Retroviral Gene Therapy

1. Retroviral Gene Therapy

2. What is Gene Therapy?

3. Gene Therapy Strategies

4. Minimal Requirements for Retrovectors

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

6. Gene Delivery Using Retroviral Vectors Poly A site + 1  IRES CMV R U5 TRANSGENE GFP U3 R 5’ LTR 3’ LTR INTEGRATION  GAG POL ENV TRANSCRIPTION R U5  IRES U3 R TRANSGENE AAAAA GFP             AA AA AA AA AA AA AA AA AA AA AA AA Vector AP2 Transfection Packaging Cell line (provides gag, pol, env)

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 RNA vector genome U3 R U5 U3 R U5 Reverse transcription LTR LTR Integration of provirus       AA AA AA AA AA AA B16 melanoma cells Transduction 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