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

描述（由申请人提供）：该项目的目标是使用患者特异性诱导多能干细胞（iPSCs）作为开发先天性角化不良（DC）患者新疗法的平台，DC是一种骨髓衰竭综合征，表现为预期寿命差和多系统组织缺陷，包括再生障碍性贫血和肺纤维化。为了实现这一目标，将使用一系列技术，包括基因校正、高通量测序、小分子药物筛选和DC iPS细胞定向分化到造血命运。所有DC患者的端粒都非常短，与其他人群相比，端粒长度通常低于第一个百分位数。到目前为止，在DC中发现的所有突变都是与端粒稳态相关的基因，或在端粒酶中，或直接与端粒结合。端粒酶是哺乳动物细胞中负责端粒合成的多酶复合物。在端粒酶缺失的情况下，端粒会逐渐缩短，这与小鼠和人类的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这些患者的成体干细胞区室的自我更新丧失，这是由于端粒酶突变环境下端粒加速缩短引起的。该项目的中心假设是，疾病特异性iPS细胞提供了一种新的、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，并可用于寻找纠正这种表型的策略。我之前已经证明，来自患有不同严重程度DC的患者的iPS细胞忠实地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。在这里，我建议使用这些患者特异性的iPS细胞来详细了解由功能失调的端粒引起的自我更新表型的丧失，并将这些细胞作为药物发现和靶向分化的平台，这可能为未来的临床治疗提供新的方案。具体目标是：(1)通过对端粒极短的DC iPS细胞的遗传互补和高通量基因表达分析来逆转DC患者特异性iPS细胞的自我更新缺陷；(2)通过寻找端粒酶稳定药物和特异性分化这些细胞进入造血细胞，将DC iPS细胞作为开发针对DC的新疗法的平台。这个项目将通过首先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症的患者，从而显著增加目前对骨髓衰竭综合征的了解。

DESCRIPTION (provided by applicant): 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 Specifi 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.