Exosome Therapeutics to Dissect HFpEF Mechanisms

项目总结 目前还没有有效的药物或医疗设备干预措施来治疗心力衰竭 射血分数(HFpEF)。我们率先提出了对HFpEF进行细胞治疗的概念：心脏球源性细胞 (CDCs)显著改善舒张期功能，减少心律失常，同时减轻纤维化和 发炎。这些有益作用中的大部分(如果不是全部)是由CDC(CDCEXO)分泌的外切体(Exosome)介导的。 在这里，我们试图建立HFpEF的详细分子特征；使用这些分子特征作为 通过剖析对CDCEXO的反应，确定关键的、潜在的因果途径的路线图；并发现 新定义的分子实体，基于CDCEXO Cargo，在HFpEF中具有疾病修改生物活性。我们的 有强劲的初步数据支持的假设是： ·HFpEF背后是心肌转录和蛋白质组学方面令人困惑的广泛变化。分选 关联变化的因果关系是一个重大挑战，但它是可行的。 ·这些与HFpEF相关的蛋白质组变化的一部分被CDC或CDCEXO逆转，并且一些相关 以逆转HFpEF的关键功能异常。我们假设，专注于CDCEXO反应 路径将有助于寻找因果异常，使有针对性的假设检验成为可能。 ·通过挖掘CDCEXO的RNA和蛋白质含量，我们有可能精确定位哪些特定因素 HFpEF具有抗病生物活性。这些确定的因素本身可能是可行的治疗方法。 候选，或者可以激励创造新的化学实体作为治疗候选。 这项建议的总体目标是更好地了解HFpEF的发病机制，并开发新的细胞- 治疗这种疾病的免费方法。提出了三个目标。在目标1中，我们将执行和分析组织和 单细胞转录组学和蛋白质组学(包括大量蛋白质翻译后修饰) 在代表不同并存的三种不同模型(猪、小鼠和大鼠)中，HFpEF，并比较其 人类HFpEF心脏组织中的疾病特征。OMICS答复的共同点如下： 物种将有助于区分HFpEF发病中的因果路径和关联路径。AIM 2将分析 比较有无转录组和蛋白质组学对疗效的分子标记 暴露在CDCEXO中。由于CDC和它们的外体显著逆转了HFpEF表型，鉴定 CDCEXO诱导的分子变化将进一步细化途径优先顺序 关联性。在这里，我们将研究来自大鼠、小鼠和猪模型的心室组织和单个细胞(和 对照)，体内暴露和不暴露于CDCEXO。在目标3中，我们将挖掘CDCEXO货物以确定关键 在HFpEF大鼠模型中影响疾病改变生物活性的因素。这将允许我们定义和/或创建 特定的分子实体(RNA物种或蛋白质)可能比复杂的生物制品(细胞， Exosome)在机械上的离散性、制造的简易性和治疗的一致性。

PROJECT SUMMARY No effective pharmacological or medical device interventions are available to treat heart failure with preserved ejection fraction (HFpEF). We have pioneered the concept of cell therapy for HFpEF: Cardiosphere-derived cells (CDCs) dramatically improve diastolic function and reduce arrhythmias, while attenuating fibrosis and inflammation. Most, if not all, of these beneficial effects are mediated by exosomes secreted by CDCs (CDCEXO). Here we seek to establish detailed molecular signatures of HFpEF; to use those molecular signatures as roadmaps to identify key, potentially causal pathways by dissecting the responses to CDCEXO; and to discover novel defined molecular entities, based on CDCEXO cargo, with disease-modifying bioactivity in HFpEF. Our hypotheses, backed by strong preliminary data, are: • Underlying HFpEF are bewilderingly extensive changes in myocardial transcriptomics and proteomics. Sorting causal from associative changes presents a major challenge, but it is doable. • A subset of these HFpEF-related proteome changes are reversed by CDCs or CDCEXO and some correlated to the reversal of the key functional abnormalities of HFpEF. We posit that focusing on CDCEXO-responsive pathways will facilitate the search for causal abnormalities, enabling targeted hypothesis testing. • By mining the RNA and protein contents of CDCEXO, we have the potential to pinpoint defined factors which have disease-modifying bioactivity in HFpEF. Such defined factors may themselves be viable therapeutic candidates, or can inspire the creation of new chemical entities as therapeutic candidates. The overall goal of this proposal is to understand better the pathogenesis of HFpEF, and to develop novel cell- free approaches to treat this disease. Three aims are proposed. In Aim 1 we will perform and analyze tissue and single cell transcriptomics and proteomics (including numerous protein post-translational modifications) of HFpEF, in three different models (pig, mouse and rat) that represent different comorbidities and compare their disease signature to those in human HFpEF heart tissue. Commonalities in the OMICS responses among species will help distinguish causal versus associative pathways in HFpEF pathogenesis. Aim 2 will analyze molecular signatures of therapeutic efficacy by comparing transcriptomics and proteomics with and without exposure to CDCEXO. Because CDCs and their exosomes strikingly reverse the HFpEF phenotype, identification of CDCEXO-induced molecular changes will further refine pathway prioritization in terms of causal versus associative. Here we will study ventricular tissue and single cells from rat, mouse and pig models of HFpEF (and controls), with and without in vivo exposure to CDCEXO. In Aim 3 we will mine CDCEXO cargo to identify critical factors underlying disease-modifying bioactivity in a rat model of HFpEF. This will allow us to define and/or create specific molecular entities (either RNA species or proteins) that may be preferable to complex biologicals (cells, exosomes) in terms of mechanistic discreteness, ease of manufacturing, and therapeutic consistency.