EVALUATION OF INHIBITION OF SHIV REPLICATION BY SIRNA VECTORS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。将能够抑制艾滋病毒复制的基因引入造血干细胞，为长期免疫重建提供了可能性。然而，多重伦理和实际考虑大大限制了在人类临床试验中解决有关艾滋病干细胞基因治疗的基本问题的能力。因此，在非人类灵长类动物身上的实验提供了在活体实验模型中严格解决这些问题的机会。最近的一些报告强调了小干扰RNA(SiRNA)分子抑制病毒复制的潜在用途。在与J.Rossi的合作下，我们开始了针对TAT和REV的siRNA对各种SHV毒株的抑制研究。我们获得了两个siRNA(一个针对TAT和REV，另一个只针对REV)和一个对照序列，每个序列都由聚合酶III启动子U6转录调控。使用瞬时转染法，si(I)和si(II)对HIV-1(如上所述)、Shiv 89.6p和Shiv Hxbc2 3.2p(具有同源包膜的病毒)都有很强的抑制作用。使用第二代表达位点I和位点II的干扰RNA作为发夹序列(ShRNA)，已经显示出对SHIV复制的更有效的抑制超过1000倍。这些令人鼓舞的结果支持了进一步研究siRNA在猕猴模型中抑制体内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.