Telomere extension using nucleoside-modified mRNA and exosomes as a novel therape

描述(申请人提供)：端粒包括染色体末端的DNA序列，由于细胞周期的S期的氧化和DNA复制不完整，端粒每年缩短约30-100bp。当端粒变得非常短时，染色体形成染色体-染色体融合，导致癌症，并被认为是双链断裂，激活DNA损伤修复反应，导致细胞死亡或衰老，从而导致组织和器官功能障碍。端粒酶是一种延伸端粒的酶，大多数细胞中端粒酶的限制性成分是端粒酶逆转录酶(TERT)。短端粒长度与许多疾病有关，包括在我们的实验室中，杜氏肌营养不良症(DMD)和血管疾病，包括动脉粥样硬化、血管性痴呆和心脏病。我们(BLAU实验室)在1983年发现了第一个涉及DMD细胞衰老的证据，并且非常令人信服的证据将短端粒与DMD联系起来，当时我们最近表明，具有突变的dystrophin和端粒缩短的小鼠(我们的MDX/MTR小鼠)忠实地概括了人类DMD，而不像只有突变的dystrophin的小鼠(Cell，2010)。进一步我们发现，短的端粒导致肌肉干细胞(MUSC)复制衰竭，从而导致无法修复突变的Dstrophin造成的损伤。的确，DMD患者的肌肉端粒较短。因此，需要一种安全、可靠的方法来延长人类的端粒。然而，所有现有的人类相容的方法都是零星和缓慢的，因为TERT在多个水平上被广泛调控，使得内源性TERT通过端粒延伸高度依赖于细胞类型、细胞周期阶段和外部条件。虽然之前的工作(库克实验室)显示，增加端粒酶活性可以避免人类心血管细胞的衰老，但这些研究需要逆转录病毒技术，这在临床上并不理想，而且需要长期治疗，因为它们没有解决内源性TERT抑制问题。我们建议通过两种高影响力和广泛适用的工具来克服这些限制：一种是旨在克服TERT调节以安全、快速和可靠地延伸端粒的瞬时疗法，另一种是允许我们的TERT疗法被输送到肌肉干细胞进行治疗的输送工具。 DMD和其他组织中的细胞，通过静脉注射。注射。我们将使用我们的DMD小鼠模型在人类DMD患者的细胞中演示这些工具，这是第一个忠实地概括DMD的模型，包括它的致命性。我们的实验室在开发具有广泛适用性的创新技术方面有着悠久的历史。拟议的研究将使端粒在体外和体内的快速延伸成为可能，由此产生的工具将有助于预防、预防或治疗与端粒短有关的许多重大疾病。 与公共健康相关：长的端粒保护染色体的末端，而端粒短的人患心脏病、癌症、血管性痴呆、阿尔茨海默氏症、肌肉营养不良和其他疾病的风险更高。我们的项目将首次使用一种简单、罕见的治疗方法来实现安全、可靠的端粒延伸。我们的目标是帮助预防、延迟或治疗所有与短端粒有关的疾病。

DESCRIPTION (provided by applicant): Telomeres comprise DNA sequences at the ends of chromosomes which shorten by about 30-100 bp per year due to oxidation and incomplete DNA replication during S phase of the cell cycle. When telomeres become critically short, chromosomes form chromosome-chromosome fusions which lead to cancer, and are recognized as double-stranded breaks that activate DNA damage repair responses that lead to cell death or senescence and consequent tissue and organ dysfunction. The enzyme telomerase extends telomeres, and the limiting component of telomerase in most cells is telomerase reverse transcriptase (TERT). Short telomere length has been linked to many diseases, including, in our labs, Duchenne muscular dystrophy (DMD) and vascular disease, which includes atherosclerosis, vascular dementia, and heart disease. We (the Blau lab) found the first evidence implicating cellular senescence in DMD in 1983, and very convincing evidence linking short telomeres to DMD when we recently showed that mice with mutant dystrophin and shortened telomeres (our mdx/mTR mice) faithfully recapitulate human DMD, unlike mice with mutant dystrophin alone (Cell, 2010). Further we showed that short telomeres lead to muscle stem cell (MuSC) replicative exhaustion, and consequent inability to repair damage caused by mutant dystrophin. Indeed, DMD patients have short muscle telomeres. Thus there is a need for a safe, reliable method to extend telomeres in humans. However, all existing human- compatible methods are sporadic and slow because TERT is extensively regulated at many levels, making telomere extension through endogenous TERT highly dependent on cell type, cell cycle stage, and extrinsic conditions. Although previous work (Cooke lab) revealed that increasing telomerase activity can avert senescence in human cardiovascular cells, these studies required retroviral technology, which is suboptimal clinically, and required chronic treatment because they did not address endogenous TERT inhibition. We propose to overcome these limitations with two high-impact and broadly-applicable tools: a transient therapeutic designed to overcome TERT regulation to extend telomeres safely, rapidly, and reliably, and a delivery vehicle that will allow our TERT therapeutic to be delivered to muscle stem cells to treat DMD, and cells in other tissues, via i.v. injection. We will demonstrate these tools in cells from human DMD patients using our mouse model of DMD, the first model to faithfully recapitulate DMD, including its lethality. Our laboratories have a long history of developing innovative technologies of broad applicability. The proposed studies will enable rapid telomere-extension in vitro and in vivo in human cells, and the resulting tools will be useful in helping to prevent, dely, or treat the many major diseases in which short telomere length is implicated. PUBLIC HEALTH RELEVANCE: Long telomeres protect the ends of chromosomes, and people with short telomeres are at greater risk of heart disease, cancer, vascular dementia, Alzheimer's, muscular dystrophy, and other diseases. Our project will for the first time enable safe, reliable extension of telomeres using a brief, infrequent treatment. Our goal is to help prevent, delay, or treat all of the many diseases in which short telomeres are implicated.