1. Gene Therapy 张咸宁 zhangxianning@zju.edu.cn Tel ： 13105819271; 88208367 Office: A705, Research Building 2012/09

2. Learning Objectives 1. Traditional managements 2. Gene therapy

3. InterventionSubstance or Technique Disease Drug/diatary avoidanceAntimalarial drugsG6PD deficiency Dietary restrictionPhe Gal Cholesterol PKU Galactosemia Familial hypercholesterolemia Replacement of deficient product ThyroxineCongenital hypothyroidism Protein drug therapyPenicillamineWilson disease Replacement of deficient enzyme/protein Blood transfusionSCID Treatment of Genetic Disease by Metabolic Manipulation

4. Wilson disease: Cu toxicity, AR Wilson SAK. Brain, 1912; 34:295-507

5. Wilson disease:Before/After therapy

6. Gene therapy The medical procedure involves either replacing, manipulating, or supplementing nonfunctional genes with healthy genes. OR “Everyone talks about the human genome, but what can we do with it?”

7. Impact of the Genome Project on Medicine Facilitate identification of genes associated with complex disorders ie. Cardiovascular disease, cancer provides more therapeutic targets-in turn enhances our ability to treat cause of disease instead of symptoms bioinformatics, array technology, proteomics -enable a systems approach to biomedical research

8. Monogenic Diseases Which May Be Candidates For Gene Therapy Sickle cell anemia/Thal Bone Marrow Congenital immune deficiencies Bone Marrow Lysosomal storage and metabolic Bone Marrow --------------------------------------------------------------- Cystic fibrosis Lung - airways --------------------------------------------------------------- Muscular dystrophy Muscle --------------------------------------------------------------- Hemophilia A or B Liver Urea cycle defects Liver Familial hypercholesterolemia Liver

9. Types Of Conditions That May Be Treated By Gene Therapy Monogenic Diseases (>1,000 known) Cancer, Leukemia Infectious (AIDS, Hep C) Cardiovascular Neurologic

11. Technical Requirements for Successful Gene Therapy 1. Normal gene must be cloned. 2. Effective method for gene delivery to cells. 3. Inserted gene expressed at appropriate level. 4. Safe for patient and public. 5. More medically beneficial and/or cost- effective than other treatments.

12. Gene Delivery Can Be: I. Ex vivo – gene into isolated cells II. In vivo – gene directly into patient a) Systemic injection +/- targeted localization +/- targeted expression b) Localized 1) Percutaneous 5) Bronchoscope 2) Vascular catheter 6) Endoscope 3) Stereotactic 7) Arthroscope 4) Sub-retinal

13. General considerations for the use of somatic gene therapy (approved in 1988) 1. Compensate for a mutation resulting in the loss of function examples of monogenic disorders: cystic fibrosis, hemophilia

14. General considerations for the use of gene therapy 1.Compensate for a mutation resulting in the loss of function examples of monogenic disorders: cystic fibrosis, hemophilia stage of the research: http://clinicaltrials.gov/ct2/results?term=gene+therapy+cystic+fibrosis

15. General considerations for the use of gene therapy 1.Compensate for a mutation resulting in the loss of function 2. Replace or inactivate a dominant mutant gene

16. General considerations for the use of gene therapy 1.Compensate for a mutation resulting in the loss of function 2. Replace or inactivate a dominant mutant gene example: Huntington disease (expanded CAG repeat) ? Ribozymes or siRNA to degrade mRNA

17. General considerations for the use of gene therapy 1.Compensate for a mutation resulting in the loss of function 2. Replace or inactivate a dominant mutant gene example: Huntington disease (expanded CAG repeat) ? Ribozymes or siRNA to degrade mRNA state of research – no open studies for Huntington’s

18. General considerations for the use of gene therapy 1.Compensate for a mutation resulting in the loss of function 2. Replace or inactivate a dominant mutant gene 3. Pharmacologic gene therapy example: cancer

22. General considerations for the use of gene therapy 1.Compensate for a mutation resulting in the loss of function 2. Replace or inactivate a dominant mutant gene 3. Pharmacologic gene therapy example: cancer state of research: clinicaltrials.gov website currently lists 35624 gene therapy trials for cancer; 10649 are open to enrollment

23. General considerations for the use of gene therapy 1.Compensate for a mutation resulting in the loss of function 2. Replace or inactivate a dominant mutant gene 3. Pharmacologic gene therapy Yet, it is important to note that there is not yet a single FDA-approved use of gene therapy!

