Gao G P, Qu G, Faust L Z, Engdahl R K, Xiao W, Hughes J V, Zoltick P W, Wilson J M
Institute for Human Gene Therapy, Department of Molecular and Cellular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Hum Gene Ther. 1998 Nov 1;9(16):2353-62. doi: 10.1089/hum.1998.9.16-2353.
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 E1 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.
腺相关病毒（AAV）是一种用于体内基因治疗的潜在载体。对其效用的关键分析受到生产方法的阻碍，这些方法效率低下、难以扩大规模，并且常常产生大量具有复制能力的AAV。我们描述了一种解决这些问题的生产AAV的新方法。通过利用内源性AAV启动子将含rep/cap的质粒稳定转染到HeLa细胞中，创建了一种名为B50的细胞系。AAV的生产分两步进行。用E2b缺陷型腺病毒感染B50，以诱导Rep和Cap表达并提供辅助功能，随后用一种杂交病毒感染，其中AAV载体克隆在复制缺陷型腺病毒的E1区域。这导致AAV基因组扩增100倍并拯救出来，从而产生高产的不含具有复制能力的AAV的重组AAV。将编码促红细胞生成素的载体肌肉注射到小鼠骨骼肌中，导致血清中激素水平超过生理水平，并持续存在且引起红细胞增多症。这种AAV生产方法在扩大规模用于包括人类在内的大型动物研究中应该是有用的。
