6. Viral Vectors Flashcards
What does gene therapy require?
An understanding of molecular and cellular virology as viruses are used to deliver the genes.
What can gene therapy be used to do?
- Replace genes
- To add new genes to a cell like loss of function or vaccines
What needs to be understood about viruses to use them as a vector in gene therapy?
- How does the virus enter the cell
- How does the virus modify the cell
- What does the virus do
What is the use of viral gene therpy?
- It is used to treat human diseases with genetically altered viruses.
- This is used to treat inherited diseases, cancer, and in vaccines.
How long has viral gene therapy been used to make vaccines?
- They have been used for a long time.
- Genetically modified adenovirus based vaccine have been used since the 90s.
What kinds of viruses can be used as viral vectors?
- Viruses that do something or make something useful for us.
- By now, most viruses have been tested to see if they can produce something for us.
What are the most common viruses used as viral vectors?
- Retroviruses
- Adenoviruses
- Adenovirus associated viruses
- Herpesviruses
What are adenovirus associated viruses?
- They only replicate in cells that are already infected by an adenovirus.
- They rely on the adenovirus to provide certain functions.
- They don’t really have a disease burden.
- If they get in a cell without an adenovirus they insert their genome into the host cell’s.
- Over 50% of the time they insert it at a specific location on Chr12.
- This is a key reason they are being looked at for gene therapy.
Why are herpesviruses good candidates as viral vectors?
- They are very good at infecting neuronal cells which most other viruses don’t.
- This creates some specificity.
Why are retroviruses good candidates as viral vectors?
They permanently insert their genes into the host genome.
What is important to remember about different viral gene delivery systems?
- Each viral system has its advantages and its drawbacks.
- Every virus group overcomes problems of different viruses but also brings its own problems.
- Each viral system will find its treatment niche.
What characteristics would the perfect viral vector have?
- It will be safe and not replicate in the host.
- It will have no damaging side effects in the host.
- It will have near perfect cell targeting.
- It has ideal gene expression levels.
- It persists only for as long as required.
(This currently doesn’t exist)
Why are gene expression levels from viral vectors important?
- You need to have correct gene expression levels for the host and for the function.
- For vaccines ideal expression is lots of gene expression.
- You need enough but not too much gene expression.
Why do adenoviruses make good viral vectors?
- They don’t cause serious infections.
- They are easily genetically modifiable.
- There is lots of evidence and research into this vector.
What infections do adenoviruses cause?
- Most common colds
- Some gastro problems
- Can cause pink eye
What is the structure of adenoviruses?
- It’s an icosahedral structure made up of hexons which have a fibre for binding to cells.
- It is non-enveloped.
- Cement proteins to stabilise the structure of the virus particle.
- Core proteins like TP, Mu, pV and pVII
- TP is the terminal protein which is covalently attached to the ends of the DNA.
- Mu, pV and pVII wrap up the genome and act a bit like histones.
- It has a dsDNA genome.
What happens when the adenovirus genome enters the nucleus?
It activates a transcription cascade.
What is the adenovirus transcription cascade?
- The E1 promoter and the major late promoter (MLP) are switched on.
- the MLP is weakly switched on so the RNA pol falls off after a few 1000 nt.
- The E1 promoter makes the E1 or early proteins which turn on the E2 promoter.
- The E2 promoter activates the transcription of mostly E4 and some E2.
- The E4 transcript makes the E4 proteins which, with E1 proteins, up regulate the promoter for the E2 transcript.
- The E2 transcript is very long and encodes the replication proteins.
- This activates viral replication which massively up regulates the MLP and RNA pol produces the whole transcript.
- the MLP transcript encodes most of the viral proteins and is subject to a complex array of splicing and polyadenylation events to create the late proteins.
- The late proteins are mostly structural proteins.
What happens if you inhibit any stage in the adenovirus transcription cascade?
It stops transcription and stops the viral replication cycle.
What are 293 cells?
- A human cell line that contains the adenovirus E1 region.
- This means that you can grow adenoviruses in these cells that don’t have the E1 region
- This cell line was created in the 70s.
- They grow very quickly as the E1 proteins dysregulate the cell cycle.
- They work well in the lab.
Why is creating cell lines containing the adenovirus E1 region useful?
- It means you can delete the E1 region from the adenovirus and insert a gene of interest in its place.
- When these viral vectors are inserted into 293 cells they replicate and carry the gene of interest.
- When these viral vectors are inserted into normal human cells, the genetic information is delivered and nothing else.
What are first-generation adenovirus vectors?
Adenovirus vectors with the E1 region and usually the E3 region removed.
What does the adenovirus E3 region do?
- It generally messes with the human immune system.
- It interferes with MHC production and T cell and other immune signalling.
- This region can be deleted as well and put into a cell line
- This means you can insert other genes of interest.
What is the principle of genetically modified adenovirus vectors?
- Take bits out of the viral genome
- Permanently integrate these genes into a cell line.
