High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus
High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus
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DOI：
10.1089/hum.1998.9.16-2353
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发表时间：
1998-11-01
影响因子：
4.2
通讯作者：
Wilson, JM
中科院分区：
文献类型：
--
作者：
Gao, GP;Qu, G;Wilson, JM
腺相关病毒(AAV)是一种潜在的体内基因治疗载体。我们描述了一种生产AAV的新方法，该方法解决了这些问题，称为B50，是通过利用内源性AAV启动子稳定地将含有rep/帽的质粒导入HeLa细胞而建立的，AAV的生产分两步进行。B50感染E2b缺陷的腺病毒，以诱导Rep和Cap的表达并提供辅助功能，然后是一种混合病毒，其中AAV载体被克隆在复制缺陷型腺病毒的E1区。这导致了AAV基因组的100倍扩增和挽救，导致了不具有复制能力的AAV的高产量重组AAV，肌肉注射编码促红细胞生成素的载体到小鼠骨骼肌导致血清中激素的超生理水平持续并导致红细胞增多症，这种AAV生产的方法在包括人类在内的大型动物的研究中应该是有用的。
Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV, We describe a novel method for producing AAV that addresses these problems, A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters, Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the El region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV, Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia, This method of AAV production should be useful in scaling up for studies in large animals, including humans.