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, Gary Selwyn Hayward, B. J. Byrne

Research output: Contribution to journalArticle

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

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Dependovirus
Simplexvirus
Cell Line
Helper Viruses
Proviruses
Coinfection
Genes
Transfection
Plasmids
Cell Culture Techniques

Keywords

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

ASJC Scopus subject areas

  • Genetics

Cite this

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 rep and cap. / Conway, J. E.; Ap Rhys, C. M J; Zolotukhin, I.; Zolotukhin, S.; Muzyczka, N.; Hayward, Gary Selwyn; Byrne, B. J.

In: Gene Therapy, Vol. 6, No. 6, 06.1999, p. 986-993.

Research output: Contribution to journalArticle

Conway, J. E. ; Ap Rhys, C. M J ; Zolotukhin, I. ; Zolotukhin, S. ; Muzyczka, N. ; Hayward, Gary Selwyn ; Byrne, B. J. / High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 rep and cap. In: Gene Therapy. 1999 ; Vol. 6, No. 6. pp. 986-993.
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