24. Minimal requirements that must be met: Identification of the affected gene A cDNA clone encoding the gene

25. Minimal requirements that must be met: Identification of the affected gene A cDNA clone encoding the gene A substantial disease burden and a favorable risk-benefit ratio Sufficient knowledge of the molecular basis of the disease to be confident that the gene transfer will have the desired effect

26. Minimal requirements that must be met: Identification of the affected gene A cDNA clone encoding the gene A substantial disease burden and a favorable risk- benefit ratio Sufficient knowledge of the molecular basis of the disease to be confident that the gene transfer will have the desired effect Appropriate regulation of the gene expression: tissue specific and levels Appropriate target cell with either a long half life or high replicative potential Adequate data from tissue culture and animal studies to support the use of the vector, regulatory sequences, cDNA and target cell

27. Minimal requirements that must be met: Identification of the affected gene A cDNA clone encoding the gene A substantial disease burden and a favorable risk-benefit ratio Sufficient knowledge of the molecular basis of the disease to be confident that the gene transfer will have the desired effect Appropriate regulation of the gene expression: tissue specific and levels Appropriate target cell with either a long half life or high replicative potential Adequate data from tissue culture and animal studies to support the use of the vector, regulatory sequences, cDNA and target cell Appropriate approvals from the institutional and federal review bodies.

28. Gene therapy In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA.

29. Gene Transfer Methods Non-viral: Expression plasmid or other nucleic acid (mRNA, siRNA). Challenge: Naked DNA or RNA does not enter cells. a) Transfer into cells using physical methods such as direct micro-injection or electroporation. b) complex to carrier to allow cross of cell membrane liposomes, cationic lipids, dextrans, cyclohexidrins (aka nanoparticles)

30. Gene Transfer Methods Viral vectors = viruses that have been adapted to serve as gene delivery vectors include: retrovirus Lenti-virus adenovirus adeno-associated virus (AAV) herpes virus

31. In Vivo Gene Transfer By AAV Vector

32. Characteristics of the Ideal Vector for Gene Therapy Safe Sufficient capacity for size of therapeutic DNA Non-Immunogenic Allow re-administration Ease of manipulation Efficient introduction into target cells/tissues Efficient and appropriate regulation of expression Level, tissue specificity, transient, stable?

33. Types of viral vectors Retrovirus Lenti-virus Adenovirus Adeno-Associated virus (AAV) Herpes virus

34. Types of viral vectors stable/transient infect non-dividing cells Retrovirusstableno Lenti-virusstableyes Adenovirustransientyes Adeno-Associated virus ?yes Herpes virustransientyes

35. Which of the following gene-therapy vectors preferentially infects nerve cells? A. Adeno-associated virus B. Retrovirus C. Herpes virus D. Adenovirus E. Liposome

36. Which of the following gene-therapy vectors preferentially infects nerve cells? A. Adeno-associated virus B. Retrovirus √ C. Herpes virus D. Adenovirus E. Liposome

37. Which of the following vectors targets both dividing and non-dividing cells? A. Retrovirus B. Adenovirus C. Adeno-associated virus D. Herpes virus E. Liposome

38. Which of the following vectors targets both dividing and non-dividing cells? A. Retrovirus √ B. Adenovirus C. Adeno-associated virus D. Herpes virus E. Liposome

39. Choice of target cells is critical Stem cells Choice of target cells: ● Long life or substantial replicative potential bone marrow ● Must express an additional proteins needed for biological activity ● Some approaches employ neighboring cells growth factors stimulating repair of nearby heart muscle

40. In vivo and ex vivo gene therapy

41. Two strategies for introducing foreign genes into patients In vivo gene therapy Gene therapy vector + therapeutic gene Advantages: cells and organs not available ex vivo (lining of the lung) Disadvantages: virus could spread to other cells/tissues Less control over titer and conditions of exposure

42. Two strategies for introducing foreign genes into patients Ex vivo gene therapy Stem cells Gene therapy vector + Normal gene Advantages: More controlled infection higher titer virus Disadvantages: technically difficult

43. Types of viral vectors stable/transient infect non-dividing cells Retrovirusstableno Lenti-virusstableyes Adenovirustransientyes Adeno-Associated virus ?yes Herpes virustransientyes

44. Use of retroviral vectors to introduce therapeutic genes into cells

46. Severe Combined Immunodeficiency Syndrome (SCID)——adenine deaminase (ADA) deficiency

47. Severe Combined Immune Deficiency (SCID) SCID is popularly known as “bubble baby disease” after a boy with SCID was kept alive for more than a decade in a germ-free room. SCID is a fatal disease, with infants dying from overwhelming infection due to the congenital absence of a functioning immune system. More than a dozen genes have been found to be able to cause human SCID. The first “SCID gene” to be identified in humans is ADA, which makes an enzyme needed for Immune cells to survive.

