iget - SsFa

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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
GLP principles in ATMPs
Patrizia Cristofori and Paola Albertini
A new challenge
in GTMP development
XXIV CONGRESSO NAZIONALE GIQAR
Napoli, 27-29 Maggio 2015
Advanced Therapy Medicinal Products
(ATMPs)
ATMPs are ‘innovative, regenerative therapies which
combine aspects of medicine, cell biology, science and
engineering for the purpose of regenerating, repairing or
replacing damaged tissues or cells’.
- Gene therapy
involves the use of normal genes to correct defective ones
- Cell therapy
components of whole cells are injected into the patient
- Tissue engineering
wholes tissues manipulated in vitro used therapeutically
Regulation (EC) on advanced therapies N.1394/2007
Gene Therapy Medicinal Product
(GTMP)
• Biological medicinal product
which contains or consists of a
recombinant nucleic acid used
or administered to human beings
with a view to regulating,
repairing, replacing, adding or
deleting a genetic sequence
• Its effect relates to the
recombinant nucleic acid
sequence or to the transgene
expression
Gene therapy strategies: current approaches
Muscle, Eye, Skin etc.
Duchenne Muscular
distrophy
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Hematopoietic system,
SNC and SNP
ADA-SCID
Wiskott-Aldrich sindrome
Leber Congenital
Amaurosis
SCID-X1
Epidermolisis bullosa
Beta-thalassemia
Hemophilia
Metachromatic
Leukodistrophy
Ex vivo Gene Therapy of Autologous HSCs*
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Bone Marrow
Bone Marrow
purified for
CD34+ cells
“Conditioning”
Transduction
* Haematopoietic Stem Cells
Alliance between GSK, Fondazione Telethon and
OSR
6
TIGET: Telethon Institute for Gene Therapy
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Joint-venture of San Raffaele Hospital and the Telethon Foundation
to perform and translate research results
into the clinical application of gene therapy for genetic diseases
• Perform innovative research on gene transfer and cell transplant
• Develop pre-clinical models to establish efficacy and safety
• OSR Pediatric Clinical Research Unit
diagnosis, treatment, follow up of patients
• primary immunodeficiency (ADA-SCID)
• hematologic and metabolic disorders (WAS, MLD)
Preclinical Support of Clinical Trials
Development
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
• Maximise the early collection of proof-of concept data and provide adequate
preclinical information to support the safety and the scientific basis for the
administration of an investigational product in the target patient population
• Provide data of high regulatory standard with assurance of scientific integrity,
validity and reliability
• The evaluation of GTMPs requires very specific expertise beyond traditional
pharmaceutical field
• Minimise the use of animals (3Rs)
Combine expertise of personnel trained in research, experimental
pathology, safety assessment and quality assurance
GLP and Nonclinical studies
General Principles
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
The GLP regulations apply to nonclinical laboratory studies that support
research or marketing applications: ICH M3(R2)
Toxicity studies are expected to be performed in compliance with Good
Laboratory Practice (GLP): ICHS6 (R1)
GLP non clinical safety studies are recommended to support human
clinical trials of a given scope and duration (FTIH, Phase II and III)
according to guidelines, EMEA, ICH, 21 CFR 58
GLP non clinical safety studies are considered “regulatory studies”
Part of IMPD (clinical phases) and CTD (marketing authorisation)
However.......
Some studies employing specialised (research) procedures may not be
fully in compliance with GLP
Study reconstruction should be ensured through adequate
documentation of the study conduct and archiving of data (ICH S7A2000)
.
