Characterization Of Myoblast-derived Exosomes And Evaluation In Duchenne Muscular Dystrophy Mouse Models

Objective:Exosomes are membrane-bound vesicle with diameters ranging from 40-100 nm.Different cell-derived exosomes contain different proteins and RNAs,which confer them different properties.Exosomes play an important role in intercellular communication as cellular messengers and are natural carriers for endogenous proteins and RNAs,including mRNA and microRNA.Therefore in our previous study,we tried to explore the potential of exosomes as a drug delivery vehicle for exogenous drugs e.g.siRNA by conferring its brain-targeting property with a neuron-specific peptide.Subsequently we successfully delivered siRNAs to targeted neurons and mediated specific gene knock-down in mice and proved its feasibility as an exogenous gene delivery vector.Based on our previous work,in this report,we wish to further exploit the potential of exosomes as the drug delivey vehicle by loading different cargoes i.e.antisense oligonucleotides(AOs)and utilizing the Duchenne muscular dystrophy(DMD)as the prototype disease model.DMD is a lethal neuromuscular disorder caused by the absence of dystrophin protein,which encoded by the dystrophin gene,one of the largest human gene.Currently there is no effective treatment available in clinic.AO-mediated exon skipping is one of the most promising approaches for DMD and is in phase III clinical trial with some exciting outcome.However there is limitation to AOs tested in clinical trials,particularly the low systemic delivery efficiency,which will require high doses.In this study,we wish to evaluate the potential and feasibility of exosomes as delivery vectors for AOs in DMD.Methods:1.Isolation of exosomes:Differential centrifugation was used for isolating exosomes from culture medium.Exosomes were centrifuged for 1000 g,15 mins to remove cell debris and the supernatant was carefully collected for centrifugation at 12,000 g for 20 mins further filtered via a 0.22 ?m filter.Followed by ultracentrifugation at 100,000 g for 70 mins,the pellets were resuspended in PBS.2.Biochemical analysis of exosomes:Using the protein involved in the formation of multivesicular bodies e.g.Alix and TSG101 as markers for characterizing exosomes.Western blot was applied for confirming the existence of exosomes and also Cytochrome C contained in mitochondria was used as a control marker for examining the contamination of cell debris in exosomes.3.Morphological examination of exosomes:Transmission eletron microscopy(TEM)was used to examine the morphology of exosomes and exosomes were pipetted on carbon-coated grids for 5 mins,followed by natural drying after discarding the waste.Subsequently exosomes were negatively stained with 1%phosphotungstic acid for 2 mins and observed under microscopy after drying.4.Density confirmation of exosomes:Sucrose density gradient centrifugation was perfomed to validate the density of exosomes as reported.Sucrose gradient solution of 1.07-1.25 g/mL was prepared and added to ultracentrifugal tubes based on the density gradient and exosomes were layered on the top.The ultracentrifugation was carried out for 16 h at 100,000 g,fractions from different layers were collected and diluted with PBS,and re-spun at 100,000 g for 1 h,followed by resuspension in PBS.5.Uptake of exosomes in its parental cells:A membrane dye-PKH67 was utilized to label exosomes derived from myoblasts-C2C 12.Labeled exosomes were co-incubated with their parental cells to examine the capability of exosomes entering their parental cells.The uptake was monitored with fluorescence microscopy at different time points.6.Localization of exosomes in C2C12 cells:PKH67 labeled exosomes were co-incubated with C2C12 cells and confocal fluorescence microoscopy was used to visualize the localization of exosomes.7.Muscle cell-or tissue tropism of exosomes:PKH67 labeled exosomes were co-incubated with C2C12,L1(preadipocytes)and NIH3T3(fibroblasts)cells and fluorescence microscope and flow cytometry were used to detect and quantify the uptake of exosomes in different cells,thereby to determine whether myoblast-derived exosomes show muscle cell tropism.Furthermore,we systemically delivered PKH-labeled exosomes into mdx mice via tail vein injection and harvested body-wide tissues 24 h after perfusion.Fluorescence microscope was used to examine tissue distribution of exosomes in mdx mice.8.In vitro evaluation of exosomes loaded with AO cargos:Electroporation was used as a method to load cargos into exosomes as reported earlier.Different parameters were optimized to define the optimal condition for cargo loading,followed by assays in in vitro system.9.In vivo evaluation of exosomes loaded with AO cargos via local intramuscular injection:Exosomes loaded with different cargos were injected into tiabilis anterior(TA)muscle of mdx mice intramuscularly.Restoration of dystrophin expression and exon skipping efficiency were tested using IHC and R.T-PCR assays.Results:1.Exosomes can be harvested from culture medium at the yield of 5 ?g/ml via differential ultracentrifugation based on total protein assay.2.We confirmed that harvested vesicles are indeed exosomes in the absence of cell debris with Western blot.3.After sucrose gradient centrifugation,we confirmed that the density range of exosomes we harvested is consistent with those reported,the majority of exosomes floating in density range of 1.13-1.19 g/ml..4.With fluroscence microscope and flow cytometry(FACS)analysis,we determined that the peak time point for exosomes entering their parental cells is 48 h,though substantial amount of labeled exosomes can be found at 72 h after incubation.5.We observed that a large portion of labeled exosomes localized in lysosomes and early endosomes after co-incubation with C2C12 cells via confocal fluroscence microscope and no fluroscence in nuclei,which is consistent with previous report.6.Compared with L1 and NIH3T3,there was significantly higher uptake observed in C2C12,suggesting that C2C12-derived exosomes might bear muscle cell tropism.Meanwhile,we observed more fluorescence in muscle than in non-muscle tissues i.e.kidney and liver after systemic administration of labeled exosomes to mdx mice,implying C2C12-derived exosomes possibly have muscle-targeting property.7.After a series of optimizaton including electic capacity and resistance,we defined that 400 V/125 ?gF and 600 V/50 ?gF are optimal parameters for MOE and 2'-O me,respectively.8.In vivo results showed that exosomes can efficiently deliver AOs into TA muscle of mdx mice and induce exon23 skipping and restore the expression of dystrophin protein.Conclusion:1.Exosomes were successfully harvested and characterized with morphologic,biochemical assays and density measurement.2.C2C12-derived exosomes can be readily uptaken by their parental cellsI and the peak time is 48 h after co-incubation.C2C12-derived excsomes show muscle cell and tissue.3.Exosomes were found mainly localized in lysosomes and a portion in early endosomes.4.Different cargo loading requires different condition.Our results showed that optimal parameters MOE and 2,-O-me are 400 V/125 ?F and 600 V/50 ?F,respectively.5.Exosome can deliver AO efficiently and induce effective exon skipping and dystrophin restoration.