Combinatorial Use of Anti-HIV RNA-based Therapeutics

描述（由申请人提供）：针对不同HIV编码蛋白的多药治疗方案的使用极大地改变了艾滋病的病程。高活性抗逆转录病毒疗法（HAART）的概念是增加抗病毒效力，同时尽量减少突变病毒耐药性的可能性。尽管对艾滋病有影响，但HAART也有并发症，包括新出现的耐药突变体、HAART与其他药物的毒性以及终生服药的长期费用。HAART疗法的一种替代或可能的辅助疗法是使用造血细胞的组合基因治疗修饰。这个项目的研究导致了一种慢病毒载体的发展，它含有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过的抗hiv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年进行t淋巴细胞试验。本提案利用前一个资助期的发现，并建议开发新的抗hiv组合rna，增强效力和安全性，以防止或尽量减少病毒逃逸突变体的出现。本研究将探索一种新的多顺反子微RNA系统，作为抗hiv sirna和核仁定位抑制RNA联合表达的可能平台。拟议的研究包括新的抗hiv靶向方法，这些方法将单独进行测试，并与已经建立的抑制rna组合进行测试。抑制基因的传递将通过慢病毒载体将组合构建体转导到造血干细胞中，用于体外和体内抗hiv功效和潜在毒性的评估。所提出的研究将使我们能够批判性地验证一种假设，即通过基因修饰的HSC移植可以实现治疗HIV感染的组合基因疗法。作为与Kiem博士和Hu博士通过华盛顿大学灵长类动物中心合作的一部分，体内试验将在长尾猕猴（M. nemestrina）中使用转导的造血干细胞和骨髓移植进行。该系统将严格评估构建物的安全性和有效性。本研究的具体目的是：1)开发抗hiv基因多路表达的新策略；2)表达策略及作用机制研究；3)评价载体转导的猕猴CD34+造血祖细胞的体内造血再生能力和对病毒攻击的抗性。这些研究将首次在灵长类动物HSC基因治疗环境中对抗病毒RNAi和其他抑制RNA组合进行详细的体内研究，并应提供将主要组合结构引入人体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.