EVALUATION OF INHIBITION OF SHIV REPLICATION BY SIRNA VECTORS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。将能够抑制HIV复制的基因引入造血干细胞提供了长期免疫重建的可能性。然而，多个伦理和实际的考虑显着限制的能力，以解决有关艾滋病的干细胞基因治疗在人体临床试验的基本问题。因此，在非人灵长类动物中的实验提供了在体内实验模型中严格解决这些问题的机会。许多最近的报道强调了小干扰RNA（siRNA）分子抑制病毒复制的潜在效用。与J. Rossi合作，我们启动了针对达特和Rev的siRNA对各种SHIV毒株的抑制研究。我们获得了两种siRNA（一种靶向达特和Rev，另一种仅靶向Rev）和一种对照序列，每种均受聚合酶III启动子U6的转录调控。使用瞬时转染，si（I）和si（II）均强烈抑制HIV-1（如前所示）、SHIV 89.6p和SHIV Hxbc 2 3.2P（具有同源包膜的病毒）。使用表达位点I和位点II干扰RNA作为发夹序列（shRNA）的第二代构建体已经证明了对SHIV复制的超过1000倍的甚至更有效的抑制。这些令人鼓舞的结果支持进一步研究siRNA在猕猴模型中体内抑制SHIV复制的效用。

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. Introduction of genes able to inhibit HIV replication into hematopoietic stem cells offers the potential for long-lived immune reconstitution. However, multiple ethical and practical considerations significantly constrain the ability to address basic questions regarding stem cell gene therapy for AIDS in human clinical trials. Experiments in nonhuman primates therefore offer the opportunity to rigorously address these issues in an in vivo experimental model. A number of recent reports have highlighted the potential utility of small interfering RNA (siRNA) molecules to inhibit viral replication. In collaboration with J. Rossi, we initiated inhibition studies of various SHIV strains with siRNA targeting Tat and Rev. We obtained two siRNA (one targeting both Tat and Rev and the other targeting only Rev) and a control sequence, each transcriptionally regulated by the polymerase III promoter U6. Using transient transfection both si(I) and si(II) strongly inhibited HIV-1 (as previously shown), SHIV 89.6p and SHIV Hxbc2 3.2P (viruses with homologous envelopes). Using a second generation construct expressing the site I and site II interfering RNAs as a hairpin sequence (shRNA) has demonstrated even more potent inhibition of SHIV replication by over 1000-fold. These encouraging results support further studies of the utility of siRNA to inhibit SHIV replication in vivo in the macaque model.