1. Dr. Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

2. Course Contents Gene Therapy
Introduction to gene therapy
Delivery of Therapeutic Genes
Gene Therapy Targets
Delivery Modes
Steps in Gene Therapy
Gene Therapy Viral & Non Viral vectors
Liposomes

3. Course Contents Cell bases therapies Cancer gene therapy
Gene therapy in plants
Ethical issues in gene therapy
Therapeutic Gene Regulation
Control of therapeutic gene expression
Prospects for gene therapy

4. GENE THERAPY
Any procedure intended to treat or alleviate disease by genetically modifying the cells of a patient

5. History of gene therapy
1930’s “genetic engineering” - plant/animal breeding
60’s First ideas of using genes therapeutically
50’s-70’s Gene transfer developed
70’s-80’s Recombinant DNA technology
First GT in humans (ADA deficiency)
GT clinical trials (3464 patients)

6. What is gene therapy? Why is it used?
Gene therapy is the application of genetic principles in the treatment of human disease
Gene therapy = Introduction of genetic material into normal cells in order to:
counteract the effect of a disease gene or
introduce a new function
GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized  to improve a genetic disorder
Can be applied as therapy for cancers, inherited disorders, infectious diseases, immune system disorders

7. Amenability to gene therapy
Mode of inheritance
Identity of molecular defect
Nature of mutation product
Accessibility of target cells and amenability to cell culture
Size of coding DNA
Control of gene expression

8. Gene Therapy

9. First gene therapy trial for ADA deficiency -1990

10. GENE THERAPY Delivery mechanism Type of cells modified
Ex vivo
In vivo
Type of cells modified
Germ-line cells
Somatic cells
Mechanism of modification
Gene augmentation/supplementation
Gene replacement
Targeted inhibition of gene expression
Targeted killing of specific cells

11. Three types of gene therapy:
Monogenic gene therapy
Provides genes to encode for the production of a specific protein
Cystic fibrosis, Muscular dystrophy, Sickle cell disease, Haemophilia, SCID
Suicide gene therapy
Provide ‘suicide’ genes to target cancer cells for destruction
Cancer
Antisense gene therapy
Provides a single stranded gene in an’antisense’ (backward) orientation to block the production of harmful proteins
AIDS/HIV

12. TYPES OF CELLS MODIFIED
Germ-line gene therapy
Modification of gametes, zygote or early embryo
Permanent and transmissible
Banned due to ethical issues
Somatic cell gene therapy
Modification of somatic cells, tissues etc
Confined to the patient

13. Getting genes into cells
In vivo versus ex vivo
In vivo = intravenous or intramuscular or non-invasive (‘sniffable’)
Ex vivo = hepatocytes, skin fibroblasts, haematopoietic cells (‘bioreactors’)
Gene delivery approaches
Physical methods
Non-viral vectors
Viral vectors

14. In vivo techniques In vivo techniques usually utilize viral vectors
Virus = carrier of desired gene
Virus is usually “crippled” to disable its ability to cause disease
Viral methods have proved to be the most efficient to date
Many viral vectors can stable integrate the desired gene into the target cell’s genome
Problem: Replication defective viruses adversely affect the virus’ normal ability to spread genes in the body
Reliant on diffusion and spread
Hampered by small intercellular spaces for transport
Restricted by viral-binding ligands on cell surface therefore cannot advance far.

15. DELIVERY MECHANISMS
(In vivo)
(ex vivo)

16. Vectors in GT Viral Non-viral Retro- Adeno- Adeno-associated-
Herpes simplex-
Non-viral
Naked DNA/Plasmid
liposomes

17. Viral vectors “Viruses are highly evolved natural vectors
for the transfer of foreign genetic information
into cells”
But to improve safety,
they need to be replication defective

18. Viral vectors Compared to naked DNA, virus particles
provide a relatively
efficient means of
transporting DNA into cells, for expression in the nucleus as recombinant genes
(example = adenovirus). [

19. Viral vectors Adenoviruses Herpes simplex
Retroviruses
eg Moloney murine leukaemia virus
(Mo-MuLV)
Lentiviruses (eg HIV, SIV)
Adenoviruses
Herpes simplex
Adeno-associated viruses (AAV)

20. Engineering a virus into a viral vector
Therapeutic
gene
Packaging
Vector DNA
Helper DNA
essential viral genes
wildtype virus
replication
proteins
Viral vector
Packaging cell
structural
proteins

21. Y Gene transfer vector Vector uncoating Integrated expression cassette
Therapeutic mRNA
and protein
Episomal vector
Integrated expression
cassette
Target cell

22. Delivery System of Choice = Viral Vectors
A. Rendering virus vector harmless
Remove harmful genes  “cripple” the virus
Example – removal of env gene  virus is not capable of producing a functional envelope
Vectors needed in very large numbers to achieve successful delivery of new genes into patient’s cells
Vectors must be propagated in large numbers in cell culture (109) with the aid of a helper virus

23. Delivery System of Choice = Viral Vectors
B. Integrating versus Non-Integrating Viruses
Integrating viruses
Retrovirus (e.g. murine leukemia virus)
Adeno-associated virus (only 4kbp accommodated)
Lentivirus
Non-Integrating viruses
Adenovirus
Alphavirus
Herpes Simplex Virus
Vaccinia

24. Adenovirus Advantages High transduction efficiency
Insert size up to 8kbHigh viral titer ( )
Infects both replicating and differentiated cells
Disadvantages
Expression is transient (viral DNA does not integrate)
Viral proteins can be expressed in host following vector administration
In vivo delivery hampered by host immune response

25. Herpes Simplex Virus Advantages Large insert size
Could provide long- term CNS gene expression
High titer
Disadvantages
System currently under development
Current vectors provide transient expression
Low transduction efficiency

26. Ex vivo Ex vivo manipulation techniques Electroporation Liposomes
Calcium phosphate
Gold bullets (fired within helium pressurized gun)
Retrotransposons (jumping genes – early days)
Human artificial chromosomes

27. Electroporation

28. Ex vivo Electroporation

29. Risks Factors gene therapy
Adverse response to the vector
Insertional mutatgenesis resulting in malignant neoplasia
Insertional inactivation of an essential gene
Viruses may infect surrounding health tissues
Overexpression of the inserted gene may lead to so much protein that it may become harmful

30. The End