect other areas of the body
(mouth, throat, eyes), causing discomfort, and in
most of the cases, dificulties in performing vital
activities, like eating.
The most severe cases are often complicated by
the development of aggressive skin cancer and
LHSB-CT-200-12073
e฀2079000
1April200
36months
www.debra-international.org/researche1a.htm
premature death. In addition to physical suffering, patients are faced with social dificulties:
they cannot perform physical activities, and they
need permanent assistance and regular medical
treatments. EB patients are also excluded from
the workforce, not only owing to their physical
appearance, but also because the public is uninformed and unaware of the condition.
Skintherapy will recruit patients, and perform diagnoses and genetic analyses of DEB patients. A
total of 120 patients have already been recruited.
The consortium will develop viral vectors (oncoretroviral, lentiviral, and hybrid adeno/AAV
vectors) to integrate and irmly express the collagen VII cDNA in DEB epidermal stem cells ex
vivo; work is in progress to deine the gene transfer eficiency into clonogenic keratinocyte stem
cells. Skin equivalents made with genetically corrected DEB cells to assess the full morphological
and functional reversion of the DEB phenotype
will be constructed. Skintherapy will graft the
skin equivalents expressing the recombinant
collagen VII on appropriate immune competent
animal models. The consortium is already grafting and carrying out an analysis of the fate of the
transplanted recombinant skin equivalents in
additional dogs.
The project partners will develop a technology
for the production and downstream processing
of clinical-grade viral vectors preparations, under
41
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SKINTHERAPY
Good Manufacturing and Laboratory Practices
(GMP/GLP) standards. Different cell lines are being evaluated to obtain the best transduction
rate and lentiviral production.
Expected outcome:
Skintherapy’s immediate goal is to develop a
gene therapy approach to DEB and design appropriate phase I/II trials. The expected result is
a model system for DEB treatment using ex vivo
gene therapy. Speciically, the project aims at
developing a gene therapy technology model
based on autologous transplantation of skin
made in vitro, using genetically modiied epidermal stem cells.
Major publications
Mavilio, F., Pellegrini, G., Ferrari, S., Di Nunzio, F., Di
Iorio, E., Recchia, A., Maruggi, G., Ferrari, G., Provasi,
E., Bonini, C., Capurro, S., Conti, A., Magnoni, C., Giannetti, A., De Luca, M., ‘Correction of junctional
epidermolysis bullosa by transplantation of genetically modiied epidermal stem cells’, Nat Med,
2006, Dec;12(12):1397-402. Epub 2006 Nov 19.
Carretero, M., Escámez, M.J., Prada, F., Mirones, I.,
García, M., Holguín, A., Duarte, B., Podhajcer, O.,
Jorcano, J.L., Larcher, F., Del Río, M., ‘Skin gene
therapy for acquired and inherited disorders’,
Histol Histopathol, 2006, Nov;21(11):1233-47. Review.
This model can be extended to other genetic
skin diseases requiring local or systemic delivery
of active molecules. The model system particularly targets the development of signiicantly improved, eficient and safe delivery systems, which
are urgently needed.
Main findings:
The feasibility of a phase I/II gene therapy trial
has been evaluated on a JEB patient. Epidermal
stem cells isolated from a patient affected by
JEB were transduced using a retroviral vector,
to make engineered epithelial grafts that were
transplanted back onto the patient. Synthesis,
proper assembly and long-term expression of
the recombinant laminin 5, together with the
development of a irmly adherent epidermis
with no blisters and/or infection episodes, were
observed for the duration of the follow-up (i.e.
12 months). The results will be used to establish
guidelines for eficient preclinical and clinical trials for gene therapy of epidermal cells, and also
for the development of a similar trial for RDEB
and DDEB patients.
42
11/8/07 10:49:03 AM
Coordinator
GuerrinoMeneguzzi
INSERM Unité 634
27 Avenue Valombrose
06107 Nice, Cedex 02, France
Partners
MicheleDeLuca
Centro Regionale di Recerca Sulle
Cellule Staminali Epiteliali
Fondazione Banca Degli Occhi del Veneto
Venice, Italy
FulvioMavilio
Università di Modena e Reggio Emilia
Department of Biomedical Sciences
Modena, Italy
LeenaBruckner-Tuderman
University Hospital Freiburg
Freiburg, Germany
MarcelaDelRio
Centro de Investigaciones Energeticas
Medioambiantales y Technologicas
Department of Epithelial Damage, Repair and Tissue
Engineering
Madrid, Spain
AnnaStornaiuolo
Molmed S.p.A
Discovery Division
Milan, Italy
JohnDart
DEBRA Europe
Crowthorne, UK
KarineBaudin
INSERM-TRANSFERT SA
Paris, France
43
11/8/07 10:49:04 AM
THERAPEUSKIN
Ex vivogenetherapyforrecessivedystrophicepidermolysisbullosa:preclinical
andclinicalstudies
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LHSB-CT-200-11974
e฀130000
26June200
36months
www.lyon.inserm.fr/therapeuskin
/download.htm
Individuals with recessive dystrophic epidermolysis bullosa (RDEB) suffer from life-long severe
skin and mucosal blistering followed by scarring.
The disorder causes considerable morbidity, as
well as premature mortality associated with an
increased risk of squamous cell carcinomas. RDEB
is caused by loss-of-function mutations in the
type VII collagen gene (COL7A1) encoding the
major structural component of anchoring ibrils
at the junction between the epidermis and the
dermis. No speciic treatment is currently available. The objective of the THERAPEUSKIN project
is to prepare an ex vivo gene therapy approach
by transplantation of genetically modiied skin
equivalents.
The THERAPEUSKIN project partners have developed safe [Self INactivating (SIN)] amphotropic
MFG-derived retroviral vectors in which COL7A1
expression is driven by the tissue speciic COL7A1
or the ubiquitous EF1alpha promoter. The MFG
vector was modiied due to the large size (8.9 kb)
of the COL7A1 cDNA. Viruses were produced by
tri-transfection (pCOL7A1-COL7A1 and pEF1aCOL7A1), or by a packaging cell line (InsertAgene
pEF1alpha-COL7A1).
Good transduction eficiencies were obtained
with one batch of the SINpCOL7A1-COL7A1 vector (prepared by tri-transfection) and batches
44
Figure 1. General overview of the ex vivo gene therapy approach for RDEB using SIN-COL7A1 retroviral vectors.
of the SIN-pEF1alpha-COL7A1 vector (prepared
from the packaging cell line). The SINpCOL7A1COL7A1 vector was subsequently used to genetically correct primary RDEB keratinocytes
and ibroblasts ex vivo, which were then used to
prepare skin equivalents (SE). The SE were made
of a human plasma-derived ibrin gel incorporating genetically corrected RDEB ibroblasts, onto
which genetically corrected epidermal cells were
grown.
Three months after grafting the genetically corrected SE onto nude mice, skin biopsies showed
a fully differentiated epithelium and no signs
of blisters, as well as positive, linear type VII collagen immunostaining at the dermal-epidermal
junction, as well as ultrastructural evidence of
anchoring ibril formation. Assessment of the
11/8/07 10:49:05 AM
graft after six months demonstrated the transduction of a proportion of epidermal stem cells
and ibroblasts suficient for the self-renewal of
the genetically corrected graft. These grafting
experiments are currently being repeated using
the SIN-pEF1alpha-COL7A1 vector produced by
the packaging cell line.
Expected outcome:
Reproduction of these results with the SIN-pEF1alpha-COL7A1 vector will lead to the production of a master cell bank suitable for the preparation of clinical grade GMP vector production.
Main findings:
The COL7A1-SIN vectors the partners have developed have a strong therapeutic potential for
clinical application: they have no selection markers; they are self-inactivating (reducing the risk
of oncogenic events); they provide a human promoter driving COL7A1 expression; and they can
be used in an autologous SE system. A cohort of
individuals expressing some type VII collagen
protein will then be selected for the grafting of
genetically corrected skin equivalents onto limited skin areas.
Figure 2/ A-F. In vivo restoration of type VII expression and
epidermal adherence in RDEB reconstructed skin 12 weeks
post-grafting, using COL7A1 retroviral vectors Histological
analysis (A,C,E) and type VII collagen immunostaining (B,D,F)
at three months post-grafting of skin equivalents (SE) made
of normal keratinocytes (NK) and ibroblasts (NF) (A,B), Krdeb
and Frdeb (C,D), and genetically corrected Krdeb and Frdeb
(E,F) with COL7A1 vector. Type VII collagen accumulates along
the dermal-epidermal junction in normal SE and in genetically
corrected SEs, while it is completely absent in SE made of uncorrected RDEB cells. Note the dermo-epidermal cleft ( ) in the
SE made of uncorrected RDEB cells (C,D), while no blistering is
seen in genetically corrected SE (E-F).
4
11/8/07 10:49:06 AM
THERAPEUSKIN
Coordinator
AlainHovnanian
INSERM U563
Purpan Hospital
Department of Genetics
31300 Toulouse, France
PatriciaJoseph-Mathieu
INSERM Transfert
Paris, France
Partners
MariaAntoniettaZanta-Boussif
Généthon
Evry, France
OlivierDanos
INSERM U781
Necker Hospital
Paris, France
YannBarrandon
EPFL/CHUV
IreneLeigh
QMUL
London, UK
JohnMcGrath
KCL
St Thomas Hospital
London, UK
ChristinaBodemer
Necker Hospital
Paris, France
LuisMoroder
Max Planck Institute of Biochemistry
Martinsried, Germany
JohnDart
DebRA Europe
Crowthorne, UK
46
11/8/07 10:49:06 AM
BETACELLTHERAPY
BetaCellProgrammingforTreatmentofDiabetes
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Diabetes is a prevalent chronic disease that reduces quality of life and increases the risks of lifethreatening complications. Its onset in younger
patients is caused by a massive loss of insulinproducing beta cells. Regenerating a functional
beta cell mass is thus a major goal, both in biomedicine and for society. Beta cell grafts prepared from human pancreases can cure the disease, but this form of cell therapy is hindered by
a shortage of donor organs.
The BetaCellTherapy consortium, with leading
teams in molecular, developmental and functional biology, is conducting an integrated programme to generate insulin-producing beta cells
in therapeutic quantities. This FP6-supported
programme is carried out with the R&D platform
of the JDRF (Juvenile Diabetes Research Foundation) Centre for Beta Cell Therapy in Diabetes. The
Centre organises transfer of knowledge to associated bio-industry and multicentre clinical trials,
as well as to the general public. It is an international consortium composed of an R&D platform,
a multicentre clinical trial team and associated
facilities, bio-industry and reference centres; its
central unit consists of a coordination core and
a beta cell bank.
The R&D platform explores the normal biological processes that preserve and maintain an adequate beta cell mass, and applies this knowledge
to produce functional beta cells in the laboratory
LSHB-CT-200-1214
IntegratedProject
e฀12000000
1April200
60months
www.betacelltherapy.org
and to regenerate them in vivo. The central unit,
reference centres and bio-industry function as
translation components towards clinical implementation. A multicentre team of clinicians carry
out clinical trials on the prevention and treatment of diabetes. Two trials are currently being
conducted:
• antibody intervention in recent-onset
type 1 diabetes;
• beta cell transplant trial in advanced
diabetes.
The Centre develops new diagnostics and therapeutics for these trials. It deines quality control
and safety criteria as a guide to preclinical testing. It also provides training, and interacts with
the scientiic and medical community. The Centre informs patients and the public of progress
and perspectives in the ield of its activities and
objectives. It has also established a collaborative
link to the EuroStemCell consortium, another 6th
FP integrated project
Nature’s biological programme for developing and preserving a functional beta cell mass
throughout life, is taken as a platform for directing strategies towards the laboratory production of a therapeutic beta cell mass (Fig 2). Beta
cells will be derived from embryonic stem cells
and from transdifferentiating liver, intestinal and
pancreatic exocrine cells.
Functional genomics will be used to compare
phenotypes of beta cells from new sources with
47
11/8/07 10:49:06 AM
BETACELLTHERAPY
Embryonic Stem Cells
Endodermal Stem Cells
H epatic Pancreatic Intestinal
P r ogenitor C ells
Duct/A cinar C ells
Beta Cells
Functional Beta Cell Mass
Therapeutic B eta C ell Mass
for C linical T r ials of B eta C ell T her apy in Diabetes
Fig 2 - Plan of integrated programme. The numerals refer to
the three projects.
those isolated from the pancreas. This analysis will direct further research, and determine
the start of preclinical testing. It will also generate new tools and quality control criteria that
will allow the standardisation of ongoing trials
and adjustments in graft biology, to increase its
long-term survival and function in patients. This
project should help develop a cure for diabetes
by (re)programming cells for beta cell therapy.
The overall objectives are addressed by three
interactive projects with the following speciic
objectives:
• Project I: Programming stem cells towards beta cells;
• Project II: Transdifferentiation of endodermal cells to beta cells;
48
• Project III: Programming a therapeutic
beta cell mass.
These projects are based on knowledge on the
embryonic development of beta cells; indings
demonstrating plasticity of somatic cells of endodermal origin, suggesting their potential
transdifferentiation to beta cells; and analysis of
key mechanisms that generate and maintain a
functional beta cell mass.
Expected outcome:
BetaCellTherapy expects the following outcomes:
• to use the physiological programming
of the pancreatic beta cell mass as template for generating beta cells with therapeutic potential in the laboratory;
• to collect comparative data on the eficacy and safety of beta cell generation by
embryonic stem cell and transdifferentiation strategies;
• to increase the availability of beta cell
grafts for transplantation in diabetic patients;
• to deine and disseminate quality control criteria for a standardised composition of therapeutic beta cell grafts in clinical protocols;
• to transfer knowledge for bioindustrial
and clinical implementation;
• to establish a scientiic and technologic platform for adjusting graft biology so
as to optimise its long-term survival and
function in patients;
• to organise a training programme on
the consortium’s state of the art.
Main findings:
Major achievements are presented below:
ProjectI. Programming stem cells towards beta
cells:
11/8/07 10:49:06 AM
• identiication of steps in the transcription factor cascade during embryonic formation of pancreatic cells: HNF1ß > HNF 6
> Pdx 1 > Nkx 6.1;
• identiication of Ngn 3 targets with endocrinogenic properties: MyT1 and IA1;
• the role of FGF4, FGF10 and retinoic
acid in driving endoderm to pancreas;
• development of an in vitro model that
recapitulates the steps of beta cell formation in rat embryonic pancreas;
• proof-of-concept that hES cells can be
induced into beta-like cells.
ProjectII.Transdifferentiating endodermal cells
towards beta cells:
• development of culture media for deriving beta-like cells from exocrine pancreas;
• transdifferentiation of beta-like cells
from human foetal liver cells;
• discovery of insulin-expressing cells in
the biliary duct system;
• transcriptome of pancreatic duct cells
driven to neuroendocrine phenotype following ngn3-transfection.
Project III. Programming therapeutic beta cell
mass:
• assay developed to measure beta cell
death in vitro and in vivo;
• transcriptome of adult rat and human
beta cells, and use to detect two novel
beta cell, as well as speciic markers, and to
identify beta-cell speciic genes in ngn3transdifferentiated human duct cells;
• regulation of phenotype at translational level;
• development and phenotyping mice
with Irs2-deletion in pancreatic endocrine
or beta cells;
• In vitro model for screening neoformed cells for susceptibility to cytokines.
Major publications:
Duvillie, B., Attali, M., Bounacer, A., Ravassard, P.,
Basmaciogullari, A., Scharfmann, R., ‘The Mesenchyme Controls the Timing of Pancreatic BetaCell Differentiation’, Diabetes, 2006, 55(3):582-9.
Pedersen, J.K., Nelson, S.B., Jørgensen, M.C.,
Henseleit, K.D., Fujitani, Y., Wright, C.V.E., Sander,
M., Serup, P., ‘Endodermal expression of Nkx6
genes depends differentially on Pdx1’, Developmental Biology, 2005, 288(2):487-501
Brolen, G.K., Heins, N., Edsbagge, J., Semb, H., ‘Signals from the embryonic mouse pancreas induce
differentiation of human embryonic stem cells
into insulin-producing beta-cell-like cells’, Diabetes, 2005, 54(10):2867-74.
Poll, A.V., Pierreux, C.E., Lokmane, L., Haumaitre, C.,
Achouri, Y., Jacquemin, P., Rousseau, G.G., Cereghini, S., Lemaigre, F.P., ‘A vHNF1/TCF2-HNF6 cascade
regulates the transcription factor network that
controls generation of pancreatic precursor cells’,
Diabetes, 2006, 55(1):61-9.
Baeyens, L., De Breuck, S., Lardon, J., Mfopou, J.K.,
Rooman, I., Bouwens, L., ‘In vitro generation of insulin-producing beta cells from adult exocrine
pancreatic cells’, Diabetologia, 2005, 48(1): 49-57.
Mellitzer, G., Bonné, S., Luco, R.F., Van De Casteele,
M., Lenne, N., Collombat, P., Mansouri, A., Lee, J.,
Lan, M., Pipeleers, D., Nielsen, F.C., Ferrer, J., Gradwohl, G., Heimberg, H., ‘IA1 is Ngn3-dependent
and essential for differentiation of the endocrine
pancreas’, Embo J, 2006; 25(6):1344-52.
Zalzman, M., Anker-Kitai, L., Efrat, S., ‘Differentiation of human liver-derived insulin-producing
cells towards the beta-cell phenotype’, Diabetes,
2005, 54(9):2568-75.
Bogdani, M., Suenens, K., Bock, T., PipeleersMarichal, M., In’t Veld, P., Pipeleers, D., ‘Growth and
49
11/8/07 10:49:07 AM
BETACELLTHERAPY
Functional Maturation of {beta}-Cells in Implants
of Endocrine Cells Puriied From Prenatal Porcine
Pancreas’, Diabetes, 2005, 54(12):3387-94.
Casellas, A., Salavert, A., Agudo, J, Ayuso, E., Jimenez, V., Moya, M., Munoz, S., Franckhauser, S., Bosch,
F.,‘Expression of IGF-I in pancreatic islets prevents
lymphocytic iniltration and protects mice from
type 1 diabetes’, Diabetes, 2006, 55(12):3246-55.
Coordinator
DanielPipeleers
Vrije Universiteit Brussel
Diabetes Research Centre
Laarbeeklaan 103
1090 Brussels, Belgium
Partners
Jackerott, M., Moldrup, A., Thams, P., Galsgaard,
E.D., Knudsen, J., Lee, Y.C., Nielsen, J.H., ‘STAT5 activity in pancreatic beta-cells inluences the severity of diabetes in animal models of type 1 and
2 diabetes’, Diabetes, 2006, 55(10):2705-12.
DanielPipeleers,HarryHeimberg,LucBouwens
Brussels, Belgium
FrédéricLemaigre
Christian de Duve Institute of Cellular Pathology
Brussels, Belgium
BernardPeers
Université de Liège
Liege, Belgium
JensH.Nielsen
University of Copenhagen
Copenhagen, Denmark
ShimonEfrat
Tel Aviv University
Tel Aviv, Israel
FatimaBosch
Universitat Autònoma de Barcelona
Barcelona, Spain
HenrikSemb
Lund University
Lund, Sweden
PedroHerrera
University of Geneva
Geneva, Switzerland
0
11/8/07 10:49:07 AM
JonathanSlack
University of Bath
Bath, UK
PetterBjörquist
Cellartis AB
Göteborg, Sweden
DominicWithers
University College London
London, UK
FinnC.Nielsen
University of Copenhagen Hospital Rigshospitalet
Copenhagen, Denmark
ChristelHendrieckx
JDRF Centre for Beta Cell Therapy in Diabetes
Brussels, Belgium
ZhidongLing
Academic Hospital VUB
Brussels, Belgium
PhilippeRavassard
Centre National de la Recherche Scientiique
Paris, France
YuvalDor
The Hebrew University
Hadassah Medical School
Jerusalem, Israel
RaphaelScharfmannandGérardGradwohl
INSERM
Paris, France
JorgeFerrrer
Institut d’Investigacions Biomediques A. Pi I Sunyer
Barcelona, Spain
AnneGrapin-Botton
Swiss Institute for Experimental Cancer Research
SueSwift
Beta-Cell NV
Brussels, Belgium
LucSchoonjans
Thromb-X NV
Louvain, Belgium
OleMadsenandPalleSerup
Novo Nordisk A/S
Gentofte, Denmark
ThomasMandrup-Poulsen
Steno Diabetes Centre
Gentofte, Denmark
1
11/8/07 10:49:07 AM
EUROSTEC
EuropeanprogrammeonSoftTissueengineeringforChildren
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The aim of the EuroSTEC project is to use modern
tissue engineering approaches to treat children
with structural disorders present at birth, such
as spina biida, urogenital defects, gastroschisis,
diaphragmatic hernia and esophageal atresia.
The project strives to take a translational route
through in vitro and animal experiments to early
clinical trials. Tailor-made ‘smart’ biomatrices
(scaffolds) will be prepared, using natural scaffold molecules (collagen, elastin) and/or manmade polymers (polylactic/glycolic acid), and will
be substituted with regulatory molecules such as
growth factors and glycosaminoglycans.
A variety of cells, including stem cells, ibroblasts,
muscle cells and urothelial/epithelial cells will be
cultured in vitro and seeded into biomatrices. For
major congenital birth defects, biomatrices, with
or without cells, will be prepared and implanted
using novel animal models, and evaluated for
their capacity to regenerate the correct tissues.
Biomatrices will degrade in time, and then be replaced by the bodies’ own tissues, thus assuring
compliance with growth which is especially important in young children. Prenatal and postnatal
reconstructive procedures will improve the inal
outcome of reconstructive surgery. Clinical trials
for diaphragmatic hernias will form the start of
the patient registry and protocol development
for future clinical studies.
2
LSHC-CT-2006-037409
IntegratedProject
e฀782800
1January2007
60months
www.eurostec.eu
Ethical and regulatory issues will be fully addressed before inal clinical application, and parents and children will have to be able to understand these new treatment options. A dialogue
with the general public, including patient’s associations, will be sought. Demonstration activities
will be carried out to increase the awareness of
new treatment modalities based on tissue engineering. Finally, surgeons will be trained to use
the new operation techniques.
The project combines European leaders in the
ield of biomatrices, cell culture, animal models,
surgery, and ethical and regulatory issues.
The EuroSTEC project has 5 research areas that
cover different aspects of soft tissue engineering for congenital birth defects in children. The
irst research area focuses on ‘biomatrices for tissue engineering’. Each tissue has its own unique
set and content of scaffold biomolecules. By
selectively incorporating biologically active
molecules, such as morphogens into tissue-engineering constructs, cellular behaviour may be
ine-tuned. The synthesis of biodegradable support polymers can be achieved using approved
monomers, following established procedures in
the laboratory. In this project, the aim is to develop polymers that can carry a mechanical load
for periods spanning 3 to 6 months, and that
are processed to form suturable compliant supports for the above bioactive extracellular matrix
(ECM).
11/8/07 10:49:07 AM
In research area 2, ‘cell culture systems and biomatrix interactions’, in vitro cell culture systems,
cell lines and human cell culture systems are applied to study epithelial and skin tissues, muscle
tissues, urogenital, esophagus and amniotic cells
and tissues. Interactions between vascular supply and biomatrices have been investigated for
extracellular matrices.
Now, various cell lines with different characteristics are available for study. This allows comparative analyses of scaffolds of different composition to construct cell-matrix implants.
Several of the university research centres have
developed different animal models for severe
congenital structural anomalies which can be
used for research area 3, ‘models for severe congenital anomalies’. These models can be used to
study new treatment strategies, including tissue engineering techniques. Currently, models
for foetal or postnatal investigations are available for spina biida (sheep), skin and epithelial
investigations (rat), urogenital reconstructions
(rabbit, sheep), Gastroschisis and Congenital dia-
Figure 1: Flowchart showing the interactions of the different research areas (RAs) and management overview of
the EuroSTEC project
phragmatic Hernia (sheep, rabbit) and oesophagus atresia (sheep). In foetal models and clinical
invasive procedures the disruption of amniotic
membranes plays an important role in secondary morbidity.
Currently, children with severe structural anomalies such as spina biida, bladder extrophy, gastroschisis, congenital diaphragmatic hernia (CDH),
and oesophageal atresia need extensive surgical
procedures with long-term additional morbidity. Most reconstructions consist of local skin lap
coverage of the defects, local tissue growth of the
organ after closure of the defects, or excision of
dysplastic tissues and additional reconstructions.
Scar tissue, dysplastic and ischemia can prevent
a good functional outcome and may result in
long-term complications. In EuroSTEC, ‘Clinical
treatments, pilot studies and instruments’ are
part of research area 4, which includes a study of
intervention for CDH.
3
11/8/07 10:49:07 AM
EUROSTEC
In the last few decades, many European countries
have had an ongoing debate about the following question: to what extent is it desirable and
morally acceptable to treat these children with
all kinds of technological means? This issue will
be further studied in research area 5, ‘ethical and
clinical registry and study management.’
Expected outcome:
EuroSTEC forms a strategic combination of basic
molecular, cellular and tissue structure scientiic
knowledge with clinical paediatric patient care
and surgical experience, as well as the development of new treatment strategies. The project
combines scientiic information and cooperation
between different individual scientiic groups
working in the ield of tissue engineering in Europe. Technically, this will lead to new products
which can be evaluated in a preclinical setting
for applications in children with severe congeni-
4
Figure 2: Graphical presentation of the different research
areas and the partners involved in the work.
tal structural anomalies needing life-long medical care. The involvement of industrial partners
will improve the technical aspects, offer larger
production facilities and a distribution network,
as well as quality control systems. The new ‘smart’
biomaterials will be distributed by European
companies in the ield of tissue engineering,
with worldwide applications both for children
with different congenital structural anomalies
and also for markets in the ield of biomedical
devices involving all children’s hospitals.
The project should lead to a database with disease-speciic information concerning children
with severe structural anomalies that can be correlated with environmental epidemiological and
genomic databases for further elucidation of
underling disease courses. The development of
this speciic patient registration and study proto-
11/8/07 10:49:08 AM
cols will enable the start of phase II studies (and
phase II/III studies).
EuroSTEC will also lead to socio-ethical discussions and protocol development concerning
the use of new biomaterials in children and the
ethical aspects involved. The inventory of the different ethical aspects and diverse social implications in various European countries, in addition
to the evaluation and analysis of this information,
will lead to applicable protocols in all involved
clinical settings, and solutions for regulatory
problems before the start of clinical trials.
Coordinator
WouterFeitz
Radboud University Nijmegen Medical Centre
Department of Urology (659)
PO Box 9101
6500 HB Nijmegen, Netherlands
Partners
JönsHilborn
Uppsala University
Polymer Chemistry,
Dept. of Materials Chemistry
Uppsala, Sweden
PeterFrey
Centre Hospitalier Universitaire Vaudois
Dept. of Paediatric Urology and Surgery
JanDeprest
Katholieke Universiteit Leuven
Department of Gynaecology and Obstetrics
Leuven, Belgium
GerardBarki
Karl Storz GmbH & Co.KG
Tuttlingen, Germany
MartinMeuli
University Children’s Hospital Zurich
Department of Surgery
Zurich, Switzerland
RolandZimmermann
University Hospital Zurich
Department of Obstetrics
Zurich, Switzerland
AmulyaSaxena
Medizinische Universität Graz
Department of Paediatric Surgery
Graz, Austria
11/8/07 10:49:08 AM
EUROSTEC
WimWitjes
CuraTrial SMO & Research BV
Arnhem, Netherlands
NoesdeVries
European Medical Contract Manufacturing (E.M.C.M.) B.V.
BenjaminHerbage
Symatese Biomateriaux
Chaponost, France
PaulvandenBerg
University Medical Centre Groningen
Department of Obstetrics and Gynaecology
Groningen, Netherlands
EduardGratacos
Institute d’Investigacions Biomediques August Pi I
Sunyer
Foetal Medicine + Therapy Research Group
Barcelona, Spain
KyprosNicolaides
Foetal Medicine Foundation / King’s College Hospital
Harris Birthright, Foetal Medicine Unit
London, UK
IngoHeschel
Matricel GmbH
Herzogenrath, Germany
6
11/8/07 10:49:09 AM
SC&CR
Application and process optimisation of human stem cells for myocardium
repair
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LHSB-CT-2004-02988
e฀194200
1February2004
48months
www.SC-CR.eu
Sudden untreated occlusion of a coronary artery,
following plaque rupture and/or vasospasm, may
result in extensive necrosis of the myocardium,
i.e. a transmural myocardial infarction. Because
remaining cardiomyocytes are not able to replicate, the loss of cardiomyocytes after infarction is
irreversible, and the tissue defect heals by ibrotic
scarring after eight weeks. Moreover, post-infarction geometric disarrangement of the myocytes
and extracellular matrix, dilation of ventricular
cavity and compensatory hypertrophy of the remaining myocardium, a process deined through
ventricular remodelling, may contribute to development of ventricular dysfunction.
Autologous transplantation of bone marrow
stem cells into the infarcted myocardium is an innovative and promising strategy for the therapy
of heart failure due to ischemic heart disease. The
SC&CR project proposes a strongly integrated approach to verify human stem cells’ safety and eficacy for the treatment of ischemic myocardium,
and to identify interventions that can be rapidly
translated to clinical practice, in order to prevent
or reduce damage to ischemic tissue.
peutic potential of these cells. In general, there
are several prerequisites to the validation of
large-scale use of stem cells for therapeutics,
namely:
• identifying the most suitable stem cell
type(s) to be used, to repair/regenerate a
given organ;
• knowing the combination of stimuli
that might drive differentiation of stem
cells toward a speciic lineage;
• identifying genes important for stem
cell conversion, as well as setting up safe
and effective vectors and protocols to
induce cardiac myocyte differentiation
pathway(s) by gene transfer;
• assessing the functional properties of
stem cell-derived differentiated cells;
• unravelling the action of factors that
might be involved in recruitment and
homing of endogenous stem cell in tissues where regeneration is needed;
• evaluating the mid- and long-term
effects of stem cell administration in patients, after having performed exhaustive
preliminary analysis of stem cell use in
animal preclinical models.
Expected outcome:
Despite the number of observations reporting “transdifferentiation” of tissue-derived stem
cells, there is no conclusive evidence on the
mechanism(s) underlying changes in stem cell
fate, and insuficient information on the thera-
It is expected that autologous HSC transplantation into the infarcted or chronically ischemic
myocardium may be highly effective in wound
repair, in terms of heart muscle regeneration and
improvement of coronary blood low. The con-
7
11/8/07 10:49:09 AM
SC&CR
sortium notes that while the approach described
in SC&CR — i.e. pharmacological mobilisation,
collection by means of apheresis and intra-myocardial or intra-coronary injection of stem cells
under direct vision — has not been used by other groups, it offers several advantages compared
to other methods such as the collection of stem
cells from the bone marrow by direct aspiration,
and from the skeletal muscle by biopsy.
Firstly, it is painless, it does not require general
anaesthesia and it does not imply any blood loss.
This is a major advantage because it reduces the
overall procedural risk in certain subsets of patients, like those affected by heart failure and refractory angina pectoris. Thus far, no major complication has emerged following the clinical use
of the granulocyte colony stimulating factor, in
patients affected by ischemic heart disease.
There is a great deal of clinical experience of HSC
mobilisation and collection by apheresis in the
haematological ield. This technique is adopted
even in healthy donors without signiicant complications. Additionally, HSCs do not require any
ex vivo pharmacological manipulation or culture
in order to modulate their differentiation potential. Moreover, the number of collected cells is
signiicantly higher than after bone marrow aspiration (60-80 x106 vs. 10-30 x106 BMSC).
The intra-myocardial injection under direct vision, during a CABG procedure, allows precise anatomic identiication of the target area, and even
of the distribution of the injections. By contrast,
the intravenous route is limited by the irst-pass
attenuation effect on the BMSC into the pulmonary circulation, and by the fact that the myocardium receives only 3-5% of the whole cardiac
output. Consequently, the number of stem cells
populating an infarcted or ischemic area may be
signiicantly reduced.
Finally, the use of autologous adult BMSC does
not imply any ethical issues, in contrast with em-
8
bryonic stem cells. The clinical use of HSCs in the
treatment of severe forms of ischemic heart disease may represent an additional or alternative
therapeutic option that may beneicially modify
the unfavourable course of the disease. HSCs
transplantation may be combined with conventional therapies in order to add further beneit. Repopulating areas of muscle loss, restoring
¬— at least partly ¬— the systolic and diastolic
properties of the left ventricle, and stimulating
the growth of new blood vessels may reduce the
high incidence of mortality and complications
associated with heart disease. Both a signiicant
improvement in the quality of life of the patients,
and a decrease in the social-economic burden of
ischemic heart disease are expected.
Main findings:
During the irst reporting period of this project,
work was carried out in order to understand
molecular events associated with cardiac myocyte differentiation of stem cells using cell culture, physiology, genetic transfer, preclinical and
clinical approaches. This work has involved the
development of individual activities sustained
by a high intra-network dissemination of results,
and the sharing of tools and common platforms.
Work has been carried out in four areas:
Clinical Activities: In this part of the project, two
independent phase I trials of autologous bone
marrow-derived stem cells, in patients suffering
chronic ischemic heart disease are being carried
out.
Physiologic assessment of stem cell differentiation: Differentiation of stem cells in the myocardium was assessed morphologically in several
studies in different preclinical models. However,
the extent of functional integration into the myocardium is still a matter of debate. In this part
of the project, the consortium targeted clarifying
at a functional level the differentiation of several
stem cell types, using cell culture techniques.
11/8/07 10:49:09 AM
In vivo preclinical studies: The consortium’s goal
was to assess the effect of factors and genes potentially involved in the recruitment, homing, differentiation and proliferation of stem cells in the
infarcted heart.
In vitro studies: A number of groups belonging
to the SC&CR consortium sought to analyse the
differentiation of stem cells by cell culture, gene
manipulation, genomic analysis and biopolymers
embedding.
Major publications
Minetti, G.C., Colussi, C., Adami, R., Serra, C.,
Mozzetta, C., Parente, V., Fortuni, S., Straino, S.,
Sampaolesi, M., Di Padova, M., Illi, B., Gallinari, P.,
Steinkuhler, C., Capogrossi, M.C., Sartorelli, V., Bottinelli, R., Gaetano, C., Pure, P.L., ‘Functional and
morphological recovery of dystrophic muscles
in mice treated with deacetylase inhibitors’, Nat
Med, 2006, 12:1147-1150.
Brugh, S.A., Zahanich, I., Rüschenschmidt, C., Bekc,
H., Blyszczuk, P., Czyz, J., Heubach, J.F., Ravens, U.,
Horstmann, O., St-Onge, L., Braun, T., Brustle, O.,
Boheler, K.R., Wobus, A.M., ‘Signals from embryonic ibroblasts induce adult intestinal epithelial
cells to form nestin-positive cells with proliferation and multineage differentiation capacity in
vitro’, Stem Cells, 24:2085-2097.
De Boer, T.P., Van der Heyden, M.A.G., Rook,
M.B., Wilders, R., Broekstra, R., Kok, B., Vos, M.A.,
De Bakker, J.M.T., Van Veen, T.A.B., ‘Pro-arrhythmogenic potential of immature cardiomyocytes
is triggered by low coupling and cluster size’, Cardiovasc Res, 2006, 71:704-714.
De Boer, T.P., Van Veen, T.A.B., Bierhuizen, M.F.A.,
Kok, B., Rook, M.B., Boonen, K.J.M., Vos, M.A., Doevendans, P.A., De Bakker, J.M.T., Van der Heyden,
M.A.G., ‘Connexin 43 repression following epithelium-to-mesenchyme transition in embryonal
carcinoma cells requires Snail 1 transcription factor’, Differentiation, 2007, Mar;75(3):208-18.
D’Arcangelo, D., Ambrosino, V., Giannuzzo, M.,
Gaetano, C., Capogrossi, M.C., ‘Axl receptor activation mediates laminar shear stress anti-apoptotic
effects in human endothelial cells’, Cardiovasc
Res, 2006, 71:754-63.
Lagostena, L., Avitabile, D., De Falco, E., Orlandi,
A., Grassi, F., Iachininoto, M.G., Ragone, G., Fucile,
S., Pompilio, G., Eusebi, F., ‘Electrophysiological
properties of mouse bone marrow c-kit cells cocultured onto neonatal cardiac myocytes’, Cardiovascr Res, 2005, 66:482-492.
Nikolova, T., Wu, M., Brumbarov, L., Alt, R., Opitz,
H., Boheler, Cross M., Wobus, A.M., ‘WNT-conditioned media differentially affect the proliferation and differentiation of cord blood-derived
CD133+cells in vitro’, Differentiation, 2007, 74
Feb;75(2):100-11.
Wiese, C., Rolletscheck, A., Kania, G., NavarreteSantos, A., Anisimov, S.V., Steinfarz, B., Tarasov, K.V.,
9
11/8/07 10:49:09 AM
SC&CR
Coordinator
MaurizioC.Capogrossi
Istituto Dermopatico dell’Immacolata – IDI – IRCCS
Laboratorio di Patologia Vascolare
Via dei Monti di Creta
104 – 00167 Rome, Italy
MichaelHallek
University of Cologne Clinical Department
Cologne, Germany
Partners
MaurizioPesce
Milan, Italy
AntonioZaza
Università degli Studi Milano-Bicocca
Milan, Italy
AldonaDembinska-Kiec
The Jagiellonian University
Krakow, Poland
AnnaMagdalenaWobus
Institute of Plant Genetics and Crop Plant Research
CarstenWerner
Institut für Polymerforschung Dresden e.V.
Dresden, Germany
JacquesDeBakker
Interuniversity Cardiology Institute of the Netherlands
Utrecht, Netherlands
NadiaRosenthal
MariaLuisaNolli
Areta International
Gerenzano, Varese, Italy
60
11/8/07 10:49:09 AM
STEMSTROKE
Towardsastemcelltherapyforstroke
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Stroke is a major cause of long-term disability
in humans, and there is a lack of effective treatments. Stroke affects about 3.5 million people
in the EU, with 700 000 new cases every year.
Although most stroke survivors spontaneously
recover to some degree, more than half of stroke
patients suffer signiicant residual impairments,
creating enormous economic and societal burdens. For example, Sweden reports an incidence
of 213 irst-ever strokes per 100 000 individuals,
indicating that the total excess direct and indirect cost of stroke would be 1.5 billion, with almost 45% of the direct costs attributed to social
services.
The extension of the average human lifespan has
resulted in a steady increase in the incidence of
stroke among the expanding elderly population,
creating a serious hazard to quality of life and
also increasing health care costs. From this perspective, all efforts to bring the latest advances of
science and technology a step closer to the development of therapies against stroke, is of great
importance.
The possibility to isolate and propagate neural
stem cells (NSCs) with self-renewal and multipotential properties from the foetal and adult nervous system, as well as embryonic stem (ES) cells
and their potential applications in cell therapy,
have attracted a lot of research interest in recent
years. This ield is just starting to reveal its poten-
LHSB-CT-2006-03726
STREP
e฀24708
1January2007
36months
www.stemstroke.eu
tial, and many aspects related to basic biological
properties of NSCs, and their possible application
as therapeutic tools, still has to be established.
The limited capacity of NSCs to be expanded in
vitro, and the enormous need to develop reproducible technology to obtain a stable and suficient number of NSCs as a source for cell therapy
in neurodegenerative diseases, makes it important to characterise NSCs obtained from different
sources, and to explore their capacity to survive
and integrate into the damaged brain.
A novel mechanism for neuronal replacement
after stroke, i.e., the formation of new neurons
from the adult brain’s own NSCs, was demonstrated recently. New neurons are generated
in the subventricular zone and migrate to the
striatum, where they differentiate into mature
neurons with the characteristics of those which
have died. This potential self-repair mechanism
has raised a lot of interest in both the scientiic
and clinical communities. Moreover, recent data
indicate that stroke-induced neurogenesis is
long-lasting and that endogenous NSCs in the
adult brain produce new mature neurons over
several months following a stroke. The long-lasting neurogenesis occurs concomitantly with the
spontaneous recovery of motor function, which
is observed over several months in both animals
and humans affected with stroke. Whether there
is any causal relationship between striatal neurogenesis and behavioural recovery after stroke is
as yet unknown.
61
11/8/07 10:49:09 AM
STEMSTROKE
cognitive deicits. Finally, the transplantationand endogenous neurogenesis-based strategies
will be optimised in animal models of stroke, and
with close collaboration between basic scientists
and clinicians, a preclinical protocol for stem cell
application in stroke will be developed.
Expected outcome:
Strategies for stem cell therapy in stroke patients explored by StemStroke
The main aim of StemStroke is to develop novel
strategies to repair the brain and restore function
after stroke based on the transplantation of NSCs
or their derivatives, or stimulation of the production of new neurons from the adult brain’s own
NSCs.
Two main strategies to generate neurons for replacement in the stroke-damaged brain will be
used in StemStroke. In the irst strategy, NSCs isolated from human ES cells, or from human foetal
or adult brain tissue, are expanded in culture, genetically modiied (if needed) and subsequently
transplanted into the recipient subjected to
stroke. In the second strategy, neurogenesis from
endogenous NSCs is stimulated using tools developed in the project, leading to the increased
survival, migration or maturation of newly
formed neurons.
The morphological and functional integration of
grafted and endogenously generated NSCs and
their progeny in the stroke-damaged brain, will
be assessed using immunocytochemistry, anatomical tracing techniques and patch-clamp electrophysiology. A new in vivo MR-based imaging
and behavioural test battery, developed within
the proposed project, will be used to assess stem
cell function and recovery of sensory, motor and
62
By providing a virtually unlimited source of different types of neurons and glia, this stem cell
technology may become the scientiic breakthrough that will render cell replacement a useful treatment strategy for stroke patients.
Involving top European scientists with complementary expertise and one SME, StemStroke,
using NSCs to repair the stroke-damaged brain,
can make this research state-of-the-art through
unique and diverse means:
• by demonstrating the capacity of stem
cells of various sources to expand ex vivo
and to differentiate into speciic neuronal
phenotypes;
• by identifying tools and molecules that
can stimulate endogenous neurogenesis
based on detailed knowledge about its
regulatory mechanisms;
• by determining whether neurons generated from NSCs can be anatomically and
functionally integrated into the strokedamaged brain;
• by developing MRI-based imaging for
monitoring the survival, migration and differentiation of endogenous, and grafted
NSCs and their progeny, as well as the alteration of the lesion in response to stem
cell therapy;
• by showing the level of functional recovery after stroke that can be induced by
grafted and endogenous NSCs;
• by developing the irst, scientiicallybased preclinical protocol for the application of stem cells in stroke patients.
11/8/07 10:49:10 AM
Coordinator
ZaalKokaia
University Hospital
Lund Strategic Research Centre for Stem Cell Biology and
Cell Therapy
Laboratory of Neural Stem Cell Biology and Neuroscience Programme, BMC B10
SE-221 84 Lund Sweden
Partners
AustinSmith
Wellcome Trust Centre for Stem Cell Research
Cambridge, UK
MathiasHoehn
Max-Planck-Institute for Neurological Research
In-vivo-NMR Laboratory
Cologne, Germany
LilianaMinichiello
Mouse Biology Unit
StephenB.Dunnett
Cardiff University
Brain Repair Group at School of Biosciences
Cardiff, UK
LilianWikström
NeuroNova AB
Stockholm, Sweden
63
11/8/07 10:49:10 AM
STEMS
Pre-clinicalevaluationofstemcelltherapyinstroke
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The use of stem cells with multipotent properties
has become a challenging area of research for
most clinical ields. It is of particular importance
in disciplines that desperately lack treatment options, such as brain disorders and lesions. In this
context, stroke or ischemic cerebrovascular disease is an important target. Stroke accounts for
roughly half of the patients hospitalised for neurological diseases, and is associated with a large
proportion of the healthcare costs in Europe.
Until now, all neuroprotective approaches that
have yielded positive results in animal models
of stroke have proved ineffective in clinical trials.
Given their expected capacity to self-renew and
differentiate eficiently into the desired cell type,
clonal populations of stem cells (SC) promise to
produce beneicial effects in a number of diseases. Several studies already indicate that SC transplantation has therapeutic potential for stroke,
using embryonic, foetal and adult SC sources,
and lines derived from teratoma.
However, a great deal of crucial information must
be found before SC transplantation becomes a
clinical reality. For instance, the standardisation
of the conditions to regulate SC proliferation and
differentiation so as to produce region-speciic
grafts needs to be better deined; and changes in
the properties of SC, induced by transplantation
into lesioned brain structures, are poorly understood, as is the full extent of functional improve-
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ment at long-term post-stroke delays. STEMS
aims speciically at determining the extent and
limits of SC therapy in stroke, in order to pave the
way for clinical therapeutic trials.
To deine the therapeutic potential of SC in stroke,
a multidisciplinary study from human stem cell
culture to the analysis of functional recovery in
animal models of stroke will be implemented.
The main objectives are to deine the following:
• the most eficient cell source, by a
comparison of embryonic and adult neural stem cells;
• the ideal differentiation stage for
transplantation;
• the window of opportunity for transplantation;
• the set of behavioural tests and imaging parameters to improve animal model
predictiveness of functional recovery.
Expected outcome:
By addressing a fundamental roadblock in the
development pathway for stroke therapy, STEMS
will make available a series of validated methods,
models and analytical tools that will greatly enhance the capability of consortium members, as
well as stimulate the development of new avenues of research for stroke therapy. The validated
methods include a culture of speciic hESC and
hANSC cell lines, up-scaling and shipping con-
11/8/07 10:49:10 AM
ditions for both cell types, and transplantation
protocols in the stroke-injured adult brain. The
validated models will include development of a
nonhuman primate model of stroke, allowing an
evaluation of higher cognitive functions. The validated analytical tools will include homogenised
guidelines for functional evaluation among the
groups, and the development of new MRI sequences for studies in monkeys.
STEMS will provide the Community with a unique
set of quantiied data in stroke research, new
standards, and the proof of concept of a new
therapeutic approach for stroke. It is the consortium’s belief that the results will encourage groups
of researchers to cooperate, and stimulate industrial interest for the ield, so as to rapidly regenerate European forces on stroke experimental and
clinical research.
Figure A Nestin-positive cells from hESC-derived progenitors transplanted into a rat model of stroke indicate differentiation into neural cell types (picture Inserm).
Firgure B Co-localisation of Nestin (red) and human
nuclear marker (green) identiies the cells as transplanted
ones (yellow indicates the co-localisation (picture Inserm).
Figure C Magnetic resonance imaging of the brain lesion
induced by 2 hours occlusion of the middle cerebral
artery in rats (picture Inserm/CEA)
6
11/8/07 10:49:11 AM
STEMS
Coordinator
BrigitteOnteniente
INSERM Unit 549
Neurobiologie de la Croissance et de la Sénescence
2 Ter, rue d’Alésia
75014 Paris, France
EvaSykova
Institute of Experimental Medicine
Academy of Sciences of the Czech Republic
Prague, Czech Republic
Inserm Transfert SA
Department of European and International Affairs
Marseille, France
Partners
PatrikBrundin
Lunds Universitet
Faculty of Medicine
Department of experimental medical science
Lund, Sweden
BenteFinsen
Medical Biotechnology Centre
Odense, Denmark
JonasFrisen
Karolinska Institute
Department of Cell and Molecular Biology
Stockholm, Sweden
PhilippeHantraye
Centre à l’Energie Atomique
URAD2210 CEA/CNRS: Unité d’imagerie isotopique,
biochimique et pharmacologique
Orsay, France
JohanHyllner
Cellartis AB
Goteborg, Sweden
KlausReymann
Leibniz-Institut für Neurobiologie
Neuropharmacology Group
Magdeburg, Germany
66
11/8/07 10:49:11 AM
STROKEMAP
Multipotentadultprogenitorcellstotreatstroke
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Stroke is caused by the occlusion of a cerebral
artery, resulting in irreversible tissue damage for
which there is no curative treatment available as
yet. Each year, approximately one million people suffer strokes in the EU. Approximately 25%
of men and 20% of women experience a stroke
if they live to 85 years, and stroke is the second
most common cause of death worldwide.