48. Severe Combined Immunodeficiency Disease (SCID) is due to a defective gene for Adenosine Deaminase (ADA). A retrovirus, which is capable of transferring it's DNA into normal eukaryotic cells (transfection), is engineered to contain the normal human ADA gene. Isolated T-cell stem line cells from the patient are exposed to the retrovirus in cell culture, and take up the ADA gene. Reimplantation of the transgenic cells into the patient's bone marrow establishes a line of cells with functional ADA, which effecitvely treats SCID. Ex vivoSomatic Therapy for SCID

49. ADA deficiency (SCID): Ashanti de Silva ， 1990

50. Father of GT: Anderson WF, 1990

51. Geneticist guilty of molestation, 2006

53. Clinical Trial of Stem Cell Gene Therapy for Sickle Cell Disease Bone Marrow HarvestIsolate Stem Cells Add Normal Hemoglobin Gene Myeloablate with Busulfan ( 16 mg/kg ) Transplant Gene- Corrected Stem Cells Freeze Certify Follow: Safety Efficacy

54. Gene Therapy Approaches To Cancer a. Replace missing tumor suppressor genes. b. Block over-active oncogenes (e.g. siRNA). c. Insert “suicide genes” (e.g. HSV TK) into tumors. d. Insert genes to induce anti-tumor immune responses (e.g. IL-2, GM-CSF, CD80). e. Express genes which impede tumor neo- vasculature. f. Add chemotherapy resistance genes to HSC to allow chemotherapy dose intensification.

55. Suicide gene therapy for brain tumors in vivo Inject HSV thymidine kinase (tk) gene into tumor cells Gancyclovir (nucleoside analog) binds viral gene to block DNA synthesis Bystander effect kills surrounding tumor cells Takes advantage of the fact that tumor cells are dividing tk gancyclovir

56. Cancer Vaccine Approach Ex vivo gene therapy tumor cells Gene therapy vector + Cytokine (immune modulator) gene Irradiated tumor cells transduced with cytokine gene Time of surgery

57. Other methods to introduce therapeutic DNA: (approved in 1993) Naked DNA DNA packed in liposomes （脂质体） Protein-DNA conjugates (targeting to cell surface receptor ++++ easy to prepare, inexpensive, avoids problems of viral vectors, no size limitations ------- low efficiency in vivo, only transient expression

58. Risks of Gene Therapy 1.Adverse reaction to vector or gene 1999/9/17: reaction to an adenovirus caused death of 18-yo man, Jesse Gelsinger, Arizona, the first victim of gene therapy. OTC (ornithine transcarbamylase) important for metabolism of N Injection of viral particles triggered massive inflammatory response in an individual with mild form of disease being treated with drugs and diet. Subsequent FDA audit revealed protocol and IRB violations.

59. Risks of Gene Therapy 2. Activation of harmful genes by viral promoters/enhancers stably integrated into the genome. 2002 retrovirus-induced leukemia Children with otherwise fatal X-linked SCID injected with ex vivo HSC modified by introduction of the g-c chain cytokine receptor in 2000 (affects lymphocyte maturation) Initial immune function was good 2/11 patients developed leukemia-like disorder at 2 years. Clonal analysis shows insertion and activation of LMO2 gene. FDA-cannot be used as first line therapy if BMT is an option

60. What factors have kept GT from becoming an effective treatment for genetic disease? Short-lived nature of gene therapy Immune response Problems with viral vectors Multigene disorders

61. RNAi

63. Patient Tissue Sample (e.g. skin biopsy) Gene Addition or Gene Correction De-Differentiation to Induced Pluripotent Stem cells (iPS) Differentiation to Hematopoietic Stem Cells (HSC) Autologous Transplant Gene therapy using Autologous HSC Made from Induced Pluripotent Stem Cells

64. Gene Therapy CurrentFuture ExperimentalProven Limited ScopeCurative High TechOff the Shelf

65. Gene Therapy Clinical Trials Worldwide (updated list of all gene therapy protocols) www.wiley.com/legacy/wileychi/genmed/clinical/

66. Acknowledge （ PPT 特别鸣谢！） UCLA David Geffen School of Medicine www.medsch.ucla.edu/ANGEL/ Prof. KohnDepartment of Microbiology, Immunology and Molecular Genetics (MIMG)Prof. ( ), et al.Prof. Kohn DB (Department of Microbiology, Immunology and Molecular Genetics (MIMG) ), Prof. Gasson JC (UCLA Jonsson Comprehensive Cancer Center ), et al.