Seppen J, Barry S, Lam G M, Ramesh N, Osborne W R
Department of Pediatrics, University of Washington School of Medicine, Seattle 98195, USA.
Hum Gene Ther. 2000 Mar 20;11(5):771-5. doi: 10.1089/10430340050015662.
Obtaining high expression levels of a therapeutic gene in target cells could be achieved by integrating multiple copies of a recombinant retrovirus. However, we observed that cells retrovirally infected at high multiplicities of infection (MOIs) carried only single or double integrated proviral copies, suggesting that maximum retroviral transduction was achieved at relatively low MOIs. The same results were obtained when purified virus, free of most medium components, was used. Retroviral infection was shown to be inhibited by supernatants of other viral producer cell lines, and this inhibition could be removed by a centrifugation step that also removed more than 90% of infectious virus. Quantitative-competitive PCR of retroviral preparations showed that the amount of retroviral vector RNA present was similar to the amount expected on the basis of virus titers. Our data suggest that retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with retroviruses and do not contain viral vector RNA. We postulate that these inhibitor particles cannot achieve a productive infection but interfere with transduction of the target cells by infectious virions. This study might define an important criterion for the selection of more effective packaging cell lines.
通过整合重组逆转录病毒的多个拷贝，可以在靶细胞中获得治疗性基因的高表达水平。然而，我们观察到，在高感染复数（MOI）下进行逆转录病毒感染的细胞仅携带单拷贝或双拷贝整合的前病毒，这表明在相对较低的MOI下可实现最大程度的逆转录病毒转导。使用不含大多数培养基成分的纯化病毒时，也得到了相同的结果。已证明其他病毒生产细胞系的上清液可抑制逆转录病毒感染，并且这种抑制作用可通过离心步骤消除，该离心步骤还可去除90%以上的感染性病毒。对逆转录病毒制剂进行的定量竞争PCR表明，存在的逆转录病毒载体RNA量与根据病毒滴度预期的量相似。我们的数据表明，源自PA317包装细胞的逆转录病毒制剂含有与逆转录病毒共纯化的抑制剂，并且不含有病毒载体RNA。我们推测这些抑制剂颗粒无法实现有效感染，但会干扰感染性病毒粒子对靶细胞的转导。这项研究可能为选择更有效的包装细胞系定义一个重要标准。
