AAV5-EF1a-mCherry

The past decade has seen the transformation of adeno-associated virus (AAV) from a gene transfer vector to a clinically viable therapeutic candidate. AAV was discovered in the mid-1960s as a contaminant in adenoviral preparations, but initially did not attract much clinical attention due to its complete lack of pathogenicity. AAV is helper-dependent, meaning that its replication relies on factors from a helper virus, such as adenovirus or herpes simplex virus. A member of the genus Dependovirus in the family Parvoviridae, AAV has a T = 1 icosahedral capsid with a diameter of approximately 25 nanometers. The 4.7 kb single-stranded DNA genome contains two open reading frames: Rep and Cap. Rep encodes four splicing proteins required for viral genome packaging and replication, while Cap encodes the capsid structural proteins VP1, VP2, and VP3 and the assembly activation protein (AAP). VP1, VP2, and VP3 share a common β-barrel domain but differ in their N-terminal extensions.

The AAV genome is flanked by inverted terminal repeats (ITRs), which are essential for packaging, replication, and integration into the host genome. ITRs are the only essential cis-acting elements in the AAV genome, allowing different transgenes to be inserted between the ITRs and then packaged into the AAV capsid to generate recombinant AAV vectors (rAAV). Recombinant adeno-associated virus (rAAV) can be generated by transfecting plasmids containing the ITR-flanked transgene, the AAV capsid gene, and necessary accessory genes, followed by harvesting the transfected cells and purifying the virus. The use of rAAV in gene therapy has developed rapidly over the past decade.