Genetic Manipulation of Viruses Flashcards
What is viral gene therapy principally aimed at
Treating human disease with genetically altered viruses
3 main areas:
- Inherited disease
- Cancer
- Vaccines
What are the candidate viruses that have been examined for utility in viral gene therapy
- Retroviruses
- Adenoviruses
- AAV
- Herpesviruses
What is the criteria of the perfect vector
1) Be safe and not replicate in the host
2) Will have no damaging side effects in the host
3) Have near perfect cell targeting
4) Have ideal gene expression levels
5) Persist only for as long as required
What is the transcription cascade for Adenoviruses
1) E1 protein is on as well as the Major Late promoter but not full length
2) E1 proteins transactivates E4 and E2
3) E1 and E4 proteins upregulates E2 (replication proteins)
4) DNA replication ensues, Major Late Promoter is massively upregulated and proceeds to end of genome
What are 293 cells
- Human cells containing the E1 region of human adenovirus Type 5
- Provide the E1 products in trans, allowing E1-deleted adenoviruses to grow
Why are 293 cells important for adenovirus research
- They support the growth of E1-deleted adenovirus
- Most widely used cells for adenovirus-based experiments
What are first-generation adenovirus vectors
- Adenoviruses that lack the E1 region
- Typically also lack the E3 region, which helps viruses evade the immune system (not needed in the cell culture)
What are second generation adenovirus vectors
- Have additional adenovrial genes (E2 and E4) inserted into the 293 cell genome
- Improve vector stability and gene expression
What are the features of this 293 cell system
It efficiently generates recombinant viruses
It is able to place about 8Kbp of foreign DNA into first generation vectors and slightly more in second generation vectors
What is a disadvantage to this 293 cell system
There is a low level expression of the other viral genes in normal cells
What are ITRs
Inverted Terminal Repeats - the very ends of the viral genome
This can be linked head to head in a plasmid and this arrangement will re-generate the linear genome in the presence of adenovirus replication proteins.
What is the packaging signal
This is a short sequence of DNA about 200 bp from the left hand end of the viral genome
Packaging signal is 200bp and acts in cis
Any linear DNA containing a packaging signal near either end of the linear molecule will be packaged into the virus particle.
How to make a plasmid for making adenoviruses
1) E3 and E1 is deleted from the plasmid but the E1 proteins are supplied by the 293 cells.
2) This would then activate E4 which would consequently activate E2
3) The replication proteins then convert DNA into a linear genome
What is the Adeno-X Adenoviral System 3
It is the most advanced commercially available adenoviral gene delivery system
Provides the simplest, fastest and more efficient method for constructing recombinant adenoviral vectors
Why is there an immune response to recombinant adenoviral vectors
While the vectors cannot replicate, they can still express viral proteins, making it a target for the immune system
Everybody has been exposed to the adenovirus and therefore there is an anti-adenovirus immune response
This is because some sequences present in the E1 deleted adenoviruses are present in the 293 cells.
What are Replication Competent Adenoviruses (RCA)
A double recombination even can lead to the virus regaining the E1 region and becoming the wild type
Several cell lines have been developed to side step this problem but they are usually heavily commercially protected
What are third generation Helper dependent ‘gutless’ vectors
These vectors only contain the ITRs and a packaging signal to prevent other viral genes from being made
Allows it to be filled with 36,000bp of any DNA desired
What is a ‘Helper virus’
The helper virus genome grows alongside the gutless vectors and provides all the virus proteins that are needed.
The packaging of the helper genome is selected against to favour packaging of the gutless genome
It is grown in normal 293 cells in bulk
What do these third generation vectors rely on
Cre recombinase (engineered into helper virus on either side of its packaging signal) , which targets IoxP sequences
Cre recombinase removes the packaging signal from the helper virus, preventing it from being packaged into a new viral particles
What are the advantages of helper virus
While the virus would contaminate the vector preparation but this is less than 0.1%
It also has E1 and E3 deleted and therefore is safe to use as traditional vectors
What are the drawbacks to this system
It is more demanding than the 1st and 2nd generation vectors and requires a constant supply of helper virus.
What is another approach to this disadvantage
Provide the helper functions on a plasmid and just keep transfecting plasmid with virus
What are the drawbacks to adenovirus vectors
1) It is not easy to alter the tropism of the virus fibre
2) There are still issues around long term expression
3) The virus genome could be mobilised by infection with another adenovirus
4) Even 35Kbp of space may not be enough
5) The immune response is still a problem but gutless is a help
What issues arise when growing Chimpanzee adenovirus Y25 in 293 cells and how is this resolved
Human E1 proteins (from 293) do not interact properly with chimpanzee E4 proteins, leading to inefficient virus production
The chimpanzee E4 region is replaced with the human E4 region
Why does adding an intron before the ORF increase protein expression
It enhances mRNA processing, stability, and nuclear export, leading to much higher protein levels
Why can adding an intron be problematic in some cases
- Protein expression can become too high, to the point of being toxic to the cell (e.g., SARS-CoV-2 spike protein)
How does the Tet repressor system control toxic over-expression
Tet repressor binding sites are added to the CMV promoter
The virus is grown in 293TREX cells, which express Tet-repressor proteins which binds to and represses S glycoprotein expression but it is not present in normal human cells.
This allows for the benefit of increased expression only in target cells
