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 muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP -ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a "glycan remodelling" process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase.

Stem Cell-Mediated Exon Skipping of the Dystrophin Gene by the Bystander Effect.

Duchenne muscular dystrophy (DMD) is characterized by the loss of a functional dystrophin protein; the muscles of DMD patients progressively degenerate as a result of mechanical stress during contractions, and the condition eventually leads to premature death. By means antisense oligonucleotides (AONs), it is possible to modulate pre-mRNA splicing eliminating mutated exons and restoring dystrophin open reading frame. To overcome the hurdles in using AONs for therapeutic interventions, we exerted engineered human DMD stem cells with a lentivirus, which permanently and efficiently delivered the cloned AONs. Here we describe for the first time the exosome-mediated release of AONs from engineered human DMD CD133+ stem cells allowing the rescue of murine dystrophin expression. Finally, upon release, AONs could be internalized by host cells suggesting a potential role of exosomes acting as vesicular carriers for DMD gene therapy.