Viruses%20and%20Gene%20Therapy

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Viruses and Gene Therapy

• The good news about viruses

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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

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Useful vectors

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

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Virus Insert size Transduction efficiency Integration Target cells 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
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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)

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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

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• 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?

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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

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• 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)

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Tumor destruction by adenovirus

• E1B region binds and inactivates p53 protein
• mutant adenovirus (dl1520) that cannot produce
  E1B wont reproduce
• tumor cells lacking functional p53 can support
  replication of this virus

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• C33A lacks p53
• U2OS has p53
• Circles WT
• Squares mutant

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Which side has the Wt? The mutant?
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Tumors in mice after treatment with Wt or mutant
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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

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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

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

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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

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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

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