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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 各种遗传策略，包括shRNA或病毒进入抑制剂，已被证明可以抑制HIV-1的体外复制。 将这些有效的体外策略转化为临床有效的治疗在很大程度上受到限制，这是由于体内可获得的遗传修饰细胞水平低。 然而，在治疗性基因的表达可以保护转导的细胞免受致病作用的临床情况下，转导的细胞可以具有选择性增殖优势并重新填充细胞区室。 为了评估各种HIV基因治疗策略保护表达遗传抑制剂的细胞免受病毒诱导的细胞病变并提供体外选择性优势的潜力，用表达GFP的慢病毒载体与HIV-1达特/Rev特异性小发夹（sh）RNA或病毒进入抑制剂M87 o一起沿着转导CD 4+细胞系CEMx 174。 在用HIV-1感染后，CEMx 174-M870群体中GFP+细胞的百分比从30%增加到大于95%，但在未感染的细胞中以及在感染的CEMx 174-GFP细胞和CEMx 174-sh（达特/rev）细胞中没有变化。 为了评估选择性生长的下限，在HIV-1 NL 4 -3感染后，在CEMx 174-M870和未转导细胞的混合物中检查病毒复制和GFP表达，范围为1%至100%。 21天后，CEMx 174-M870混合物中GFP+细胞的百分比从1%增加到大于95%。 HIV-1 p24 Gag的细胞内表达在转导细胞中增加至第9天，但随后在第21天消退。 这些结果证明了用病毒进入抑制剂M87 o非常有效地抑制HIV-1病毒复制，并且重要的是，体外转导细胞的存活优势。

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.