Clinical Phenotyping and Disease Specific Sampling to Identify Non-coding RNAs for Human Therapeutics in PAD

摘要 周围动脉疾病(PAD)是系统性动脉粥样硬化的主要并发症之一。 闭塞导致两种主要的临床表现：腿部疼痛伴行走/行走能力下降(一种情况 称为间歇性跛行(IC)或严重肢体缺血(CLI)，伴有休息时腿痛、缺血性溃疡和 面临每年10%的重大截肢风险的患者。目前还没有药物治疗来改善心脏的血流灌注。 PAD中的肢体缺血，显然仍需要开发新的治疗方法。的核心教条 分子生物学认为DNA制造核糖核酸，核糖核酸制造蛋白质。然而，绝大多数人 哺乳动物基因组不编码蛋白质。有两种类型的非编码RNA：Micro-RNA(MiR) 和长的非编码(LncRNA)RNA。不同物种的MIR的多样性远远大于编码的基因 与miRs相比，lncRNAs的多样性要高出一个数量级；这就需要战略研究人类 疾病。为此，这项提议汇集了一位研究PAD的翻译心血管科学家 他的工作包括microRNAs(附件)，一位专注于体外和体内研究的科学家 PAD模型(甘塔)，一位研究骨骼肌对损伤的适应性的翻译科学家， 开发了一个无与伦比的人类PAD组织生物库(McClung)，以及一位计算生物学家 (Keene)提供RNA测序数据的生物信息学分析。这项提议的核心假设是 规定根据对井中临床疾病状况的洞察进行特定采样- 表型人类、生物信息学分析和特定疾病的非临床测试将解锁非编码 核糖核酸新人类疗法。为此，具体目标是：1：确定用于治疗的微循环 通过确定间歇性疾病受试者血浆中的差异表达来调节 跛行(IC)和严重肢体缺血(CLI)患者和年龄/性别匹配的无PAD对照 研究对象。确定缺血骨骼肌是否来自症状最严重的缺血小腿 显示miR的表达升高。候选人的miR将通过体外研究在 PAD相关条件下，通过体内疗效观察。2.通过确定要调制的lncRNA LncRNA在非PAD和PAD(IC和CLI)患者小腿肌肉中的差异表达 我们将首先寻找在PAD(IC和CLI)中上调的lncRNA，然后检查是否上调- 监管发生的方式不同(IC或CLI)。在新的队列中，我们将检查来自以下初始列表的预测 候选的lncRNAs，并将确定靶标上调是否也发生在血液中。3)利用鼠标 HLI模型，概括了人类IC与CLI名称的“主要”区别 识别额外的候选lncRNA的条件，这些候选lncRNA可能在老鼠和人之间保守 作为治疗的候选对象。

Abstract Peripheral arterial disease (PAD) is one of the major complications of systemic atherosclerosis where arterial occlusions result in two major clinical manifestations: leg pain with walking/reduced walking ability (a condition termed intermittent claudication, IC) or critical limb ischemia (CLI) with leg pain at rest, ischemic ulcers, and patients that face a >10% risk/year of major amputation. No medical therapy exists to improve perfusion to the ischemic limb in PAD and there remains a clear need to develop new treatments. The central dogma of molecular biology states that DNA makes RNA and RNA makes protein. However, the vast majority of the mammalian genome does not encode proteins. There are two types of non-coding RNAs: micro-RNA (miR) and long non-coding (lncRNA) RNAs. The diversity of miRs across species is far greater than encoded genes and lncRNAs are an order of magnitude more diverse that miRs; this necessitates that strategies study human disease. To that end, this proposal brings together a translational cardiovascular scientist who studies PAD and whose work has included micro-RNAs (Annex), a research scientist who focuses on in-vitro and in-vivo models of PAD (Ganta), a translational scientist who studies skeletal muscle adaptability to injury and has developed an unparalleled biobank of human PAD tissues (McClung), as well as a computational biologist (Keene) to provide bioinformatics analysis of RNA sequencing data. The central hypothesis of this proposal states that condition specific sampling based on insights into the clinical disease condition from well- phenotyped humans, bioinformatics analysis, and disease specific non-clinical testing will unlock non-coding RNA novel human therapeutics. To that end, the Specific Aims are: 1: Identify miRs for therapeutic modulation by determining differential expression in plasma across subjects with intermittent claudication (IC) and critical limb ischemia (CLI) patients and age/sex-matched non-PAD control subjects. Determine whether the ischemic skeletal muscle from the most symptomatic ischemic leg demonstrates elevated expression of the miR. Candidate miR's will be examined through in-vitro studies in PAD relevant conditions, through in-vivo efficacy. 2. Identify lncRNAs for modulation by determining the differential expression of lncRNA in calf muscle from non-PAD and PAD (both IC and CLI) patients. We will first look for lncRNA's that are up-regulated in PAD (IC and CLI) and will then examine whether up- regulation occurs differentially (IC or CLI). In a new cohort, we will examine predictions form an initial list of candidate lncRNAs and will determine whether target up-regulation also occurs in blood. 3) Utilize mouse models of HLI that recapitulate the “major” differential for the IC vs. CLI designation in the human condition to identify additional candidate lncRNAs that may be conserved from mouse to man and serve as therapeutic candidates.