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

使用抗 HIV 包膜反义分子治疗艾滋病的干细胞基因疗法

• 批准号: 7349499
• 负责人: Stephen E. Braun
• 金额: $ 13.48万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7349499
• 关键词: STEM; CELL; GENE; THERAPY; AIDS

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。将抑制基因引入造血干细胞，为子代细胞抵抗 HIV-1 复制提供了长期免疫重建的潜力。然而，成功开发艾滋病干细胞基因疗法可能需要在恒河猴模型中进行临床前测试。我们检测了编码 937 bp 反义 HIV-1 包膜序列的 HIV-1 慢病毒载体 (VRX494) 抑制病毒复制的能力。由于 HIV-1 不能在恒河猴中复制，因此使用带有 HIV-1 包膜的嵌合 SIV/HIV-1 病毒来确定 VRX494 的功效。 VRX494 转导的 CEMx174 细胞的攻击导致对 HIV-1 和几种 SHIV 毒株的有效抑制。为了评估 VRX494 对转导 CD34+ 细胞衍生的 CD4+ T 细胞的功效，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以诱导 T 细胞分化。对 CD34+ 细胞的转导条件进行了优化，以产生相对较高水平的转导效率（大于 50%），同时有效感染复数最小。与对照相比，来自 VRX494 转导细胞的纯化 CD4+GFP+ T 细胞强烈抑制 SHIV HXBc2P 3.2 和 SHIV 89.6P 复制。对转导的 CD34+ 细胞衍生的 T 细胞克隆进行 Southern 印迹分析，揭示了一个细胞子集，每个细胞具有多个原病毒拷贝，这凸显了优化转导条件以尽量减少每个细胞发生多个整合事件的可能性的重要性。这些结果表明，表达HIV-1反义序列的慢病毒载体强烈抑制HIV-1和SHIV复制，并且SHIV猕猴模型应作为评估艾滋病干细胞基因治疗的有用临床前模型。