OPTIMIZATION OF ONCORETROVIRAL VECTORS ENCODING RNA DECOYS

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。 RNA诱骗在抑制HIV复制方面有许多优点，包括它们缺乏免疫原性，以及它们能够靶向病毒复制所必需的保守基因。然而，通过RNA诱骗抑制病毒复制的最佳方法通常是使用多聚体RNA诱骗，这显著增加了使用逆转录病毒载体运送转基因不稳定的风险。因此，我们研究了影响肿瘤逆转录病毒载体稳定传递HIV-1RNA诱饵和抑制病毒复制的能力的一些参数。对于逆转录病毒骨架，我们选择了不包含可选择标记基因的肿瘤逆转录病毒载体MMP，并产生了一系列带有和不带有完整剪接供体和剪接受体信号的载体，以及具有转录调控聚合TAR和RRE RNA诱骗的肿瘤逆转录病毒LTR或内部HIV-1 LTR。通过减少TAR诱饵的数量，提高了载体的稳定性，从而更有效地抑制了转导细胞中的病毒复制。在逆转录病毒载体中加入内部HIV启动子可以提供更一致的病毒抑制。这些不同载体的效率已经在从转导细胞和稳定的高滴度产生细胞系中获得的多个T细胞克隆中进行了评估。用其中一种载体转导自体CD34+骨髓细胞，获得了0.1%~1%的稳定基因标记。这些优化的载体将有助于在非人灵长类动物模型中分析干细胞基因的RNA诱骗对艾滋病的有效性。

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. RNA decoys have a number of advantages for the inhibition of HIV replication, including their lack of immunogenicity and their ability to target conserved genes essential for viral replication. However, optimal inhibition of viral replication by RNA decoys has generally been obtained with multimeric RNA decoys, which significantly increase the risk of transgene instability when delivered using retroviral vectors. We therefore examined a number of parameters affecting the ability of oncoretroviral vectors to stably deliver HIV-1 RNA decoys and inhibit viral replication. For the retroviral backbone, we chose the oncoretroviral vector MMP, which does not contain a selectable marker gene, and generated a series of vectors with and without intact splice donor and splice acceptor signals, and with the oncoretroviral LTR or an internal HIV-1 LTR transcriptionally regulating the polymeric TAR and RRE RNA decoys. By decreasing the number of TAR decoys, vector stability was increased, resulting in more efficient inhibition of viral replication in transduced cells. Inclusion of an internal HIV promoter within the retroviral vector provided more consistent viral inhibition. The efficiency of these different vectors has been evaluated in multiple T cell clones derived from transduced cells and stable high titer producer cell lines generated. Transduction of autologous CD34+ bone marrow cells with one of these vectors has resulted in stable gene marking of 0.1 percent  1 percent of lymphocytes. These optimized vectors will facilitate analysis of the efficacy of RNA decoys for stem cell gene for AIDS in a nonhuman primate model.