1. TOPICS IN (NANO) BIOTECHNOLOGY
Gene Therapy
Lecture 11
27th November, 2006
PhD Course

2. 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

3. What is gene therapy?

4. 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)

5. 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

6. Different Delivery Systems are Available
In vivo versus ex vivo
In vivo = delivery of genes takes place in the body
Ex vivo = delivery takes place out of the body, and then cells are placed back into the body

7. 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

8. 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.

9. Viral vectors “Viruses are highly evolved natural vectors
for the transfer of foreign genetic information
into cells”
Kay et al 2001
But to improve safety,
they need to be replication defective

10. 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).
[figure from Flint et al. Principles of Virology, ASM Press, 2000]

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

12. 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

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

14. 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

15. B. Integrating versus Non-Integrating Viruses
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

16. 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

17. 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

18. 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

19. Electroporation

20. Ex vivo Electroporation

21. Liposomes
A phospholipid
Lipid Organization
Phopholipid Hierarchal Structures
In aqueous solution, polar phospholipids form ordered aggregates to minimize hydrophobic interactions
Lipid shape and conditions of formation affect the final lipid organized structure

22. Liposomes Liposomes are not limited by size or number of genes safe
easy to produce
short-term expression

23. Liposomes
liposome
DNA
complexes

24. Liposomes Diverse manners of ‘lysing’ the liposome
Temperature sensitive
Target sensitive
pH sensitive
Electric field sensitive

25. Limitations of Gene Therapy
Gene delivery
Limited tropism of viral vectors
Dependence on cell cycle by some viral vectors (i.e. mitosis required)
Duration of gene activity
Non-integrating delivery will be transient (transient expression)
Integrated delivery will be stable
Patient safety
Immune hyperresponsiveness (hypersensitivity reactions directed against viral vector components or against transgenes expressed in treated cells)
Integration is not controlled  oncogenes may be involved at insertion point  cancer?

26. Limitations of Gene Therapy
Gene control/regulation
Most viral vectors are unable to accommodate full length human genes containing all of their original regulatory sequences
Human cDNA often used  much regulatory information is lost (e.g. enhancers inside introns)
Often promoters are substituted  therefore gene expression pattern may be very different
Random integration can adversely affect expression (insertion near highly methylated heterogeneous DNA may silence gene expression)

27. Limitations of Gene Therapy
Expense
Costly because of cell culturing needs involved in ex vivo techniques
Virus cultures for in vivo delivery
Usually the number of patients enrolled in any given trial is <20
More than 5000 patients have been treated in last ~12 years worldwide 3
Other
196
Cancer 21
HIV 18
Genetic disease
# Trials (total = 338)
Diagnosis
Gene Therapy Trials in U.S.
(Information from US NIH, Office of
Recombinant DNA Activities – 1999)

28. Applications of gene therapy

29. Example: Severe Combined Immunodeficiency Disease (SCID)
Before GT, patients received a bone marrow transplant
David, the “Boy in the Bubble”, received BM from his sister  unfortunately he died from a a form of blood cancer

30. Example: Severe Combined Immunodeficiency Disease (SCID)
SCID is caused by an Adenosine Deaminase Deficiency (ADA)
Gene is located on chromosome #22 (32 Kbp, 12 exons)
Deficiency results in failure to develop functional T and B lymphocytes
ADA is involved in purine degradation
Accumulation of nucleotide metabolites = TOXIC to developing T lymphocytes
B cells don’t mature because they require T cell help
Patients cannot withstand infection  die if untreated

31. Example: Severe Combined Immunodeficiency Disease (SCID)
September 14, NIH, French Anderson and R. Michael Blaese perform the first GT Trial
Ashanti (4 year old girl)
Her lymphocytes were gene-altered (~109) ex vivo  used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene (billions of retroviruses used)
Cynthia (9 year old girl) treated in same year
Problem: WBC are short-lived, therefore treatment must be repeated regularly

