Gene therapy

Zasady i perspektywy
terapii genowej
Prof. dr hab. Józef Dulak
Zakład Biotechnologii Medycznej
Wydział Biochemii, Biofizyki i Biotechnologii UJ
Email: jozef. [email protected]
Web page: http://biotka.mol.uj.edu.pl/zbm
05.06.2010
What is gene therapy?
Gene therapy
Protein
Therapeutic
gene
(transgene)
Vector
Patient
Expression of therapeutic gene
Gene therapy is also silencing or replacing
the bad genes or overexpression
of good genes
Which diseases could be cured with gene therapy?
Gene therapy was born in... 1962
HPRT-/- cells
HAT medium
Prof.Wacław Szybalski
McArdle Laboratory
for Cancer Research,
Wisconsin, Madison,
USA
HPRT+/+ cells
Inborn error of metabolism – deficiency of HPRT
HPRT
allopurinol
Lesch-Nyhan syndrome
Children suffering from deficiency
of HPRTLesh-Nyhan syndrome
Children suffering from deficiency of HPRT- Lesh-Nyhan syndro
Development of gene therapy
Development of tools (vehicles)
Vectors
Carriers of the therapeutic nucleic acids
Retroviral expression system
DMB
Gag – core proteins, matrix, nucleocapsid
Pol – reverse transcriptase and integrase
Env – envelope glycoproteins
Retroviral vectors
DMB
• gag – structural proteins
• pol – reverse transcriptase
• env – envelope proteins
ITR
gag pol
env
• long-term expression due to
integration into cellular genome
ITR
retrovirus
ITR
ITR
transgen
Retroviral vector
First controlled trial of gene therapy - 1990
DMB
HPRT
ADA
ADA deficiency– results in severe immunodeficiency syndrome
Gene therapy of ADA deficiency
ADA
ADA-
ADA+
ADA+
DMB
First clinical trial of gene therapy - 1990
Retroviral vector containing
correct ADA gene (cDNA) has been
transduced into blood lymphocytes
This first clinical trial was not „pure” from the methodological
point of view.
Ashanti De Silva (patient)
The patients have been treated concomitantly with enzyme injections –
ADA-PEG.
Nevertheless, the marker transgene (neo) could be detected in the
blood cells of the patients even more than 5 years after injection of
modified cells.
Gene therapy
DMB
Ex vivo
Cells are isolated,
modified in vitro
and injected back to
the patient
Eg. Leucocytes, hepatocytes
In vivo
Vectors are injected
into the diseased tissue
eg. into the tumor
or healthy tissue
Vectors are injected
into the blood
Vectors
DMB
Non-viral/plasmids
„naked” DNA
Lipoplexes
Viroplexes
(lipoplexes enhanced
in proteins from viral
capsids)
viral
RNA
Retroviral
(including
Lentiviral)
Complexes
With chemical
vehicles
DNA
Adenoviral
AAV
Herpes
Vectors for gene therapy
DMB
Plasmids
Transfection
efficacy
Capacity
Cytotoxicity
Expression
Retroviral
Lentiviral
low
low
unlimited
4-5 kb
9 kb
low
low
low
Very low
Days-weeks
Long-term
Long-term
AAV
moderate
4-5 kb
low
Long-term
Adenoviral
„Gut-less”
Adenoviral
high
high
7-10 kb
30 kb
high
Weeks-months
low
Long-term
Gene therapy clinical trials
Types of vectors used in clinical trials of gene therapy
DMB
liposomes
retroviruses
naked DNA
AAV
Six years ago...
adenoviruses
DMB
DMB
Clinical trials in gene therapy
Phases of clinical trials
disease
Some Phase II and most Phase III drug trials are randomized, double-blin
26
and placebo-controlled
Clinical trials
The strongest evidence for therapeutic interventions is provided by
systematic review of randomized, double-blind, placebo-controlled trials
involving a homogeneous patient population and medical condition.
Randomized: Each study subject is randomly assigned to receive either
the study treatment or a placebo.
Double-blind: The subjects involved in the study and the researchers
do not know which study treatment is being given.
Placebo-controlled: The use of a placebo (fake treatment) allows
the researchers to isolate the effect of the study treatment.
