Mutations in the gene coding for Fukutin related protein (FKRP) can lead to different diseases, ranging from very severe multi-organ syndromes like Walker Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB), to muscle defects as Congenital Muscle Dystrophy (CMD) and the mildest but more frequent form Limb Girdle Muscle Dystrophy type 2i (LGMD2i or LGMD-R9 FKRP-related). These diseases, all part of the dystroglycanopathies group, are characterized by the defective glycosylation of alpha-dystroglycan (aDG), a membrane glycoprotein involved in the cell/matrix anchoring of muscle fibers. FKRP is one of the multiple proteins involved in the aDG glycosylation process, acting with its partner Fukutin as a ribitol-transferase.
We previously published a proof-of-principle of the efficiency of AAV-mediated transfer of the FKRP gene to correct the myopathology in a KI mouse model reproducing the most frequent mutation (L276I) in LGMD-R9. Since the phenotype of this model is relatively modest, we developed a muscle specific FKRP knock-out mouse model, based on Cre-lox recombination technology. This new mouse model, named HSA-FKRPdel, has a normal life span but present a severe dystrophic process in the skeletal muscle. Defects of glycosylation of aDG and of its binding to laminin were observed, as well as progressive histological dystrophic signs as centronucleation, inflammation, necrosis and fibrosis. Functional evaluation also revealed a reduced force of HSA-FKRPdel mice muscles. Seric and in situ biomarkers of muscle dystrophy pathways were assessed and highlight activation of pathways relative of regeneration, inflammation and fibrosis. The HSA-FKRPdel mouse model was then used for a dose effect study of an AAV9 expressing FKRP under the expression of a muscle promoter. The results of the study indicate that a dose of 5e12 vg/kg corrects to the wild-type level the myopathology at the histological and functional levels. No adverse events or signs of toxicity was observed even at 20x the efficient dose. These preclinical studies demonstrated efficacy and safety in AAV-mediated transfer of FKRP and are relevant for defining the doses to be used in clinical trials for FKRP deficiencies.