scAAV6-CAG-GFP

Adeno-associated virus (AAV) is a non-enveloped virus with a single-stranded linear DNA genome of approximately 4.7 kb that requires a helper virus for replication. Recombinant AAV (rAAV) vectors provide stable long-term expression and low immunogenicity. However, AAV vector transduction involves multiple steps, including cell binding, intracellular trafficking, capsid uncoating, and second-strand synthesis. Any of these steps may limit transgene expression. In particular, second-strand synthesis has been widely demonstrated to be the rate-limiting step for efficient transgene expression by AAV vectors. Transient genomic instability following double-stranded DNA (dsDNA) conversion results in significant loss of gene expression. In addition, failure to complete second-strand synthesis in a timely manner results in loss of single-stranded AAV (ssAAV) genomes. To address these limitations, self-complementary AAV (scAAV) vectors were developed.

scAAV vectors skip the second-strand synthesis step and have a 5- to 140-fold increase in transduction efficiency compared to conventional ssAAV vectors in vitro. In animal models, scAAV vectors have demonstrated rapid onset of efficacy and higher transgene expression in the liver, muscle, brain, and central nervous system. Unlike ssAAV vectors, expression of scAAV is independent of second-strand synthesis. Aphidicolin and hydroxyurea DNA replication inhibitors do not affect scAAV vector transduction. In a recent preclinical study, genomic RNA (gRNA) carrying scAAV significantly improved CRISPR-Cas9-mediated gene editing, achieving at least a 20-fold improvement at lower doses compared to ssAAV-gRNA vectors. This increased efficiency means that scAAV vector doses can be significantly reduced to achieve similar results, potentially improving safety.