Combinatorial Use of Anti-HIV RNA-based Therapeutics

描述(由申请人提供)：针对不同艾滋病毒编码蛋白的多种药物方案的使用极大地改变了艾滋病的进程。高效逆转录病毒疗法(HAART)的概念是在增加抗病毒效力的同时将变异病毒耐药性的可能性降至最低。尽管HAART对艾滋病有影响，但也存在并发症，包括新出现的耐药突变、HAART与其他药物的毒性以及终生药物的长期成本。HAART疗法的一种替代或可能的辅助治疗是使用组合基因疗法修饰造血细胞。该项目的研究导致了一种慢病毒载体的开发，该载体携带三种不同的治疗基因，针对艾滋病毒生命周期的不同步骤。这种三重RNA抑制剂组合已被证明是迄今为止我们测试的抗艾滋病毒方法中最有效的。该载体将于2006年底进入艾滋病/淋巴瘤患者细胞造血干细胞转导(HSC)的I期临床试验，并于2007年进入T淋巴细胞试验。本提案利用上一个资助期的研究结果，并建议开发新的抗艾滋病毒组合RNA，以增强效力和安全性，以防止或最大限度地减少病毒逃逸突变的出现。建议的研究将探索一种新的多顺反子microRNA系统，作为一种可能的平台，用于共表达抗HIV siRNA和核仁定位抑制RNA的组合。拟议的研究纳入了新的抗艾滋病毒靶向方法，这些方法将单独进行测试，并与已经建立的抑制性RNA进行组合测试。抑制基因的传递将通过慢病毒载体将组合构建物导入HSCs，用于体外和体内抗HIV疗效和潜在毒性的评估。拟议的研究将使我们能够批判性地检验这样一种假设，即组合基因疗法可以通过基因修饰的造血干细胞移植来治疗艾滋病毒感染。体内测试将使用转导的造血干细胞和骨髓移植在小尾猴(M.nomestrina)身上进行，这是通过华盛顿大学灵长类中心与Kiem和Hu博士合作的一部分。在这个系统中，将对结构的安全性和有效性进行严格的评估。本研究的具体目的是：1)开发抗HIV基因表达的新策略；2)表达策略和作用机制研究；3)评价载体转导的猕猴CD34+造血祖细胞的体内造血再增殖能力和抗病毒攻击能力。这些研究将提供灵长类HSC基因治疗环境中抗病毒RNAi和其他抑制性RNA组合的第一次详细的体内研究，并将提供将领先组合结构带入人类临床试验所需的安全性和有效性数据。

DESCRIPTION (provided by applicant): The use of multi-drug regimens which target different HIV encoded proteins has dramatically changed the course of AIDS. The concept of highly active retroviral therapy (HAART) is to increase antiviral potency while minimizing the probability of mutant viral resistance. Despite the impact on AIDS, there are complications of HAART, including emerging resistant mutants, toxicities of HAART with other medications, and the long term cost of a lifetime of medication. An alternative or possible adjuvant to HAART therapy is the use of combinatorial gene therapy modification of hematopoietic cells. Research from this program has led to the development of a lentiviral vector harboring three different therapeutic genes targeting different steps in the HIV life cycle. This triple RNA inhibitor combination has proven to be the most potent of our tested anti-HIV approaches to date. This vector will enter a phase I clinical trial for hematopoietic stem cell transduction (HSC) of AIDS/lymphoma patient cells sometime in late 2006, and T-lymphocyte trials in 2007. The present proposal capitalizes upon the findings of the previous funding period, and proposes to develop new anti-HIV combinatorial RNAs with enhanced potency and safety to prevent or minimize the emergence of viral escape mutants. The proposed studies will explore a novel polycistronic micro RNA system as a possible platform for co-expressing combinations of anti-HIV siRNAs and nucleolar localizing inhibitory RNAs. The proposed studies incorporate novel anti-HIV targeting approaches which will be tested alone and in combinatorial fashion with already established inhibitory RNAs. Delivery of the inhibitory genes will be via lentiviral vector transduction of the combinatorial constructs into HSCs for in vitro and in vivo evaluations of anti-HIV efficacy and potential toxicities. The proposed studies will allow us to critically test the hypothesis that combinatorial gene therapy for treatment of HIV infection can be effected via genetically modified HSC transplantation. The in vivo testing will be conducted using transduced HSCs and bone marrow transplantation in the pigtail macaque (M. nemestrina) as part of a collaborative effort with Drs Kiem and Hu through the University of Washington Primate Center. The safety and efficacy of the constructs will be critically evaluated in this system. The Specific Aims of this study are: 1) Development of new strategies for multiplexing anti-HIV gene expression;2) Expression strategies and mechanism of action studies; 3) Evaluation of in vivo hematopoietic repopulation capabilities and resistance to viral challenge of vector transduced macaque CD34+ hematopoietic progenitor cells. These studies will provide the first detailed, in vivo investigations of antiviral RNAi and other inhibitory RNA combinations in a primate HSC gene therapy setting, and should provide the type of safety and efficacy data required to bring the lead combinatorial constructs to human clinical trials.