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Clearance and characterization of residual HSV DNA in recombinant adeno-associated virus produced by an HSV complementation system

Gene Therapy volume 18, pages 135–144 (2011)Cite this article

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Abstract

Encapsidation of cellular- or plasmid-derived DNA sequences during recombinant adeno-associated virus (rAAV) production has been well documented. However, most of the published data were generated from rAAV vectors manufactured by the plasmid transient transfection method. We previously reported a novel, scalable method for rAAV manufacturing based on a recombinant herpes simplex virus (rHSV) complementation system. In this report, we evaluated clearance of DNA impurities during rAAV purification, by determining the quantity of residual herpes simplex virus and cellular DNA at each process step. A single Benzonase treatment during the upstream process effectively reduced unprotected HSV and cellular DNA to <300 bp fragments, and subsequent chromatography steps completely removed these small DNA fragments. Further analysis showed that trace amounts of residual, DNase-resistant HSV and cellular DNA were present at static concentrations during subsequent purification steps, and the residual HSV DNA sequences were single stranded, ranging from 0.8 to 4.2 kb. After transduction of human embryonic kidney 293 cells with purified rAAV, the residual HSV DNA fragments were neither transcribed nor translated into HSV proteins. In summary, this manufacturing process for rAAV production was effective in removing DNA and protein contaminants and achieving a highly purified product, suitable for human clinical application.

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Acknowledgements

We thank Dr Bernard Roizman (Department of Molecular Genetics and Cell Biology, the University of Chicago) for cosmids containing HSV DNA, Dr Nicholas Muzyczka (Department of Molecular Genetics and Microbiology, the University of Florida), and Dr Jeffery D Chulay (AGTC) for technical discussions and constructive comments on the manuscripts. We also thank Dr Darby L Thomas (AGTC) for technical assistance in BHK cellular DNA quantification.

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

    G-J Ye, M M Scotti, J Liu, L Wang, D R Knop & G Veres

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  1. G-J Ye
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Correspondence to G-J Ye.

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G-J Ye, J Liu and DR Knop hold share options in AGTC and have a conflict of interest to the extent that this work potentially increases their personal financial interests.

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Ye, GJ., Scotti, M., Liu, J. et al. Clearance and characterization of residual HSV DNA in recombinant adeno-associated virus produced by an HSV complementation system. Gene Ther 18, 135–144 (2011). https://doi.org/10.1038/gt.2010.102

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