STROKEMAP hypothesises that a universal population of stem cells that can restore bloodlow
through the ischemic region, and that can differentiate in vivo into neurons and astrocytes that
died due to cerebral ischemia will be the superior
cell population for the vascular and neural repair
needed for therapy of stroke.
The overall hypothesis is that allogeneic Multipotent Adult Progenitor Cells or MAPCs, a novel
bone marrow stem cell population irst described
by the Verfaillie lab in 2002, are an ideal candidate stem cell population, and this will be tested
in STROKEMAP. Indeed, MAPCs can generate arterial the endothelial cells and vascular smooth
muscle cells needed to restore the vasculature,
as well as the neural cells and neuroprogenitors
that could be used to restore neural circuitry.
LSHB-CT-2006-037186
e฀2400000
1October2006
36months
MAPCs endothelial cells and neural cells;
• to compare the eficacy of MAPCs or
their differentiated progeny with goldstandard cell populations (that is, bone
marrow cells currently used in animal
models) to restore circulation and neural
circuitry damaged by stroke;
• to develop innovative, non-invasive
imaging techniques that will allow it to
follow cell survival, migration and engraftment following transplantation, as well
as to gain insight into the mechanism(s)
through which stem cells repair the blood
supply and neural circuitry in the brain,
and also to provide quantitative assessment of such repair;
• to develop strategies allowing transplantation of universal donor human
stem cells, and strategies to circumvent
possible immunological rejection;
• to develop clinical scale and grade
stem cell populations that could be used
in clinical trials upon completion of the
translational and preclinical studies proposed in this project;
• to develop an ethical and legal framework in which to initiate clinical trials with
stem cells for stroke.
The objectives of STROKEMAP are:
• to carry out studies to further understanding in processes that underlie
the differentiation and specialisation of
Two work packages will determine the mechanisms that underlie differentiation of rodent
and human MAPC to arterial versus venous en-
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STROKEMAP
dothelium and to neural stem cells. This involves
differentiation in vitro, gene array studies, and for
the endothelium testing, certain principles in zebraish.
Another two work packages are aimed at the
immunological consequences of grafting MAPC
in vivo: one evaluating the principles in mouse,
and a second where tests will be carried out to
establish whether mice can be given a human
immune system using MAPC. The consortium
will then test the immunological consequences
of MAPC themselves, or MAPC-derived endothelium and neural cells, in the autologous and allogeneic setting.
A ifth work package will evaluate whether
MAPC can restore blood low in the brain and
neural circuitry. This will be performed chiely on
rodents. A sixth work package will evaluate different methods for labelling stem cells to allow
for their visualisation via PET, MRI and BLI, while a
seventh work package will focus on cell populations that will be developed, and that conform to
GMP guidelines. These populations could then be
used at the end of the granting period in phase I
or II trials, if the results obtained are encouraging.
Finally, there is also an ethics work package.
Expected outcome:
The anticipated outcome of the STROKEMAP
project is to:
• establish the possible beneicial effects of MAPC therapy in the setting of
stroke;
• develop tools to image the fate of
stem cells in the brain;
• understand the immunological consequences of MAPC transplantation;
• develop GMP procedures to expand
MAPC or their differentiated progeny;
• develop an ethical framework for
MAPC storage and use.
68
Coordinator
CatherineVerfaillie
Stamcelinstituut, K U Leuven
Onderwijs & Navorsing 1
Herestraat 49
3000 Leuven, Belgium
Partners
AernoutLuttun,StefaanVanGool,LucMortelmans,Paul
Schotsmans,BartDeMoor
Leuven, Belgium
PeterCarmelietandMiekeDewerchin
Vlaams Interuniversitair Instituut voor Biotechnology
Leuven, Belgium
FelipeProsperandIvanPenuelas
Universidad de Navarra
Pamplona, Spain
JoséManuelGarcia-Verdugo
Universidad de Valencia
Valencia, Spain
MarkusManz
Institute for Research in Biomedicine
Bellinzona, Switzerland
ErnestArenas
Stockholm, Sweden
GilVanBokkelen
ReGenesys
Brussels, Belgium
Jean-MarcIdee
Guerbet
Roissy, France
11/8/07 10:49:11 AM
RESCUE
Fromstemcelltechnologytofunctionalrestorationafterspinalcordinjury
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LHSB-CT-200-18233
e฀200000
1December200
36months
www.rescueproject.eu
According to the International Campaign for
Cures of spinal cord injury Paralysis (ICCP), more
than 130 000 people worldwide survive a traumatic spinal cord injury (SCI) each year, leading
to permanent paralysis and a lifetime of disability. Thus, with an average age at injury of 33.4
years and a nearly normal life expectancy due to
advances in healthcare, it is clear that the population of people with SCI is steadily increasing
globally.
It is estimated that by 2005, over 2.5 million people worldwide were living with SCI-induced paralysis. In Europe, there are approximately 330
000 people suffering from SCI, with more than 10
000 new cases occurring each year.
SCI has long been regarded as intractable, largely
due to the alleged inability of the central nervous
system (CNS) to regenerate. However, over the
last two decades, technological advances, combined with the understanding of the pathophysiology of SCI, have progressed to the point where
it is now conceivable to develop therapeutic
intervention strategies aimed at reconstructing
the neuronal circuitry damaged by the lesion.
One of the most powerful tools for this objective
is based on stem cells, which can be used in the
following ways:
• to introduce permissive molecules
and/or trophic agents at the level of the
lesion to improve regeneration of the severed axons;
• to replace damaged cells, grafted locally to stimulate speciic circuits such as
the central pattern generator;
• to enhance the therapeutic potential
through the activation of intrinsic stem
cells.
Rescue focuses on the combination of the most
eficient technologies to direct the fate
of intrinsic, as well as extrinsic stem cells,
and/or their transformation, in order to
obtain appropriate cell types at the right
time and in the right place, to promote repair of the injured spinal cord.
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RESCUE
RESCUE will investigate a number of different approaches that are currently available, to try to restore function of the injured spinal cord through
the use of human stem cells from bone marrow
and the central nervous system. These approaches are set out below.
• Use of stem cells during the acute
phase (within one week of the injury in
man) to reduce inlammation and secondary degeneration in the spinal cord. This
also reduces the formation of scar tissue
which constitutes a major barrier to axonal regrowth.
• Enhanced regeneration of neurons
and axons in the spinal cord through increased availability of axon growth-permissive molecules and/or trophic agents
produced by stem cells or their progeny.
Such molecules may be produced by unmodiied cells, or by cells speciically engineered to do so.
• Regeneration/restoration of function
by supporting and monitoring the activation of intrinsic spinal cord stem cells.
• Restoration of function after replacement of the damaged cells, following local
grafting.
of RESCUE can therefore be used as templates for
the elaboration of therapeutic strategies whose
application should be broadened to other CNS
traumatic damages, such as traumatic brain injuries and stroke.
Murine and human adult spinal cord stem cells
Expected outcome:
RESCUE’s inal objective is to translate experimental studies using human stem cells in preclinical animal models into the clinic. This will be
achieved through the elaboration of a series of
therapeutic tools for stem cell therapy to be used
in a wide variety of clinical paradigms of SCI.
Spinal cord lesions represent an ideal model for
the development of regenerative therapy for
traumatic lesions of the central nervous system
(CNS), as they are more prone to precise functional characterisation and follow-up than brain
injuries. Preclinical results obtained in the context
70
11/8/07 10:49:12 AM
Coordinator
AlainPrivat
INSERM
Unit 583 Physiopathology and treatment of sensorial
and motor deiciencies
Institut de Neuroscience
Hôpital Saint-Eloi
80 rue Augustin Fliche
34295 Montpellier Cedex 05, France
JackPrice
Centre for the Cellular Basis of Behaviour
London, UK
Jean-PhilippeHugnot
Université Montpellier 2
Montpellier, France
Partners
JeanSchoenen
University of Liège
Department of Neurology
Liège, Belgium
EvaSykova
Institute of Experimental Medicine
JacquesMallet
Centre national de la recherché scientiique - CNRS
Laboratoire Génétique de la Neurotransmission
Paris, France
ManuelGaviria
Neuréva
Montpellier, France
Inserm-Transfert SA
Department of European and International Affaires
Marseille, France
GaryBrook
University Hospital Aachen
Aachen, Germany
MinervaGimenezyRibotta
Consejo Superior de Investigaciones Cientiicas
Alicante, Spain
71
11/8/07 10:49:12 AM
STEM-HD
Stem cells for therapeutics and exploration of mechanisms in Huntington’s
disease
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Huntington’s Disease (HD) is a rare monogenic
disorder. Age of onset of clinical symptoms of HD
is quite variable, although the disease starts, in
the majority of cases, at adulthood (average 35
years of age). Pathological lesion primarily affects
the GABAergic output medium-spiny neurons of
the striatum. Clinical symptoms are characterised by a rapidly progressive alteration of motor
abilities (chorea, bradykinesia), psychological disturbances (depression and irritability) and cognitive impairment (of most functions, by the later
stages), leading to dementia. On average, death
occurs 15 to 20 years later.
HD is an autosomal, dominant monogenic disease. The mutation consists of an enlarged CAG
triplet repeat that encodes an elongated stretch
of polyglutamine in the N-terminal portion of the
large protein huntingtin. Neither the physiological role of huntingtin, nor the molecular mechanisms of the pathology are currently known, but
since its discovery, a number of cellular pathways,
interacting proteins and nucleic acids have been
identiied.
HD research has beneited from a direct genetic
screen for more than 12 years, and preimplantation genetic diagnosis has been proposed to
at-risk couples for more than 6 years. However,
no validated treatment — neither curative nor
symptomatic — is available for HD today.
The fundamental objective of STEM-HD is to con-
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www.stemhd.eu
tribute in a decisive manner to the understanding of the mechanisms of HD, as a necessary
step toward inding a cure. The consortium aims
at reaching two main complementary goals: to
decipher molecular mechanisms of HD, and to
identify compounds endowed with therapeutic
potential for HD.
To realise its goals, STEM-HD will perform the following activities:
• use an available human embryonic
stem (ES) cell line, based upon the hypothesis that ES cells expressing a disease-related mutant gene may be used for molecular modelling of that monogenic
disease;
• implement large-scale technological
resources, and combine resource-driven
and hypothesis-driven analyses at all stages of the project.
Expected outcome:
The identiication of new biomarkers and of the
molecular pathway of HD may lead to possible
therapeutic applications, and the development of
new diagnostic tools. These tools will allow specialists to follow the development of the disease
in patients known as carriers of the mutation,
helping them to determine the best time to start
the palliative treatments (psychological care, exercise, etc), or the treatments, once developed. It
11/8/07 10:49:13 AM
Figure A Colony of undifferentiated human embryonic
stem cells (200X, passage 19) on a monolayer of feeder
cells. hES marker proteins Oct4 and Nanog stained in red
and green, respectively; nuclear staining in blue (DAPI).
Figure B hES cells differentiated into neuronal progenitor cells (day 45). Immature neural cells stained in red
(Nestin), young neurons in green (Tuj1); nuclear staining
in blue (DAPI).
will also be of help to facilitate the identiication
of the disease in patients not known as carriers of
the mutation. Finally, it will be a valuable tool to
evaluate in vitro in patients, the effectiveness of
any new treatment in clinical trials for HD.
Although the focus of the consortium will be
dedicated fully to HD, the participants’ common
goal is to extend the value of their achievements
by making it a model disease for designing protocols and infrastructures applicable to many
other monogenic diseases. Because each rare
monogenic disease affects only a limited number
of people (less than 5 in 10 000), it has been dificult until now to allocate to each affected individual, the investments required by large-scale
approaches, that are used for non-rare diseases
both for the exploration of mechanisms and for
high throughput/high content drug screening.
The fact that these monogenic diseases are diverse in terms of their phenotype, and that each
one is relatively rare, have made them comparatively unattractive targets for drug discovery.
STEM-HD aims at directly addressing these issues, by reducing the speciicity of the process
for each disease — through the standardisation
of common protocols and infrastructures for all
Figure C Mature GABAergic neurons (day 63 of differentiation). GABAergic striatal neurons stained in red
(DARPP-32), neuronal cells in green (MAP2); nuclear staining in blue (DAPI).
© Photos Inserm
— and by restricting its workload to a minimum
through systematic automation, thus providing
the opportunity to search for therapeutics of several rare monogenic diseases.
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11/8/07 10:49:14 AM
STEM-HD
Coordinator
MarcPeschanski
INSERM Unit 421, I-STEM
BP 118, 1 rue de l’Internationale
91004 Evry cedex, France
Inserm-Transfert SA
Department of European and International Affairs
Marseille, France
Partners
JosephItskovitz-Eldor
Technion - Israel Institute of Technology
Stem Cell Centre, Faculty of Medicine
Haifa, Israel
DoroteaRigamonti
Dialectica srl
Nerviano, Italy
JacquesHaiech
Université Louis Pasteur Strasbourg
Faculté de Pharmacie
LC1 UMR7175 - Groupe Chimiogénomique et Pharmacogénomique
Illkirch, France
NicholasAllen
Cardiff University
School of Biosciences
Cardiff, UK
ElenaCattaneo
Universita degli Studi di Milano
Department of Pharmacological Sciences and Centre of
Excellence on Neurodegenerative Diseases
Laboratory of Stem Cell Biology and Pharmacology of
Neurodegenerative Disorders
Milan, Italy
KarenSermon
Research Group Reproduction and Genetics
Brussels, Belgium
74
11/8/07 10:49:14 AM
NEUROSCREEN
Thediscoveryoffutureneuro-therapeuticmolecules
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
LHSB-CT-2006-0300
SME-SpeciicTargetedResearchProject
e฀287100
1March2007
36months
NEUROscreen is an industry-led project combining novel neural stem cell bioassays and postgenomic chemical genetics, so as to discover
innovative therapies in the ields ofneurological
diseases, regenerative medicine and cancer. A key
objective is to join ledgling and well-established
European commercial partners in a pre-competitive, collective drive to translate fundamental
stem cell biology into a state-of-the-art discovery
platform, and successfully demonstrate the potential exploitability of the concept while simultaneously strengthening European bio-industry.
The project work plan consists of 6 interlinked
work packages (WPs). WP1 focuses on the procurement, derivation, banking, annotation and
establishment of quality control standards for
a range of rodent and human neural stem cell
lines, including brain stem cell tumour lines. WP2
is the bioassay design component covering the
development of key genetic modiication technology (BAC) and the establishment of a repertoire of lines containing reporter constructs. WP2
will provide a valuable chemical ‘hit’ validation
platform involving the veriication of protein
function using RNA interference.
Using modiied cell lines to design bioassays requires a number of key validatory steps to be performed in order to assess the integrity of the cells.
Novel neural stem (NS) cells stained with antibodies
speciic for a deining marker of the cells
WP3 and WP4 provide essential conirmation of
speciication potential for cell lines in use, via in
vitro (WP3) and in vivo (organotypic slice culture;
WP4) assessment. WP5 focuses on the provision
of stem cell lines of adequate quality and quantity for screening, as well as the compilation of
technology components and processes for the
automated handling of cells for the screening
activity. Cell formatting will be the precursor to
the screening activities in WP6. A two-site, twoplatform approach will be used for the screens
using a large number of chemicals pre-selected
by in silico-based design. Leads will be validated
through WP2, WP3 and WP4 activities.
7
11/8/07 10:49:15 AM
NEUROSCREEN
Expected outcome:
Partners
NEUROscreen will present a detailed description
of the comparative properties of human neural
stem cells derived either from tumor or normal
biopsies, and will offer a comparative idelity of
functional properties of neural stem cell-derived
neurons, versus those isolated acutely ex vivo.
Procedures for the automated expansion of stable, proliferating human neural stem cells, as well
a repertoire of bioassays to measure differentiation and subtype speciication from neural stem
and progenitor cells, will be set up. The NEUROscreen project will establish a repository of annotated stem cell lines, with product release criteria
based on deined QA/QC characteristics, as well
as a deined set of bioactive, optimal compounds
to be validated as contenders for prospective regenerative medicines.
Coordinator
TimAllsopp
Stem Cell Sciences Plc
Minerva Building 250, Babraham Research Campus
Babraham, Cambridge, CB22 4AT, UK
DoroteaRigamonti
Dialectica s.r.l.
Milan, Italy
PasqualeDeBlasio
BioRep s.r.l.
Milan, Italy
StefanieTerstegge
Life & Brain GmbH
Biomedizinische & Neurowissenschaftliche
Technologie-Plattform
Bonn, Germany
AustinSmith
The Wellcome Trust Centre for Stem Cell Research
Cambridge, UK
LucianoConti
Università Degli Studi di Milano
Dipartimento di Scienza Farmacologiche
Milan, Italy
OliverBrüstle
University of Bonn
Institute of Reconstructive Neurobiology
Bonn, Germany
Scientific coordinator
LilianHook
European Research Programme Manager
Stem Cell Sciences UK Ltd
Roger Land Building
Kings Buildings
West Mains Rd
Edinburgh, EH9 3JQ, UK
76
FrançoisGuillemot
National Institute Medical Research
Molecular Neurobiology Division
London, UK
CesareSpadoni
Albany Medical Research Inc
(formerly ComGenex)
Budapest, Hungary
11/8/07 10:49:15 AM
MYOAMP
Ampliicationofhumanmyogenicstemcellsinclinicalconditions
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Many groups have used animal models to investigate the possibilities of using autologous cell
therapy for muscular dystrophies, but these data
are dispersed, not always comparable and little
attention has been focused on the transfer of this
knowledge towards applications for therapeutic
trials.
Data exist on injecting murine cells into mouse
muscle, but information regarding human cells
is sparse. The feasibility of autologous myoblast
transfer therapy has already been demonstrated
for cardiac repair, even if in cardiac therapy, injected cells were mainly used to counteract the
development of ibrosis in patients devoid of any
defect in skeletal muscle.
The fact that preclinical trials developed in mouse
models of muscular dystrophies have been successful as compared to clinical trials, which used
mostly allogenic cells and resulted in very limited
clinical beneit for the patients, illustrates the urgent need for preclinical studies using human
cells.
Myoamp will aim at deining conditions and
guidelines to produce transduced human stem
cells as vectors for clinical trials. Myoamp will synergise expertises from European leaders in their
respective ield to set up conditions for autologous transfer of human stem cells in GMP conditions for the treatment of DMD by exon-skipping.
LSHB-CT-2006-037479
e฀2480000
1December2006
36months
It will ensure that these conditions and guidelines
are transferred to SME and clinicians, deining eficient integration through dedicated partners
within the 3-years duration of this project.
Many clinical trials using muscle cells have been
developed in the past for Duchenne Muscular
Dystrophy with very limited success. The recent
emergence of new therapeutic venues, based
upon post-transcriptional genetic corrections
called “exon-skipping”, have raised new hope for
this disease. Using viral transfer approaches it has
given very promising results but cannot reach
every muscle of the body and trigger a immune
response to the vector. Autologous cell therapy
may bypass this reaction and be used as a complement or alternative if the cell type used fulilled both being an eficient vector and bringing
a functional beneit to the diseased muscle.
Autologous muscle cells cannot be used since
these are already defective in dystrophic muscle, while stem cells from other origins are ideal
candidates, as long as their myogenic and proliferative potentials are ensured. In this perspective mesoangioblasts, which have already been
used in a mouse model of muscular dystrophy,
and AC133 cells have a therapeutic potential
as demonstrated in the mouse, but very little is
known about the conditions required to amplify
in GMP conditions these stem cells isolated from
humans, which is an essential step required before any clinical trial.
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MYOAMP
Myoamp will address the question of the ampliication of these cells used as autologous cell therapy vectors and their safety. The cells transduced
with a lentiviral construct allowing exon-skipping
will be selected and further ampliied. It should
be noted that transduced cells may be cloned so
that their integration site is determined), as will
be proposed in the part of myoamp that focus on
safety and ethics. In vitro and in vivo approaches
will bring understanding on the regulation of
proliferation, while the telomere length (relecting the mitotic clock status of the cell) will be
monitored, as well as a combination of receptors
known to trigger proliferation, (FGF, IGF1, ).
The stability of the parameters initially examined
in non-GMP conditions, will be checked through
ampliication in various conditions, to allow the
deinition of both guidelines for GMP production
and key-parameters to be followed during the
GMP ampliication.
The number of cells to be injected at each implantation, which is purely empirical in many clinical
trials, will be tested and optimised in a model of
implantation of human cells in immuno-deicient
mice, in order to deine the maximum number of
cells to be inally ampliied in GMP conditions.
In addition to basic knowledge on the ampliication mechanisms, myoamp will bring guidelines
and standard operative procedures to obtain
these cells in a reproducible and safe manner,
that can be directly transferred to SMEs and clinicians for clinical applications.
These guidelines will therefore address technical,
ethical and safety issues in a GMP environment.
Potential applications:
• preclinical protocols, standard operating procedures to characterise, amplify
and assess myogenic human stem cells
for autologous cell therapy treatments in
muscle disease.
• ethical and safety procedures to cover
the protocols.
• speciic culture medium with a deined
set of growth factors.
Role of SMEs
1. 3H Biomedical cell provider and responsible for deining SOP for cell handling;
2. CELLGENIX development of adapted
serum-free culture medium and deinition
of SOP;
3. GENOSAFE development of safety procedures and assays for the process.
Expected results:
The main expected result is to obtain the inal
product, i.e. protocols to obtain ampliied human
stem cells, in a state that will allow an optimised
eficiency in injections in vivo.
78
11/8/07 10:49:15 AM
VincentMouly
INSERM
UMR S 787 Myologie
Institut de Myologie
105 bd de l’Hôpital
75634 Paris Cedex 13, France
AntonOttavi
Inserm-Transfert
Paris, France
Partners
LuisGarcia
INSERM
Paris, France
YvanTorrente
University of Milan
Milan, Italy
GiulioCossu
Fondazione Centro San Raffaele del Monte Tabor
Milan, Italy
JennyMorgan
Imperial College, London
London, UK
OttoMerten
Généthon,
Evry, France
MallenHuang
3H Biomedical
Uppsala, Sweden
RolandBosse
Cellgenix Gmbh,
Freiburg, Germany
VincentGiuliani
Genosafe SA
Evry, France
79
11/8/07 10:49:15 AM
CRYSTAL
Cryo-bankingofstemcellsforhumantherapeuticapplication
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Stem cells are currently at the centre of biomedical research. Besides advancing the basic understanding of the human development and the
cellular differentiation processes, stem cells hold
the unique potential for novel therapies of degenerative diseases such as ischemia of the heart,
Parkinson’s disease, diabetes, and certain types of
tumours.
Future human stem cell therapy, however, will
have to build on a readily available, safe and reliable supply of high-quality human stem cells that
must be assured by cell banking. Current banking
approaches still rely on storing sources of stem
cells rather than on the banking of deined, wellcharacterised stem cell populations (both adult
and embryonic). It should be noted that there is
room for improvement of the conditions for reliable outgrowth, since they are still at a rudimentary stage. The isolation, identiication and culture
of stem cells are not standardised among laboratories and reproducibility of protocols is limited.
As the culture of human embryonic stem cells
routinely requires the use of animal products or
cells, their therapeutic use is ruled out. In vitro culture and expansion of haematopoietic stem cells,
either from adult bone marrow or cord blood, is
far from optimal, necessitating further research in
order to overcome problems related to the insuficient numbers of obtained stem cells and the
ageing of the stem cell population.
80
LSHB-CT-2006-037261
e฀2400000
1February2007
36months
www.crystal-eu.org
In addition, for many stem cell types cryopreservation itself is neither optimised nor validated for
different cell types, and there are multiple cellular and biophysical challenges to be addressed,
in order to deine optimised cryopreservation
protocols. Current methods represent a trade-off
between preventing formation of damaging ice
crystals and toxic effects of cryoprotectants. In
particular, the amenability of different stem cell
populations to freezing conditions is not well
understood. The viability of human embryonic
stem cells is low after freezing, and short- and
mid-term effects of freezing on cellular properties remain to be investigated.
The aim of CRYSTAL is to develop tools and procedures to enable the cryopreservation of different stem cell types for the generation of suficient
numbers of high-quality cells suitable for safe human stem cell therapy. To this end, CRYSTAL will
conduct focused research on the methods, tools
and protocols required for optimal cryopreservation and banking of stem cells.
In order to achieve this objective, the following
unresolved methodological and experimental
aspects of stem cell banking will be addressed:
• scientiic validation and optimisation
of protocols for identiication, characterisation, maintenance and expansion of
stem cells;
• establishment and validation of the
cryopreservation of stem cells;
• deinition of validation methods of the
11/8/07 10:49:15 AM
properties of stem cells after defreezing,
including maintenance and expansion,
engraftment capability, etc.
The CRYSTAL project pursues an integrated approach, using both somatic and embryonic stem
cells to address existing shortcomings that limit
the routine application of stem cell banking with
a therapeutic perspective. The consortium will
develop tools and optimised procedures to enable cryopreservation of different stem cell types
and allow for the safe production of suficient
numbers of high-quality cells for future human
therapy. This will comprise standardised protocols and tools for stem cell isolation, identiication and culture, novel approaches to their cryopreservation (e.g. novel cryoprotectants, freezing
in different conformations) and an automated
quality control system for stem cell preparations.
Five stem cell research laboratories providing
four different sources of adult (from cord blood,
bone marrow and placenta) and human embryonic stem cells have teamed up with two partners specialising in applied banking and fundamental cryobiological research. The scientiic
experimental work is supported by a unit that
will create a common knowledge base for integrating pre-existing know-how from both inside
and outside the consortium, and for guiding
partner laboratories in the implementation and
reinement of standard operating procedures for
culturing techniques, cryopreservation and validation of protocols.
The scientiic work is to be supported by professional project management and a team ensuring the effective dissemination and use of the
obtained results. CRYSTAL is thus in a position
to solve existing problems using an integrated,
systematic approach, and to provide standardised, reproducible methods and tools to advance
therapeutic stem cell research in Europe.
Figure (© IBMT): Cryopreservation issues and novel approaches to cryopreservation.
Fig. 1 - combined block face scanning electron microscopy
(back scattered mode) with freeze substitution of cryopreserved adherent murine ibroblasts. The arrow indicates an
intracellular ice domain.
Fig. 2 - scanning electron micrograph (secondary electron
mode) of adherent cells (murine ibroblasts, gold) on carbon
based nanostructured ibres (blue). The image is manually
colored, horizontal image width about 120 microns.
Expected outcome:
CRYSTAL plans to deliver a set of optimised, validated protocols, covering the core aspect of stem
cell banking, and achieving signiicant innovation
in the three areas of preparation and cultivation
methods, preservation methods and validation
methods. These optimised, validated methods
and tools will be made available to the scientiic
community to underpin initiatives on stem cell
banking, thus providing a solid foundation for
the future development of stem cell therapies.
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11/8/07 10:49:16 AM
CRYSTAL
Coordinator
JürgenHescheler
University of Cologne / Clinical Centre of the University
of Cologne
Institute of Neurophysiology
Robert-Koch-Str. 39
50931 Cologne, Germany
Partners
GuyWouters
Life-Sciences Group N.V.
HB Zutphen, Netherlands
HeikoZimmermann
Fraunhofer Institute for Biomedical Engineering (IBMT)
St. Ingbert, Germany
AndreaKolbus
Medical University of Vienna
Department of Obstetrics and Gynaecology
Vienna, Austria
AndreasZisch
University Hospital Zurich
Department of Obstetrics
Zurich, Switzerland
CatherineVerfaillie
Interdepartementaal Stamcelinstituut
Leuven, Belgium
PeterPonsaerts
University of Antwerp
Laboratory of Experimental Hematology
Antwerp, Belgium
AnnetteRingwald
ARTTIC
Paris, France
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84
New Therapies – Gene Therapy
11/8/07 10:49:17 AM
GENE THERAPY
CLINIGENE
Europeannetworkfortheadvancementofclinicalgenetransferandtherapy
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
As the ield of gene therapy has matured, opportunities have been created that are both exciting and promising, and some treatments have
already been proven clinically effective. However,
precise quality and safety standards for clinical
gene transfer have yet to be deined.
In this context, establishing optimal methods for
the production of both standard and innovative
vector systems would pave the way for accelerated product development and improved safety.
This would be of enormous value to patients,
individual investigators, industry and regulatory
authorities alike.
The move from the preclinical to the clinical
phase calls for clinical-quality grade products
manufactured for human use. In addition, the
safety proile of these products must ensure that
the potential beneits outweigh potential side
effects, thus providing patients with a safe and
eficacious treatment option.
The role of CLINIGENE is to eficiently mobilise all
interested parties, involving academic research
and production centres in particular, alongside
companies, patients’ groups and regulatory bodies. Its goal is to integrate multidisciplinary research so as to decipher the key elements that
can lead to improved safety and clinical eficacy
of gene transfer/therapy products. Control and
test methods will be established, and can then be
applied as platforms for particular gene transfer
products.
LSHB-CT-2004-018933
NetworkofExcellence
e฀12000000
1April2006
60months
www.clinigene.eu
CLINIGENE aims to:
• foster interaction between all stakeholders: regulators, preclinical and clinical
investigators, scientists, companies (otherwise competitors), as well as patients’
groups, in order to streamline integration
of multidisciplinary expertise;
• establish quality, safety, eficacy and
morally acceptable standards for clinical
gene transfer products;
• identify the ‘critical path’ to accelerate
the transit phase from the preclinical to
the clinical phase by integrating expertise
and generating new knowledge;
• improve European competitiveness by
spreading excellence and disseminating
knowledge;
• gain a clinically signiicant improvement in the treatment of human disease
through gene therapy.
The network initially analysed the bottlenecks that
exist. From this assessment, partners were able to
better understand current and future limitations,
and could then examine and offer potential solutions and methods (Table 1).
The CLINIGENE project will not establish standards in retrospect, based on previous trials; it
will rather augment the development of entirely
novel protocols using cutting-edge technology.
Practical examples of new knowledge applications and, in the longer term, of clinical success
8
11/8/07 10:49:17 AM
CLINIGENE
Technological
platforms
AAV
Adenovirus
Retrovirus
Bottlenecks
to be addressed
Current
limitations
Solutions to propose
to be tested & if
possible exempliied
by the NoE
Other bottlenecks
(not addressed by
CLINIGENE)
• Serotype production
• Reproducibility
• Titers
• Scale up
• Non-speciic
encapsidation
• Stable cell-lines
for each selected
serotype
•
•
•
•
• Gutless vector
production
•Eficiencyof
oncolytic Ad-vectors
•Nonspeciicuptake/
Targeting
• Lackof
standardized high
yield production
system
• Unspeciic
interaction of
vector with nontarget cellular
and extracellular
compartments
• Contamination by
helper virus
• Low degree of
Tumour spread
• Establish SOPs and
improved monitoring
methods to achieve
improved production and
puriication
• Improved production cell
lines
• Capsidmodiication/
polymercoating/speciic
targeting
• Genome optimisation
(Oncolytic)
• Free access to
transcomplementation celllines
• Integration
• Non-characterized
packagingcell-lines
• Targeting
Lentivirus
Besides
Retroviruses
Safety issues
Partial recombination
Validation of RCL assays
Stablepackagingcell-lines
and toxicity of viral proteins
• Lentivirus associated
tumour development in liver
• Exclusion of regulatory viral
genes (rev sequences)
• Quality of vector
preparation for in vivo use
• Mode of
production (stable
versustransient),
puriicationand
quality
• Low-yield
• Plasmid
contaminants
• Synthesis of viral
proteins by target
cells
Cell Therapy
(genemanipulated
cells)
• Differentiation
• Long-term function
•Immuneattackto
the cells
• Homogeneity of
source & phenotype
• Viability/Survival
• Distribution and
Homing
Non-viral
technologies
General
• Bacterial sequences
(CpG,selection
genes)
• Mitotic stability
• Replicating
episomes
No vectorisation
Naked DNA
• Limitation to a few
speciicsituations
•Eficacy
Physical
vectorology
Electroporation
• Safety
• Perception of the
technology (fears of
the electricity)
Chemical
vectorology Present
DNA formulations
• Purity of the products
• Too large number of
options
•
•
•
•
86
• Vector titers
•Puriication
• Stability
• Characterizedpackaging
cell-lines
• Packagingcellswith
speciicintegrationsites
• Novelpuriication,
formulation & storage
improving titers
• Pseudotyping with a
variety of virus envelopes
• Locus insultators in target
cells
• Speciicintegration
• Chromatography
basedpuriication
• Test methods &
reference material
for replication
competent
recombinant
• Testing methods for
homing & distribution
• Cell-marking
• Optimised culture
conditions
•SMAR/ORI
• Minicircles
Size of the insert
Intracellular barriers
Route of administration
Vector targeting
• Insertional
mutageneis in target
cells
• Vector titers
• Size of transgenic
sequences
• Number and
distribution of vector
integration sites per
cell
• Number and
distribution of
vector integration
sites per cell
• Cell-transformation
• Heterogeneity of
autologous primary cells
• Adventitious viruses
• Acute toxicity of
delivery method
• Potential genotoxicity
of physicochemical
methods (e.g. DNA
damage due to electric
forces in electroporation)
• Speciicelectrodes
• Control algorithms
• SOPpreparation,
evaluation,diffusion
• Training
• Lackofstandards
• Lackof
comparisons
• Reference
materials
• Standard procedures
for material preparation
11/8/07 10:49:17 AM
GENE THERAPY
are to be provided. The network will
address orphan diseases, such as
Fanconi’s anaemia or Duchenne’s, as
well as more prevalent pathologies,
including cancer, cardiovascular and
neurodegenerative disorders.
Common standards of reference must
be shared in order to establish quality. Firstly, such standards must be
tested, broad consensus must then
be reached and subsequently agreed
upon by the academic centres and the
companies specialising in the manufacture of gene transfer vectors for
the purpose of human gene therapy.
Due to the diversity of the emerging
needs, the standards will be worked
out along distinct timelines from one
vector system to the next, during the
course of the programme (Fig 1a&b).
CLINIGENE is developing platform
databases for particular vectors with
respect to their safety, eficacy and
quality standards in order to accelerate the transition from research to
clinical trials (Fig 1a&b).
Safety will be considered both in
terms of pharmaco-toxicology and
viral safety. Whatever the technology, viral safety
assessment includes a series of common checkpoints to consider and also standardise. Another
important safety issue relates to vector integration with a concurrent risk of insertional mutagenesis. The EMEA/CHMP-GTEG (now GTWP) has
reported on serious adverse events in clinical trials
involving X-linked severe combined immunodeiciency patients. More work is needed on a caseby-case basis in order to evaluate the risks according to the nature/function of the gene of interest
and the disease to be treated. The network is addressing potential improvements, using a variety
of experimental approaches. CLINIGENE recently
submitted its comments to the FDA-Long Term
Follow-Up guideline, the relevance of which is rec-
Figure 1a & 1b: The European Network Structure and
outcomes
ognised by regulatory authorities.
Pharmaco-toxicology proiles can be drawn only
in a case-by-case manner, combining the vector
system of choice and the speciic gene addressing the disease in question. A challenge for the
network is to successfully mobilise partner expertise in order to obtain useful clinical trial information, which will form the basis on an international effort to use the existing technology for
the treatment of rare diseases in particular. Practical clinical protocols will be considered, dealing
mainly with the deinition of the requirements
for patients’ clinical monitoring. These include:
• thorough deinitions of biological samples to be taken and of those to be stored
where retrospective studies are required;
87
11/8/07 10:49:18 AM
CLINIGENE
Fig. 2
• points to be considered and steps to
be taken towards the long-term followup of patients, in particular those patients
treated with integrating vectors at risk for
insertional mutagenesis.
The CLINIGENE network has adopted an integrated approach targeting high-level education,
training and human mobility of post-doctoral
scientists, doctors and pharmacists in order to
support a European dimension of highly specialised researchers, health professionals and
managers. The clinical development process is
expanded with this approach in collaboration
with the European Society of Cell & Gene Therapy. Contacts have already been established with
other EC-funded gene therapy-related research
88
programmes to streamline innovation and optimise dissemination (Fig 2).
To successfully integrate its efforts — given the
breadth of the technological approaches and applications anticipated within CLINIGENE — the
network coordinator has developed the innovative scientiic and strategic management software called Clinisoft, accessible online through
a conidential and restricted-access website. It
serves several purposes, and functions in the following ways:
• a virtual and highly hierarchic workspace supporting information exchange
and collaborative work on documents;
• an archive for all important networkproduced information and documents;
11/8/07 10:49:18 AM
GENE THERAPY
• an online coordination, planning and
network monitoring tool; and
• a direct communication tool between
deined working groups or technology
platforms.
Expected outcome:
The partnership will combine its efforts and skills
to initiate clinical approaches and decipher the
key elements that can lead to clinical success.
Implementing benchmarking activities will effectively establish good practices as well. In order to promote safe and high quality clinical
gene transfer treatments, CLINIGENE will apply
a series of imaging technologies in collaboration
with the DiMI FP6 NoE on Molecular Imaging and
Industry partners. CLINIGENE aims to achieve the
following:
• conducting of phase I trials addressing
previously-unasked questions;
• prevention of failures at the clinical
stage, as they would have negative consequences for patients entering the study
and would penalise the ield in general;
• delivery of practical results that would
open new opportunities for funding research and clinical development in this
ield, as well as promoting the expansion
of the high-tech industrial sector.
In developing the actions initiated by the Euregenethy2 network, CLINIGENE seeks to reinforce
links to the international community. The network will address NIH/OBA-RAC and FDA-CBER,
contribute to the International Liaison Group,
and promote interaction through the international committee of the American Society of
Gene therapy. By encouraging and favouring
contacts, cooperation and data sharing with
members of the international community, such
as the USA and Japan, CLINIGENE will also advance the strengthening and creation of jobs in
the biotechnology sector.
Coordinator
OdileCohen-Haguenauer
École Normale Supérieure de Cachan & AP-HP
Laboratoire de Biotechnologies et Pharmacologie génétique Appliquée (LBPA)
61, avenue du President Wilson
94235, Cachan Cedex, France
Partners
AlbertoAurricchio
TIGEM - Telethon Institute of Genetics and Medicine
Naples, Italy
RobinAli
Institute of Ophthalmology - University College London
Division of Molecular Therapy Bath St
London, UK
SegoleneAyme
Orphanet - INSERM SC11
Hôpital Broussais
Paris, France
FatimaBosch
Universitat Autònoma de Barcelona
Centre de Biotecnologia Animal i Teràpia (CBATEG)
Bellaterra, Spain
JanBubenik
Institute of Molecular Genetics
ManuelCarrondo-PedroCruz
Instituto de Biologia e Tecnologia (IBET)
Oeiras, Portugal
ChristopherBaum
Hannover Medical School
Department of Experimental Haematology
Hannover, Germany
89
11/8/07 10:49:19 AM
CLINIGENE
KlausCichutek
Paul-Ehrlich-Institut
Langen, Germany
NicoleDeglon
CEA - MIRCen
Orsay, France
GeorgeDickson
Royal Holloway & Bedford New College
School of Biological Science
Egham (Surrey), UK
GöstaGahrton
Department of Medicine, Huddinge University Hospital
Stockholm, Sweden
BerndGänsbacher
Klinikum rechts der Isar der Technischen Universität
München
Institut für Experimentelle Onkologie und Therapieforschung
Munich Germany
HansjörgHauser
Helmoltz Centre for Infection Research
AndreasJacobs
Klinik für Neurologie der Universität zu Köln
Labors für Gentherapie & Molekulares Imaging am MPI
für Neurologische Forschung
Cologne, Germany
DavidKlatzmann
UMR 7087-UPMC/CNRS-CERVI
Hôpital de la Pitié Salpêtrière
Paris, France
StefanKochanek
University of Ulm
Division of Gene Therapy
Ulm, Germany
90
NicolasMermod
Université de Lausanne
Institut de Biotechnologie
LluisMir
CNRS - UMR 8121
Institut Gustave-Roussy
Villejuif, France
PhilippeMoullier
CHU Hôtel Dieu Nantes
Laboratoire Thérapie Génique
Nantes, France
AmosPanet
The Hebrew University
Hadassah Medical School
Jerusalem, Israel
MarcPeschanski
ISTEM (Unité INSERM)
Evry Cedex, France
Maria-GraziaRoncarolo
FCSR-TIGET
San Raffaele Telethon Institute for Gene Therapy
Milan, Italy
DanielScherman
INSERM
Paris, France
RichardVile
Mayo Clinic - Gene Therapy Programme
Rochester (Minnesota), USA
ChristofVonKalle
German Cancer Research Centre (DKFZ)
National Centre for Tumour Diseases (NCT) and
Department of Translational Oncology,
Heidelberg, Germany
11/8/07 10:49:19 AM
GENE THERAPY
SeppoYla-Herttuala
University of Kuopio
A.I.Virtanen Institute
Kuopio, Finland
HeinzZwierzina
Innsbruck University
Department of Internal Medicine
Innsbruck, Austria
Anne-MarieMasquelier
Généthon
Evry, France
MartinWisher
Bioreliance
Scotland, UK
VincentZuliani
Genosafe
Evry, France
LuçaySautron
Science Pratique SA
Cachan, France
GillesAvenard
BioAlliance Pharma SA
Paris, France
CharlotteDalba
Epixis
Paris, France
FredericHenry
Clean Cells
Bouffere, France
CharlesIrving
Theravir
Jerusalem, Israel
MonicaLuskyandJean-YvesBonnefoy
Transgene
Strasbourg, France
KyriMitrophanous
Oxford Biomedica
Oxford, UK
FeliciaM.Rosenthal
CellGenix GmbH
Freiburg, Germany
MartinSchleef
Plasmid Factory
Bielefeld, Germany
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11/8/07 10:49:19 AM
CONSERT
ConcertedSafetyandEficiencyEvaluationofRetroviralTransgenesisforGene
TherapyofInheritedDiseases
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The CONSERT project integrates leading European activities for a structured implementation of
novel therapies, using genetically modiied postnatal stem cells, with a focus on the treatment of
monogenic immunodeiciencies, haemoglobinopathies, anaemias and storage disorders.
CONSERT develops and evaluates methods for
genetic stem cell modiication with wide implications for many other disorders, including viral
infection and cancer.
A central theme of the project is an unbiased
safety and eficiency evaluation of the key technology used in the genetic modiication of replicating somatic cells: retroviral vector-mediated
transgenesis. This is made possible only through
concerted multi-centre studies. Lenti-, spumaand gamma-retroviral vectors are tailor-made
for target disorders, and tested for potency and
safety in preclinical disease models. Designed
with a translational aim, basic studies in stem cell
biology and selectable marker technology complement this research.
A highly important aspect of CONSERT is the
molecular and clinical monitoring of currently
active and successful clinical trials of stem cell
gene therapy in inherited diseases. This creates
a paradigmatic data-mining activity to obtain
insights into crucial issues of clonal kinetics of
gene-modiied cells in vivo. Molecular studies in
precise cell systems and animal models provide
92
LSHB-CT-2004-00242
IntegratedProject
e฀1163000
1November2004
48months
www.gene-therapy.eu
the mechanistic understanding of transgenehost interactions. The project generates the basis
for technology development and promotes patient safety.
Translational dissemination of know-how from
academia to industry creates a network of cell
processing manufacturers with large economic
potential, and prepares researchers for future
clinical studies with improved predictability.
Genetic enhancement of cellular therapies is expected to have a major impact on the treatment
of numerous inherited and acquired disorders.
Introducing deined genes into transplantable
cells will ultimately result in a better control of
their expansion, migration, differentiation and
elimination in vivo, according to the individual’s
unique medical condition. Also, novel effectorfunctions can be introduced, such as the restoration of defective genes or protection against
drug toxicity, side effects or virus infection. Lessons learnt in the context of selected inherited
disorders will have immediate intellectual and
practical consequences for the development of
novel treatment options against acquired diseases such as cancer or viral infection.
European scientists and SMEs that hold key patents in gene transfer technologies and diagnostic assays for patient monitoring have pioneered
this ield. All are members of CONSERT, underlining its high proile; they have also provided the
irst clinical evidence of a cure for inherited dis-
11/8/07 10:49:19 AM
GENE THERAPY
orders of haematopoiesis by corrective gene transfer into haematopoietic stem/progenitor cells.
Stem cell Biology and Transduction
In vivo Selection approaches
Safety
Fundamental
Knowledge
Manufacturing
Interface
projects
Given this background, CONSERT’s
major objective is the concerted
A Genomic Interface Program
safety and eficiency evaluation of
retroviral transgenesis. This is exempliied with selected inherited
disorders as clinical targets, which
Disease Specific
Projects
Outreach &
are in urgent need of the developRed cell disorders
Training
Ethics
ment of novel curative approaches.
Wiskott-Aldrich Syndrome
Regulatory, IPR and legal issues
Leukodystrophies & Lysosomal storage disorders
CONSERT’s work programme reA tailored Education and Exchange programme
Chronic Granulomatous Disease
lects that the full potential of gene
therapy can only be developed
through an integrated approach that targets ex- seminations and ethics component, can be visuisting biological, clinical, technological, commer- alised, in a nutshell, as demonstrated below:
cial and ethical hurdles.
Main findings:
CONSERT’s scientiic structure of integrating
work packages (WPs) directed at fundamental Highlights of the work completed are presented
knowledge to treat speciic diseases with cura- below.
tive intent, as well as with a strong genomics and
Schematic representation of gene therapy for inherited
manufacturing component, and a proactive disdiseases. Areas shaded in blue, mark pharmaceutical
involvement and potential.
93
11/8/07 10:49:20 AM
CONSERT
Members of CONSERT have developed breakthroughs in basic vector technology, molecular
analysis of gene insertion proiles, and upscaling
of cell manipulation procedures for clinical use.
As with any new therapeutic approach, gene
transfer may also induce novel kinds of side effects. Somewhat relecting the leading status of
the project’s research and application, members
of CONSERT have also demonstrated the irst
cases of serious adverse events of gene transfer
into haematopoietic cells, and designed preclinical as well as clinical approaches to address and
prevent the underlying mechanisms.
The population of stem cells that allows for the
complete restoration of lymphohaematopoiesis
is dificult to enrich and maintain in vitro. Several
participants have made a breakthrough by demonstrating that growth factor stimulated stem
cells ex vivo under serum free conditions do not
lose their repopulating capacity and actually can
be ampliied ex vivo. This is now further implemented in preclinical evaluation protocols. Stateof-the-art preclinical assay systems have been
made available to the project, which include primary cell culture models, murine models of regular and genetically altered haematopoiesis, and
immunodeicient mice for the preclinical study of
human haematopoiesis, to be validated by studies in non-human primates if ethically justiied.
