1. Gene Therapy Dr. Aws Alshamsan & Dr. Nermin Hassan Department of Pharmaceutics aalshamsan@ksu.edu.sa nhassan@ksu.edu.sa

2. Objectives of this lecture By the end of this lecture you will be able to: 1.Define the term “Gene Therapy” 2.Use the correct terminology for gene transfer 3.Realize the significance of gene therapy research

3. Objectives of this lecture By the end of this lecture you will be able to: 1.Describe the different strategies for gene therapy 2.Select the suitable strategy based on the clinical case 3.Understand the complexity of clinical application of gene therapy 4.Evaluate proposed strategies according to the therapeutic need

4. What is gene therapy?

5. Introduction of new genetic material into a cell for therapeutic purposes Genetic Materials: DNA RNA

6. ProblemExampleSolution Defect in protein expression G6PD deficiency Introduce a correct version of the gene Expression of harmful proteins Cancer Inhibit or block the harmful proteins

7. Advantages of gene therapy Specificity Can be either temporary or permanent Localization Low immunogenicity

8. Gene Therapy Germ-Line Gene Therapy: Introduction of genes into germ cells (sperm and ovum) Somatic Gene Therapy: Introduction of genes into somatic cells

9. Germ-Line Gene Therapy Hypothetically, germ-line therapy prevents transfer of defective genes to subsequent generation Due to ethical and safety reasons, it is not presently accepted for human application However, it is used for the transgenic laboratory animals production

10. Gene therapy In vivoEx vivo

11. AdvantageDisadvantage SpecificityTime and labor consuming High transfection efficiencyInvasive Contamination Not every cell type is growable

12. Ex vivo gene therapy Most widely used in clinical trials Some attempts in practice

13. AdvantageDisadvantage Low costNone specificity None invasive Low transfection efficiency Less contamination

14. Ballistic DNA Injection (gene guns) Invented for DNA transfer to plant cells Fully applicable to eukaryotic cells plasmid DNA shown here

15. Liposomes Next level idea – why naked DNA? Lets’ wrap it in something safe to increase transfection rate Therapeutic drugs Lipids – are an obvious idea !

16. DNA delivery of genes by liposomes Cheaper than viruses No immune response Especially good for in-lung delivery (cystic fibrosis) 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector

17. In vivo gene therapy Some organs are less suited for ex vivo e.g. (brain, heart and lungs) More practical approach

18. Gene Therapy Considerations What gene will be delivered or targeted? What method will be used? Genetic material either for gene expression or downregulation Vector to carry the genetic material inside the cells Elements for gene transfer

19. Expression Vector (Plasmid)

22. Gene Transfer Transformation: Transformation: introduction of genetic materials into bacteria Transfection: Transfection: introduction of genetic materials into eukaryotic cells (e.g. fungi, plant, or animal cells) Transduction: Transduction: introduction of genetic materials using viruses Lipofection: Lipofection: introduction of genetic materials using liposomes

23. Stable vs. Transient Gene Transfer Stable Gene Transfer: Stable Gene Transfer: achieved by plasmid integration in the host genome or episomal replication of the transferred plasmid. Transient Gene Transfer: Transient Gene Transfer: the foreign DNA is usually not integrated into the nuclear genome and will be degraded or diluted through mitosis

24. Gene Therapy Strategies Replacement of a missing or defective gene Introduction of gene(s) to influence cellular process Interference with gene products

25. Replacement strategy Applies to diseases caused by single gene defects Transfer of a functional copy of the defective or missing gene Examples: enzyme deficiencies

26. Replacement strategy To apply this strategy, three requirements must be met: 1.The specific gene defect must be known 2.A functional copy of the gene must be available 3.Target cells must be available and amenable to transfection methods resulting in long- term expression

27. Replacement strategy Gene with defectDisease/Disorder Adenosine deaminase (ADA)SCID  -1-antitrypsin Emphysema CF transmembrane regulatorCystic fibrosis Clotting factor VIIIHemophilia A Clotting factor IXHemophilia B  -chain of hemoglobin Sickle cell anemia

28. Mucus in bronchi is thick, interfering with lung function 1 in 25 are carriers One of the first disorders to be actively studied for gene therapy. Most lethal autosomal recessive disorder in U.S. Cystic Fibrosis

29. Based on: Harvard Family Health Guide, 1999 Sickle Cell Disease- recessive allele Red blood cells are sickle shaped, issues with circulation causing anemia and pain

30. Hemophilia A disorder in which a person’s blood does not clot properly. It is a recessive sex-linked, X- chromosome disorder. 1 in 10,000 males born are afflicted. “Royalty Disease”

31. ADA-deficient persons are affected by severe immunodeficiency, with recurrent infections that might be life-threatening. First disease approved for gene therapy. Autosomal recessive disorder. The drug exists but is very expensive, needs to be injected in vein for life. Adenoside Deaminase (ADA) Deficiency Ashanti Disilva

33. Bubble Boy David Phillip Vetter (September 21, 1971 – February 22, 1984)

34. Gene therapy trial First successful clinical trial in gene therapy was initiated in September 14, 1990 Hematopoietic stem cells were isolated from the patient (4 y/o girl) and transduced with retroviral vector containing ADA gene 25% recovery of normal ADA in patient T cells

