scAAV7-CAG-GFP

After decades of preclinical development, adeno-associated virus (AAV) vectors have emerged as a promising delivery system for clinical gene therapy, with six therapies approved by the U.S. Food and Drug Administration since 2017. As these and other potential AAV-based therapies are introduced, issues related to vector manufacturing have become increasingly important. The native AAV virus encodes two essential genes (rep and cap) that encode nine proteins but is incapable of self-replication and requires co-infection with a helper virus for replication.

Recombinant AAV vectors are typically (but not exclusively) produced by co-transfecting HEK293 cells with three plasmids containing: (1) the AAV rep/cap genes, (2) adenoviral helper genes, and (3) a proviral plasmid containing the AAV inverted terminal repeats (ITRs) flanking the therapeutic gene of interest (sometimes referred to as an AAV transfer plasmid). The ITR sequences define the AAV genome and contain the AAV packaging signal. Thus, a DNA sequence cloned between two AAV ITR sequences can be packaged within an AAV capsid with a maximum size of ∼4.7 kilobase pairs for single-stranded AAV (ssAAV) and ∼2.3 kilobase pairs if a self-complementary double-stranded genome (scAAV) is used. For ssAAV, both ITRs are 145 base pairs (bp) in size, while the scAAV genome is created by deleting a portion of the terminal resolution sequence (also known as Δtrs) in one of the ITRs.