#17 Virus as therapeutics Flashcards
A virus can:
gene replacement therapy
made recombinant vaccines (immunity against tumors and infections)
act as targted killers of cancer cells
transfer of new genetic material to cells of ind w/ benefical results
Gene therapy
modifies gene that may be passed onto subsequent generations
Germline gene therapy
genetic mods restricted to somatic cells, NO effect on germline
Somatic gene therapy
Two delivery options for gene therapy
ex vivo:
in viv:
cells removed from pt and exposed to vector in cell culture
-vector introduced directly into pt
Example of in vivo gene therapy:
two methos
Replace deficient factor (like giving CF gene to pts with CF)
Expression of foreing gene (would be beneficial to infect brain tumor with a virus so that when we give meds to tx virus, tx tumor)
Gene therapy trials are adressing:
lots of cancer disorders
Gene types transfered in gene therapy trials, main ones
see antigen, cytokine, tumor suppresor and suiciced gene transfer
Gene delivery agents
Viruses!!! they are great at delivery
-also see chemicals/DNA loaded vesicles/ DNA protein complexes
What viruses are being used for gene therapy trials
Adenovirus, Retrovirus, Poxvirus~ we’ve talked of these
retrovirus gene therapy history:
terminal melonma
given lymphocytes marked with ‘neo’ gene via a retrovirus vector
Ex of Ashanti with T cell deficiency
Tcells genetically corrected, then returned to her and now 20–25% of all her T cells ahve the gene the retrovirus introduced
SCID story
used retroviral gene transver to CD34+ cells
X-linked severe combined immunodeficiency
• Patients present with no T or NK lymphocytes and non- functional B cells
• If untreated, X-SCID is fatal in the first few years of life due to overwhelming susceptibility to infections
• X-SCID is due to molecular defects in the gene for the
common gamma chain (γc)
Fixing SCID
kids with no T/NK and nonfnx B cells
Replace viral genes with exogenous genes
During Gene Delivery for SCID:
Retain LTRs (_______,) psi sequence (_______) Supply deleted gene products in trans
– packaging cell lines
– helper virus, helper plasmids
Infect cells to introduce exogenous genes
integration
for packaging
Basically, you delete the gag/pol/env and put in what you need, but we keep the LTR region which includes
U3-R-U5
With our modified product from the retrovirus, we do all the same steps up to ________ and _________ but NO virions made, just the proteins of interest
circulization and integration
We start with a culture cell then transfect with:
gag, pol and env
Creation of packaging cell line
start with normal cultured cells and
transfect plasmid DNAs that will express
gag, pol, and env proteins in the cells
Creating packaging cell line:(post transfection)
genes encoding gag, pol, and env will _______ into the genome of the packaging cells and will be constitutively expressed in the packaging cells
integrate
Transfect vector plasmid with gene of interest into the packaging cell line to produce
vector particles
Vector plasmid will integrate into the genome of the packaging cells vector
RNA will be transcribed by __________
host-cell RNA polymerase
_________will be packaged into vector particles by the viral proteins that are being synthesized in the producer cells
vector RNA
Transfect vector plasmid with gene of interest into the packaging cell line to produce vector particles
…….
vector plasmid will integrate into the genome of the packaging cells vector RNA will be transcribed by host-cell RNA polymerase
vector RNA will be packaged into vector particles by the viral proteins that are being synthesized in the producer cells
The cells will die after time… but they have made
vector containing virus that can infect a target cell –> reverse transcriptase–> integrate gene
Advantages of Retrovirus vectors
– efficient delivery of foreign genes
– control over host range, cell and tissue tropism
Disadvantages of retrovirus vectors
– “random” integration into chromosomes, insertional mutagenesis
– maintaining high levels of gene expression
– dependence on target cell proliferation with MMLV-based vectors
____LTR contains strong, T cell specific enhancers
MLV
—-integrates in region
we can get over expression of U3 regions because our gene inserts a a MLV enhancer region?
Lentivirus Vectors:
preintegration complex with Vpr, integrase, and matrix protein will cross the nuclear membrane in _______
nondividing cells
preintegration complex with Vpr, integrase, and matrix protein will cross the nuclear membrane in nondividing cells
Lentivirus Vectors:
Key parts of lentivirus
Vpr, integrase and matrix proteins
Lentivirus Vectors Addressing Safety Concerns
- Multi-plasmid systems for creating packaging cells
- Self-inactivating vectors – Deletion in U3 region of right LTR
- Control of vector tropism
Lentivirus is pretty sweet bc it has this deleted in the right of the LTR
U3 region that causes ehnaced expression
has multi-plasmid systems for creating packaging cells
lentivirus
Basic Biology – linear, dsDNA genome – replicates in the nucleus, no integration
– gene transcription regulated with immediate early, early and late classes of mRNAs
Adenovirus Vectors
Adenovirus Vectors
lindear dsDNA and replicates in the ______
does it have integration?
