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

2. i). Metabolic manipulation ii). Manipulation of the protein
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. Synthesis of a retroviral gene therapy vector
Selectable marker for transduced cells
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. Stem Cell Transplantation
Harvest marrow
Radiation/Chemotherapy
Infuse normal donor cells
Donor
Patient

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

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

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
bs chains
Non-sickled red cell
Survives 120 days
LCR g b
g chains

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

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

29. Approaches to Improving the Efficiency of Gene Therapy Targeting the Stem Cell
Use selection to exponentially expand stem cells carrying the therapeutic gene.
Use repeated treatments to additively expand stem cells carrying the therapeutic gene.

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

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

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

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
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 combining human reproductive cloning and genetic engineering.