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

4. Dividing cells (pseudo- typed) Insertional mutagenesis
Virus
Insert size
Transduction
efficiency
Integration
Target
cells
Problems
Retrovirus
~ 8 kb
High
Yes
Dividing cells (pseudo- typed)
Insertional mutagenesis
Adeno-virus
~ 30 kb No
Cells w/ receptor
Immunity
AAV
~ 4kb
Random or site sp.
Small insert size
HSV
~ 40 kb
Low
Neurons
Latency/ immunity
Vaccinia
~ 25 kb

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