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Updated: Jan 18, 2026
Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System
Published on: January 13, 2022
Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant
Yang Wu1, Ting Mei1, Liangyu Jiang1
1State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, Brain Research Center, Wuhan Institute of Physics and Mathematics, Center for Excellence in Brain Science and Intelligent Technology, Chinese Academy of Sciences, Wuhan, P.R. China.
Abstract:
Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 105 vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.
