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。由于骨髓移植并不是一种有效的DC治疗方法，因此必须设计新的策略。这项应用验证了X连锁DC源于特定端粒酶RNA缺失的假设，并研究了淋巴细胞中的端粒酶功能，以期了解疾病的分子机制(S)并开发有效的疾病治疗方法。目的I：在表达突变的dyskerin的X连锁DC患者成纤维细胞端粒酶功能恢复的同时，表达野生型dyskerin的家族匹配的X连锁DC载体成纤维细胞的端粒酶活性也将恢复。为端粒酶功能而挽救的DC患者细胞将接受测试，以获得正常的增殖能力和维持端粒长度。这些发现将有助于理解疾病的发病机制，也可能为端粒酶激活作为临床治疗方法提供理论依据。目的：目的研究表达DC或正常dyskerin的端粒酶激活细胞株在端粒酶以外的RNA生物发生中潜在的DC dyskerin缺陷。与目前可用的DC患者细胞培养不同，+TERT细胞系具有强劲、均匀的生长，因此将避免因增殖率或经历活跃增殖的细胞百分比的变化而导致的RNA生物发生缺陷的误导性特征。将检测核糖体RNA前体修饰和切割过程中Dyskerin相关RNA的积累和Dyskerin酶的功能。目的III：利用细胞培养系统研究正常淋巴细胞端粒酶功能的机制，该系统解决了端粒酶在促进细胞生长和存活中的端粒长度依赖和非依赖作用。还将评估端粒酶在防止细胞死亡方面的第三个潜在作用。这些研究将揭示端粒酶在DC中最受影响的细胞中的新角色。

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.