1. Retroviral Gene Therapy

2. What is Gene Therapy?

3. Gene Therapy Strategies

4. Minimal Requirements for Retrovectors

5. target gene AP2 GFP IRES CMV LTR 293 GPG target cells Transduction of target cells Cell sorting or drug selection Re-infusion/inoculation Screen for biological effect Transfection of packaging cells - expresses gag, pol, env Production of virus vector stock Cloning of the target gene into retroviral vector Retroviral Gene Therapy

6. Packaging Cell line (provides gag, pol, env) Transfection Vector AP2 TRANSCRIPTION R U5U3 R AAAAA TRANSGENE IRES GFP   INTEGRATION GAG POL ENV  AA            5’ LTR 3’ LTR CMV GFP IRES TRANSGENE R U5U3 R  Poly A site + 1 Gene Delivery Using Retroviral Vectors

7. Retroviral Pseudotyping Substitution of the retroviral ENV glycoprotein with a different ENV, encoding a viral receptor with a broad host range eg. VSV G protein

8. Gene Delivery Using Retroviral Vector B16 melanoma cells RNA vector genome  AA      Transduction Reverse transcription LTR U3 R U5 Integration of provirus Retroviral Pseudotyping Substitution of the retroviral ENV glycoprotein with a different ENV, encoding a viral receptor with a broad host range e.g.. VSV G protein

9. Gene Therapy Strategies 1. Apoptosis-related gene 2. p53 tumor suppressor 3. dominant negative protein 4. immunomodulatory protein (cytokine, adhesion, MHC) 5. toxin eg. HSV Thymidine Kinase gene TK + gancyclovir >>> gancyclovir-phosphate toxic to HSV viral replication and induces cell death in viral TK expressing cells TK + gancyclovir strategy effective in gene therapy of brain cancer where only the tumor cells are dividing