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

Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further develo pment of rAAV vectors for clinical use requires significant technological i mprovements in large-scale vector production. In order to facilitate the pr oduction of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the A AV-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 uni ts (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/cel l of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 d erived cell line. Effective amplification of rAAV vectors introduced into 2 93 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 (> 10(9) cell s) 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 provide s 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 preparation s from any rAAV proviral construct. The efficiency and potential for scalab le delivery of d27.1-rc to producer cell cultures should facilitate the pro duction of sufficient quantities of rAAV vectors for clinical application.