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 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.

Conway JE ，Rhys CM ，Zolotukhin I ，Zolotukhin S ，Muzyczka N ，Hayward GS ，Byrne BJ ... -  《GENE THERAPY》

Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids.

Booth MJ ，Mistry A ，Li X ，Thrasher A ，Coffin RS ... -  《GENE THERAPY》

Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

Conway JE ，Zolotukhin S ，Muzyczka N ，Hayward GS ，Byrne BJ ... -  《-》

Selective Rep-Cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines.

Liu X ，Voulgaropoulou F ，Chen R ，Johnson PR ，Clark KR ... -  《MOLECULAR THERAPY》

Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome.

Chadeuf G ，Favre D ，Tessier J ，Provost N ，Nony P ，Kleinschmidt J ，Moullier P ，Salvetti A ... -  《JOURNAL OF GENE MEDICINE》