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

J. E. Conway, C. M.J. Ap Rhys, I. Zolotukhin, S. Zolotukhin, N. Muzyczka, G. S. Hayward, B. J. Byrne

Research output: Contribution to journalArticlepeer-review

126 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)986-993
Number of pages8
JournalGene Therapy
Volume6
Issue number6
DOIs
StatePublished - Jun 1999
Externally publishedYes

Keywords

  • Adeno-associated virus
  • Gene therapy
  • Herpes simplex virus
  • Vector production
  • rAAV

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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