Some Phase II and most Phase III drug trials
27
Evidence-based medicine (EBM)
According to the Centre for EBM:
"Evidence-based medicine is the conscientious, explicit and j
of current best evidence in making decisions about the care
EBM integrates best available research evidence with clinical
expertise
28
Registered drugs are only a portion of all tested
29
Succesful gene therapy
David Vetter - „Bubble Boy”
David has spent 12 years in a foilprotected environment. Finally has
received the bone marrow transplantation
from his sister, but unfortunately died
due to Epstein-Barr virus infection
X-SCID deficiency
X-linked severe combined immunodeficiency (X- SCID)
Lack of γc gene
Restoration of B and T lymphocytes
and NK cells
D. Kohn et al., Nature Rev Cancer July 2003
Cytokines receptors
D. Kohn et al., Nature Rev Cancer July 2003
DMB
Cavazzana-Calvo M et al .
Gene therapy of human severe combined
immunodeficiency (SCID)-X1 disease
Science 2000: 28 April: 288: 669-672
Gene therapy is efficient in treatment of X-SCID
Gene therapy is efficient in treatment of X-SCID
DMB
Stem cells without
correct γc gene
Retroviral vector with
a correct γc gene
DMB
Gene therapy has been beneficial
to most treated SCID-X1 patients!!!
- they can now cope with environment microorganisms and have a normal life in
the absence of any specific therapy
- no evidence for γc transgene silencing has been observed
DMB
Potential risk of application of retroviral vectors
• gag – structural proteins
• pol – reverse transcriptase
• env – envelope proteins
ITR
gag pol
env
•long-term expression &
integration into cellular genome
ITR
retrovirus
ITR
ITR
transgen
Retroviral vector
random integration –
risk of insertional mutagenesis
Integration of retroviral vector into the promoter of LMO2 gene
42
Serious side effects of SCID-X1 gene therapy
-development of uncontrolled clonal T lymphoproliferative syndrome, similar to
acute lymphoblastic leukemia (ALL) in 4 out of 10 treated children in Paris
- due to the integration of a vector into an LMO2 gene either close to the promoter
or in the first intron
Reasons: 1. LMO-2 locus is a frequent site for retroviral integration – BUT NOT
SUPPORTED BY DATA
2. Cells with aberrant expression of LMO-2 could have been selected
because the provide a clonal growth advantage
December 18, 2007 – a leukemia case has been reported in
one of boys treated in hospital in London…
Gene therapy has been beneficial
to most treated SCID-X1 and ADA
patients!!!
SCID-X1:
1. French trial – 10 treated, 9 benefited. Unfortunately, four of those
who benefited in the begining developed leukemia and
one boy died this year because of leukemia.
2. British trial – 10 treated, 10 benefited –one developed leukemia
Gene therapy of ADA deficiency
Gene therapy of ADA deficiency
Spośród 10 pacjentów poddanych leczeniu, naprawę funkcji
układu odpornościowego i ochronę przed infekcjami
osiągnięto u 9 osób!
Podczas obserwacji (średnio 4 lata!) nie zaobserwowano
poważnych efektów ubocznych.
DMB
Enhancing
enhancement
of gene expression
Acquired diseases
Caused by endogenous
and exogenous factors
Gene therapy
Substituting
delivery of
the missing gene
Inherited diseases
Suppresive
inhibition of
gene expression
Acquired diseases
DMB
Cancer gene therapy
DMB
p53-induced cell-cycle arrest in
response to DNA damage.
