scAAV3-GFP

Self-complementary AAV vectors (scAAV) are widely recognized for their superior efficiency in transgene expression. These vectors were constructed using the mITR strategy to increase the proportion of vectors carrying double-stranded genomes. During scAAV generation, the Rep78/68 protein complex is unable to cleave the trs at one end until the replication complex at the other end approaches it. This results in the displacement of the strand originating from the dimeric replication intermediate template. This unique mechanism produces a double-stranded genome with two ITRs flanking the central mITR. However, it is important to note that scAAV vector preparations may still contain particles with monomeric DNA, and the proportion of such particles depends on the size of the packaged genome. During AAV genome replication, a critical step in generating monomeric ssAAV DNA for packaging is the efficient cleavage of the covalently linked AAV ITRs in the monomer. This cleavage results in the formation of an open double-stranded structure by the Rep78/68 protein at specific trs. Successful cleavage requires specificity in the trs sequence and structure. When trs and its adjacent sequences were mutated, Rep-mediated nicking activity was reduced, suggesting that cleavage may still occur at a lower level. Based on previous studies, it is clear that the mITR in scAAV gDNA can function as a replication primer like the ITR and also allow nonspecific nicking.