.:Research
Viral Vectors for Gene Therapy
Adeno-Associated Virus
AAV is a non-pathogenic human parvovirus. Productive AAV infection requires helper functions that can be supplied by coinfection with helper viruses such as Ad and herpesvirus. AAV can also replicate in cells that have been put under stress such as irradiation or treatment with genotoxic agents. In the absence of a permissive environment that will support AAV replication, the viral DNA can become integrated into the host chromosomal genome to establish a latent infection.
Many different serotypes of AAV have been isolated and the list continues to increase (58-63). They have in common a similar size and genomic configuration of replication and structural genes. AAV serotype 2 (AAV2) is the best characterized. AAV virions are small non-enveloped particles (20-25 nm) that carry a linear single stranded DNA (ssDNA) genome, which is approximately 4.7 Kb in size. There are 2 viral ORFs, rep and cap, flanked by T-shaped inverted terminal repeats (ITRs). The ITRs are important for replication, packaging and integration, and these are the only genetic elements from the virus that are retained in rAAV vectors. There are 4 regulatory Rep proteins that are required for replication and packaging. The cap ORF encodes for 3 structural proteins (VP1, VP2 and VP3) that form the capsid.
The different serotypes of AAV utilize a variety of approaches for cell entry, and this results in different host ranges. The primary attachment site for AAV2 is the ubiquitous heparan sulfate proteoglycan. The fibroblast growth factor receptor 1 and integrin v5 have both been implicated as co-receptors that facilitate internalization by endocytosis. AAV4 and AAV5 use sialic acid, although different carbohydrate linkages determine specificity and the PDGF receptors are also involved in AAV5 infection. After binding to its receptor the virus enters the cell through receptor-mediated endocytosis and is subsequently transported to the nucleus. Viral uncoating in the nucleus releases the single-stranded genome that then needs to be converted to a double-stranded form to enable gene expression.
The self-complementary sequences in the ITRs fold back on themselves to form hairpin structures that contain the replication origins. The large viral Rep proteins bind to a specific sequence within the ITRs and mediate replication. In addition, the virus relies upon the host cell replication machinery as well as helper functions supplied by the co-infecting virus. The helper proteins promote initiation of transcription, viral gene expression and DNA replication. Replication proceeds at discrete sites in the nucleus and strand displacement produces both plus and minus genomes that are packaged with equal efficiency into the icosahedral capsid.
