Molecular Mechanism of X-linked Dyskeratosis Congenita

描述（由申请人提供）： 在先天性角化不良（DC）和许多其他疾病状态中，患者的健康和寿命受到造血更新不足的限制。由于骨髓移植不是一种有效的DC治疗方法，因此必须设计新的策略。本申请测试了X连锁DC来源于特定端粒酶RNA缺陷的假设，并研究了淋巴细胞中的端粒酶功能，其长期目标是了解疾病分子机制并开发有效的疾病治疗方法。目标一：端粒酶功能将在表达改变的dyskerin的X连锁DC患者成纤维细胞中恢复，与表达野生型dyskerin的家族匹配的X连锁DC载体成纤维细胞中的端粒酶活化平行。将测试针对端粒酶功能拯救的DC患者细胞获得正常增殖能力和端粒长度维持。这些发现将有助于理解疾病的机制，也可能为端粒酶激活作为临床治疗提供理论依据。AIM II：目的研究端粒酶激活的表达DC或正常dyskerin的细胞系在端粒酶以外的RNA生物合成中可能存在的DC dyskerin缺陷。与目前可用的DC患者细胞培养物不同，细胞系+TERT具有稳健、均匀的生长，因此将避免继发于增殖速率或经历活跃增殖的细胞百分比变化的RNA生物发生缺陷的误导性表征。将测定dyskerin相关RNA蓄积和dyskerin酶在核糖体RNA前体修饰和切割中的功能。AIM III：端粒酶在正常淋巴细胞中的作用机制将通过一个细胞培养系统来研究，该系统可以解决端粒酶在促进细胞生长和存活中的端粒长度依赖性和非依赖性作用。端粒酶在防止细胞死亡中的第三个潜在作用也将被评估。这些研究将揭示端粒酶在DC中受影响最显著的细胞中的新作用。

DESCRIPTION (provided by applicant): In dyskeratosis congenita (DC) and many other disease states, patient health and longevity are limited by insufficient hematopoietic renewal. Because bone marrow transplantation has not been an effective DC therapy, new strategies must be devised. This application tests the hypothesis that X-linked DC derives from a specific telomerase RNA deficiency and investigates telomerase function in lymphoid cells, with the long-term goal of understanding disease molecular mechanism(s) and developing effective disease therapies. AIM I: Telomerase function will be restored in X-linked DC patient fibroblasts expressing altered dyskerin, in parallel with telomerase activation in family-matched X-linked DC carrier fibroblasts expressing wild-type dyskerin. DC patient cells rescued for telomerase function will be tested for the acquisition of normal proliferative capacity and telomere length maintenance. These findings will be relevant for understanding disease mechanism and also may provide proof-of-principle for telomerase activation as a clinical therapy. AIM II: Aim I telomerase-activated cell lines expressing DC or normal dyskerin will be used to investigate potential DC dyskerin defects in the biogenesis of RNAs other than telomerase. Unlike currently available DC patient cell cultures, cell lines +TERT have robust, uniform growth and will therefore avoid misleading characterization of RNA biogenesis defects secondary to changes in proliferation rate or the percentage of cells undergoing active proliferation. Dyskerin-associated RNA accumulation and dyskerin enzyme function in ribosomal RNA precursor modification and cleavage will be assayed. AIM III: Mechanisms of telomerase function in normal lymphoid cells will be studied using a cell culture system that resolves telomere length -dependent and -independent roles for telomerase in promoting cell growth and survival. A potential third role for telomerase in protection against cell death will also be evaluated. These studies will reveal new roles for telomerase in the cells that are most significantly affected in DC.