05.06.2010

Zasady i perspektywyterapii genowej

Prof. dr hab. Józef Dulak

Zakład Biotechnologii Medycznej Wydział Biochemii, Biofizyki i Biotechnologii UJ

Email: jozef. dulak@uj.edu.pl

Web page: http://biotka.mol.uj.edu.pl/zbm

What is gene therapy?

Gene therapy

Therapeutic gene

(transgene)

Vector Patient Expression of therapeutic gene

Protein

Gene therapy is also silencing or replacingthe bad genes or overexpression

of good genes

Which diseases could be cured with gene therapy?

HPRT-/- cells

HAT medium

Prof.Wacław Szybalski

McArdle Laboratory for Cancer Research, Wisconsin, Madison,

USA

Gene therapy was born in... 1962

HPRT+/+ cells

Lesch-Nyhan syndrome

HPRTHPRT

Inborn error of metabolism – deficiency of HPRT

allopurinol

Children suffering from deficiency of HPRT-

Lesh-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

Gag – core proteins, matrix, nucleocapsid Pol – reverse transcriptase and integraseEnv – envelope glycoproteins

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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

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First controlled trial of gene therapy - 1990

HPRT ADA

ADA deficiency– results in severe immunodeficiency syndrome

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Gene therapy of ADA deficiency

ADA

ADA- ADA+

ADA+

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.

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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

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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

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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

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Gene therapy clinical trials

retroviruses

adenoviruses

AAV

naked DNA

liposomes

Six years ago...

Types of vectors used in clinical trials of gene therapyDMB

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Clinical trials in gene therapyDMB

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Phases of clinical trials

disease

Some Phase II and most Phase III drug trials are randomized, double-blinand placebo-controlled

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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.

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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

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Registered drugs are only a portion of all tested

Succesful gene therapy

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-SCID deficiency

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

Cytokines receptors

D. Kohn et al., Nature Rev Cancer July 2003

Cavazzana-Calvo M et al .

Gene therapy of human severe combinedimmunodeficiency (SCID)-X1 disease

Science 2000: 28 April: 288: 669-672

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Gene therapy is efficient in treatment of X-SCID

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

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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

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random integration –risk of insertional mutagenesis

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Integration of retroviral vector into the promoter of LMO2 gene

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!!!

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.

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

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Cancer gene therapy

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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

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Application of adenoviral vectors for gene therapy

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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 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

Advexin – adenoviral vector expressing p53 gene effective in patients with head and neck cancer

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

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Suicide gene therapy

Vector with a suicide gene (eg. Thymidine kinase)

Pro-drug (eg. ganciclovir)

Phosphorylated ganciclovir

Inhibition of DNA synthesis in cancer cells

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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

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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

Take home messages...

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Every disease can be (potentially) treatedwith gene therapy, because every disease has a genetic background!

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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

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