These are needed to address how the quality of
cell puriication, novel culture conditions, vector
improvements and cell application protocols can
be improved, to achieve long-term polyclonal reconstitution of haematopoiesis in vivo.
Leukaemia induction is still a rare complication
of stem cell gene therapy, which requires the
simultaneous existence of clonal dominance
(which might even contribute to the therapeutic effect of retroviral gene transfer) of otherwise
benign haematopoietic cells and additional
cancerogenic signal alterations. Importantly, the
partners have successfully established various
94
mouse models in which the impact of vector
design on insertional mutagenesis can be evaluated directly.
Ever-increasing data obtained by CONSERT teams
suggest that the risk of insertional mutagenesis
can be strongly reduced by improving the vector
design (using lentiviral vectors and other types
of integrating vectors with redesigned transgene
cassettes), highly puriied and limited numbers
of cells used, and the use of cell lineage speciic
expression of the transgenes. The consortium
has succeeded in developing a new generation
of self-inactivating gammaretroviral and lentiviral vectors, that are currently tested for eficacy
and safety.
In the safety evaluation and genomics work
packages, the consortium has unravelled the relationship between gammaretroviral integration
and gene expression proiles of immature stem
cells. The results have provided novel insights
into retroviral transgenesis and the effects of
retroviral integrations on stem cell behaviour, as
well as yielded novel insights into stem cell biology. These results provide a solid basis for safety
evaluation of the new generation of vectors
constructed.
Elaborating from our paradigms of successful
clinical studies and side effect mana¬g¬¬e¬ment,
the project is addressing a number of inherited
disorders for which a suficient conventional
therapy is not available besides allogeneic bone
marrow transplantation (alloBMT). Several of
the diseases chosen are paradigms for a selective advantage of gene-modiied cells (SCID-X1,
ADA-SCID, Wiskott-Aldrich-Syndrome, Fanconi
anemia), while others (chronic granulomatous
disease, thalassemia, Diamond-Blackfan anemia)
require stronger preparation of the patient for
cell transplantation or the co-expression of a lineage-restricted therapeutic gene with a selectable gene operative at the level of stem cells.
11/8/07 10:49:20 AM
GENE THERAPY
Animal models are available for all disorders, allowing disease-speciic preclinical potency and
safety evaluation. Among the clinical targets
chosen, thalassemia ultimately has the greatest
social and healthcare impact. The metabolic storage disorder metachromatic leukodystrophy
and Wiskott-Aldrich-Syndrome, have reached
the stage of pharmaceutical development preparing for clinical trial. Members of the consortium actively conduct clinical trials for SCID-X1
and ADA-SCID.
In future commercialisation development, the
leading role of European science in gene therapy
development for inherited diseases as represented in CONSERT, also translates into SME activities
with signiicant potential for intellectual property management and product development.
Three SMEs or non-proit organisations engaged
with CONSERT have documented their expertise
in retroviral technology upscaling, clinical application of advanced cell products and process
standardisation. Moreover, insertion site inventories arising from current preclinical and clinical
data mining approaches of retroviral transgenesis, create the basis for further SME participations,
in which key technologies of functional genomics are developed and implemented. The IPR position of the consortium has been fully evaluated,
and alternative models for exploitation of the results are under development.
Public interactions, ethical relections and teaching activities are integral components of CONSERT’s scientiic and technological objectives.
Expanding the well-documented activities of individual members with national and EU regulatory agencies (including EMEA), patient interest
groups, ethical committees and gene therapy societies (including ESGT), CONSERT is implementing a central ethics project to address potential
conlicts arising from cutting-edge technology
developments in somatic transgenesis.
The annual scientiic meetings are designed to
be a major educational event for the post-docs
and PhD students working within the project. In
those meetings, the ethical relection has been
implemented as a plenary workshop for all participants. Two ethics conferences, directed at establishing a European consensus for the ethical considerations involved in the development of gene
therapy for inherited diseases, are scheduled for
2007. In addition, the consortium participates in
European training courses in collaboration with
other European projects and patient organisations, and periodically approaches the lay press.
The European umbrella patient organisation
EGAN has been included as a partner in the second phase of the project, to lead the patient and
public information platform of the consortium.
The consortium is maintaining a website for access to public and professional information.
Major publications
Berglin-Enquist, I., Nilsson, E., Ooka, A., Månsson,
J.E., Olsson, K., Ehinger, M., Brady, R.O., Richter, J.,
Karlsson, S., ‘Effective cell and gene therapy in a
murine model of Gaucher disease’, Proc. Natl. Acad.
Sci. USA, 2006, 103:13819-13824. E-publ Sept 5.
Fuchs, M., ‘Gene therapy. An ethical proile of a
new medical territory,’ Journal of Gene Medicine,
2006, 8(11): p. 1358-1362.
Kustikova, O., Fehse, B., Modlich, U., Düllmann, J.,
Kamino, K., von Neuhoff, N., Schlegelberger, B., Li,
Z., Baum, C., ‘Clonal dominance of haematopoietic stem cells triggered by retroviral gene marking’, Science, 2005, 308: 1171-1174
Kustikova, O., Geiger, H., Li, Z., Brugman, M.H.,
Chambers, S.M., Shaw, C.A., Pike-Overzet, K., de
Ridder, D., Staal, F.J.T., Keudell, G., Cornils, K., Nattamai, K.J., Modlich, U., Wagemaker, G., Goodell,
M.A., Fehse, B., Baum, C., ‘Retroviral vector insertion sites associated with dominant haematopoietic clones mark “stemness” pathways’, Blood,
2006, E-publ. Nov 21.
9
11/8/07 10:49:20 AM
CONSERT
Montini, E., Cesana, D., Schmidt, M., Sanvito, F.,
Ponzoni, M., Bartholomae, C., Sergi Sergi, L., Benedicenti, F., Ambrosi, A., Di Serio, C., Doglioni, C.,
von Kalle, C., Naldini, L., ‘Haematopoietic stem cell
gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration’, Nature Biotechnol, 2006, (6):687-96.
Ott, M.G., Schmidt, M., Schwarzwaelder, K., Stein,
S., Siler, U., Koehl, U., Glimm, H., Kuhlcke, K., Schilz,
A., Kunkel, H., Naundorf, S., Brinkmann, A., Deichmann, A., Fischer, M., Ball, C., Pilz, I., Dunbar, C., ,Du
Y., Jenkins, N.A., Copeland, N.G., Luthi, U., Hassan,
M., Thrasher, A.J., Hoelzer, D., von Kalle, C., Seger, R.,
Grez, M., ‘Correction of X-linked chronic granulomatous disease by gene therapy, augmented by
insertional activation of MDS1-EVI1, PRDM16 or
SETBP1’, Nature Medicine, 2006, Apr;12(4):401-9.
Pike-Overzet, K., de Ridder, D., Weerkamp, F., Baert,
M.R., Verstegen, M.M., Brugman, M.H., Howe, S.J.,
Reinders, M.J., Thrasher, A.J., Wagemaker, G., van
Dongen, J.J., Staal, F.J., ‘Gene therapy: is IL2RG oncogenic in T-cell development?’ Nature, 2006, Sep
21;443(7109):E5; discussion E6-7.
Thrasher, A.J., Gaspar, H.B., Baum, C., Modlich, U.,
Schambach, A., Candotti, F., Otsu, M., Sorrentino,
B., Scobie, L., Cameron, E., Blyth, K., Neil, J., Abina,
S.H., Cavazzana-Calvo, M., Fischer, A., ‘Gene therapy: X-SCID transgene leukaemogenicity’, Nature,
2006, Sep 21;443(7109):E5-6; discussion E6-7.
Verhoeyen, E., Wiznerowicz, M., Olivier, D., Izac, B.,
Trono, D., Dubart-Kupperschmitt, A., Cosset, F.L.,
‘Novel lentiviral vectors displaying “early acting cytokines” selectively promote survival and
transduction of NOD/SCID repopulating human
haematopoietic stem cells’, Blood, 2005, Nov
15;106(10):3386-95.
Yanez-Munoz, R.J., Balaggan, K.S., MacNeil, A.,
Howe, S.J., Schmidt, M., Smith, A.J., Buch, P., MacLaren, R.E., Anderson, P.N., Barker, S.E., Duran, Y.,
Bartholomae, C., von Kalle, C., Heckenlively, J.R.,
96
Kinnon, C., Ali, R.R., Thrasher, A.J., ‘Effective gene
therapy with nonintegrating lentiviral vectors’,
Nature Medicine, 2006, Mar;12(3):348-53.
Coordinator
GerardWagemaker
Erasmus University Medical Centre
Faculty Building, Department of Haematology, Ee 1314
Dr. Molewaterplein 50
3015 GE Rotterdam, Netherlands
PO Box 2040, 3000 CA Rotterdam, Netherlands
Partners
DidierTrono
École Polytechnique Fédérale de Lausanne
Laboratory of Virology and Genetics
ChristopherBaum
Department of Haematology Haemostaseology and
Oncology
Laboratory of Experimental Cell Therapy
Hannover, Germany
ChristofVonKalle
National Centre for Tumour Diseases (NCT)
Heidelberg, Germany
KlausKuehlcke
European Institute for Research and Development of
Transplantation Studies AG
Oberstein, Germany
MichaelFuchs
Institut für Wissenschaft und Ethik
Section of Biomedical Ethics
Bonn, Germany
11/8/07 10:49:20 AM
GENE THERAPY
ThomasM.Pohl
GATC Biotech AG
Konstanz, Germany
JordiBarquinero
Centre de Transfució i Banc de Teixit
Unitat de Diagnostic i Terapia Molecular
Barcelona, Spain
JuanA.Bueren
Centro de Investigaciones Energeticas Mediambientales
Y Technolgicas
Haematopoiesis Programme
Madrid, Spain
LuigiNaldini
DIBIT – TIGET
Milan, Italy
MariaGraziaRoncarolo
Università Vita-Salute San Raffaele
Faculty of Medicine
Milan, Italy
LouisevandenBos
Science & Technology Transfer
Rotterdam, Netherlands
François-LoïcCosset
INSERM Unit 412
Laboratoire de Vectorologie Retrovirale et
Therapie Genique –ENS
Lyon, France
StefanKarlsson
Lunds Universitet
Laboratory of Medicine
Medical Faculty
Moleculair Medicine and Gene Therapy
Lund, Sweden
AlainFischerandMarinaCavazzana-Calvo
INSERM Unit 429
Hopital Necker Enfants Malades
Paris, France
AdrianThrasher
Molecular Immunology Unit, Institute of Child Health
London, UK
AnneGaly
Généthon
Evry, France
MaryCollins
Immunology and Molecular Pathology
London, UK
WilliamSaurin
Genomining
Montrouge, France
NicholasAnagnou
National and Kapodistrian University of Athens
Basic Sciences, University of Athens School of Medicine,
Laboratory of Biology
Athens, Greece
FulvioMavilio
Molecular Medicine S.p.A
Discovery
Milan, Italy
GeorgeVassilopoulos
Foundation of Medical and Biological Research of the
Academy of Athens
Laboratory of Cell and Gene Therapy, Centre of Basic
Research
Athens, Greece
ManuelGrez
Georg-Speyer Haus
Frankfurt, Germany
CorOosterwijk
European Genetic Alliances Network
Soestdijk, Netherlands
97
11/8/07 10:49:20 AM
GIANT
Genetherapy:anintegratedapproach
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Screening for prostate cancer is likely to have
an impact on the spectrum of disease, changing
the distribution towards organ-conined disease
that is susceptible to radical surgery, radiotherapy, and in 80% of cases, anti-androgen therapy.
However, even with such improvements and advancements, the disease burden in Europe in an
ageing male population will be substantial, considering the expense involved in screening as
well as in radical surgery (with impotence as the
most common consequence and incontinence
encountered in 3-5% of cases).
LSHB-CT-2004-12087
IntegratedProject
e฀9700000
1January200
60months
www.giant.eu.com
the transgene, and duration and stability of its
expression all inluence inal vector choice and
design. With ongoing research and discoveries in
the ield of molecular genetics, it is expected that
new generations of genetic vectors will be essential for the translation of molecular research into
clinical practice.
In the longer term, when the tumour has escaped
the prostatic capsule, and anti-androgen therapy
has failed, the tumour returns and is almost invariably fatal within 18-24 months. At this stage,
most conventional cytotoxic therapies are ineffective, although immunotherapeutic approaches and taxotere treatment have shown some
promise. Even these improvements result in little
more than a two-month extension of life.
A novel gene-based therapy, which can take account of the heterogeneity of the disease, will offer the possibility of extended life of good quality,
even if all of the tumour cells are not destroyed.
To be a successful vehicle for gene therapy, each
vector type (both viral or non-viral) has its own
particular qualities and limitations, and its degree
of development also depends on the intended
medical application. The choice of the target,
98
EGFP expressing virus Infected xenografts
Adenovirus CMV EGFP
The GIANT project will provide an international
resource to apply innovative technologies for the
modiication of existing gene therapy vectors, focusing on increased prostate targeting and de-
11/8/07 10:49:21 AM
GENE THERAPY
creased vector immunogenicity by ‘stealthing’.
The pre-existing and innate immune response
against viruses requires the inoculation of large
amounts of virus to produce a therapeutic effect
with a high cost per treatment. The GIANT modiied vectors will be tested in uniform in vitro and
in vivo models of human prostate for improved
eficacy, prior to GIANT sponsored and organised
Phase I trials.
However, this translational action will also serve
as a template for the development of innovative
treatments based on gene therapy, which can be
applied in generic terms to other tumour types,
using appropriate, tumour-speciic targeting.
The project also seeks to harmonise the regulations for clinical trials in Europe, to encourage
multicentre, multinational clinical trials of cancer
gene therapy using common stocks of GLP grade
viruses as a ‘gene medicine’ in the same way as
small molecules can now be tested in the clinic.
Expected outcome:
The GIANT consortium seeks to secure the
following:
• generation of vectors which preferentially attach to and penetrate prostate
tumour cells;
• preferential expression of optimised
therapeutic genes in prostate tumour
cells, relative to other cell and tumour
types;
• combination of the above to eliminate
the expression of the prototype therapeutic genes (HSVtk), and growth of oncolytic
adenoviruses in adjacent tissues;
• optimisation of therapeutic gene/prodrug combinations to maximise therapeutic effects;
• further development of advanced
non-viral vector systems, and assessment
of their activity preclinically in controlled
comparisons with the currently accepted
gold standard vector system for cancer
gene therapy (human adenovirus vectors);
• prevention of attachment of retargeted vectors to cells and matrix elements
within blood vessels and other tissues and
organs, to permit ultimate intravenous application of gene therapy, and minimise
treatment side effects;
• promotion of gene therapy as a valid
anti-cancer strategy by early design and
conduction of a Phase I trial, using the irst
generation of viral vectors.
Main findings:
The project partners have signiicantly improved
the technology to incorporate polypeptide-ligands in the HI loop of the adenovirus ibre. Furthermore, adenovirus ibres have been entirely
replaced by a single-chain TCR and the viral protein IX molecules have been fused with scTCRs,
with afibodies, as well as with scFv fragments.
This technology allows the modiied viruses to
bind with enhanced afinity to new target cells
(e.g.prostate cancers) and to target the delivery
of therapeutic genes.
New molecules have been designed using advanced polymer chemistry, to de- and re-target
adenoviruses by coating the viral particles. The
effectiveness of these novel polymer coated
vectors has been determined in vivo. A novel human phage display library has been generated
to provide new anti-PSMA scFv, Afibodies and
MHC-restricted Fab fragments directed against
the Prostate-Speciic Membrane Antigen. Good
manufacturing practice grade polyethyleneimine has been generated to initiate clinical trials
of non-viral vectors.
GIANT has also achieved the retargeting of nonviral vectors with cell-speciic receptors and hydrolytic degradation/reduction. Eficacy of the
Ad[I/PPT-E1A] prostate-speciic oncolytic adenovirus has been determined in vitro and in vivo as
a prelude to the irst GIANT clinical trial. Finally,
logistics for a three-country phase I trial of the
99
11/8/07 10:49:21 AM
GIANT
Ad[I/PPT-E1A] prostate-speciic oncolytic adenovirus are mostly complete, and clinical grade material has been ordered for the trial.
Major publications
Kreppel, F., Gackowski, J., Schmidt, E., Kochanek, S.,
‘Combined Genetic and Chemical Capsid Modiications Enable Flexible and Eficient De- and
Retargeting of Adenovirus Vectors’, Molecular
therapy: The Journal of the American Society of
Gene Therapy, 2005, July: 12(1): 107-17
Kraaij, R., van Rijswijk, P., Oomen, M.H.A., Haisma,
H.J., Bangma, C.H., ‘Prostate Speciic Membrane
Antigen (PSMA) Is a Tissue-Speciic Target for Adenoviral Transduction of Prostate Cancer In vitro’,
The Prostate, 2005, February 15; 62(3): 253-9
Vellinga, J., De Vrij, J., Myhre, S., Uil, T., Martineau,
P., Lindholm, L., Hoeben, R.C., ‘Eficient Incorporation of a Functional Hyper-Stable Single-Chain
Antibody Fragment Protein-IX Fusion in the Adenovirus Capsid’, Gene Ther, 2007, Apr;14(8):66470. Epub 2007 Feb 1.
Dissemination
‘New treatments for an old man’s disease’ The
House, Maitland, N.J., 2007, 32: 36-37.
Patents
100
11/8/07 10:49:21 AM
GENE THERAPY
Coordinator
NormanJ.Maitland
University of York
YCR Cancer Research Unit
Department of Biology
York YO10 5YW, UK
EllenSchenk-Braat
Erasmus MC University Medical Centre
Department of Urology H1072
Dr Molewaterplein 40
3015 GD Rotterdam, Netherlands
LeifLindholm
Got-a-Gene AB
Kullavik, Sweden
WystkevanWeerden
Scuron
NormanMaitland
Procure
York, UK
ErnstWagner
Ludwig-Maximilians-Universitat
Munich, Germany
Partners
Jean-PaulBehr
Université Louis Pasteur de Strasbourg
Illkirch, France
ChrisBangma
Erasmus MC University Medical Centre
Rotterdam, Netherlands.
PatrickErbacher
Polyplus Transfection
Illkirch, France
MagnusEssand
Uppsala University
Uppsala, Sweden
KerryFisher
Hybrid Systems
Oxford, UK
LenSeymour
University of Oxford
Oxford, UK
RobHoeben
Leiden, Netherlands
StefanKochanek
University of Ulm
Ulm, Germany
KarelUlbrich
101
11/8/07 10:49:21 AM
BACULOGENES
Useofbaculovirusasavectorforgenetherapy
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
LHSB-CT-2006-03741
e฀2499746
1January2007
36months
Gene therapy is a technique that delivers therapeutic nucleic acids into somatic cells; it is one
of the most promising therapeutic methods
under development for treating a large scope
of pathologies, ranging from genetic disorders
(e.g. myopathies) to degeneration syndromes
or cancers. The potential of gene therapy has
not yet been fully exploited, mainly because of
signiicant limitations related to the safety, gene
delivery capacities and several other properties
of the currently used vectors.
Baculoviruses (BVs) are insect pathogenic DNA viruses and are not known to replicate in mammalian cells; this gives them an advantage in terms
of safety over classical mammalian viruses currently used as vectors, like the adeno-associated
virus (AAV), the adenovirus, murine retroviruses
and lentiviruses. The most promising baculovirus
for gene therapy is the well-known Autographa
californica multiple nucleopolyhedrovirus (AcMNPV). It is inherently safe and can deliver large
pieces of DNA on its genome (≥50 Kbp).
BV replication and virus production does not occur
in mammalian cells and BV is not known to be associated with any human disease. However, by using
a vertebrate active expression cassette as a part of
baculovirus genome, eficient gene expression can
also be directed in non-target cells. A large range of
vertebrate cells has been shown to be permissive
for AcMNPV transduction, both in vitro and in vivo.
102
BV technology has been used for years for producing recombinant proteins, and thus largescale production technology is readily adaptable
for the exploitation of gene therapy approaches.
In addition, BV vectors can be used eficiently for
producing other gene therapy vectors such as
AAVs. The BV genome is well-known, and several
selective targeting approaches engineered into
the virus envelope and capsid have been developed.
BACULOGENES aims to develop clinically suitable methods for the development, production,
testing and validation of next-generation stabilised and selective BV vectors for gene therapy
applications, as well as to optimise the production of new AAV serotype vectors. Target diseases
for in vivo gene delivery with selectively targeted
BV include muscle disorders, age-related macular
degeneration and prostate cancer.
The BACULOGENES consortium consists of eight
partners from six countries, including pioneers in
the use of BVs for mammalian gene transfer applications, and two major established gene therapy
vector-producing companies in the EU. The consortium will devote its efforts not only to BV gene
therapy applications, but also to the development
of large-scale production, downstream processing, puriication and analysis methods. The quality
control and validation assays, and all issues related to regulatory aspects required for the clinical
exploitation of BV technology, will be covered.
11/8/07 10:49:22 AM
GENE THERAPY
The innovative strategy of BACULOGENES relies
on the original engineering of BVs (AcMNPV) to
make them safe, speciic and eficient vectors for
gene therapy. BACULOGENES’ approach consists
of stabilising the BV genome through deletion
and insertion of speciic sequences, in combination with transgenes relevant to the disease
targeted. The ‘stabilised’ BVs will then be engineered as follows: (a) to enhance the capacity to
deliver the therapeutic gene(s) to the right cells,
(b) to optimise the therapeutic gene expression
in the right cells, and (c) to make BVs less immunogenic and potentially invisible for the immune
system (stealth virus). Such improvements will be
obtained through envelope protein, capsid and
genome modiications.
In parallel, the baculovirus expression system
will be optimised for the production of different
serotypes of AAV by using stabilised constructs.
In addition, the development of baculovirus constructs allowing the production of recombinant
AAV without the concomitant production of baculovirus is planned.
Expected outcome:
BACULOGENES will strengthen European leadership, knowledge and competitiveness in BV
technology, through the delivery of novel and
validated eficient vectors, as well as in platform
technology, for the exploitation of potential clinical applications of gene therapy. This project will
thus pave the way for BVs to be used in clinical
applications, an area not yet explored with experiments, and will provide an optimised BVbased AAV platform for gene therapy purposes.
The project will also offer the biotech community a highly eficient manufacturing process, and
associated QC methods for BVs processing.
Coordinator
JohnMartin
Ark Therapeutics Ltd
79 New Cavendish Street
London W1W 6XB, UK
AnssiMähönen
Ark Therapeutics Oy
Kuopio, Finland
Partners
NickHunt
Altonabiotec
Hamburg, Germany
PaulaAlves
Instituto de Biologia Experimental e Tecnologica (IBET)
Oeiras, Portugal
MoniqueM.vanOers
Wageningen University
Laboratory of Virology
Wageningen, Netherlands
KariAirenne
Kuopio, Finland
LindsayGeorgopoulos
University of York
YCR Cancer Research Unit
York, UK
Otto-WilhelmMerten
Généthon
Evry Cedex, France
103
11/8/07 10:49:22 AM
THOVLEN
Targeted herpesvirus-derived oncolytic vectors for liver cancer European
network
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
THOVLEN seeks to develop safe and eficient
herpes simplex virus type 1 (HSV-1)-derived oncolytic vectors, designed to strictly target and
eradicate human hepatocellular carcinomas
(HCCs), the most common liver cancer in adults.
HSV-1 is certainly one of the most promising viral platforms for the development of improved
oncolytic vectors, as anticipated by the unique
biological properties of this virus and conirmed
by the encouraging results generated by clinical
trials in gliomas. However, the irst generations of
oncolytic HSV-1 vectors have also shown limitations regarding eficacy and safety.
New generations of innovative HSV-1 vectors,
with improved potency and safety, are required
before the oncolytic strategy using HSV-1 becomes a standard therapeutic reality in the ight
against cancer. This is the goal of THOVLEN. One
of the most important innovative contributions
of this project concerns the overall approach towards the advancement of HSV-1-based oncolytic
viruses. Instead of focusing on the development
of vectors carrying deletions, and in particular
virus genes, THOVLEN will engineer competent,
but replication-restricted HSV-1 vectors, strictly
targeted to HCC. These vectors will combine
multiple HCC-targeting approaches, both at the
level of entry and at the level of gene expression
and replication, and will be able to multiply and
spread only in HCC, while displaying no virulence
in normal healthy tissues.
104
LSHB-CT-200-018649
e฀2494460
1January2006
36months
http://www.thovlen.eu
Another novelty is related to the ability of the
HSV-1 vectors to permit a sophisticated and lexible combined approach against HCC. That is, in
addition to optimising the oncolytic properties
of HSV-1 vectors, THOVLEN will exploit the very
large transgenic capability of HSV-1, to generate
vectors that will simultaneously display multiple
and multimodal anti-tumour activities acting
either locally or systemically. These include combined expressions of anti-angiogenic, immunemodulatory, and oncolytic proteins.
THOVLEN will design HCC-targeted virus vectors that will simultaneously display multiple
targeting elements acting at different steps of
the virus life cycle, in order to ensure maximum
aggressiveness for HCC cells with minimum or
no virulence for healthy tissues. Moreover, the
unique advantage of the HSV genome to carry
about 40 kbs of foreign DNA will be exploited in
the context of designing a multimodal approach
for cancer therapy, required for improvement of
the inherent anti-tumour activity of the virus.
The availability and expertise in the use of several well-deined animal models for liver cancer,
will allow the consortium to evaluate the safety
and eficacy of their vectors in relevant systems.
Through fundamental research, they will generate novel genomic and proteomic information
on the interactions between the oncolytic vectors and the normal and cancer cells, which will
11/8/07 10:49:22 AM
GENE THERAPY
guide the rational design of vectors targeted to
HCC cells. The emerging results will be the improvement of the vector oncolytic potency and
of safety. Different ways of producing HSV-1 vectors conceived to speciically penetrate, express
genes, and replicate into HCC will be investigated, thus killing these cells and allowing the virus
to spread and infect other tumour cells.
These vectors should be unable to replicate and
destroy normal surrounding cells. In addition
to their inherent targeted oncolytic potential,
these vectors will express enhancing transgenic
sequences, encoding immune-modulators, antiangiogenic molecules, fusion proteins, or toxic
proteins, which are expected to have an additive
negative effect on tumour growth.
Expected outcome:
Once the project ends, the consortium expects
to produce a number of HSV-1-based oncolytic
vectors speciically targeted to treat HCC. These
vectors will combine targeting and enhancing
functions, which will be evaluated for eficacy
and toxicity on different HCC animal models,
including standard and transgenic mice, as well
as woodchucks. The potential applications of the
results and observations generated by THOVLEN
concern the development of novel strategies
for the treatment of primary liver cancers of
humans.
The irst of these recombinant vectors has already been achieved and THOVLEN is currently
studying their biological properties. At the same
time, animal models and immune tools that will
allow assessments of the anti-oncogenic properties, the toxicity and the immunogenicity of the
vectors, have been created.
Lastly, THOVLEN is now conducting proteomic
and transcriptomal studies to compare infected
versus non-infected hepatomas, in order to identify changes in the expression patterns of the
infected cells, as well as a way to identify novel
activated promoters that could eventually allow
improving the targeting of the oncolytic vectors.
With several collaborations forged with hospitals
in France and Greece, fresh human hepatocytes
have been secured, helping to serve to evaluate
the impact of the oncolytic vectors in these cells,
and to conirm that the vectors can replicate and
disseminate in cultured hepatomas, but not in
cultured hepatocytes.
Main findings and results:
In the irst year of the project, the consortium
succeeded in constructing the majority of transcription units expressing essential HSV-1 genes
under the control of cancer-speciic promoters.
They also set up transcription units expressing
HSV-1 glycoproteins that were modiied to allow
speciic entry of the vectors in liver cells. These
genes are currently being introduced into the
HSV-1 genome so as to generate the recombinant viruses.
10
11/8/07 10:49:22 AM
THOVLEN
Coordinator
AlbertoEpstein
Centre de Génétique Moléculaire et Cellulaire
Université Claude Bernard Lyon 1
Lyon, France
Partners
RobertoManservigi
Dipartimento di Medicina Sperimentale e Diagnostica
Sezione di Microbiologia
Università degli Studi di Ferrara
Ferrara, Italy
ThomasBrocker
Ludwig-Maximilians-Universitaet Muenchen
Institute for Immunology
Munich, Germany
PenelopeMavromara
Hellenic Pasteur Institute
Athens, Greece
RubenHernandezAlcoceba
Fundacion para la investigacion medica aplicada (FIMA)
Pamplona, Spain
FernandoCorrales
Fundacion para la investigacion medica aplicada (FIMA)
Pamplona, Spain
AndresCrespo
Genopoietic
Miribel, France
Jean-JacquesDiaz
Centre de Génétique Moléculaire et Cellulaire
CNRS – UMR 5534
Université Claude Bernard Lyon I (UCBL)
Villeurbanne, France
106
11/8/07 10:49:22 AM
GENE THERAPY
THERADPOX
Optimisedandnoveloncolyticadenovirusesandpoxvirusesinthetreatment
ofcancer:virotherapycombinedwithmolecularchemotherapy
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Today, cancer is the second-leading cause of disease-induced mortality worldwide. Among the
innovative treatments for cancer, virotherapy
holds great promise. Oncolytic viruses (OVs) are
replicating micro-organisms (viruses) that have
been selected or engineered, so as to grow inside and kill tumour cells. It is well known that as
a tumour evolves, mutations in multiple genes
contribute to the malignant phenotype. OVs
speciically target cancer cells, because they are
able to exploit the very same cellular defects that
promote tumour growth.
Although the number of different types of OVs
that have been tested in preclinical trials is growing, only a few have made the transition into the
clinic, limiting the available clinical experience.
These new therapeutics are therefore still the
subject of research as the scientiic community
develops means to optimise their eficacy.
The THERADPOX project focuses on speciic oncolytic vectors such as pox virus and adenovirusbased vectors, owing to their inherent strong
oncolytic potencies and safety records. THERADPOX aims to improve the safety and therapeutic
eficacy of OVs in vivo.
Ultimately, THERADPOX targets the improvement of cancer treatment, for which there is a
high unmet medical need and where available
effective modalities are missing. Owing to their
LSHB-CT-200-018700
e฀2411006
1December200
36months
www.theradpox.org
inherent strong oncolytic potencies and safety
records, pox virus- and adenovirus-based vectors
have been chosen as the vehicle for pursuing the
goal of the project, which is to improve the safety
and therapeutic eficacy of OVs in vivo. Novel and
improved OVs will be engineered, with the following traits in vivo:
• to speciically target colorectal, pancreatic and ovarian cancer cells;
• to replicate exclusively in cancer cells
that are armed with therapeutic genes
rendering only tumour cells sensitive to
chemotherapy;
• to widely spread within the tumour to
permit total tumour eradication.
Through the proposed multidisciplinary work
plan and the strong and complementary expertise of the 10 European partners involved,
THERADPOX will succeed in performing the following objectives:
• generate advanced knowledge that
could be translated in safer cancer treatments with an increased therapeutic index;
• contribute to the improved quality of
life of cancer patients by offering fewer
treatments with no toxic side effects;
• propose new guidelines and standards
for the development of OVs;
• strengthen the competitiveness of Europe in the war against cancer.
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THERADPOX
Common strategies and methodologies
were deined and adapted for OVs in the
THERADPOX project. The establishment
and harmonisation of common strategies
related speciically to the following: vector
engineering, production and puriication of
oncolytic Pox and Ad; reporter genes; quality controls; standardisation of therapeutic
potency and tumour selectivity; methods
for tumour cell infection in vitro; common
in vitro evaluation of the functionality of
FCU1; tumour models; and standardised
analysis of data (all partners).
Expected outcome:
It is anticipated that THERADPOX will
achieve the development of oncolytic
vectors with increased tumour selectivity
of infection, replication and viral spread
through the tumour tissue that will subsequently enter clinical development.
Adenovirus and Pox viruses
Normal cells
Cancer cells
De-targeting
• Mutations viral surface proteins
Normal cells
Cancer cells
Targeting
• Surface expression of ligands which bind
to cancer cells receptors
Normal cells
Cancer cells
Replication
•P
Promoter mutations
• Gene deletion
Normal cells
Cancer cells
Spreading
• Mutations
• Gene insertion
• Gene deletion
Tumour cell fusion
Arming
• Gene insertion: suicide FCU1 gene
Normal
cells
Testing
Cancer
cells
Prodrug
Drug
Main findings:
Tumour cell lines
In vivo: Human tumours and
The indings of the project are set out beand biopsies in vitro
metastasis in mice
low:
• Enhanced tumour infectivity and
tumour selective replication con• Conirmed selective replication of the
cerning oncolytic adenoviruses through
myxoma virus in tumour cells and their
liver de-targeting and tumour targeting.
lack of replication in normal cells concern• Engineering of various optimised oning oncolytic Pox viruses. This set the basis
colytic adenoviruses. These chimeric viral
for further molecular engineering of these
capsids were constructed in such a way
viruses, so as to enhance their safety and
as to target transcriptional deregulated
selectivity for selective tumour cell killpathways in tumours; speciically, the E2F
ing. Oncolytic vaccinia viruses were engipathway was targeted in most tumour
neered, harbouring gene deletions, with
types and the Wnt pathway in colon canincreased selectivity for tumour cells.
cer cells. These engineered adenoviruses
• Increased spreading of an oncolytic
also exhibit increased replication in tuadenovirus carrying a fusogenic protein
mour cells as well as a signiicantly imthrough tumour cells in culture, aiming
proved tumour-to-liver ratio in vitro and in
for increased spreading of OVs through
vivo.
the tumour tissue. Oncolytic adenoviruses
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11/8/07 10:49:23 AM
GENE THERAPY
targeting stromal barriers in tumour tissue have also been engineered.
• “Armed” oncolytic adenoviruses with a
gene coding for a non-toxic prodrug converting enzyme, FCUI, targeting improved
therapeutic eficacy of OVs.
• Testing of various formulations to allow for controlled release of OVs for enhanced safety of OVs.
• Selection of various tumour models for
the in vivo testing of selected candidates.
• Development of a software application, allowing for a fast, sharp and standardised analysis of data. The website www.
theradpox.org will enable the dissemination of results to the scientiic, clinical and
citizen communities.
Coordinator
MonikaLusky
TRANSGENE SA
11 rue de Molsheim
67082 Strasbourg Cedex, France
Partners
RamonAlemany
Institut Català d’Oncologia
Barcelona, Spain
StéphaneBertagnoli
Institut National de la Recherche Agronomique
Toulouse, France
Conclusion:
Various model oncolytic Pox and adenoviruses
have been generated as proof of concept towards the development of oncolytic vectors with
increased tumour selectivity of infection, replication and viral spread through the tumour tissue.
The results obtained during the irst year of the
project have set the groundwork for the development of viral vectors with improved therapeutic eficacy. Therefore, selected oncolytic pox and
adenoviruses will be used to screen their tumour
selectivity in a panel of colorectal, ovarian and
pancreatic tumour models during the period following the THERADPOX project.
RichardIggo
University of St Andrews
Bute Medical School
St Andrews, UK
HarryJalonen
DelSiTech Ltd
Turku, Finland
AkseliHemminki
University of Helsinki
Helsinki, Finland
MohammedBenbouchaib
NewLab
Villers Les-Nancy, France
GerdSutter
Paul Ehrlich Institut
Langen, Germany
VictoriaSmith
Oncotest GmbH
Freiburg, Germany
109
11/8/07 10:49:23 AM
RIGHT
RNAinterferencetechnologyashumantherapeutictool
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The research initiative RIGHT aims at exploiting
and further developing the vast potential of RNA
interference (RNAi) to provide effective therapeutic tools for the treatment of severe diseases,
based on an advanced understanding of their
underlying mechanisms.RNA interference is a
naturally occurring mechanism that regulates
gene expression and is naturally employed by the
organism as defence against viruses. Short (2123 bp) double-stranded RNA is used to induce
the sequence-speciic degradation of mRNA and
thereby block the synthesis of the corresponding
protein.
LSHB-CT-2004-00276
IntegratedProject
e฀11202230
1January200
48months
www.ip-right.org/
There are two main possibilities to apply RNAi:
small interfering RNAs (siRNAs) that are delivered directly into cells as naked siRNAs or with
the help of a transfection reagent are used, while
the other possibility is to use small hairpinRNAs
(shRNA) that are encoded by a vector and delivered into the cell, and are thereby expressed
using the machinery of the cell itself. As a new
technology, RNAi has revolutionised basic research by enabling gene function analysis and
by providing potential new therapeutic strategies. Only recently, the Nobel Prize in Medicine
was awarded to Craig C. Mello and Andrew Z. Fire
for ‘RNA interference – gene silencing by doublestranded RNA’.
As a technique to control gene expression, RNAi
has the potential to speciically regulate the production of disease-associated genes. For application as a therapeutic tool in vivo, it is necessary
to overcome technical challenges, such as insuficient uptake or low stability of the inhibitors,
undesired interferon response or non-speciic
silencing of other genes. To address the multiple facets of therapy development, the RIGHT
project is divided into 5 competence domains.
Experts from various scientiic ields are working
together to exploit the vast potential of this interesting new technology and to make the application of RNAi as a therapeutic tool possible,
in a multidisciplinary way.
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11/8/07 10:49:24 AM
GENE THERAPY
1)MolecularMechanismsandTechnologies:
The understanding of the molecular processes
associated with RNAi and the naturally occurring
counterpart microRNA is improved. This knowledge serves as a basis for the development of
novel molecular strategies enabling the successful application of RNAi technology for human
therapy. Use is made of large-scale RNAi libraries
and high-throughput screening technologies
to identify new target genes for therapeutic approaches and to analyse identiied inhibitors.
2)ChemicalTools:
New chemically modiied siRNAs are synthesised
and extensively tested in cell culture and living
organisms in order to increase sensitivity, speciicity, delivery, stability and cost-effectiveness, and
to reduce side effects.
3)GeneticTools:
Potent viral or non-viral RNAi delivery vectors
are generated and their features evaluated in
relation to their chemical counterparts. Special
emphasis is given to the development of tissuespeciic and inducible systems.
4)Pharmacokinetics:
For the development of a drug, synthetic or genetic RNAi, reagents are tested in cultured cells
Overview of the ive competence domains in RIGHT
with pharmacokinetic methods. Successful candidates are then assessed in animal models, and
extensive phenotyping of treated animals will be
performed.
)CellBiologyandDiseaseModels:
Selected disease models are used for the paradigmatic assessment of RNAi as a therapeutic
tool. This generates RNAi leads for clinical tests. In
particular, RIGHT focuses on infectious diseases
and genetic defects causing degenerative diseases and cancer.
Expected outcome:
Within 4 years, the potential of RNAi to diagnose
and successfully treat diseases will be demonstrated and proof of principle will be provided
for the value of RNAi as a therapeutic tool in living organisms.
Main findings:
In the RIGHT project, new tools for the application
and evaluation of RNAi, such as random libraries
or high throughput techniques have been developed. New microRNAs could be identiied and
their function analysed in more detail. It could
be shown that miR-181 participates in muscle
111
11/8/07 10:49:24 AM
RIGHT
models, like inluenza and HBV. For these disease
models, speciic inhibitors were identiied in vitro.
These inhibitors will now be optimised for application in vivo and their effects tested in animal
models.
Major publications
Press conference at Max Planck Institute in Berlin, from left
to right: Prof. Dr. Joachim W. Engels, Prof. Dr. Thomas F. Meyer,
Prof. Dr. Arndt Borkhardt
cell differentiation (Naguibneva et al. 2005) and
that miR-223 is involved in human granulocyte
differentiation (Fazi et al., 2005).
In the ield of chemically-modiied siRNAs, the
invention of a new design for these inhibitors
shows promising results. This new design leads
to higher stability of the inhibitors and allows additional modiications that are under evaluation
in the consortium. These modiications could
help to increase targeting and reduce undesired
side effects of the compounds. This new design
has already been submitted for patenting.
Besides the construction of synthetic inhibitors,
efforts are undertaken to develop new vector
systems for the expression of shRNAs. Here, some
irst promising results show the possibility to
use lentiviral vectors that include miRNA target
sequences to regulate gene expression in different organs (Brown et al., 2006). With these vectors, the delivery and segregated expression of
shRNAs among different tissues is possible and
should provide a new tool for more eficient and
safe expression of shRNAs.
In the irst two years of the project several disease
models were established to assess the potential
of RNAi approaches in vivo. These models include
different cancer models and infectious disease
112
Piva, R., Pellegrino, E., Mattioli, M., Agnelli, L., Lombardi, L., Boccalatte, F., Costa, G., Ruggeri, B.A.,
Cheng, M., Chiarle, R., Palestro, G., Neri, A., Inghirami, G., ‘Functional validation of the anaplastic
lymphoma kinase signature identiies CEBPB and
BCL2A1 as critical target genes’, J Clin Invest, 2006,
116(12):3171-82.
Pal, A., Severin, F., Lommer, B., Shevchenko, A., Zerial, M., ‘Huntingtin-HAP40 complex is a novel
Rab5 effector that regulates early endosome motility and is up-regulated in Huntington’s disease’,
J. Cell Biol, 2006, 13;172(4):605-18.
Brown, B.D., Venneri, M.A., Zingale, A., Sergi, L.S.,
Naldini, L., ‘Endogenous microRNA regulation
suppresses transgene expression in haematopoietic lineages and enables stable gene transfer’,
Nat Med, 2006, 12(5):585-91.
Naguibneva, I., Ameyar-Zazoua, M., Polesskaya, A.,
Ait-Si-Ali, S., Groisman, R., Souidi, M., Cuvellier, S.,
Harel-Bellan, A., ‘The microRNA miR-181 targets
the homeobox protein Hox-A11 during mammalian myoblast differentiation’, Nat Cell Biol, 2006,
8(3):278-84.
Varghese, O.P., Barman, J., Pathmasiri, W., Plashkevych, O., Honcharenko, D., Chattopadhyaya,
J., ‘Conformationally constrained 2’-N,4’-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies
of aza-ENA-T-modiied oligonucleotides’, J Am
Chem Soc, 2006, 128(47):15173-87.
Casares, N., Pequignot, M.O., Tesniere, A., Ghiringhelli, F., Roux, S., Chaput, N., Schmitt, E., Hamai, A.,
11/8/07 10:49:25 AM
GENE THERAPY
Coordinator
ThomasF.Meyer
Max-Planck-Institute for Infection Biology
Charitéplatz 1
D 10117 Berlin, Germany
Partners
ThomasRudel
Max Planck Institute for Infection Biology
Berlin, Germany
JørgenKjems
University of Aarhus
Aarhus, Denmark
OlliKallioniemi
VTT Technical Research Centre of Finland
Turku, Finland
Poster from a public symposium organised by RIGHT in
Paris, Institut Pasteur in October 2006
Hervas-Stubbs, S., Obeid, M., Coutant, F., Métivier,
D., Pichard, E., Aucouturier, P., Pierron, G., Garrido,
C., Zitvogel, L., Kroemer, G., ‘Caspase-dependent
immunogenicity of doxorubicin-induced tumor
cell death’, J Exp Med, 2005, 19;202(12):1691-701.
Fazi, F., Rosa, A., Fatica, A., Gelmetti, V., De Marchis,
M.L., Nervi, C., Bozzoni, I., ‘A minicircuitry comprised of microRNA-223 and transcription factors
NFI-A and C/EBPalpha regulates human granulopoiesis’, Cell, 2005, 123(5):819-31.
Patents
One patent application was submitted within the
RIGHT project by Jesper Wengel (Southern Denmark University) and Jørgen Kjems (University
of Aarhus) on a new design for small interfering
RNAs (Danish Patent Application 2006 00433).
JesperWengel
Odense, Denmark
JyotiChattopadhyaya
University of Uppsala
Uppsala, Sweden
AnnickHarel-Bellan
Institut André Lwoff, CNRS
Villejuif, France
CarolaPonzettoandGiorgioInghirami
University of Torino
Turin, Italy
MarinoZerial
Max Planck Institute of Molecular Cell Biology and
Genetics
Dresden, Germany
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11/8/07 10:49:25 AM
RIGHT
MichaelRoberts
Regulon
Athens, Greece
PatrickBrianArbuthnot
University of the Witwatersrand
Johannesburg, South Africa
JoachimEngels
Johann Wolfgang Goethe-Universität
Frankfurt, Germany
GuidoKroemer
INSERM
Villejuif, France
PietHerdewijn
Leuven, Belgium
ArndtBorkhardt
Heinrich-Heine- Universität Düsseldorf - Universitätsklinikum
Düsseldorf, Germany
LuigiNaldini
Milano, Italy
WlodzimierzKrzyzosiak
Polish Academy of Sciences
Poznan, Poland
JörnGlökler
RiNA GmbH
Berlin, Germany
IreneBozzoni
University of Rome “La Sapienza”
Rome Italy
JohanAuwerx
Insitut Clinique de la Souris (ICS)
Illkirch, France
GeorgeMosialosandGeorgeKollias
Biomedical Sciences Research Centre “Al. Fleming”
Vari (Attica), Greece
PatrickErbacher
PolyPlus-transfection SA
Illkirch, France
SaraSkogsäter
ARTTIC
Paris, France
114
11/8/07 10:49:26 AM
GENE THERAPY
ZNIP
Therapeuticin vivoDNArepairbysite-speciicdouble-strandbreaks
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
To date, more that 6 000 human single gene disorders have been identiied, affecting about 2%
of the population. To cure or prevent disorders
caused by single gene defects, direct in situ repair would be an attractive strategy, particularly
for diseases where the therapeutic approach
could be based on short sequence substitutions,
deletions or insertions.
Currently, most gene therapy approaches are
based on the delivery of a therapeutic gene
coupled to promoter sequences. Some of the
therapeutic sequences may be replacements for
a mutated gene, while other approaches aim to
kill or protect particular cells. However, the delivery of genes that are taken from their natural
context poses a number of problems and limitations. Thus, a strategy that corrects, or disrupts,
a gene in situ is a central biomedical challenge
that unlocks entirely new strategies for functional genomics, and ultimately, for gene therapy.
Direct and site-speciic modiication of a gene at
its natural locus, without the introduction of additional sequences like selection cassettes, offers
an appealing novel strategy in gene therapy.
Such gene targeting is based upon the cells’ capacity to carry out homologous recombination
(HR), a process which results in an accurate and
precise exchange of genetic material between
the introduced donor DNA and the homologous
genomic target DNA. The eficiency of conven-
LSHB-CT-2006-037783
e฀2349996
1January2007
36months
tional gene targeting by HR increases with the
length of homology between the donor DNA
and the target sequence. Although conventional
gene targeting is an invaluable tool for creating
transgenic laboratory animals, the technique is
hampered by major limitations, including low
targeting frequencies, generally with only one
targeted cell in every 105-107 treated cells.
Moreover, in most mammalian cells, the ratio between site-speciic homologous recombination
and non-homologous end joining (NHEJ) is unfavourable. Consequently, until now, gene targeting has depended on sensitive selection systems
in order to identify the few cells where gene targeting has occurred. Such selection strategies
normally require the donor DNA to contain extra
non-homologous sequences coding for selection markers (often derived from bacteria or viruses). These can result in undesired side effects
on the cells, as exempliied by down regulation
of expression due to de novo methylation.
Designed endonucleases have recently provided
a revolutionary tool for achieving high rates of
targeted genome alteration. By stimulating homologous recombination through the creation
of site-speciic targeted double strand breaks, in
vivo sequence alteration rates can be obtained.
