title: High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.
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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Conway
authors.forename: C M
authors.initials: CM
authors.lastname: Rhys
authors.forename: I
authors.initials: I
authors.lastname: Zolotukhin
authors.forename: S
authors.initials: S
authors.lastname: Zolotukhin
authors.forename: N
authors.initials: N
authors.lastname: Muzyczka
authors.forename: G S
authors.initials: GS
authors.lastname: Hayward
authors.forename: B J
authors.initials: BJ
authors.lastname: Byrne
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D000229:Dependovirus; D005455:Fluorescent Antibody Technique; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D018139:Simplexvirus; D014162:Transfection; D014773:Virology / Q000379:methods*; D014779:Virus Replication