SURVIVAL OF THE FITTEST: CHALLENGING TRANSDUCED CELLS WITH HIV-1 REPLICATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 各种遗传策略，包括shRNA或病毒进入抑制剂，已被证明在体外抑制HIV-1复制。将这些有效的体外策略转化为临床有效的治疗方法在很大程度上受到限制，这在很大程度上是因为体内可以实现的转基因细胞水平很低。然而，在临床情况下，治疗性基因的表达可能会保护转导细胞免受致病效应的影响，转导细胞可能具有选择性增殖优势，并重新填充细胞室。为了评估各种HIV基因治疗策略保护表达基因抑制物的细胞免受病毒诱导的细胞病变的可能性，并在体外提供选择性优势，将表达GFP的慢病毒载体与HIV-1 TAT/rev特异性小发夹(Sh)RNA或病毒进入抑制剂M87o一起转导到CD4+细胞系CEMx174。感染HIV-1后，CEMx174-M87o细胞中GFP+细胞的百分比从30%增加到95%以上，但在未感染细胞、感染的CEMx174-GFP细胞和CEMx174-sh(Tat/rev)细胞中GFP+细胞的百分比没有变化。为了评估选择性生长的下限，检测了HIV-1NL4-3感染后CEMx174-M87o和未转导细胞的混合细胞中的病毒复制和GFP表达。21天后，CEMx174-M87o混合物中GFP+细胞的百分率从1%上升到95%以上。在转导细胞中，HIV-1 p24 Gag的细胞内表达在第9天增加，但在第21天减弱。这些结果表明，病毒进入抑制剂M87o非常有效地抑制了HIV-1病毒的复制，重要的是，转导细胞在体外具有生存优势。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A variety of genetic strategies, including shRNA or viral entry inhibitors, have been documented to inhibit HIV-1 replication in vitro. Translation of these effective in vitro strategies to clinically efficacious treatments has been limited in large part due to the low levels of genetically modified cells that can be achieved in vivo. However, in clinical scenarios where expression of the therapeutic gene may protect transduced cell from pathogenic effects, the transduced cells may have a selective proliferative advantage and repopulate the cellular compartment. To assess the potential of various HIV gene therapy strategies to protect cells expressing genetic inhibitors from virus-induced cytopathicity and to provide for a selective advantage in vitro, the CD4+ cell line CEMx174 was transduced with lentiviral vectors expressing GFP along with either a HIV-1 tat/rev-specific small hairpin (sh)RNA or the viral entry inhibitor M87o. After infection with HIV-1, the percentage of GFP+ cells in the CEMx174-M87o population increased from 30% to greater than 95%, but was unchanged in uninfected cells, and in infected CEMx174-GFP cells and CEMx174-sh(tat/rev) cells. To assess the lower limit for selective outgrowth, viral replication and GFP expression were examined in mixtures of CEMx174-M87o and untransduced cells ranging from 1% to 100% following HIV-1 NL4-3 infection. After 21 days, the percentage of GFP+ cells in the CEMx174-M87o mixture increased from 1% to greater than 95%. Intracellular expression of HIV-1 p24 Gag increased in transduced cells through day 9, but then receded by day 21. These results demonstrate very potent inhibition of HIV-1 viral replication with the viral entry inhibitor M87o and, importantly, a survival advantage of transduced cells in vitro.