- You can then insert genes of interest into the space you have created by removing viral genes.
- These viruses can grow fine in the cell lines but cannot grow in human cells.
What are the main features of these adenovirus vectors?
- There is fairly efficient generation of recombinant viruses.
- You can place around 8Kbp of foreign DNA in 1st generation vectors and a bit more in 2nd generation vectors.
What are the downsides of the adenovirus vector systems?
- There is a limit to how much genetic material you can fit in a virus.
- The more genes you delete the more space you have for other genes.
- Some viral proteins are still produced in normal human cells. It is not enough to kill a human cell but it can still trigger the immune system.
What are ITRs?
- Inverted terminal repeats
- They are at the very end of viral genomes.
- They have an identical DNA sequence just inverted.
How can ITRs turn the viral genome into a plasmid?
- ITRs are identical so have homology.
- They can randomly join up and turn the viral genome into a plasmid.
- It is then opened up again by replication proteins.
What is the packaging signal?
- It is a short sequence of DNA.
- Normally around 200bp and on the left hand end of the viral genome.
- Any piece of DNA with this signal ends up in the final viral particle.
- You can put the packaging signal on any piece of linear DNA, and it will end up in the viral particle.
What are the components of simple plasmid that make a recombinant adenovirus?
- Use the ITR to make the DNA a plasmid
- Delete the E1 and E3 region.
- Contains a packaging signal.
- You can then add a bacterial origin of replication and amp resistance.
- This creates a plasmid you can make, grow and manipulate.
- You then transfect this plasmid into 293 cells to replicate your virus.
What happens once the recombinant viral plasmid is transfected into 293 cells?
- The plasmid and host cell work together to mimic the virus.
- The E1 region in the 293 cells starts the transcription cascade.
- This switches on the E4 region.
- This ensures the E2 region is switched on to produce replication proteins.
- The replication proteins open up the plasmid into a linear piece of DNA.
- This can then be replicated, and you have all the right components to produce adenoviruses.
How can you get your gene of interest into the viral genome using the Adeno-X adenoviral system 3?
- Select your gene of interest.
- Use a PCR primer with homology to the viral plasmid.
- Use this to amplify the gene.
- The gene of interest now has homology to the viral plasmid.
- Transform both plasmid and gene into a bacteria to allow recombination to occur.
- Then grow up the recombined plasmid in cells that contain the E1 region.
- You can then harvest the virus
- This system also uses Pac1 restriction enzymes to linearise the genome improve efficiency.
What are the main downsides of viral vectors?
- Unwanted expression of viral genes
- Replication competent adenoviruses
Why is unwanted expression of viral genes a problem?
- They are expressed at low levels and not working efficiently.
- This can eventually make the cell a target for the immune system.
- As adenoviruses are very common, we all have had an anti-adenovirus immune response, which can be reactivated by the vector.
What are replication competent adenoviruses?
- There are regions of homology in the viral genome with bits of the host cell genome surrounding the inserted E1 region.
- This can recombine and put the E1 region back into the virus.
- This creates a mix of viruses that can and cannot replicate independently.
- This is a big problem.
How have some companies side stepped the problem of replication competent adenoviruses (RCA)?
- They have created special cell lines that only contain specific regions of E1.
- These are the regions of E1 needed for viral replication.
- It also removed the homology between the viral plasmid and the host genome.
- This eliminates RCA.
What are 3rd generation or “gutless” vectors?
- These are viral vectors that have had as many of the viral genes removed as possible.
- This prevents RCA and other viral genes from being made.
- These gutless vectors only contain the ITRs and packaging signal.
- This means these vectors can be filled with 36,000bp of any DNA you want.
What do gutless vectors require to grow?
A helper virus.
What are helper viruses?
- These are viruses that grow alongside the gutless virus genome
- It provides the gutless vector with all the proteins needed for replication.
- They are normally grown in bulk in normal 293 cells
What is the issue with using gutless vectors and helper viruses?
- You need to select against the helper virus.
- This is needed to prevent the helper virus DNA from ending up in the final virus particle.
How are helper viruses selected against?
- Putting a LoxP restriction site either side of the packaging signal
- Use a helper plasmid instead of a helper virus
How do you use a helper virus and a gutless vector then select against it using LoxP?
- You grow the virus and the vector in 293 cells.
- When you want to select the gutless vector you move to using 293 cells that express Cre recombinase.
- LoxP removes the packaging signal from the help virus genome while it still provides it helper functions.
- This stops the helper virus genome being packaged into the viral particle.
- You need to ensure the gutless vector has no LoxP sites.
- You do this for several rounds constantly feeding the system with helper virus from normal 293 cells.
Is using LoxP sites to remove the packaging signal 100% effective?
- No
- But contamination is usually less than 0.1%
- The helper virus normally also has E1 and E3 deleted, so they are “safe”
- It is also technically demanding.
How do you use helper plasmids with gutless vectors?