The normally unstable p53 protein is stabilized by damaged DNA, so its
concentration increases. Acting as a transcription factor, p53 induces expression of
p21CIP, a cyclin-kinase inhibitor that inhibits all Cdk1-, Cdk2-, Cdk4-, and Cdk6cyclin complexes. Binding of p21CIP to these Cdk-cyclin complexes leads to cell
cycle arrest in G1 and G2
DMB
Application of adenoviral vectors
for gene therapy
Binding and internalization of adenovirus
DMB
Adenoviruses and adenoviral vectors
•
~40 serotypes of adenoviruses (for gene therapy type 2 and 5 were used),
causing usually mild illness in humans
•
Genome consists a 36 kb double-stranded linear DNA with ITR sequences at each
end, with:
Early genes (responsible for viral gene transcription, DNA replication, host immune
suppression and host cell apoptosis
Late genes (coding proteins required for virus assembly)
•
E1 early gene is essential for the subsequent adenoviral gene expression
region E1
Adenoviral DNA
DNA of 1st generation vector
leczniczy gen
Adenoviral delivery of p53 gene
DMB
Adenoviral vector
with correct p53 gene
TP53
Cancer cell
expression of p53 in
cancer cell
apoptosis
First registered drug
for gene therapy of cancer
2004
patients with head and neck
cancer
Gendicine (SiBiono GeneTech, Chiny)
Adenoviral vector with p53 gene
Advexin – adenoviral vector expressing p53 gene
effective in patients with head and neck cancer
DMB
Cancer gene therapy
1. Direct attack on tumor cells
a) transfer of tumor suppressor gene
b) inhibition of oncogenes
- antisense therapy
- ribozymes
c) suicide genes
d) oncolytic viruses (replication-competent viruses)
2. Harnessing immune response to tumor antigens
3. Chemoprotection
4. Anti-angiogenic therapy
DMB
Suicide gene therapy
Pro-drug
(eg. ganciclovir)
Vector with a suicide gene
(eg. Thymidine kinase)
Phosphorylated
ganciclovir
Inhibition of DNA synthesis
in cancer cells
Enzyme-prodrug combination for suicide gene therapy
Enzyme
HSV-tk
Prodrug
ganciclovir
acyclovir
valacylcovir
Product
ganciclovir triphosphate
cytosine deaminase
5-fluorocytosine
5-fluorouracil (5-FU)
cytochrome P450
cyclophosphamide
phosphoramide mustard
Mechanism
blocks DNA synthesis
blocks DNA and RNA
synthesis (pyrimidine antagonist)
DNA alkylating agent;
blocks DNA synthesis
DMB
1.Glioblastoma multiforme - Infiltrative; rapidgrowing; occurs: most frequently in mid-aged; apt to
involve both cerebral hemispheres via the corpus
callosum; Average Survival: 1 year
Gene therapy for glioblastoma multiforme
Kuopio University
Mol Ther. 2004 Nov;10(5):967-72
- surgical resection of tumor followed by radiotherapy –
all patients
- delivery of AdvHSV-tk (3 x 10(10) pfu) by local injection into the wound
bed after tumor resection, followed by intravenous ganciclovir
5 mg/kg twice daily for 14 days
AdvHSV-tk treatment produced a clinically and statistically significant increase in
mean survival from 39.0 +/- 19.7 (SD) to 70.6 +/- 52.9 weeks (P = 0.0095, log-rank
regression vs. randomized controls). The median survival time increased from
37.7 to 62.4 weeks.
AdvHSV-tk gene therapy with intravenous ganciclovir improves
survival in human malignant glioma: a randomised, controlled study
Next step for suicide gene therapy in glioblastoma multiforme
Results presented by Prof. Seppo Yla-Herttuala from
AI Virtanen Institute, Kuopio, Finland at Gene & Cell Therapy Conference,
Brugge, November 2008
Phase III trial – Adv-Tk - 3 x 1010 pfu/ml
Vector injected into tumor cavity at the depth of 10 mm
250 patients from Germany, France, Belgium, UK, Poland, Finalnd, Czech, Hungary, Israel
1) Standard therapy:
mean survival – 308 days
2) + temodar (temozolamide)
- 307 days
3) Standard therapy + gene therapy
- 300 days
4) + temodar + gene therapy
- 350 days
DMB
Take home messages...
DMB
Every disease can be (potentially) treated
with gene therapy, because every
disease has a genetic background!
Summary
-there are different sorts of vectors used in gene therapy
- vectors have their advantages and drawbacks
- the most serious limitations of vectors is the risk of
induction of oncogenesis (retroviral) or strong inflammatory
response (adenoviral)
-the aim of gene therapists is to enhance the efficiency and
to limit (eradicate) the risk o side effects
- however, there is no drug without side effects!
Gene therapy can cure the disease not only in
experimental animals but also in human – SCID trials
Gene therapy will never be a simple method effective
in treatment of every disease
DMB