In ZNIP, a major effort is in progress to generate
tools, both improved zinc inger endonucleases
and second generation TFOs (Triplex-forming
oligonucleotides) for targeted sequence altera-
11
11/8/07 10:49:26 AM
ZNIP
encounter DNA damage that challenges their
genomic integrity, they generally activate DNA
repair pathways and cell cycle checkpoint signalling in order to counteract the damage. Moreover, if the DNA damage can not be repaired, cells
can activate apoptotic signalling pathways.
Finally, the technology will be tested in primary
stem cells and transgenic reporter mice as an important step towards clinical trials.
tion in living cells. The goal of the ZNIP consortium is to take gene repair protocols to a level
where high rates can be obtained with consistency, high eficacy and speciicity, as well as with
strongly reduced levels of side effects. To achieve
this goal, the consortium will proceed as follows:
• improve and test sequence-speciic
DNA zinc inger endonucleases (ZFNs);
• develop triple helix-forming oligonucleotides (TFOs) that induce high rates;
• elucidate and constructively improve
the cellular mechanisms involved in the
process;
• test the targeted sequence alteration
in primary cells (including stem cells) and
in living test animals.
Only limited knowledge is available on the cellular pathways and proteins that are involved in
and/or regulate targeted gene correction after
site-speciic DNA double strand breaks. A balance between HR and NHEJ may determine the
eficiency of gene correction. However, other
pathways may also be involved. To improve targeting frequencies and targeting safety (i.e. to
reduce genotoxic side-effects by non-homologous end-joining, for instance), it is of paramount
importance to understand the role of cellular factors in the correcting pathway(s); this work will
be carried out extensively by the consortium.
Furthermore, it is important to establish whether
cellular pathways that could impair the viability
of the targeted cells are activated. When cells
116
ZNIP will proceed by the following steps:
• computer-assisted design of new endonuclease speciicities and in vitro validation;
• development of new bioassays for in
vivo optimisation of zinc inger endonucleases;
• analysis of pathways involved in targeted sequence correction with DNA
double strand breaks;
• application of these pathways for
improvement and increase of targeting
rates;
• analysis of the cellular responses to
targeted sequence alteration: aberrant integration and toxicity;
• analysis of targeted sequence correction in animal models and natural stem
cell populations;
• analysis and improvement of delivery
and dose control.
Expected outcome:
ZNIP anticipates further development of a novel
technique for targeted sequence alteration in living cells as a tool for gene therapy. Designed endonucleases are a revolutionary new tool for inducing site-speciic genome alterations. They will
have a signiicant impact on future gene therapy
protocols, as well as on a broad spectrum of biotechnological applications.
11/8/07 10:49:26 AM
GENE THERAPY
Coordinator
StefanKrauss
Rikshospitalet
Section for cellular and genetic therapy
Forskningparken
Gaustadalleen 21
0349 Oslo, Norway
RalfWagner
GENEART GmbH
D-93053 Regensburg, Germany
Partners
ToniCathomen
Institute of Virology
Charité Universitätsmedizin Berlin
Campus Benjamin Franklin
Berlin, Germany
KlausSchwarz
University Hospital Ulm
Transfusion Medicine
Ulm, Germany
LuisSerrano
Centre de Regulació Genomica
Barcelona, Spain
TomBrown
University of Southampton
School of Chemistry
Highield, Southampton, UK
RolandKanaar
Erasmus Medical Centre
Department of Cell Biology and Genetics
BengtNordén
Chalmers University of Technology
Physical Chemistry
Gothenburg, Sweden
ATDBioLtd
117
11/8/07 10:49:26 AM
SNIPER
Sequencespeciicoligomersforin vivoDNArepair
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Gene targeting can be deined as a method for
introducing site-speciic sequence alterations in
the genomes of living cells. Successful gene targeting would have far reaching implications for
the construction of mutant cell lines and animals,
both for studies of gene function as well as for
the development of disease models. Furthermore, direct modiication of a target gene at its
genomic location, without the introduction of
additional redundant sequences, would offer an
appealing strategy for gene therapy.
Use of gene targeting in order to create transgenic cell lines does not necessarily require high
targeting rates as long as powerful selection bioessays can be applied to facilitate isolation of the
targeted cells. However, for a clinical application
in gene therapy protocols, the introduction of
selective markers in the target genome is not desirable and, consequently, such strategies require
higher targeting rates. For example, it has been
estimated that in order to observe a beneicial effect from the correction of the mutation underlying the monogenetic disease haemophilia, at
least 1%-5% of the treated cells need to express
the corrected protein.
Since targeted sequence alteration in living cells
is increasingly turning into a promising tool for
gene therapy applications, it is important to analyse the various strategies needed for site speciic DNA alterations. The SNIPER consortium is
118
LSHB-CT-2004-00204
e฀203000
1January200
36months
focusing on strategies based on singles stranded
oligonucleotides (ssODN) and triple helix forming oligonucleotides (TFO).
TFOs have demonstrated genome altering activity in vitro and in vivo. Improving selective sitespeciic DNA recognition by TFOs is a central
requirement, because the lack of a general recognition code restricts TFO applicability to a limited
number of loci in the genome. The consortium
is working towards modiied bases in the triplehelix-forming oligonucleotides that tolerate
pyrimidine interruptions and that contribute to
increased triplex stability and binding kinetics.
A second class of molecules capable of sequence-speciic DNA recognition is the peptide
nucleic acids (PNAs). PNAs can recognise DNA by
three modes: triplex invasion; duplex invasion; or
double duplex invasion. Of these, the triplex and
double duplex invasion approaches are of particular interest for targeted gene repair and are
being analysed by the consortium. Using various chemical modiications, the project partners
have improved DNA binding properties of TFOs
and PNAs. The consortium has also strongly improved the properties of the sequence template
for the targeted repair reaction by a combination
of structural improvements and a novel strategy
based on an active group transfer.
By using different mechanisms, ssODN have the
potential to achieve site-speciic alterations in
the sequence of a genomic DNA template in liv-
11/8/07 10:49:26 AM
GENE THERAPY
in the observed targeted repair;
• develop improved cellular delivery
systems;
• provide a thorough analysis of potential side effects.
Expected outcome:
ing cells. The consortium has shown that ssODN
induce signiicant rates of in vivo sequence correction rates.
The project will further develop strategies for
targeted sequence alteration in living cells as a
tool for biotechnology and gene therapy.
Main findings:
A critical aspect for the further development of
gene repair approaches is the elucidation of the
cellular responses needed to achieve targeted
sequence alteration. For this purpose, siRNAbased screens are carried out in order to identify
factors involved in gene repair. The consortium
also seeks to gain an undestanding of the cellular components of the process by studying those
components that make up the cellular mismatch
repair machinery, as well as those of the replication machinery and of the cell cycle checkpoints.
The consortium successfully demonstrated the
role of the cell cycle on in vivo targeted sequence
alteration.
Despite the substantial challenges that remain,
targeted sequence alteration based on ssODN
promises to become a part of the standard toolkit
for functional gene alterations.
High rate site-speciic targeted sequence alteration is likely to prove a viable strategy. Strategies
based on a combination of site speciic endonucleases and selected DNA templates are offering
substantial promise for future biotechnological
and gene therapy applications.
Major publications
Olsen, P.A., Randol, M., Luna, L., Brown, T., Krauss, S.,
‘Genomic sequence correction by single-stranded DNA oligonucleotides: role of DNA synthesis
and chemical modiications of the oligonucleotide ends’, J Gene Med, 2005, Dec;7 (12):1534-44.
Olsen, P.A., Randol, M., Krauss, S., ‘Implications of
cell cycle progression on functional sequence
correction by short single-stranded DNA oligonucleotides’, Gene Ther, 2005, 12:546-51.
SNIPER aims to:
• improve platform technology for targeted sequence alteration in living viable
cells using single strand oligonucleotides;
• improve TFO modules by chemical
modiications;
• design and implement new reactions
to induce single base pair alterations;
• deine the cellular pathways involved
Fox, K.R., Brown, T., ‘An extra dimension in nucleic
acid sequence recognition’, Q Rev Biophys, 2005,
Nov;38 (4):311-20.
Alzeer, J., Scharer, O.D., ‘A modiied thymine for
the synthesis of site-speciic thymine-guanine
DNA interstrand crosslinks’, Nucleic Acids Res,
2006, 34(16):4458-66.
Frykholm, K., Morimatsu, K., Norden, B.,‘Conserved
conformation of RecA protein after executing the
119
11/8/07 10:49:27 AM
SNIPER
DNA strand-exchange reaction. A site-speciic
linear dichroism structure study’,
Biochemistry, 2006, Sep 19;45(37):11172-8.
Tom Brown
Hanada, K., Budzowska, M., Modesti, M., Maas, A.,
Wyman, C., Essers, J., Kanaar, R., ‘The structurespeciic endonuclease Mus81-Eme1 promotes
conversion of interstrand DNA crosslinks into
double-strands breaks’, EMBO J, 2006, 25:4921-32.
KeithR.Fox
School of Biological Sciences
Southampton, UK
RolandKanaar
Li, H., Broughton-Head, V.J., Peng, G., Powers, V.E.,
Ovens, M.J., Fox, K.R., Brown, T.,
‘Triplex staples: DNA double-strand cross-linking
at internal and terminal sites using psoralen-containing triplex-forming oligonucleotides’, Bioconjug Chem, 2006, Nov-Dec;17 (6):1561-7.
BengtNordén
Chalmers University of Technology
Gothenburg, Sweden
Wyman, C., Kanaar, R., ‘DNA double-strand break
repair: all’s well that ends well’, Annu Rev Genet,
2006; 40:363-83.
PeterE.Nielsen
Copenhagen, Denmark
Kim, K.H., Fan, X.J., Nielsen, P.E.,‘Eficient SequenceDirected Psoralen Targeting Using Pseudocomplementary Peptide Nucleic Acids’, Bioconjug
Chem, 2007, Jan 26.
OrlandoD.Schärer
Institute of Molecular Cancer Research
University of Zurich
Zurich, Switzerland
Coordinator
DorcasBrown
ATDBio Ltd
Highield, Southampton, UK.
StefanKrauss
Rikshospitalet
Section for cellular and genetic therapy
Forskningparken
Gaustadalleen 21
0349 Oslo, Norway
RalfWagner
GENEART GmbH
Regensburg, Germany
Partners
UlfEllervik
Lund Institute of Technology
Lund University
Lund, Sweden
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11/8/07 10:49:27 AM
GENE THERAPY
IMPROVED PRECISION
Improvedprecisionofnucleicacidbasedtherapyofcysticibrosis
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Cystic ibrosis (CF) is a recessive congenital disease with a high incidence in Caucasian populations (1 in 3 000 newborns is affected). It is caused
by mutations in the gene coding for CFTR (cystic
ibrosis transmembrane conductance regulator),
a chloride channel, which results in decreased
chloride secretion and hyper absorption of sodium across epithelia. These disturbances in ion
luxes result in water hyper-absorption from the
airway surface liquid into the cells, which produce a highly viscous and elastic mucus. These
changes result in chronic bacterial infection of
the airways, leading to chronic lung disease.
Despite recent developments in the treatment of
CF there is no deinite cure for this disease, and
life expectancy is around 30 years. Several observations suggest that a downregulation of ENaC
restores the perciliary liquid layer, thereby rehydrating the mucus and improving ciliary clearance in the lung. Therefore, the members of the
consortium propose to speciically downregulate ENaC expression by RNA interference.
The intention is to develop compounds able to
modulate ENaC expression, either transiently by
synthetic siRNA constructs or plasmids expressing siRNA constructs, or long-term by lentiviral
gene transfer for stable chromosomal integration, or minichromosomes expressing siRNA
constructs. After testing these compounds in
murine cell lines, the consortium members will
LHSB-CT-2004-00213
e฀304000
1December2004
26months
www.improvedprecision.com
use optimised compounds in established foetal
and adult F 508del mice and in a new ( -ENaC
overexpressing transgenic mouse) animal model
of cystic ibrosis.
This comprehensive approach of interfering with
ENaC expression by delivering siRNA to the airways, includes the following objectives:
1. Develop compounds able to modulate ENaC
expression by RNA interference:
• novel formulations of synthetic siRNA
and plasmid expression systems,
• minichromosomes for expressing siRNA,
• lentiviral vectors for expressing siRNA;
2. Establish aerosolisation of such compounds
without damaging the compounds.
3. Formulate such compounds as magnetic
vectors, in order to make them susceptible to
magnetic ield guidance (“Magnetofection” technology) and combine the magnetic approach
with aerosolisation.
4. Develop instrumentation to generate magnetic gradient ields for magnetic lung targeting.
. Evaluate and validate the novel formulations
and delivery technologies using meaningful
models in vitro and in vivo.
Expected outcome:
The expected outcome of this project is to demonstrate that the downregulation of ENaC subunits by RNA interference in airway epithelial
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IMPROVED PRECISION
cells in vitro and in vivo is feasible. Evidence for
eficient downregulation should be shown on
the level of mRNA- and protein concentration of
the three ENaC subunits: ,- ,-, and -. In addition, a correction of the pathologic sodium hyper
absorption which characterises cystic ibrosis airway epithelia is expected by downregulation of
ENaC-subunits. For the in vivo experiments, a CFmouse model, as well as -ENaC-overexpressing
transgenic mouse model, will be tested. At the
end of the project, enough evidence should be
available to initiate a clinical trial on RNA interference for cystic ibrosis lung disease by targeting
ENaC.
Main findings:
Downregulation of ENaC mRNA and sodium
transport function in cell culture systems resulting in signiicant electrophysiologic effects
on ENaC-mediated sodium uptake, could be
achieved by RNA interference. Currently, experiments in animal models are ongoing to conirm
these data.
OZ Biosciences, a SME partner in this project, as
in this project, commercialised three novel magnetic nanoparticle formulations based on Magnetofection (SilenceMag, ViroMag and ViroMag
R/L), and one lipid-based formulation speciic to
siRNA applications (Lullaby® siRNA transfection
reagent).
Xenariou, S., Griesenbach, U., Ferrari, S., Dean, P.,
Scheule, R.K., Cheng, S.H., Geddes, D.M., Plank, C.,
Alton, E.W., ‘Using magnetic forces to enhance
non-viral gene transfer to airway epithelium in
vivo’, Gene Ther, 2006.
Babincova, M., Babinec, P., ‘Aerosolized VEGF in
combination with intravenous magnetically targeted delivery of DNA-nanoparticle complex
may increase eficiency of cystic ibrosis gene
therapy’ Med Hypotheses, 2006, 67(4):1002.
Schillinger, U., Brill, T., Rudolph, C., Huth, S., Gersting, S., Krotz, F., Hirschberger, J., Bergemann, C.,
Plank, C., ‘Advances in magnetofection - magnetically guided nucleic acid delivery’, Journal of Magnetism and Magnetic Materials, 2005, 293(1):501508.
Rudolph, C., Ortiz, A., Schillinger, U., Jauernig, J.,
Plank, C., Rosenecker, J., ‘Methodological optimization of polyethylenimine (PEI)-based gene delivery to the lungs of mice via aerosol application’,
Journal of Gene Medicine, 2005, 7(1):59-66.
Rudolph, C., Schillinger, U., Ortiz, A., Plank, C., Golas, M.M., Sander, B., Stark, H., Rosenecker, J., ‘Aerosolized nanogram quantities of plasmid DNA
mediate highly eficient gene delivery to mouse
airway epithelium’, Molecular Therapy, 2005,
12(3):493-501.
Major publications
Mykhaylyk, O., Vlaskou, D., Tresilwised, N., Pithayanukul, P., Moller, W., Plank, C., ‘Magnetic nanoparticle formulations for DNA and siRNA delivery’,
Journal of Magnetism and Magnetic Materials,
2007, in press.
Dames, P., Ortiz, A., Schillinger, U., Lesina, E., Plank,
C., Rosenecker, J., Rudolph, C., ‘Aerosol gene delivery to the murine lung is mouse strain dependent’, J Mol Med, 2006, Dec 8.
122
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GENE THERAPY
Coordinator
JosephRosenecker
Klinikum der Universität München
Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen
Kinderspital
Lindwurmstraße 4
D-80337 München, Germany
Partners
MassimoConese,ElenaCopreni
San Raffaele University
Institute for Experimental Treatment of Cystic Fibrosis
- DIBIT
Milan, Italy
HolgerSchankinandHansSchreier
MCS Micro Carrier Systems GmbH
Neuss, Germany
OlgaZegarra-Moran
Laboratorio di Genetica Molecolare
Istituto Giannina Gaslini
Genoa, Italy
PeterBabinecandMelaniaBabincova
Comenius University
Department of Nuclear Physics and Biophysics
Bratislava, Slovakia
DetlefEricHinzandLenaGrimm
Fraunhofer Patent Centre
Munich, Germany
FiorentinaAscenzioni
University of Rome “La Sapienza”
Dipartimento di Biologia Cellulare e dello Sviluppo
Rome, Italy
OlivierZelphati
OZ Biosciences
Parc Scientiique et Technologique de Marseille-Luminy
Marseille, France
CharlesCoutelleandSuzyBuckley
Imperial College London
Faculty of Medicine
Division of Biomedical Sciences
Gene Therapy Research Group
London, UK
ChristianPlankandOlgaMykhaylyk
Institut für Experimentelle Onkologie der TUM
Klinikum r. d. Isar
Munich, Germany
BobJ.Scholte,PatriciaSpijkers
Cell Biology Department
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11/8/07 10:49:27 AM
INTHER
Development and application of transposons and site-speciic integration
technologiesasnon-viralgenedeliverymethodsforevvivogene-basedtherapies
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Considerable effort has been devoted to the
development of gene delivery strategies for the
treatment of inherited and acquired disorders in
humans. Ex vivo gene therapies are based on removing cells from a patient, introducing a therapeutic gene construct into the cells, and implanting the engineered cells back into the patient. Ex
vivo gene therapy is less cost-effective and more
labour-intensive than in vivo gene therapy, but
from a safety perspective, it is potentially more
attractive.
124
LSHB-CT-200-018961
e฀2800000
1November200
36months
www.mdc-berlin.de/izsvak/eng
Transposable elements are recombinases that can
be considered as natural, non-viral delivery vehicles, capable of eficient genomic insertion. The
use of transposable elements can address one of
the main problems of non-viral technologies: stable genomic integration provides long-term expression of therapeutic genes. Using transposon
vectors, integration from plasmid DNA into chromosomes provides the basis for long-term expression of therapeutic genes in treated cells.
This project will evaluate the available recombinase systems in terms of their eficacy and safety
features. INTHER proposes to establish optimal
delivery protocols for the therapeutic/recombinase-systems into a variety of cells. It will determine what type of cells and diseases models are
best suited to the recombinase technology that
can offer an alternative to existing viral and nonviral technologies.
Currently, both viral and non-viral methods are
used for gene delivery. Adapting viruses for
gene transfer is a popular approach, but safety,
immunogenicity and production issues hamper
clinical progress. The establishment of non-viral,
integrating vectors generated considerable interest in developing eficient and safe vectors for
human gene therapy.
INTHER aims to establish and apply methods
and protocols for eficient nucleic acid/protein
delivery into therapeutically relevant target cells,
in the context of recombinase technologies. The
overall objective of INTHER is to develop non-viral, recombinase-based technologies for ex vivo
gene therapeutic applications as an alternative
to current viral/non-viral gene delivery technologies, with the aim of circumventing the toxicity
and immunogenicity problems raised by viral
delivery systems.
Novel gene transfer technologies will be established by developing transposon vectors that
mediate eficient and targeted integration of
therapeutic genes into the genome. INTHER aims
at developing recombinases tailored for gene
therapeutic purposes, by using three different
recombinase systems: SB, FP and PhiC31. The
project will evaluate which recombinase system
holds the greatest promise for further development, by performing head-to-head comparisons
among the three recombinase systems.
11/8/07 10:49:28 AM
GENE THERAPY
Figure 1. Experimental strategies for targeting Sleeping
Beauty transposition. The common components of the targeting systems include a transposable element that contains
the IRs (arrowheads) and a gene of interest equipped with
a suitable promoter. The transposase (purple circle) binds to
the IRs and catalyzes transposition. A DNA-binding protein
domain (red oval) recognizes a speciic sequence (turquoise
box) in the target DNA (parallel lines). (a) Targeting with
transposase fusion proteins. Targeting is achieved by fusing
a speciic DNA-binding protein domain to the transposase.
(b) Targeting with fusion proteins that bind the transposon
DNA. Targeting is achieved by fusing a speciic DNA-binding
protein domain to another protein (white oval) that binds to
a speciic DNA sequence within the transposable element
(yellow box). In this strategy, the transposase is not modiied.
(c) Targeting with fusion proteins that interact with the
transposase. Targeting is achieved by fusing a speciic DNAbinding protein domain to another protein (light green oval)
that interacts with the transposase. In this strategy, neither
the transposase nor the transposon is modiied.
Figure 2. Transposon targeting using a strategy based on
protein-protein interactions between a targeting fusion protein and the SB transposase. (a) The targeting fusion protein
consists of the tetracycline repressor (TetR) that binds to
the TRE, a nuclear localization signal (NLS), a glycine-bridge
(G) and the N-terminal protein interaction domain of the
SB transposase (N-57). (b) Cells were cotransfected with
the components of the transposon targeting system, and
genomic DNA of pooled transformant cells was subjected
to PCR as described in Fig. 5c. The agarose gel shows PCR
products obtained from cells transfected with a vector expressing TetR/NLS/N-57 or with TetR/NLS/LexA with primers
amplifying the left or the right IR of the transposon. M: size
marker. (c) Mapping of targeted SB insertions (arrows) with
respect to the TRE-EGFP target isolated from ive independent experiments is shown. Multiple arrows represent
independent insertions into the same site. Positions of the
insertions are indicated below; the numbers correspond to
the base pair numbering of the pTRE-d2EGFP plasmid (Clontech). (d) Frequency of targeted transposition. The agarose
gel shows PCR products obtained from individual, transgenic cell clones, using primer pairs amplifying the right IR
of the transposon. A PCR product recovered from pooled (P)
DNA samples serves as a reference. M: size marker.
Figure 3. Bioenginered muscle implants
Figure 4. Compared to Mo-MuLV-LTR, Sleeping Beauty IRs
exhibit moderate promoter activity. Luciferase-reporter
experiments.
Figure 5. The expression of the neighbouring genes is inluenced primarily by the promoter activity of the transgene.
The shielding effect of the SH4 insulator sequences inluences. Luciferase-reporter experiments.
12
11/8/07 10:49:29 AM
INTHER
INTHER proposes to improve the performance
of transposon vectors by enhancing their transpositional activity and by strengthening their biosafety proiles. The project intends to improve
the performance of the SB and FP systems, to ensure that the expressed therapeutic gene does
not inluence the genes at the integration site.
It also aims to enhance the activity of the transposase by in vitro evolution (DNA shufling of hyperactive versions of the transposases).
The project will target SB/FP transposon integration into speciic locations in the human genome
in order to improve the safety proile of transposon vectors for gene therapeutic applications.
Molecular strategies of targeted transposition
employed by naturally occurring transposable
elements will be adapted to the SB/FP systems.
INTHER seeks to establish the optimal conditions to deliver the recombinase system/therapeutic gene combinations for the different cell
types. Since the transposon is a plasmid based
non-viral delivery vehicle, cutting edge non-viral
nucleic acid delivery methods will be utilised. In
the project’s framework, Nucleofection technology would be optimised speciically for each of
the recombinase systems in the context of the
relevant cell types targeted in this project. Simultaneously, the capabilities of cell-penetrating
peptides (CPPs), in combination with the transposon systems, will be evaluated. In addition, the
application of reversible implantation systems
(collagen implants, skin biopumps, and encapsulated cells) will be explored.
A disease model system will be identiied, where
the recombinase technology has a clear advantage over viral approaches and current non-viral
approaches. To ascertain whether recombinasebased technologies might offer an alternative
to treatments based on viral transduction, the
recombinase systems will be probed in seven
different ex vivo disease models, previously treated by viral vectors. The selected ex vivo model
126
systems are well established, ‘single therapeutic
gene’ models. Selective advantage of the treated
cells either exists or can be provided, using the
human ABCG2 multidrug transporter protein
as a selectable marker for several models. The
treatment of the selected diseases has not been
solved by conventional treatments in these models, and they are currently established to use viral
vectors. The animal disease models of INTHER include copper metabolism diseases, anaemia, hypercholesterolemia, bleeding disorders, chronic
granulomatous disease (CGD) and neurological
disorders.
INTHER will address the interaction between the
recombinase systems and the treated cell, in particular. Safety issues of transposon vector administration and the characteristics of genomic integration will also be tackled. Extensive datasets
with viral vectors are available in these models
in order to evaluate the potential of the transposon-based, non-viral approach.
Expected outcome:
INTHER is expected to establish new therapeutic
tools for somatic gene therapy as an alternative
to existing viral and non-viral technologies. The
project will evaluate the potential of recombinase-based (Sleeping Beauty, Frog Prince and
PhiC31-mediated) vectors for safe and eficient
transgene expression in ex vivo models. It will develop and optimise delivery techniques suiting
the transposon technology.
INTHER is investigating seven animal disease
models to establish the advantages and limitations of the recombinase-mediated therapeutic
approach. To address the problems of eficacy using non-viral technology, the experimental model systems are carefully selected. In most of the
disease models, the treated cell population has a
selective advantage. The use of selectable markers, particularly ABCG2, is a promising strategy
that will enhance the selection of engineered
11/8/07 10:49:29 AM
GENE THERAPY
cells. The experimental data will provide a strong
basis for further validation of transposon-based
vectors in preclinical and clinical settings.
INTHER expects to deliver the results set out
below.
• signiicantly more active recombinase
systems compared to the prototype versions, to ensure the feasibility of the recombinase-based gene therapeutic approach;
• proof of principle experiments targeting the integration sites of Sleeping
Beauty and Frog Prince to predetermined
genomic loci in human cells;
• better penetration of the transposon
into cells, through the use of cell-penetrating peptides (CPPs);
• development of optimal protocols for
the combination of the transposons and
Nucleofector® technology;
• utilising transposon vectors and ex
vivo therapeutic approaches, INTHER will
test the potential of clinically feasible and
potentially safer approaches for the following: somatic gene therapy of disorders
in copper metabolism; Biopump laser
beam gene transfer (BPLBGT) technology
as a cell factory for the production of human secretory proteins; gene therapy of
the genetic bleeding disorders haemophilia and hereditary thrombocytopenia;
for gene therapy of CGD, to develop a
method to provide a selective advantage
for modiied stem cells, using the human
ABCG2 multidrug transporter protein as
a selectable marker; the use of encapsulated human cell lines and human neural
progenitor cells; and cell therapy in LDL
receptor-deicient mice;
• evaluation of safety and toxicity issues
associated with the use of transposons as
gene vectors in human cells.
Main findings:
Breakthroughs: In the irst year of the project, the
proof of principle of manipulating the target site
of integration was established.
Major publications
Liu, J., Jeppesen, I., Nielsen, K., Jensen, T.G., ‘Phi
c31 integrase induces chromosomal aberrations
in primary human ibroblasts’, Gene Ther, 2006,
Aug;13(15):1188-90.
Thorrez, L., Vandenburgh, H., Callewaert, N.,
Mertens, N., Shansky, J., Wang, L., Arnout, J., Collen,
D., Chuah, M., Vandendriessche, T., ‘Angiogenesis
enhances factor IX delivery and persistence from
retrievable human bioengineered muscle implants’, Mol Ther, 2006, Sep;14(3):442-51.
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INTHER
Coordinator
ZsuzsannaIzsvák
Max-Delbrück-Center for Molecular Medicine
Robert Rössle Strasse 10
13125 Berlin, Germany
PéterKrajcsi
Solvo Biotechnology, Inc.
Szeged, Hungary
LarsWahlberg
NsGene A/S
Ballerup, Denmark
Partners
ÜloLangel
University of Stockholm
Department of Neurochemistry
Stockholm, Sweden
ThomasG.Jensen
The Kennedy Institute
National Eye Clinic
Glostrup, Denmark
EithanGalun
Goldyne Savad Institute of Gene Therapy
Hadassah Medical Organisation
Jerusalem, Israel
SeppoYlä-Herttuala
A.I.Virtanen Institute
Department of Biotechnology and Molecular Medicine
Kuopio, Finland
MarineeChuah
Flanders Interuniversity Institute for Biotechnology vzw
University of Leuven
Department of Transgene Technology and Gene Therapy
Leuven, Belgium
BalázsSarkadi
National Medical Centre
Institute of Haematology and Immunology
Budapest, Hungary
HerbertMüller-Hartmann
AMAXA GmbH
Cologne, Germany
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GENE THERAPY
EPIVECTOR
Episomalvectorsasgenedeliverysystemsfortherapeuticapplication
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LSHB-CT-2004-1196
e฀2100000
1January200
36months
www.ls.manchester.ac.uk/epivector
There are many chronic human diseases that
cause great suffering as a result of inherited or
acquired mutations in our genetic material. Once
present, these changes to the structure of DNA
are passed from one cell to the next as cells divide. Inherited and acquired mutations are responsible for many diseases, common examples
being cystic ibrosis and muscular dystrophy. In
these cases, the debilitating consequences of
mutations in DNA arise because of changes to
the structure of the protein that is expressed
from the mutated gene.
The concept of gene therapy is to overcome the
damage caused by genetic mutations in human
cells by providing, to the appropriate cells, a normal copy of the damaged gene. In principle, if the
normal protein is then expressed in these target
cells it will be possible to replace the malfunctioning protein with a fully functioning counterpart. However, this is technically very challenging,
and systems that are currently being evaluated
in clinical trials suffer from potential deiciencies
that compromise safety.
The most challenging aspects of gene therapy
arise from two main sources. First, available protocols for delivering DNA to the target cells are
generally ineficient. The most eficient systems
use viral particles, but the best systems are also
associated with immunological side effects. Second, even when the DNA is delivered to target
Figure 1: Distribution of pEPI-1 in CHO cells, during and
immediately following mitosis. The localisation of the
episome was studied by FISH on spreads of metaphase
chromosomes (A), and the equal distribution of vector
molecules was monitored in postmitotic nuclei of dividing cells (B). The episome (green) was visualised by pEPI
FISH. To-Pro-3 was used for DNA counterstaining (red). A
maximum intensity projection was rendered from a set
of 5 mid serial sections in (B). Arrows in (A) indicate a pEPI
signal pair, each signal localised on a sister chromatid.
Note the extremely high eficiency with which episomes
are segregates to daughter nuclei - in (B) both daughters
have 7 single plasmids deined by FISH signals. The FISH
spots also show low grade mirror symmetry, similar to
that seen for chromosome territories following mitosis.
cells, it is by no means certain that the gene will
be expressed faithfully. In fact, all too often the
protein is only made for a short time. One way
of providing long-term expression is to use vectors that integrate into the chromosome of the
target cell; this ensures that the new gene is not
lost as cells divide. However, a potential problem
with this is that by interfering with the cell genome, it is possible to alter the normal patterns
of gene expression, and in some cases this can
lead to cancer.
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11/8/07 10:49:29 AM
EPIVECTOR
Figure 2: Reporter gene expression in transgenic pig foetuses generated by sperm mediated gene transfer of epi-vectors.
Shows an analysis of enhanced-green luorescent protein expression in embryonic pig tissues after transfer of epi-vectors to
oocytes using sperm mediated gene transfer. The episomal status of the epivectors was determined by blotting and plasmid
rescue. Confocal images show reporter gene expression in six tissues (from left to right: skeletal muscle, heart, liver, kidney,
lung, and skin) from a representative positive foetus (Top) and the same tissues from a negative control foetus (Below).
This project is designed to evaluate the possibility of developing extra-chromosomal gene
delivery systems for gene therapy and evaluate
protocols for their safe use in pre-clinical model
systems. The episomal systems under study do
not interfere with host cell chromosomes and so
do not have any secondary genetic effects. The
key features of EpiVector are to deine the genetic elements that are required for persistent,
long-term gene expression and maintenance of
the extra-chromosomal DNA molecules in appropriate cells.
Key approaches are to use vector construction
and engineering to deine the behaviour/properties of different vector constructs in different
cellular environments. Models systems used include: general cell culture models; model systems
for gene therapy (e.g. muscular dystrophy); gene
expression in primitive haematopoietic stem
cells; mouse embryonic stem cells; and sperm
mediated gene transfer to explore development
behaviour.
The key feature of epi-vectors is their exceptional mitotic and expressional stability in cells
130
with no selection pressure. The main limitation
is the (low) eficiency with which highly mitotically stable vectors are maintained. This appears
to be a general property of extra-chromosomal
DNA vectors. Establishing a stable maintenance
phenotype does not involve genetic changes
(shown by plasmid rescue and sequencing), so
it is clearly dependent on epigenetic programming. This problem has been studied in detail
and important observations are beginning to
pinpoint the key features:
1. The Lipps and Jackson collaboration has
shown that when stably maintained, vectors are stably associated with the most
active nuclear compartment and retain an
association with some chromosomal landmarks throughout mitosis. This provides
a means of ensuring eficient inclusion
into daughter nuclei but also – though
the molecular mechanism is unknown – a
means of uniform segregation during cell
division.
2. The Lipps and Azorin collaboration has
revealed that the eficiency of establishing stable clones is increased 5-10fold if
DNA is assembled into chromatin before
delivery to cells.
3. Bode and colleagues have shown that
11/8/07 10:49:30 AM
GENE THERAPY
generation of minicircles that do not contain bacterial DNA also improved establishment eficiencies by 5 to10 times.
4. Studies by Lipps and collaborators have
shown that epi-vectors can be introduced
into pig oocytes by sperm-mediated gene
transfer to give an extremely high eficiency of episomal gene transfer to all tissues
of pig embryos.
5. Establishment of human stem cell clones
and mouse embryonic stem cell clones is
ongoing.
observations made using sperm mediate transfer of epi-vectors to pig oocytes.
The oocyte is a permissive environment for epigenetic re-programming and epi-vectors have
been shown to pass to all tissues of an embryo
following their introduction into oocytes by
sperm mediated gene transfer. This implies that
mouse embryonic stem cells will likely provide a
route for making transgenic animals with a stable expression of genes from epi-vectors.
Major publications
Expected outcome:
The expected outcome of the project is to
deliver a detailed understanding of the genetic
and epigenetic features that support the stable
mitotic behaviour of episomal DNA molecules in
proliferating human cells. The project has deined
the genetic elements required for stable mitotic
inheritance and is now attempting to analyse
the epigenetic features. An important aspect
of this activity is to understand in molecular
detail how epi-vectors interact with functional
nuclear compartments and deine the dynamic
behaviour of these interactions. Live cell imaging
of tagged epi-vectors is being used to address
vector dynamics.
Main findings:
Signiicant indings have included a phenotypic
description of the molecular behaviour of epivectors in proliferating cells. This has provided
early insight into the molecular behaviour and
provides a system that should allow the molecular mechanism to be deined. It is clear that
epigenetic programming of the vector is key to
the way it behaves in proliferating cells. Modifying the sequence or chromatin architecture of
the vector prior to introducing into target cells
signiicantly inluences the eficiency with which
mitotically stable clones are established. A particular breakthrough in this regard comes from
Jenke, A.C.W., Eisenberger, T., Baiker, A., Stehle,
I.M., Wirth, S., Lipps, H.J., ‘The nonviral episomal
replicating vector pEPI-1 allows long-term inhibition of bcr-abl expression by shRNA’, Human
Gene Therapy, 2005, 16:533-539.
Bode, J., Winkelmann, S., Götze, S., Spiker, S., Tsutsui, K., Bi, C., Benham, C., ‘Correlations between
Scaffold/Matrix Attachment Region (S/MAR)
Binding Activity and DNA Duplex Destabilisation
Energy’, J. Mol. Biol, 2006, 358:597-613. http://
dx.doi.org/10.1016/j.jmb.2005.11.073
Jackson, D.A., Juranek, S., Lipps, H.J., Designing nonviral vectors for eficient gene transfer
and long term gene expression’, Mol. Ther, 2006,
14:613-626.
Winkelmann, S., Klar, M., Benham, C., A.K., P.,
Goetze, S., Gluch, A., Bode, J., ‘The
positive aspects of stress: Strain Initiates Domain
Decondensation (SIDD)’, Briefings in Functional
Genomics and Proteomics, 2006, 5, 24-31 http://
dx.doi.org/10.1093/bfgp/ell003
Manzini, S., Vargiolu, A., Stehle, I., Bacci, M.L., Cerrito, M.G., Giovannoni, R., Forni, M., Donini, P., Papa,
M., Lipps, H.J., Lavitrano, M., ‘Genetically modiied
pigs produced with a non vital episomal vector’,
Proc. Natl, 2006, Acad. Sci. USA 103:17672-17677.
131
11/8/07 10:49:30 AM
EPIVECTOR
Nehlsen, K., Broll, S., Bode, J., ‘Replicating minicircles: Generation of nonviral episomes for the eficient modiication of dividing cells’, Gene Ther
and Mol Biol, 2006, 10B: 233-243.
Coordinator
DeanAJackson
Manchester Interdisciplinary Biocentre
University of Manchester
Faculty of Life Sciences
131 Princess St
M1 7DN Manchester, UK.
E-mail: [email protected].
Partners
RoelvanDriel
University of Amsterdam
Science Faculty
Swammerdam Institute for Life Sciences
Amsterdam, Netherlands
HansLipps
University of Witten
Department of Cell Biology
Witten, Germany
GeorgeDickson
Royal Holloway - University of London
Centre for Biomolecular Sciences
Egham, UK
FernandoAzorin
CSIC Institute of Molecular Biology
Barcelona, Spain
JürgenBode
GBF
German Research Centre for Biotechnology
AriellaOppenheim
Hebrew University-Hadassah Medical School
Department of Haematology
Jerusalem, Israel
BenDavis
genOway Germany
Hamburg, Germany
132
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GENE THERAPY
POLEXGENE
Biocompatible non-viral polymeric gene delivery systems for the ex vivo
treatmentofocularandcardiovasculardiseaseswithhighunmetmedicalneed
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
PolExGene aims to develop a non-viral ex vivo
gene therapy that can ind potential applications
in the ield of ocular and cardiovascular diseases
with high unmet medical need. One of the key
innovative aspects of PolExGene is the development of novel gene vectors from non-toxic and
non-immunogenic, biodegradable polymeric
carrier materials based on multifunctional poly-aminoacids.
Overall DNA delivery will be improved through
the combination of polyplexes with cell penetrating peptides (CPP). The internalisation eficiency is expected to be enhanced through the
use of Penetratin-like CPP, while the membranecell interaction will be improved by functionalis-
LSHB-CT-2006-019114
e฀2132607
1June2006
36months
ing the polymer membrane with cell interacting
peptides (CIP).
Two different strategies will be developed and
compared. In a irst strategy (A), cells will be transfected using CPP containing polyplexes, after
which the cells will be seeded on a biodegradable polymer membrane, functionalised with CIP.
Polymer membranes will be prepared by solvent
casting or electrospinning.
Alternatively (strategy B), the polymer membrane
will be surface coated with CPP-containing polyplexes prior to cell seeding. In both cases, the cellFigure 1. Application of an ex vivo gene therapy approach
for the treatment of ocular and cardiovascular diseases.
133
11/8/07 10:49:31 AM
POLEXGENE
seeded biodegradable polymer membrane will
be implanted into the retina of the eye or used as
coating for cardiovascular prosthesis. The biodegradable polymers selected will be biocompatible: non-toxic and non-immunogenic.
In order to obtain the technological objectives, the following scientiic objectives will be
achieved by implementing seven Work Packages
(WP):
• WP1: selection of CIP and CPP;
• WP2: development of CPP-containing
polymers;
• WP3: development of CIP-containing
polymer membranes;
• WP4: preparation of plasmids and CPPcontaining polyplexes;
• WP5: characterisation of polyplex-cell
and polymer membrane-cell interactions;
• WP6: study on immunological properties of polyplexes and polymer membranes;
• WP7: polymer membrane implantation in test animals.
134
Coordinator
EtienneSchacht
Polymer Chemistry & Biomaterials Research Group
Department Organic Chemistry
Ghent University Krijgslaan 281 S-4
9000 Ghent, Belgium
Partners
ArtoUrtti
University of Helsinki
Faculty of Pharmacy/Drug Discovery and Development
Technology Center
Helsinki, Finland
HagenThielecke
Fraunhofer Institute for Biomedical Engineering
St. Ingbert, Germany
Prof.Blanca.Rihova
Institute of Microbiology
Prague, Czech Republic)
An eighth Work Package, WP8, will be devoted to
the project management to ensure that the overall project objective is met.
AlainJoliot
Ecole Normale Supérieure
Paris, France
The technological objectives addressed for the
development of CPP containing biocompatible
non-viral polymeric gene delivery systems for ex
vivo treatment are very ambitious:
• development of non-toxic and nonimmunogenic biodegradable polymeric
CPP-containing DNA-carriers;
• development of a biohybrid cell-based
CIP-containing implantation system for
ocular and cardiovascular applications;
• development of a cell-based micro array technology for the quantiication of
the biological activity (transfection eficiency and toxicity) of the DNA-carriers.
EberhartZrenner
University Eye Hospital of Tübingen
Tübingen, Germany
AlainYvorra
Epytop
Nîmes, France
DavidEckland
Ark Therapeutics
London, UK
SeppoYlä-Herttuala
A.I. Virtanen Institute
Kuopio, Finland
11/8/07 10:49:31 AM
GENE THERAPY
MAGSELECTOFECTION
Combinedisolationandstablenonviraltransfectionofhaematopoieticcells–a
novelplatformtechnologyforex vivohaematopoieticstemcellgenetherapy
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The feasibility of ex vivo gene therapy in humans
has been demonstrated with retrovirally transduced haematopoietic stem cells. At the same
time, risks associated with the use of retroviral
vectors became apparent. In order to circumvent problems associated with viral vectors, we
will develop a novel, nonviral, combined ex vivo
cell separation/transfection platform for haematopoietic cells, suitable for site-speciic genomic
integration of transfected nucleic acids into noncoding regions of the host genome. This concept
will be applied to haematopoietic stem cells and
will be validated with established preclinical
models of SCID-X1.
This platform technology for integrated cell isolation and transfection will be based on a clinically approved magnetic cell separation technique
(MACS Technology), combined with magnetically enhanced transfection (Magnetofection).
It will also be based on nucleic acid constructs
that provide site-speciic genomic integration
— either the phage phiC31 integrase system or,
alternatively, a drug-inducible AAV-derived replicase/integrase system.
Technology validation includes the following:
analysis of genomic integration sites; transcriptom proiling; characterisation of stable and inducible trans-gene expression, and evaluation
of homing; and engraftment and persistence in
transgenic animal models using molecular bio-
LSHB-CT-2006-019038
e฀2800000
1May2006
36months
www.magselectofection.eu
logical tools, as well as magnetic resonance imaging. The therapeutic potential will be examined
in a SCID-X mouse model in direct comparison
with established retroviral technology. Besides
contributing to the progress of health care, Magnetoselectofection will foster the competitiveness of Europe’s biotechnology industry.
Magnetoselectofection seeks to combine magnetic cell sorting and transfection based on
Miltenyi’s clinically approved MACS Technology
and Magnetofection (magnetically guided nucleic acid delivery), for manipulation of haematopoietic cells. It also aims to achieve a stable and
regulatable transfected gene expression in haematopoietic stem cells, by site-speciic genomic
integration of delivered nucleic acids upon
Magnetoselectofection, with plasmid constructs
harbouring the phiC31 integrase system, and alternatively, a drug-inducible AAV-derived replicase/integrase system.
Magnetoselectofection will characterise this technology through an analysis of genomic integration
sites, transcriptom proiling, and a characterisation
of stable and inducible transfected gene expression. The project will validate the technology in
transgenic SCID-X mouse models by evaluating
homing, engraftment and persistence in transgenic animal models, via molecular biological
tools and magnetic resonance imaging. Magnetoselectofection will demonstrate the therapeutic
eficacy, as well as assess the associated risks of the
technology in transgenic mouse models.
13
11/8/07 10:49:31 AM
MAGSELECTOFECTION
The dissemination and use of Magnetoselectofection will be made possible from its transfer
into research and clinics through the participating companies.
The clinical success in curing patients suffering
from SCID-X has demonstrated the great potential of gene therapy. In the same study, the shortcomings and serious biological risks associated
with the use of current viral gene vector technology became evident.
Of the 11 patients treated with retrovirally transduced haematopoietic stem cells, 2 developed a
lympho-proliferative disease due to insertional
mutagenesis caused by the uncontrolled integration properties of the vector. Hence, safe alternatives are required to realise gene therapeutic
concepts involving stable genetic modiication
of cells.
Members of this consortium have previously developed independent technologies for the following: magnetic cell separation; magnetic ieldassisted nucleic-acid delivery to cells; nucleic acid
constructs suitable for stable integration into
eukaryotic genomes; disease-relevant transgenic
mouse models, to evaluate the biological characteristics and therapeutic potential of transgenic
cells under pre-clinical settings; and tools for expression proiling.
There are strong personal and scientiic links to
the EU-funded CONSERT project. The fundamental objective of Magselectofection is to combine
the named independent technologies and skills
so as to generate a novel, yet simple, eficient and
safe integrated platform technology for the genetic modiication of cells in clinical and research
applications. This innovative technology is the
irst developed towards the application in SCIDX1 gene therapy; clinical and research protocols
are well established.
136
Expected outcome:
Magselectofection will generate a novel technology for nucleic acid delivery which combines the
ease of application of magnetic nanoparticlebased cell isolation and sorting, with the advantages of magnetic nanoparticle-based nucleic
acid delivery technology. While this novel platform technology is expected to be broadly applicable to a variety of clinical and research applications, this project focuses on its use in gene
therapy of a rare hereditary disease, SCID-X1.
Therefore, Magselectofection comprises detailed
analyses in the stable genetic modiication and
transplantation of haematopoietic stem cells,
while avoiding the use of viral gene vectors.
Expected results include novel nucleic acid constructs for stable genetic modiication of cells
and novel technology for transfecting such constructs. In addition, they include insights into the
molecular biology of such constructs once transfected, into the cell biology of haematopoietic
stem cells modiied in this manner, and into the
biodistribution, engraftment and homing of such
cells once transplanted in animal models.
Apart from the primary intended application, i.e.
gene therapy of SCID-X1, there are multiple nucleic acid therapy applications that can be envisaged, as is indicated below.
• using the method to induce RNA interference (RNAi) in hematopoietic stem cells
to ight HIV infections;
• using the method for the transfection
of lymphocytes for adoptive immunotherapy of cancer;
• transfection of neuronal stem cells for
ex vivo gene therapy of central nervous
system disorders;
• ex vivo transfection of stem cells or
progenitor cells for tissue engineering
(bone, cartilage reconstruction, tendon
and wound healing etc.);
• cell tracking, molecular imaging. The
11/8/07 10:49:31 AM
GENE THERAPY
magnetic nanoparticles used in Magselectofection are suitable contrast agents
for magnetic resonance imaging. Thus, the
method can be used to introduce these
particles in any cell of interest, optionally
along with other agents for molecular imaging (e.g. luorescent dyes).
PeterBabinec
Comenius University
Bratislava, Slovakia
OlivierZelphati
OZ Biosciences S.A.R.L.