32. Ornithine transcarbamylase (OTC) deficiency
September 17, 1999
Ornithine transcarbamylase (OTC) deficiency
Urea cycle disorder (1/10,000 births)
Encoded on X chromosome
Females usually carriers, sons have disease
Urea cycle = series of 5 liver enzymes that rid the body of ammonia (toxic breakdown product of protein)
If enzymes are missing or deficient, ammonia accumulates in the blood and travels to the brain (coma, brain damage or death)

33. Ornithine transcarbamylase (OTC) deficiency
Severe OTC deficiency
Newborns  coma within 72 hours
Most suffer severe brain damage
½ die in first month
½ of survivors die by age 5
Early treatment
Low-protein formula called “keto-acid”
Modern day treatment
Sodium benzoate and another sodium derivative
Bind ammonia  helps eliminate it from the body

34. Ornithine transcarbamylase (OTC) deficiency
Case study: Jesse Gelsinger
GT began Sept. 13, 1999, Coma on Sept. 14, Brain dead and life support terminated on Sept. 17, 1999
Cause of death: Respiratory Disease Syndrome
Adenovirus (a weakened cold virus) was the vector of choice (DNA genome and an icosahedral capsid)
Chain reaction occurred that previous testing had not predicted following introduction of “maximum tolerated dose”
Jaundice, kidney failure, lung failure and brain death
Adenovirus triggered an overwhelming inflammatory reaction  massive production of monokine IL-6
 multiple organ failure

35. Single Gene Defects = Most Attractive Candidates
Cystic fibrosis
“Crippled” adenovirus selected (non-integrating, replication defective, respiratory virus)
Gene therapy trials – 3 Research teams, 10 patients/team
2 teams administered virus via aerosol delivery into nasal passages ad lungs
1 team administered virus via nasal passages only
Only transient expression observed  because adenovirus does not integrate into genome like retroviruses

36. Single Gene Defects = Most Attractive Candidates
AIDS
HIV patients  T lymphocytes treated ex vivo with rev and env defective mutant strains of HIV
Large numbers of cells obtained
Injected back into patient
Stimulated good CD8+ cytotoxic T cell responses (Tcyt)

37. Single Gene Defects = Most Attractive Candidates
Familial Hypercholesterolemia
Defective cholesterol receptors on liver cells
Fail to filter cholesterol from blood properly
Cholesterol levels are elevated, increasing risk of heart attacks and strokes
1993  First attempt
Retroviral vector used to infect 3.2 x 109 liver cells (~15% of patients liver) ex vivo
Infused back into patient
Improvement seen
Has been used in many trials since then

38. HOW STEM CELLS AND GENE THERAPY MIGHT WORK TOGETHER
A sample of bone marrow is removed.
Stem cells are isolated and allowed to multiply in culture.
Cells are treated with a modified virus containing a therapeutic gene
           

39. HOW STEM CELLS AND GENE THERAPY MIGHT WORK TOGETHER
The virus is taken up by individual cells and the therapeutic gene goes into the cell's nucleus.
Treated ("corrected") cells are injected into the bloodstream.
Treated cells respond to injury signals from degenerating muscle or other tissues and migrate out of the bloodstream.
Treated cells patch damage and build healthy tissue
                       
                   
             
                    

40. Stem cells for Gene Therapy

41. Recent Developments in Gene Therapy
Liposomes coated in polymer PEG – can cross the blood-brain barrier (viral vectors are too big) (January 2003)
Case Western Uni. & Copernicus Therapeutics able to create tiny liposomes 25nm across to carry therapeutic DNA through pores in nuclear membrane
New gene approach repairs errors in mRNA
Thalassaemia
Cystic fibrosis
Some cancers
(Please refer to Newscientist.com)

42. Future?
2003 – temporary hold on all gene therapy trials including retroviral vectors in blood stem cells
Too early to tell
$200 million/year by NIH on clinical trials
Desperately need improved DELIVERY …could liposomes be the answer?