Regulation and manipulation of the telomerase RNA component in hematopoiesis

项目摘要/摘要 端粒酶对健康和长寿至关重要，但人们对端粒酶的基本原理缺乏了解。 在人类细胞中受到调节。这种知识差距阻碍了针对不断增长的谱系开发治疗方法的能力 与端粒酶功能障碍有关的疾病。该项目的长期目标是能够 操纵人类细胞中的端粒酶以达到治疗的目的，其关键目标是造血系统。 非编码端粒酶RNA组分TERC的水平是端粒酶功能的关键决定因素 细胞。基因突变导致的低TERC水平会导致广泛的退行性疾病， 包括先天性角化不良、再生障碍性贫血、MDS/白血病、心血管疾病、肺疾病 纤维化和肝硬变。最近的研究发现，非正则聚合酶PAPD5是一个关键的负调控因子 TERC的。因此，PAPD5是小分子抑制剂修复人类端粒和端粒的新靶点 疾病。尚不清楚的是PAPD5选择性调控非编码RNA(NcRNAs)的基础，也不是 抑制PAPD5是否具有治疗效果。这项建议的总体目标是(1) 了解PAPD5如何选择性地调节ncRNAs，以及(2)确定PAPD5的体内疗效 用于恢复造血干细胞(HSC)功能的抑制剂。中心假设是PAPD5 选择性地调节TERC和少量的ncRNA，为系统性红斑狼疮提供治疗窗口 抑制PAPD5以恢复干细胞的端粒和自我更新能力。我们工作的基本原理 是了解小分子PAPD5的选择性机制并展示其治疗效果 抑制剂将为它们作为治疗骨髓衰竭的药物的发展提供强有力的科学框架。 以及一系列疾病。将通过追求两个具体目标来检验中心假设：(1)确定 PAPD5选择性调节NcRNAs的机制，以及(2)决定了PAPD5的治疗效果 体内的PAPD5抑制剂。在第一个目标下，生化和遗传方法将被严格用于 识别真正的PAPD5靶点，并破译依赖于PAPD5的转录组的调控逻辑 人类细胞，重点是造血干细胞。已经开发和演示的工具和技术 在申请者手中可行的将被使用。在第二个目标下，小分子药物的治疗效果 恢复体内端粒维持和HSC功能的PAPD5抑制剂将被确定。新方法 为了克服端粒生物学和人类HSC模型的物种间差异，将应用HSC失败。这个 方法是创新的，因为PAPD5是一个长期寻求的、新颖的和易处理的目标，调节TERC， 抑制该基因可能会使系统性端粒酶调节疗法能够选择性地影响干细胞。 这项拟议的研究意义重大，因为它有望产生操纵端粒酶的新策略。 越来越多的造血和退行性疾病，包括再生障碍性贫血、MDS/白血病、 肺和心血管疾病，与端粒酶功能障碍有关，但很少涉及 如果有什么根治疗法。

PROJECT SUMMARY/ABSTRACT Telomerase is critical for health and longevity, but there is a fundamental lack of understanding of how it is regulated in human cells. This knowledge gap impedes the ability to develop therapies for a growing spectrum of disorders in which telomerase dysfunction is implicated. The long-term goal of this project is to be able to manipulate telomerase in human cells for therapeutic benefit, with a key target being the hematopoietic system. The level of the noncoding telomerase RNA component TERC is a critical determinant of telomerase function in cells. Low TERC levels resulting from genetic mutations cause a wide spectrum of degenerative disorders, including dyskeratosis congenita (DC), aplastic anemia, MDS/leukemia, cardiovascular disease, pulmonary fibrosis, and cirrhosis. Recent work identifies the non-canonical polymerase PAPD5 as a key negative regulator of TERC. PAPD5 is thus a novel target for small molecule inhibitors to restore TERC and telomeres in human diseases. What is not known is the basis of selective regulation of non-coding RNAs (ncRNAs) by PAPD5, nor whether inhibiting PAPD5 will be therapeutically effective. The overall objectives of this proposal are (1) to understand how PAPD5 selectively regulates ncRNAs, and (2) to determine the in vivo efficacy of PAPD5 inhibitors in restoring hematopoietic stem cell (HSC) function. The central hypothesis is that PAPD5 selectively regulates TERC and a small number of ncRNAs, providing a therapeutic window for systemic PAPD5 inhibition to restore telomeres and self-renewal capacity in stem cells. The rationale for our work is that understanding mechanisms of selectivity and demonstrating therapeutic efficacy of small molecule PAPD5 inhibitors will provide a strong scientific framework for their development as treatments for bone marrow failure and a range of diseases. The central hypothesis will be tested by pursuing two Specific Aims: (1) Identify the mechanisms of selective regulation of ncRNAs by PAPD5, and (2) Determine the therapeutic efficacy of PAPD5 inhibitors in vivo. Under the first aim, biochemical and genetic approaches will be used to rigorously identify bona fide PAPD5 targets, and to decipher the regulatory logic of the PAPD5-dependent transcriptome in human cells, emphasizing HSCs. Tools and techniques that have been developed and demonstrated to be feasible in the applicants’ hands will be used. Under the second aim, the therapeutic efficacy of small molecule PAPD5 inhibitors to restore telomere maintenance and HSC function in vivo will be determined. New approaches to overcome interspecies differences in telomere biology and model human HSC failure will be applied. The approach is innovative because PAPD5 is a long-sought, novel and tractable target regulating TERC, the inhibition of which may enable a systemic telomerase-modulating therapy that selectively impacts stem cells. The proposed research is significant, because it is expected to yield new strategies to manipulate telomerase in a growing number of hematopoietic and degenerative disorders including aplastic anemia, MDS/leukemia, pulmonary and cardiovascular disease, in which telomerase dysfunction is implicated but for which there are few if any curative therapies.