GLP HSR-TIGET Test Facilities
HISTORY
2011 - Start the Set up GLP Test Facilities
Facilities (laboratories, animal rooms)
Personnel, GLP Training
SOPs and Documentation
Instruments
2012 - GSK QA Advisory Visit
2013 - Italian Monitoring Authority Visit
2013 - GSK QA Advisory Visit
GLP CERTIFICATION
28th March 2014 from Italian Ministry of Health
To perform Gene Therapy studies
according to GLP OECD principles
-Toxicology Studies
- Biotechnology and Molecular biology
studies
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Non Clinical Studies OBJECTIVES
EMEA/CHMP/GTWP/125459/2006
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
The objectives of preclinical studies are:
• Demonstrate Proof of Biological Principle of GT
• Define toxicological effects
• Provide information for clinical trials
– Safe dose/adverse effects
– Route of administration
– Duration of dose
– Identify target organs for toxicity and parameters for monitoring in
patients
Gene Therapy nonclinical studies
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
• Biodistribution study
– Distribution of test item/vector to target and non-target tissues
– Germline trasmission
• Toxicology and Tumorigenicity study
– to evaluate the potential toxicity and tumorigenicity induced by
transplantation of murine HSPCs transduced in recepient mice
• Type of Vector
• Transgene expression
• Adverse immune response
• Insertional mutagenesis
Presentation title in footer
00 Month 0000
12
Gene Therapy
Safety issues
Gene therapy has inherent risks such as:
• The higher concern on gene therapy relates to
• vector integration and insertional mutagenesis
• malignancy development
Preclinical development relies on:
• One / two nonclinical studies
Usually a single dose in patient
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Challenge:
Non-standard preclinical
study design
Non-standard Non Clinical
Study Design
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
• Test System
- Animal Models of disease vs healthy common laboratory animals used in
conventional toxicology studies
- Immunodeficient animals to evaluate safety of genetically modified human cells
• Conditioning agent
- Busulfan or irradiation
• Duration of the study: single dose and long term monitoring
Test Item production and characterization as study phase
• Parameters to be included
• Molecular and biological methods validation
Toxicity and Tumorigenicity Study
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Day Day Day Day
-4 -3 -2 -1
Termination
Day
16 + 2 weeks
12 mo
1
-----Daily monitoring of clinical signs, weekly body weight, food consumption---ANALYSIS
VIRAL VECTOR
4 X 105
Lin- cells
Gene Therapy
n=30 (M+F)
Test System
BU 25mg/kg/day
4 X 105
Lin- cells
•
•
•
•
•
Engraftment (FACS)
VCN
Integration sites
Transgene expression
Hematology
• Engraftment (FACS)
• Hematology
Mock
n=30 (M+F)
• Hematology
Test System
untreated
•
•
•
•
Engraftment
Transgene expression
Hematology
Clinical chemistry
•
•
•
VCN
Lineage reconstitution
Integration sites
•
Necropsy Histology
Pathology
•
•
•
Engraftment
Hematology
Clinical chemistry
•
Lineage reconstitution
•
Necropsy
Pathology
•
•
Hematology
Clinical chemistry
•
Lineage reconstitution
Histology
n=30 (M+F)
•
Necropsy Histology
PPathology
Tox-Tumorigenicity study: serial transplantation
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Viral vector.LV
Murine HSPCs
Test Item
Phase I
disease model
After 5 weeks
* dose proof
*Haematology
Test Item
production and
characterization
Transplant
6 months
follow up:
Clinical
monitoring
Termination
Necropsy
OrganWeight
Disease Model
Termination
6 months
follow up:
Clinical
monitoring
After 5
weeks: dose
proof
Tot BM
Secondary
transplantation
Phase II
17
Biodistribution Study: CD34+ cells
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
NSG
(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)
~ 8 week old
Day 1
Day -2
Day -1
Irradiation
Week 8
Week 12
Busulfan
Busulfan
Transplantation
PB analysis
Termination
Busulfan
Transplantation UCB
Endpoints:Survival, Clinical Signs and Body Weight
or
Test Item or
FACS
200 rad TBI
Control Item (mock)
VCN (human CD45+ purification of BM and Spleen) Pathology
3 groups of NSG mice
12mice/group
(male + female)
Take Home Messages
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
• Study designed on a case-by-case basis
It is important to understand the product
– Employ study designs that address safety and the scientific basis for conducting a clinical trial
– Some questions to be answered:
• What is/are the biologically relevant animal species for testing your product?
