558. Second Generation Vaccinia Carrier for Scalable and Efficient Production of Recombinant AAV Vectors

Abstract

Recent successes of recombinant adeno-associated virus (rAAV) vectors in clinical trials demand a more efficient system for scalable production of high-quality rAAV vectors. Our recently developed rAAV production employed a novel concept by utilizing cytoplasmic carriers, vaccinia viruses carrying AAV rep and cap genes; nuclear carrier, an adenovirus carrier with AAV transgene expressing cassette and suspension Hela cells to produce rAAV vectors. In this prototype production system, two vaccinia virus carriers are necessary to provide the required Rep and Cap functions, with one containing Rep78 and VP2(Rep78-VP2) and another containing Rep52 and VP1(Rep52-VP1). This scheme is necessary due to the sequences identity between rep genes and cap genes. To further reduce the vaccinia carriers necessary for rAAV production, we developed a novel vaccinia carrier (VW22) which is capable of all necessary rAAV production gene products, Rep 78, Rep52, VP1, VP2 and VP3. Despite the VP1 and VP2, Rep78 and Rep52 all have identical amino acids in the C-termini, VW22 is stable for at least 12 continuous replication and production cycle. The rAAV packaging efficiency using VW22 is 2≈3 times higher than the previous generation using two vaccinia carriers expressing Rep78-VP2 and Rep52-VP1 separately.In the initial system, a wild type adenovirus was shown to increase rAAV yield significantly in the production cell line, HeLa cells. We made first improvement of the production system by making a HeLa cell line expressing adenovirus E1A and E1B (HE1 cells). HE1 cells were able to support rAAV replication efficiently without supplying wtAd. Higher rAAV yield in HE1 cells than that in Hela cells in the presence of wtAd were observed.Our new progresses reduced the numbers of vaccinia carrier and adenovirus helper. This new simplified system not only reduce the production cost, but also improved rAAV production yield and make the vector purification easier. We are able to further optimize other parameter for rAAV production such as the MOI of VW22 and Ad, the infection interval between Ad and VW22, the serum level and also the harvest time. In summary, the new development in using cytoplasmic carrier for packaging rAAV vectors showed great promise for a new system which is scalable, reliable, efficient and versatile for producing any serotype of AAV vectors.