基因治疗

1. 基因治疗 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: A705, Research Building 2013/09

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

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 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. 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

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 • example: 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’s

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 • example: cancer • state of research: • clinicaltrials.govwebsite currently lists 35624 • gene therapy trials for cancer; 10649 are open to • enrollment

21. 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!

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

23. 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

24. 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

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 • 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.

26. 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.

27. 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)

28. 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

29. In Vivo Gene Transfer By AAV Vector

30. 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?

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

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

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 vectors targets both dividing and non-dividing cells? A. Retrovirus B. Adenovirus C. Adeno-associated virus D. Herpes virus 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. 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

39. In vivo and ex vivo gene therapy

40. 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

41. 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

42. 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

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

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

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

46. 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.

47. 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.

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

49. Father of GT: Anderson WF, 1990

50. Geneticist guilty of molestation, 2006