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

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.

The process of muscle degeneration in Duchenne muscular dystrophy (DMD) has been extensively studied in mice. In recent years a new type of cell called fibro-adipogenic precursor cell (FAPs) has been discovered. These cells have a pivotal role in both muscle regeneration and degeneration regulating the function of other muscle stem cells and producing fibrotic and fat tissue contributing to muscle fiber lose. Research in murine models have generated candidate molecular targets for therapies although there is still a considerable 'translational gap' between these putative targets and effective therapies for patients. This is in part due to limited definition of the functional heterogeneity and interactome of cell lineages that contribute to the muscle degeneration, which is imperfectly recapitulated by rodent models. We have developed a new protocol to isolate FAPs and other muscle cells from muscle biopsies. We have already identified that there are different subpopulations of FAPs in DMD with a different gene expression profile whose function in completely unknown. We will isolate these subpopulations and study their biological properties and how these cells interact with other muscle resident cells. Additionally, we will study the temporal dynamics of FAP subpopulations through the progression of the disease and the molecular pathways governing changes in their phenotype. These experiments will identify molecular targets for the development of new therapies that could influence FAP cells function. In this sense, we have recently identified a series of drugs that slowed down disease progression in mice. We will test these drugs in the cells obtained from patients to confirm their effectivity in humans as a preliminary step to trials. In summary, we aim to characterize the role of different subpopulations of FAP cells in DMD patients to discover new putative molecular targets for developing new therapies to slow down the progression of this devastating disorder.