AAV6-EF1α-GFP

AAV is a single-stranded DNA parvovirus whose genome consists of the rep gene and the cap gene, flanked by two inverted terminal repeats (ITRs). The rep gene encodes Rep78, Rep68, Rep52, and Rep40 from a single ORF, which facilitate AAV genome replication and virion assembly. The three capsid proteins (virion protein 1 (VP1), VP2, and VP3) are produced from a single cap ORF but are regulated by transcription from a rare start codon (ACG) and alternative splicing. Therefore, the C-terminal amino acids of VP1 and VP2 are identical to VP3. In addition, the assembly-activating protein (AAP), which is essential for capsid assembly, is encoded by a frameshifted ORF within the cap gene. All AAV virions are composed of 60 VP subunits with a 1:1:10 ratio of VP1:VP2:VP3. Each subunit has nine variable regions on the virion surface that determine the primary tropism and intracellular trafficking of AAV vectors and are often the domains recognized by neutralizing antibodies (NAbs).

Genetic modification of these variable regions can alter the transduction efficiency of AAV and the ability of NAbs to bind to the virion surface.

In vitro studies support that AAV infects target cells by binding to primary and coreceptors on the cell surface, triggering their entry into endosomes. Following structural changes that expose the N-termini of VP1 and VP2, AAV virions are released from endosomes and accumulate in the perinuclear region of the cell. Once in the nucleus, AAV virions uncoat and release their single-stranded genome, which is converted to a double-stranded DNA (dsDNA) template from which transgenes can be transcribed and translated.