Adeno-associated virus (AAV) is a single-stranded DNA parvovirus with a genome of 4700 nucleotides, the genome of which comprises two open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The proteins responsible for replication (rep) are encoded by the left ORF, while the right ORF encodes for the structural proteins of the capsid (cap). The rep gene from the ORF encodes Rep78, Rep68, Rep52 and Rep40, which aid AAV genome replication and virion assembly. Virion protein 1 (VP1), VP2 and VP3 are three capsid proteins that generated from the cap gene from which translation is initiated at different start codons. As a result, these structural proteins have the same amino acids in their C-terminus. In addition, assembly-activating protein (AAP), which is essential for capsid assembly, is encoded from an in-frameshifted ORF within the cap gene. AAV virions are composed of 60 VP subunits. Each subunit has nine variable regions on the virion surface that determine the primary tropism and intracellular trafficking of the AAV vector and are typically the domains recognized by neutralizing antibodies (NAbs). Genetically modifying these variable regions can alter the transduction efficiency of AAV and the ability of NAbs to bind to the virion surface.
The process of AAV infection begins with viral binding to the cell surface and is followed by viral uptake, intracellular trafficking, nuclear localization, uncoating, and second-strand DNA synthesis. AAV infects target cells by binding to primary receptors and co-receptors on the cell surface, thereby triggering endocytosis of target cells into endosomes. When the N-terminal structures of VP1 and VP2 are altered, AAV virions are released from endosomes and accumulate in the perinuclear region of the cell. Once in the nucleus, AAV virions uncoat and release their single-stranded genome which is converted into a double-stranded DNA (dsDNA) template. The transgene from the dsDNA template can be transcribed and translated. Only the 145bp AAV ITRs are required for the propagation of recombinant AAV (rAAV), because these ITRs induce transgene expression and play an important role in vector production and ensuring sustained transduction of cells.
At least 12 natural serotypes and more than 100 variants of AAV have been isolated and developed as gene delivery vehicles. Different AAV serotypes have different binding receptors and tissue tropisms. AAV-1 and AAV-7 are the most efficient serotypes transduced into skeletal muscle. AAV-3 is superior for the transduction of megakaryocytes. AAV-2, AAV-4, and AAV-5 transduce different cell types in the central nervous system. AAV-5 and AAV-6 are effective in infecting apical airway cells. AAV-8 and AAV-5 can transduce hepatocytes better than AAV-2. AAV mutants from these serotypes are continuously developed to optimize the use of AAV for gene delivery applications.
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