35. Why was ADA suitable? Single gene defect Gene was isolated and cloned in 1983 HSC are easy to obtain and maintain in vitro

36. Influence strategy Applies to complex disorders were more than one gene is involved Based on in vitro cloning of human genes that were derived from human tissue Examples: cancer

37. Areas of investigation Enhancement of anti-tumor response Introduction of drug-resistance genes Introduction of drug-sensitivity genes Replacement of tumor suppressor genes

38. Introduction of drug- sensitivity genes Suicide gene therapy Gene that converts non-toxic prodrug into a toxic metabolite Bystander effect Gancyclovir triphosphate Problem: it can transfect normal cells too

39. Gene Therapy Strategies Replacement of a missing or defective gene Introduction of gene(s) to influence cellular process Interference with gene products

40. Interference strategy Downregulation of gene expression at the mRNA level Inhibition of mRNA translation

41. Interference nucleic acids DNA Antisense oligodeoxynucleotide (ODN) DNAzyme RNA Antisense RNA Ribozyme Small interfering RNA (siRNA) Short hairpin RNA (shRNA) microRNA (miRNA) RNA interference (RNAi)

42. DNAzyme

43. DNAzyme binds target mRNA mRNA cleavage and degradation DNAzyme AAAAAA….A Target mRNA protein X © American Society for Investigative Pathology

44. Ribozyme

45. Antisense ODN Sequence-selective oligonucleotide that can bind to a target mRNA to inhibit gene expression i.e. to inhibit translation

46. Antisense ODN

47. mRNA RNase H

48. We don’t have antisense ODN for every disease The main barrier to antisense strategy is optimal delivery in sufficient quantities to the correct target and for the desired time frame to achieve the desired level of gene inhibition ODNs are polyanionic macromolecule (large and charge) Stability issues in vivo

49. Designing Biologically Stable ODNs

52. Fomivirsen Sodium (Vitravene)® FDA-approved for the local treatment of CMV retinitis in AIDS patients

53. Fomivirsen Sodium (Vitravene)® Dose 150-330 μg intravitreal injection Every other week for 2 doses Cleared locally by exonucleases 1-2 hr after injection

54. Antisense RNA

55. What is RNAi? Post-transcriptional phenomenon that was initially discovered in plants double-stranded RNA Mediated by double-stranded RNA

56. siRNA RISC siRNA is bound by RISC and unwound by RNA helicase RISC Sense RNA strand degraded © American Society for Investigative Pathology

57. RISC AAAAAA….A Target mRNA mRNA cleavage and degradation RISC-bound antisense strand directed to target mRNA AAAAAA….A Target mRNA RISC © American Society for Investigative Pathology

58. siRNA www.nature.com/focus/rnai/ animations/rnai_revised_320 x180.mov

59. Antisense ODN v.s. siRNA Antisense ODNsiRNA Nucleotide sugarDeoxyriboseRibose StructureSingle strandedDouble stranded Length16-30 bp19-21 bp Molecular weight~ 6-9 kDa~ 13-14 kDa Precursor vailabilityNoYes Site of actionCytoplasm / NucleusCytoplasm mRNA cleavageRNase HRISC Degradation upon activityYesNo Effective concentration50-400 nM5-100 nM

60. shRNA Plasmid DNA

61. miRNA

62. cytoplasmic processing Dicer (RNase III) pre-miRNA miRNA Interact with target mRNA RISC ribosome mRNA A(n) RISC RNA-Induced Silencing Complex © American Society for Investigative Pathology

63. miRNA RNA-induced silencing complex

64. General mechanisms of siRNA and miRNA

65. siRNA v.s. miRNA

67. Production and Regulation Strict adherence to GLP and GMP principles Effectiveness in an appropriate animal model Safety in rodents and larger animals such as monkeys Clinical Trials Good Laboratory Practices Good Manufacturing Processes

68. Gene Therapy Successes approved by the FDA for sale, some diseases have been experimentally successful: Melanoma (skin cancer) Severe Combined Immunodeficiencies Hereditary Blindness Sickle Cell Anemia

69. 2006: Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients. This study constitutes one of the first demonstrations that gene therapy can be effective in treating cancer. 2007- 2011: Research is still ongoing and the number of diseases that has been treated successfully by gene therapy increases. Retinal disease Colour blindness Adrenoleukodystrophy 2011: Medical community accepted that it can cure HIV as in 2008, Gero Hutter has cured a man from HIV using gene therapy

70. Gene Therapy Disappointments In 1999 a boy died due to an immune response to an adenovirus gene therapy vector. Four children have developed cancer due to a retrovirus gene therapy vector

71. You are now able to: Define the term “Gene Therapy” Use the correct terminology for gene transfer Realize the significance of gene therapy research

72. Now you are able to: Describe the different strategies for gene therapy Select the suitable strategy based on the clinical case Understand the complexity of clinical application of gene therapy Evaluate proposed strategies according to the therapeutic need

73. http://www.wellesley.edu/Biology/Courses/219/Gen_news/i3_Gene_Therapy.jpg