scAAV DJ-CAG-GFP

Adeno-associated virus (AAV) vectors are the leading platform for gene delivery. AAV is used to deliver therapeutic genes to express or silence target proteins. AAV vectors consist of a protein capsid that surrounds and protects a single-stranded DNA genomic cargo of approximately 4.8 kilobases (kb). The AAV genome includes rep and cap genes encoding seven proteins. The rep genes encode four nonstructural proteins (Rep78, Rep68, Rep 52, and Rep 40) involved in replication, transcriptional control, integration, and encapsidation. The products of the three cap genes (Vp1-3) combine into 50 Vp3, five Vp1, and five Vp2 proteins to form the capsid. Capsid assembly is assisted by assembly activator proteins, which are nonstructural proteins encoded within the cap genes that promote capsid stability and interactions between capsid proteins.

Gene shuffling relies on high homology of cap genes between serotypes, which enables homologous recombination to generate novel capsid variants composed of different regions and motifs with unique targeting properties. Capsid gene shuffling generated cA2, a chimera of AAV1, 2, 6, 8, and 9, which transduced heart, liver, and muscle at higher rates than AAV2 following intravenous injection into mice. In vitro recombination of related parental cap genes from serotypes with >50% homology increased the efficiency of gene shuffling, generating AAV-DJ, a chimeric variant composed primarily of AAV2, 8, and 9 capsid sequences. This variant was able to bind to HSPGs and transduce various human cell systems, including primary hepatocytes, at higher efficiencies than the parental serotypes. Transduction levels of mouse liver were comparable to AAV8 and AAV9, but superior to AAV2 when injected intravenously. Transduction levels were similar to those of AAV8 and AAV9 in the heart, kidney, and spleen, and lower levels were observed in the lung, central nervous system, pancreas, and intestine.