Scalable Generation of High-Titer Recombinant Adeno-Associated Virus Type 5 in Insect Cells

Recombinant adeno-associated virus (rAAV) is being developed as a gene transfer vector. rAAV based on serotype 2 (rAAV2) successfully transduces nondividing cells, including muscle, liver, and brain cells (29). Conventional rAAV production requires packaging of rAAV DNA into type 2 capsids by transient transfection of HEK293 cells with two or three plasmids: an AAV helper plasmid encoding rep and cap genes devoid of inverted terminal repeat (ITR) sequences, a vector plasmid harboring the therapeutic gene between ITRs, and an adenovirus helper plasmid expressing E2A, virus-associated (VA) RNA, and E4orf6. Transient cotransfection is the major limitation for scale-up of rAAV production. Since rAAV can be purified using column chromatography, which can result in highly purified rAAV while eliminating other contaminating viruses, some efforts were made to develop rAAV production systems by using recombinant mammalian viruses such as adenovirus (10) or herpes virus (4) which do not rely on the plasmid transfection and therefore may be amenable to scale-up production. Recombinant baculoviruses based on the Autographa californica nuclear polyhedrosis virus are widely employed for production of heterologous proteins in cultured insect cells. The highly active, late A. californica nuclear polyhedrosis virus promoters, such as polyhedrin and p10 promoters, regulate the expression of heterologous proteins, resulting in large amounts of foreign proteins. Insect cells may be grown in suspension cultures in volumes ranging from shake flasks of sizes from, e.g., 50 to 400 ml, up to commercial-size bioreactors, e.g., 1,000 liters and larger. Recently, we described a highly scalable and efficient method for packaging rAAV2 in insect cells by use of baculovirus expression vectors (31). The ease of scale-up production is perhaps the most attractive feature of this production system. Infection of insect cells in suspension culture with recombinant baculoviruses eliminates the transfection process. Standard downstream processing to recover rAAV, such as tangential flow filtration and column chromatography, is readily applied. In addition to vectors derived from serotype 2, other serotypes, utilizing different cell surface receptors, constitute a vector set from which an appropriate vector can be selected for a specific application. AAV5 is the most divergent dependovirus characterized (2), and type 5 AAV vectors have desirable properties that differ from other serotype vectors. AAV5 utilizes different receptors from other serotypes (14, 30), and rAAV5 has demonstrated different tropism from AAV2 (5), thus making it worthwhile to establish a method to produce rAAV5 in insect cells. AAV is a member of the family Parvoviridae. The genome of AAV is a linear, single-stranded DNA of 4.7 kb in length. The ITRs flank the unique coding sequences for the nonstructural replication initiator proteins, Rep, and the structural capsid proteins, VP. The ITRs serve as origins of DNA replication and may also function as the packaging signal. Type 2 Rep78 is generated by the p5 promoter, while Rep68 is translated from spliced mRNA from the p5 promoter. The small Rep polypeptides Rep52 and Rep40 are expressed by the p19 promoter with nonspliced or spliced mRNA. The p40 promoter regulates expression of capsid proteins VP1, VP2, and VP3. Alternate usage of two splice sites and translation of VP2 at a non-AUG codon results in a stoichiometry of 1:1:10 of VP1, VP2, and VP3. Both p5 proteins Rep78 and Rep68 are AAV origin binding proteins, and the presence of either is required for AAV DNA replication and processing replicative intermediates of the virus DNA (13). Also, either Rep52 or Rep40 is necessary for packaging the single-stranded, linear virion genome into preformed empty capsids (17). The transcriptional map of type 5 AAV differs from that of type 2; the p7 promoter or p19 promoter transcribes mRNA for Rep78 or Rep52. Type 5 AAV does not encode the spliced form of Rep polypeptides Rep68 and Rep40 (25). Structural protein VP1 is a minor constituent in the AAV capsid. But the VP1-unique portion of approximately 140 amino acid residues is highly conserved among different serotypes and has a phospholipase A2 motif. The YXGGX and HDXXY motifs (where X is any amino acid residue) in phospholipase A2 indicate the catalytic site and Ca2+ binding loop, respectively (see Fig. ​Fig.3A).3A). Enzymes classified into the secretory phospholipase A2 family hydrolyze the ester bond at the 2-acyl ester position of glycerophospholipids in the presence of Ca2+ and are involved in many aspects of cellular pathways, such as lipid membrane metabolism and signal transduction pathways (1, 21). The VP1-unique portion of parvovirus is required for transfer of the virus from late endosomes to the nucleus (36). A mutant virus lacking the VP1-unique portion or the activity of phospholipase is not processed properly, and thus no virus or vector genes are expressed. FIG. 3. (A) Chimeric VP genes constructed. The portions derived from type 2 are indicated in gray, while those from type 5 are in white. The common portions are indicated in black. The phospholipase A2 motifs are shaded. The YXGGX and HDXXY motifs (where X is ... In the present study, we describe a rAAV5 production system based on recombinant baculovirus and insect cells. In order to achieve high production levels of rAAV5 particles, we replaced a portion of the VP1 polypeptide with the corresponding portion of type 2. The VP1 substitution did not alter the tropism of rAAV5, which behaved indistinguishably from rAAV5 with wild-type VP1. In an attempt to improve the yields of rAAV5 particles, we used type 1 Rep52 instead of type 5, which resulted in the production of more than 5 × 104 vector genomes (vg) per insect cell.