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Gene Therapy (IV) “Strategies and Applications” Dr. Aws Alshamsan Department of Pharmaceutics Office: AA87 Tel: 4677363 [email protected] 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 Gene Therapy Strategies •Replacement of a missing or defective gene •Introduction of gene(s) to influence cellular process •Interference with gene products Replacement strategy Applies to diseases caused by single gene defects Transfer of a functional copy of the defective or missing gene Examples: enzyme deficiencies 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 longterm expression Replacement strategy Gene with defect Adenosine deaminase (ADA) Disease/Disorder SCID a-1-antitrypsin Emphysema CF transmembrane regulator Cystic fibrosis Clotting factor VIII Hemophilia A Clotting factor IX Hemophilia B b-chain of hemoglobin Sickle cell anemia Bubble Boy David Phillip Vetter (September 21, 1971 – February 22, 1984) 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 Why was ADA suitable? Single gene defect Gene was isolated and cloned in 1983 HSC are easy to obtain and maintain in vitro 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 Areas of investigation Enhancement of anti-tumor response Introduction of drug-resistance genes Introduction of drug-sensitivity genes Replacement of tumor suppressor genes Introduction of drugsensitivity 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 Interference strategy Downregulation of gene expression at the mRNA level Inhibition of mRNA translation 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) DNAzyme Ribozyme Antisense ODN • Sequence-selective oligonucleotide that can bind to a target mRNA to inhibit gene expression i.e. to inhibit translation Antisense ODN Antisense ODN Antisense ODN mRNA RNase H Disadvantages of Conventional Therapy • Requires screening of thousands of compounds to find an active molecule. • Lacks specificity of action. 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 Designing Biologically Stable ODNs Designing Biologically Stable ODNs Fomivirsen Sodium (Vitravene)® • FDA-approved for the local treatment of CMV retinitis in AIDS patients Fomivirsen Sodium (Vitravene)® • Dose 150-330 μg intravitreal injection • Every other week for 2 doses • Cleared locally by exonucleases 1-2 hr after injection Antisense RNA What is RNAi? • Post-transcriptional phenomenon that was initially discovered in plants • Mediated by double-stranded RNA siRNA Antisense ODN v.s. siRNA Antisense ODN Nucleotide sugar Structure siRNA Deoxyribose Single stranded Ribose Double stranded Length 16-30 bp 19-21 bp Molecular weight ~ 6-9 kDa ~ 13-14 kDa Precursor vailability Site of action mRNA cleavage Degradation upon activity Effective concentration No Cytoplasm / Nucleus RNase H Yes 50-400 nM Yes Cytoplasm RISC No 5-100 nM shRNA Plasmid DNA miRNA miRNA siRNA v.s. miRNA siRNA v.s. miRNA 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 Next Lecture Protein pharmaceuticals