scAAV4-CAG-GFP

scAAV is a viral vector engineered from the naturally occurring AAV for use as a gene therapy tool. This laboratory-made AAV offspring is referred to as "self-complementary" because the coding regions are engineered to form an intramolecular double-stranded DNA template. The scAAV vector genome is engineered as a single-stranded inverted repeat that folds back on itself to form a double-stranded genome upon entry into infected cells. As a result, a genome smaller than 2.5 kilobases can be packaged as a dimer with two inverted repeats paired along its length and covalently closed by a hairpin at the terminal repeat. Complementary strand synthesis is therefore not required and this rate-limiting step can be bypassed. Genome dimerization in this manner can be stabilized by mutating or deleting one of the two terminal resolution sites (trs; these are Rep binding sites contained within each inverted terminal repeat), thereby preventing cleavage of the AAV Rep protein to form monomers. Replication forks initiate at the wild-type trs and traverse the genome and the mutant trs, which are unable to promote resolution, resulting in the replication fork traversing the genome and back, terminating at the wild-type trs. Thus, the resulting self-complementary molecule is flanked by wild-type trs and has a mutant trs in the middle, which dimerizes along its length when packaged into AAV. The rate-limiting step for standard AAV genomes involves second-strand synthesis, as the typical AAV genome is a single-stranded DNA template. However, this is not the case for scAAV genomes. Upon infection, the two complementary halves of scAAV do not wait for cell-mediated synthesis of the second strand, but instead combine to form a double-stranded DNA (dsDNA) unit that is immediately available for replication and transcription. Upon transduction of target cells, scAAV genomes exist as circular genomes or concatemers, with the former being more efficient in transgene expression. Compared to single-stranded AAV (ssAAV) vectors, scAAV vector genomes are more stable and more easily circularized upon tissue transduction in vivo.