Marseille, France
Coordinator
JosephRosenecker
Ludwig-Maximilians-Universität
Munich, Germany
ChristianPlank
Klinikum rechts der Isar der TU München
Institute of Experimental Oncology
Ismaninger Str. 22
D-81675 Munich, Germany
UlfJohann
Fraunhofer Gesellschaft zur Förderung der angewandten
Wissenschaft e.V.
Munich, Germany
Partners
MicheleCalos
Stanford University
Palo Alto (CA), USA
GerardWagemaker
FulvioMavilio
Milan, Italy
TsveeLapidot
Weizmann Institute of Science
Rehovot, Israel
ZygmuntPojda
M. Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology
Warsaw, Poland
MichaelApelandIanJohnston
Miltenyi Biotec GmbH
Bergisch-Gladbach, Germany
PeterSteinlein
Research Institute of Molecular Pathology
Vienna, Austria
137
11/8/07 10:49:31 AM
SYNTHEGENEDELIVERY
Ex vivogenedeliveryforstemcellsofclinicalinterestusingsyntheticprocesses
ofcellularandnuclearimportandtargetedchromosomalintegration
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LHSB-CT-200-018716
e฀2400000
1December200
36months
http://lepg.univ-tours.fr
The project will develop SyntheGeneDelivery
(SGD), a new ex vivo gene delivery (evGD) protocol to provide stable long-term expression of
integrated transgenes. The clinical objective is
to provide gene therapy solutions for some genetic diseases of the neuromuscular and skeletal
systems, as well as for circulating polypeptide
deiciencies that together aflict more than 35
million patients in Europe.
In gene therapy for inherited disorders, the main
area of application of an eficient evGD process lies in the modiication of stem cells. Indeed,
the advantage of such systems is that cells can
be transfected and veriied in vitro, before being
grafted in a patient with a debilitating genetic
deiciency.
The main limitation in developing these therapeutic strategies is the availability of eficient and safe
processes. The SGD protocol is designed to overcome these problems and to ensure long-term
maintenance of transgene expression by chromosomal integration, bypassing several barriers:
• the extra-cellular matrix and the cell
membrane;
• the trafic from endosomes to lysosomes and subsequent degradation;
• the dissociation of DNA from its carrier;
• the transfer into the nucleus;
138
Murine mesenchymal stem cells transfected with
plasmid DNA encoding GFP complexed with BGTC/DOPE
liposomes.
• the access to “good” target sites to ensure healthy integrations in chromosomes;
• avoidance of undesirable transgene
integrations.
To achieve these goals and fully develop the
technology, SyntheGeneDelivery will use two
populations of stem cells. The irst is a mesenchymal stem cell (MSC), which can be simply recovered from bone marrow and manipulated for
the correction of some genetic disorders, such
as those involving haematopoietic and mesenchymal derivative cells. The second will be muscle stem cells that have the potential to correct
some muscle genetic disorders, but that can also
be used, after the intramuscular graft of the modiied stem cells, as a factory for the production of
polypeptides that are deicient in genetic and
acquired disorders, and that can be secreted by
the muscle to diffuse toward some target organs
of the patient.
For DNA vector internalisation in cells, the consortium will use the BGTC-DOPE lipoplex system
11/8/07 10:49:32 AM
GENE THERAPY
in the irst version, and the Poloxamer block copolymer in the two latest versions. Transgene integration will be achieved using vectors derived
from the mariner Mos1 transposon. These DNA
vectors will be improved for their key properties
using molecular engineering methods.
First, their integration eficiency will be improved,
not only by optimising the vector nuclear import,
but also by obtaining hyperactive transposases
(the enzyme assuming the transposon mobility),
and ITR (the end of the transposon). Second, SyntheGeneDelivery will deine the coniguration of
vectors for which integration eficiency will not
be dependent on the transgene size. Finally, sitespeciic integration vectors will be developed
by fusing mariner Tpases with polydactyl Zinc
Finger Domains, thus targeting the DNA integration at close proximity of a predeined site. The
eficiency and the idelity of integration of the
chimeric Tpases will be modelled in vivo before
their use for clinical purposes.
At each step, the modiications to the SyntheGenTransfer process will be evaluated in foetal
and adult MSC, and in muscle stem cells. Their effect on retaining the dual stem cell properties on
self renewal and multilineage differentiation will
be evaluated, in comparison to standard lentiviral integration systems.
Expected outcome:
EvGD strategies for therapy purposes are of high
interest when they concern stem cells, due to
the potential of differentiating them ex vivo and
in vivo in a wide range of cell types. Indeed, this
opens key possibilities that meet challenges
posed by many diseases. The SGD process is well
adapted to develop evGD on stem cells because
of the properties of the BGTC-DOPE, which is the
only identiied chemical product able to internalise DNA molecules in these cells. Developing the
project, the innocuity of the cellular import will
be improved by replacing the BGTC-DOPE with
The mariner Mos1 transposon.
the fully non-cytolytic product approved by the
FDA — the Propoplex block copolymer — using
procedures adapted to ex vivo transfection.
The consortium will use the SGD process on muscle or mesenchymal stem cells, in an attempt to
assay its therapeutic potential in several diseases:
Duchenne Muscular Dystrophy (DMD), type 1 Osteogenesis imperfecta, Hemophilia A and Diabetes mellitus type 1. The properties of SGD and its
evolutions are adequate to the therapeutic strategies targeted on these diseases. It can be used
with cassettes containing the therapeutic transgene that are less than 5 kpb for the DMD (using
highly engineered microdystrophin constructs),
and less than 3-4 kbp for other diseases. Moreover, if mariner vector is able to carry larger transgenes, the consortium should then use cassettes
containing therapeutic transgenes of 20-30 kpb.
Main findings:
Foetal mesenchymal stem cells and a clone of
murine muscle stem cells from a conditionally immortilised mouse have been selected for
evaluation of all the SGD process. Human muscle
stem cells will be used to evaluate the SGD V3.
DNA internalisation in stem cells is actually performed using the nucleofection system from
Amaxa. A more eficient transfection reagent, a
lipidic aminoglycoside derivative, is now available from In Cell Art (Partners 2 and 7), and will
be used to develop SGD V2.
MRNA encoding the transposase internalisation
in HeLa cells is actually eficiently performed using PEI. The Kozak modiied and codon optimised
Mos1 transposase has little activity in transfected
139
11/8/07 10:49:32 AM
SYNTHEGENEDELIVERY
stem cells. A second version, which has no selfannealing properties was developed by Partner
1 and will be used in SGD V2. Hyper-active transposases are still available and will be used in SGD
V2. Similarly, more eficient ITR were designed
and will be used in SGD V2.
In conclusion, it must be underlined that SGD V1
was made from few optimised components (cellular import, nuclear import, protein and DNA
counterparts). Results were encouraging but not
yet at the level expected at the end of the programme. However, the necessary elements are in
place, to proceed with evaluation of the SGD V2.
Major publications
Sinzelle, L., Jegot, G., Brillet, B., Rouleux-Bonnnin,
F., Bigot, Y., Augé-Gouillou, C., ‘Factors controlling
Mos1 transposition in vitro and in bacteria’, Submitted.
Svahn, M.G., Hasan, M., Sigot, V., Valle-Delgado,
J.J., Rutland, M.W., Lundin, K.E., Smith, C.I., ‘Self-assembling supra-molecular complexes by singlestranded extension from plasmid DNA’, Oligonucleotides, 2007, Spring;17(1):80-94.
Ge, R., Heinonen, J.E., Svahn, M.G., Mohamed, A.J.,
Lundin, K.E., Smith, C.I., ‘Zorro locked nucleic acid
induces sequence-speciic gene silencing’, FASEB
J, 2007, Apr 10.
140
Coordinator
YvesBigot
Université François Rabelais de Tours
LEPG FRE 2969
UFR Sciences & techniques
37200 Tours, France
Partners
CyrilleGrandjean
In Cell Art
Nantes, France
OscarSimmonson
Avaris
Stockholm, Sweden
KarinLundin
Stockholm, Sweden
RonaldChalmers
Oxford, UK
DominicWells
Imperial College
London, UK
BrunoPitard
INSERM
Nantes, France
11/8/07 10:49:32 AM
GENE THERAPY
MOLEDA
Molecularoptimisationoflaser/electrotransferDNAadministrationintomuscle
andskinforgenetherapy
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LHSB-CT-2004-12034
e฀2447972
1January200
36months
www.moleda.org
For gene therapy, the use of non-viral DNA offers
the advantage of lack of immunogenicity, absence of size limit for the therapeutic expression
cassette, simpler GMP production, and improved
safety/toxicity proiles. However, the eficient,
precise and safe delivery of plasmids or other
forms of non-viral DNA remains to be improved.
Two different in vivo local tissue plasmid delivery
techniques were recently introduced, and are
currently the most eficient in terms of gene expression level: electrotransfer using mild electric
pulses (ET) and laser beam gene transfer using
femtosecond infrared titanium sapphire laser energy (LBGT).
MOLEDA aims to determine the optimal conditions for precise and selective plasmid transfer
into skeletal muscle and skin, in the frame of a
head-to-head comparison between the two
most promising in vivo plasmid DNA delivery
technologies at present, and to introduce molecular strategies to improve these technologies.
Optimised conditions will then be used on animals with four different therapeutic applications.
The overall objective is to develop non-viral DNA
technology into a preclinical phase. Consequently, MOLEDA will address safety issues (tissue damage, inlammation, etc.) to ensure the success of
its efforts.
Moleda’s Team - Brussels
More precisely, MOLEDA aims at identifying the
optimal conditions for plasmid delivery into
muscle and skin. This optimisation will be accompanied by a head-to-head comparison, using the same DNA preparations, between ET and
LBGT. This optimisation/comparison will be performed in two different tissues: muscle and skin.
MOLEDA will, for each tissue and each model of
delivery (ET or LBGT), assess which is the best
promoter: cytomegalovirus (CMB) or two different tissue-speciic promoters. For muscle, two
strong skeletal muscle promoters will be examined. For skin vaccination, one strongly active
keratinocyte promoter, and one dendritic cell
speciic promoter will be used.
The researchers will assess and select the preferential form of non-viral DNA to be used, from the
following options:
• conventional plasmid;
• prokaryotic-backbone deleted, ‘miniplasmid’ devoid of antibiotic resistance
141
11/8/07 10:49:37 AM
MOLEDA
Moleda’s big team
gene (an approach pioneered by Team 1);
• linear PCR produced eukaryotic expression cassette (technology mastered
by Team 2).
MOLEDA will assess, for the secreted transgenic
protein part of the programme, the usefulness of
an optimised enhancing secretion discovered recently (Tauler et al., 1999), as well as examine for
each tissue (muscle and skin), and for each mode
of delivery (ET and LBGT), what the best DNA formulation is. The options are ‘naked’ versus selfassociated to cationic lipid, or to non cationic
lipid (discovered by Team 1; Patent Herscovici et
al. 2002). To date, cationic lipids have proved ineficient for naked DNA intramuscular injection,
but except for that particular case, little is known
about the effect of cationic lipids, and even less
of the effect that neutral DNA formulation has on
ET and on LBGT in different tissues.
The researchers will study if pre- or post-iontophoresis may be beneicial for ET or LBGT. Iontophoreisis involves applying low intensity electrical currents, <0,5mA/cm2, for several minutes. It
has been shown to enhance transdermal drug
delivery and promote intratissular migration of
charged molecules.
142
These optimisation results will be applied to
three different therapeutic paradigms, for four
different medical applications:
• long-term intracellular expression of
the dystrophin gene in skeletal muscle for
the therapy of Duchenne muscular dystrophy (an inherited neuromuscular disease);
• long-term blood secretion of a circulating protein, with erythropoietin (EPO)
and secreted monoclonal antibodies as
the selected applications;
• short-term transgene expression in
skin for raising humoral and cellular immune response: antitumour vaccination
(i.m. ET has been shown to enhance DNA
vaccine response).
It is likely that each speciic therapeutic application will require an adapted protocol of DNA
administration. Internal data among the consortium’s participants conirm this assumption. For
instance, electrotransfer might represent the
best technology for skeletal muscle DNA delivery
when maximal production and blood secretion
of the transgenic protein is needed. Converesely,
LBGT might represent a more eficient technology for skin administration or for the production
11/8/07 10:49:39 AM
GENE THERAPY
by muscle of a very potent hormone. Also, a muscle speciic promoter might prove more adaptive
(i.e. leading to high level sustained transgene
expression), only to ET or LBGT. In addition, the
precision of gene delivery might depend on the
delivery technology used.
Expected outcome:
Optimised conditions and constructs for ET and
LBGT will be applied in the context of the three
different gene therapy paradigms, and four different medical applications. The safety issues (tissue damage, inlammation, integration, etc.) will
be speciically addressed as well. The overall objective is to develop non-viral DNA technology
into a preclinical phase.
Coordinator
DanielScherman
U640 Inserm, UMR 8151 CNRS
Faculté de Sciences Pharmaceutiques et Biologiques,
4 avenue de l’Observatoire
75270 Paris Cedex 06, France
Partners
EithanGalun
Hadassah Medical Organisation
Jerusalem, Israel
VéroniquePréat
Université Catholique de Louvain
Louvain, Belgium
Main findings:
In the irst part of the programme, ET and LBGT
were optimised in parallel by local DNA delivery
into skeletal muscle and skin, by assessing the
following:
• what the best promoter (ubiquitous or
tissue-speciic) is for each model of delivery (ET or LBGT);
• the preferential form of non-viral DNA
— either conventional plasmid or linear
PCR produced expression cassette;
• the usefulness of an optimised secretion sequence for secreted transgenic
proteins;
• what the best DNA formulation is —
‘naked’ versus associated to cationic or to
non-cationic lipid;
• if pre- or post-iontophoresis is beneicial.
In addition, MOLEDA has made great progress by
constructing a new generation of high eficiency
plasmids devoid of antibiotic resistance genes
and their corresponding genetically modiied
bacterial host.
GeorgeDickson
Royal Holloway & Bedford New College
University of London
London, UK
VolkerSchirrmacher
Deutsches Krebsforschungszentrum
Heidelberg, Germany
IacobMathiesen
INOVIO
Oslo, Norway
PaulParren
GENMAB
StéphaneBlot
Ecole Nationale Vétérinaire d’Alfort
Maison-Alfort, France
AlainCimino
Inserm Transfert
Paris, France
143
11/8/07 10:49:40 AM
ANGIOSKIN
DNAelectrotransferofplasmidscodingforantiangiogenicfactorsasaproofof
principleofnon-viralgenetherapyforthetreatmentofskindisease.
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The ANGIOSKIN consortium wants to establish
the proof of concept that therapeutic genes can
be safely delivered to skin by DNA electrotransfer (electrogenetherapy), in order to prevent or
to treat acquired or inherited skin diseases.
The ANGIOSKIN project is based on the results of
the earlier CLINIPORATOR (Analysis of the mechanisms of DNA electrotransfer, and elaboration of
a CE labelled pulse generator), and ESOPE (Preparation of the Standard Operating Procedures
of Electrogenetherapy, electrotransferring of a
reporter gene into humans) projects and on a
proprietary gene coding for a potent human antiangiogenic factor. This factor is the disintegrin
fragment of the Metargidin, also referred to as
AMD-15 (MDC 15 in mouse).
144
LSHB-CT-200-12127
e฀2780683
1May200
48months
http://dl.ltfe.org/login_angioskin.asp
trodes for safety and eficacy on normal
skin in animals, and analyse the effects of
the electrotransfer of the antiangiogenetic factor on models of skin disease related
to excessive angiogenesis, using non nonvasive biophysical, as well as histological
methods to follow changes in the vascularisation of the lesion.
• electrotransfer the therapeutic gene
to a benign lesion in humans, as a proof
of concept of the use of non-viral gene
therapy to treat acquired or inherited skin
diseases.
Final goal:
ANGIOSKIN will bring the proof of concept of
therapeutic gene non-viral electrotransfer into
skin, to treat inherited or acquired diseases.
Main findings:
ANGIOSKIN will do the following:
• electrotransfer the proprietary therapeutic antiangiogenesis gene to melanoma cutaneous metastasis in humans, using the procedures validated in the ESOPE
project, in order to show its clinical eficacy. Noninvasive biophysical methods
(Doppler ultrasounds, Near InfraRed Spectroscopy, etc.) will be used to monitor the
antiangiogenic effects.
• develop new speciic electrodes for
skin lesions treatment, validate the elec-
The consortium has been actively working on
the gene therapy vector that will be used in the
clinical trials foreseen in the project. The issues
related to the GMP production of the DNA for the
clinical trials have also been cleared with subcontractors. The sequences required for gene expression (promoters) have also been worked out,
resulting in the latest data showing improved
expression and eficacy of the vector.
The preparation of the clinical trials has started.
The clinical partners involved in the irst clinical
11/8/07 10:49:40 AM
GENE THERAPY
trials (related to malignant tissues, i.e. melanomas) have deined the protocols. Despite the
delay in the submission of the protocols to the
ethics committees and regulatory agencies, the
preparation of these documents and contacts
has continued.
The technological aspects have resulted in prototypes for new devices. The extent of their future
use is still under evaluation by the consortium.
Moreover, other items are also under experimental evaluation for eventual inclusion in future
patent applications. Portions of the work of the
consortium will soon be presented in specialised
meetings.
Major publications
Patents:
Two patent applications have been prepared and
submitted. They cover some of the microluidics
aspects of the project, as well as the pulses delivered by the new type of electrodes on which
the consortium is working. However, their summaries have yet to be published and no details
can be provided at the moment.
Mir, L.M., Miklavcic, D., ‘Combinations of electric
pulses for DNA electrotransfer’.
Jullien, M.C., Hoel, A., Mir, L.M., ‘Fluid dispensing
system’.
Coordinator
LluisM.Mir
UMR 8121 CNRS - Institut Gustave-Roussy
Laboratory of Vectorology and Gene Transfer
39, rue C. Desmoulins
F- 94805 Villejuif Cédex, France
Partners
GonzaloCabodevila
Franche Comté Electronique Mécanique Thermique et
Optique
Besançon, France
DamienGrenierandCarolineJullien
CNRS UMR 8029 SATIE (Systèmes et Applications de
Technologies de l’Information et de l’Energie) Team
BIOMIS (biomicrosystèmes)
ENS Cachan Campus de Kerlann
Bruz, France
RuggeroCadossi
IGEA S.r.l.
Carpi (MO), Italy
CarolineRobert
Institut Gustave-Roussy
Dermatology Department of Medicine
Villejuif Cédex, France
VéroniquePréat
Université catholique de Louvain
Unité de Pharmacie galénique
Brussels, Belgium
JulieGeh
Herlev Hospital
Deptarment of Oncology, 54B1
Herlev, Denmark
14
11/8/07 10:49:40 AM
ANGIOSKIN
MichaelP.Schön
University of Würzburg
Rudolf Virchow Zentrum für Experimentelle Biomedizin
Würzburg, Germany
DominiqueCostantini
BioAlliance Pharma SA
Paris, France
DamijanMiklavcic
University of Ljubljana
Faculty of Electrical Engineering
Ljubljana, Slovenia
Subcontractor SC2 to partner University of Ljubljana:
GregorSersa
Institute of Oncology
Laboratory of Radiation Biology
Ljubljana, Slovenia
LoneSkov
Afdelingslæge
Dermatologisk afdeling
KAS Gentofte
Hellerup, Denmark
146
11/8/07 10:49:40 AM
GENE THERAPY
INVIVOVECTORTRAIN
European labcourse: towards clinical gene therapy: preclinical gene transfer
assessment
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LSSB-CT-2003-03219
SpeciicSupportAction
e฀16100
1June2003
21months
www.vecteurotrain.org
Main findings:
The InVivoVectorTrain event, sponsored by the
EC, was the third in a series of training initiatives
on gene and cell therapy that Généthon developed, with the participation of other centres active in this ield in the EU. Launched in 2001 with
the extensive GVPN conference (Evry of France,
4-5 October), the InVivoVector Train project continued with three workshops: the Eurolabcourse
on vectorology in Evry, 14-26 April 2002, and two
in 2004 in Bellaterra (Spain, this report) and again
in Evry, 14-26 June . This educational activity addresses a primary need of the scientiic community to update scientiic concepts and technologies on gene therapy, in order to implement its
developmental process towards a new medical
practice.
About 150 researchers hailing from different institutions across the EU and abroad participated
in the last three events. The success of the conference and course in Bellaterra was also due to the
new training format, that combined the typical
research conference (including invited lectures,
short communications and poster sessions) with
a round table discussion and a practical course
(see the table in the annex). After the basic techniques of vector production, puriication and
characterisation were delivered with the irst
Eurolabcourse in 2002 in Evry, the InVivoVectorTrain consortium thought it necessary to provide
training on the basic techniques of gene transfer
in vivo into animals, which is the second essential step in gene therapy development. Therefore,
lectures, short communications and posters on
the principles of gene delivery into different organs (including gene expression assessment in
Scheme of the training events in 2002 and 2004 (see
www.vecteurotrain.org for more information)
4 days
8 days
Conference
Practical course
Deliverables:
Concepts
Examples
Good practices (1),
business opportunities
Experience
Activities:
Tutorials
Research Data
Public round table
Lectures, discussions
Posters, short
communications
Discussions with large public
exhibits, video-shows
Practical training
International keynote
speakers
Researchers
Industries, regulatory agencies,
patients’ associations, opinion groups
Teachers:
Experts/ teachers/tutors
(2)
Participants from all institutions
Participants:
(1)
32 selected young researchers(3)
(2)
GLP, GMP, GCP, ethics, regulations... 200 max PhD students, post docs, research staff, industries, regulators, other professionals
PhD students, post docs, young researchers, selected on the basis of scientific excellence and motivation to participate
(3)
147
11/8/07 10:49:50 AM
INVIVOVECTORTRAIN
GVPNConference
4-5 October 2001
EVRY
TOTAL
participants
nb
%
Country
1. Algeria
2. Austria
3. Belgium
4. Brazil
5. Bulgaria
6. Canada
7. China
8. Czech Republic
9. Denmark
10. Finland
11. France
12. Germany
13. Greece
14. Hungary
15. Iceland
16. Iran
17. Ireland
18. Israel
19. Italy
20. Japan
21. Korea
22. Latvia
23. Lithuania
24. Luxembourg
25. Mexico
26. Netherlands
27. Poland
28. Portugal
29. Romania
30. Russia
31. Slovenia
32. Spain
33. Sweden
34. Switzerland
35. Turkey
36. Taiwan
37. United Kingdom
38. Uruguay
39. USA
Total
1
13
1
223
8
4
4
3
5
2
3
0,36
4,63
EUROLABCOURSE
14-27 April 2002
EVRY
TOTAL
Participants in the
participants
practical course
%
nb
nb
%
CONFERENCE & COURSE
1-14 February 2004
BELLATERRA
TOTAL
Participants in the
participants
practical course
nb
nb
%
nb
1
10
0,70
7,10
3
1
1,97
0,66
1
0,70
3,13
3,13
3,13
2,10
0,66
1,32
1,32
0,66
0,66
1
1
1
3
1
2
2
1
1
42
17
30,00
12,10
8
2
25,0
6,25
12,5
1,40
2
6,25
29,61
1,97
1,32
3,29
0,66
0,66
4
2
45
3
2
5
1
1
1
3
1
3,13
9,38
3,13
3
2
1
1,97
1,32
0,66
2
6,25
2
1,32
2
6,25
1
3
1
4
0,66
1,97
0,66
2,63
1
1
2
1
0,66
46
3
3
2
30,26
1,97
1,97
1,32
5
1
6
152
0,36
79,36
2,85
1,42
1,42
1,07
1,78
0,71
1,07
2
6
1,40
4,30
1
2
3,13
6,25
1
1
2
1
0,70
0,70
1,40
0,70
1
3,13
6
4,30
1
3,13
1
1
0,70
0,70
1
1
3,13
3,13
7
11
5
1
5,00
7,90
3,60
0,70
7
4
1
1
12
4,27
12
8,60
2
0,71
7
5,00
281
100
140
100
13 countries
45 % women
22 countries
44 % women
32
21,88
12,5
3,13
3,13
100
13 countries
75 % women
CONFERENCE & COURSE
14-26 June 2004
EVRY
TOTAL
Participants in the
participants
practical course
nb
%
nb
%
1
0,6
1
0,6
2
1,2
1
0,6
2
1,2
1
3,13
2
1,2
1
0,6
1
3,13
1
64
8
1
6
0,6
38,3
4,8
0,6
3,6
1
4
1
1
1
3,13
12,5
3,13
3,13
3,13
1
3
3
14
1
0,6
1,8
1,8
8,4
0,6
1
1
2
5
1
3,13
3,13
6,25
15,63
3,13
3,13
3,13
6,25
5
1
2
3,0
0,6
1,2
1
1
3,13
3,13
1
3,13
6
1
18,75
3,13
12,5
9,38
1
3
3,13
9,38
3,29
0,66
3,95
0,6
0,6
6,0
1,8
3,6
4,2
0,6
6,0
4
3
1
1
10
3
6
7
1
10
1
3,13
100
32
100
29 countries
49.5 % women
18 countries
50 % women
8
4,8
2
6,25
167
100
32
100
29 countries
39 % women
18 countries
65 % women
131 institutions
87 institutions
73 institutions
101 institutions
(105 academic, 22
industries, 4 other
organisations(1))
(53 academic, 29 industries, 5 other
organisations)
organisations)
organisations)
TOTAL number of institutions: 293 (226 academic and 67 industries)
SPONSORS
EC-MC-FP5, AFM,
Industries
(1)
EC-MC-FP5, AFM, INSERM,
GENOPOLE, EMBO, Industries
EC-LSH-FP6, FEBS, INSERM,
GENOPOLE, Industries
EC-LSH-FP6, GENOPOLE, CGEssonne, INSERM, Industries
patient’s associations, ethical/regulatory agencies, scientific press
Statistics on the participants in the training events on
gene therapy since 2001
148
11/8/07 10:50:00 AM
GENE THERAPY
live animals and the immune response), were
followed by a practical training on gene delivering in vivo, i.e. administration into liver, lung, brain
and muscle.
In this course, preclinical gene transfer, and the
theoretical and practical issues of gene vectors
as potential new bio-pharmaceuticals were presented. For the irst time, 32 researchers from 18
countries were trained on the techniques of in
vivo gene transfer, based on the criteria of good
laboratory practices and the ethical principles of
animal experimentation.
Coordinator
MauroMezzina
Généthon-CNRS FRE 3018
1bis Rue de l’International
91002 Evry, France
Major publications
Mezzina, M., ‘Towards clinical gene therapy: preclinical gene transfer assessment’, Gene Therapy,
2004, vol. 11, S1 pp 1-172
149
11/8/07 10:50:01 AM
INDUSTRYVECTORTRAIN
Europeanlabcourse:advancedmethodsforindustrialproduction,puriication
andcharacterisationofgenevectors
10
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LSSB-CT-2003-016
e฀172000
1January2004
21months
www.vecteurotrain.org
The topics developed in IndustryVectorTrain
focused on the biotechnological issues of gene
therapy (GT) products (vectors, plasmids, cell
lines, etc.), and when they must be prepared at
pharmaceutical scale. It is a mandatory step in
the development process of gene therapy. A total of 114 scientiic communications (36 full lectures, 23 short communications and 55 posters)
were presented at a conference, highlighting the
biology, use, and industrial development of the
main gene vectors with general and expert information on the following:
• RVV, LVV, AdV and parvovirus-based,
non-viral and other viral (alphavirus and
herpes-based) vector systems;
• some applications in gene transfer
into liver, muscle, CNS and HSC;
• biotechnological issues of scaling up,
i.e. theory and praxis of puriication, cell
cultures techniques and devices for industrial production of RTV, AdV and AAV
vectors, as well as quality control analysis;
• the regulatory and ethical issues of
gene transfer and GT;
• in a special session or round table , a
number of vector production facilities
and services currently available to the
European and US scientiic communities
were presented.
During the practical course, 32 selected researchers acquired hands-on experience in advanced
methods for the following:
• the production of AAV (i.e. the transient transfection of 293 cells or infection
of insect cells with recombinant baculoviruses) and puriication with simple and
fast methods using new ion-exchange
chromatographic devices;
• the preparation of large batches
of AdV puriied with chromatographic
procedures;
• the production of LVV vectors at a
large scale with improved packaging cell
lines;
• new strategies to purify RVV and LVV
pseudotyped with VSV-G protein;
• the characterisation of viral vectors for
basic bio-safety parameters (i.e. physical
and functional titration and assessment
of adventitious contaminants and RCPs).
Main findings:
Researchers and other GT professionals attending this course were trained in the scientiic,
technological and regulatory issues concerning
the evolutionary process of gene therapy products towards new biopharmaceuticals. This could
be achieved by integrating the skills of different professions: the researchers, who devise the
gene transfer prototypes and protocols, and per-
11/8/07 10:50:01 AM
GENE THERAPY
form animal experimentation to validate proofs
of principle; the regulators, who deine the legal
frame where gene therapy products must be
used in clinical trials; the industrialists, who allow
technology transfers from the research benches
to the biotech factories in order to transform the
prototypes into exploitable manufactures; and
the physicians, who are deeply involved in the
last steps which lead to the completion of a clinical trial.
Coordinator
MauroMezzina
Généthon-CNRS FRE 3018
1bis Rue de l’International
91002 Evry, France
Major publications
Bagnis, C., Merten, O.W., Mezzina, M., ‘Advanced
methods for industrial production puriication
and characterisation of gene vectors’, Gene Therapy, 2005, vol. 12, S1 pp 1-176
11
11/8/07 10:50:01 AM
12
New Therapies – Immunotherapy and Transplantation
11/8/07 10:50:02 AM
11/8/07 10:50:02 AM
I
14
11/8/07 10:50:03 AM
ALLOSTEM
Thedevelopmentofimmunotherapeuticstrategiestotreathaematologicaland
neoplasticdiseasesonthebasisofoptimisedallogenicstemcelltransplantation
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LHSB-CT-2004-03319
IntegratedProject
e฀8000000
1April2004
48months
www.allostem.org
Diseases of the blood, be they malignant or not,
are often terminal. Over the last 30 years, improvements in chemotherapy and the use of
external agents in cancer treatments have been
progressing steadily. Despite these advances,
however, haematopoietic stem cell transplantation remains the only therapy that can result in
long-term disease-free survival of many patients,
especially those who have had relapses following initial sessions of chemotherapy.
It is also now possible to approach the treatment
of these diseases through the exploitation of the
genetic differences between individuals, to produce targets through which the immune system
can eliminate the diseased cells. This treatment,
immunotherapy, involves the stem cell transplant
delivering speciic immune effector cells expanded from the donor. The scientiic and technological objectives of AlloStem are to develop new
strategies for optimising the use of allogeneic
haematopoietic stem cell transplantation, based
on the prediction and modulation of the immune
response in order to achieve the following:
• reduce the risk of graft versus host disease (GvHD);
• generate selective anti-tumour immune responses;
• provide protective immunity against
opportunistic infections;
• extend the applicability of transplantation to a larger population of patients.
AlloStem is developing protocols for the treatment of patients with haematological disease,
and for the effective delivery of immunotherapy
to those patients.
The AlloStem consortium has brought together
29 clinical, research and industrial partners from
the EU and abroad. The 24 biomedical research
teams and 5 SMEs involved as full partners are
devoted to the advance of translational research
and new technology for the development of immunotherapeutic strategies to treat haematological and neoplastic diseases. In line with their
objectives, the consortium’s research efforts are
organised in three main work packages (WPs),
wherein the different participating groups cooperate and constitute integrated task forces (TFs)
to address the following topics:
• development of immunotherapeutic
strategies to mediate anti-tumour activity;
• the application of immunotherapeutic
strategies to control infectious diseases
which threaten the lives of transplant recipients;
• the optimisation of the transplant procedure as a basis for subsequent immunotherapy.
1
11/8/07 10:50:03 AM
I
ALLOSTEM
Expected outcome:
AlloStem targets improved healthcare for EU citizens through new treatment protocols and pharmaceuticals. As a result of the AlloStem training
programme, knowledge in this important ield
will be broadened and developed in those countries involved.
Main findings:
The results for the irst 33 months have been impressive across each work package. The tumour
immunotherapy work package (WP1) has characterised several new potentially immunogenic
peptides. Algorithms have been developed to
allow for the determination of new potential
targets of immunotherapy. Several important
signals from toll-like receptors were identiied,
marking these pathways in dendritic cells as potential targets, thus allowing manipulation of the
induction of a cellular immune response.
Protocols have been developed for the purposes
indicated below:
• selecting and enriching antigen speciic T cells;
• allowing the modiication of malignant cell populations, which result in the
generation of malignant antigen presenting cells;
• selecting and enriching antigen specific T cells following the activation by these
cell populations, using cytokine capture
reagents;
• conducting important studies to better understand the interaction of Natural
Killer (NK) cells with target tumour cells.
It should be noted that a number of crucial steps
have also been taken to ensure new immunotherapeutic strategies in the near future.
The infectious disease work package (WP2) has
achieved the prediction and veriication of new
16
HLA class I and class II ligands of HCMV, EBV and
other relevant infectious pathogens. These new
ligands have been incorporated into assays for
the monitoring of virus-speciic immune reconstitution following allogeneic HSCT, as well as
in protocols of T cell stimulation and priming,
to generate virus-speciic T cell lines and clones
for adoptive immunotherapy. Expression of tolllike receptors on dendritic cells has been studied
after stimulation with various infectious pathogens. Protocols were developed for the selection
of pathogen-speciic T cells using the cytokinecapture assay. Standard operating procedures
were developed for the generation of clinical
grade CMV- and EBV-speciic T cell lines.
The transplant modalities work package (WP3)
has deined new conditioning regimens of reduced toxicity and suficient immuno-suppressive qualities that allows more therapeutically
targeted transplants of haematopoietic progenitors from HLA mismatched donors, using different strategies of cell manipulation that enhance
the contributions of regulatory T cells, veto cells,
mesenchymal stem cells or NK cells. Protocols
have been developed for the following purposes:
• transplants of umbilical cord blood
supported by third party cells;
• cell selection and cell purging and/or
enrichment, using methods based on the
immune recognition of speciic cell surface or functional markers.
Standard operating procedures for post-transplant assessment of tolerance (both in terms of
rejection and of GvHD), as well as for the recovery of protective immune functions were also
deined. Clinical assays based on these protocols
were initiated.
Major publications
Ghiringhelli, F., Puig, P.E., Roux, S., Parcellier, A., Schmitt, E., Solary, E., Kroemer, G., Martin, F., Chauffert,
B., Zitvogel, L., ‘Tumor cells convert immature my-
11/8/07 10:50:03 AM
eloid dendritic cells into TGF-beta secretors that
stimulate the proliferation of CD4+CD25+ regulatory T cells’, Journal of Experimental Medicine,
2005, 202:919-929 (Epub Sept 26).
Marcenaro, E., Della Chiesa, M., Bellora, F., Parolini, S., Millo, R., Moretta, L., Moretta, A., ‘IL12 or
IL4 prime human Natural Killer cells to mediate
functionally divergent interactions with dendritic cells or tumors’, Journal of Immunology, 2005;
174:3992-3
Orabona, C., Puccetti, P., Vacca, C., Bicciato, S., Luchini, A., Fallarino, F., Bianchi, R., Velardi, E., Perruccio, K., Velardi, A., Bronte, V., Fioretti, M.C.,
Grohmann, U., ‘Towards the identiication of a
tolerogenic signacells’, Blood, 2005; Dec 8 (Epub
ahead of print).
Taieb, J, Chaput, N, Ménard, C, Apetoh, L, Péquignot,
M, Casares, N, Terme, M, Flament, C, Maruyama, K,
Opolon, P, Lecluse, Y, Métivier, D, Tomasello, E, Vivier, E, Ghiringhelli, F, Martin, F, Klatzmann, D, Poynard, T, Yagita, H, Ryffel, B, Kroemer, G., Zitvogel,
L., ‘A novel dendritic cell subset involved in tumor immunosurveillance’, Nature Medicine, 2006,
12:214-219 (Epub Jan 29).
Stewart, C.A., Laugier-Anfossi, F., Vely, F., Saulquin, X., Riedmuller, J., Tisserant, A., Gauthier, L.,
Romagne, F., Ferracci, G., Arosa, F.A., Moretta, A.,
Sun, P.D., Ugolini, S., Vivier, E., ‘Recognition of peptide-MHC class I complexes by activating killer
immunoglobulin-like receptors’, Proceedings National Academy of Sciences, USA, 2005, 102:1322413229.
Shaw, B., Marsh, S.G.E., Mayor, N.P., Russell, N.H.,
Madrigal, J.A., ‘HLA-DPB1 matching status has
signiicant implications for recipients of unrelated donor stem cell transplants’, Blood, 2006,
107:1220-1226.
Einsele, H., Reusser, P., Bornhäuser, M., Kalhs, P.,
Ehninger, G., Hebart, H., Chalandon, Y., Kröger,
N., Hertenstein, B., Rohde, F., ‘Oral valganciclovir leads to higher exposure to ganciclovir than
intravenous ganciclovir in patients following allogeneic stem cell transplantation’, Blood, 2006,
107: 3002-3008.
Coordinator
AlejandroMadrigal
The Anthony Nolan Trust
The Royal Free Hospital
Pond Street
Hampstead, London NW3 2QG, UK.
E-mail [email protected]
Partners
AlloStemLtd
London, UK
LeonidAlexeev
State Research Centre Institute of Immunology
Moscow, Russia
JirinaBartunkova
Univerzita Karlova V Praze
JavierBordone
ITMO Fundacion Mainetti
Buenos Aires, Argentina
DominiqueCharron
HLA et Médecine
Paris, France
Hans-GeorgRammensee
Eberhard Karls-Universitaet
Tuebingen, Germany
FredFalkenburg
University of Leiden Medical Centre
Leiden, Netherlands
17
11/8/07 10:50:03 AM
I
ALLOSTEM
ManuelFernandez
Universidad Autonoma de Madrid
Madrid, Spain
NikolaiSchwabe
Proimmune Ltd
Oxford, UK
JuanGarcia
Centre de Transfusio I Banc de Teixits
Barcelona, Spain
RobertRees
Nottingham Trent University
Nottingham, UK
ElsGoulmy
University of Leiden Medical Centre
Leiden, Netherlands
YairReisner
Rehovot, Israel
FrançoisRomagne
Innate Pharma
Marseille, France
DoloresSchendel
GSF Forschungszentrum fuer Umwelt und Gesundheit
GmbH
Munich, Germany
AndrzejLange
Ludwik Hirszfeld Institute
Wroclaw, Poland
FrancoLocatelli
IRCCS Policlinico San Matteo
Pavia, Italy
MarioAssenmacher
Bergisch Gladbach, Germany
AlessandroMoretta
Università degli studi di Genova
Genoa, Italy
LorenzoMoretta
Istituto Giannina Gaslini
Genoa, Italy
AndreaVelardi
Università Degli Studi di Perugia
Perugia, Italy
EricVivier
Marseille, France
LaurenceZitvogel
Institut Gustave Roussy
Villejuif Cedex, France
HermannEinsele
Julius-Maximilians-University
Wuerzburg, Germany
OysteinAamellem
Dynal Biotech ASA
Oslo, Norway
RicardoPasquini
Universidade Federal do Parana
Parana, Brazil
PavelPisa
Stockholm, Sweden
18
11/8/07 10:50:03 AM
DC-THERA
Dendriticcellsfornovelimmunotherapies
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LHSB-CT-2004-12074
NetworkofExcellence
e฀7600000
1January200
60months
www.dc-thera.org
Dendritic cell (DC) immunobiology has enormous potential for the development of new immunotherapies for cancer and infectious disease.
Europe is home to a critical mass of leaders in
the ield, who have pioneered many advances
and provided initial proof of principle for the
approach. DC-THERA is a Network of Excellence
(NoE) whose goal is to promote the integration
of the activities of 26 participant groups of scientists and clinicians, along with 6 expert SMEs
across Europe, over its ive-year duration.
DC-THERA will incorporate additional groups,
particularly from new and candidate Member
States, as Associated Members of the Network.
Their collective expertise and resources will be
forged into an ambitious joint programme of activities, so as to restructure the ield. The project
will translate genomic, proteomic and bioinformatic information, with knowledge from molecular cell biology and preclinical models, into
therapeutic endpoints: clinical trials of DC-based
therapies for cancer and HIV.
Four thematic S&T Clusters have been deined,
with a ifth for horizontal activities. The latter
includes development of synergistic links with
other networks, providing added value to EC
programmes by underpinning all projects developing new vaccine strategies for major killer
Fig 1. Langerhans cells (a type of dendritic cell) and
gamma-delta T cells in skin epidermis
diseases. The network will provide a centralised
European resource of databases for the ield.
DC-THERA will develop new research tools, integrate existing and new technological platforms,
recruit additional support staff, and make these
available as shared resources for all partners. It will
implement an ambitious education and training
programme, including new PhD studentships, a
visiting scholars scheme, and high-quality training courses, with the possibility of a postgraduate degree in translational DC immunobiology.
DC-THERA will contribute to the European biotechnology sector and have a major impact on
European policymaking for the future.
19
11/8/07 10:50:04 AM
I
DC-THERA
Fig 2. Scanning electron micrograph of dendritic cells (e.g.
the cell with ‘veils’ at the right hand side) interacting with
resting T cells and an activated T cell (small round cells
and large round cell, respectively)
Expected outcome:
DC-THERA will evolve into a centre of excellence
for DC Biology, with a lasting and global impact.
Main findings:
By the end of its second year, the network has
made considerable progress in all areas. Some
highlights are as follows:
Cluster 1, Genes and Proteins, comprises three
work packages (WPs): genomics (WP1), proteomics (WP2) and bioinformatics (WP3). In WP1, transcriptional proiling of DCs exposed to different
microbial stimuli has resulted in the observation
that surprisingly similar patterns of gene expression may be involved in cellular responses to different agents. In WP2, initial proteomic mapping
of subcellular organelles of DC, including phagosomes and exosomes, is in progress. And in WP3,
testing of a database that will ultimately link genomic and proteomic information is under way.
Cluster2, Molecular Cell Biology, also comprises
three WPs: advanced imaging (WP4), intracellular signalling (WP5) and in vitro activation of DC
(WP6). In WP4, a variety of imaging techniques,
including confocal and two photon microscopy,
160
and MRI, has been used to visualise intracellular
signalling events, intercellular communication
in real time, and cell migration in living tissues.
In WP5, intracellular signalling pathways have
been investigated in different subsets of DC, and
during their responses to different stimuli. Small
inhibitory RNA (siRNA) constructs have been prepared and delivered to DC to explore the effect
of interfering with these pathways. In WP6, the
responses of DC to ‘intrinsic’ stimuli (e.g. inlammatory cytokines, CD40 ligation) and ‘extrinsic’
stimuli (e.g. toll-like receptor, TLR, agonists) have
been studied in depth, and some of the indings
will facilitate the generation of more potent immunostimulatory DC for clinical use.
Cluster 3, Global Responses, comprises two
WPs: DC as adjuvants in vivo (WP7) and pre-clinical studies (WP8). In WP7, two approaches have
been taken to enhance immune responses induced by DC, either by targeting particular receptors on DC themselves, or by targeting different cell types, such as natural killer (NK) cells,
NKT cells and gamma-delta T cells, to exploit
their bidirectional interactions with DCs. In WP8,
a variety of preclinical models of infectious disease and cancer have been used, respectively, to
investigate the role of DCs in immunity against
viruses (LCMV and HSV-1, the latter in neonates)
and bacteria (Mycobacterium tuberculosis and
listeria), as well as to explore various strategies
to enhance anti-tumour responses that include
depletion of regulatory T cells.
Cluster 4, Therapeutic Applications, comprises
three WPs: clinical trials (WP9), immunomonitoring (WP10) and regulatory affairs (WP11). In WP9,
clinical trials of DC-based immunotherapy for a
variety of cancers including chronic lymphocytic
leukaemia, melanoma and renal cell carcinoma
are continuing, while others are in advanced
stages of planning, and a retrospective critical review of published trials will be undertaken in the
next period. In WP10, a critical review of immunomonitoring approaches has also been under-
11/8/07 10:50:05 AM
taken, and initial consideration has been given to
the future standardisation of immunomonitoring
approaches. Finally, in WP11, standard operating
procedures for DC generation and quality control, and templates for clinical trial protocols and
case report forms have been prepared. These will
be made available to different centres as models
before the preparation of master standard operating procedures and templates commences, in
the next period.
As an NoE, DC-THERA seeks to enhance not
only the integration of joint research activities,
as exempliied by the four S&T clusters, but
also the infrastructure underpinning the
ields of DC immunobiology, vaccinology and
immunotherapy in Europe. Cluster 5 has been
structured to meet the latter goal,and it comprises
four WPs.The irst,‘promoting integration’ (WP12),
is designed to enhance the research capabilities of
the network, and to remove obstacles hampering
progress, by establishing core technological
platforms and promoting the development of
shared tools and protocols. The second, ‘ensuring
excellence’ (WP13), focuses on the development
of education and training activities, the sharing
and dissemination of expertise and resources
with groups from outside of the network, and
the forging of collaborative links with other
networks.
Three technological platforms have now been
established, as part of WP12, for genomics, imaging and cell therapeutics in order to enhance the
research infrastructure for S&T clusters 1, 2 and 4,
respectively. For example, the genomics platform
has provided microarray analyses as a service to
other partners who would not necessarily normally have access to such specialised expertise
and resources. The platforms are also responsible,
as part of WP13, for delivering the training activities for the network, which include theoretical
courses, practical workshops and individual training for speciic purposes. To date, the network
has provided a total of six high quality courses or
Fig 3. Magnetic resonance (MR) imaging of lymph node
region before and after DC vaccination. The patient was
injected with superparamagnetic iron oxide(SPIO)-labeled DC in a lymph node of the inguinal lymph node
region under ultrasound guidance. MR imaging of this
region was performed before (ig 1a,c,e) and 48 hours
after (ig 1b,d,f ) the injection of the DC. Arrows indicate
lymph nodes that show a decreased signal intensity after
vaccination indicating the presence of SPIO-DC.
workshops on gene expression proiling and bioinformatics: DC migration and dynamic imaging
techniques; good manufacturing practice (GMP)
and good clinical practice (GCP).
As part of its education activities in WP13, DCTHERA has created 13 new PhD positions for
young researchers who target collaborative research projects between 21 of the network’s 32
partners. These positions are incanced for a total
of 37 person years. It has also organised an annual
Graduate School for Young Investigators, the irst
of which was held in Celerina, St Moritz of Switzerland, in March 2006. The remaining activities
in WP13 include an associated partner scheme in
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DC-THERA
which speciic groups from outside the network
are invited to participate in its activities for the
mutual beneit of both, and for the exploration
of potential collaborative interactions with other
networks in related ields.
To date, a total of ~40 additional groups and SMEs
working in the area have been integrated as associated partners, including several from new
and candidate member states. Moreover, close
interactions with a STREP in the area, DC-VACC,
has resulted in two joint meetings between the
networks. WP13 also deals with communications
issues and includes the development of the network website at www.dc-thera.org, which is now
integrated with a new management and communications software tool for all partners and
associated partners. The remaining two WPs of
Cluster 5, ‘exploiting results’ (WP14) and ‘maintaining infrastructures’ (WP15) deal with management issues per se and look forward to the
future sustainability and continued impact of the
network after the end of the contract.
dendritic cells in melanoma patients for monitoring of cellular therapy’, Nat Biotechnol, 2005,
Nov;23(11):1407-13.
