1. TOPICS IN (NANO) BIOTECHNOLOGY Gene Therapy Lecture 8 31st March, 2004 PhD Course

2. What is an antibody?

3. How do we produce polyclonal and monoclonal antibodies? Polyclonal antibodies - larger quantities may be produced at a time - sometimes better selectivity and sensitivity due to recogintion of multiple epitopes - no guarantee of batch to batch reproducibility Monoclonal antibodies - long and expensive process - sometimes lower selectivity and sensitivity in comparison to Pabs observed - once cell line established constant reproducible supply of antibodies …. forever

4. 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

5. What is gene therapy?

6. 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 1990 first GT in humans (ADA deficiency) 2001 596 GT clinical trials (3464 patients)

7. 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

8. 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

9. 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

10. 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 In vivo techniques –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.

11. “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 Viral vectors

12. 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] Viral vectors

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

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

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

16. 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 #s to achieve successful delivery of new genes into patient’s cells Vectors must be propagated in large #s in cell culture (10 9 ) with the aid of a helper virus

17. 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 Delivery System of Choice = Viral Vectors

18. Advantages Disadvantages High Transduction Effciency Requires dividing cells for infectivity Insert Size up to 8kBLow Titers (10 6 - 10 7 ) Integrates into host genome resulting in sustained expression of vector Integration is random Extremely well studied system In vivo delivery remains poor. Effective only when infecting helper cell lines Vector protiens not expressed in host

19. Advantages High transduction efficiency Insert size up to 8kbHigh viral titer (10 10 -10 13 ) Infects both replicating and differentiated cells Disadvantages Expression is transient (viral DNA does not integrate) Viral protiens can be expressed in host following vector administration In vivo delivery hampered by host immune response Adenovirus

20. 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 Herpes Simplex Virus

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

22. Electroporation

23. Ex vivo Electroporation

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

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

26. DNA liposome complexes Liposomes

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

28. 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 agains transgenes expressed in treated cells) –Integration is not controlled  oncogenes may be involved at insertion point  cancer?

29. 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) Limitations of Gene Therapy

30. 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 Limitations of Gene Therapy 3Other 196Cancer 21HIV 18Genetic disease # Trials (total = 338) Diagnosis Gene Therapy Trials in U.S. (Information from US NIH, Office of Recombinant DNA Activities – 1999)

31. Applications of gene therapy

32. 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

33. 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 Example: Severe Combined Immunodeficiency Disease (SCID)

34. September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial –Ashanti (4 year old girl) Her lymphocytes were gene-altered (~10 9 ) 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 Example: Severe Combined Immunodeficiency Disease (SCID)

35. 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)

36. 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 Ornithine transcarbamylase (OTC) deficiency

37. 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 Ornithine transcarbamylase (OTC) deficiency

38. 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

39. 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 (T cyt ) Single Gene Defects = Most Attractive Candidates

40. 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 10 9 liver cells (~15% of patients liver) ex vivo –Infused back into patient –Improvement seen –Has been used in many trials since then Single Gene Defects = Most Attractive Candidates

41. Strategies #1: Strengthening of the immune response against a tumor –B7 expression on tumors may provide necessary second signal (co-stimulation) required for T cyt cell activation –Monoclonal antibody binding to tumor antigens can stimulate: NK cell killing via antibody-dependent cell- mediated cytotoxicity Phagocytosis via FcR-binding on macrophages Complement activation  opsonization Gene Therapy of Cancer

42. Watch video

43. #2: Repair of cell cycle defects caused by losses of tumor suppressor genes –e.g. p53 = DNA repair enzyme – “Guardian of the genome” Prevents replication of damaged DNA in normal cells and promotes apoptosis of cells with abnormal DNA Faulty p53 allows cells carrying damaged DNA to survive when they would normally die –Mutations passed to progeny –Can accumulate additional mutations  lethal tumor In most human cancers, the p53 gene appears defective Gene Therapy of Cancer

44. Normal p53 Abnormal p53 Active p53 DNA damaged Apoptosis Inactive p53 DNA damaged Tumor cells proliferate Gene Therapy of Cancer

45. Viral vector treatment –Virus is engineered so that they reproduce in cells with abnormal p53 but not in healthy cells (selective replication) Adenovirus p53-binding protein (E1B-55kDa) is mutated  therefore cannot shut down p53 in normal cells (p53 binding protein binds and inactivates p53 in normal cells in order to initiate virus replication) Virus reproduces in tumor cells (not healthy cells)  Tumor cells die (only cells which have lost p53 are permissive for virus replication because there is no requirement for p53 binding protein to switch off p53) Gene Therapy of Cancer

46. #3: Introduce genes for IL-4, IL-6 and TNF –TNF – kills tumours –IL-4 – growth factor for producing AB producing cells –IL-6 – stimulates certain cells to secrete antibodies & stimulates red blood cell production Gene Therapy of Cancer

47. Liposomes coated in polymer PEG – can crss 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) Recent Developments in Gene Therapy

48. 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? Future?