Molecular Biology of the Adeno-Associated Virus
Adeno-associated viruses are non-enveloped and non-pathogenic parvoviruses comprising a double-stranded 5.5 kb genome packaged into a capsid assembly of proteins. At less than 50 nm, this virus is one of the smallest used in gene therapy applications. Consequently, its genome is comprised of only two genes, rep and cap encoding for non-structural and structural genes respectively, which are flanked by two 145 bp ITRs (see figure one). The virus is capable of infecting both dividing and non-dividing cells and can only replicate in the presence of a helper virus, (e.g. adenovirus or herpes virus). Uniquely, it can integrate specifically into the mammalian cell genome at position 19q13, a property conferred by its rep protein. Typically, the AAV maintains a latent infection by integrating in this site, and is subsequently mobilised after secondary infection with helper virus. Its ability to mediate long-term expression in the absence of a significant immune response, combined with its lack of pathogenicity, makes this an ideal candidate gene transfer vector.
Diagram One: AAV Genome
Recombinant Adeno-Associated Viral Vectors
Recombinant vectors based on the AAV are limited in that only 4.5 kb of DNA can be effectively packaged into the capsid. Despite this drawback, its lack of pathogenicity has meant that this virus has been extensively used as a gene transfer agent. Typically, the rep and cap genes are replaced with an expression cassette encoding the therapeutic gene and the vector is propagated in HEK 293 cells by co-transfection with two extra plasmids; one expressing rep, cap and the second expressing adenoviral E1, E2, E4-orf6 and VA RNA (see diagram two).
Diagram Two: AAV Vectors
Recombinant AAV vectors are unable to site-specifically integrate into the host genome, due to the absence of the Rep protein, however, the vector genome forms head-to-tail concatamers that persist in infected cells for many months. AAV vectors can be repeatedly administered without a severe immune response and as such represent the safest viral-based gene transfer vector evaluated to date.