Van Hall, T., Wolpert, E.Z., van Veelen, P., Laban, S.,
van der Veer, M., Roseboom, M., Bres, S., Grufman,
P., de Ru, A., Meiring, H., de Jong, A., Franken, K.,
Teixeira, A., Valentijn, R., Drijfhout, J.W., Koning,
F., Camps, M., Ossendorp, F., Karre, K., Ljunggren,
H.G., Melief, C.J., Offringa, R., ‘Selective cytotoxic
T-lymphocyte targeting of tumor immune escape variants’, Nat Med, 2006, Apr;12(4):417-24.
Sporri, R., Reis e Sousa, C., ‘Inlammatory mediators are insuficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function’, Nat Immunol,
2005, Feb;6(2):163-70.
Frentsch, M., Arbach, O., Kirchhoff, D., Moewes, B.,
Worm, M., Rothe, M., Scheffold, A., Thiel, A., ‘Direct
access to CD4+ T cells speciic for deined antigens according to CD154 expression’, Nat Med,
2005, Oct;11(10):1118-24.
Major publications
Savina, A., Jancic, C., Hugues, S., Guermonprez, P.,
Vargas, P., Moura, I.C., Lennon-Dumenil, A.M., Seabra, M.C., Raposo, G., Amigorena, S.,‘NOX2 controls
phagosomal pH to regulate antigen processing
during crosspresentation by dendritic cells’, Cell,
2006, Jul 14;126(1):205-18.
Koch, M., Stronge, V.S., Shepherd, D., Gadola,
S.D., Mathew, B., Ritter, G., Fersht, A.R., Besra, G.S.,
Schmidt, R.R., Jones, E.Y., Cerundolo, V., ‘The crystal structure of human CD1d with and without
alpha-galactosylceramide’, Nat Immunol, 2005,
Aug;6(8):819-26.
Napolitani, G., Rinaldi, A., Bertoni, F., Sallusto,
F., Lanzavecchia, A., ‘Selected Toll-like receptor
agonist combinations synergistically trigger a
T helper type 1-polarizing program in dendritic
cells’, Nat Immunol, 2005, Aug;6(8):769-76.
Taieb, J., Chaput, N., Menard, C., Apetoh, L., Ullrich,
E., Bonmort, M., Pequignot, M., Casares, N., Terme,
M., Flament, C., Opolon, P., Lecluse, Y., Metivier,
D., Tomasello, E., Vivier, E., Ghiringhelli, F., Martin,
F., Klatzmann, D., Poynard, T., Tursz, T., Raposo, G.,
Yagita, H., Ryffel, B., Kroemer, G., Zitvogel, L., ‘A
novel dendritic cell subset involved in tumor immunosurveillance’, Nat Med, 2006, Feb;12(2):214-9.
De Vries, I.J., Lesterhuis, W.J., Barentsz, J.O., Verdijk, P., van Krieken, J.H., Boerman, O.C., Oyen,
W.J., Bonenkamp, J.J., Boezeman, J.B., Adema, G.J.,
Bulte, J.W., Scheenen, T.W., Punt, C.J., Heerschap,
A., Figdor, C.G., ‘Magnetic resonance tracking of
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Coordinator
JonathanMAustyn
John Radcliffe Hospital
Nufield Department of Surgery
Headington
Oxford OX3 9DU, UK
E-mail (Project Manager): [email protected]
Partners
CarlG.Figdor
University Hospital
Medical Centre Nijmegen
MurielMoser
Université Libre de Bruxelles
Brussels, Belgium
AndreaSplendiani
Leaf Bioscience S.R.L.
Milan, Italy
CharlesNicolette
Argos Biosciences, Inc
Durham, US
FilippoPetralia
Sekmed S.R.L.
Milan, Italy
SebastianAmigorena
Institut Curie
Paris, France
ThierryBoonandPierreCoulie
Christian de Duve Instite of Cellular Pathology
Brussels, Belgium
AnneO’Garra
Medical Research Council
London, UK
DuccioCavalieri
Preclinica e Clinica Università degli studi di Firenze
Dipartimento Di Farmacologia
Florence, Italy
FrancescaGranucci
University of Milano-Bicocca
Milan, Italy
SandraGessani
Istituto Superiore di Sanita
Rome, Italy
GeroldSchulerandAlexanderSteinkasserer
Friedrich Alexander Universitat
Erlangen, Germany
NicolasGlaichenhaus
Universite Nice-Sophia Antipolis
INSERM
Valbonne, France
RobertCofin
Biovex
Abingdon, UK
CatherineDeGreef
BruCells SA
Brussels, Belgium
UgoD’Oro
Chiron Vaccines S.R.L.
Siena, Italy
RolfKiessling,HakanMellstedt,PavelPisa
Stockholm, Sweden
CornelisJ.M.Melief
Leiden, Netherlands
PhilippePierre
Centre CNRS-INSERM d’Immunologie de Marseille-Luminy
Marseille, France
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DC-THERA
MariaPiaProtti
Fondazione Centro San Raffaele Del Monte Tabor
Milan, Italy
MatthiasMann
Max-Planck Institute of Biochemistry
Martinsried, Germany
AndreasRadbruch,AlexanderScheffold,
AndreasThiel
Deutsche Rheuma-Forschungszentrum Berlin
Berlin, Germany
AlbertoMantovani
Istituto Clinico Humanitas
Milan, Italy
CaetanoReiseSousa
Cancer Research UK
London, UK
BeneditaRocha
Université René Descartes
Faculté de Médecine Necker
Paris, France
AntonioLanzavechia,MarkusManz,
FedericaSallusto,MariagraziaUguccioni
Insitute for Research in Biomedicine
Bellinzona, Switzerland
MarkSuter
Zurich, Switzerland
KrisThielemans
Brussels, Belgium
HermannWagner
Technische Universitaet Muenchen
Munich, Germany
LaurenceZitvogel
Institut Gustave Roussy
Paris, France
PaolaRicciardi-Castagnoli
Genopolis – Consortium for Functional Genomics
Milan, Italy
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DC-VACC
Therapeuticin vivoDNArepairbysite-speciicdouble-strandbreaks
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
The immune system of vertebrate animals
evolved to respond to different types of perturbations, such as pathogens, whilst limiting
self-tissue damage. Initiation of the immune response is accomplished by unique antigen presenting cells called dendritic cells (DC) –highly
phagocytic sentinels of the immune system, resting until they encounter foreign microorganisms
or inlammatory stimuli.
Early-activated DC trigger innate immune responses that represent the irst line of defence
against pathogens and provide effective antitumour immunity. Activated DC subsequently
prime antigen-speciic immune responses (T
and B lymphocytes) to clear the infections and
give rise to immunological memory. Activation
of Natural Killer (NK) cells is also key during an
anti-pathogen response. A direct involvement of
NK cells has also been shown in anti-tumour responses in different systems and unequivocally
observed in patients with cancer.
The aim of DC-VACC, a Speciic Targeted Research
project (STREP), was to develop novel vaccine
technologies and to use DC as natural adjuvants
with speciicity and minimum side effects. Early
clinical trials have shown that antigen-pulsed DC
have potential in the treatment of cancer – also
applicable in the eradication of infectious diseases. The objective of the proposed project was
part of the priority area 1.2.4-6: ‘Development
LSHB-CT-2003-03037
e฀2000000
1January2004
36months
of Vaccine Technologies Targeted to Dendritic
Cells’, proposed as a STREP for the Framework
Programme 6. The basis of this proposal was to
develop in situ DC targeting for use as vaccines
in infectious diseases and cancer.
The consortium has effectively generated improved reagents and protocols for antigen delivery and targeting, which improved antigen
processing and presentation by DC and can be
used for vaccine and therapeutic technology.
They have also deined optimal reagents and
protocols for maturation and activation of mouse
and human DC in vitro for use in vaccination and/
or therapeutic intervention. Optimisation of protocols was compared across both species, which
is essential for use in preclinical models and clinical trials in future NoEs and/or IPs.
Main findings:
DC-VACC targeted two speciic objectives in
Work Packages (WP) 1 and 2.
WP1 generated the tools and methods required
for appropriate and eficient targeting and antigen delivery for the development of DC vaccine
technology. This included the construction of
viral and bacterial vectors, modiication of RNA,
peptides and proteins, and antibody development for speciic targeting of DC receptor repertoire. Excellent research progress was made in
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DC-VACC
Time (min)
the following:
• microbial vectors development (bacterial and viral);
• modiication of RNA, peptides and
proteins;
• antibody development for speciic targeting of DC receptor repertoire
WP2 deined optimal reagents and protocols for
maturation and activation of mouse and human
DC in vitro for use in vaccination and/or therapeutic intervention, such that optimisation of
protocols for both species are compared so as to
promptly facilitate information from preclinical
models being transferred rapidly to clinical trials
in future NoEs and/or IPs. The DC-VACC consortium determined the following:
• signals for activation/maturation of
mouse and human DC;
• optimised human DC-vaccines: reagents and procedures;
• transcriptome analysis of mouse and
human DC;
• DC-based bioassays.
The humanised antibody to DC-SIGN hD1 G2/G4 (hD1)
was cross-linked to a model antigen, keyhole limpet
hemocyanin (KLH). Using confocal microscopy it was
observed that the chimeric antibody-protein complex
(hD1-KLH).bound speciically to DC-SIGN, was rapidly
internalised and translocated to the lysosomal compartment as shown in the above igure over time (minutes).
There has been great productivity with respect
to the development of microbial vectors, and the
ability to deliver antigen and target DC, as well as
approaches. These include the discovery of new
pathways to target the enhancement of anti-tumour therapy and therapeutic intervention, as
well as for vaccination in infectious diseases.
The knowledge from this project has been widely
disseminated via publications and international
immunology conferences. During the duration
of the project, meetings (held together with an
NoE, DC-THERA) were held at various sites to exchange scientiic knowledge and via the website
created by Partner Biopolo (http://www.biopolo.
it/), where the list of publications generated from
DC-VACC are displayed.
Ultimately, the studies from DC-VACC have resulted in the discovery of new adjuvant approaches.
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Major publications
Smith, C.L., Dunbar, P.R., Mirza, F., Palmowski, M.J.,
Shepherd, D., Gilbert, S.C., Coulie, P., Schneider, J.,
Hoffman, E., Hawkins, R., Harris, A.L., Cerundolo, V.,
‘Recombinant modiied vaccinia Ankara primes
functionally activated CTL speciic for a melanoma tumor antigen epitope in melanoma patients
with a high risk of disease recurrence’, Int J Cancer,
2005, Jan 10;113(2):25.
Boonstra, A., Rajsbaum, R., Holman, M., Marques,
R., Asselin-Paturel, C., Pereira, J.P., Bates, E.M., Akira,
S., Vieira, P., Liu, Y-J., Trinchieri, G., O’Garra, A. ‘Macrophages and myeloid DC, but not plasmacytoid
DC, produce IL-10 in response to MyD88- and
TRIF- dependent TLR signals, and TLR-independent signals’, J.Immunol, 2006, 177, 7551 - 7558.
Hermans, I., Silk, J., Gileadi, U., Masri, H.S., Shepherd,
D., Farrand, K.J., Salio, M., Cerundolo, V., ‘Dendritic
cell function can be modulated through co-operative actions of TLR ligands and invariant NKT
cells’, J. of Immunology, 2007, Mar 1; 178(5):2721-9.
Granucci, F., Zanoni, I., Pavelka, N., van Dommelen,
S.L.H., Andoniou, C.E., Belardelli, F., Degli Esposti,
M.A., Ricciardi-Castagnoli, P., ‘A Contribution of
Mouse Dendritic Cell-Derived IL-2 for NK Cell Activation’, J. Exp. Med., Aug 2004; 200: 287-295.
Foti, M., Granucci, F., Pelizzola, M., Beretta, O., Ricciardi-Castagnoli, P., ‘Dendritic cells in pathogen
recognition and induction of immune responses:
a functional genomics approach’, J Leukoc Biol,
2006, May;79(5):913-6. Review.
Schaft, N., Dorrie, J., Thumann, P., Beck, V.E., Muller, L., Schultz, E.S., Kampgen, E., Dieckmann, D.,
Schuler, G., ‘Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by
transfecting deined RNAs after rather than before maturation’, J. Immunol, 2005, 174 (5):30873097.
Prechtel, A.T., Turza, N.M., Kobelt, D.J., Eisemann,
J.I., Cofin, R.S., McGrath, Y., Hacker, C., Ju, X., Zenke,
M., Steinkasserer, A., ‘Infection of mature dendritic cells with herpes simplex virus type 1 dramatically reduces lymphoid chemokine-mediated
migration’, J Gen Virol, 2005, Jun;86(Pt 6):1645-57.
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DC-VACC
Coordinator
AnneO’Garra
National Institute for Medical Research Council
The Ridgeway
Mill Hill
London NW7 1AA, UK
Partners
LeonardoBiondi
Biopolo SCRL
Milan, Italy
VicenzoCerundolo
Oxford, UK
CarlFigdor
University Medical Centre
MurielMoser
Brussels, Belgium
PaolaRicciardiCastagnoli
University of Milano-Bicocca
Milan, Italy
GeroldSchuler
University Hospital of Erlangen
Erlangen, Germany
FilippoPetralia
Sekmed Srl
Milan, Italy
KaiZwingenberger
Brucells SA
Brussels, Belgium
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THERAVAC
Optimiseddeliverysystemforvaccinestargetedtodendriticcells
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
LSHB-CT-2004-0382
e฀2267000
1March2004
4months
In response to urgent medical and societal needs
for novel immunotherapies for cancer and chronic
infections, as well as for prophylactic vaccination,
optimised delivery systems for vaccines targeting dendritic cells will be developed and clinically
evaluated. The approach relies on two new antigen delivery vectors: the detoxiied adenylate cyclase toxoid (ACT) and the porcine parvovirus-like
particles (PPV-VLP), which were recently shown to
target dendritic cells very eficiently and speciically, allowing for a highly effective presentation
of delivered antigens to T cells.
These vaccine vectors enable the induction of
strong, speciic and protective immune responses, and have an established record of safety and
eficacy in preclinical animal models. Under this
project, academic experts in immunology, vaccinology, and molecular and structural biology
have joined forces with toxicologists, clinicians
and vaccine production experts from two companies, to translate these novel vaccine technologies from research into clinical application.
Electron microscopy image of PPV-VLP particles, one of
the two vaccine delivery vehicles developed by THERAVAC
in-depth analysis of the cellular and molecular
mechanisms, as well as of the structural basis of
ACT interaction with dendritic cells will be conducted. Particular emphasis will be placed on
gaining new knowledge for furthering the delivery capacity of the ACT molecule towards enhanced eficiency and broader versatility in clinical use. The PPV-VLP vector will be developed in
parallel, by deining its cellular receptor and traficking inside dendritic cells, preclinical eficacy
and toxicology, in order to bring this alternative
vaccine carrier to the level of clinical trial maturity.
Based on the preclinical record of ACT-based
vaccines in animal models, the step into the
safety and eficacy phase I/II human trial will be
made with an ACT-based construct delivering
the tyrosinase A.2 epitope as a therapeutic vaccine for metastasic melanoma. Simultaneously
with Good Manufacturing Practice (GMP) batch
production, development and clinical testing, an
THERAVAC combines basic research into the
mechanism of function of these novel vaccine
targeting systems with their preclinical application, including a phase I/II trial. The irst two work
packages form a demonstration activity centred
on the production of the GMP lot and the handling of the phase I/II trial. The remaining four
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THERAVAC
work packages correspond to the research and
development component of the project, aimed
at understanding the basic mechanisms at the
molecular level. The latter work package employs
cell biology, immunology, biochemistry and biophysics, including structural biology, to address
the questions of speciicity and eficacy of the
vaccine vehicle molecules used.
Expected outcome:
THERAVAC aims to achieve:
• production of a GMP batch of ACT carrying the melanoma tyrosinase epitope
suitable for a phase I/II trial;
• the detailed toxicology assessment of
this GMP batch of ACT;
• a phase I/II trial in melanoma patients
of this GMP batch of ACT carrying the tyrosinase melanoma CD8+ T cell epitope;
• the understanding of the interaction
of ACT with dendritic cells at molecular
and atomic details;
• the detailed understanding of the
mechanism of PPV-VLP speciicity and
eficacy;
• the engineering of improved vaccine delivery molecules based on our improved understanding of their functional
mechanism.
Main findings:
The Bordetella adenylate cyclase toxin (ACT)
binds the complement receptor 3 (CD11b/CD18,
CR3 or Mac-1) of host myeloid phagocytes, penetrates their cytoplasmic membrane, and upon
translocation into cytosol and binding of calmodulin, it delivers into the MHC Class I/II antigen presentation pathway its AC domain. Furthermore, ACT can form small cation selective
channels in target membranes, bind calcium,
promote its inlux into cells and induce a cascade
of signalling events leading to the maturation of
dendritic cells.
170
These various activities associated with the ACT
molecule synergise to bring about the potency
of ACT as an antigen delivery vector into dendritic cells and induction of speciic T cell mediated responses against delivered antigens. The
complex activity of ACT is dissected into individual contributions by characterising properties
of ACT variants, which are selectively blocked by
speciic mutations in individual steps of toxoid
penetration into cells and in channel-forming
and signalling properties of ACT. A novel mechanism of calcium mobilisation into myeloid cells
by ACT was discovered, and shown to be linked
to AC domain translocation across cytoplasmic
membrane of cells. Over the past 36 months,
important progress was made in understanding
the mechanisms underlying the toxoid interaction with CD11b, signalling activity on myeloid
cells and the capacity to deliver the AC domain
with antigens into cells. This was eficiently used
to manipulate the antigen delivery potency of
toxoid forms of ACT.
Important progress was also made in the understanding of the mechanisms of how PPV-VLPs
interact with dendritic cells. Importantly, these
VLPs were shown to possess a strong adjuvant
activity. THERAVAC demonstrates that the baculovirus (BV) is responsible for this adjuvant effect
and that it plays a major role in the strong immuScheme of antigen delivery by recombinant ACT.
The toxoid inserts into cellular membrane of antigen
presenting cells in two different conformations that give
rise to molecules with the AC domain translocated into cell
cytosol and to molecules forming oligomeric membrane
channels with the AC domain stuck at the external face of
cellular membrane. The translocated AC domain bearing the
inserted antigen is processed inside the cytosol of antigen
presenting cells by proteasome. The peptides are then
transported by TAP into the endoplasmic reticulum and bind
to newly synthesised MHC I molecules. The MHC I – peptide
complexes are transported to the cell surface and presented to CD8+ T cells, thereby promoting their activation.
Endocytosis of membrane associated CyaA may also occur
and CyaA molecules can be processed to antigenic peptides
that bind to MHC II molecules. These MHC II complexes are
presented to CD4+ T cells. Reviewed in Simsova et al. (2004)
Int. J. Med. Microbiol. 293, 571-576. © Urban & Fischer Verlag.
11/8/07 10:50:08 AM
nogenicity of PPV-VLPs produced in the baculovirus-insect cell expression system. This adjuvant
behaviour of BVs is mediated primarily by IFN / ,
although mechanisms independent of type I interferon signalling are also involved.
Basler, M., Masin, J., Osicka, R., Sebo, P., ‘Pore-forming and enzymatic activities of Bordetella adenylate cyclase synergise in promoting lysis of
monocytes’, Infect Immun, 2006, 74, 2207-14.
The irst pre-GMP batch has been prepared on a
small scale by CellGenix, GmbH, the subcontractor chosen for this task. Once this batch is validated, a GMP-like batch will be provided for toxicology, as well as a larger-scale GMP preparation
for the clinical trial, starting in 2008.
Mascarell, L., Bauche, C., Fayolle, C., Diop, O.M.,
Dupuy, M., Nougarede, N., Perraut R., Ladant, D.,
Leclerc, C., ‘Delivery of the HIV-1 Tat protein to
dendritic cells by the CyaA vector induces speciic Th1 responses and high afinity neutralising
antibodies in non human primates’, Vaccine, 2006,
24, 3490-9.
Major publications
a) Publications in peer-reviewed journals
Prior, S., Fleck, R. A., Gillett, M., Rigsby, P., Corbel, M.
J., Stacey, G., Xing, D. K. L., ‘Evaluation of adenylate
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THERAVAC
cyclase toxin constructs from Bordetella pertussis as vaccine candidate antigens in an in vitro
complement-dependent opsonophagocytic-killing model’, Vaccine, 2006, 24:4794-4803.
Cheung, Y.C.G., Xing, D., Prior, S., Corbel, M.J., Parton, R., Coote, J.G., ‘Adjuvant effect of different
forms of adenylate cyclase toxin of Bordetella
pertussis on protection afforded by acellular pertussis vaccine in a murine model. Infect. Immu,
2006, 74:6797-6805.
Bauche, C., Chenal, A., Knapp, O., Bodenreider, C.,
Benz, R., Chaffotte, A., Ladant, D., ‘Structural and
functional characterisation of an essential RTX
sub-domain of Bordetella pertussis adenylate cyclase toxin’, J Biol Chem, 2006, 281:16914-16926.
Coordinator
ClaudeLeclerc
Institut Pasteur
25 rue du Dr. Roux
75014 Paris, France
Partners
AnitaLewit-Bentley
CNRS
Cachan, France
RinoRappuoli
Novartis
Sienna, Italy
PalomaRueda
INGENASA
Madrid, Spain
Fiser, R., Masin, J., Basler, M., Krusek, J., Spulakova,
V., Konopasek, I., Sebo, P., ‘Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin:
Membrane translocation of AC domain polypeptide promotes calcium inlux into CD11b+ monocytes independently of the catalytic and hemolytic activities’, J. Biol. Chem, 2007, 282 2808-2820.
PeterSebo
Institute of Microbiology
Vojtova, J., Kamanova, J., Sebo, P., Bordetella adenylate cyclase toxin: a swift saboteur of host defense. Curr. Op. Microbiol, 2006, 9, 69-75.
DanielLadant
Institut Pasteur
Paris, France
Hervas-Stubbs, S., Rueda, P., Lopez, L., Leclerc, C.,
Insect baculovirus strongly potentiate adaptive immune responses through the induction of type 1 interferon. J. Immunol, 2007, Feb
15;178(4):2361-9.
BenoîtVandenEynde
ICP
Brussels, Belgium
b) Patents
DorothyXing
NIBSC
Potters Bar, UK
Leclerc, C., Boisgerault, F., Rueda, P., ‘Empleo de
partículas pseudovirales como adyuvante. No.
P200403077’ Presented in the Spanish Patent and
Trademark Office, 23/12/2004.
Leclerc, C., Hervas-Stubbs, S., Rueda, P., Lopez, L..
Viral adjuvants. EP 06380240.9, 1.09.06
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DENDRITOPHAGES
Therapeuticcancervaccines
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Cancer has been considered as a fatal disease
for many years and has been selected as a priority disease throughout successive European
research programmes. Cancer is a group of over
100 related diseases characterised by uncontrolled cell growth that expands locally by invasion and systemically by metastases. Cancer is the
second most common cause of death in western
countries accounting for 2.5 million deaths in the
developed world.
Existing therapies have been able to increase
the survival rate, though only marginally. Current cancer treatments allow for the treatment
of approximately 50% of the patients, but these
therapies are associated with potentially serious
or intolerable adverse side effects. Thus, the quality of life of cancer patients is presently not satisfactorily preserved with surgery, chemotherapy
and radiotherapy. However, the advent of new
therapies, such as cancer vaccines, is expected
to enhance the quality of life since the dendritic
cell (DC) vaccines present an alternative immunotherapy approach with very few side effects.
Quality of life is therefore preserved during treatment and no hospitalisation is required.
The goal of cancer therapeutic cell vaccine is
to prevent progression and tumour recurrence.
Adoptive therapy in adjuvant settings will complement classical anti-cancer treatments. In this
technology the patient’s blood monocytes are
LSHB-CT-2003-0383
e฀1999940
1January2004
42months
transformed into effector monocyte-derived DCs
(dendritophages) which ight the patient’s own
disease. The therapeutic cell drug comprises DCs
loaded with cancer-speciic antigens to activate
the patient’s immune system after re-injection.
This ex vivo approach is compared to the in vivo
injection of the tumour antigen plus adjuvant.
This project aims to demonstrate on a
multinational level, the immunogenicity and
eficacy, as well as the reproducibility and
feasibility of anticancer vaccine by sequential
steps: choosing the best DC vaccination strategy
through adequate pre-clinical studies (DC
differentiation and maturation, tumour antigens
selection and loading, dose delivered, and site
and vaccination schedule); and monitoring the
immune response in correlation to the clinical
response after deining the most relevant
immuno-monitoring techniques.
This requires the establishment of quality control
criteria, the design for the production of the cellular product, as well as for the proteic tumour
antigen and its formulation, and to optimise a
GMP process. DENDRITOPHAGES initiated a clinical trial to evaluate the vaccines on progressing
prostate cancer patients.
On a short-term basis, the new treatments directed to residual cancer diseases resistant to conventional treatments will be used in an adjuvant
setting as second line therapy. On a long-term
basis, these patient-oriented therapeutic strate-
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DENDRITOPHAGES
Investigations in mouse models and recent evidence from the use of the cell therapeutic vaccine to treat and prevent cancer recurrence will
therefore be comprised of adequately differentiated and maturated DCs derived from the
patient’s blood monocytes loaded in vitro with
tumour antigens. The resulting cellular vaccine
is re-injected into the patient to stimulate an immune response against their cancer. In a clinical
setting, DC should be differentiated ex vivo, loaded with the chosen antigenic formulation, and
inally, maturation should be initiated by a short
treatment.
Dendritophage Copyright: IDM Author: J. Davous
gies should replace some irst line conventional
treatments of cancer.
Dendriticcells
Discovered in the early 1970s by Steinman and
Collin, DCs, key actors of the immune system,
are now considered as being the most potent
adjuvant for cancer immunotherapy to present
and stimulate speciic T cell responses. Capable
of taking up the antigen at the pathogen entry
site, to migrate into secondary lymphoid tissues
and to activate both helper and cytotoxic T cells,
DC can also interact with B cells, and probably
natural killer cells, and thus direct the speciicity
of the immune response.
However, it is only upon maturation that they acquire full expression of costimulatory molecules
and major histocompatibility peptide complexes; increase cytokine production; and migrate
to draining lymphoid organs. Despite a growing interest in the use of autologous DC for the
management of cancer and infectious diseases,
the relation between the type of DC infused in
humans and the type of immune response obtained, is not yet clear.
174
DENDRITOPHAGES anticipates that, once injected,
DC will migrate to T cell areas during the spontaneous progression of their activation and reproduce the physiological process of maturation.
The goal of these therapeutic vaccines is to prevent metastasis development and to provide
long-term protection. This immunotherapeutic
approach can be applied to diseases other than
cancer, in particular to stimulate an immune reaction against one or several antigens associated
with virus-infected cells.
In view of the urgent need to provide adapted
therapy to treat cancer, improved standardised
DC protocols will enable rationally designed
clinical studies to give cancer vaccine immunotherapy further credibility and cancer patients a
new therapeutic option associated with a better
quality of life. Sequential steps and objectives are
required.
The methodology developed and optimised during the project is adoptive cancer vaccine where
DCs are differentiated from apheresis of mononuclear cells, then matured and loaded with tumour antigen before being puriied, frozen, aliquoted and re-injected into the patients.
The participating teams compared a number of
11/8/07 10:50:10 AM
differentiation and maturation methods, and selected IL-13, GM-CSF and then IFNg + FMKp as
GMP processes, thus producing more than one
billion DCs.
KSA (Epcam) was chosen as a tumour proteic antigen and loaded on DCs as a protein, encaplulated in liposomes or in virosomes. The stimulation of KSA speciic cytotoxic T lymphocytes by
these DCs was achieved. Such loaded DCs have
already been injected in prostate cancer patients
in Vienna, and will be compared to the KSA-liposomes subcutaneous vaccine.
Main findings:
DENDRITOPHAGES has already inalised many
steps required for the evaluation of an anticancer vaccine:
• standardised preparation, differentiation and maturation of DCs: three distinct
processes for DC differentiation (IL-13 +
GM-CSF 7 days; IL-4 + GM; IFN 3 days) and
maturation factors were compared by ive
partners for differentiated DCs, matured
or not. DCs differentiated in the presence
of IL-13 appear to be the most robust in a
GMP process and the only ones to secrete
high IL-12. Maturation is induced within
six hours of being in the presence of IFNg
and of Klepsiella bacterial membrane
fraction.
• KSA is the choice of the adequate antigen: The KSA (EpCam) antigen was chosen
as a pancarcinoma tumour Ag protected
by IDM I.P.
• antigen loading: Loading of immature
DCs with KSA liposomes/KSA, virosomes/
KSA, and following a 24-hour stimulation period of T8 expansion, the indings
showed that virosomes were effective
only in stimulating CD4 T cells. KSA liposomes were the most effective in inducing
a CD8 T cell response. They were therefore
selected for clinical approach.
• injection site: The migration of immature versus mature DCs were followed in
patients according to the injection route,
using Cu64 labelled DCs. Intradermal injection delivers a better accumulation in
lymph nodes. The tracking and migration
of DCs were also studied in mice for the
different DC types produced. They were
labelled with Ittrium 86.
• the development of an emulsion with
adjuvant: Loading with liposomes also
containing lipid A as adjuvant and resuspended in water in oil emulsion appeared
the most effective, in comparison with
KSA alone or in virosomes.
• immunological monitoring of the
response to the vaccine: Speciic CD8 T
cell and antibody responses to KSA are
measured.
• generation of a GMP process acceptable by regulatory authorities: Based on
the GMP industrial cellular process developed by IDM, DENDRITOPHAGES determined the optimal strategy of DCs immunotherapy against prostate cancer. The
DC vaccine was elaborated and tested in
phase I/II studies on prostate cancer using
the selected autologous DCs loaded with
the KSA antigen. The researchers evaluated its tolerance and immune response
generated against KSA, and looked for
signs of clinical vaccine eficiency, probably based on serum PSA level and evolution. A second study with liposomal KSA
plus adjuvant was initiated in prostate
cancer patients for comparison.
GMP processes and clean room for cell therapy
were achieved in Paris, Rome, Melbourne and Vienna. A GMP lot of KSA incorporated in liposomes
was prepared, qualiied and released for clinical
use. A phase I clinical trial initiated in Vienna by
the end of 2005 on advanced prostate cancer patients compared the immune response obtained
after vaccination with DCs loaded with free KSA
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DENDRITOPHAGES
or with a liposomal KSA-lipid A emulsion. A second clinical trial was initiated in Regensburg
on less-advanced prostate cancer patients vaccinated with liposomal KSA emulsion plus lipid
A. The duration of the project was extended by
six months to launch this clinical protocol after
regulatory approval.
Coordinator
JacquesBartholeyns
IDM SA Immuno-Designed Molecules
172 rue de Charonne
75011 Paris, France
Partners
MilesPrince
Centre for Blood Cell Therapies
c/o Peter MacCallum Cancer Centre
Victoria, Australia
RheinardGlueck
Etnabiotech
c/o Università di Catania
Facoltà di Medicina
Dipartimento di Farmacologia Sperimentale e Clinica
Catania, Italy
ThomasFelzmann
Children’s Cancer Research Institute
Vienna, Austria
FilippoBelardelli
Istituto Superiore di Sanità
Laboratori di Virologia
Rome, Italy
AndreasMackensen
University of Regensburg
Department of Haematology/Oncology
Regensburg, Germany
Anne-CéciledeGiacomoni
Alma Consulting Group
Lyon, France
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11/8/07 10:50:10 AM
GENOMES TO VACCINES
Translatinggenomeandproteomeinformationintoimmunerecognition
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Genome information is accumulating rapidly and
it encompasses a wide range of species, including humans and many pathogens, among others.
It is now possible to completely sequence microbial genomes within a period ranging from days
to months, and genomics can therefore be used
as a powerful starting point to investigate the biology of any pathogen of interest. The translated
protein sequence information constitutes a signiicant input to the immune system.
Indeed, the immune system considers peptides
as one of its key targets, and it has devoted an entire arm — that of T cells, which essentially control
speciic immune responsiveness — to peptide
recognition. T cells are speciic for peptides presented in the context of major histocompatibility complexes (MHC). Prior to their presentation,
these peptides were generated in the cytosol by
limited proteolytic fragmentation of all available
protein antigens, then translocated to the endoplasmic reticulum (ER) and speciically sampled
by the MHC for subsequent presentation.
The MHC is extremely polymorphic and the peptide binding speciicity varies for the different
polymorphic MHC molecules. The net effect of
this complicated system is that each individual
presents to their T cells a unique and highly diverse peptide imprint of the ongoing protein
metabolism. The scientiic rationale of Genomes
To Vaccines is one of understanding, describing
LSHB-CT-2003-03231
e฀2000000
1January2004
48months
www.cbs.dtu.dk/researchgroups/
immunology/Genomes2vaccines/index.php
and predicting how the immune system handles
proteins; or more speciically, of how it generates, selects and recognises peptides. Generating
these predictive tools amounts to translating genomes/proteomes into immunogens, and it will
enable a new and rational approach for vaccination and immunotherapy.
Genomes To Vaccines focuses on MHC class I
mediated antigen presentation to cytolytic T
cells. While several mechanisms involved in antigen processing and presentation have been
described in general terms, only a few of them
have been described in suficient detail to allow
for the accurate prediction of their outcome; it is
a key requirement so as to achieve an overall (integrated) prediction of the inal result of a series
of processing events.
To produce ample and accurate data, Genomes
To Vaccines will generate biochemical highthroughput assays, representing the most important events in antigen processing and presentation. Focused on peptides and peptide
recognition, the research team seeks to develop
a high throughput peptide-array technology,
and it aims to generate accurate and quantitative predictions through the following data-driven approach:
• build and extend databases containing information on natural ligands and
immune epitopes;
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GENOMES TO VACCINES
• use these databases to generate preliminary predictions;
• use these predictions to select a set of
data points that will complement existing
data (i.e. the most information-rich new
data);
• perform the experiment to actually
generate the new quantitative data;
• add the new data to the existing
databases;
• update and improve the bioinformatics resources.
The approach constitutes an iterative and interactive loop between bioinformatics and wet
biochemistry, leading to successively improved
predictions. Eventually, this will allow Genomes_
To_Vaccines to search the entire sequence space
(more than 1012 members) computationally (fast
and cheap), while reserving the experimental
work (slow and costly) to the most informationrich data points. The researchers believe that this
integrated and iterative approach will generate
accurate prediction algorithms with a minimum
of effort (i.e. in the most cost-eficient manner).
Expected outcome:
Genomes To Vaccines believes that the integration of experiments and bioinformatics will
lead to an overall tool-generation process that
requires fewer and more pointed experiments,
with more accurate predictions. The resulting
predictors will be able to computationally (i.e.
rapidly and exhaustively) translate and evaluate genomic data for the presence of immunogenic epitopes. These resources will be hosted at
the Genomes To Vaccines website and be made
publicly available. This will enable any interested
basic or clinical scientist to perform searches for
immune targets from any microbial organism, or
tumour, of their choice.
The researchers expect that this tool will become
an essential part of medicine in the future, solv-
178
ing one of the bottlenecks in the current stateof-the-art search for vaccine candidates and
targets for immunotherapy. Only with computational bioinformatics tools and concurrent high
throughput life-science tools will it be possible
to search entire genomes for the presence of immunogenic epitopes and validate them. Eventually, it will enable a rational approach to population-wide vaccination, as well as individualised
immunotherapy.
Main findings:
Genomes To Vaccines has generated eficient biochemical assays for three different major steps
in the generation of T cell epitopes. This includes
the proteolytic fragmentation of protein antigens
(proteasome), the peptide translocation event
(TAP), and the peptide selection and presentation event (MHC). New biochemical assays were
used to generate data representing the above
events. Signiicant progress in generating a new
and improved method for the generation of
high-density peptide arrays was also made, and
the researchers hope to implement this technology in the generation of orders of magnitude for
more biochemical data in the future.
The data obtained so far has been used to generate bioinformatics tools capable of predicting the outcome of these events in silico and
to initiate the iterative process leading to improved predictions. For validation purposes, the
researchers have generated a set of data using
peptides extracted from cellular MHC, as representative examples of how the immune system
works under natural conditions. They integrated
the prediction tools and demonstrated that such
an integrated predictor is more successful in predicting natural epitopes.
Genomes To Vaccines has made these tools generally available as web-based services, and will
eventually also host the data itself. Finally, the
utility of these tools was demonstrated through
11/8/07 10:50:10 AM
the prediction of human T cell responses. Initially,
the researchers showed the speed of these tools
by performing a complete epitope scanning of
the SARS virus.
Subsequently, they performed a complete
epitope scanning of Inluenza A isolates, and
tested the predicted peptides both in biochemical MHC binding assays, but, more importantly, in
healthy inluenza convalescents. The latter study
identiied 13 new inluenza epitopes, which
covered virtually the entire population. These
epitopes were selected for being highly conserved, and notably, they all encompassed the
current bird lu isolates.
The ability to translate genomic information
into immune recognition will form the basis for
a rational approach to immunotherapy (including vaccination), which the researchers believe
will serve the policy objectives of enhancing the
quality of life for EU citizens.
Major publications
transport eficiency, and proteasomal cleavage
predictions’, Eur J Immunol, 2005, Aug;35(8):2295303.
Sylvester-Hvid, C., Nielsen, M., Lamberth, K., Roder,
G., Justesen, S., Lundegaard, C., Worning, P., Thomadsen, H., Lund, O, Brunak, S., Buus, S., ‘SARS CTL
vaccine candidates; HLA supertype-, genomewide scanning and biochemical validation’, Tissue
Antigens, 2004, May;63(5):395-400.
Lund, O., Nielsen, M., Kesmir, C., Petersen, A.G.,
Lundegaard, C., Worning, P., Sylvester-Hvid, C.,
Lamberth, K., Roder, G., Justesen, S., Buus, S., Brunak, S., ‘Deinition of supertypes for HLA molecules using clustering of speciicity matrices’,
Immunogenetics, 2004, Mar;55(12):797-810. Epub
2004 Feb 13.
Nielsen, M., Lundegaard, C., Worning, P., Hvid,
C.S., Lamberth, K., Buus, S., Brunak, S., Lund, O.,
‘Improved prediction of MHC class I and class II
epitopes using a novel Gibbs sampling approach’,
Bioinformatics, 2004, Jun 12;20(9):1388-97. Epub
2004 Feb 12.
Wang, M., Lamberth, K., Harndahl, M., Roder, G.,
Stryhn, A., Larsen, M.V., Nielsen, M., Lundegaard,
C., Tang, S.T., Dziegiel, M.H., Rosenkvist, J., Pedersen, A.E., Buus, S., Claesson, M.H., Lund, O., ‘CTL
epitopes for inluenza A including the H5N1 bird
lu; genome-, pathogen-, and HLA-wide screening’, Vaccine, 2006, Dec 29; [Epub ahead of print]
Peters, B., Bui, H.H., Frankild, S., Nielson, M., Lundegaard, C., Kostem, E., Basch, D., Lamberth, K., Harndahl, M., Fleri, W., Wilson, S.S., Sidney, J., Lund, O.,
Buus, S., Sette, A., ‘A community resource benchmarking predictions of peptide binding to MHC-I
molecules’, PLoS Comput Biol, 2006, Jun 9;2(6):e65.
Epub 2006 Jun 9.
Larsen, M.V., Lundegaard, C., Lamberth, K., Buus,
S., Brunak, S., Lund, O., Nielsen, M., ‘An integrative
approach to CTL epitope prediction: a combined
algorithm integrating MHC class I binding, TAP
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GENOMES TO VACCINES
Coordinator
SørenBuus
Institute of Medical Microbiology and Immunology
Panum 18.3.12
Blegdamsvej 3
DK-2200 Copenhagen N, Denmark
Partners
StefanStevanovic
University of Tübingen
Tübingen, Germany
PetervanEndert
INSERM U25
Hôpital Necker
Paris, France
HansjörgSchild
University of Mainz
Mainz, Germany
SørenBrunak
Technical University of Denmark
Lyngby, Denmark
ClausSchafer-Nielsen
Symbion Science Park
Copenhagen, Denmark
180
11/8/07 10:50:11 AM
COMPUVAC
Rationaldesignandstandardisedevaluationofgeneticvaccines
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LSHB-CT-2004-00246
IntegratedProject
e฀7969442
1January200
48months
www.compuvac.org
CompuVac has assembled 16 European teams
from both academia and industry, in 9 European
countries, with multidisciplinary expertise in the
ields of vaccine development, immunology, virology, vectorology, biomathematics and computer sciences. Recombinant viral vectors and viruslike particles are considered the most promising
vehicles to deliver antigens in prophylactic and
therapeutic vaccines against infectious diseases
and cancer. Several potential vaccine designs exist, but their cost-effective development cruelly
lacks a standardised evaluation system.
CompuVac is devoted to the standardisation of
vaccine evaluation and the rational development
of a novel platform of recombinant vaccines, with
HCV as a target disease. The CompuVac consortium has several primary objectives:
• to standardise the qualitative and
quantitative evaluation of genetic vaccines using deined ‘gold standard’ antigens and methods;
• to rationally develop a platform of
novel genetic vaccines using genomic
and proteomic information, together with
the project’s gold standards;
• to generate and make available to the
scientiic community a ‘tool box’ and an
‘interactive database’ allowing for a comparative assessment of future vaccines
to be developed with the project’s gold
standards.
CompuVac members : The CompuVac 2nd Annual
Meeting, November 2006, Athens, Greece
As end products, the consortium’s vector platform and ‘gold standard’ tools, methods and
algorithms will be made available to the scientiic and industrial communities as a toolbox and
interactive database. CompuVac seeks to apply
these vectors, tools and methods to the development of a preventive and/or therapeutic vaccine
against Hepatitis C Virus, incorporating one or
more of the project’s platform vectors expressing
the HCV envelope protein.
CompuVac recognises that a uniform method
for side-by-side qualitative and quantitative vaccine evaluation is lacking. Thus, standardising
the evaluation of vaccine eficacy and molecular signature by using ‘gold standard’ tools, and
molecular and cellular methods in virology and
immunology, as well as algorithms based on genomic information, is key.
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COMPUVAC
Main findings:
182
iii. Measles virus/Measles-VLP,
iv. MVA/MVA-VLP,
v. Polyoma virus;
Within the irst 2 years of its development, 4 major platforms have been set up by CompuVac.
These are presented below:
•BCG;
•DNA/DNA-VLP.
1.The CompuVac vector provider platform. This
platform is dedicated to the engineering and/or
optimisation of vectors expressing and/or containing the chosen model epitopes. A rational
vector design is carried out based on up-to-date
genomic and proteomic information. This platform assembles a wide range of viral vectors and
virus-like particles that are among today’s most
promising vaccine candidates:
•inertparticlevaccines—antigendisplay:
i. VLPs derived from retrovirus, polyomaviruses, hepatitis B virus, bacteriophage,
ii. Polyepitope peptides;
• gene expression vectors — antigen/transgene/VLPexpression:
i. Adenovirus/adeno-VLP,
ii. Herpes Simplex Virus 1/HSV-VLP,
There are two selected ‘gold standard’ antigens:
• T-cell antigen: GP33-41 epitope from
LCMV, to study the T cell response. The
LCMV glycoprotein G1 peptide p33-41 is
an extremely well-characterised model
antigen that has been widely used in numerous studies for evaluating humoral
response. In addition, this is a virus that is
mainly controlled by cellular immune responses in vivo.
• B-cell antigen: VSV-G protein, to study
the B cell response. The VSV envelop protein is the viral envelop that is best pseudotyped on retroviral-based particles
used for neutralisation assays. In addition,
this is a virus that is mainly controlled by
humoral immune responses in vivo.
11/8/07 10:50:14 AM
These ‘gold standard’ antigens have been introduced in the set of vectors of CompuVac.
2. The CompuVac immunological testing platform. Once produced, the immunological response will be studied by the immunological
testing platform regarding T and B cell response
and molecular signature. Data have been generated and entered in the ‘Genetic Vaccine Decision
Support system’ (GeVaDSs). ‘Gold standard’ algorithms for the intelligent interpretation of vaccine eficacy and molecular signature are built
into CompuVac’s interactive GeVaDSs, which
generates the following:
• vector classiication according to induced immune response quality, accounting for gender and age;
• vector combination counsel for primeboost immunisations;
• vector molecular signature according
to genomic analysis.
A GeVaDSs user manual for the current version
Fig 1 & 2: Radar and Bar charts - CompuVac, Genetic
Vaccines Decision Support System : Results
of the system has already been created. Scientiic
forms for immunisation protocols, animal models
and T cell responses were created, and the immunisation protocols were then analysed and implemented in GeVaDSs. A public test of GeVaDSs
took place at the CompuVac’s second executive
committee meeting in Stockholm in June 2006.
There were 2 reports generated by GeVaDSs:
• The T cell response project report: in
this project, CompuVac compared the
following: (i) interferon g production, (ii)
memory phenotype induction, (iii) clonal
expansion, (iv) peak of clonal response,
and (v) cytotoxic T lymphocytes response;
• The molecular signature report.
3. A CompuVac training and dissemination platform. The exchange programme was effectively
launched, allowing CompuVac’s young scientists
to learn new technologies and beneit from the
partners’ expertise. An expert immunologist and
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COMPUVAC
mathematician were hired, to train and connect
the scientists involved in the building-up of GeVaDSs.
Four training sessions dedicated to the use of
GeVaDSs by the data producers and the CompuVac team leaders, were coordinated by the computer scientists in order to improve data integration into GeVaDSs. A dissemination meeting was
organised by the CompuVac management team
at the 2006 annual meeting of the European Society of Gene Therapy on ‘Perspectives in Vaccine
Development’.
The CompuVac tool for dissemination is a 26minute ilm dedicated to the project and new
vaccine development. A ilm team (Actes&Avril
Productions) was contracted for the CompVac
ilm, and sequences were shot between
November and December 2006 in the following
locations:
• Hellenic Pasteur Institute, Athens,
Greece (CompuVac annual meeting, ESGT
dissemination meeting, and participating
institute/immunological testing platform);
• University of Munich, Munich, Germany (participating institute/immunological
testing platform);
• University of Zürich, Zürich, Switzerland (participating institute/immunological testing platform);
• CNRS – Pierre et Marie Curie University,
Paris, France (participating institute/scientiic coordination/management platform);
• Poznan University of Technology,
Poznan, Poland (participating institute/
GeVaDSs platform);
• Cytos Biotec, Zürich, Switzerland (participating biotechnology company/vector
platform),
• Epixis SA, Lyon, France, (participating
biotechnology company/vector platform).
4. The CompuVac management platform has
been set up, and is dedicated to the day-to-day
184
management of the scientists, the administrative
and inancial management, and the intellectual
property management, with a major feature being the study of the future of GaVaDSs.
Major publications
Bellier, B., Dalba. C., Clerc, B., Desjardins, D., Drury,
R., Cosset, F.L., Collins, M., Klatzmann, D., ‘DNA vaccines encoding retrovirus-based virus-like particles induce eficient immune responses without
adjuvant’, Vaccine, 2006, Mar 24;24(14):2643-55.
