AUTOLOGOUS INTRAMUSCULAR TRANSPLANTATION OF ENGINEERED SATELLITE CELLS INDUCES EXOSOME-MEDIATED SYSTEMIC EXPRESSION OF FUKUTIN-RELATED PROTEIN AND RESCUES DISEASE PHENOTYPE IN A MURINE MODEL OF LIMB-GIRDLE MUSCULAR DYSTROPHY TYPE 2I

α-Dystroglycanopathies are a group of heterogeneous dystrophic phenotypes associated to reduced levels of α-DG glycosylation. Since the glycosylated domains of α-DG exert a key role in extracellular matrix proteins (ECM) binding to the myofiber cytoskeleton, the hypoglycosylation leads to the disruption of this linkage and, consequently, to sarcolemma fragility and myofiber necrosis. To date, any successful therapeutical approach has been developed in the field of α-dystroglycanopathies. Many genes have been found to be implicated in the α-DG glycosylation process; all these genes encode for glycosyltransferase enzymes. Of note, mutations occurring in one of these genes, the fukutin related protein (FKRP) give rise to different subtypes of clinical phenotypes, ranging from the mild limb girdle muscular dystrophy 2I (LGMD2I), to the severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome (WWS) and Muscle-Eye-Brain disease (MEB). FKRP glycosyltransferase exerts its glycosyltransferasic function by conveying a ribitol phosphate group from a CDP –ribitol to α-DG. Recent findings have reported cells ability to release FKRP in vitro. In line with this evidence, it has also been reported that some glycosyltransferases, generally referred to as ‘’extracellular glycosyltransferases’’, freely circulate in the bloodstream or, alternatively, packed with a subpopulation of microvesicles (MVs) physiologically secreted by cells, named exosomes. Thus, we hypothesize that freely or exosome-carried FKRP might circulate as an extracellular glycosyltransferase, reaching distal compartments and acting as a “glycan remodeller”. Interestingly, we firstly established a successful transduction of blood-derived CD133+ multipotent stem cells isolated from an MDC1C patient and FKRP L276IKI murine satellite cells, exploiting a lentiviral vector expressing the wild-type isoform of human FKRP gene. Furthermore, we reported FKRP expression in infected LV-FKRP cells- derived exosomes. Subsequently, we performed an autologous intramuscular transplantation of LV-FKRP infected SCs in the L276IKI mouse model and we evaluated the recovery of the exogenous protein expression and function. Similarly, we investigated the presence of FKRP positive exosomes in the plasma of transplanted FKRP L276IKI mice. The exosome-mediated systemic distribution of FKRP glycosyltransferase favoured its rescue at distal sites, determining an overall recovery of α-DG glycosylation and improved muscle strength, as suggested by functional measurement performances. An in vitro model based on an optically accessible microfluidic bioreactor allowed us to mimic the exosome diffusion between cells in vivo, providing further details of functional kinetic and mechanisms underlying exosome uptake. Overall, observed data suggest the possibility to develop a trivalent therapeutical approach, based on the combination of: cell therapy, gene therapy, leading to a physiological exosome-mediated therapy. The autologous transplantation of engineered stem cells would simultaneously provide the recovery of the wild-type isoform of the mutated protein and tissue regeneration, thus overcoming the limits related to single gene therapy. Moreover, intramuscularly transplanted engineered cells- derived exosomes would allow toobtain a systemic amelioration of the dystrophic phenotype. This exosome-based approach is extremely pioneering and promising, particularly in the field of metabolic myopathies and all muscular dystrophies presenting an enzymatic defect, rather than a structural protein disruption. Indeed, vesicles carrying the exogenous therapeutic enzyme rapidly diffuse through the bloodstream reaching target cells and promoting a fast response mediated by the stabilization of the enzymatic activity.