19. Treatment of Genetic Diseases

1. 19.Treatment of Genetic Diseases Somatic Cell Gene Therapy

2. a).Treatment strategies i).Metabolic manipulation ii).Manipulation of the protein iii).Modification of the genome b).Strategies for gene transfer

8. Three categories of somatic cell gene therapy: • Ex vivo – cells removed from body, incubated with vector and gene-engineered cells returned to body. • In situ – vector is placed directly into the affected tissues. • In vivo – vector injected directly into the blood stream.

9. Example of ex vivo somatic cell gene therapy • Usually done with blood cells because they are easiest to remove and return. • Sickle cell anemia

10. Examples of in situ somatic cell gene therapy • Infusion of adenoviral vectors into the trachea and bronchi of cystic fibrosis patients. • Injection of a tumor mass with a vector carrying the gene for a cytokine or toxin. • Injection of a dystrophin gene directly into the muscle of muscular dystrophy patients.

11. Example of in vivo somatic cell gene therapy • No clinical examples. • In vivo injectable vectors must be developed.

12. Barriers to successful gene therapy: • Vector development • Corrective gene construct • Proliferation and maintenance of target cells • Efficient transfection and transport of DNA to nucleus for integration into genome • Expansion of engineered cells and implantation into patient

13. Types of vectors • RNA viruses (Retroviruses) 1. Murine leukemia virus (MuLV) 2. Human immunodeficiency viruses (HIV) 3. Human T-cell lymphotropic viruses (HTLV) • DNA viruses 1. Adenoviruses 2. Adeno-associated viruses (AAV) 3. Herpes simplex virus (HSV) 4. Pox viruses 5. Foamy viruses • Non-viral vectors 1. Liposomes 2. Naked DNA 3. Liposome-polycation complexes 4. Peptide delivery systems

14. Advantages: • Randomly integrates into genome • Wide host range • Long term expression of transgene • Disadvantages: • Capacity to carry therapeutic genes is small • Infectivity limited to dividing cells • Inactivated by complement cascade • Safety

15. Adenovirus • Advantages: • Efficiency of transduction is high • High level gene expression • Slightly increased capacity for exogenous DNA • Disadvantages: • Expression may be transient • Cell-specific targeting difficult to achieve • Virus uptake is ubiquitous • Safety

16. Other viral vectors • Adeno-associated virus– infects wide range of cells (both dividing and non-dividing), able to integrate into host genome, not associated with any human disease, high efficiency of transduction. • Herpes simplex virus, vaccinia virus, syndbis virus, foamy viruses– not yet widely studied • Onyx virus– limited replicating adenovirus that replicates mainly in tumor cells.

17. Non-viral vectors 1. Liposome 2. Cationic polymers 3. Naked DNA 4. Peptide-mediated gene delivery May overcome limitations with viruses including small capacity for therapeutic DNA, difficulty in cell-type targeting and safety concerns.

18. Selectable marker for transduced cells Synthesis of a retroviral gene therapy vector Site of insertion of therapeutic gene

19. Percent effort directed towards different gene therapy trials.

20. Examples of Gene Therapy Trials • Adenosine deaminase gene transfer to treat Severe Combined Immuno-Deficiency (SCID) • CFTR gene transfer to treat Cystic Fibrosis (CF) • Advanced Central Nervous System (CNS) Malignancy • Mesothelioma • Ornithine Transcarbamylase Deficiency • Hemophilia • Sickle Cell Disease

21. Harvestmarrow Donor Infuse normal donor cells Radiation/Chemotherapy Patient Stem Cell Transplantation

22. Make gene Put into vehicle for delivery into cell Harvest marrow Introduce therapeutic gene Reinfuse corrected cells Radiation/Chemotherapy Stem Cell Gene Therapy

23. Sickled red cell Survives 15 - 25 days The Molecular Basis of Sickle Cell Anemia achains z a2 a1 Polymerization a2 bs2 LCR e g g bs bs chains

24. Preferential Survival of Normal Red Blood Cells in Sickle Cell Anemia Normal 120 days Sickle Cell 20 days

25. Gene Therapy for Sickle Cell Anemia a chains z a2 a1 No polymerization a2 bsg LCR e g g bs bschains LCR Non-sickled red cell Survives 120 days g b gchains

26. Mixed Chimerism following BMT for b Thalassemia and Sickle Cell Disease • Occurs in a minority of patients (5 - 10%). • A minority of donor-origin progenitors (10 - 20%) is sufficient to ameliorate disease. Thus, it may be possible to achieve therapeutic effects by gene transfer into only a fraction of stem cells.

27. TURNOVER RATE HIGH LOW 20 / 120 = 1/6th normal or corrected stem cells = 50% corrected mature red cells Preferential Survival of Normal Red Blood Cells

28. Therapeutic effects from small numbers of normal stem and progenitor cells in the marrow BLOOD BONE MARROW N S S 120 days S S S S 20 days

29. Approaches to Improving the Efficiency of Gene Therapy Targeting the Stem Cell • Useselectiontoexponentially expand stem cells carrying the therapeutic gene. • Userepeated treatmentstoadditively expand stem cells carrying the therapeutic gene.

30. Therapeutic gene Selectable gene In Vivo Selection Selectable gene = MDR1 (taxol, navelbine, vinblastine) DHFR (methotrexate) Other (MGMT, aldehyde dehydrogenase, cytidine deaminase)

31. Drug Treatment In Vivo Selection of Genetically Modified Bone Marrow

32. GCSF Mobilize Stem Cells Introduce gene Re-infuse REPEAT In vivo selection Gene Therapy for Sickle Cell Disease

33. One developing technology that may be utilized for gene therapy is nuclear transfer (“cloning”).

36. What’s in a Name? – Nuclear Transplantation vs. Therapeutic Cloning vs. Human Reproductive Cloning.

37. Ethical Considerations • Use of technology for non-disease conditions such as functional enhancement or “cosmetic” purposes– for example, treatment of baldness by gene transfer into follicle cells , larger size from growth hormone gene, increased muscle mass from dystrophin gene. • In utero somatic gene therapy– only serious disease should be targeted and risk-benefit ratios for mother and fetus should be favorable. • Potential for real abuse exists by combininghuman reproductivecloningandgenetic engineering.