Combinatorial Use of Anti-HIV RNA-based Therapeutics

描述（由申请人提供）：艾滋病毒/艾滋病仍然是人类健康的主要威胁。在高效抗逆转录病毒疗法（HAART）中使用小分子药物的组合来阻止或阻碍HIV传播，这对延迟从HIV-1感染发展为AIDS具有重大影响。尽管取得了这一进展，但仍存在与终身使用抗病毒药物治疗相关的问题。这些因素包括毒性、对多种药物产生耐药性的病毒的出现以及每日药物的成本。拟议的研究利用了基因疗法治疗HIV-1感染的最新进展。人血祖细胞的基因治疗可以被认为是目前抗HIV-1治疗的潜在替代或补充。上一个资助期的一个成果是开发了一种三管齐下的基因方法来抑制病毒进入和复制。这种三重小RNA基因疗法已经在艾滋病/淋巴瘤患者的首次人类血液干细胞基因疗法试验中测试了可行性和安全性。基于这四名患者试验的结果，我们认为必须开发新的和不同的小RNA抗病毒组合。拟议的研究利用了一组新的HIV-1感染抑制剂，它们使用不同的作用机制。我们假设，这种组合将防止病毒耐药突变体的出现，同时无毒。具体目标1将选择体外进化的“选择性结合HIV-1整合酶并阻断整合酶功能的RNA适体”。适体将在造血细胞中表达，作为设计用于阻断病毒感染的组合方法的一部分。特异性目标2将“在慢病毒载体中结合联合收割机核仁定位抗HIV RNA、microRNA模拟物、tRNA-抗CCR 5 shRNA、U 5 tRNA shRNA融合物和抗整合酶适体，用于造血祖细胞的转导”。这项研究的结果将为体内功能测试提供新的抗HIV RNA组合。具体目标3解决了“人源化Rag 2-/-3c-/-（RAG-hu）小鼠模型中组合构建体的体内分析”。这种人源化小鼠模型代表了一种用于测试抗HIV基因治疗策略的功效和安全性的新颖且简易的系统，因为基因修饰的人造血干细胞和祖细胞可以移植并分化成体内可被HIV-1感染的造血谱系。总的来说，该计划旨在为临床带来新的基于小RNA的基因治疗组合，用于未来的人类基因治疗应用。

DESCRIPTION (provided by applicant): HIV/AIDS continues to be a major threat to human health. The use of combinations of small molecule drugs in highly active anti-retroviral therapy (HAART) to stop or thwart HIV propagation ahs had a major impact on delaying the progression from HIV-1 infection to the development of AIDS. Despite this progress, there are problems associated with a lifelong use of anti-viral drug therapy. These include toxicity, the emergence of virus resistant to multiple drugs, and the cost of a daily medication. The proposed studies take advantage of recent advances in gene therapy for the treatment of HIV-1 infection. Gene therapy of human blood progenitor cells can be considered as a potential replacement or supplement to the current anti-HIV-1 therapies. An outcome of the previous funding period was the development of a three pronged genetic approach for inhibiting viral entry and replication. This triple small RNA gene therapy has been tested for feasibility and safety in a first in human blood stem cell gene therapy trial in AIDS/lymphoma patients. Based upon the results of this four patient trial we believe it is imperative to progress to new and different small RNA anti-viral combinations. The proposed studies take advantage of a set of novel inhibitors of HIV-1 infection which use different mechanisms of action. We hypothesize that such combinations will prevent the emergence of viral resistant mutants and at the same time be non-toxic. Specific Aim 1 will select for in vitro evolved "RNA aptamers that selectively bind HIV-1 integrase and block integrase function". The aptamers will be expressed in hematopoietic cells as part of a combinatorial approach designed to block viral infection. Specific Aim 2 will "combine nucleolar localizing anti-HIV RNAs, microRNA mimics, tRNA-anti CCR5 shRNA, U5 tRNA shRNA fusions and the anti-integrase aptamers in a lentiviral vector for transduction of hematopoietic progenitor cells". The results from this research will provide novel combinations of anti-HIV RNAs for functional testing in vivo. Specific Aim 3 addresses the "in vivo analyses of combinatorial constructs in the humanized Rag2-/- 3c-/- (RAG-hu) mouse model". This humanized mouse model represents a novel and facile system for testing the efficacy and safety of anti-HIV gene therapy strategies since gene modified human hematopoietic stem and progenitor cells can engraft and differentiate into hematopoietic lineages that are infectible by HIV-1 in vivo. Overall, this program is designed to be a path towards bringing new small RNA based gene therapy combinations to the clinic for use in future human gene therapy applications.