A Recombinant Baculovirus Efficiently Generates Recombinant Adeno-Associated Virus Vectors in Cultured Insect Cells and Larvae

Abstract

Current large-scale recombinant adeno-associated virus (rAAV) production systems based on the baculovirus expression vector (BEV) remain complicated and cost-intensive, and they lack versatility and flexibility. Here we present a novel recombinant baculovirus integrated with all packaging elements for the production of rAAV. To optimize BEV construction, ribosome leaky-scanning mechanism was used to express AAV Rep and Cap proteins downstream of the PH and P10 promoters in the pFast.Bac.Dual vector, respectively, and the rAAV genome was inserted between the two promoters. The yields of rAAV2, rAAV8, and rAAV9 derived from the BEV-infected Sf9 cells exceeded 105 vector genomes (VG) per cell. The BEV was shown to be stable and showed no apparent decrease of rAAV yield after at least four serial passages. The rAAVs derived from the new Bac system displayed high-quality and high-transduction activity. Additionally, rAAV2 could be efficiently generated from BEV-infected beet armyworm larvae at a per-larvae yield of 2.75 ± 1.66 × 1010 VG. The rAAV2 derived from larvae showed a structure similar to the rAAV2 derived from HEK293 cells, and it also displayed high-transduction activity. In summary, the novel BEV is ideally suitable for large-scale rAAV production. Further, this study exploits a potential cost-efficient platform for rAAV production in insect larvae.

Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production

“…17 In 2018, Wu et al created a single BEV/Cap-(ITR-GOI)-Rep that integrated all of the elements for rAAV production in both cultured Sf9 cells and armyworm larvae. 18 The rAAV yield level in this system was similar to the current highest state upon infection of Sf9 cells. Moreover, this research was the first to exploit a potential low-cost rAAV production method in insect larvae.…”

“…16 The pFD/Rep, pFD/Cap2, pFD/Cap8, and pFD/Cap9 and pFD/Cap2-(ITR-GFP) constructs were described previously. 18 In brief, the Cap gene was inserted downstream from the P10 promoter in the pFast.Bac.dual (pFD) plasmid, and translated using atypical initiation codons (CTG for VP1, ACG for VP2, and ATG for VP3) to generate VP1, VP2, and VP3 in a stoichiometry of around 1:1:10 from one transcript without alternate splicing. 9,12 Meanwhile, we digested the pFD plasmid with Mro I and Spe I to insert the ITR-GOI adjacent to the Cap gene.…”

AAV9-Retro mediates efficient transduction with axon terminal absorption and blood–brain barrier transportation

“…The various AAV serotype vectors, including AAV2-Retro-CaMKIIa-EGFP, AAV9-Retro-CaMKIIa-EGFP and AAV2-Retro-CaMKIIa-mCherry, were produced by a baculovirus-AAV expression vector system [59] and purified by iodixanol gradient ultracentrifugation [60,61]. The purified rAAVs were titred by qPCR using the iQ SYBR Green Supermix kit (Bio-Rad) and diluted to 1.0 × 10 13 viral particles/mL.…”

AAV9-Retro mediates efficient transduction with axon terminal absorption and blood–brain barrier transportation

“…The various AAV serotype vectors, including AAV2-Retro-CaMKIIa-EGFP, AAV9-Retro-CaMKIIa-EGFP and AAV2-Retro-CaMKIIa-mCherry, were produced by a baculovirus-AAV expression vector system [59] and puri ed by iodixanol gradient ultracentrifugation [60,61]. The puri ed rAAVs were titred by qPCR using the iQ SYBR Green Supermix kit (Bio-Rad) and diluted to 1.0 × 10 13 viral particles/mL.…”