OPTIMIZATION OF ONCORETROVIRAL VECTORS ENCODING RNA DECOYS

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。RNA诱饵对于抑制HIV复制具有许多优点，包括它们缺乏免疫原性和它们靶向病毒复制所必需的保守基因的能力。然而，通常用多聚体RNA诱饵获得RNA诱饵对病毒复制的最佳抑制，当使用逆转录病毒载体递送时，其显著增加转基因不稳定性的风险。因此，我们研究了一些参数影响的能力，稳定提供HIV-1 RNA诱饵和抑制病毒复制的逆转录病毒载体。对于逆转录病毒骨架，我们选择了肿瘤逆转录病毒载体MMP，它不包含选择标记基因，并产生了一系列载体，有和没有完整的剪接供体和剪接受体信号，并与肿瘤逆转录病毒LTR或内部HIV-1 LTR转录调节聚合TAR和RRE RNA诱饵。通过减少TAR诱饵的数量，载体稳定性增加，导致转导细胞中病毒复制的更有效抑制。在逆转录病毒载体内包含内部HIV启动子提供了更一致的病毒抑制。这些不同载体的效率已经在源自转导细胞的多个T细胞克隆和产生的稳定高滴度生产细胞系中进行了评估。用这些载体之一转导自体CD 34+骨髓细胞已导致0.1%-1%淋巴细胞的稳定基因标记。这些优化的载体将有助于在非人灵长类动物模型中分析RNA诱饵对艾滋病干细胞基因的功效。

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.