An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes

被引：44

作者：

Kang, W. ^{[1
]}

Wang, L. ^{[1
]}

Harrell, H. ^{[1
]}

Liu, J. ^{[1
]}

Thomas, D. L. ^{[1
]}

Mayfield, T. L. ^{[1
]}

Scotti, M. M. ^{[1
]}

Ye, G. J. ^{[1
]}

Veres, G. ^{[1
]}

Knop, D. R. ^{[1
]}

机构：

[1] Appl Genet Technol Corp, Alachua, FL 32615 USA

来源：

GENE THERAPY | 2009年 / 16卷 / 02期

关键词：

adeno-associated virus; herpes simplex virus; viral vector; co-infection; scalable rAAV production; HERPES-SIMPLEX-VIRUS; ADENO-ASSOCIATED VIRUS; MEDIATED GENE-TRANSFER; ADENOASSOCIATED VIRUS; REP GENE; CELL-LINES; FACTOR-IX; EXPRESSION; AAV; TYPE-1;

D O I：

10.1038/gt.2008.158

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14: 1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human alpha-1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 x 10(11) DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 x 10(4) and 9.4 x 10(5) ng ml(-1) serum respectively, the latter being clinically relevant.

引用

页码：229 / 239

页数：11

共 59 条

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