Download Oncogenic Viruses

Viruses and Gene Therapy
The good news about viruses
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
Useful vectors
•
•
•
•
•
•
Retroviruses including HIV
Adenovirus
Adeno-associated virus (AAV)
Herpes simplex virus
Vaccinia virus
DNA vectors
Transduction
efficiency
Integration
Target
cells
Problems
Retrovirus ~ 8 kb
High
Yes
Dividing
cells
(pseudotyped)
Insertional
mutagenesis
Adenovirus
~ 30
kb
High
No
Cells w/
receptor
Immunity
AAV
~ 4kb
High
Random or Cells w/
site sp.
receptor
Small insert
size
HSV
~ 40
kb
Low
No
Neurons
Latency/
immunity
Vaccinia
~ 25
kb
High
No
Cells w/
receptor
Immunity
Virus
Insert
size
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)
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
• 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?
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
• 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)
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
•
•
•
•
C33A lacks p53
U2OS has p53
Circles = WT
Squares = mutant
Which side has the Wt? The mutant?
Tumors in mice after treatment with Wt or
mutant
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
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
• Correcting hemophilia in dogs AAV vector with factor IX
• Tumor reduction - AAV vector
with angiostatin
HSV vectors come in three varieties
• Recombinant virus made
replication deficient w/o IE
gene
• Replication conditional
replicate in certain cell lines
• Amplicon - bacterial plasmidbased
• Neurotropic but problems with
immunity
• Large virus with many
nonessential genes - can add
150KB
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