Bowles N E, Eisensmith R C, Mohuiddin R, Pyron M, Woo S L
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Hum Gene Ther. 1996 Sep 10;7(14):1735-42. doi: 10.1089/hum.1996.7.14-1735.
Although recombinant retroviruses have been widely used for the transduction of target organs in vivo, the viral titers achieved by current production methods are often too low to achieve therapeutic levels of gene expression. To overcome this limitation, a simple method for the efficient concentration and purification of amphotropic retrovirus particles was developed. After portal vein infusion into partially hepatectomized rats of 5.5 x 10(7) cfu of a beta-galactosidase (beta-gal)-expressing retrovirus (LX/beta geo) concentrated by this method, up to 25% of hepatocytes stained positive for beta-Gal activity. Measurement of human alpha 1-antitrypsin (hAAT) levels after infusion of various doses of a similarly concentrated retrovirus encoding hAAT (LX/hAAT) demonstrated that viral transduction increased proportionally with titer, up to a dose of 7.5 x 10(7) cfu per rat. The ability to concentrate retroviral virion efficiently from large volumes of supernatant has allowed the further purification of virus particles by sucrose banding ultracentrifugation. This procedure results in a greater than 50% recovery of infectious virus particles, with titers up to 500-fold higher than in the original supernatant. These methods may have significant utility in both ex vivo and in vivo retroviral applications in human gene therapy.
尽管重组逆转录病毒已被广泛用于体内靶器官的转导，但目前生产方法所获得的病毒滴度往往过低，无法达到治疗水平的基因表达。为克服这一限制，开发了一种高效浓缩和纯化嗜双性逆转录病毒颗粒的简单方法。将通过该方法浓缩的表达β-半乳糖苷酶（β-gal）的逆转录病毒（LX/βgeo）5.5×10⁷cfu经门静脉注入部分肝切除的大鼠后，高达25%的肝细胞β-Gal活性染色呈阳性。在注入不同剂量的类似浓缩的编码人α1-抗胰蛋白酶（hAAT）的逆转录病毒（LX/hAAT）后，对hAAT水平的测量表明，病毒转导与滴度成比例增加，每只大鼠的剂量高达7.5×10⁷cfu。能够从大量上清液中高效浓缩逆转录病毒粒子，使得通过蔗糖密度梯度超速离心进一步纯化病毒粒子成为可能。该程序可使感染性病毒粒子的回收率超过50%，滴度比原始上清液高500倍。这些方法在人类基因治疗的体外和体内逆转录病毒应用中可能具有重要用途。
