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High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap

Gene Therapy volume 6, pages 986–993 (1999)Cite this article

Abstract

Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27.1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health to BJB, POI, HL59412 and NCI T32-CA-09243 to JEC. We gratefully acknowledge the gifts of d27.1, the V27 cell line and P Ab 3–83 from D Knipe, and the C12 cell line from PR Johnson. We also appreciate the assistance of Jennifer Hussey in infectious center assays and the suggestions of Kye Chesnut in the preparation of this manuscript.

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Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    J E Conway, CMJ ap Rhys & G S Hayward

  2. Department of Molecular Genetics and Microbiology, Gene Therapy Center, Gainesville, FL, USA

    I Zolotukhin, S Zolotukhin, N Muzyczka & B J Byrne

  3. Pediatrics, University of Florida, Gainesville, FL, USA

    I Zolotukhin & B J Byrne

  4. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    G S Hayward

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  1. J E Conway
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Conway, J., Rhys, C., Zolotukhin, I. et al. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. Gene Ther 6, 986–993 (1999). https://doi.org/10.1038/sj.gt.3300937

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