• Are there potentially relevant animals models of disease/ injury that can be used?
• What preclinical study design will provide the most useful information to assess long term risks?
• Will repeat administration be needed?
• Will immuno-suppression be needed?
• What happens to the cells in vivo following delivery?
• What is the expected in vivo persistence profile of the cells?
• Hybrid pharmacology-toxicology study design
– Incorporate activity & safety endpoints in animal model of disease. Local microenvironment &
pathophysiology status of the model may impact the safety/bioactivity of the product
• It is important to work closely to Regulators
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Challenge:
Test Item
Production & Characterisation
Test Item
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
(EMA/CAT/GTWP/671639/2008)
• DEFINITION (OECD GLP Principles)
- CD34+ cells
- Lin- cells
Test Item
Vectors with
gene
Haemopoietic Stem Cells (HSC) transduced with viral
vector expressing the gene
Reference item
Haemopoietic Stem Cells (HSC) non transduced
(mock–transduced)
• PRODUCTION
BM collection from
donor mice
Isolation HSPC
Daily collection of BM cells from donors
Staggered start of treatment
Cell count, viability, purity
Different batches in one study
Transduction
Batch of mock-transduced cells
Reference item (Control)
Batch n: STUDY CODE-CI-nn
Batch of transduced cells
Test item
Batch n: STUDY CODE-TI-nn
Test Item
Characterisation
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Test item:
Batch n: “STUDY CODE _TI_nn”
Control item: Batch n: “STUDY CODE _CI_nn”
Purity
Identity
Cell count and viability
of HSPCs
cytofluorimetric assay
(FACS)
Test to detect presence of
the intended genetic
modification and assay
specific for the cell
population
Cell count and viability
Vector Copy Number
qPCR
Potency
Functional properties achieved
by the genetic modification (gene
copy number and expression
level of the transgene, stemness
and differentiation capacity)
Clonogenic
Assay/qPCR/FACS
Test Item
Characterisation
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
• Toxicology and Tumorigenicity study
– TI and CI production and characterisation in GLP as
Phases of GLP study
– TI and CI characterisation performed during the study
with production of certificate of analyses
– Acceptance of test Item versus defined
acceptance criteria (SOP and study protocol)
• Biodistribution study
–TI provided and characterised by GMP supplier *
23
Take Home Messages
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
• TI and CI production and characterisation as Phases of GLP study
• TI and CI characterisation performed during the study with production of
certificate of analyses
• Acceptance of test item versus defined acceptance criteria (SOP and study
protocol)
• Sample identification and traceability to allow study reconstruction and
interpretation should be ensured through adequate documentation of study
conduct
• Method validation is complex for assays such as qPCR and FACS but feasible,
and provides confidence and reliability of obtained results
• Performing and using validated methods early during GTMP development (VCN)
may provide better comparability between preclinical and clinical phases later on
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
GLP test Facility set up
Challenges
Challenges
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
GLP Facility/laboratories within Research environment /OSR*
Research activities performed within GLP Test Facility
OSR structures / functions
GLP TF
Financial constrains
Turnover of personnel
Traceability of study data *
internal supplier of
Alliance with industry
training
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GLP HSR-TIGET Test Facilities
Necropsy and Animal Rooms
FACS & Pathology
SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
GLP Office
Animal Room
Molecular & Cellular
Labs
Animal Room
Archive
GLP HSR-TIGET Test Facilities
GLP FACILITY
• 2 molecular and biological laboratories
• 5 animal rooms within SPF animal faciility
• GLP Archive
• 1 laboratory for cytofluorimetry FRACTAL
• 1 laboratory for pathology
GLP Personnel
• 50 people : 1 archivist; 5 Study Directors; 3 QA
*
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Challenge :
Traceability of study data
&
Sample identification
Design of nonclinical studies at HSRHSR-TIGET
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Identification
Test System
Use of specific strain of
mouse
• Depends on the
disease
• Generally use
transgenic animals,
modelling the disease
• Biodistribution study:
immunocompromised
mouse (NSG)
Prodution and
characterization
of Test Item
Stem cell isolation and
preparation
• In vitro transduction of
the harvested cells
using a vector (e.g.
