| Duchenne Muscular Dystrophy(DMD)is a lethal,progressive muscle wasting disease,caused by frame-disrupting mutations in the DMD gene,which result in the deficiency of functional dystrophin protein.There is no cure in the clinic.Antisense oligonucleotide(AO)-mediated exon-skipping therapy represents a promising therapeutic approach with a few AO drugs conditionally approved in the clinic.However,low systemic delivery efficiency and lack of muscle-targeting hinder clinical application of this approach.Recently,small compound-based novel drug delivery systems have been under intensive scrutiny.Our group previously demonstrated that hexose can enhance delivery of phosphorodiamidate morpholino oligomer(PMO)to muscle and PMO activities;however hexose showed limited effect in aged mdx mice.In this study,we performed screening in tibialis anterior(TA)muscles of mdx mice with a small compound library consisting of amino acids and citric acid,sodium pyruvate and succinic acid.These compounds have been shown to stabilize protein degradation and provide energy.Co-administration of these small compounds with PMO in TA muscles of mdx mice resulted in significantly increased numbers of dystrophin-positive fibres and dystrophin protein expression compared to the saline group,particularly for glycine,arginine and serine.Systemic evaluation in mdx mice revealed that glycine significantly promotes uptake and activities of PMO in body-wide muscles compared to saline and other amino acids.Long-term repeated administration of glycine and PMO in mdx mice elicited phenotypic rescue with marked functional improvement and up to 15-fold increase of dystrophin in quadriceps,triceps and abdominal muscles compared to PMO in saline.Also,glycine boosted satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1(m TORC1)and replenishing the one-carbon unit pool.The expanded regenerating myofiber population then resulted in increased PMO uptakes and activities.Moreover,glycine was able to promote PMO uptake in aged mdx mice,indicating the applicability of glycine in different ages of mdx mice and potentially DMD subpopulations.Further comparison on different dosing routes of glycine revealed that oral delivery is comparable to intravenous injection and thus oral delivery was chosen for subsequent studies.Systemic investigation in dystrophin/utrophin double knock-out mice(DKO)indicated that glycine can enhance PMO activity and dystrophin expression in body-wide muscles except for the heart.However,cardiomyopathy is the major cause for the mortality of DMD patients.And our previous study showed that metformin can improve heart functions.Therefore,in this study,we examined the synergistic effect of glycine,PMO and metformin in DKO mice for 3 weeks and 6months,in which glycine,PMO and metformin were given via oral delivery,intravenous injection and drinking water,respectively.The results showed that the combined treatment induced significantly increased dystrophin expression in body-wide muscles and extended lifespan of DKO mice,and resulted in functional improvements including cardiac functions,reflected by significantly improved EF and FS,respiratory and muscle functions,reflected by muscle strength and endurance,and spinal deformity index.In addition,low cell transplantation efficiency is the major challenge for cell therapy in DMD.Considering the ability of glycine to boost muscle satellite cell proliferation and muscle regeneration,we transplanted the primary myoblasts or EGFP-positive muscle satellite cells isolated from wild-type or EGFP transgenic C57BL/6 mice into cardiotoxin-injured TA muscles of mdx mice and glycine was intravenously administered during the period of experiments.The results showed that glycine also augmented the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice.Our data provide evidence that glycine enhances satellite cell proliferation,cell transplantation and oligonucleotide efficacy in mdx mice,and thus has therapeutic utility for cell therapy and drug delivery in muscle wasting diseases.In summary,in this study,we identified that glycine,as a novel delivery formulation,can enhance AO delivery to muscle.And glycine functions via replenishing one-carbon unit donor pool and upregulating m TORC1 activation,resulting in muscle satellite cell proliferation and muscle regeneration and leading to increased PMO uptake in regenerating myofibres.The combined treatment of glycine,PMO and metformin is able to elicit phenotypic rescue of DKO mice and thus provide a new treatment option for DMD patients.Also glycine can enhance cell transplantation efficiency in mdx mice.These findings demonstrate the huge potential of glycine in nucleic acid and cell therapies for DMD. |
