Production of adeno-associated virus mediated by Bac-to-Bac baculovirus insect expression system

Objective To establish an efficient and safe method for production of recombinant adeno-associated viruses (rAAV) of clinical scale.Methods The 2053 bp of rAAV inverted terminal repeat (ITR) and foreign gene expression cassette was cloned into baculovirus vector as cis-acting vector.The capsid and replication proteins of rAAV serum type 2 were expressed in a trans-acting baculovirus vector (pFastBacDual) by leaky scanning mechanism of eukaryotic gene transcription.Both constructs were made into baculovirus which infected insect cells st9 for packaging rAAV.Enhanced green fluorescent protein (EGFP) was used as reporter gene to verify the feasibility of system.Results Transposition of cis-acting and trans-acting vector into bacmid was carried out in DH10BacTM reswctively.Two baculoviruses were prepared in insect cells sf9 and the titers were about 1×108-3×108 v.g./mL.rAAV were rescued,replicated and packaged by co-infection of two baculoviruses into sf9.The biological activity of rAAV-EGFP was proved by infecting 293T cells.Conclusion This system provides an experimental data for making rAAV of clinical scale and a good basis for future gene therapy. Key words: Baculovirus insect expression system; Adeno-associated viruses; sf9 cells