Dalba, C., Bellier, B., Kasahara, N., Klatzmann, D.,
‘Replication-competent Vectors and Empty Virus-like Particles: New Retroviral Vector Designs
for Cancer Gene Therapy or Vaccines’, Mol. Ther,
2007, Jan 23.
Dreux, M., Pietschmann, T., Granier, C., Voisset, C.,
Ricard-Blum, S., Mangeot, P.E., Keck, Z., Foung, S.,
Vu-Dac, N., Dubuisson, J., Bartenschlager, R., Lavillette, D., Cosset, F.L., ‘High density lipoprotein
inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of
the scavenger receptor BI’ J Biol Chem, 2006, Jul
7;281(27):18285-95.
Tegerstedt, K., Franzen, A., Ramqvist, T., Dalianis,
T., ‘Dendritic cells loaded with polyomavirus VP1/
VP2Her2 virus-like particles (VLPs) eficiently
prevent outgrowth of a Her2/neu expressing
tumour’, Cancer Immunology Immuntherapy, In
press, 2007.
Sominskaya, I., Alekseeva, E., Skrastina, D.,
Mokhonov, V., Starodubova, E., Jansons, J., Levi,
M., Prilipov, A., Kozlovska, T., Smirnov, V., Pumpens,
P., Isaguliants, M.G., ‘Signal sequences modulate the immunogenic performance of human
hepatitis C virus E2 gene’, Mol Immunol, 2006,
May;43(12):1941-1952.
Bartosch, B., Cosset, F.L., ‘Cell entry of hepatitis C
Virus’, Virology, 2006, Apr 25;348(1):1-12.
11/8/07 10:50:15 AM
Lavillette, D., Bartosch, B., Nourrisson, D., Verney,
G., Cosset, F.L., Penin, F., Pecheur, E.I., ‘Hepatitis C
virus glycoproteins mediate low pH-dependent
membrane fusion with liposomes’, J Biol Chem,
2006, Feb 17;281(7):3909-17.
CharlotteDalba
Epixis SA
Paris, France
AlbertoEpstein
Université Claude Bernard Lyon 1
Villeurbanne, France
Coordinator
DavidKlatzmann
CNRS and Université Pierre et Marie Curie
Laboratoire de Biologie et Thérapeutique des Pathogies
Immunitaires
UMR 7087 – UPMC-CNRS
Groupe Hospitalier Pitié-Salpêtrière
Bat. CERVI, 83, boulevard de l’Hôpital
75651 Paris Cedex 13 - France
E-mail : [email protected]
StefanKochanek
Universitätsklinikum Ulm
Ulm, Germany
PenelopeMavromara
Hellenic Pasteur Institute
Athens, Greece
AlbertusOsterhaus
Erasmus MC Rotterdam
Partners
AnatolySharipo
ASLA Biotech Ltd
Riga, Latvia
JacekBlazewicz
Poznan University of Technology
Poznan, Poland
ThomasBrocker
Institute of Immunology - Ludwig-Maximilians-Univerität
München
Munich, Germany
PaulPumpens
BioMedical Research and Study Centre
Riga, Latvia
KestutisSasnauskas
Institute of Biotechnology
Vilnius, Lithuania
RudolfMartinZinkernagel
Institute of Experimental Immunology
Zurich, Switzerland
François-LoïcCosset
INSERM
Lyon, France
GaryThomasJennings
Cytos Biotechnology AG
Zurich-Schlieren, Switzerland
TinaDalianis
Stockholm, Sweden
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HEPACIVAC
New preventative and therapeutic Hepatitis C vaccines: from preclinical to
phaseI
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Liver disease caused by Hepatitis C Virus (HCV)
infection is a major medical problem, affecting
an estimated 123 million people worldwide. No
vaccine is available and the best antiviral therapy
— a combination of interferon alpha and ribavirin — is only effective in a minority of patients.
An increasing body of data suggests that virus-speciic T cell responses are associated with
clearance of HCV in acutely infected humans and
chimpanzees.
Based on these observations, the long-term
objective of the HCV vaccine programme is
to develop both prophylactic and therapeutic
vaccines that elicit antiviral CD4+ and CD8+ responses capable of one of the following:
• reducing the rate of incidence and/or
persistence of HCV infection following exposure (for prophylactic vaccine);
• increasing the rate of clearance of
HCV infection as a monotherapy, and/or
in combination with current therapy or
novel anti-viral therapy (for therapeutic
vaccine).
The HEPACIVAC project is being carried out by a
large consortium, which includes two major vaccine manufacturers, and several academic and
research groups with expertise in clinical vaccine development, human immunity and animal
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LSHB-CT-2007-03743
IntegratedProject
e฀8800000
1January2007
60months
models. The consortium will focus on the skills of
the individual participants towards the development of new preventative and therapeutic strategies against HCV infection, integrating different
approaches and disciplines.
The HEPACIVAC proposed work is focused on the
development of two HCV vaccine candidates,
independently identiied by IRBM (Istituto di
Ricerche di Biologia Molecolare) and Novartis.
This study will be performed with a strong commitment to translate the results into effective
approaches for the prevention and therapy of
HCV. The irst vaccine candidate is gene-based,
encodes for the 2 000 amino acid-long HCV Non
Structural region (from NS3 to NS5B) and utilises adenoviral vectors for delivery. These vectors have been shown to elicit potent CD4+ and
CD8+ T cell responses in rodents and primates.
Recently, a proof-of-concept vaccination and heterologous challenge experiment in chimpanzees
showed that potent, broad and long-lived T-cell
responses to HCV were elicited in vaccinated animals. The gene-based vaccine protected against
acute and chronic disease induced by challenge
with a high dose of an heterologous HCV strain.
The second vaccine candidate consists of recombinant HCV glycoproteins, gpE1- and gpE2associated, to resemble a pre-virion envelope
structure. Protection against homologous and
heterologous challenge, mediated by CD4+ T cell
response and antibodies, was observed in experiments in chimpanzees.
11/8/07 10:50:16 AM
It follows that both vaccine candidates have the
potential to protect humans from a large number
of viral strains.The complementary action of these
vaccines might be highly instrumental in making
a vaccine against HCV, which is able to stimulate
several components of the immune system and
to elicit eficacious immune responses, both in
preventative and therapeutic vaccination settings. Furthermore, since the two vaccines target
different arms of the acquired immune response,
HEPACIVAC will offer the opportunity to analyse
in detail immune correlates of protection from
HCV infection, as well as to standardise immunogenicity assays, and to establish benchmark references for future preclinical and clinical studies
of HCV candidate vaccines.
• demonstration of immunogenicity of
the gene-based vaccine, in HCV chronically infected chimpanzees;
• demonstration of immunogenicity of
the two vaccines administered in combination in macaques;
• standardisation of the immunological
assays;
• evaluation of safety and immunogenicity of each of the two candidate vaccines, in healthy volunteers and in chronically infected individuals.
During the irst part of the project and after the
production of GLP & GMP vaccines, the work will
focus on several areas: animal safety and tolerability studies for the gene-based vaccine; and a
preclinical evaluation of compatibility, synergistic or antagonistic actions of the two vaccines
administered in combination in macaques. These
studies, aimed at verifying the possibility of using
this vaccine in humans, will be accompanied by
speciic activities on preclinical and clinical samples, in order to identify and standardise suitable
immunological assays to be used in the following HCV vaccine clinical trials.
In the second part of the project, HEPACIVAC
plans to perform Phase I trials with the vaccines
separately, to assess safety, tolerability and immunogenicity in healthy volunteers and in chronically infected individuals.
Expected outcome:
HEPACIVAC anticipates the following results:
• production of vaccine lots in suficient
amounts to conduct preclinical and clinical studies; demonstration of safety in animals, of the gene-based vaccine;
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HEPACIVAC
Coordinator
RiccardoCortese
CeInge Biotecnologie avanzate s.c.a.r.l.
via Pansini 5
80131 Naples, Italy
SergioAbrignani
Novartis Vaccine & Diagnostics
via Fiorentina 1
53100 Siena, Italy
KrystinaBienkowska-Szewczyk
University of Gdansk
Gdansk, Poland
SayedF.Abdelwahab
The Egyptian Company for Diagnostics
Agouza Giza, Egypt
Cristiana Tozzi
ALTA Srl
Siena, Italy
AdrianHill
Oxford, UK
Partners
AlfredoNicosia
Istituto di Ricerche di Biologia Molecolare (IRBM)
P.Angeletti
Pomezia (RM), Italy
FerruccioBonino
Fondazione IRCCS Ospedale Maggiore Mangiagalli e
Regina Elena
Milan, Italy
AlbertD.M.E.Osterhaus
GeertLerouxRoels
University of Ghent
Ghent, Belgium
JaneMcKeating
University of Birmingham
Birmingham, UK
StefanZeuzem
University of Saarland
Saarland, Germany
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BACABS
Assessment of structural requirements in complement-mediated bactericidal
events:‘Towardsaglobalapproachtotheselectionofnewvaccinecandidates’
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
High throughput cloning and expression of large
sets of genomic open reading frames (ORFs) has
become the preferred industrial strategy for genome-wide searches of new vaccine candidates.
For invasive infections in particular, the aim is
to ind proteins eliciting antibodies capable of
binding to the bacterial cell surface, and through
interaction with the complement system effectively killing the bacteria.
However, current data from reverse vaccinology
studies (the targeting of possible vaccine candidates, beginning with genomic information)
show that only a small fraction of surface-exposed proteins appears to elicit antibodies with
bactericidal activity. Using information generated by reverse vaccinology projects within the
consortium, the BacAbs project will apply a novel
multidisciplinary approach that will help to single out the structural requirements for viable
bactericidal vaccine candidates, and will develop
bioinformatics tools to predict compliance with
such structural requirements.
To this end, a systematic analysis of the sequence,
structure, dynamics and interactions of selected
protein targets will be conducted, using the
serogroup-B Neisseria meningitidis as a model
system. The consortium consists of an industrial
partner with extensive experience in vaccine
development, three SMEs with strong expertise
in several key technological aspects of the
LSHB-CT-2006-03732
e฀2269999
1January2007
36months
www.bacabs.org
project, and ive academic partners that are
internationally recognised for their experimental
and theoretical studies of protein structure and
interactions.
The development of antibiotic resistance constitutes one of the most potentially serious threats
in modern medicine. One approach to minimising the use of antibiotics, is to vaccinate against
pathogenic strains of bacteria. A clear candidate
for this approach is Neisseria meningitidis, a major cause of bacterial septicemia and meningitis against which no effective vaccine exists. N.
meningitidis is a Gram-negative bacterium that
is capsulated in its invasive form, and classiied
into ive major pathogenic serogroups on the
basis of the chemical composition of distinctive
capsular polysaccharides. Although a promising
candidate has entered clinical development, no
effective vaccine against serogroup-B N. meningitidis (MenB), which is responsible for more than
50% of all meningococcal diseases in Europe, has
reached the market yet. The capsular polysaccharide of MenB is identical to that of a widely
distributed human carbohydrate, making its use
as the basis of a vaccine for prevention of MenB
diseases problematic. Consequently, most efforts
have turned to the development of vaccines
based on surface-exposed or exported proteins.
A bactericidal response, i.e. one that leads to
bacterial-cell death, can be triggered through a
variety of methods. For meningococcal infections,
in vitro bactericidity assays with immune sera,
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I
BACABS
correlate with protection in humans. Although
in vivo protection against MenB may not be
solely achieved by complement-dependent
bacteriolysis, an antigen that elicits (murine)
antibodies capable of triggering bacterial-cell
death in vitro in a complement-dependent
manner, is normally considered a candidate for
human vaccine development.
In this context, one major obstacle to vaccine development, besides sequence and antigenic variability, is the dificulty to identify antigens that
will generate a bactericidal response. Typically,
only a very small fraction of the antibodies raised
in large-scale antigen-screening studies are bactericidal. Thus, while potential antigens can be
readily identiied, this information is of little use
unless we are also able to predict the antigens
that can lead to the production of bactericidal
antibodies.
In principle, the capacity of a protein antigen to
raise bactericidal antibodies may depend on (a
combination of ) the following factors:
• size, shape, structural complexity,
abundance, solubility, propensity and oligomerisation;
• sequence, structure, dynamics and
location of speciic protein regions
(epitopes).
These factors may in turn modulate the properties
of the Ag-Ab complex and its susceptibility to be
recognised by the C1q component. The BacAbs
project aims at deciphering possible correlations
between these factors and bactericidity.
of non-bactericidal antibodies.
To achieve this goal, the consortium will investigate the requirements for a productive AgAb-C1q complex formation. It proposes to ind
relevant answers using a multidisciplinary and
comparative approach to study the structure of
a number of these complexes. The MenB vaccine
development project of Novartis Vaccines & Diagnostics is to serve as a model, while a source
of useful data and reagent molecules will be
employed as well. To single out possible structural determinants, the focus is being placed on
groups of antigens with similar size, abundance,
solubility, etc. (both eliciting and not eliciting
bactericidal antibodies).
Although initially centred around group-B N.
meningitidis, the speciic target of the project
is the development of tools that can be applied
effectively to the genome-wide identiication of
vaccine candidates against any bacterial pathogen susceptible to complement-mediated lysis.
RoleofSMEs
The three SMEs involved in the BacAbs project
have a principal technological role. The IT company INFOCIENCIA SL will be operating the management and dissemination web servers of the
Consortium, carrying out bioinformatics analysis
of antigen and epitope sequences, implementing
algorithms, protocols and data emerging from
the Consortium’s work into computational tools
and databases within a web-based technological
platform, and evaluating the commercial interest
of this platform via demonstration.
Following the framework outlined above, the
BacAbs project is concerned with the identiication of (surface-exposed or exported) protein
antigens that may elicit complement-mediated
bactericidity in vitro. This includes early discrimination of antigens that may induce production
190
Two biotechs will be working on sample preparation and protein-structure determination. ASLA
Biotech Ltd will be performing protein expression and labelling; screening and optimisation of
sample conditions for NMR analysis; monoclonal
antibody generation; and sequential backbone
assignment and structure determination via NMR.
11/8/07 10:50:16 AM
Bio-Xtal SA will be performing protein expression and puriication, especially in connection
with derivatives (seleno-methionine labelled),
for X-ray analysis, expression and solubilisation
screens on highly hydrophobic or poorly soluble
targets. The consortium will also explore protein
crystallogenesis using nanodrop scale robotics techniques based on commercially available
and proprietary screening approaches; develop
and optimise robotic processes for crystallisation
plate storage and drop visualization; reine crystal growth conditions for selected hits to yield
diffraction quality crystals; and collect X-ray data
and structure solutions.
Potential applications:
The results of the BacAbs project could eventually be applied in the selection of new vaccine
candidates against group-B Neisseria meningitidis and other bacterial pathogens.
Expected outcome:
The BacAbs project will provide the following:
• Structural information on a set of proteins that are components of the cell surface (the bacterial organ for interaction
with eukaryotic host cells) of a major human pathogen;
• Improved experimental protocols and
techniques, bioinformatics tools and databases to assist the development of vaccines against human bacterial pathogens
in general, and group-B Neisseria meningitidis in particular;
• A framework in which experimental
and in silico methods for determining
protein structure and studying macromolecular recognition, immunological response mechanisms, and sequence-structure-function relationships can be further
developed;
• A web-based technological platform
integrating this knowledge, with the potential to improve the effectiveness and
reducing the costs of vaccine-candidate
searches.
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BACABS
Coordinator
XavierDaura
Universitat Autònoma de Barcelona Campus UAB s/n
Institución Catalana de Investigaciòn y
Estudios Avanzados
08193 Bellaterra (Cerdanyola del Vallès), Spain
Partners
GuidoGrandi
Novartis Vaccines and Diagnostics
Siena, Italy
AnatolySharipo
ASLA BIOTECH, Ltd.
Riga, Latvia
GhericiHassaine
Bio-Xtal SA
Mundolsheim, France
GiorgioColombo
Consiglio Nazionale delle Ricerche
Istituto di Chimica del Riconoscimento Molecolare
Milan, Italy
MartinZacharias
International University Bremen GmbH
School of Engineering and Science
Bremen, Germany
MartinoBolognesi
Università degli Studi di Milano
Milan, Italy
AlexandreM.J.J.Bonvin
Universiteit Utrecht
Department of Chemistry
JoséManuelMasBenavente
INFOCIENCIA SL
Barcelona, Spain
192
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MIMOVAX
Alzheimer’s disease-treatment targeting truncated Aß 40/42 by active
immunisation
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Alzheimer’s disease (AD) is the most common
form of dementia in humans. The Alzheimer Association has observed that there are currently
12 million patients worldwide suffering from this
disease, with estimated social costs for every patient reaching 40 000 per year. Currently, there
is no effective treatment available to stop the
progressive neurodegeneration and associated
cognitive decline in human patients. The current
cases are expected to almost double within the
next 20 years. All the treatment strategies presently being applied are focusing on the use of
small molecular drugs that inhibit the activity
of cholinesterase, to alleviate disease symptoms.
These drugs, however, have not been proven to
effectively halt or to revert disease progression
after prolonged treatment.
Alzheimer’s disease is caused by the deposition
of Beta-Amyloide (BA) into plaques within the
patients’ brain. The main constituent of BA is protein fragments, of 40-42 amino acids. These fragments derive from the Amyloid Precursor Protein
(APP), which is expressed on various cell types in
the nervous system. In humans, the majority of
amyloid plaque material is formed by Aß40/42
derivatives which are frequently truncated and
modiied. Aß peptides are considered to be directly involved in the pathogenesis and progression of AD.
Consequently, reduction of Aß burden in the
LSHB-CT-2006-037702
e฀23701
1October2006
36months
www.mimovax.eu
brain is predicted to slow down or halt disease
progression, and could also stop cognitive decline in AD patients.
Indeed, immunotherapeutic treatment using
active and passive immunisation strategies to
target full length Aß led to the reduction of Aß
plaques and had a beneicial impact on disease
progression in animal models of AD. However,
the irst phase II vaccination trial in AD patients
using full length Aß42 as an antigen had to be
discontinued, due to severe neuroinlammatory
side effects, including brain iniltration by autoreactive T cells.
MimoVax — Alzheimer’s vaccine — is a speciic
targeted research project (STREP) for the development and optimisation of a irst treatment to
stop the progression of AD. This project aims at
developing a vaccine against modiied forms of
BA. The immune system of AD patients will be activated to attack and remove BA, and will be able
to ight the cause of the disease directly.
The innovative technology presented in MimoVax
has been developed to create antigens mimicking
the structure of neo-epitopes which do not contain sequences of the native Aß peptide. A Mimotope-based AD vaccine would induce antibody
responses, exclusively reacting with the pathological Aß molecules but not with parental structures
like APP. Furthermore, Mimotopes do not contain
potential T cell self-epitopes and they avoid induction of detrimental autoreactive T cells.
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MIMOVAX
didates will be tested for eficacy in animal models of AD, altering disease progression.
This evaluation will include analysis of AD-pathology in the brain, as well as the testing of memory
and learning in these animals by cognitive tests
in vivo. The successful candidates will then be
tested for tolerability and safety in a irst phase
I trial in AD patients. Additionally, new diagnostic methods will be developed and evaluated in
order to monitor treatment eficacy in vivo in animal models.
Amyloid plaque staining in the brain of an AD mouse.
One of the hallmarks of Alzheimer’s disease is the accumulation of amyloid plaques in the brain. These amyloid
plaques can be visualised on sections of these brains by
staining with amyloid speciic antibodies. This staining
reaction results in a red amyloid plaque (indicated by a
white arrow) surrounded by blue nuclei of surrounding
cells like neurons or astrocytes. A Mimotope-based AD
vaccine as described in this abstract would induce such
speciic antibody responses reacting with the pathological A molecules and could therefore be a safe treatment
regimen to eficiently ight AD in patients.
Despite the fact that similar to full length Aß,
truncated and modiied Aß peptides seem to be
involved in the pathogenesis and progression of
AD, no relevant development programme has
been initiated to date. The goal of MimoVax is
the development of a safe and eficacious Alzheimer vaccine, which can prevent or even revert
cognitive decline in AD patients. In addition, new
diagnostic methods will be developed in order
to monitor treatment eficacy.
Expected outcome:
The expected outcome of the project will comprise a proof of concept for active immunisation
using MimoVax vaccines in animal models. Successful treatment should result in a reduction of
pathologic alterations in the brain, as well as in
improved learning and memory of the treated
animals. Furthermore, the researchers expect to
demonstrate the safety and tolerability of the
vaccine in patients, in initial phase I testing.
In summary, vaccines based on Mimotope peptides mimicking neo-epitopes which are derived
from the highly pathological and abundant Amyloid- derivatives, provide an innovative, inexpensive and eficient way to treat this widespread
disease with its severe personal and economic
impact on patients, their families and society.
This novel vaccine-technology is based on Mimotope peptides mimicking neo-epitopes which
are present on the above-mentioned highly
pathological and abundant Amyloid-ß derivatives, but not on the parental Amyloid Precursor
Protein (APP). The irst step in this project will be
the identiication and evaluation of novel mimotopes mimicking epitopes present on different
Aß derivatives. Subsequently, these vaccine can-
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Coordinator
FrankMattner
Afiris GmbH
Campus Vienna Biocentre
Viehmarktgasse 2a
A-1030 Vienna, Austria
MarkusMandler
Afiris GmbH
Campus Vienna Biocentre
Viehmarktgasse 2a
A-1030 Vienna, Austria
Partners
IrisGrünert
Biolution gruenert & co KEG
Vienna, Austria
AntónAlvarez
EUROESPES, SA
Department of Neuropharmacology
A Coruna, Spain
ManfredWindisch
JSW – Research GmbH
Graz, Austria
FritzAndreae
piCHEM research development
Graz, Austria
RichardDodel
Philipps-Universität Marburg
Marburg, Germany
AlexanderDrzezga
Technische Universität München –
Klinikum rechts der Isar
Munich, Germany
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I
PHARMA-PLANTA
Recombinantpharmaceuticalsfromplantsforhumanhealth
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Pharma-Planta aims to build a plant-based production platform for pharmaceuticals in Europe
and to enter the irst candidate pharmaceuticals
into clinical trials. The programme will develop
robust risk-assessment and risk-management
practices based on health and environmental impact and will work with EU regulatory authorities
to ensure safety and acceptance.
Plants have enormous potential for the production of recombinant pharmaceutical proteins as
they are inexpensive and versatile, in addition to
them being amenable to rapid and economical
scale-up. A major goal will be to address the necessary biosafety and regulatory requirements
for the use of plant-derived pharmaceuticals
through a process of engagement and consultation with the regulatory bodies involved in GM
plants, as well as new medicines. The project addresses pharmaceuticals for the prevention of
HIV, rabies, tuberculosis and diabetes, which remain signiicant health problems, both in Europe
and the developing world.
The Pharma-Planta consortium partners represent many of the major laboratories in Europe focusing on the creation of transgenic plants that
express important pharmaceuticals for human
health. Collectively, the consortium has a wide
range of expertise spanning the areas of molecular and plant biology, immunology, recombinant
protein expression technology, vaccinology, plant
196
LSHB-CT-2003-036
IntegratedProject
e฀12million
1February2004
60months
www.pharma-planta.org
biotechnology, risk assessment and IP management. This is a unique opportunity to make an
impact on EU and global health, through the responsible development of plant biotechnology.
The Pharma-Planta project is divided into 6 work
packages (WPs), focusing on distinct aspects of
pharmaceutical production in plants. WP1 examines targets and is responsible for producing
expression constructs and assays for the disease
target proteins to be expressed in plants. In this,
the third year of the programme, the role of
WP1 is all but complete. WP2 is concerned with
regulatory guidance and engagement with the
regulatory authorities. WP3 focuses on the major
production crops — maize and tobacco — which
were chosen early in the project after consultations carried out in WP2.
Of the 8 targets, 2 were chosen in WP1, and were
‘fast-tracked’ in WP3, which means they were
selected for breeding, scale-up and eventual
production under good manufacturing practice
(GMP) conditions. It was decided at the start of
the project that these targets would be 2 antiHIV antibodies. However, WP3 is concerned only
with the upstream production and the generation of plant material ready for extraction and
processing of these antibodies. WP3 also looks at
alternative expression platforms, including plastid transformation in tobacco, tomato and lettuce. WP4 is called the development loop, and its
11/8/07 10:50:18 AM
role is to examine various different strategies to
improve production yield and product quality.
Different workgroups within WP4 are looking at fusion proteins to enhance stability, protease knockouts, genetic modiication of glycan
structures, protein targeting and even the use
of plant viruses as expression vectors. Novel discoveries and innovations in WP4 will be beneicial to molecular farming as a whole and will be
exploited in future programmes. WP5 and WP6
have gained importance as we enter the second
half of the project. WP5 is concerned with downstream processing – extraction, clariication, puriication and formulation of the antibodies as a
clinical batch under GMP conditions. WP6, which
will run until the end of the project, is the clinical
trial itself.
Expected outcome:
The overall aim of the project is to complete a
phase I trial in humans, using either or both of
the 2 anti-HIV antibodies selected for fast-tracking through production, processing and regulatory compliance. At this stage of the project, the
Pharma-Plant consortium remains convinced
that this outcome will be achieved.
Main findings:
In the irst year of the project, the 2 anti-HIV antibodies were chosen as fast-track molecules and
the other 6 targets were consigned to the development loop. Another signiicant decision was to
focus on maize and tobacco as dual production
systems. Pharma-Plant has achieved high levels
of antibody expression in both systems, and fully
expects to achieve a clinical batch of antibody for
clinical trials by 2009. The project partners have
achieved signiicant progress in the area of process development, to allow the extraction and puriication of antibody molecules from plant tissue
under GMP conditions. This has been facilitated
not only by process development work in WP5,
but also through a signiicant number of interactions between WP2 and the regulatory authorities in Europe and South Africa.
Major publications
Sparrow, P.A.C., Irwin, J.A., Dale, P.J., Twyman, R.M.,
Ma, J.K.C., ‘Pharma-Planta: Road testing the developing regulatory guidelines for plant-made
pharmaceuticals’, Transgenic Res., 2007, Feb 7,
16(2):147-61. Epub.
Van Droogenbroeck, B., Cao, J., Stadlmann, J., Altmann, F., Colanesi, S., Hillmer, S., Robinson, D.G.,
Van Lerberge, E., Terryn, N., Van Montagu, M.,
Liang, M., Depicker, A., Jaeger, G.D., ‘Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds’, Proc Natl Acad Sci USA,
2007, 104(4):1430-1435.
Dunkley, T.P., Hester, S., Shadforth, I.P., Runions,
J., Weimar, T., Hanton, S.L., Grifin, J.L., Bessant, C.,
Brandizzi, F., Hawes, C., Watson, R.B., Dupree, P., Lilley, K.S. ‘Mapping the Arabidopsis organelle proteome’, Proc Natl Acad Sci USA, 2006, 103(17):651865123.
Ma, J.K., Barros, E., Bock, R., Christou, P., Dale, P.J.,
Dix, P.J., Fischer, R., Irwin, J., Mahoney, R., Pezzotti,
M., Schillberg, S., Sparrow, P., Stoger, E., Twyman,
R.M., ‘European Union Framework 6 PharmaPlanta Consortium Molecular farming for new
drugs and vaccines. Current perspectives on the
production of pharmaceuticals in transgenic
plants. EMBO Rep., 2005, 6(7):593-599.
Ma, J.K., Chikwamba, R., Sparrow, P., Fischer, R.,
Mahoney, R., Twyman, R.M., ‘Plant-derived pharmaceuticals - the road forward’, Trends Plant Sci.,
2005, 10(12):580-585.
Nuttall, J., Ma, J.K., Frigerio, L., ‘A functional antibody lacking N-linked glycans is eficiently
folded, assembled and secreted by tobacco
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PHARMA-PLANTA
mesophyll protoplasts’, Plant Biotechnol J.,,2005,
3(5):497-504.
Twyman, R.M., Schillberg, S., Fischer, R., ‘Transgenic plants in the biopharmaceutical market’, Expert
Opin Emerg Drugs., 2005, 10(1):185-218.
Coordinator
RainerFischer
Fraunhofer Gesellschaft
Hansastrasse 27c
D-80686 Munich, Germany
JulianMa
St. George’s Hospital Medical School
Department of Cellular and Molecular Medicine (CMM)
Cranmer Terrace
London SW17 0RE, UK
Partners
198
PhilipJ.DaleandGeorgeLomonossoff
John Innes Centre
Norwich, UK
LaurenceDedieuandRogerFrutos
Centre de Coopération Internationale en Recherche
Agronomique pour le Développement (CIRAD)
Paris, France
ChrisHawes
Oxford Brookes University
Oxford, UK
LorenzoFrigerio
University of Warwick
Coventry, UK
FriedrichAltmann
Universität für Bodenkultur
Vienna, Austria
FriedemannHesse
Polymun Scientiic Immunbiologische Forschungs GmbH
Vienna, Austria
MarioPezzotti
Università Degli Studi di Verona
Verona, Italy
RainerFischer,JuergenDrossardandStefanSchillberg
Fraunhofer Institute of Molecular Biology and Applied
Ecology
Aachen, Germany
EugenioBenvenuto
Ente per le Nuove Technologie, l’Energia e l’Ambriente
Rome, Italy
JulianMaandDavidLewis
St George’s Hospital Medical School
London, UK
ChristianVivares
Université Blaise Pascal Clermont-Ferrand II
Aubiere, France
EvaStogerandRainerFischer
Rheinisch-Westfälische Technische Hochschule (RWTH)
Aachen, Germany
HenriSalmon
Institut National de la Recherche Agronomique
Paris, France
PhilipJ.DixandJackieNugent
National University of Ireland
Maynooth, Republic of Ireland
JohnC.GrayandKathrynLilley
Cambridge, UK
11/8/07 10:50:18 AM
PaulGarside
University of Glasgow
Glasgow, UK
DavidRobinsion
Ruprecht-Karls-Universität Heidelberg
Heidelberg, Germany
MarcBoutry
Lovain-la-Neuve, Belgium
JurgenDenecke
University of Leeds
Leeds, UK
AlessandroVitale
Consiglio Nazionale Delle Ricerche
Rome, Italy
RachelChikwambaandEugeniaBarros
Council for Scientiic and Industrial Research (CSIR)
Pretoria, South Africa
JohnathanA.Napier
Rothamstead Research Ltd
Harpenden, UK
AnnDepicker
Vlaams Interuniversitair Instituut voor Biotechnologie
VZW
Zwijnaarde, Belgium
NikosLambrou
Agricultural University of Athens
Athens, Greece
RobEissandHarryThangaraj
Centre for the Management of Intellectual Property in
Health Research and Development (MIHR)
London, UK
EricvanWijk
Mosaic Systems BV
Prinsenbeek, Netherlands
PaulChristou
Univeritat de Lleida
Lleida, Spain
OscarReif
Sartorius AG
Gottingen, Germany
Jean-MarcNeuhaus
Université de Neuchatel
Neuchatel, Switzerland
RalphBock
Max Planck Institute
Postdam, Germany
TonyKavanagh
Trinity College
Dublin, Republic of Ireland
UdoConrad
Institut für Planzengenetik und
Kulturplanzenforschung
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SAGE
SME-ledantibodyglycol-engineering
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
The use of plant systems for the production of
pharmaceutical glycoproteins (including antibodies) offers an attractive alternative to the current state-of-the-art in protein production, given
their cost eficiency and overall safety. Producing
large quantities of antibodies for use in cancer
diagnostic and therapeutic systems using plant
systems could therefore prove a viable and inancially sound approach.
However, the differences in glycan structures
added by plants in comparison to those found in
humans pose signiicant obstacles and in fact researchers recognise that the plant species used
as well as the tissue and cell type and the age
have a huge impact on the inal glycoform of an
antibody.
The SAGE project targets an improved plant production platform for pharmaceutical glycoproteins. The partners, a high-calibre team of experts
in plant-based production technology and immunology, will use four plant-based expression
systems (including transgenic plants, virus-infected plants and transformed plant cell lines)
and mammalian cells as a control to generate a
therapeutic antibody that recognises the wellcharacterised carcinoembryonic antigen (CEA),
as well as their experience and know-how to
launch protein therapeutics on the market.
200
LSHB-CT-2007-037241
SME-SpeciicTargeted
ResearchProject
e฀1843427
1April2007
36months
The partners, part of an international consortium
of four research organisations, one small
and medium-sized enterprise (SME) and two
companies, will produce an antibody as a panel
of different glycoforms, which will be tested for
the following:
• stability;
• eficacy (e.g. in Fc-receptor binding
assays, tumour cell binding assays and tumour grafting);
• pharmacokinetic properties, such as
serum half life and antibody-dependent
cellular cytoxicity (ADCC).
The project partners will use the results to develop safer and more active glycoform varieties
for therapeutic applications.
By using the plant expression systems and BY-2
cells to generate the same recombinant antibody,
the consortium targets the production of H10, a
human full-length immunoglobulin G (IgG) that
recognises the CEA.
SAGE will also conduct a comprehensive, comparative study to establish how the structural,
functional and clinical properties of the antibody are inluenced by the glycan structures. The
project partners will compare the properties of
the H10 antibodies with those of a control H10
molecule generated in Chinese Hamster Ovary
(CHO) cells.
11/8/07 10:50:18 AM
Expected outcome:
The use of advanced plant-based expression
technologies will help SAGE generate innovative
therapeutic and diagnostic antibodies. The consortium will establish which best plant-based
expression platform should be used to produce
therapeutic antibodies, as well as identify the
glycan structures that give antibodies superior
properties in a clinical setting.
Coordinator
StefanSchillberg
Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie IME
Department of Plant Biotechnology
Forckenbeckstrasse 6
52074 Aachen, Germany
Partners
The team will also determine the effects the various plant-derived glycans have on the physical
and functional properties of antibodies. This action will support SAGE’s intention to produce improved antibody-based therapeutics with superior performance, as well as to provide treatment
for many more people who need it. Ultimately,
SAGE will be instrumental in improving health
care for everyone and in bringing down the costs
for treatment.
With strong business support, SAGE will convert
any new knowledge or product developed during the duration of the three-year project directly into strategic advantage. The project partner
Bayer BioScience will utilise IP management support to conduct IP searches and secure extra IP
protection, if needed.
SAGE will also transfer the acquired knowledge to
other scientiic groups working in the same ield.
Results and information will be disseminated via
electronic mail, peer-reviewed articles, abstracts
and posters. The SAGE consortium website will
also make key indings available to the public.
YuriGleba
ICON Genetics
Halle, Germany
GilbertGorr
greenovation Biotech GmbH
Freiburg, Germany
GerbenvanEldik
Bayer BioScience
Gent, Belgium
DirkBosch,DionFlorack,GerardRouwendal
Plant Research International BV
Wageningen, Netherlands
EvaStogerandRainerFischer
RWTH
Aachen, Germany
HardevSPandhaandDavidLewis
St George’s Hospital Medical School
London, UK
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I
BMC
Bispeciicmonoclonalantibodytechnologyconcept
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Among protein-based drugs, monoclonal antibodies (mAb) have a particular characteristic,
acting both as a drug and as a targeted delivery
system. MAb have recently shown great potential in the treatment of human cancer because
they are much more speciic than conventional
chemotherapy.
Antibodies are large protein molecules comprising two parts: First, the two variable binding sites
confer the unique speciicity to the molecule, and
can send a signal of growth inhibition or of programmed death, called apoptosis. Second, a constant part can trigger the activation of host defence cytotoxic molecules or killer lymphocytes,
directed against the cancer cells. However, a great
deal of progress is needed before we can understand the mechanism of binding and the activity
of antibodies, in order to improve their targeting
capabilities and their eficiency.
BMC aims to do the following:
• develop a recombinant bispeciic mAb
with tetrameric binding sites, directed
against two different antigens expressed
on the same target tumour cell, which will
add more speciicity and also double its
chances to target tumour cells and to recruit more effector natural killer cells;
• develop new bispeciic or bifunctional
molecules with the property of cross-linking two different receptors on the surface
202
LHSB-CT-200-1818
STREP
e฀240000
1November200
36months
of the cell, an event known to enhance the
signal transduction pathway of apoptosis;
• modify the carbohydrate moiety of
bispeciic antibodies and the tumour targeting the complement regulator molecule, properdin, to trigger the activation
of the complement enzymatic cascade at
the tumour site;
• establish the chemical conjugation
of antibodies of different speciicities on
emulsion nanoparticles.
The development of novel approaches to optimise the targeting and destruction of tumoural
cells is ensured in the following ways: by engineering new constructs of recombinant bispeciic or bifunctional antibodies reacting with
two different antigens on the same tumour cells;
by developing new strategies for the induction
of complement-dependant cytotoxicity (CDC),
either through modiications of the glycosylation of bispeciic monoclonal antibodies, or by
genetically fusing properdin to recombinant
monoclonal antibodies; and by creating a chemical conjugation of antibodies of different speciicities on emulsion nanoparticles.
The consortium will evaluate and validate the
novel tumour targeting strategies proposed in
the project, on tumour cells from patients with
CD5+ B-cell lymphoproliferative diseases, as well
as in an experimental model of SCID mice graft-
11/8/07 10:50:19 AM
ed with CD5+ B-cell lines. The optimisation of the
beneit/risk ratio in B CD5+ B-cell leukaemias and
lymphomas will also constitute a key approach.
Expected outcome:
A doubling of the avidity of bispeciic antibodies
for the BCLL targets tumour cells, in comparison
with state-of-the-art monospeciic monoclonal
antibodies, can be made possible with through
the following activities:
• induction of apoptosis in B-CLL cells
previously unresponsive to monomeric
anti- CD5 mAb;
• induction of lysis of at least 50% of
BCLL cells in the presence of human complement and speciic mAbs with carbohydrate modiication or fused properdin;
• induction of C3b opsonisation in 100%
of BCLL cells in the presence of the same
reagents;
• increase of vascular permeability in
subcutaneous tumour xenografts of human BCLL in SCID mice, after systemic
injection of mAb with a complement activating property;
• inhibit tumour growth in a model of
subcutaneous xenografts of human BCLL in SCID mice, by systemic injection of
selected bispeciic or bifunctional molecules.
Coordinator
JeanKadouche
Monoclonal Antibodies Therapeutics
Génopole Campus 1
5, rue Henri Desbruères
91030 Evry cedex, France
Partners
Jean-PierreMach
University of Lausanne
Bruno Robert Institute of Biochemistry
ChristianBerthoud
Université de Brest
Brest, France
MartineCerutti
Montpellier, France
SimonBenita
Hebrew University of Jerusalem
School of Pharmacy
Jerusalem, Israel
JoséeGolay
Ospedali Riuniti di Bergamo
Bergamo, Italy
JurgenBorlak
Fraunhofer – Institut Toxikologie und Experimentelle
Medizin
Hannover, Germany
CandiceOuinon
ALMA Consulting Group
Lyon, France
PatrickHenno
MABGENE S.A.
Alès, France
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I
AUTOCURE
Curingautoimmunediseases.Atranslationalapproachtoautoimmunediseasesin
the post-gnomic era using inlammatory arthritis and myositis as prototypes and
learningexamples
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The objective of the AutoCure project is to transform knowledge obtained from molecular research, particularly within genomics, into a cure
for the increasing number of patients suffering
from inlammatory rheumatic diseases. Rheumatoid arthritis (RA) is used as a prototype, since
this disease offers unique opportunities to deine
and evaluate new therapies.
Development of the irst generation of targeted
therapies (anti-TNF and anti-IL-1) in chronic inlammatory disease used RA as the prototype
disease, following work by European investigators included in the current project. This work
demonstrated the following:
• that targeted therapies can be
eficient;
• that a cure, while not yet achieved, is
within the reach of a strong international
consortium covering translational research and molecular technology.
Potential key molecular mechanisms determining the course of RA and myositis are deined
from genetic studies in humans, from relevant
animal models and from basic cell and molecular biology. Predictors of disease development
and therapeutic responses, which could lead to
future individualised therapies, are developed
with the help of the consortium’s unique large
patient group and biobanks. Development and
evaluation of new therapies is performed using
204
LSHB-CT-2006-018661
IntegratedProject
e฀11000000
1March2006
60months
www.autocure.org
combinations of novel molecular tools, and precise deinition of disease phenotypes.
AutoCure’s aims involve producing the following:
• an increased understanding of the
causes of RA and myositis, enabling better
prevention;
• new potential targets for therapy in arthritis and myositis, which can be further
tested in other rheumatic inlammatory
diseases;
• a prototype system for translational
research in Europe, enabling collaborative development of targeted therapies in
many inlammatory diseases and allowing
European SMEs to rapidly develop ideas
and patents into targeted therapies in inlammatory diseases.
The project targets available knowledge in genetics and genomics, to gain a better understanding
of rheumatoid arthritis and myositis. This will be
achieved through the appropriate use of largescale genetic investigations to characterise subsets of rheumatoid arthritis and myositis, and
the use of genetic, as well as other molecular
characteristics, to predict disease development
in these different subsets. Subsequently, knowledge about molecular mechanisms in different
genetically deined subsets would be used to
develop new targeted therapies, suitable for different subsets of these diseases.
11/8/07 10:50:19 AM
Expected outcome:
The AutoCure consortium expects to deliver new
and original knowledge concerning molecular
pathways used for different kinds of rheumatic
diseases, and also to provide a platform for the
development of new preventive measures and
therapies.
Main findings:
Findings have been made within the areas of etiology and pathogenesis of arthritis. Using large
biobanks with information on genetics and environmental factors, and immunity, the consortium
has been able to deine new risk factors for RA,
such as smoking and other environmental risk
factors, how they interact with genes, and how
they give rise to immune reactions that may
cause arthritis. Immunity against citrullinated
antigens were initially described by scientists
within this consortium, and studies on how such
immunity can cause arthritis is now very actively
ongoing within the consortium.
• 21 academic institution
Factors triggering the development of myositis
are investigated in one work package, and new
indings have been made concerning the molecular mechanisms of development of the symptoms of myositis, such as the characteristic weakness that causes disability. Using experimental
models for arthritis, new modes of using siRNa
for inhibiting cytokine production have been
used both in vitro and in vivo. Many groups are
now collaborating on the greater use of siRNa for
therapy of arthritis.
The development of therapies continues in many
of the clinics that are participating in the project.
In particular, studies on the individualisation of
therapy are ongoing, where biomarkers and genetic determinants are used to determine the eficacy, as well as the adverse effects of targeted
therapies against arthritis and myositis.
• 5 SME’s
• The Karolinska Institute (Co-ordinating institution)
• Prof Lars Klareskog (Coordinating Investigator)
Who are AutoCure
A major effort is being made apropos the harmonisation of guidelines for collection, and the use
of large clinical data bases and biobanks in relation to bioethical rules and regulations in Europe.
The guidelines to be developed will effectively
help scientists — both within rheumatology and
in other ields — to work eficiently together
while adhering strictly to guidelines and ethical rules. Collaboration is ongoing between the
clinical and experimental academic laboratories,
as well as the companies contributing to the consortium. Here, in particular, Genmab has been developing active new therapies with support from
scientists active in the consortium.
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11/8/07 10:50:21 AM
I
AUTOCURE
Grundtman, C., Salomonsson, S., Dorph, C., Bruton,
J., Andersson, U., Lundberg, I.E., ‘Immunolocalization of interleukin-1 receptors in the sarcolemma
and nuclei of skeletal muscle in patients with
idiopathic inlammatory myopathies’, Arthritis
Rheum, 2007, Jan 30;56(2):674-687
Hultqvist, M., Olofsson, P., Gelderman, K.A., Holmberg, J., Holmdahl, R., ‘A new arthritis therapy
with oxidative burst inducers’, PLoS Med, 2006,
Sep;3(9):e348.
Overview
Major publications
Van der Pouw Kraan, T.C., Wijbrandts, C.A., van
Baarsen, L.G., Voskuyl, A.E., Rustenburg, F., Baggen,
J.M., Ibrahim, S.M., Fero, M., Dijkmans, B.A., Tak, P.P.,
Verweij, C.L.,‘Rheumatoid Arthritis subtypes identiied by genomic proiling of peripheral blood
cells: Assignment of a type I interferon signature
in a subpopulation of patients’, Ann Rheum Dis.,
2007, Jan 18;
Michou, L., Croiseau, P., Petit-Teixeira, E., du Montcel, S.T., Lemaire, I., Pierlot, C., Osorio, J., Frigui, W.,
Lasbleiz, S., Quillet, P., Bardin, T., Prum, B., ClergetDarpoux, F., Cornelis, F., ‘European Consortium
on Rheumatoid Arthritis Families. Validation of
the reshaped shared epitope HLA-DRB1 classiication in rheumatoid arthritis’, Arthritis Res Ther,
2006, 8(3):R79.
Working model
206
Canete, J.D., Santiago, B., Cantaert, T., Sanmarti,
R., Palacin, A., Celis, R., Graell, E., Gil-Torregrosa, B.,
Baeten, D., Pablos, J.L., ‘Ectopic lymphoid neogenesis in psoriatic arthritis’, Ann Rheum Dis, 2007, Jan
12;
Wesoly, J., Hu X., Thabet, M.M., Chang, M., Uh, H.,
Allaart, C.F., Toes, R.E., Houwing-Duistermaat, J.J.,
Begovich, A.B., Huizinga, T.W., ‘The 620W allele is
the PTPN22 genetic variant conferring susceptibility to RA in a Dutch population’, Rheumatology,
Oxford, 2006, Nov 29.
Bendtzen, K., Geborek, P., Svenson, M., Larsson, L.,
Kapetanovic, M.C., Saxne, T., ‘Individualized monitoring of drug bioavailability and immunogenicity in rheumatoid arthritis patients treated with
the tumor necrosis factor alpha inhibitor inliximab’, Arthritis Rheum, 2006, Dec; 54(12):3782-9.
Michou, L., Lasbleiz, S., Rat, A.C., Migliorini, P.,
Balsa, A., Westhovens, R., Barrera, P., Alves, H., Pierlot, C., Glikmans, E., Garnier, S., Dausset, J., Vaz, C.,
Fernandes, M., Petit-Teixeira, E., Lemaire, I., Pascual-Salcedo, D., Bombardieri, S., Dequeker, J.,
Radstake, T.R., Van Riel, P., van de Putte, L., LopesVaz, A., Prum, B., Bardin, T., Dieude, P., Cornelis, F.,
‘European Consortium on Rheumatoid Arthritis
Families. Linkage proof for PTPN22, a rheumatoid arthritis susceptibility gene and a human
autoimmunity gene’, Proc Natl Acad Sci USA, 2007,
Jan 30;104(5):1649-54.
11/8/07 10:50:21 AM
Coordinator
LarsKlareskog
Department of Medicine
Rheumatology Unit
Stockholm, Sweden
E-mail [email protected]
or [email protected]
RenateGay
Centre of Experimental Rheumatology
and WHO Collaborating Centre
Zurich, Switzerland
ToreSaxneandRikardHolmdahl
Lund University
Lund, Sweden
Partners
TomHuizinga
Leiden, Netherlands
GerdBurmesterandAndreasRadbruch
Charité Universitätsmedizin Berlin
Berlin, Germany
JosefSmolen
Vienna, Austria
BarryBresnihan
St Vincent’s University Hospital
Dublin, Ireland
PaulPeterTakandDominiqueBaeten
Univesrity of Amsterdam
AndrewCope
Imperial College
London, UK
WimvandenBergandWalthervanVenrooij
University Medical Centre
ChristianJorgensen
Institut de la santé et de la recherche medicale
Paris, France
SolbrittRantapää-Dahlqvist
Umeå University
Umeå, Sweden
JiriVencovsky
Institute of Rheumatology
JaneWorthington
University of Manchester
Manchester, UK
JanvandeWinkel
GENMAB A/S
Copenhagen, Denmark
ChristopherBuckley
University of Birmingham
Birmingham, UK
FrançoisCornelis
Univeristé d’Evry
Evry, France
WlodzimierzMaslinski
Institute of Rheumatology
Warsaw, Poland
DimitriosBoumpas
University of Crete
GerdBurmester
EULAR Genomics Study Group
Zurich, Switzerland
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11/8/07 10:50:21 AM
I
AUTOCURE
CorVerweij
VU University Medical Centre
PeterOlofsson
Biovitrum
Gothenburg, Sweden
KarineMignon-Godefroy
BMD
Marne-la-Vallée, France
MargrietVervoordeldonk
Arthrogen B.V.
