Gene Therapy for HIV Using Aptamers that Target Reverse Transcriptase

描述：仍然缺乏有效的疫苗，以及目前常规艾滋病毒-1药物治疗的局限性，迫使开发替代方法。使用改良的造血干细胞(HSC)的干细胞策略提供了多年至终生保护病毒的潜力，因为改良的HSC不断再生，而且来自这些HSC的T细胞和巨噬细胞表达保护性转基因。在一名HIV+患者接受了32(CCR5-/-)捐赠者的骨髓移植后，对干细胞策略的兴趣增加了。这名患者随后停止了抗逆转录病毒治疗，已经多年没有感染病毒了。干细胞疗法的当前前沿 HIV-1将保护性的基因修饰引入人CD34 HSC，最终移植到患者体内。小的、非编码的抗病毒RNA，如适配子、核酶和siRNA，特别适合运送到这些细胞，部分原因是它们的效力和非免疫原性。小动物模型，如Rag-Hu免疫缺陷小鼠，支持人类T细胞分化，对于评估候选治疗性转基因货物的体内疗效具有很高的价值。我们工作的长期目标是优化抗HIV治疗的适配子，特别是结合HIV-1逆转录酶(RT)的适配子。与RT高亲和力结合的适配子在体外通过与引物/模板(p/t)竞争RT来抑制该酶。重要的是，它们还在基于细胞的检测中抑制病毒。近年来，我们定义了几个适配子类别对RT氨基酸序列变化的敏感性，确定了以前未知的RNA和单链DNA适配子的结构类别，定义了几个适配子-RT复合体的分子界面，开发并实现了一个用于评估来自适配子选择的高通量测序(HTS)数据的生物信息学平台，开发了新的RNA适配子细胞内表达平台，并展示了从这些平台表达的RNA适配子的体外抗病毒效果。在这些坚实的基础和自最初提交以来获得的大量新数据的基础上，这项修订的合作提案寻求将经过验证的适配子转移到已建立的HSC免疫重建的Rag-Hu小鼠模型中，并加强适配子的开发管道。有趣的是，目前还没有对复制病毒过程中出现的适体抗性进行仔细的评估。为了应对其对抗病毒治疗策略的潜在影响，我们将彻底评估艾滋病毒的 在细胞培养和体内对表达的适体产生抵抗力的能力，除了 探索了几条克服阻力的途径。

DESCRIPTION: The continued absence of an effective vaccine and current limitations of conventional HIV-1 drug treatments compel the development of alternative approaches. Stem cell strategies involving modified hematopoietic stem cells (HSC) offer the potential for multi-year to lifetime protection from the virus, because the modified HSC continually regenerate and because T cells and macrophage derived from these HSC express protective transgenes. Interest in stem cell strategies has increased following the apparent cure of an HIV+ patient who received a bone marrow transplant from a ?32 (CCR5-/-) donor. This patient has subsequently been off ART and free of virus for a number of years. A current frontier in stem cell therapies for HIV-1 is to introduce protective genetic modifications into human CD34 HSC for eventual transplantation into patients. Small, non-coding antiviral RNAs, such as aptamers, ribozymes and siRNA, are especially attractive cargo to deliver to these cells, in part because of their potency and non-immunogenicity. Small animal models such as Rag-hu immunodeficient mice support human T cells differentiation and are highly valuable for evaluating in vivo efficacy of candidate therapeutic transgene cargos. The long term objective of our work is to optimize aptamers for anti-HIV therapies, particularly aptamers that bind HIV-1 reverse transcriptase (RT). Aptamers that bind RT with high affinity inhibit the enzyme in vitro by competing with primer/template (p/t) for access to RT. Importantly they also suppress the virus in cell-based assays. In recent years we have defined the sensitivity of several aptamer classes to RT amino acid sequence variation, identified previously-unrecognized structural classes of RNA and single-stranded DNA aptamers, defined the molecular interfaces for several aptamer-RT complexes, developed and implemented a bioinformatics platform for evaluating high-throughput sequencing (HTS) data from aptamer selections, developed new platforms for intracellular expression of RNA aptamers and demonstrated in vitro antiviral efficacy of RNA aptamers expressed from within those platforms. Building from these strong foundations and on substantial new data obtained since its original submission, this revised collaborative proposal seeks to move validated aptamers into an established Rag-hu mouse model of HSC immune reconstitution and to strengthen the aptamer developmental pipeline. Interestingly, there has been no careful evaluation of the emergence of aptamer resistant in replicating viruses. To address its potential impact on antiviral therapeutic strategies, we will thoroughly evaluate HIV's ability to evolve resistance to expressed aptamers both in cell culture and in vivo, in addition to exploring several routes to overcome resistance.