Studying a bone marrow failure disease using patient-specific iPS cells

7.项目摘要/摘要 该项目的目标是利用患者特定的诱导多能干细胞(IPSCs)作为一个平台 骨髓先天性角化不良(DC)患者新疗法的开发 以低预期寿命和包括再生障碍性贫血在内的多系统组织缺陷为表现的失败综合征 贫血和肺纤维化。将使用多种技术组合来实现这一目标，包括 基因校正、高通量测序、小分子药物筛选和靶向分化 DC诱导的iPS细胞走向造血祖细胞。 所有DC患者的端粒相对于他们的年龄来说都非常短，通常低于第一个百分位数的长度 与其他人口相比。到目前为止，在DC中发现的所有突变都是与 端粒动态平衡，或者在端粒酶中，或者直接与端粒结合。端粒酶是由多种酶组成的 哺乳动物细胞中负责端粒合成的复合体。在没有端粒酶的情况下，端粒将 逐渐缩短，这与老鼠和人类的干细胞功能受损有关。因此， DC中观察到的组织缺陷可能是由于成体干细胞室失去自我更新所致。 这些患者是由端粒加速缩短引起的端粒酶突变。中环 这个项目的假设是，疾病特异性的iPS细胞提供了一个新的和合适的系统来研究 端粒酶突变和DC多能细胞自我更新丧失的后果 寻找纠正这种表型的策略。 我之前已经证明，iPS细胞来源于表现出不同严重程度的DC的患者 总结一下在人类患者中观察到的端粒缩短和自我更新表型的丢失。在这里，我 建议使用这些患者特有的iPS细胞来详细了解自我更新表型的丧失 并利用这些细胞作为药物发现和靶向的平台 差异化，这可能使未来临床治疗的新方案成为可能。具体目标是：(一) 通过基因互补和高表达逆转DC患者特异性iPS细胞的自我更新缺陷 端粒过短的DC iPS细胞的基因表达谱分析 作为开发抗DC新疗法的平台，通过搜索端粒酶稳定药物和通过 特别是将这些细胞分化为造血细胞。 该项目将极大地增加目前对骨髓衰竭综合征的了解，首先 了解人类多能细胞中端粒功能失调的有害影响，然后通过 设计新的策略来治疗患有先天性角化不良的患者，这种疾病目前没有 解药。

7. PROJECT SUMMARY/ABSTRACT The goal of this project is to use patient-specific induced pluripotent stem cells (iPSCs) as a platform for the development of novel therapies for patients suffering with dyskeratosis congenita (DC), a bone-marrow failure syndrome that presents with poor life expectancy and multi-systemic tissue defects that include aplastic anemia and pulmonary fibrosis. A combination of technologies will be used to achieve this goal, including genetic correction, high-throughput sequencing, small-molecule drug screening and targeted differentiation of DC iPS cells to hematopoietic fates. All patients with DC have very short telomeres for their age, typically below the first percentile length when compared to the rest of the population. All mutations discovered in DC so far were in genes related to telomere homeostasis, either in telomerase, or directly binding to telomeres. Telomerase is the multi-enzymatic complex responsible for telomere synthesis in mammalian cells. In the absence of telomerase, telomeres will progressively shorten, which has been linked to impaired stem cell function in mice and humans. Thus, the tissue defects observed in DC likely result from the loss of self-renewal in adult stem cells compartments of these patients, caused by accelerated telomere shortening in settings of mutant telomerase. The central hypothesis of this project is that disease-specific iPS cells offer a novel and suitable system to study the consequences of mutant telomerase and the loss of self-renewal in DC pluripotent cells and can be used to search for strategies to correct this phenotype. I have previously shown that iPS cells derived from patients showing variable severity of DC faithfully recapitulate the telomere shortening and loss of self-renewal phenotypes observed in human patients. Here, I propose to use these patient-specific iPS cells to understand in detail the loss of self-renewal phenotype arising from dysfunctional telomeres and to use these cells as a platform for drug discovery and targeted differentiation, which could enable novel protocols for clinical therapy in the future. The Specific Aims are (I) to reverse the self-renewal defect in DC patient-specific iPS cells by genetic complementation and high- throughput gene expression analysis of DC iPS cells with critically short telomeres and (II) to use DC iPS cells as a platform for developing new therapies against DC, by searching for telomerase stabilizing drugs and by specifically differentiating these cells into hematopoietic fates. This project will significantly increase the current knowledge on bone-marrow failure syndromes, by first understanding the deleterious effects of dysfunctional telomeres in human pluripotent cells, and then by devising novel strategies to treat patients afflicted with dyskeratosis congenita, a disease that currently has no cure.