Study Of Exosomes In The Diagnosis And Therapy Of Muscular Dystrophy

Aim: Muscular dystrophy is composed of a group of lethal hereditary disorders.Among them,Duchenne Muscular dystrophy(DMD)is one of the most common muscular dystrophy,caused by frame-disrupting mutations in the DMD gene,resulting in the absence of functional dystrophin protein,with clinical manifestations of progressive muscle wasting and cognitive defects.Another common muscular dystrophy is dysferlinopathy,caused by the loss or down-regulation of dysferlin,a transmembrane protein encoded by the DYSF gene,with late onset and high heterogeneity in the clinic.The common clinical practice for the diagnosis of muscular dystrophy in the clinic is muscle biopsy,which is invasive and imposes harmful effects on patients psychologically and physically.Therefore,accurate and non-invasive diagnostic approaches are urgently required.In addition,there is no effective treatment for muscular dystrophies available in the clinic,thus it is important to develop novel,effective and safe therapeutic strategies for muscular dystrophies.Recently,exosomes are emerging as natural biological nanovesicles and have drawn much attention as a tool for diagnosis and therapeutics.The low immunogenicity and stability,particularly as cellular messengers,confer exosomes superiority to other synthetic nanovesicles.In the present study,we plan to explore the feasibility and potential of exosomes in the diagnosis and therapy of muscular dystrophies and aim to provide a new approach and strategy for muscular dystrophies.Methods: Ultracentrifugation was used to isolate exosomes from healthy and dysferlinopathy patients' serum and urine.Transmission electron microscopy,nanosight and western blotting were performed to characterize exosomes.Western blotting and ELISA were undertaken to measure the expression of dysferlin protein in serum and urine-derived exosomes between healthy and patients.Also western blot was employed to determine the expression of other functional proteins in serum and urine-derived exosomes from other muscular dystrophy patients.Dysferlin-deficient muscle cells(DYSF-/-)were used as in vitro models toevaluate the feasibility and potential of exosomes as nano-carriers for transmembrane protein-dysferlin.Western blotting,immunocytochemistry and laser confocal fluorescence microscopy were used to examine the transfer and localization of dysferlin protein in DYSF-/-cells after incubation with dysferlin-expressing exosomes(EXOdysf).Two-photon laser scanning confocal microscopy and immunocytochemistry were performed to measure the capability of EXOdysf to restore the membrane repair capacity of DYSF-/-myotubes upon stimuli after 48 hr incubation and the involvement of lysosomes in the repair process.Fluorescence probe(Fluo-3 AM)was used to monitor the calcium flux in EXOdysf-treated DYSF-/-cells upon membrane injury.CRISPR-Cas9 genome-editing system was used to generate human DYSF gene exon 22 and 55 deletions in human primary myoblasts.Western blotting and two-photon laser scanning confocal microscopy were applied to examine the transfer of dysferlin protein and membrane repair process in human dysferlin-deficient myotubes treated with human serum-derived exosomes.To investigate the therapeutic effect of exosomes on DMD,we applied exosomes derived from different cells and injected into mdx mice at the dose of 150?g/mouse twice a week for 3 weeks intraperitoneally.Grip strength and serum creatine kinase(CK)levels were monitored to reflect the functional improvement in exosome-treated mdx mice.Body-weight,biochemical and histopathological analysis were used to examine the pathological improvement and toxicities.Serum immunoglobulin(Ig G)staining was used to meansure the integrity of muscle fibers.Western blotting and immunohistochemistry were used to evaluate tprotein expression and localization of different dystrophin-associated protein complex(DAPC)in muscle from exosome-treated mdx mice.Results: 1.Measurement of dysferlin protein in serum and urine exosomes derived from healthy volunteers and dysferlinopathy patients indicated that no or trace amounts of dysferlin protein were detected in serum or urine exosomes derived from dysferlinopathy patients,whereas a clear band of dysferlin was found in serum or urine exosomes derived from healthy volunteers,suggesting that levels of dysferlin protein expression in serum or urine exosomes can be used as diagnosticmarkers for dysferlinopathy.Furthermore,an ELISA approach with specific detection limits was established for measuring dysferlin in serum or urine exosomes from dysferlinopathy patients.2.Examination of related functional proteins in serum or urine exosomes from dysferlinopathy patients revealed that serum or urine exosomes can potentially be used as a diagnostic approach for other muscular dystrophies.3.Measurement of dysferlin protein in exosomes derived from different cells demonstrated that myotube-derived exosomes(EXOdysf)carry the most abundant dysferlin protein compared to other exosomes with the expression of exosomal biomarkers such as CD63,Alix and CD9.EXOdysf showed a typical sauce-cup shape and dysferlin protein was localized in cell membrane and cytoplasma as revealed by transmission electron microscopy and immunogold staining.EXOdysf could effectively deliver dysferlin protein into DYSF-/-cells and restore the expression of dysferlin protein and localization in cell membrane in DYSF-/-cells as demonstrated by western blot and immunocytochemistry.EXOdysf-treated DYSF-/-myotubes showed improved membrane repair ability compared to untreated controls and lysosomes contributed to the membrane repair process as demonstrated by the expression of lysosomal marker protein LAMP-1.EXOdysf-treated DYSF-/-myotubes showed increased Ca2+ regulation capacities.Evaluation of serum-derived exosomes in human dysferlin-deficient myoblasts,generated by deletion of human DYSF exon 22 and 55 via CRISPR-Cas genome editing system,revealed that serum derived exosomes can restore the expression of dysferlin protein and the membrane repair capability of human dysferlin-deficient myoblasts irrespective of mutation types.4.Study of exosomes in the treatment of DMD revealed that a significant functional recovery was observed in mdx mice treated with exosomes from different cells compared to untreated mdx controls as demonstrated by significant increases in the grip strength and reductions in the levels of serum CK.Monitoring of body weight and serum biochemical parameters showed that no significant change was found for body weight and levels of AST,ALT and other biochemical parameters.Dramatically reduced fibrosis,inflammation and serum Ig G staining wereobserved in peripheral muscles from exosome-treated mdx mice compared to untreated mdx controls,suggesting that exosomes can halt the pathological progression without any detectable toxicity.Importantly,exosomes promoted muscle regeneration and increased the expression of dystroglycan and sarcoglycan and their re-localization in muscle membrane in treated mdx mice.Further elucidation on the underlying mechanism revealed that exosomes likely improve the expression and re-localization of DAPC by inhibiting the expression of proteasome expression and activities.Conclusions: 1.Serum and urine derived exosomes can be used as a non-invasive diagnostic approach for the diagnosis of dysferlinopathy and other muscular dystrophies.2.Exosomes can be efficient natural nano-carriers for transmembrane protein and can effectively transfer dysferlin protein and promote its correct localization in cell membrane and restore cellular functions.Thus our study provides a new therapeutic strategy and modality for dysferlinopathy.3.Exosomes can elicit phenotypic rescue and restore dystrophin associated protein complex and promote muscle regeneration in mdx mice and thus our findings provide a new approach for the treatment of DMD.4.The mechanistic investigation revealed that exosomes possibly increase and stabilize the expression and re-localization of dystrophin associated protein complex in body-wide muscles through the inhibition of proteasome expression and activities and thus shed light on the new role of exosomes in treating DMD.