replicates in nucleus
NO NO integration so no insertional mutagensis
What vector virus replicates in the nucleus and doesn’t integrate into host DNA
Adenovirus vector
How is gene trascription regulated with adenovirus
early, early and late classes of mRNAS
Create adenovirus vector construct
- delete certain viral genes to make room for therapeutic gene of interest
- some deleted viral genes are essential for the replication of adenovirus so they must be
provided in trans by the complementing cell line
(adenovirus vecotr contstruct)
Transfect vector DNA construct into the complementing cell line (293 cells) to produce
vector particles
Once we’ve made adenoviris vector particles, what happens
Vector particles deliver the therapeutic gene of interest into the target cells of the patient
therapeutic product is synthesized in the cells that have been infected by the adenoviral vectors but the vector DNA does not
integrate
(Adenovirus)
vector DNA enters the nucleus of the target cell but does not integrate
therapeutic product is synthesized by what?
the cells that have been infected by the adenoviral vectors
Adenovirus Vectors - Problems
Short term gene expression:
Immune response
Size restrictions on inserted genes
- -no integration of recombinant DNA into host cell genome
- limits success of repeated use of vectors and E3 region deltions will increase immune response
Solutions to Adenovirus issues
better packaging cell lines, “gutless vectors”
– better understanding of immune response to adenovirus
Vaccinia Virus Expression Vectors
• Related Biology –
large, dsDNA genome (187,000 bp) – flexibility in the size DNA that can be packaged
– cytoplasmic replication
– virus encoded enzymes
– viral promoters
Basic Strategy for Foreign Gene Expression with Vaccinia Vectors
• Plasmid Construction – foreign gene + vaccinia promoter
• Chimeric gene –>vaccinia genome (homologous recombination)
• Foreign gene –>non-essential site in vaccinia genome
– TK gene is the most popular site
TK gene is most popular because it’s non-essential
How i believe this whole recombinant plasmid shit works
have plasmid DNA that we added our foregin gene/vaccinia DNA–>
transfected a cell with this + infectious vaccinia virus –> get recombination of vaccinia sequences–>
replication of recombiant vaccina DNA with foreign gene–> maturation –> release of recombinat vaccinia virus containing the foreign gene
The Foreign gene thats recombinant of vaccinia virus is used to make vaccination with a live virus capable of:
producing foreign immunizing antiG
Chimps that got the recombinant vaccine and were then infected with HBV
had a primed immune response and did great
stable integration, long-term gene expression
potential for insertional mutagenesis replication incompetent
Retrovirus vectors:
Key point
retrovirus have________ integration and______ gene expression
stable
long term
these have potential insertional mutagensis
Retrovirus
these are replication incompetent
retrovirs
vector has no integration and short term gene expression
adenovirus
this vector sucks because we see vigours immune response to the vector
adenovirus
replication incompetent vectors
retro
adeno
pox
_____ vectors: no integration
replication competent live, attenuated vaccines
Poxvirus (vaccinia)
which vector makes live attenuated vaccines
poxvirus
Canarypox is useful for
HIV vaccine because they dont complete replication in human cells and no lysis
- Myxomavirus
- Bovine herpesvirus 4
- Reovirus
- Newcastle disease virus
- Coxsackie virus
- Vesicular stomatitis virus
- Parvovirus
Oncolytic Wild Viruses - some wild-type viruses have natural oncolytic activity in human tumors:
Genetically manipulated viruses specifically targeted to infect and kill cancer cells:
- Adenoviruses
- Herpes simplex viruses
- Vaccinia virus
adenovirus, HSV and vaccinia all are manipulated to
target, infect and kill cancer cells
Conditionally Replicative Adenovirus (CRAd)
take normal adeno
mutate it so that it prefers to grow in cancer cells
will rapidly grow and lyse cancer cells
Modifications in adenovirus that allow tumor- specific tropism:
- Delet E1A or E1B genomic regions: thus cells only replicate in cells with dysfunctions
- incorporate tumor specific promoters
- alter virus cell attachment protein so it can cause trasnduction better in tumor cells
- Delet E1A or E1B genomic regions: thus cells only replicate in cells with dysfunctions
- incorporate tumor specific promoters
- alter virus cell attachment protein so it can cause trasnduction better in tumor cell
modification made in the adenovirus for tumor tropism
Benefit of modifying fiber protein to alter cell attachment on adenovirus
or example RGD (arginine-glycine-aspartic acid) motif mediates efficient binding to integrins that are present on many tumor cells
Adenovirus wants to take over cell to use it’s DNA apparatus, so wants to be in a cell that is dividing a lot… what kind of cell would that be?
cell with p53 mutation! aka, cancer cell bc these cells are constituitvely active and adenovirus needs to use that machinary..
- Human telomerase reverse transcriptase (hTERT) promoter
* Highly active in
tumor cells like glioma, inactive in normal brain cells
We create an adenovirus that has it’s E1A and E1B replicated and activated by ________ so that it goes to places with lots of it
hTERT
Reovirus (wild-type strain) no modifications by recombinant DNA
• Inherent tumor selectivity for cells with an activated______ activity
Ras
• In normal cells reovirus replication is restricted by the activation of the
RNA-activated protein kinase (PKR)
—tumor cells dont have this!!! thus reovirus is like, fuck yeah, i’m partying here
