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

7.项目总结/摘要 该项目的目标是使用患者特异性诱导多能干细胞（iPSC）作为平台， 为患有先天性角化不良（DC）的患者开发新的治疗方法， 衰竭综合征，表现为预期寿命低和多系统组织缺损，包括再生障碍性贫血 贫血和肺纤维化。为实现这一目标，将采用多种技术，包括 基因校正、高通量测序、小分子药物筛选和靶向分化 DC iPS细胞的造血命运。 所有DC患者的端粒相对于其年龄而言都非常短，通常低于第一百分位数长度 与其他人相比。到目前为止，在DC中发现的所有突变都与以下基因相关： 端粒稳态，或者在端粒酶中，或者直接结合到端粒。端粒酶是多酶 在哺乳动物细胞中负责端粒合成的复合物。在没有端粒酶的情况下，端粒 逐渐缩短，这与小鼠和人类干细胞功能受损有关。因此 在DC中观察到的组织缺陷可能是由于成体干细胞区室中自我更新的丧失， 这些患者是由突变端粒酶加速端粒缩短引起的。中央 该项目的假设是，疾病特异性iPS细胞提供了一种新的和合适的系统来研究 突变端粒酶的后果和DC多能细胞中自我更新的丧失，并可用于 寻找纠正这种表型的策略。 我以前已经证明，来自患者的iPS细胞忠实地显示了DC的不同严重程度， 概括了在人类患者中观察到的端粒缩短和自我更新表型的丧失。这里我 我建议使用这些患者特异性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治疗的平台。 分化，这可能使新的协议，在未来的临床治疗。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细胞中的自我更新缺陷， 具有极短端粒的DC iPS细胞的通量基因表达分析和（II）使用DC iPS细胞 作为开发针对DC的新疗法的平台，通过寻找端粒酶稳定药物， 特别是将这些细胞分化成造血细胞。 这个项目将显著增加目前对骨髓衰竭综合征的认识， 了解人类多能细胞中功能失调的端粒的有害影响，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，这种疾病目前还没有 疗方

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.