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

摘要 外周动脉疾病（PAD）是全身性动脉粥样硬化的主要并发症之一，其中动脉粥样硬化是动脉粥样硬化的主要并发症。 闭塞导致两种主要的临床表现：伴随行走的腿疼痛/行走能力降低（一种病症 称为间歇性跛行（IC）或严重肢体缺血（CLI）伴休息时腿部疼痛、缺血性溃疡，以及 面临>10%风险/年的大截肢患者。没有医学治疗存在以改善灌注到 PAD中的缺血肢体，并且仍然明显需要开发新的治疗方法。的中心法则 分子生物学认为DNA制造RNA，RNA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：微RNA（miR） 和长链非编码（lncRNA）RNA。物种间miR的多样性远远大于编码基因 lncRNA的多样性比miR高出一个数量级，这就需要研究人类的策略。 疾病为此，这项建议汇集了一位研究PAD的翻译心血管科学家 他的工作包括微RNA（附件），一位专注于体外和体内研究的科学家， PAD模型（Ganta），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 根据对临床疾病状况的深入了解， 人类的表型、生物信息学分析和疾病特异性非临床测试将解开非编码基因的密码子。 RNA新型人类治疗剂。为此，具体目标是：1：鉴定用于治疗的miR 通过测定间歇性高血压受试者血浆中的差异表达进行调节， 跛行（IC）和严重肢体缺血（CLI）患者和年龄/性别匹配的非PAD对照 科目确定是否缺血骨骼肌从最有症状的缺血腿 证明miR的表达升高。候选miR将通过体外研究进行检查， PAD相关疾病，通过体内疗效。2.通过测定lncRNA的表达来鉴定lncRNA的调节作用。 来自非PAD和PAD（IC和CLI）患者的小腿肌肉中lncRNA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。 调节发生差异（IC或CLI）。在一个新的队列中，我们将检查来自初始列表的预测， 候选lncRNA，并将确定目标上调是否也发生在血液中。3)使用鼠标 HLI模型，概括了IC与CLI在人体中指定的“主要”差异 条件以鉴定可能从小鼠到人保守的另外的候选lncRNA， 作为治疗候选人。

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.