Adeno associated vector Flashcards
What are the characteristics of adeno associated vector?
Parvoridae family. Infects humans and some other primates but not known to cause disease. Non-pathogenic. Can deliver to target cells. Doesn’t integrate into genomes. Long lasting expression.
When and how was it discovered?
1960s through basic research. Realised AV had contaminants that were a helper virus to replicate effectively. Without this they become latent or dormant. Found in vertebrates, humans and non-human primates. Subfamily of parvovirus called dependoparvovirus.
What is the structure of AAV?
Icosahedral (20 side polygon) capsid, 26nm diameter. ssDNA genome around 4.7kb.
What are the different domains of adenovirus?
Rep 78/68 for replication. Rep 52/40 for packaging, ITR inverted terminal repeat 145bp replication priming, VP 1/2/3 capsid proteins.
What are the advantages of AAVs?
Lack of pathogenicity, infects non-dividing cells, low immunogenicity/toxicity , non-replicating (piggyback on helper virus HS/AdV), episomal, low level of integration (integrations at AAVS1 chromosome 19 not associated with disease).
What are the disadvantages of AAVs?
Small packaging capacity, contamination with helper virus, low yield (high cost).
What is the method of the most common production of recombination AAV?
Triple transfection of HEK293 producer cells (endogenous adenovirus E1a and E1b). HEK293 are human embryonic kidney cells that are transformed with a portion of the adenovirus genome containing the E1A and E1B genes. These provide essential functions that adenovirus supply during natural AAV infection which stimulates viral gene expression and replication in AAV. Allows replication without adenovirus being present.
What are the three plasmids used?
Cis plasmid with transgene for packaging into AAV capsid. Trans plasmid expressing cap and rep. Helper plasmid with other Adv genes needed for replication, mRNA processing and translation.
What are rAAVs requirements?
Capsid, transgene and ITRs.
Why are the three different plasmids used?
Safety minimal risk of producing replication competent AAV from separating them into different plasmids, flexibility different cap genes (capsid serotypes) can be used to produce rAAVs tailored for different tissue types. Efficiency having cis/trans separation facilitates optimal expression of rep and cap proteins.
What are the other three less common methods of rAAV production?
Producer and packing stable cell line, herpervirus-based system and baculovirus expressing vector and insect cell.
What is the producing and packaging stable cell line method?
Rely on stable cell lines that have been engineered to continuously express some or all of the necessary components for AAV production.
What is the herpervirus based system?
Has two recombinant HSV vectors which are typically used to introduce the necessary components into producer cells. HSV vector 1 contains the AAV transgene flanked by inverted terminal repeats. This transgene will be packaged into the AAV capsid. HSV vector 2 contains the Rep and Cap genes of AAV which are needed for genome replication and capsid assembly.
What is the baculovirus expressing vector and insect cell method?
Recombinant baculoviruses are engineered to contain the genetic elements necessary for AAV production. Baculovirus 1 carries the AAV transgene flanked by inverted terminal repeats. Baculovirus 2 contains the Rep and Cap genes of AAV required for replication and capsid formation. Then used to infect insect cells, which are optimal hosts for infection and AAV production.
How does rAAV transduction into mammalian cells occur?
Infect both dividing and non dividing cells. Bind to receptors: heparin sulphate proteoglycan, abv5 integrin and FGR. Endosome escape dependent on pH and confirmational change in VP1/2. Internalize and form early endosome then late endosome then lysosome then escape to enter nuclear pore. Single stranded ssAAV and self-complementary scAAV. Most remain episomal.
