FAP-DMD: Elucidating the role of FAP cells in the process of muscle degeneration in patients with Duchenne muscular dystrophy

杜氏肌营养不良症（DMD）是一种破坏性的疾病所产生的突变的肌营养不良基因。疾病的发病伴随虚弱的发作发生在5岁之前，并且在青春期期间进展导致肌肉松弛。患者在生命的第三个十年内因心脏或呼吸系统并发症而死亡。迄今为止唯一批准的治疗方法是皮质激素，它可以减缓疾病进展，但对自然史没有真实的影响。尽管投入了巨大的努力来开发新的治疗方法，但大多数都失败了。目前，基于向肌肉纤维释放健康的抗肌萎缩蛋白基因拷贝的基因疗法显示出有希望的结果，但似乎很明显，它们无法阻止疾病的进展，肌肉退化的过程将保持活跃。DMD的肌肉变性过程已经在该疾病的动物模型中研究了多年。肌营养不良蛋白的缺乏使纤维在正常肌肉收缩时更容易受到损伤。受损的纤维最初由称为卫星细胞的细胞修复。然而，重复性损伤在组织中引发了一系列后果，包括炎症细胞的持续浸润，这些炎症细胞激活了一种称为纤维脂肪形成前体细胞（FAP）的细胞。FAP导致肌肉退化，因为它们产生脂肪和纤维化组织，替代受损的肌肉纤维。在疾病的进展过程中，纤维的损失与纤维化和脂肪组织的扩张有关，这损害了卫星细胞再生受损肌纤维的能力。尽管已经确定了几种分子是调节小鼠这一过程的关键，但人类对限制新治疗方法发展的因素知之甚少。我们已经开发了一种新的协议，从肌肉活检中分离卫星细胞和FAP，这些肌肉活检用于诊断并储存在生物库中。我们分析了从DMD和健康人获得的细胞，并鉴定了不同类型的FAP细胞。总而言之，我们已经确定了两个主要群体，一个是积极增殖和保留干性性质，另一个是已经致力于产生纤维化组织。在本提案中，我们将使用不同临床阶段患者的肌肉活检来探索这些不同人群何时出现在DMD患者的进展中，并将其存在与组织特征相关联，以了解这些亚群是否与纤维化，炎症或肌纤维死亡等变化相关。然后，我们将分析在对照和DMD肌肉中表达的基因，这些基因由纤维以及其他成分（如炎症细胞）表达，这些成分可以指导FAP亚型的变化。了解控制FAP亚群变化的分子途径将为旨在对抗纤维脂肪组织扩张的治疗提供潜在的新靶点。在第三阶段，我们将分离FAP的亚群并研究它们的特性，包括它们如何增殖，移动或分化成纤维化或脂肪产生细胞以及它们如何与卫星细胞相互作用。这些后来的研究提供了关于两个细胞如何在培养皿中相互作用的信息，因为它们在组织中时会发生这种情况。我们将在标准的2D培养皿中研究这种相互作用，也将在使用打印模具的3D系统中研究这种相互作用，这些模具能够更持久和结构化地生成人造肌肉。这些实验将提供有价值的信息，以了解FAP是否以及如何影响卫星细胞功能。最后，我们将测试能够抵消先前实验中确定的关键分子通路功能的药物库，以了解它们是否能够调节FAP的功能。

Duchenne muscular dystrophy (DMD) is a devastating disease produced by mutations in the dystrophin gene. Disease's onset with onset of weakness occurs before the age of 5 years and progresses leading to loose of ambulation during adolescence. Patients die during the third decade of life because of cardiac or respiratory complications. The only treatment approved so far is corticoids that slow down progression but without having a real impact in natural history. Despite huge efforts invested in developing new treatments, most of them have failed. At the moment, gene therapies based on the release of a healthy copy of the dystrophin gene to the muscle fibers are showing promising results, but it seems clear that they are not going to be able to stop disease's progression and that the process of muscle degeneration will remain active. The process of muscle degeneration in DMD has been studied for many years in animal models of the disease. Lack of dystrophin makes the fibers more susceptible to damage during normal muscle contraction. Damaged fibers are initially repaired by cells named satellite cells. However, repetitive damage triggers a series of consequences in the tissue including persistent infiltration by inflammatory cells that activates a type of cell known as fibro-adipogenic precursor cells (FAPs). FAPs contribute to muscle degeneration as they produce fat and fibrotic tissue which substitutes the damaged muscle fibers. During disease's progression, loss of fibers is associated to expansion of fibrotic and fatty tissue which impairs the capability of satellite cells to regenerate damaged muscle fibers. Although several molecules have been identified as key to regulate this process in mice, little is known in humans what is limiting the development of new treatments. We have developed a new protocol to isolate satellite cells and FAPs from muscle biopsies that were taken for diagnosis and are stored in biobanks. We have analyzed the cells obtained from DMD and healthy people and have identified different types of FAP cells. To summarize we have identified two main populations, one that is actively proliferating and retain stemness properties and another that is already committed to produce fibrotic tissue. In this proposal we will explore when these different populations appear in the progression of DMD patients using muscle biopsies of patients at different clinical stages and correlate their presence with tissue features to understand if these subpopulations are associated to changes such as fibrosis, inflammation or muscle fiber death. Then we will analyze the genes that are expressed in control and DMD muscle by fibers but also by other components, such as inflammatory cells, that could guide the changes in FAP subtype. Understanding the molecular pathways governing the changes in FAP subpopulations will provide potential new targets for therapies aimed to counteract the expansion of fibro-fatty tissue. In a third stage we will isolate the subpopulations of FAPs and study their properties including how they proliferate, move, or differentiate into fibrotic or fat producing cells and how they interact with satellite cells. These later studies provide information about how two cells interact in a dish, as it would happen when they are in the tissue. We will study this interaction both in standard 2D culture dishes, but also in 3D systems using printed molds that enable a more perdurable and structured generation of artificial muscles. These experiments will provide valuable information to understand if and how FAPs influence satellite cell function. Finally, we will test libraries of drugs able to counteract the function of key molecular pathways identified in the previous experiments to understand if they are able to modulate the function of FAPs.