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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.
A new OneBac system enhances recombinant adeno-associated virus (rAAV) production. This improved system offers flexibility for large-scale gene therapy vector manufacturing with high yield and quality.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Recombinant adeno-associated viruses (rAAVs) are key vectors for gene delivery.
- Current OneBac systems using Sf9/Cap-Rep packaging cell lines lack versatility for large-scale rAAV production.
Purpose of the Study:
- To develop an improved OneBac system for versatile and flexible large-scale rAAV production.
- To enhance the efficiency and applicability of rAAV manufacturing.
Main Methods:
- Developed a novel dual-function baculovirus expression vector (BEV/Cap-(ITR-GOI)) carrying AAV Cap gene and ITR-flanked gene of interest.
- Created a versatile Sf9-GFP/Rep packaging cell line with silent AAV2 Rep gene copies and GFP reporters.
- Utilized BEV reconstruction for switching Cap gene serotypes and assessed cell line versatility.
Main Results:
- The improved OneBac system demonstrated flexibility for switching rAAV serotypes.
- The Sf9-GFP/Rep cell line enabled production of various rAAV serotypes with yields exceeding 10^5 vector genomes per cell.
- rAAVs produced using the new system exhibited biophysical properties, quality, and activity comparable to HEK293 cell-derived rAAVs.
Conclusions:
- The novel Sf9-GFP/Rep packaging cell line-dependent OneBac system facilitates large-scale rAAV production.
- This system offers a versatile platform for advancing rAAV-based gene therapy manufacturing.
