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

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。在造血干细胞中引入抑制基因为抗HIV-1复制的后代细胞的长寿免疫重建提供了可能性。然而，艾滋病干细胞基因疗法的成功开发可能需要在恒河猴模型中进行临床前测试。我们检测了基于HIV-1的慢病毒载体(VRX494)编码937bp的HIV-1反义包膜序列抑制病毒复制的能力。由于HIV-1不会在恒河猴体内复制，因此使用带有HIV-1包膜的SIV/HIV-1嵌合病毒来确定VRX494的疗效。VRX494转导的CEMx174细胞对HIV-1和几种SHV毒株有较强的抑制作用。将VRX494导入恒河猴CD34+骨髓细胞，在恒河猴胎胸腺基质细胞上诱导T细胞分化，以评价VRX494对转导CD34+细胞的CD4+T细胞的作用。对CD34+细胞的转导条件进行了优化，以获得相对较高的转导效率(大于50%)，并将有效感染的多样性降至最低。与对照组相比，VRX494转导细胞来源的纯化的CD4+GFP+T细胞对SIV HXBc2P 3.2和SIV 89.6P复制有较强的抑制作用。从转导的CD34+细胞获得的T细胞克隆的Southern杂交分析显示，每个细胞具有多个前病毒拷贝的细胞亚群，强调了优化转导条件的重要性，以最大限度地减少每个细胞发生多个整合事件的可能性。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SIV的复制，SIV猕猴模型可以作为评估艾滋病干细胞基因治疗的有用的临床前模型。

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.