Combination of Baculovirus-Mediated Gene Delivery and Packed-Bed Reactor for Scalable Production of Adeno-Associated Virus
- 1 November 2007
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 18 (11), 1161-1170
- https://doi.org/10.1089/hum.2007.107
Abstract
The production of recombinant adeno-associated virus (rAAV) commonly requires plasmid cotransfection, which hinders its mass production. Herein we describe the development of a novel process for rAAV production by combining the advantages of baculovirus-mediated gene delivery and BelloCell bioreactor (a novel packed-bed reactor for animal cell culture; CESCO Bioengineering, Hsinchu, Taiwan). We constructed three baculoviral vectors: Bac-LacZ carries the lacZ gene flanked by AAV inverted terminal repeats, Bac-RC harbors AAV rep and cap genes, and Bac-Helper carries helper genes derived from adenovirus. Cotransduction of HEK-293 cells with these three baculoviruses resulted in successful production of rAAV, and the protein and rAAV yield did not decrease with Bac-RC passage for up to four passages. By adjusting the dose ratio of Bac-LacZ to Bac-RC, adding sodium butyrate, and transferring the production process to the BelloCell-500-AP (500 ml), which allowed for high-density culture and effective baculovirus-mediated transduction of HEK-293 cells, the maximal specific rAAV yield reached ≈3.8 × 104 vector genome (VG) or 247 infectious viral particles (IVP) per cell, which corresponded to ≈1 × 1014 VG or 8.5 × 1011 IVP per reactor run. The yield was comparable or superior to those obtained with other production systems. Baculoviral transduction is simple and cost-effective and the BelloCell-500-AP offers high-density culture of HEK-293 cells. Altogether, the combination of baculoviral transduction and BelloCell reactor culture provides a novel and economically viable approach for rAAV production.This publication has 35 references indexed in Scilit:
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