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

Gene Therapy volume 16, pages 229–239 (2009)Cite this article

Abstract

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 α-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 × 1011 DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 × 104 and 9.4 × 105 ng ml−1 serum respectively, the latter being clinically relevant.

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Acknowledgements

We thank Dr Barry Byrne (Department of Pediatrics), Irene Zolotukhin, Dr Nicholas Muzyczka, Dr Richard O Snyder and Dr Brian Cleaver (Molecular Genetics and Microbiology Department) of the University of Florida Powell Gene Therapy Center for technical discussions and helpful comments on the manuscript.

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  1. H Harrell

    Present address: 2Current address: Florida Biologix, 13706 Innovation Drive, Alachua, FL 32615, USA.,

  2. T L Mayfield

    Present address: 3Current address: Monsanto Company, 800 N. Lindbergh Blvd., St Louis, MO 63167, USA.,

Authors and Affiliations

  1. Applied Genetic Technologies Corporation, Alachua, FL, USA

    W Kang, L Wang, H Harrell, J Liu, D L Thomas, T L Mayfield, M M Scotti, G J Ye, G Veres & D R Knop

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Correspondence to D R Knop.

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Kang, W., Wang, L., Harrell, H. et al. An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes. Gene Ther 16, 229–239 (2009). https://doi.org/10.1038/gt.2008.158

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