Lentivirus) containing
the gene of interest
• Production and
characterisation of test
item and control item
Test System
conditioning
Recipient conditioning
• Use of either
BUSULFAN or
IRRADIATION
• to induce
myeloablation and
allow the graft
Administratio
n of Test Item
Transplantation
• Of the
transduced
cells in the
conditioned
recipient
• Day1
Experimental
phases
Analysis
•
•
•
•
Engraftment
Transgene expression
Hematology
Clinical chemistry
• VCN
• Lineage reconstitution
• Integration sites
• Necropsy
• Histology
• Duration variable, up to
12 months
Lin- cells
th3/+ CD45.1
30
Toxicity/tumorigenicity study for gene therapy
Test and control item production
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Bone collection from
donor mice
large-scale vector
TIGET GLP Test Facility/BSL2
(SPF animal facility)
Isolation lin- cells from bones
SOP LAB-002
Lin – cells ID: “Study Code lin-nn”
“Study Code lin-nn”
“Study Code lin-nn_FC”
Transduction
Lin - Purity
FACS staining (Purity)
FACS acquisition
SOP FC-009
Production of:
Batch of mock-transduced cells =
Control item
Batch n.: STUDY CODE-CI-nn
Production of:
Batch of transduced cells
= Test item
Batch n.: STUDY CODE-TI-nn
Toxicity/tumorigenicity study for gene therapy
Test and control item characterization
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
TIGET GLP Test Facility/BSL2
Test item:
Batch n: “STUDY CODE _TI_nn”
Control item: Batch n: “STUDY CODE _CI_nn”
“STUDY CODE _ TI/CI_nn”
“STUDY CODE _ TI/CI_nn_CFU”
Cell count and
viability and FACS
Clonogenic assay
(CFU)
“STUDY CODE _ TI/CI_nn_LC”
Liquid Culture
“STUDY CODE _
TI/CI_nn_LC_Day...”
Cell freezing
viable
“STUDY CODE _ TI/CI_nn_LC_xx”
Cell pellet
“STUDY CODE _ TI/CI_nn_LC_xx”
IDUA Activity
“STUDY CODE _ TI/CI_nn_CFU_yy”
“STUDY CODE _ TI/CI_nn_CFUpool_yy
Single colony
Pool of colonies
“STUDY CODE_TI/CI_nn_CFUpool_yy”
“STUDY CODE _ TI/CI_nn_CFU_yy”
“STUDY CODE _ TI/CI_nn_CFU_yy”
PCR Transduction eff.
