prof. dr hab. józef dulak - jagiellonian...
TRANSCRIPT
05.06.2010
Zasady i perspektywyterapii 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
HPRT-/- cells
HAT medium
Prof.Wacław Szybalski
McArdle Laboratory for Cancer Research, Wisconsin, Madison,
USA
Gene therapy was born in... 1962
HPRT+/+ cells
Retroviral expression system
Gag – core proteins, matrix, nucleocapsid Pol – reverse transcriptase and integraseEnv – envelope glycoproteins
DMB
Retroviral vectors
• long-term expression due to integration into cellular genome
gag pol envITR ITR
retrovirus
transgenITRITR
Retroviral vector
• gag – structural proteins
• pol – reverse transcriptase
• env – envelope proteins
DMB
First controlled trial of gene therapy - 1990
HPRT ADA
ADA deficiency– results in severe immunodeficiency syndrome
DMB
Ashanti De Silva (patient)
First clinical trial of gene therapy - 1990
Retroviral vector containing correct ADA gene (cDNA) has beentransduced into blood lymphocytes
This first clinical trial was not „pure” from the methodological point of view.
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.
DMB
Gene therapy
Ex vivo In vivo
Cells are isolated, modified in vitro
and injected back to the patient Vectors are injected
into the bloodVectors are injected into the diseased tissue
eg. into the tumor or healthy tissue
Eg. Leucocytes, hepatocytes
DMB
Vectors
Non-viral/plasmids viral
RNA DNA Retroviral(including Lentiviral)
AdenoviralAAV Herpes
„naked” DNA
Lipoplexes
Viroplexes(lipoplexes enhancedin proteins from viral capsids)
ComplexesWith chemical vehicles
DMB
Vectors for gene therapy
Transfectionefficacy
Capacity
Expression
Cytotoxicity
Plasmids Retroviral Lentiviral AAV Adenoviral
unlimited 4-5 kb 9 kb 4-5 kb 7-10 kb
Very low low low moderate high
low
Days-weeks Long-term Weeks-months
low low low high
„Gut-less”Adenoviral
30 kb
low
Long-term
high
Long-term Long-term
DMB
retroviruses
adenoviruses
AAV
naked DNA
liposomes
Six years ago...
Types of vectors used in clinical trials of gene therapyDMB
26
Phases of clinical trials
disease
Some Phase II and most Phase III drug trials are randomized, double-blinand placebo-controlled
27
The strongest evidence for therapeutic interventions is provided bysystematic review of randomized, double-blind, placebo-controlled trialsinvolving a homogeneous patient population and medical condition.
Clinical trials
Some Phase II and most Phase III drug trials
Randomized: Each study subject is randomly assigned to receive eitherthe study treatment or a placebo.
Double-blind: The subjects involved in the study and the researchersdo not know which study treatment is being given.
Placebo-controlled: The use of a placebo (fake treatment) allowsthe researchers to isolate the effect of the study treatment.
28
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
David Vetter - „Bubble Boy”
David has spent 12 years in a foil-protected environment. Finally has received the bone marrow transplantation from his sister, but unfortunately died due to Epstein-Barr virus infection
X-linked severe combined immunodeficiency (X- SCID)
Restoration of B and T lymphocytes and NK cells
D. Kohn et al., Nature Rev Cancer July 2003
Lack of γc gene
Cavazzana-Calvo M et al .
Gene therapy of human severe combinedimmunodeficiency (SCID)-X1 disease
Science 2000: 28 April: 288: 669-672
DMB
Retroviral vector with a correct γc gene
Stem cells without correct γc gene
Gene therapy is efficient in treatment of X-SCIDDMB
Gene therapy has been beneficialto 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
•long-term expression & integration into cellular genome
gag pol envITR ITR
retrovirus
transgenITRITR
Retroviral vector
• gag – structural proteins
• pol – reverse transcriptase
• env – envelope proteins
DMB
random integration –risk of insertional mutagenesis
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…
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 has been beneficialto most treated SCID-X1 and ADA
patients!!!
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.
Gene therapy
Enhancing Substituting Suppresive
enhancement delivery of inhibition ofof gene expression the missing gene gene expression
Acquired diseasesCaused by endogenous and exogenous factors Inherited diseases Acquired diseases
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 Cdk6-cyclin complexes. Binding of p21CIP to these Cdk-cyclin complexes leads to cell cycle arrest in G1 and G2
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 apoptosisLate genes (coding proteins required for virus assembly)
• E1 early gene is essential for the subsequent adenoviral gene expression
leczniczy gen
region E1Adenoviral DNA
DNA of 1st generation vector
TP53
Cancer cell
Adenoviral vector with correct p53 gene
expression of p53 incancer cell
apoptosis
Adenoviral delivery of p53 geneDMB
First registered drug for gene therapy of cancer
2004
patients with head and neckcancer
Gendicine (SiBiono GeneTech, Chiny)
Adenoviral vector with p53 gene
Cancer gene therapy
1. Direct attack on tumor cells a) transfer of tumor suppressor geneb) inhibition of oncogenes
- antisense therapy - ribozymes
c) suicide genes d) oncolytic viruses (replication-competent viruses)
2. Harnessing immune response to tumor antigens 3. Chemoprotection4. Anti-angiogenic therapy
DMB
Suicide gene therapy
Vector with a suicide gene (eg. Thymidine kinase)
Pro-drug (eg. ganciclovir)
Phosphorylated ganciclovir
Inhibition of DNA synthesis in cancer cells
DMB
Enzyme-prodrug combination for suicide gene therapy
Enzyme Prodrug Product Mechanism
HSV-tk ganciclovir ganciclovir triphosphate blocks DNA synthesisacyclovirvalacylcovir
cytosine deaminase 5-fluorocytosine 5-fluorouracil (5-FU) blocks DNA and RNA synthesis (pyrimidine antagonist)
cytochrome P450 cyclophosphamide phosphoramide mustard DNA alkylating agent;blocks DNA synthesis
1.Glioblastoma multiforme - Infiltrative; rapid-growing; occurs: most frequently in mid-aged; apt to involve both cerebral hemispheres via the corpus callosum; Average Survival: 1 year
DMB
Gene therapy for glioblastoma multiforme
- surgical resection of tumor followed by radiotherapy –all patients
- delivery of AdvHSV-tk (3 x 10(10) pfu) by local injection into the woundbed after tumor resection, followed by intravenous ganciclovir5 mg/kg twice daily for 14 days
Kuopio University
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.
Mol Ther. 2004 Nov;10(5):967-72
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
Every disease can be (potentially) treatedwith gene therapy, because every disease has a genetic background!
DMB
Summary -there are different sorts of vectors used in gene therapy
- the most serious limitations of vectors is the risk of induction of oncogenesis (retroviral) or strong inflammatory response (adenoviral)
- vectors have their advantages and drawbacks
-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