scAAV2-Syn-GFP

The AAV genome is flanked by two inverted terminal repeats (ITRs), each containing a palindromic hairpin (HP) structure and a 20-nucleotide D sequence segment. When the single-stranded virion DNA enters the host cell nucleus, the 3′ ITR serves as a primer for the DNA replication complex, converting it into a dsDNA molecule with a covalently closed end (ITR) at the downstream end. Subsequently, the Rep endonuclease 78/68 cuts the terminal resolution site (trs) of the downstream ITR to form a 5′ overhang. The DNA replication complex then fills in the 5′ overhang to produce a blunt end with two open double-stranded ITRs, thus forming a completely linear monomeric genome. In this monomer, the 3′ ITR restarts to synthesize the second complementary strand, replacing the original strand, which can then be packaged or enter a new replication cycle.

AAV genome packaging is based on a strand displacement mechanism. Only single-stranded (ss) genomes produced during the AAV replication cycle can be packaged into AAV capsids. However, if Rep fails to cleave the trs before reaching the other end of the replication complex, replication continues through the ITR. This generates a dimeric DNA molecule with a covalently closed ITR downstream. The end cut by the Rep78/68 protein is then filled in by the replication complex to generate a complete linear dimeric DNA. One strand initiates a new round of DNA synthesis, while the other strand is displaced, resulting in a single-stranded dimeric inverted repeat genome. Once this genome is released from the virion, it self-anneals to double-stranded DNA. Although when the genome is small enough, the dimeric inverted repeat sequence itself can be packaged as scAAV. To generate dsAAV genomes more efficiently, scAAV vectors were designed to lack the trs site in one of the ITRs, enabling the packaging of single-stranded molecules that can rapidly self-anneal after uncoating.