Gene Therapy for Treatment of Genetic Diseases

1. 基因治疗 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: C303, Teaching Building 2014/09

2. Learning Objectives 1. Traditional managements（传统的治疗方法） 2. Gene therapy（基因治疗）

3. 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 （青霉胺）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 • i.e. 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. Gene Delivery Can Be: I. Ex vivo（离体）– gene into isolated cells II. In vivo（体内）– 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

12. 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

13. General considerations for the use of gene therapy • 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

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

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

18. General considerations for the use of gene therapy • 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

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

20. General considerations for the use of gene therapy • 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!

21. Minimal requirements that must be met（基因治疗试验的最低要求）: • Identification of the affected gene • A cDNA clone encoding the gene

22. Minimal requirements that must be met （基因治疗试验的最低要求）: • 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

23. Minimal requirements that must be met（基因治疗试验的最低要求）: • 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

24. Minimal requirements that must be met（基因治疗试验的最低要求）: • 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.

25. 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.

26. 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)

27. 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

28. 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?

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

32. In vivo and ex vivo gene therapy

33. 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

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

35. Types of viral vectors • stable/transientinfect non-dividing cells • Retrovirus stable no • Lenti-virus stable yes • Adenovirus transient yes • Adeno-Associated virus ? yes • Herpes virus transient yes

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

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

38. 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.

39. Somatic Therapy for SCID Ex vivo 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.

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

41. Father of GT: Anderson WF, 1990

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

43. Risks of Gene Therapy • 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.

44. 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

45. 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

46. RNAi

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

48. Gene Therapy CurrentFuture Experimental Proven Limited Scope Curative High Tech Off the Shelf

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