Viruses and Gene Therapy

1. Viruses and Gene Therapy The good news about viruses

2. Quality of viral vector • Size of insert • Integration or not/and where • Ability to obtain in high titer • Transduction efficiency • Target cell specificity - pseudotyping • Expression efficiency/control • Possible pathogenicity or lack • No immune response developed

3. Useful vectors • Retroviruses including HIV • Adenovirus • Adeno-associated virus (AAV) • Herpes simplex virus • Vaccinia virus • DNA vectors

5. Retroviruses • Require LTR and packaging signal • LTR modified not to be a promotor • Cloned gene inserted with strong promotor (pCMV) and may have IRES • HIV vectors may have some accessory genes if LTR promotor is used (TAT)

6. Created using a packaging cell line • Genes for gag/pol/env on separate plasmid to get packaging • May have two or three plasmids for trans genes to reduce recombination

8. Advantages: Stable integration Can design to target HIV infected cells with suicide gene Can add other promoters that respond only in specific cells Disadvantages: No replication and spread of vector Integration may be random Requires dividing cell to integrate and express What would happen to a retroviral vector with pCMV and HSV-TK injected into brain tumor and treatment with ganciclovir? No affect on neurons so can treat glioma TK activates ganciclovir and kills cells What would happen if used vector to add WT p53 gene to lung cancer tumor?

10. Adenovirus vectors • Non-enveloped so no pseudotyping • Requires elimination of early gene (E1 or E3) and other nonessential genes and becomes defective • Packaging cell line has E gene integrated and expressed (less likely crossover) • “Gutless” vectors have only the inverted terminal repeats (ITR) and a packaging signal and get all other gene products in trans in packaging cell

11. Vector-infected cells are removed by immune system so transient response but that is reduced in gutless vector • May not work at all if host starts with some Ad immunity • Infects wide range of cells (common receptors)

12. Tumor destruction by adenovirus • E1B region binds and inactivates p53 protein • mutant adenovirus (dl1520) that cannot produce E1B won’t reproduce • tumor cells lacking functional p53 can support replication of this virus

13. C33A lacks p53 • U2OS has p53 • Circles = WT • Squares = mutant

14. Which side has the Wt? The mutant?

15. Tumors in mice after treatment with Wt or mutant

16. Adenovirus carries lots of DNA • Put human hepatitis B genome into ad cloning vector (removed E1 and E3 genes) • Hep genome also had GFP linked to pCMV as marker to recognize transduced cells • Used to create HepB cell line in mouse cells that produces infectious viruses from nonintegrated DNA • Infected mouse as well - small animal model • Crossed species barrier

17. AAV - parvovirus • Requires adenovirus early genes to replicate - not related • ssDNA with promoter and two genes - capsid and replication protein • Terminal repeats allow for specific integration into genome if helper is not there • Vector has TR and Promoter - no rep and capsid; insert size is small • W/o rep integration is at random

18. Correcting hemophilia in dogs - AAV vector with factor IX • Tumor reduction - AAV vector with angiostatin

19. HSV vectors come in three varieties • Recombinant virus made replication deficient w/o IE gene • Replication conditional replicate in certain cell lines • Amplicon - bacterial plasmid-based • Neurotropic but problems with immunity • Large virus with many nonessential genes - can add 150KB

20. HSV amplicons • Plasmid contains • HSV ORI • HSV packaging signal • IE promoter and gene of interest • Selection marker • Virus made in cell that provides all other proteins in trans