208
11/8/07 10:50:21 AM
INNOCHEM
Innovative chemokine-based therapeutic strategies for autoimmunity and
chronicinlammation
Innochem
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The general objective of INNOCHEM is to develop
innovative chemokine-based therapeutic strategies for autoimmunity and chronic inlammation.
The project is based on the scientiic excellence
of the applicants, which have made major recognised contributions to the ield since the very
beginning of chemokine discovery, and on the
construction of shared technological platforms.
This includes the following:
• proteomics;
• transcriptional proiling for the outline
of the ‘chemokinome’ in pathophysiological conditions and identiication of new
antagonists;
• molecular modelling of agonist/antagonist receptor or agonist/inhibitor interaction, for pharmacology and drug design;
• gene modiied mice for target validation in autoimmune disorders.
Genetic, structural, biological, and immunopathological studies will provide a framework
for the development of innovative chemokinebased therapeutic strategies. The therapeutic approaches to be investigated are innovative and
not limited to conventional antagonists. These
include decoy receptors, agonist binders, and
non-competitive allosteric inhibitors. In addition
to academic groups, therapy-oriented research
includes 3 biotech SMEs, 1 medium- and 2 largesized pharmaceutical companies. The companies
involved are developing complementary non-
LSHB-CT-200-18167
IntegratedProject
e฀11730696
1November200
60months
www.altaweb.eu/innochem
overlapping approaches to target the chemokine system with recombinant and low molecular
weight molecules.
INNOCHEM is expected to conduct a ‘proof-ofprinciple’ clinical study in volunteers. The ambition of this project is to re-establish European
leadership in basic and applied chemokine research, by integrating academic and industrial
cutting-edge groups, to develop innovative therapeutic strategies against autoimmunity and
chronic inlammatory disorders.
The approach of INNOCHEM is based on ‘platforms’, a ‘pipeline’ and the ‘integration of excellence’. The hard core ‘platforms’ on which INNOCHEM is based, include the following:
• proteomics and whole genome transcriptional proiling for the deinition of
the chemokinome in pathophysiological
conditions, and for the identiication of
new modulators;
• gene modiied mice, including double
knock outs (KO) and KO mice in autoimmune backgrounds for pathophysiology,
target identiication and validation, and
preclinical evaluation;
• molecular modelling for agonist/antagonist-receptor interaction or agonistinhibitor interaction and pharmacology
for the design and optimisation of innovative strategies.
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11/8/07 10:50:22 AM
I
INNOCHEM
The ‘pipeline’ characteristics are presented
below:
• Development of original, anti-chemokine strategies including receptor antagonists, agonist blockers, competitors of
agonist-GAG interaction, decoy receptors,
and non-competitive allosteric inhibitors.
These strategies are original inasmuch as
they extend beyond (but include) conventional antagonists.
• Pathophysiology of the chemokine
system in preclinical and clinical conditions, as a basis for rational, targeted intervention.
• Preclinical, multistep evaluation, including target validation in gene modiied
mice and by genetic analysis in humans
• Proof-of-principle clinical studies.
The entire pipeline has been activated from the
very beginning of the study (Fig 1). ‘Integration
of excellence’ represents a major characteristic
of the INNOCHEM approach. Not only does INNOCHEM have the ambition of collecting and attracting major European groups in the ield, but
its components also have a proven strong track
record of bilateral interactions. In the context of
INNOCHEM, a quantum leap of integrating European efforts in the ield will be possible, on the
solid basis of a tradition of collaboration.
Expected outcome:
The goal of INNOCHEM is to develop innovative
chemokine inhibitors as a novel therapeutic approach for the treatment of autoimmune and
chronic inlammatory diseases up to one ‘proofof-concept’ study in humans. The development
of innovative therapeutic strategies will be based
on a deinition of pathophysiological signiicance
and target validation (Fig 2). The therapeutic approaches to be investigated are innovative, and
not limited to conventional antagonists. These
will include decoy receptors, agonist binders, and
non-competitive allosteric inhibitors.
A unique non-competitive allosteric inhibitor,
Reparixin, which has completed phase I and has
been awarded orphan drug status by FDA and
EMEA, is available for proof-of-concept studies in
humans.
The results obtained in this project will open new
perspectives in therapeutic intervention against
autoimmunity and chronic inlammation, with
social impacts measurable as improved health
and reduced health societal costs, (including direct and indirect costs for patient assistance, as
well as loss of manpower), emerge.
Figure 1. – INNOCHEM pipeline.
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11/8/07 10:50:22 AM
Main findings:
During the irst year of activity, the INNOCHEM
consortium focused its efforts along the lines
originally identiied, albeit with some adjustments. In general, the lines of activity encompassed diverse ields, ranging from the basic biology of the chemokine system to translational
efforts in the clinical phase. Potential technological approaches have included the use of whole
genome transcriptional proiling, the use of
gene-modiied mice, and original reagents generated by the group. A major thrust of the INNOCHEM efforts has been devoted to strengthening integration and collaboration. As an example,
one could emphasise the strengthening of European leadership in the ield of silent receptors for
chemokines which can act as decoys. In terms of
translational efforts, a major result obtained during the irst year is the identiication of a chemokine (CXCL8) as a surrogate endpoint for the
activity in patients in inlammatory prostatitis of
a compound currently being developed by one
of the participating SMEs.
Major publications
Pello, O.M., Moreno-Ortiz, M.D., Rodriguez-Frade,
J.M., Martinez-Munoz, L., Lucas, D., Gomez, L., Lucas, P., Samper, E., Aracil, M., Martinez, A., Bernad,
A., Mellado, M., ‘SOCS upregulation mobilises
autologous stem cells through CXCR4 blockade’,
Blood, 2006, 108:3928-3937.
Rosenkilde, M.M., David, R., Oerlecke, I., nedJensen, T., Geumann, U., Beck-Sickinger, A. G.,
Schwartz, T.W., ‘Conformational constraining of
inactive and active states of a 7TM receptor by
metal-ion site engineering in the extracellular
end of transmembrane segment V (TM-V)’, Mol
Pharmacol, 2006, 70:1892-1901.
Penna, G., Mondaini, N., Amuchastegui, S., Degli
Innocenti, S., Carini, M., Giubilei, G., Fibbi, B., Colli,
E., Maggi, M., Adorini, L., ‘Seminal plasma cy-
Figure 2. - Outline of INNOCHEM targeting chemokine/receptor interactions, emphasising the implementation of
therapeutic strategies.
tokines and chemokines in prostate inlammation: Interleukin 8 as a predictive biomarker in
chronic prostatitis/chronic pelvic pain syndrome
and benign prostatic hyperplasia’, Eur Urol, 2007;
Feb;51(2):524-33.
Schmid, H., Boucherot, A., Yasuda, Y., Henger, A.,
Brunner, B., Eichinger, F., Nitsche, A., Kiss, E., Bleich,
M., Grone, H. J., Nelson, P. J., Schlondorff, D., Cohen,
C. D., Kretzler, M., ‘Modular activation of Nuclear
Factor- B transcriptional programs in human
diabetic nephropathy’, Diabetes, 2006, 55:29933003.
Proost, P., Struyf, S., Loos, T., Gouwy, M., Schutyser,
E., Conings, R., Ronsse, I., Parmentier, M., Grillet, B.,
Opdenakker, G., Balzarini, J., Van Damme, J., ‘Coexpression and interaction of CXCL10 and CD26 in
mesenchymal cells by synergising inlammatory
cytokines: CXCL8 and CXCL10 are discriminative
markers for autoimmune arthropathies’, Arthritis
Res Ther, 2006, 8:R107.
Springael, J. Y., Le Minh, P. N., Urizar, E., Costagliola, S., Vassart, G., Parmentier, M., ‘Allosteric modulation of binding properties between units of
chemokine receptor homo- and hetero-oligomers’, Mol Pharmacol, 2006, 69:1652-1661.
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INNOCHEM
Sironi, M., Martinez, F.O., D’Ambrosio, D., Gattorno,
M., Polentarutti, N., Locati, M., Gregorio, A., Iellem,
A., Cassatella, M.A., Van Damme, J., Sozzani, S.,
Martini, A., Sinigaglia, F., Vecchi, A., Mantovani, A.,
‘Differential regulation of chemokine production
by Fc receptor engagement in human monocytes: association of CCL1 with a distinct form of
M2 monocyte activation (M2b, type 2)’, J Leukoc
Biol, 2006, 80:342-349.
Coordinator
AlbertoMantovani
Humànitas Mirasole S.p.A
Via Manzoni 56
20089 Rozzano-Milano, Italy
MariagraziaUguccioni
Istituto di Ricerca in Biomedicina
Via Vela 6
6500 Bellinzona, Switzerland
Partners
ChristopheCombadiere
Université Pierre et Marie Curie-Paris
INSERM U543
Paris, France
DominiqueEmilie
INSERM
Paris, France
AndrzejGlabinski
Uniwersytet Medyczny w Łodzi, Department
of Neurology
Lodz, Poland
MartinLipp
Max-Delbrueck-Centrum für Moleckulare Medizin
Berlin, Germany
CarlosMartinez
Centro Nacional de Biotecnologia, CSIC
Madrid, Spain
BernhardMoser
Universität Bern, Institute of Cell Biology
Bern, Switzerland
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11/8/07 10:50:23 AM
PeterJ.Nelson
Ludwig-Maximilians-University Münich
Münich, Germany
AntalRot
Novartis Institutes for Biomedical Research
Vienna, Austria
MarcParmentier
Université Libre de Bruxelles, IRIBHM
Brussels, Belgium
AmandaProudfoot
Serono International SA
Plan les Outas, Switzerland
CostantinoPitzalis
London, UK
AldoTagliabue
Alta S.r.l.
Siena, Italy
SergioRomagnani
Università degli Studi di Firenze, “MCIDNENT”
Florence, Italy
MetteM.Rosenkilde
The Panum Institute
Department of Pharmacology
Copenhagen, Denmark
JozefvanDamme
Katholieke Universiteit
Leuven, Belgium
TimothyWilliams
Imperial College of Science, Technology and Medicine
London, UK
AmnonPeled
BioKine Therapeutics Ltd.
Rehovot, Israel
LucianoAdorini
BioXell
Milan, Italy
MarcelloAllegretti
Dompe pha.r.ma Spa
L’Aquila, Italy
JanW.Vrijbloed
Polyphor Ltd.- PEMB Unit
Allschwill, Switzerland
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I
CELLAID
Europeansymposiafortheevaluationofpotentialsandperspectivesofcurative
celltherapiesforautoimmunediseases
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
Autoimmune diseases, such as inlammatory
rheumatic diseases, are currently impossible to
cure with either conventional therapies or modern biologics therapies. Existing therapies are
immune-suppressive, systemic and/or accompanied by serious side effects, which drastically
reduce quality of life and life expectancy. The socioeconomic impact of inlammatory rheumatic
diseases is approximately 15 billion per year in
Germany alone, with a trend for costs to increase
rapidly due to the market prices of emerging biologics.
The CELLAID project will establish a European
consortium to organise three symposia on immunocyte-based therapies for autoimmune
diseases. The new treatment concepts should
target pathomechanisms, especially the involved
immune cells and messenger biomolecules in
the course of the disease. CELLAID also aims at
providing a communication platform for European experts to evaluate the existing potential,
and develop integrated pan-European strategies.
Joint European efforts are required to strengthen
the European lead in respective technologies,
and also to combine experience in pan-European clinical trials.
The European Research Area can maintain excellence, and scientiic, technological and economic
competitiveness with innovative therapies for
autoimmune diseases through networking.
214
LSHB-CT-2004-00094
e฀198800
1January200
30months
www.cellaid-eu.org
Such networking of European research institutions and clinics will enhance research expertise,
the standardisation of clinical interventions and
quality assessment, e.g. immune monitoring. Patient cohorts will grow, even for rare autoimmune
diseases. The translation of concepts into clinics
will involve extensive industry cooperation: joint
efforts will boost the competitiveness of small,
medium and large biotech and pharmaceutical
companies in Europe.
The irst two CELLAID symposia were held in
Berlin in June 2005, and in Brussels in April 2006.
Cellular concepts of immune therapies for autoimmune diseases and European perspectives
towards immunocyte-based therapies were addressed by an international selection of expert
speakers, chairmen and participants. Results
(programmes and summaries) of the symposia
are published on the website of the project, www.
cellaid-eu.org. The third symposium is scheduled
to take place in Florence, Italy, on 20-22 February
2007.
Expected outcome:
Currently, no cure is available for autoimmune
diseases, which require a life-long immune suppressive treatment with a high risk of adverse
events, and lack a regenerative perspective. Immunocyte-based therapies — supported by recent advances made in genomics, immunology
11/8/07 10:50:23 AM
CellAid 2005 - Andreas Radbruch (Coordinator)
and cytometry — aim for an autoimmunity cure
and a reset of tolerance, which is a prerequisite for
regenerative therapies. They will have high translational potential for other immune diseases.
Main findings:
While research highlights of the two symposia
were numerous, only a selection is mentioned in
this report. Detailed summaries are available at
www.cellaid-eu.org. The function and dysfunction of regulatory T cells were the areas of focus
in a number of presentations. Cytokines such as
IL-6 and its transsignaling mechanisms were presented, representing potential new future therapeutic targets. Many other relevant cytokines
and their functions were discussed, highlighting
the role of special cytokine networks that perpetuate chronic inlammation, particularly in autoimmunity. B cells and their roles as plasma and
memory cells were intensively discussed in both
symposia. Currently, B cell (e.g. CD 20)-targeted
biologics do achieve the most convincing therapeutic effects. However, it remains to be seen
whether remission or a cure can be achieved
without long-term side effects.
With regard to genomics in multi-factorial diseases, such as rheumatoid arthritis, the identiication
of genes other than the major histocompatibility
complex that regulate autoimmune diseases was
discussed. One regulator of arthritis severity in
rats was identiied and then presented at the irst
CELLAID symposium. In experimental models, a
single gene such as AIRE has provided important
insight into the regulation of autoimmunity. Discussions at the two CELLAID symposia highlighted that the power and impact of genetic analysis is undisputed, and that the translation from
animal models to genetic analysis for diagnosis,
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11/8/07 10:50:25 AM
I
CELLAID
prediction and therapy of autoimmune diseases
patients is in progress.
One of the experimental therapies gaining approval is autologous stem cell transplantation,
performed for many autoimmune diseases. In
Europe, they are registered through the EULAR
and EBMT working parties. They do in some instances (e.g. Systemic Lupus Erythematosis) have
the potential to induce full remission, even in patients with a long disease history. As discussed in
the presentations at both meetings, this is seemingly based on the combination of a drastic depletion of pathogenic memory and effector cells,
and the development of a new and tolerant immune system. So far, however, autologous stem
cell transplantation is only applicable for standard treatment of refractory patients, as the side
effects can be hazardous.
Coordinator
AndreasRadbruch
Deutsches Rheuma-Forschungszentrum
Charitéplatz 1
JuttaSteinkötter
Competence network rheumatology
Luisenstr. 41
Finally, mesenchymal stem cells were introduced
as ‘targets’ and they have been applied
successfully in experimental animal models,
where they show they can provide strong
immunosuppressive effects. Results from the irst
European trials in human were presented at the
CELLAID symposia.
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STEMDIAGNOSTICS
Thedevelopmentofnewdiagnostictests,newtoolsandnon-invasivemethods
for the prevention, early diagnosis and monitoring for haematopoietic stem
celltransplantation
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Over 7 000 allogeneic haematopoietic stem cell
transplants (HSCT) are carried out each year in
Europe alone, as a treatment for leukaemia and
lymphoma. Techniques and cure rates are improving, but the overall survival rate remains between 40% and 60%.
STEMDIAGNOSTICS will develop new proteomic,
biological and genomic tests and tools for early
diagnosis and monitoring of patient response to
novel therapeutics for the most severe complication of HSCT - graft versus host disease (GvHD).
It will bring to the clinic a new generation of
diagnostics that will signiicantly improve HSCT
therapy and patient outcome.
The consortium unites 5 European SMEs with expertise and markets in genomic and proteomic
testing, diagnostic assay development and biochips, with clinical partners selected for their
world leading research in HSCT and access to
clinical samples and patient groups. The project
will focus on the role of relevant genes and biomarkers associated with acute and chronic
GvHD, using retrospective samples from established biobanks and prospective clinical trials to:
• identify novel bio and genomic markers for diagnostics;
• develop novel diagnostic tools using
genomics, proteomics, in vitro bioassays
and biochips;
• test the new diagnostics in animal
models and on clinical samples;
LSHB-CT-2007-037703
e฀200000
1June2007
36months
• exploit the new tools for commercial
use.
STEMDIAGNOSTICS will develop diagnostic tests
using single nucleotide polymorphism (SNP)
analyses (IMGM), based on results from previous
EC-funded research (Eurobank, Transeurope).
It will use proteomics via mass spectrometry
evaluation/development of diagnostic patterns
(Mosaiques), ELISA kits (Apotech) and protein
biochip prototypes (Orla) for the development of
fast, high throughput technologies. The consortium will also develop novel reagents for monitoring graft versus leukaemia, GvHD and targeted
therapy (Multimune; Nascacell). Finally, comparative studies in an autoimmune disease model of
inlammation — rheumatoid arthritis — will be
made.
Adult stem cells are starting to be used for the
therapy of diverse disorders including autoimmune disease and cancer. The potential differentiation of such stem cells into various types
of adult tissue is also a new and exciting area of
research. It will create opportunities for the innovation of a new generation of medical diagnostic tools, tests and therapies; for improving
healthcare services and patient outcomes; and
for reducing expenditure on healthcare systems.
It offers Europe’s biotech SMEs the opportunity
to strengthen their competitiveness and successfully help meet the growing demands of the
healthcare sector.
Stem cell research is essential due to the lack,
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I
STEMDIAGNOSTICS
over the past 30 years, of any general cure for
various illnesses, such as cancer, heart disease or
autoimmune disorders, among others.
Haematopoietic stem cell transplantation (HSCT),
however, is one area of stem cell research where
major advances in the cure of haematological disorders, like leukaemia and lymphoma, inherited
immune disorders, and aplastic anaemia, have
been made. Transplantation of stem cells in this
context, where immature cells develop into white
and red blood cells and platelets (i.e. haematopoiesis) from related or unrelated donors leads to
immune reconstitution and can be a life saving
therapy. Currently over 7 000 transplants are carried out each year in Europe. Despite this success
the overall survival rate after HSCT is poor.
A 40-60% survival rate is the norm for such
transplants, which involve the use of bone marrow, peripheral blood stem cells and umbilical
cord blood as the stem cell source. The cure of
patients is limited by clinical complications that
arise post-transplant. These are largely due to a
lack of understanding of acceptance and rejection mechanisms and genetic differences that
exist between a given patient and donor and
inaccurate/lack of diagnostic tests for early complications.
The application of HSCT is also hampered by the
lack of suitably matched donors. Only 25-30% of
patients will ind a HLA matched sibling donor
and, therefore, more matched unrelated donors
within International Bone Marrow Transplant
Registries are needed. There is therefore an urgent need to improve patient-donor matching at
both the biological response and genomic level,
which will increase the pool of both suitable and
more appropriate donors and reduce the most
severe complication of acute and chronic graft
versus host disease (GvHD).
It is essential to predict the development, the
severity and treatment outcome of GvHD, which
218
is an important predictor of transplant survival.
The strongest predictor of GvHD is the degree of
mismatching between patient and donor; however, there is a signiicant incidence of acute and
chronic GVHD even in patients undergoing HLA
identical sibling transplants, thus clearly indicating a role of genetic risk factors other than major
HLA differences There are also no reliable predictive or diagnostic indicators to date for either
acute or chronic GvHD and no reliable markers
that distinguish GvHD from viral or other inlammatory manifestations.
The most reliable clinical predictors of GvHD are
the donor recipient sex-mismatch (female donor
to male recipient) but no other clinical factors
can predict acute GvHD. Research therefore has
focused on the biology of GvHD and the involvement of cytokines in the “cytokine storm”). Serum
levels of cytokines, mRNA expression of candidate
cytokines in peripheral blood or target tissue, or
as in the case of several of the current partners,
a study of non HLA genetic polymorphisms for
candidate cytokines using pretransplant recipient and donor DNA have been analysed. However, none have as yet been tested effectively
for commercial use as novel diagnostic tests).
These studies will form the basis of further development of novel diagnostics based on the fact
that early detection of GvHD and its severity is
urgently needed and that its progression needs
to be monitored during the course of HSCT.
The project will focus on the role of relevant
genes and biomarkers associated with acute and
chronic GvHD, using retrospective samples from
established biobanks and prospective clinical trials to:
• identify novel bio and genomic markers for diagnostics;
• develop novel diagnostic tools using
genomics proteomics, in vitro bioassays
and biochips;
11/8/07 10:50:26 AM
• test the new diagnostics in animal
models and on clinical samples;
• exploit the new tools for commercial
use;
• undertake comparative studies in an
autoimmune disease model of inlammation; rheumatoid arthritis.
The SMEs’ speciic aims include:
• development of new tools for novel
diagnostics, novel drug targets and therapeutics for use in both the transplant and
autoimmune settings;
• access to important clinical and biological samples, as well as data for use in
accurate assessment of results and conirmatory studies for the development of
novel diagnostics;
• testing and evaluation of new diagnostics on independent cohorts and correlation of data across HSCT centres;
• testing of new prototypes against
current assays via collaboration between
SMEs;
• use of new target molecules in the
monitoring of response to therapy and
during the post transplant period to assess acute and chronic GvHD.
Expected outcome:
STEMDIAGNOSTICS will identify new diagnostics in the form of novel proteins associated with
graft versus host disease (GvHD) compared to
viral disease. The consortium will also develop
early diagnostic tools for GvHD and rheumatoid
arthritis using gene proiling. The fast throughput
development of novel monoclonal antibodies
and ELISA kits for research, diagnostics, and potentially therapeutic use emerge as well. STEMDIAGNOSTICS will develop novel peptides for use
in monitoring GvHD and graft versus leukaemia
effects in transplant patients, and it will identify
new single nucleotide polymorphisms (SNPs) for
analysis in prognostic/diagnostic indices.
Coordinator
AnneDickinson
University of Newcastle upon Tyne
School of Clinical and Laboratory Sciences
The Medical School
Newcastle upon Tyne, UK
Partners
ErnstHoller
Klinikum der Universität Regensburg
Dept. Hamatology und Internistiche Onkologie
Regensburg, Germany
HaraldMischak
Mosaiques Diagnostics GmbH
Hannover, Germany
GabrieleMulthoff
Multimmune GmbH
Munich, Germany
RalphOehlmann
IMGM Laboratories
Martinstried, Germany
LarsFrench
Zurich University Hospital
OlivierDonzé
Apotech Corporation (Headquarters)
Epalinges, Switzerland
Hans-JochemKolb
Clinical Cooperation Group Hematopoietic Cell
Transplantation, Institute of Molecular Immunology
Forschungszentrum fuer Umwelt und Gesundheit
Munich, Germany
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STEMDIAGNOSTICS
DaleAthey
Orla Protein Technologies Ltd
Nanotechnology Centre
University of Newcastle upon Tyne,
Newcastle, UK
GerardSocie
Association de Recherche sur la Greffe de CSP
Aplosies et HPN
Paris, France
HildegardGreinix
University Hospital of Vienna
Bone Marrow Transplantation Unit
Vienna, Austria
IlonaHromadníková
Charles University
3rd Medical Faculty
AmandaMcMurray
CENAMPS
The Fabriam Centre
Atmel Way
Newcastle upon Tyne, UK
220
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RISET
Reprogrammingtheimmunesystemfortheestablishmentoftolerance
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
LSHB-CT-200-12090
IntegratedProject
e฀10000000
1March200
60months
www.risetfp6.org
RISET aims at inducing tolerance, which is deined as the permanent acceptance of the transplant in the absence of continuous immunosuppression. It is based on the translation of recent
advances in post-genomic immunology that are
focused on the development of new biotechnology products to promote long-term transplant
acceptance in preclinical models.
Université Libre de Bruxelles- IMI. Gosselies. Belgique.
In order to achieve this objective, the RISET
programme anticipates developing diagnostic
tests to identify transplanted patients for whom
immunosuppressive treatment could be safely
minimised or withdrawn. Related ethical and
societal questions will be speciically addressed,
in addition to communication with patient
organisations and regulatory bodies. In parallel,
relevant models of tolerance will be used to
identify new genes, molecules or cell types that
will form the basis for novel diagnostic and
therapeutic approaches.
The use of knowledge will be facilitated by the direct involvement of SMEs in the consortium, the
building of an industry platform, and the yearly
monitoring of external reviewers with both scientiic and industrial backgrounds in the ield.
Expected outcome:
The ultimate goal of this project is to develop
safe and eficient diagnostic tools, and therapies
to induce long-term tolerance in transplanted
patients, in the absence of any immunosuppression. This ambitious objective will be achieved
through a multistep approach involving intermediate milestones:
• the development of biological tests
predictive of transplant tolerance or ‘neartolerance’;
• the clinical and biological assessment
of the outcome of patients enrolled in
clinical investigations, targeting the complete withdrawal of immunosuppression;
• the establishment of ethical guidelines
for tolerance induction protocols and educational programmes on tolerance induction for patients and their families;
• the establishment of educational programmes on tolerance induction for physicians, scientists and nurses;
• the identiication of new molecular
targets for tolerance induction in preclinical models.
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I
RISET
ary 2006, 2 more patients were transplanted, and
relevant data will be provided in the next report.
Identiication and recruitment of additional patients to be involved over the second period is
under way. In parallel, the use of a combination of
anti-CD3 and anti-CD7 immunotoxins to induce
long-standing remission of severe high-grade
graft-versus-host disease, has been investigated
(drug components have been produced and
puriied, draft protocols and inform consent prepared, phase I/II study has been registered and a
preclinical evaluation has been realised). A study
using monkeys to perform cytotoxicity tests has
been prepared.
Participants
Main findings:
A irst series of diagnostic tests have been deined and validated in-house in WP1 (work
package 1), to identify transplanted patients for
whom immunosuppressive treatment could be
safely minimised or withdrawn. The validation of
these tests and markers in clinical protocols has
started, with irst samples of tolerance induction
pilot trials already collected and distributed under strict quality control.
Basic research performed in WP2 has contributed
to the characterisation at molecular level of important actors and mediators of tolerance (markers of regulatory T cells, tolerogenic DCs). Models
of endothelial cell damage to test the effect of
endothelial cell protection on allograft survival
were initiated. Antibody therapy in human CD3
or CD52 transgenic animals was tested, with a
view to optimising anti-CD3 and anti-CD52 treatment in clinical settings.
Ethical aspects have been monitored through
WP4 and further research is ongoing for key societal, organisation, ethical, regulatory and legal
issues related to activities performed in RISET.
Several dissemination supports have been developed (website, lealet, newsletter, and presentations to scientiic and large public audiences).
Training actions related to RISET activities and
research objectives have taken place at local, national and international level. A feasibility study
to examine the opportunity of setting up an industry platform to foster exploitation aspects of
the project has been performed, and has led to
recommendations for RISET’s future activities.
Coordinator
MichelGoldman
Rue Adrienne Bolland, 8
6041 Charleroi (Gosselies), Belgium
E-mail:[email protected]
Partners
Clinical trials have started. In total, 12 patients
have been included in RISET clinical trials so far.
Safety, eficacy and biological tests have been
performed for 10 patients. In January and Febru-
222
Hans-DieterVolk
Charite, University Medicine Berlin
Berlin, Germany
11/8/07 10:50:27 AM
M.C.Cuturi
CHU Nantes
Nantes, France
M.DahaandF.Claas
Leids Universitair Medisch Centrum
Leiden, Netherlands
L.Chatenoud
Université René Descartes Paris
Hospital Necker
Paris, France
U.Janssen
Cologne, Germany
K.Wood
Oxford University
John Radcliffe Hospital
Nufield Department of Surgery, level 6, Headington
Oxford, UK
H.Waldmann
Sir William Dunn School of Pathology
Oxford, UK
M.G.Roncarolo
San Raffaele Telethon Institute for Gene Therapy
Milan, Italy
R.Lechler
Kings College
Hodgkin Building, Guy’s Campus
London, UK
M.Guillet
TC Land
Nantes, France
R.Rieben
Bern University Hospital
Bern, Switzerland
U.Kunzendorf
University of Schleswig-Holstein
Kiel, Germany
F.Fandrich
Blasticon Gmb
Kiel, Germany
B.Miranda
Organización Nacional de Transplantes
Madrid, Spain
B.Arnold
Deutsches Krebsforschungszentrum
Heidelberg, Germany
Y.Reisner
Rehovot, Israel
A.Cambon-Thomsen
INSERM U558
Toulouse, France
A.Wendel
Université Konstanz
Konstanz, Germany
J.P.Soulillou
CHU Nantes
Nantes, France
N.Schwabe
ProImmune Ltd
Oxford, UK
O.Vilkicky
Institute for Clinical and Experimental Medicine
S.Houard
Henogen
Gosselies, Belgium
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I
XENOME
Engineeringoftheporcinegenomeforxenotransplantationstudiesinprimates:
asteptowardsclinicalapplication
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Website
The ultimate goal of XENOME is to generate the
necessary data to allow xenotransplantation to
advance towards its initial clinical phase. The data
generated in this project will encompass both eficacy and safety aspects of xenotransplantation.
Tools that will be used to reach this ambitious
objective include state-of-the-art biomolecular
technologies and in vivo models.
XENOME aims to produce a ‘super-engineered’
pig, i.e. a pig with a newly generated genotype
that will improve the eficacy and safety proile of xenotransplantation. Assessments of eficacy will irst take advantage of existing pig
lines expressing human complement regulators,
thrombomodulin (TM) and knock-out for -Gal
transferase ( -GalT KO). Using the most suitable
background, further engineering of the pig genome will be undertaken. Additional transgenes
able to control immune responses, endothelial
cell activation and subsequent microangiopathy,
will be added.
The ultimate pig strain will thus combine the
already available background with novel molecules exhibiting anticoagulant and immunosuppressive properties. In addition, an effective
immuno-suppression regimen will be deined,
and new pharmaceutical agents will be tested. A
strong safety framework will also be established,
that may allow, at some stage, the progression of
xenotransplantation into the clinic. This will en-
224
LSHB-CT-2006-037377
IntegratedProject
e฀987646
1November2006
60months
www.xenome.eu
tail the development of technologies enabling
the timely diagnosis of infection, the design of
a safety plan for an eficacious containment of
an untoward infectious event, the breeding of a
herd of ‘clean’ source pigs, and the provision of
safety-related data derived from long-term in
vivo studies in primate xenograft recipients. Finally, the project will also offer a strong ethical,
social (especially regarding public communication) and regulatory framework for xenotransplantation research (and possibly for clinical application too).
Xenotransplantation addresses the growing
problem of the shortage of human organs, which
prevents many patients with terminal organ failure from receiving a human organ. As a result of
improved medical and technological interventions in the ield of transplantation, an increasing
number of people are now short-listed for transplants. The waiting lists continue to grow due
to a limited organ supply; it is, for instance, estimated that approximately 45 000 people across
the EU are currently on waiting lists for a kidney
transplant.
The fundamental objectives of this project are
presented below:
• Bring the EU back to the forefront of
xenotransplantation research, a ield with
a huge potential for the treatment of endstage organ failure. At the end of the programme, XENOME will deliver a European
‘product’ for xenotransplantation. This will
consist of a ‘super-engineered’ pig with
11/8/07 10:50:27 AM
©Shutterstock, 2007
European intellectual property rights.
• Generate in vivo eficacy data: experimental evidence that the ‘engineered’ pig,
combined with new immunosuppressive
reagents, allows long-term survival in
non-human primates.
• Assess new immunosuppressive regimens and agents for the eficient inhibition of complement activation and acute
vascular rejection.
• Gain a better understanding of the
physiology of long-term transplanted
kidney xenografts in the pig-to-primate
model.
• Establish the necessary safety framework that would allow the progression of
xenotransplantation into the clinic.
• Provide a strong ethical, social, educational (especially apropos communication
with the public) and regulatory framework, within which xenotransplantation
research (and possibly clinical application)
should take place.
Addressing safety and the eficacy of xenotransplantation is the major endeavour of this initiative. Novel constructs that could confer advantages to the xenograft will be passed on to the
pig-engineering team whose principal objective
will be to modify the pig characteristic proile
according to the desired traits. The biomolecular tools applied will allow complete abrogation
(knock-out), reduced expression (knock-down),
or over-expression of candidate genes of interest
in porcine endothelial cells (transgenesis).
The pig-engineering team in XENOME will ensure that the in vitro indings generated are adequately utilised and translated to in vivo work, resulting in novel and stable pig lines. It will also be
part of the responsibilities of the same team to
ensure that animals are appropriately screened,
bred and provided in suficient number to generate the proof-of-concept data in in vivo primate
studies. Indeed, when new lines of engineered
22
11/8/07 10:50:30 AM
I
XENOME
pigs are made available to this network, the eficacy and safety of the organs will be tested by
transplanting them into primates.
All the activities undertaken will be tightly monitored and supervised by an ethical and legal
team, whose responsibilities will include ensuring that all the initiatives conducted within the
scope of this programme are fully compliant with
existing European ethical and legal frameworks.
Furthermore, this team will target the need to
bridge the gaps between laboratories and society, and between scientists, citizens and institutions, both by creating and maintaining a continuous communication low with the scientiic
partners, as well as by establishing a connection
with the general public. This will be made possible via diverse means, such as the preparation of
videos and lealets, and the organisation of open
laboratory workshops. Public consultation activities will also be conducted.
Coordinator
EmanueleCozzi
Azienda Ospedaliera di Padova
Ospedale Giustinianeo
Via Giustiniani, 2
Padova, Italy
Partners
Jean-PaulSoulillou
ITERT
Nantes, France
PierreGianello
Leuven, Belgium
CarlosRomeo-Casabona
Universidad del Pais Vasco
Bilbao, Spain
Expected outcome:
XENOME is expected to generate the necessary set of data that may allow the transition of
xenotransplantation from the current preclinical
phase to its initial clinical phase, within the next
10 years. This is an ambitious goal and the data
that will be generated during the ive- year duration of this project will address both the eficacy
and safety aspects of xenotransplantation.
MarialuisaLavitrano
University of Milan
Milan, Italy
RainerdeMartin
Vienna, Austria
YasuhiroTakeuchi
London, UK
HeinerNiemann
Institute for Animal Breeding, Federal Agricultural
Research Centre
Neustadt, Germany
LindaScobie
University of Glasgow
Glasgow, UK
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CesareGalli
Consorzio per l’incremento Zootecnico srl
Cremona, Italy
MohamedR.Daha
Leiden, Netherlands
JiriHejner
Institute of Molecular Genetics, Academy of Sciences of
the Czech Republic
ReinhardSchwinzer
Hannover, Germany
PaoloSimoni
University of Padua
Padua, Italy
RogerBarker
Cambridge Centre for Brain Repair
Cambridge, UK
JohannesRegenbogen
GATC Biotech AG
Konstanz, Germany
ErmannoAncona
Consorzio per la Ricerca sul trapianto d’Organi
Padova, Italy
OlleKorsgren
Corline Systems AB
Uppsala, Sweden
GuilhermeDeOliveira
Biomedical Law Centre
Coimbra, Portugal
MariachiaraTallachini
Università Cattolica S.C. Milano
Milan, Italy
MiguelCheParreiraSoares
Instituto Gulbenkian de Ciência
Oeiras, Portugal
227
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I
CLINT
Facilitatinginternationalprospectiveclinicaltrialsinstemcelltransplantation
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
Autologous and allogeneic stem cell transplantation (SCT) is the treatment of choice for many
haematological diseases. Within healthcare provision, SCT is not only one of the most costly but
it is also one of the most risky procedures for patients, with transplant related mortalities of up to
50%. SCT has always been on the cutting edge
of translational medicine, and over the past decade there have been many changes to the way
in which transplant is performed, such as the introduction of new drugs and technologies. While
these innovations have the potential to improve
patient outcome, they can also increase the cost
considerably. At the same time, many new developments have emerged with respect to targeted
drug therapies; leave supportive care in these diseases and therapies may replace or delay transplant for some patients. These new treatments
are also expensive and require urgent evaluation.
It is essential that they and SCT are used wisely
and economically.
LSHB-CT-2007-037662
e฀00000
1April2007
24months
severely curtailed by the recently introduced requirement to carry out these studies in accordance with the EU Directive on Clinical Trials.
Although introduced with laudable intentions,
the effect of the directive has been to increase
the resources and therefore the expense involved in clinical trials, whilst at the same time
national differences in the interpretation of the
new legislation have rendered international
studies extremely dificult. The objective of CLINT
is to support the EBMT to further develop the infrastructure necessary to perform academicallyinitiated international prospective studies in SCT
throughout Europe. This will hasten the evaluation of new treatment strategies and improve
the outcome for European citizens. The CLINT
consortium will be assisted in this endeavour, by
advice provided from a similar initiative in the
United States.
There has always been willingness for the SCT
community to critically evaluate the role of transplants, as is exempliied by the transmission of
outcome data from individual centres to a central database held by the European Bone Marrow
Transplant Group (EBMT) for further analysis and
reporting. SCT physicians have also been enthusiastic exponents of clinical trials, and are ready
to test new hypotheses, and to compare SCT with
other treatments. Their ability to do this has been
228
11/8/07 10:50:30 AM
Coordinator
JaneApperley
Imperial College
Department of Haematology
Hammersmith Hospital
Ducane Road
London W12 0NN, UK
Partners
FionaMacDonald
European Group for Blood and Marrow Transplantation
Barcelona, Spain
DorisSchroeder
University of Central Lancashire
Preston, UK
229
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I
TRIE
TransplantationresearchintegrationacrossEurope
ContractNo
Projecttype
ECcontribution
Startingdate
Duration
The primary objectives of the project are outlined below:
1. To identify current opportunities and challenges in the ield of transplantation research,
focusing on themes common to cell and solid
organ transplantation. In order to achieve this
overall objective, the following sub-objectives
are required:
• The establishment of a respected advisory
council which includes the following:
- recognised scientists in the ield of
transplantation research, selected on the
basis of ‘peer recognition’,
- representatives of the key organisations involved in transplantation research
in Europe, i.e. the European Society for Organ Transplantation (ESOT) and the European Group for Blood and Marrow Transplant Society (EBMT),
- representatives of relevant EU-funded
research projects in the ield of transplantation (e.g. RISET, Allostem, Alliance-O,
DOPKA etc),
- a representative of the European
Agency for Evaluation of Medicinal Products (EMEA),
- representatives of industries active in
the ield (both big pharmas and SMEs);
• To identify common opportunities and
challenges on different transplantation
research themes, through discussions and
230
LSHB-CT-2007-03740
e฀424732
1March2007
24months
consultations with working groups and
members of the advisory council:
- biomarkers and pharmacogenomics,
to tailor immunosuppression,
- how to promote living donation
- regulatory aspects of novel cell-based
immunotherapies,
- standardisation and validation of immunomonitoring tests,
- training programmes in transplantation medicine;
2. To prepare a set of recommendations regarding priority actions to be implemented, so as to
foster the integration of research activities on
themes common to cell and solid organ transplantation. In order to achieve this overall objective, the following sub-objectives are required:
• to form working groups on different
research themes, to facilitate discussion
and consultation in order to reach a consensus on existing ‘gaps’;
• to prepare detailed recommendations
on optimal measures to address the gaps;
• based on the output of the working
groups, to prepare a consensual position
paper that will present in an articulated
manner the challenges, opportunities and
recommendations in terms of priority actions to integrate research activities on
cell and solid organ transplantation.
3.Organisation of a public ‘dissemination’ event
in close collaboration with media experts and
11/8/07 10:50:30 AM
patients’ representatives. This event would have
the following two sub-objectives:
• project coordinators and participants
would present concrete results of past
and current research efforts — supported
namely by the EU Commission — in the
ield of cell and organ transplantation;
• to present, discuss and debate the output of TRIE with stakeholders.
Coordinator
MichelGoldman
Institute for Medical Immunology
Rue Adrienne Bolland, 8
B-6041 Charleroi (Gosselies), Belgium
Partners
KathrynWood
Nufield Department of Surgery - Immunology
Oxford, UK
AlejandroMadrigal
The Antony Nolan Trust
London, UK
BlancaMiranda
Organización Nacional de Trasplantes
Madrid, Spain
SiobhànMcQuaid
Abu International Project Management Ltd
Dublin, Ireland
231
11/8/07 10:50:31 AM
232
New Therapies – Index of Projects
11/8/07 10:50:31 AM
NEW THERAPIES
INDEX OF PROJECTS
AlloStem
ANGIOSKIN AutoCure BacAbS BACULOGENES
BetaCellTherapy
BMC
CELLAID
CLINIGENE
CLINT
COMPUVAC CONSERT CRYSTAL DC-THERA DC-VACC DENTRITOPHAGES
EPISTEM EPI-VECTOR
EuroSTEC EuroStemCell
GenomesToVaccines
GENOSTEM
GIANT
HEPACIVAC ImprovedPrecision
IndustryVectorTrain
INNOCHEM
INTHER
InVivoVectorTrain
Magselectofection
MimoVax
MOLEDA
Myoamp
NEUROscreen
OsteoCord
Pharma-Planta
PolExGene
RESCUE
RIGHT
RISET
SAGE
SC&CR
Skintherapy
SNIPER
155
144
204
189
102
47
202
214
85
228
181
92
80
159
165
173
32
129
52
17
177
22
98
186
121
150
209
124
147
135
193
141
77
75
27
196
133
69
110
221
200
57
41
118
STEMDIAGNOSTICS
STEM-HD
STEMS
STEMSTROKE
STROKEMAP
SyntheGeneDelivery
THERADPOX
THERAPEUSKIN
THERAVAC
TherCord
THOVLEN
TRIE
UlcerTherapy
XENOME
ZNIP
217
72
64
61
67
138
107
44
169
30
104
230
37
224
115
New Therapies – Index of Projects
233
11/8/07 10:50:31 AM
INDEX OF
COORDINATORS
NETWORK OF EXCELLENCE
Austyn, Jonathan M (DC-THERA)
Cohen-Haguenauer, Odile (CLINIGENE)
163
89
INTEGRATED PROJECTS
Cortese, Riccardo (HEPACIVAC)
Cozzi, Emanuele (XENOME)
Feitz, Wouter (EUROSTEC)
Fischer, Rainer (Pharma-Planta)
Goldman, Michel (RISET)
Jorgensen, Christian (GENOSTEM)
Klareskog, Lars (AutoCure)
Klatzmann, David (COMPUVAC)
Ma, Julian
(Pharma-Planta, scientiic coordinator)
Madrigal, Alejandro (AlloStem)
Maitland, Norman J. (GIANT)
Meyer, Thomas F. (RIGHT)
Mantovani, Alberto (INNOCHEM)
Pipeleers, Daniel (BetaCellTherapy)
Schenk-Braat, Ellen
(GIANT, scientiic coordinator)
Smith, Austin (EuroStemCell)
Vandenabeele, Peter (EPISTEM)
Wagemaker, Gerard (CONSERT)
188
226
196
222
2
207
18
198
17
101
113
213
0
101
21
3
96
SPECIFIC SUPPORT ACTION
Apperley, Jane (CLINT)
Goldman, Michel (TRIE)
Mezzina, Mauro (InVivoVectorTrain)
(IndustryVectorTrain)
Radbruch Andreas (CELLAID)
234
229
231
149
11
216
New Therapies – Index of Coordinators
11/8/07 10:50:31 AM
NEW THERAPIES
SPECIFIC TARGETED RESEARCH
Allsopp, Tim (NEUROscreen)
Bartholeyns, Jacques (DENDRITOPHAGES)
Bigot, Yves (SyntheGeneDelivery)
Buus, Søren (Genomes To Vaccines)
Capogrossi, Maurizio C. (SC&CR)
Daura, Xavier (BacAbS)
Dickinson, Anne (STEMDIAGNOSTICS)
Epstein, Alberto (THOVLEN)
Genever, Paul (OsteoCord)
Hescheler, Jürgen (CRYSTAL)
Hook, Lilian
(NEUROscreen, scientiic coordinator)
Hovnanian, Alain (THERAPEUSKIN)
Izsvák, Zsuzsanna (INTHER)
Jackson, Dean A. (EpiVector)
Kadouche, Jean (BMC)
Kokaia, Zaal (StemStroke)
Krauss, Stefan (SNIPER)
Krauss, Stefan (ZNIP)
Leclerc, Claude (THERAVAC)
Lusky, Monika (THERADPOX)
Martin, John (BACULOGENES)
Mattner, Frank (MimoVax)
Meneguzzi, Guerrino (Skintherapy)
Mir, Lluis M. (ANGIOSKIN)
Mouly, Vincent
(myoamp, scientiic coordinator)
Onteniente, Brigitte (STEMS)
Peschanski, Marc (STEM-HD)
Plank, Christian (Magselectofection)
Privat, Alain (RESCUE)
Rosenecker, Joseph (Improved Precision)
Schacht, Etienne (PolExGene)
Scherman, Daniel (MOLEDA)
Schillberg, Stefan (SAGE)
Verfaillie, Catherine (STROKEMAP)
Zambruno, Giovanna (Ulcer Therapy)
7
176
140
180
60
192
219
106
29
82
7
46
128
132
203
63
120
117
172
109
103
19
43
14
79
66
74
137
71
123
134
143
201
68
40
New Therapies – Index of Coordinators
23
11/8/07 10:50:31 AM
11/8/07 10:50:31 AM
11/8/07 10:50:31 AM
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How to obtain EU publications
Our priced publications are available from EU Bookshop (http://bookshop.europa.eu/), where you
can place an order with the sales agent of your choice.
The Publications Office has a worldwide network of sales agents. You can obtain their contact
details bysending a fax to (352) 29 29-42758.
11/8/07 10:50:31 AM
11/8/07 10:50:31 AM
European Commission —
Directorate-General for Research Life Sciences, Genomics and Biotechnology for Health
NEW THERAPIES — EU-supported Research in Genomics and Biotechnology for Health
Sixth Framework Programme (2002-2006)
EUR 22841
Luxembourg: Office for Official Publications of the European Communities
2007 — 236 pp. — 17.6 x 25 cm
ISBN: 978-92-79-05562-1
Catalogue number: KI-NA-22841-EN-C
Price (excluding VAT) in Luxembourg: 25.00 EUR
11/8/07 10:50:31 AM
11/8/07 10:50:31 AM

new therapies

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