For the three serotypes tested, AAV9 yields increased ~7-fold, AAV2 yields increased ~15-fold, and AAV8 yields increased ~19-fold. AAV manufacturing amenable for preclinical and clinical applications. and Sf9 cells are grown in suspension in serum-free media and can reach higher cell densities than HEK293 cells. Third, Sf9 cells do not support the replication of potentially contaminating human agents. Lastly, baculoviruses cannot replicate in humans. These characteristics increase the biosafety profile of Sf9-derived AAV vectors for preclinical and clinical applications. Indeed, this insect cell-based expression system was used for the AAV gene-therapy drug Glybera, which was approved by the European Medicines Agency (EMA) in 2012.11 The use of baculoviruses to produce infectious AAV in insect cells was reported by Dr. Kotins group12 in 2002. Raxatrigine hydrochloride Briefly, Sf9 cells were infected with three different recombinant baculoviruses to produce AAV2: one containing the AAV2 Rep gene, another containing the AAV2 Cap gene, and the last containing the transgene cassette flanked by the AAV2 inverted terminal repeat (ITR) elements. In the following years, further modifications were made to both simplify and improve the stability of the system by placing the Rep and Cap genes into one dual cassette and removing homologous regions in the Rep construct.13 This improved system uses attenuated start codons for both VP1 and Rep78 to utilize leaky scanning of the?ribosome and promote translation of the downstream products.14, 15, 16 Other modifications were also made in between the Rep78 and Rep52 start codons. Recently, Thermo Fisher Scientific developed the ExpiSf Baculovirus Expression System, which is the first-ever, clinically applicable, chemically defined (CD) insect protein expression system, which can achieve a 3 higher protein yield.17 In this study, we assessed AAV production in the ExpiSf Baculovirus Expression System. With the same baculoviral stocks, we infected both Sf9 and ExpiSf9 cells with the same multiplicity of infection (MOI) to compare the differences in AAV vector Raxatrigine hydrochloride yield. In addition, we characterized the purified AAVs from both cell lines and for their transduction efficiency. Results Generation of a Baculovirus and Sf9 Cell-Based AAV Production System We designed and generated baculovirus constructs as shown in Figure?1A. Specifically, our two baculoviral vectors harbor either the transgene cassette flanked by the AAV2 ITRs or the AAV2 Rep (Rep2) and Cap genes of various serotypes with attenuated start codons for both VP1 and Rep78 to utilize leaky scanning of the ribosome and promote translation of the downstream products, as previously reported.13 After expansion of the baculoviruses in Sf9 cells, we obtained titers of approximately 1C2? 108 plaque forming units (PFUs) per milliliter. Open in a separate window Figure?1 Design of Constructs and Confirmation of Infectivity (A) The packaging construct (top) containing both the AAV2 Rep and Cap genes from AAV2, AAV8, or AAV9 were cloned into a pFastBac Dual vector. The Cap cassette is driven by the p10 promoter and contains the herpes simplex virus (HSV) thymidine kinase (tk) polyadenylation signal (pA). The Rep cassette is driven by the polyhedrin promoter (pH) and contains the simian virus 40 (SV40) pA. The transgene construct (bottom) contains either the ZsGreen or luciferase gene, which is driven by the cytomegalovirus (CMV) promoter. It also contains a beta-globin (B-globin) intron and a human growth hormone (hGH) pA signal. Lastly, the transgene cassette is flanked by the AAV2 inverted terminal repeat (ITR). (B) Purified Raxatrigine hydrochloride AAV2 containing the ZsGreen transgene cassette was used to infect 293T cells at an MOI Raxatrigine hydrochloride of 200. The cells were ITGA8 then imaged for ZsGreen expression 48?h.