High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap
High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap
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DOI：
10.1038/sj.gt.3300937
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发表时间：
1999-06-01
期刊：
影响因子：
5.1
通讯作者：
Byrne, BJ
中科院分区：
文献类型：
--
作者：
Conway, JE;ap Rhys, CMJ;Byrne, BJ
重组腺相关病毒2型（rAAV）载体最近已被用于实现长期，高水平的体内转导。进一步开发rAAV载体用于临床需要在大规模载体生产中进行重大技术改进。为了促进rAAV载体的生产，已经将不产生ICP 27的重组单纯疱疹病毒I型载体（rHSV-1）工程化以表达AAV-2 rep和cap基因。已经确定了该载体d27.1-rc用于AAV生产的最佳剂量，并且在用AAV-GFP质粒DNA转染后，从293细胞产生的AAV-GFP的产量为380个表达单位（EU）。此外，d27.1-rc还有效地从具有整合的AAV-GFP前病毒的细胞系产生rAAV。从细胞系GFP-92（前病毒293衍生的细胞系）可以产生高达480 EU/细胞的AAV-GFP。还证明了通过感染引入293细胞的rAAV载体的有效扩增。rAAV与d27.1-rc的传代导致载体共感染后每次传代的AAV-GFP扩增高达200倍。还实现了从前病毒细胞系高效、大规模地生产（> 10（9）个细胞）AAV-GFP，并且这些储备液不含可复制的AAV。所述rHSV-1载体提供了一种新的、简单的和灵活的方式来引入AAV-2 rep和cap基因以及从任何rAAV前病毒构建体产生高滴度rAAV制剂所需的辅助病毒功能。d27.1-rc向生产细胞培养物的可扩展递送的效率和潜力应有助于产生足够量的rAAV载体用于临床应用。
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 (> 10(9) 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.