QPCR
VCN
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Test System
TIGET GLP Test Facility/BSL2
Recipients mice
Mice conditioning
(busulfan-irradiation vs control)
Test item:
Batch n.: “STUDY CODE_TI_nn”
Control item:
Batch n.: “STUDY CODE_CI_nn””
Group allocation Group ID
Identification Animal ID
Cage Identification Cage Label
Treatment
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TIGET GLP Test Facility/BSL2
SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
from day 1 to the end:
Daily inspection
Clinical signs
BW/FC (weekly)
Day 1:
treatment of Test System
Intercurrent deaths
Periodically:
Opthalmoscopy
Day 30: After treatment of Test System
Intercurrent deaths
Peripheral Blood sample
“Study code_animal group/n._Time point_#”
Haematology # HE
Engraftment evaluation by FACS analysis # FC
IDUA activity (MPS) # PBMC
Hb electrophoresis (β-Tal) # CA
qPCR x VCN # CFU-PB
(SOP LAB 008 ; SOP MET-004)
(Lab 23)
Termination
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TIGET GLP Test Facility/BSL2
SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Analysis
“Study code_animal group/n._Time point_#”
Haematology # HE
*CRO
*Clinical chemistry # CC
FACS analysis # Tissue_FC
IDUA activity #BM_nn
Hb electrophoresis # HE
qPCR x VCN on BM #BM_nn
Observations
“Study code_animal group/n
Opthalmoscopy
consultant
Necropsy
*Histology
Pathology
Tecnnical Sheets @ example
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Documents managed according to a specific SOP
• Document used to record data and study information
• Approved by management
• Controlled distribution upon request by Study Director
• Reconciliation of distributed TS at the end of the study
Records of raw data
Drive the operator to record all equipment, material in each step of the
study
Allow the reconstruction of the study phases
Allow the traceability of study sample : cross references to TS ( origin and
destination )
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
QA Role
&
QA Monitoring Program
QA challenges
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
QA must be aware of all non standard aspects and challenges of
preclinical studies in Gene Therapy
QA must be knowledgeable in technical procedures and methods
Work in partnership with scientists and management to identify critical
phases and define QA monitoring program
Quality Assurance Program
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Risk Base Inspection Program
Risk in GLP world
“It will be an undesirable event that put a study, a project or a facility in jeopardy”
Probability that event will occur
Severity of the event
“Detectability” of the event
Design of nonclinical studies at HSRHSR-TIGET
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Identification of Critical Phases to be monitored by QA
Identification
Test System
Use of specific strain of
mouse
• Depends on the
disease
• Generally use
transgenic animals,
modelling the disease
• Biodistribution study:
immunocompromised
mouse (NSG)
Prodution and
characterization of
Test Item
Stem cell isolation and
preparation
• In vitro transduction of
the harvested cells
using a vector (e.g.
Lentivirus) containing
the gene of interest
• Production and
characterisation of test
item and control item
Test System
conditioning
Recipient conditioning
• Use of either
BUSULFAN or
IRRADIATION
• to induce
myeloablation and
allow the graft
Administratio
n of Test Item
Transplantation
• Of the
transduced
cells in the
conditioned
recipient
• Day1
Experimental
phases
Analysis
•
•
•
•
Engraftment
Transgene expression
Hematology
Clinical chemistry
• VCN
• Lineage reconstitution
• Integration sites
• Necropsy
• Histology
• Duration variable, up to
12 months
Lin- cells
th3/+ CD45.1
40
iget
SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
QA
Personnel
work closely with technical personnel to share criticality and best
practices
But
To be independent of study conduct and designed to assure Test
Facility management of compliance with GLP Principles
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Overall Conclusions
Based on Tiget experience
Overall Conclusions
iget
SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
• Study supporting GTMPs development are not standard; designed on a
case-by-case basis:
– it is important to understand the product
– areas of non-compliance should be identified and their significance evaluated
• It is important to work closely to Regulators
• QA must be aware of all the aspects of preclinical studies and
knowledgeable in GT and work closely with people working on
preclinical studies
• Experience with TIGET shows that work within the Academic
environment can be conducted to GLP standard
.................................................and.............................................................
Team work is Key
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Acknowledgements
L. Naldini
A. Sauer
I. Visigalli
A. Aiuti
A. Biffi
G. Ferrari
V. Parezanovic
F. Sanvito
P. Rodegher
N. Carriglio
R. Hernandez
S. Delai
G. Ferrari
F. Tiboni
F. Ferro
C. Rossi
C. Villa
G. Mandelli
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
C. Doglioni
E. Stradella
E. Vicentini
E. Beltrame
M. Gabaldo
E. Oriani
S. Giannelli
E. Draghici
F. Cecere
S. Ghiandoni
M. Lidonnici
S. Ungari
A. Aprile
M. Goodwin
C. Powell
F. Winder
B. Brown
A.Launchbury
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
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SAN RAFFAELE
TELETHON INSTITUTE
FOR GENE THERAPY
Thank you