L6 - Genetic manipulation of viruses Flashcards
LO: - Appreciate the qualities required of hypothetical “ideal vector” - Know the four principle gene therapy vectors - Understand the principles behind the most widely used vector -adenovirus - Know the details of how this virus is modified to make gene therapy/vaccine vectors - Understand the principle problems and advantages of this vector system - Have an overview of how the ChAdOx1 nCoV-19 was made and its transcription profile
What is the primary aim of viral gene therapy?
To treat human diseases by using genetically altered viruses to deliver therapeutic genes.
Which three major therapeutic areas are targeted by viral gene therapy?
Inherited diseases, cancer, and vaccines.
Name some of the viral families that have been examined as gene therapy vectors.
Retroviruses, adenoviruses, adeno-associated viruses (AAV), and herpesviruses.
What characteristics would an ideal viral vector have?
It should be safe, non-replicating, have no adverse side effects, exhibit precise cell targeting, provide optimal gene expression levels, and persist only as long as necessary.
How are adenovirus vectors rendered replication-deficient?
By deleting essential regions such as the E1 region—and often the E3 region—to prevent replication in normal host cells.
Why are 293 cells important in the production of adenovirus vectors?
They supply the missing E1 gene products in trans, allowing E1-deleted viruses to propagate.
What is the purpose of deleting the E3 region in these vectors?
The E3 region encodes proteins that modulate the immune response, and its deletion can reduce unwanted interactions in cell culture.
What distinguishes second-generation adenovirus vectors from first-generation ones?
Second-generation vectors have additional deletions (such as parts of the E2 or E4 regions) to further reduce viral gene expression.
What is the approximate foreign DNA capacity of first-generation adenovirus vectors?
They can typically accommodate up to about 8 kilobase pairs, with slightly larger capacity in second-generation systems.
What function do Inverted Terminal Repeats (ITRs) serve in adenovirus vectors?
ITRs act as origins of replication and enable the regeneration of the linear viral genome in the presence of replication proteins.
Describe the role of the packaging signal (ψ) in adenovirus vector construction.
The packaging signal is a short (approximately 200 bp) sequence that directs the incorporation of the viral genome into virus particles.
How is a recombinant adenovirus typically generated using plasmid-based systems?
A plasmid containing the packaging signal, deletions in the E1 and E3 regions, and the gene of interest is transfected into appropriate cells (such as 293 cells) to produce recombinant virus.
Why might adenovirus vectors still express viral genes even though they are replication-deficient?
Residual expression of viral genes can occur, which may trigger immune responses in the host.
What are Replication Competent Adenoviruses (RCAs) and how do they arise?
RCAs are viruses that regain replication ability through recombination events between sequences in the E1-deleted vector and those in the 293 cells.
How have cell lines like PER.C6 been used to address the RCA issue?
They are designed to reduce homologous recombination events, thereby minimising the emergence of RCAs.
What advantage do third-generation or “gutless” adenovirus vectors offer over earlier systems?
They contain only the ITRs and packaging signal, allowing for up to 36 kilobase pairs of foreign DNA and drastically reducing unwanted viral gene expression.
How is the helper virus used in the production of gutless vectors, and what is a potential drawback of this system?
The helper virus supplies the necessary viral proteins for replication and packaging, while site-specific recombinases ensure minimal helper contamination; however, this system is technically more demanding and requires a continuous supply of helper virus.
What is the origin of the ChAdOx1 nCoV‑19 vector?
It is derived from a chimpanzee adenovirus known as Y25, modified to be replication-deficient.
How is the ChAdOx1 nCoV‑19 vector modified to optimise compatibility with human cells?
Its E1 and E3 regions are deleted, and the chimpanzee E4 region is replaced with the human E4 region to improve interactions with human-derived E1 products in 293 cells.
What strategy is employed to enhance transgene expression for the SARS-CoV‑2 spike protein in this vector? Check
A CMV promoter with an added intron and a tet repressor binding site is used, with the vector grown in 293TREX cells that express the tet repressor to suppress toxic expression during production.
Why is the use of the tet repressor system beneficial during vector production?
It prevents excessive expression of the spike protein during virus rescue and growth, ensuring higher viability in production cells while allowing robust expression in target cells.
How are the late transcripts of adenovirus vectors organised?
They comprise a family of transcripts (designated L1 to L5) that share a common polyadenylation site and undergo complex splicing events.
What issue can arise from aberrant splicing in adenovirus vectors?
Aberrant splicing can generate unintended transcripts (e.g. aberrant pIX transcripts), although these are generally present only at low levels.
How do transcription patterns vary among different cell types infected with adenovirus vectors?
In some cell types, such as lung carcinoma cells (e.g. A549), high levels of the transgene (such as the SARS‑CoV‑2 spike) are produced relative to adenoviral transcripts, whereas in normal cells (e.g. MRC‑5), adenoviral transcripts are minimal.
