1. Gene Therapy (IV) “Strategies and Applications” Dr. Aws Alshamsan Department of Pharmaceutics Office: AA87 Tel: 4677363 aalshamsan@ksu.edu.sa

2. 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

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

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

5. 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

6. 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

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

8. Gene therapy trial First 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

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

10. 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

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

12. 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

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

14. 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)

15. DNAzyme

16. Ribozyme

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

18. Antisense ODN

19. mRNA RNase H

20. Disadvantages of Conventional Therapy Requires screening of thousands of compounds to find an active molecule. Lacks specificity of action.

21. 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

22. Designing Biologically Stable ODNs

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

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

27. Antisense RNA

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

29. siRNA

30. 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

31. shRNA Plasmid DNA

32. miRNA

33. miRNA

34. siRNA v.s. miRNA

36. 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

37. Next Lecture Protein pharmaceuticals