Title:Popularizing Recombinant Baculovirus-derived OneBac System for Laboratory Production of all Recombinant Adeno-associated Virus Vector Serotypes
Volume: 21
Issue: 2
关键词:
rAAV，重组杆状病毒，OneBac系统，规模化生产，血清型，基因治疗。
摘要:
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.
