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Updated: Nov 21, 2025
Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System
Published on: January 13, 2022
Popularizing Recombinant Baculovirus-derived OneBac System for Laboratory Production of all Recombinant
Yang Wu1, Zengpeng Han1, Mingzhu Duan1
1State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, 430071, China.
Background:
Recombinant adeno-associated virus (rAAV) has been widely used as an efficient transgenic vector in biomedical research, as well as gene therapy. Serotype-associated transduction efficiency, tissue- or cell-type tropism and immunological profile are major considerations in the various applications of rAAVs. There are increasing needs for different serotypes of rAAV, either naturally isolated or artificially engineered. However, affordable and scalable production of a desired serotype of rAAV remains very difficult, especially for researchers lacking relevant experience.
Objective:
On the basis of our previously established single recombinant baculovirus expression vector (BEV)-derived OneBac system, we have optimized the process and expanded the rAAV production range to the full range of serotypes rAAV1-13.
Methods:
Firstly, the AAV Cap gene was optimized to translate by ribosome leaky scanning and the gene of interest (GOI) was cloned into the pFD/Cap-(ITR-GOI)-Rep2 shuttle plasmid. Following the classical Bac-to-Bac method, sufficient BEV stock containing all rAAV packaging elements can be quickly obtained. Finally, we can repeatedly scale up the production of rAAVs in one week by using a single BEV to infect suspension-cultured Sf9 cells. The rAAV1-13 shows relatively high yields ranging from 5×104 to 4×105 VG/cell. More than 1×1015 VG purified rAAVs can be easily obtained from 5 L suspension-cultured Sf9 cells.
Results:
As expected, rAAV serotypes 1-13 show different potencies for in vitro transduction and cell-type tropisms.
Conclusion:
In summary, the single BEV-derived OneBac system should prove popular for laboratory scaling-up production of any serotype of rAAV.
