A Robust System for Production of Superabundant VP1 Recombinant AAV Vectors

Qizhao Wang; Zhongren Wu; Junping Zhang; Jenni Firrman; Hongying Wei; Zhengjing Zhuang; LinShu Liu; Linqing Miao; Yang Hu; Dong Li; Yong Diao; Weidong Xiao

doi:10.1016/j.omtm.2017.11.002

A Robust System for Production of Superabundant VP1 Recombinant AAV Vectors

Mol Ther Methods Clin Dev. 2017 Nov 7:7:146-156. doi: 10.1016/j.omtm.2017.11.002. eCollection 2017 Dec 15.

Authors

Qizhao Wang^{1

2

3}, Zhongren Wu², Junping Zhang², Jenni Firrman^{4

5}, Hongying Wei², Zhengjing Zhuang¹, LinShu Liu⁵, Linqing Miao⁶, Yang Hu³, Dong Li⁷, Yong Diao¹, Weidong Xiao^{1

2

4

8

7}

Affiliations

¹ School of Biomedical Sciences, Huaqiao University, Quanzhou, Fujian, China.
² Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, PA, USA.
³ Byers Eye Institute, Stanford University School of Medicine, Stanford, CA, USA.
⁴ Department of Microbiology and Immunology, Temple University, Philadelphia, PA, USA.
⁵ United States Department of Agriculture, ARS, ERRC, Wyndmoor, PA, USA.
⁶ Shriners Hospital Pediatric Research Center, Temple University, Philadelphia, PA, USA.
⁷ Department of Clinical Laboratory, Tongji Hospital of Tongji University, Shanghai, China.
⁸ Cardiovascular Research Center, Temple University, Philadelphia, PA, USA.

Abstract

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 trans 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 cis 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 × 10¹⁵ 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 in vivo after intravitreal injection.

Keywords: AAV; VP1; gene therapy; vaccinia virus; vector production.

Abstract

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