Viruses and Gene Therapy The good news about viruses.
-
date post
19-Dec-2015 -
Category
Documents
-
view
221 -
download
3
Transcript of Viruses and Gene Therapy The good news about viruses.
![Page 1: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/1.jpg)
Viruses and Gene TherapyViruses and Gene Therapy
The good news about viruses
![Page 2: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/2.jpg)
Quality of viral vectorQuality 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
![Page 3: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/3.jpg)
Useful vectorsUseful vectors
• Retroviruses including HIV
• Adenovirus
• Adeno-associated virus (AAV)
• Herpes simplex virus
• Vaccinia virus
• DNA vectors
![Page 4: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/4.jpg)
Virus Insert size
Transductionefficiency
Integration Targetcells
Problems
Retrovirus ~ 8 kb High Yes Dividing cells (pseudo- typed)
Insertional mutagenesis
Adeno-virus
~ 30 kb
High No Cells w/ receptor
Immunity
AAV ~ 4kb High Random or site sp.
Cells w/ receptor
Small insert size
HSV ~ 40 kb
Low No Neurons Latency/ immunity
Vaccinia ~ 25 kb
High No Cells w/ receptor
Immunity
![Page 5: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/5.jpg)
RetrovirusesRetroviruses
• 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)
![Page 6: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/6.jpg)
Created using a packaging cell lineCreated 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
![Page 7: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/7.jpg)
![Page 8: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/8.jpg)
• 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?
![Page 9: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/9.jpg)
![Page 10: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/10.jpg)
Adenovirus vectorsAdenovirus 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
![Page 11: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/11.jpg)
• 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)
![Page 12: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/12.jpg)
Tumor destruction by adenovirusTumor 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
![Page 13: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/13.jpg)
• C33A lacks p53
• U2OS has p53
• Circles = WT
• Squares = mutant
![Page 14: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/14.jpg)
Which side has the Wt? The mutant?Which side has the Wt? The mutant?
![Page 15: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/15.jpg)
Tumors in mice after treatment with Wt or Tumors in mice after treatment with Wt or mutantmutant
![Page 16: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/16.jpg)
Adenovirus carries lots of DNAAdenovirus 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
![Page 17: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/17.jpg)
AAV - parvovirusAAV - 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
![Page 18: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/18.jpg)
• Correcting hemophilia in dogs - AAV vector with factor IX
• Tumor reduction - AAV vector with angiostatin
![Page 19: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/19.jpg)
HSV vectors come in three varietiesHSV 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
![Page 20: Viruses and Gene Therapy The good news about viruses.](https://reader031.fdocuments.net/reader031/viewer/2022032704/56649d405503460f94a19cf3/html5/thumbnails/20.jpg)
HSV ampliconsHSV 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