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

Duchenne肌营养不良症(DMD)是一种由肌营养不良蛋白基因突变引起的毁灭性疾病。该病的发病年龄在5岁之前，起病时身体虚弱，并在青春期进展，导致行动不便。患者在生命的第三个十年期间死于心脏或呼吸系统并发症。到目前为止，唯一被批准的治疗方法是皮质激素，它可以减缓病情发展，但在自然历史上没有真正的影响。尽管在开发新疗法上投入了巨大的努力，但大多数都失败了。目前，基于将营养不良蛋白基因的健康拷贝释放到肌肉纤维上的基因疗法正在显示出令人振奋的结果，但似乎很明显，它们无法阻止疾病的进展，肌肉退化过程将保持活跃。多年来，人们在DMD的动物模型中研究了DMD的肌肉退变过程。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。受损的纤维最初由名为卫星细胞的细胞修复。然而，重复性损伤会在组织中引发一系列后果，包括炎症细胞的持续渗透，激活一种被称为纤维脂肪前体细胞(FAP)的细胞。FAP会导致肌肉退化，因为它们会产生脂肪和纤维组织，取代受损的肌肉纤维。在疾病的发展过程中，纤维的丢失与纤维和脂肪组织的扩张有关，这损害了卫星细胞再生受损肌肉纤维的能力。尽管已经确定有几种分子是调节小鼠这一过程的关键，但对人类来说，限制新疗法开发的因素知之甚少。我们已经开发了一种新的方案，从用于诊断并存储在生物库中的肌肉活检组织中分离卫星细胞和FAP。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，一个是积极增殖并保留茎干特性的群体，另一个是已经致力于产生纤维组织的群体。在这项建议中，我们将使用不同临床阶段的患者的肌肉活检来探索这些不同的人群何时出现在DMD患者的进展中，并将他们的存在与组织特征相关联，以了解这些亚群是否与纤维化、炎症或肌肉纤维死亡等变化有关。然后，我们将分析在对照和DMD肌肉中通过纤维以及其他成分(如炎症细胞)表达的基因，这些基因可以指导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、移动或分化为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两个细胞在培养皿中如何相互作用的信息，就像它们在组织中时会发生的那样。我们将在标准的2D培养皿中研究这种相互作用，也将在3D系统中研究这种相互作用，这些3D系统使用打印模具，使人造肌肉能够更持久和更有结构地生成。这些实验将为了解FAP是否以及如何影响卫星细胞功能提供有价值的信息。最后，我们将测试能够中和先前实验中确定的关键分子通路功能的药物文库，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.