Brief Introduction of Recombinant AAV

Wild type adeno associated viruses (wtAAVs) are small, non-enveloped viruses with a 4.7kb single-stranded DNA genome. Scientists have developed recombinant AAVs (rAAVs) as efficient transfer vectors that have been successfully utilized in many in vitro and in vivo applications.

There is a set of unique characteristics which make rAAVs attractive as a genetic delivery vector. 1) Unlike human pathogenic HIV-derived recombinant lentiviruses (rLVs), wtAAVs are have not been reported to be associated with any human diseases. The engineered rAAVs are safer for researchers or end users. 2) Although wtAAVs can integrate into human chromosome 19 at the locus known as AAVS1 (or PPP1R12C), rAAVs has been eliminated the integrative ability by removal of the rep and cap from the recombinant viral genome, which means rAAVs can not cause insertional mutagenesis resulting from the integration. 3) Like rLVs, rAAVs can infect both dividing and non-dividing cells. 4) rAAVs have several serotypes targeting different specific cell types or tissues.

There are multiple methods to produce rAAVs, but typical strategies include helper-free AAV production system and baculovirus-based AAV production system. The helper-free system involves transfecting packaging cells with three different plasmids, 1) vector containing ITR-flanked GOI, 2) vector containing the replication (rep) and capsid (cap) genes, and 3) vector containing essential helper virus genes such as adenovirus E4, E2A and VA. The baculovirus-based AAV production system briefly involves two steps, 1) generation of GOI and helper baculoviruses, 2) co-infection of two baculoviruses into insect cells to achieve rAAVs. The baculovirus-based system is typically used for large scale AAV manufacturing.

rAAVs can be divided into different subgroups according to the viral genome and their cap proteins.

1) Numerous AAV serotypes have been identified, but the best characterized is AAV2. So far, almost all rAAVs have utilized ITRs from AAV serotype 2.

2) AAV variants: Single-stranded AAV (ssAAV) & Self-complementary AAV (scAAV)
ssAAV is the traditional rAAVs vetors. The genome of ssAAV is a linear single-stranded (ss) DNA molecule with a length of approximately 4.7 kb. scAAV is a derivative of ssAAV and its genome is an intra-molecular double-stranded DNA. Compared with ssAAV, scAAV has faster and higher levels of gene expression as it circumvents the step of second second-strand synthesis. The disadvantage of scAAV is its smaller cargo capacity which is ~2.2 kb.

3) AAV serotypes
Serotype is determined by the type of capsid protein. So far, rAAVs have been equipped with several capsid proteins and have been successfully used in many in vitro / in vivo experiments. These serotypes include AAV1, 2, 3, 4, 5, 6, 7, 8, 9, DJ, DJ-8, PHP.B, PHP.eB, PHP.S, rh10, Retrograde, B1, 7m8, shh10, Anc80L65AAP, Anc80L65, with more under development.

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