Popularizing Recombinant Baculovirus-derived OneBac System for Laboratory Production of all Recombinant Adeno-associated Virus Vector Serotypes

Abstract

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.

Keywords: OneBac system; gene therapy; rAAV; recombinant baculovirus; scaling-up production; serotype.