A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo
A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo
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DOI：
10.1089/hum.1996.7.14-1735
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发表时间：
1996-09-10
影响因子：
4.2
通讯作者：
Woo, SLC
中科院分区：
文献类型：
--
作者：
Bowles, NE;Eisensmith, RC;Woo, SLC
尽管重组逆转录病毒已被广泛用于体内靶器官的转导，但通过当前生产方法获得的病毒滴度通常太低而不能达到治疗水平的基因表达。为了克服这种限制，开发了一种用于有效浓缩和纯化嗜异性逆转录病毒颗粒的简单方法。肝部分切除大鼠门静脉输注5.5 × 10（7）cfu β-半乳糖苷酶后（β-gal）表达逆转录病毒（LX/β geo），高达25%的肝细胞对β-Gal活性染色呈阳性，输注不同剂量的类似浓缩的编码hAAT的逆转录病毒（LX/hAAT）后人α（1）-抗胰蛋白酶（hAAT）水平的测量证明病毒转导与滴度成比例增加，剂量高达每只大鼠7.5 x 10（7）cfu。从大体积上清液中有效浓缩逆转录病毒粒子的能力允许通过蔗糖条带超离心进一步纯化病毒颗粒。该方法导致感染性病毒颗粒的回收率大于50%，滴度比原始上清液高500倍。这些方法在人基因治疗中的离体和体内逆转录病毒应用中可能具有显著的实用性。
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.