- You need to use large amount of helper plasmid.
- The plasmid has no E1 or E3, no ITR and no packaging signal.
- It is transfected into the cell along with the gutless vector that has the ITR and packaging signal.
- The helper plasmid never had a packaging signal so there is no contamination from the helper genome.
- You can keep putting them in fresh cells with fresh plasmid
Which is better to use LoxP sites or helper plasmids?
- Neither really
- LoxP is more technically challenging and has a slightly higher risk of contamination and alerting the immune system.
What are some of the problems with adenovirus vectors?
- It is not easy to alter the cell tropism of the virus fibre.
- There are still issues around long term expression.
- The virus genome could be mobilised by infection with another adenovirus.
- Even 35Kbp of space may not be enough.
- The immune response can still be a problem.
Why is co-infection with another adenovirus a problem?
- The adenovirus infection introduces the E1, E3 and other proteins to the vector.
- This means the vector can replicate.
- When the cell then dies, the adenovirus infection and the replicated vector are released from the cell.
- This is called mobilisation.
- This needs to be considered when using adenovirus vectors.
How did adenovirus and mRNA SARS-CoV-2 vaccine appear so quickly?
- Adenovirus systems are much older than the mRNA system.
- Both systems have a large evidence base with the adenovirus system slightly more.
- Both systems have been used in multiple clinical trials and have decades of animal models research behind them.
What was the ChAdOx1 nCoV-19 vaccine platform developed for?
for Ebola and MERS vaccines
What is unique about adenoviruses?
- They are very host specific.
- Human adenoviruses don’t infect other animal and visa verse.
What virus is used in the ChAdOx1 nCoV-19 vaccine?
- The chimpanzee adenovirus called Y25.
- Like most recombinant adenoviruses it has the E1 and E3 region deleted.
- They can replicate in 293 cells as the human E1 compensates for the chimp E1.
How is the ChAdOx1 nCoV-19 viral vector made more efficient?
- The human E1 in 293 doesn’t interact very well with chimp E4.
- If you replace the chimp E4 with human E4 it works much better
- The resulting virus grow really fast.
What does the genome of the ChAdOx1 nCoV-19 vector look like?
- The E1 region is replaced with the SARS-CoV-2 spike glycoprotein.
- Protein 9 stabilises the spike glycoprotein.
- the MLP produces the normal transcripts.
- On the opposite strand of the genome there are genes that encode the viral replication protein.
How can you make the S-glycoprotein expression more efficient in ChAdOx1 nCoV-19?
- You can add an intron after the transcription start point but before the open reading frame.
- This makes sure the mRNA is spliced by the host cell before its exported and this causes a massive upregulation of protein expression.
- This works because ribosomes are more efficient dealing with spliced mRNA.
- The addition of the intron ensures the mRNA undergoes polyadenylation and other modifications.
What can the addition of an intron into the ChAdOx1 nCoV-19 vector cause and how can we control it?
- It causes massive upregulation of all protein expression to the point it is toxic to the cell.
- We need to add a repressor to control the expression of the proteins.
- This is done in 293TREX cells that express the tet repressor protein.
- Tet binds to and represses the S glycoprotein expression during growth but not in normal human cells
How much S glycoprotein is made once ChAdOx1 nCoV-19 is injected into human cells?
- Loads of S glycoprotein is made in the cells.
- This alerts the muscle cells to the immune system.
- This creates an effective vaccine.
Are other viral proteins made when the ChAdOx1 nCoV-19 vaccine is injected into 293 cells?
- the rest of the adenovirus genome is very active.
- Lots of viral proteins are being made
- The S glycoprotein is spliced.
- This is what we expect.
Are other viral proteins made when the ChAdOx1 nCoV-19 vaccine is injected into A549 cells?
- A549 cells are a lung cell carcinoma line.
- The virus cannot replicate in these cells.
- The virus is still quite active and produces lots of proteins but very few late proteins.
- There are about 100 viral transcripts compared to 10,000s of S glycoprotein transcripts.
- As these are cancer cells they are very transcriptionally active.
Are other viral proteins made when the ChAdOx1 nCoV-19 vaccine is injected into MRC5 cells?
- This is a normal cell line derived from foetal lung cells.
- The virus cannot replicate in these cells.
- Many 1000s of S glycoprotein transcripts are made.
- Very few adenovirus genes are made. 3 to be precise.
- Protein IX is also made through aberrant splicing of the S glycoprotein transcript. Only a few of these are made.
What are the key points about ChAdOx1 nCoV-19?
- In different cells the transcription patterns are different.
- The adenovirus vector must be making some transcripts that ensure the virus infected cells are eventually cleared.
- Gutless vectors remove all adenovirus genes and this removes all transgene expression.
- Low levels of adenovirus protein expression draw in T cells. This is not a huge issue in vaccines but could be in other gene therapies or if multiple doses are needed.
- RCA is not an issue as there is not enough homology between the E1 and 293 cells.
