Combinatorial use of anti-HIV RNA-based therapeutics

描述(申请人提供)：高效抗逆转录病毒疗法 (HAART)使用两种或两种以上抗病毒药物的组合，目的是 逆转录酶和蛋白酶用于治疗HIV-1感染。 服用HAART的患者血液和组织水平迅速下降 艾滋病毒-1RNA和总体积极结果。尽管HAART总体上取得了成功， HIV-1的储备库继续在接受HAART的患者中复制，这使得 这种疗法的长期使用，甚至是终生使用。负性副作用 结合患者依从性问题和多种药物的出现 HIV-1的抗药性变种主张开发佐剂或 另类疗法。其中一种方法涉及造血的基因治疗。 易受HIV-1感染的细胞。该计划的总体目标是 对人类细胞进行基因改造，以抵抗艾滋病毒-1感染和 复制。为此，一系列基于RNA的抗HIV-1药物已经被 开发和测试。这些抑制性RNA针对病毒中的多个步骤 生命周期和细胞CCR5共同受体，并包括TAT的诱饵 和REV，结合REV和核酶的适体，这些核酶可以切割HIV-1的RNA或 CCR5基因的表达。先前的研究已经导致了POL III表达的发展， 嵌合RNA将抑制RNA定位于细胞质、核或 核仁。我们已经证明了一种抗HIV-1核酶的核仁定位， TAR或RBE诱饵可有效抑制病毒复制。通过组合 该抗病毒药物的组成和细胞内定位 RNA，应该有可能实现长期的、协同的抑制 HIV-1复制。重要的是，使用组合抑制性RNA疗法 与HAART结合可提供额外的协同效应，从而减少 HAART试剂的剂量。这项提案的总体目标是测试 不同组方的表达模式及抗HIV-1效果 我们的嵌合抑制RNA在整合的逆转录病毒和 慢病毒载体。我们还将探讨两国之间潜在的协同效应 抑制性RNA和几种HAART试剂。该计划是 互动R01计划(项目1)，Ramesh Akina博士(项目2)，题为 “在SCID-HU小鼠中建立基于RNA的HIV基因治疗模型。” 这项建议的具体目标是：1)构建逆转录病毒和 携带多个Polm盒的慢病毒载体能够同时 表达不同的抗HIV-1 RNA；2)检测表达水平和 组合构建物在培养细胞和原代细胞中的抗HIV活性 逆转录病毒和慢病毒载体稳定转导表达 抗HIV-1 RNA的组合；3)作用机制的研究 抑制性RNA；4)检测相互间协同作用的可能性 治疗性RNA和HAART药物在阻断HIV-1感染传播中的作用 细胞培养；5)原代CD34+细胞的转导 用于监测培养细胞分化的组合RNA表达载体 SCID-Hu小鼠模型的分化。这一目标是要实现的 与Ramesh Akina博士合作(项目2)。

DESCRIPTION (provided by applicant): Highly active anti-retroviral therapy (HAART) utilizes combinations of two or more anti-viral drugs targeted to reverse transcriptase and protease for the treatment of HIV-1 infection. Patients on HAART have witnessed rapid decreases in blood and tissue levels of HIV-1 RNA and overall positive outcomes. Despite the overall success of HAART, reservoirs of HIV-1 continue to replicate in patients on HAART, necessitating prolonged and perhaps lifetime use of this therapy. Negative side effects combined with problems of patient compliance and the emergence of multiple drug resistant variants of HIV-1 argue for the development of adjuvant or alternative therapies. One such approach involves gene therapy of hematopoietic cells susceptible to HIV-1 infection. The overall goal of this program is to genetically modify human cells for resistance to HIV-1 infection and replication. To this end a series of RNA based anti-HIV-1 agents have been developed and tested. These inhibitory RNAs target multiple steps in the viral life cycle as well as the cellular CCR5 co-receptor, and include decoys to Tat and Rev, aptamers which bind Rev and ribozymes which cleave HIV-1 RNAs or the CCR5 mRNA. Previous research has led to the development of Pol III expressed, chimeric RNAs that localize the inhibitory RNAs to the cytoplasm, nucleus or nucleolus. We have shown that nucleolar localization of an anti-HIV-1 ribozyme, TAR or RBE decoy provides potent inhibition of viral replication. By combining the composition and intracellular localization of this collection of antiviral RNAs, it should be possible to achieve long-term, synergistic inhibition of HIV-1 replication. Importantly, the use of combinatorial inhibitory RNA therapy in conjunction with HAART may provide additional synergy, allowing reduced dosing of HAART reagents. The overall objectives of this proposal are to test the expression patterns and anti-HIV-1 efficacy of different combinations of our chimeric, inhibitory RNAs in the context of integrated retroviral and lentiviral vectors. We will also address the potential for synergy between the inhibitory RNAs and several HAART reagents. This program is part of an interactive R01 program (Project 1) with Dr. Ramesh Akkina (Project 2) entitled "Modeling RNA-based HIV gene therapeutics in the SCID-hu mouse." The Specific Aims of this proposal are: 1) construction of retroviral and lentiviral vectors harboring multiple Pol m cassettes capable of simultaneously expressing different anti-HIV-1 RNAs; 2) testing expression levels and the anti-HIV activities of combinatorial constructs in cultured and primary cells stably transduced with retroviral and lentiviral vectors expressing combinations of Anti-HIV-1 RNAs; 3) examination of the mechanisms of action of the inhibitory RNAs; 4) examination of the potential for synergy between therapeutic RNAs and HAART agents in blocking infectious spread of HIV-1 in cell culture; 5) transduction of primary CD34+ cells with the single and combinatorial RNA expressing vectors to monitor differentiation in culture and differentiation in the SCID-hu mouse model. This aim is to be carried out collaboratively with Dr. Ramesh Akkina (Project 2).