Wang Qizhao, Wu Zhongren, Zhang Junping, Firrman Jenni, Wei Hongying, Zhuang Zhengjing, Liu LinShu, Miao Linqing, Hu Yang, Li Dong, Diao Yong, Xiao Weidong
School of Biomedical Sciences, Huaqiao University, Quanzhou, Fujian, China.
Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA, USA.
Mol Ther Methods Clin Dev. 2017 Nov 7;7:146-156. doi: 10.1016/j.omtm.2017.11.002. eCollection 2017 Dec 15.
Recombinant adeno-associated viral (rAAV) vectors have been widely used in human gene therapy. One major impediment to its broad application is the inability to produce high-quality vectors in mass quantity. Here, an efficient and scalable suspension cell culture system for the production of rAAV vectors is described. In this system, the AAV factors, Rep78, Rep52, VP1, VP2, and VP3, were stably integrated into a single vaccinia virus carrier by maximizing the use of alternative codons between genes with identical amino acids, and the rAAV genome was carried by an E1/E3 gene-deleted adenovirus. Infection of improved, E1 integrated, suspension-cultured cells with these two viral vectors resulted in the robust production of rAAV vectors. The newly enhanced system can consistently produce ∼1 × 10 genome containing rAAV vectors per liter of suspension cells. Moreover, the capsid composition of rAAV vectors produced by this system is markedly different from those produced using the traditional system in that the VP1 protein is more abundant than the VP2 protein (19:1 versus 1:1). The unique VP1 superabundant rAAV vectors produced in this new system exhibited improved transduction after intravitreal injection.
重组腺相关病毒（rAAV）载体已广泛应用于人类基因治疗。其广泛应用的一个主要障碍是无法大量生产高质量的载体。在此，描述了一种用于生产rAAV载体的高效且可扩展的悬浮细胞培养系统。在该系统中，通过最大限度地利用具有相同氨基酸的基因之间的替代密码子，将AAV因子Rep78、Rep52、VP1、VP2和VP3稳定整合到单个痘苗病毒载体中，rAAV基因组由E1/E3基因缺失的腺病毒携带。用这两种病毒载体感染经过改良的、整合了E1的悬浮培养细胞，可大量生产rAAV载体。新的增强系统每升悬浮细胞能够持续生产约1×10个含有rAAV载体的基因组。此外，该系统生产的rAAV载体的衣壳组成与传统系统生产的明显不同，在于VP1蛋白比VP2蛋白更丰富（19:1对1:1）。在新系统中产生的独特的VP1超量rAAV载体在玻璃体内注射后表现出改善的转导效果。
