STEM CELL GENE THERAPY FOR AIDS USING AN ANTI-HIV ENVELOPE ANTISENSE MOLECULE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。将抑制基因引入造血干细胞，为后代细胞抵抗HIV-1复制提供了长期免疫重建的潜力。然而，干细胞基因治疗艾滋病的成功发展可能需要在恒河猴模型中进行临床前试验。我们检测了基于HIV-1的慢病毒载体（VRX494）编码937 bp反义HIV-1包膜序列抑制病毒复制的能力。由于HIV-1不能在恒河猴体内复制，因此使用带有HIV-1包膜的嵌合SIV/ HIV-1病毒来确定VRX494的疗效。对vrx494转导的CEMx174细胞的攻击导致HIV-1和几种SHIV毒株的有效抑制。为了评估VRX494对CD34+细胞转导后的CD4+ T细胞的作用，我们用VRX494转导恒河猴CD34+骨髓细胞，然后在恒河猴胎儿胸腺基质细胞上培养，诱导T细胞分化。对CD34+细胞的转导条件进行了优化，以产生相对较高的转导效率（大于50%），而感染的有效多重性最小。与对照组相比，vrx494转导细胞纯化的CD4+GFP+ T细胞强烈抑制SHIV HXBc2P 3.2和SHIV 89.6P的复制。来自转导CD34+细胞的T细胞克隆的Southern blot分析显示，每个细胞具有多个前病毒拷贝的细胞亚群突出了优化转导条件以最小化每个细胞多重整合事件可能性的重要性。这些结果表明，表达HIV-1反义序列的慢病毒载体可以强烈抑制HIV-1和SHIV的复制，SHIV猕猴模型可以作为评估干细胞基因治疗艾滋病的有用临床前模型。

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. The introduction of inhibitory genes into hematopoietic stem cells offers the potential for long-lived immune reconstitution with progeny cells resistant to HIV-1 replication. However, successful development of stem cell gene therapy for AIDS is likely to require preclinical testing in the rhesus macaque model. We examined the ability of a HIV-1-based lentiviral vector (VRX494) encoding a 937 bp antisense HIV-1 envelope sequence to inhibit viral replication. Because HIV-1 does not replicate in rhesus macaques, chimeric SIV/ HIV-1 viruses with the HIV-1 envelope were used to determine the efficacy of VRX494. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the efficacy of the VRX494 in CD4+ T cells derived from transduced CD34+ cells, rhesus CD34+ bone marrow cells were transduced with VRX494 and then cultured on rhesus fetal thymus stromal cells to induce T cell differentiation. Transduction conditions for CD34+ cells were optimized so as to yield relatively high levels of transduction efficiency (greater than 50 per cent), with minimal effective multiplicity of infection. Purified CD4+GFP+ T cells derived from VRX494-transduced cells strongly inhibited SHIV HXBc2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of T cell clones derived from transduced CD34+ cells revealed a subset of cells with multiple proviral copies per cell highlighting the importance of optimizing transduction conditions to minimize the possibility of multiple integration events per cell. These results indicate that a lentiviral vector expressing an HIV-1 antisense sequence strongly inhibits HIV-1 and SHIV replication and that the SHIV macaque model should serve as a useful preclinical model to evaluate stem cell gene therapy for AIDS.