SiRNA Gene Therapy for HIV/AIDS

描述（申请人提供）：最近描述的小干扰RNA （sirna）介导的RNA干扰（RNAi）现象是高度序列特异性的基因沉默机制。最近的研究证实了sirna在体外抑制HIV-1复制方面的显著效力，并强调了其作为基因治疗强有力工具的潜力。将这项有前途的新技术转化为临床需要将siRNA基因稳定地引入造血干细胞，并衍生出病毒抗性T细胞和巨噬细胞。利用体外实验和独特的SCID-hu小鼠体内模型，我们最近发现，CD34造血祖细胞通过抗hiv sirna、核酶和RNA诱饵转导，可以分化为抗病毒的T细胞和巨噬细胞。这些概念研究的证明为在体内环境中评估新方法铺平了道路。最近在干细胞生物学、慢病毒载体和与HIV基因治疗直接相关的体内建模领域也出现了一些新的进展。在目前的建议中，我们的目标是在我们最近取得的进展的基础上进一步发展。我们的具体目标是：在体外评估针对HIV-1生命周期不同阶段的新型组合siRNA的协同效应，并分析其作用机制。2. 通过慢病毒载体将sirna转导到CD34造血祖细胞中，获得HIV-1抗性巨噬细胞，并研究其在分化细胞中的作用机制。3. 确定不同的抗HIV-1 sirna的体内保护作用，无论是单独的还是联合的，在SCID-hu小鼠的活体移植物中抵抗HIV-1的攻击，并评估转基因T细胞的功能能力。4. 将siRNA构建物转导到新描述的CD34+/KDR+原始造血祖细胞中，在体外获得巨噬细胞，在体内获得胸腺细胞，并评估其HIV-1抗性。

DESCRIPTION (provided by applicant): The recently described RNA interference (RNAi) phenomenon mediated by small interfering RNAs (siRNAs) is highly sequence specific gene silencing mechanism. Recent studies established the remarkable potency of siRNAs in suppressing HIV-1 replication in vitro and highlighted its potential as 3owerful tool for gene therapy. Translation of this promising new technology into the clinic requires stable introduction of the siRNA genes into hematopoietic stem cells and derivation of viral resistant T cells and macrophages. Using in vitro assays and a unique SCID-hu mouse in vivo model that harbors transplanted human tissue, we recently showed that CD34 hematopoietic progenitor cells transduced with anti-HIV siRNAs, ribozymes and RNA decoys could be differentiated into virus resistant T cells and macrophages. These proofs of concept studies paved the way to evaluate novel approaches in an in vivo setting. Several new developments have also occurred recently in the areas of stem cell biology, lentiviral vectors, and in vivo modeling with direct relevance to HIV gene therapy. In the current proposal our goal is to build upon our recent progress. Our specific objectives are: 1. Evaluate the synergistic efficacy of novel combinatorial siRNA constructs targeted to different stages of HIV-1 life cycle in vitro and analyze their mechanism of action. 2. Transduce siRNAs via lentiviral vectors into CD34 hematopoietic progenitor cells to derive HIV-1 resistant macrophages and investigate the mechanism of their action in differentiated cells. 3. Determine the in vivo protective effects of different anti-HIV-1 siRNAs, either individually or in combination, in SCID-hu mice thy/liv grafts against HIV-1 challenge and also assess the functional competence of transgenic T cells. 4. Transduce siRNA constructs into the newly described CD34+/KDR+ primitive hematopoietic progenitor cells and derive macrophages in vitro and thymocytes in vivo, and evaluate their HIV-1 resistance.