419. Production of Recombinant Adeno-Associated Virus Type 5 in Insect Cells

Abstract

Recombinant adeno-associated virus (rAAV) is one of the promising gene transfer vehicles. AAV vectors based on AAV type 2 successfully transduce non-dividing cells in muscle, liver, brain, and eye. rAAV derived from another serotype, type 5 is capable of delivering the gene of interest into some cells or tissues more efficiently than rAAV2. We reported a new method for production of rAAV2, which utilizes recombinant baculoviruses and insect cells (Hum Gene Ther 13:1935–1943, 2002). This system can produce rAAV2 at high titer. Moreover insect cells are maintained in suspension culture and thus easily be scalable. In the present study we developed a method to generate rAAV5 in invertebrate cells. Three recombinant baculoviruses were produced; one has GFP/Neo gene between the inverted terminal repeats of type 5 AAV, the second carries type 5 large Rep and Small Rep genes, and the third expresses type 5 VP. Spodoptera frugiperda Sf9 cells were infected with each recombinant baculovirus at an moi of 5 and were incubated at 28°C for three days. The infected cells were lysed and rAAV5 particles produced were purified by two round of CsCl density gradient ultracentrifuge. The yields of the rAAV5 were approximately 25,000 DNAse resistant particles per Sf9 cell. However, rAAV5-GFP produced with the three baculoviruses poorly transduced COS cells compared to rAAV5-GFP produced in HEK293 cells. Western analysis showed that the type 5 capsids produced in insect cells appeared to have relatively less VP1 than those produced in mammalian cells. In order to enhance the production of VP1, we constructed a chimeric VP1 consisting of the amino-terminal portion of type 2 VP1 and the type 5 VP2 – VP3 residues. The GFP/Neo gene was efficiently encapsidated into the chimeric VP capsids (rAAV5C-GFP). The buoyant density of rAAV5C-GFP produced in insect cells was 1.4g/cm2. To compare the rAAV5C-GFP with rAAV5 produced in HEK293 cells, we infected COS cells at 30,000 through 1,000 vector genomes per cell. Chimeric rAAV5 produced in Sf9 cells had equivalent biological activity as rAAV5 produced in 293 cells with wild-type VP1. To further characterize the biological activity of the chimeric rAAV5, the following experiments were performed. Since sialic acid acts as a primary co-receptor for AAV5, the addition of 3'-N-acetylneuraminyl-N-acetyl-lactosamine, an analogue of sialic acid, in culture medium inhibits rAAV5 infection. The analogue also suppressed rAAV5C-GFP transduction of COS cells or HEK293 cells. In contrast, heparin, an analogue of heparan sulfate proteoglycan (a co-receptor for AAV2), failed to block the GFP expression mediated by rAAV5C-GFP produced in insect cells. Furthermore, pretreatment of COS cells with neuraminidase inhibited GFP expression by rAAV5C-GFP. These results indicated that the rAAV5-GFP with modified capsid has similar characteristics as the wt capsid based on the ability to transduce cells as rAAV5-GFP generated in HEK293 cells does. The new method for production of rAAV5 in insect cells offers a better alternative to the existing production methods of rAAV5.