Exploring heterogeneity of cardiac fibroblasts to reverse fibrosis

描述(申请人提供)：心脏成纤维细胞(CFs)是心脏中最丰富的非心肌细胞类型，提供心肌细胞之间的连续性，维持心肌和冠状动脉血管系统的结构完整性，是心脏损伤反应中纤维化的主要细胞介质。最近，为了产生新的收缩组织和血管，诱导CFs向心肌细胞和内皮细胞的谱系转换的努力受到了低效的重编程的限制。一种稳健而有效的方法包括精确的化学计量学以及在时间上定义的序列中重新编程因子的特定水平的表达。此外，一个根本的障碍仍然是无法确定易受重新编程影响的成纤维细胞的同质性群体。这一建议的中心假设是，与心肌细胞或内皮细胞具有相同胚胎起源的离散CFs群体可能具有表观遗传的‘记忆’，这使它们易于在所需转录因子(TF)的剂量可滴定、时间控制和阶段特定的顺序递送后直接重新编程。通过使用修饰的RNA(ModRNA)作为非整合的遗传递送系统，我们可以实现核心转录因子的可控“脉冲式”表达，并避免与病毒整合相关的潜在问题。为了解决我们的假设，我们将使用谱系追踪实验来确定与心肌细胞和/或内皮细胞共享共同祖先的CFs的离散群体。编码核心重编程因子的合成modRNAs将用于体外和体内的谱系转换。我们相信，完整心脏的天然环境、周围的收缩细胞、生长因子和细胞因子将为功能重新编程提供更宽松的环境。因此，有望将modRNA运送到心脏损伤区域，在那里有成纤维细胞的活跃和动态增殖，可以导致CFs有效地重新编程为心肌细胞和内皮细胞。与该项目相关的挑战是巨大的；然而，其潜在的科学和临床影响是显著的。我们有信心 我们细致的实验设计和创新的方法将揭开CFS的起源及其对心脏发育和损伤的贡献。此外，我们的发现将开辟全新的途径，利用modRNAs在体内操纵CFs，以恢复受损心肌和逆转纤维化。

DESCRIPTION (provided by applicant): Cardiac fibroblasts (CFs), the most abundant nonmyocyte cell type in the heart, provide continuity between cardiomyocytes, maintain the structural integrity of the myocardium and coronary vasculature and are the predominant cellular mediators of fibrosis in the heart in response to injury. Recent efforts to induce lineage conversion of CFs to cardiomyocytes and endothelial cells, in an effort to generate new contractile tissue and blood vessels, have been limited by an inefficient reprogramming. A robust and efficient method involves precise stoichiometry as well as certain levels of expression of reprogramming factors in a temporally defined sequence. Additionally, a fundamental obstacle remains the inability to identify a homogenous population of fibroblasts susceptible to reprogramming. The central hypothesis of this proposal is that a discrete population of CFs that share the same embryonic origin as cardiomyocytes or endothelial cells may have an epigenetic 'memory' that predisposes them to direct reprogramming following a dose-titratable, temporally-controlled, and stage-specific sequential delivery of required transcription factors (TFs). By using modified RNA (modRNA) as a non-integrating genetic delivery system, we can achieve a controlled 'pulse-like' expression of core TFs and avoid potential issues associated with viral integration. To address our hypotheses, we will use lineage-tracing experiments to identify discrete populations of CFs that share a common ancestor with cardiomyocytes and/or with endothelial cells. Synthetic modRNAs that encode core-reprogramming TFs will be used for lineage conversion both in vitro and in vivo. We believe that the native milieu of the intact heart, surrounding contractile cells, growth factors ad cytokines will provide a more permissive environment for functional reprogramming. Therefore, it is expected that delivery of modRNA to the injured area of the heart where there is active and dynamic proliferation of fibroblasts could result in an efficient reprogramming of CFs to cardiomyocytes and endothelial cells. The challenges associated with this project are substantial; however, its potential scientific and clinical impact is significant. We are confident that our meticulous experimental design and innovative approach will unravel the origin of CFs and their contribution to cardiac development and injury. Additionally, our findings will open entirely new avenues to manipulate CFs in vivo using modRNAs to restore damaged myocardium and reverse fibrosis.