Gene Therapy for HIV Using Aptamers that Target Reverse Transcriptase

产品说明：有效疫苗的持续缺乏和目前常规HIV-1药物治疗的局限性迫使开发替代方法。涉及修饰的造血干细胞（HSC）的干细胞策略提供了多年至终身保护免受病毒侵害的潜力，因为修饰的HSC不断再生，并且因为源自这些HSC的T细胞和巨噬细胞表达保护性转基因。在接受Δ32（CCR 5-/-）供体骨髓移植的HIV+患者明显治愈后，对干细胞策略的兴趣有所增加。这名患者随后停止了抗逆转录病毒治疗，多年来没有感染病毒。干细胞治疗的最新前沿 HIV-1是将保护性遗传修饰引入人CD 34 HSC中，以最终移植到患者体内。小的非编码抗病毒RNA，如适体、核酶和siRNA，是递送到这些细胞的特别有吸引力的货物，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T细胞分化，并且对于评估候选治疗性转基因货物的体内功效非常有价值。 我们工作的长期目标是优化用于抗HIV疗法的适体，特别是结合HIV-1逆转录酶（RT）的适体。以高亲和力结合RT的适体通过与引物/模板（p/t）竞争进入RT而在体外抑制酶。重要的是，它们还在基于细胞的测定中抑制病毒。近年来，我们已经确定了几种适体类别对RT氨基酸序列变异的敏感性，鉴定了先前未识别的RNA和单链DNA适体的结构类别，确定了几种适体-RT复合物的分子界面，开发并实施了用于评估来自适体选择的高通量测序（HTS）数据的生物信息学平台，开发了用于RNA适体的细胞内表达的新平台，并证明了从这些平台内表达的RNA适体的体外抗病毒功效。 基于这些坚实的基础和自最初提交以来获得的大量新数据，该修订后的合作提案旨在将经验证的适体转移到已建立的HSC免疫重建Rag-hu小鼠模型中，并加强适体开发管道。有趣的是，一直没有仔细评估复制病毒中适体抗性的出现。为了解决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 在细胞培养物和体内都能产生对表达的适体的抗性， 探索几种途径来克服阻力。

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 Δ32 (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.