2 AAV biology
The AAV belongs to the parvoviridae family: a family of single-stranded DNA viruses that have evolved over the years, expanding a spectrum of hosts[20]. The viruses of this family share some common structural and functional features such as conserved phospholipase A2 (PLA2)-like enzymatic domain and identical replication components and pathways [20], [21]. Based on the host specificity, the parvoviridae family is divided into two subfamilies: Parvovirinae and Densovirinae, which infect mammalian and invertebrate hosts, respectively (Figure 1A). The Parvovirinae subfamily is further divided into eight genera, including Dependovirus, into which AAV is classified. The insect cells are a natural host of Densovirinae viruses, and from a taxonomical standpoint, they should provide an adequate cellular environment for replication of viruses from closely related subfamily Parvovirinae, including AAV. This relation was first recognized and later found to be applicable by Urabe et al., where the group demonstrated the first IC-BEVS based rAAV production platform [22].
AAV virus particle comprises a vector genome: the functional element of the virus, and the capsid, a metastable entity that preserves it. The vector genome is a linear single-stranded DNA with a size of about ~4.7kb and a major coding region consisting of the sequences for non-structural replicase proteins (Rep) and structural capsid proteins (Cap or VP). This coding region is flanked by an inverted terminal repeat sequences (ITR). The ITR is a segment of 145bp with a T-shaped hairpin-like structure formed via complementary base pairing of palindromic sequence of 125 bp. The remaining 20 bases stay unpaired. The ITR region also encompasses the origin of replication formed by the terminal resolution site and rep-binding element (RBE). The free 3‘ hydroxyl region acts as an initiation sequence for DNA replication.
AAV contains four non-structural Rep proteins (Rep 78, Rep 68, Rep 52, and Rep40). Rep78 and Rep52 are expressed under the transcriptional control of p5 and p19 promoters, respectively. The larger Rep78 and its spliced variant Rep68 are crucial for- and participate in vector genome integration, excision, rescue, and replication at various stages of AAV life cycle[23]–[25]. The smaller Rep proteins Rep52 and its spliced variant Rep40 play a critical role in the packaging of vector DNA in preformed capsids and regulation of replication by repressing p5 in the absence of a helper virus[26], [27]. The three structural proteins (VP1, VP2, and VP3) expressed from the right open reading frame (ORF) under p40 promoter form an icosahedron capsid that carries the viral genome and delivers it to the host cell traversing through post cell surface attachment. A representative genomic map of wtAAV2 [28] and its transcriptional profile are shown in Figure 1B.