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

描述（由申请人提供）：端粒包含染色体末端的DNA序列，由于细胞周期S期的氧化和不完全DNA复制，端粒每年缩短约30-100 bp。当端粒变得非常短时，染色体形成导致癌症的染色体-染色体融合，并被识别为激活DNA损伤修复反应的双链断裂，导致细胞死亡或衰老以及随后的组织和器官功能障碍。端粒酶延伸端粒，并且大多数细胞中端粒酶的限制性组分是端粒酶逆转录酶（TERT）。短端粒长度与许多疾病有关，在我们的实验室中，包括杜氏肌营养不良症（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DMD中细胞衰老的第一个证据，以及将短端粒与DMD联系起来的非常令人信服的证据，当我们最近显示具有突变型肌营养不良蛋白和缩短的端粒的小鼠（我们的mdx/mTR小鼠）忠实地重现人类DMD时，与仅具有突变型肌营养不良蛋白的小鼠不同（Cell，2010）。此外，我们发现短端粒导致肌肉干细胞（MuSC）复制衰竭，以及随后无法修复由突变型肌营养不良蛋白引起的损伤。事实上，DMD患者的肌肉端粒很短。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的和缓慢的，因为TERT在许多水平上被广泛调节，使得通过内源性TERT的端粒延伸高度依赖于细胞类型、细胞周期阶段和外在条件。尽管先前的工作（Cooke实验室）揭示了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，这在临床上是次优的，并且需要长期治疗，因为它们没有解决内源性TERT抑制。我们建议用两种高影响力和广泛适用的工具来克服这些限制：一种旨在克服TERT调节以安全，快速和可靠地延长端粒的瞬时治疗剂，以及一种使我们的TERT治疗剂能够被递送到肌肉干细胞以治疗肿瘤的递送载体。 DMD和其他组织中的细胞。我们将使用我们的DMD小鼠模型在来自人类DMD患者的细胞中展示这些工具，这是第一个忠实再现DMD的模型，包括其致死性。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。拟议的研究将使快速端粒延伸在体外和体内的人类细胞中，所产生的工具将有助于预防，延迟，或治疗许多重大疾病，其中涉及短端粒长度。 公共卫生相关性：长端粒保护染色体的末端，短端粒的人患心脏病、癌症、血管性痴呆、阿尔茨海默氏症、肌肉萎缩症和其他疾病的风险更大。我们的项目将首次使用简短，不频繁的治疗来安全，可靠地延长端粒。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.