Custom-engineered extracellular vesicles to counteract muscle degeneration

Rationale Muscular dystrophies are genetically inherited degenerative myopathies1.The most prevalent muscular dystrophies involve dystrophin and components of the dystrophin-associated protein complex (DAPC)2. Among them, Limb-Girdle Muscular Dystrophy type 2E (LGMD2E) is caused by mutations in the β-sarcoglycan (βSGC) gene, leading to skeletal and cardiac dysfunctions3. Intercellular communication plays a crucial role in muscle homeostasis, and growing evidence highlights the involvement of extracellular vesicles (EVs) in muscle regenerative processes4. Studies on EVs have highlighted that secreted vesicles play a role in muscle physiology. Among the cargos loaded into these vesicles are microRNAs, non-coding RNAs that play several roles in skeletal muscle physiopathology1,4,5. Recent studies have also shown that engineered EVs carrying specific miRNAs can be powerful tools in the treatment of muscle-wasting conditions6. General Objectives The main goal of this project is to develop and validate custom-engineered EVs, enriched with specific pro-myogenic microRNAs (miR-146b and miR-212), and to assess their therapeutic potential in both in vitro and in vivo models of LGMD2E. Human induced pluripotent stem cells (hiPSCs) with a CRISPR/Cas9-mediated knockout of the βSGC gene were used to recapitulate disease features in 2D and 3D muscle models. Additionally, the efficacy of engineered EVs was evaluated in βSGC-null C57BL/6 mice. Experimental Design and Methods The study involved a multi-step approach. hiPSC βSGC-null and isogenic control lines were differentiated into myotubes (2D) and skeletal muscle spheroids (3D) following previously established protocols7,8. EVs were isolated from DROSHA-null HEK293T cells, which lack endogenous miRNAs, through differential ultracentrifugation9. Characterization of EVs included nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), and Western blot for canonical EV markers. The EVs were then loaded with miR-146b and miR-212 using the ExoFect™ transfection system, and the uptake efficiency and miRNA loading were confirmed via RT-qPCR. For in vitro testing, fully differentiated 2D and 3D muscle models were treated with engineered EVs containing miR-146b and miR-212 or scramble controls. The treatment’s effects were assessed via immunofluorescence, morphometric analysis, and gene expression.In vivo, βSGC-null mice received intramuscular injections of engineered EVs every five days for 30 days. Functional tests (grip strength, treadmill performance, gait analysis) were conducted throughout the treatment period, and muscles were harvested post-sacrifice for further analysis. Results Both βSGC-null and control hiPSCs successfully differentiated into myotubes and muscle spheroids. To assess the differentiation status, terminally differentiated 2D myotubes were stained for MyoD and MF20. The total number of nuclei, MyoD+ cell, the number of myotubes and their diameter appear to be greater in the bSGCnull cell line. While there are no differences in the differentiation and fusion index. Terminally differentiated spheroids were cryosectioned and stained for MyoD and MF20. The total number of nuclei and the cross-sectional area showed a significant increase in bSGCnull spheroids compared to those of the healthy control. Molecular analyses were also performed, and the expression of pluripotency and skeletal muscle markers was evaluated in both cells lines during differentiation by RT-qPCR assay. The expression of pluripotency genes decreases as differentiation progresses while the expression of late marker of myogenesis appears to be increased throughout the differentiation. Thus, molecular analyses confirm the ability of both cell line to differentiate. EVs were characterized with nanoparticle tracking analysis (NTA) for measuring the concentration of the particles in the sample, scanning electron microscopy (SEM) to measure their dimension, and Western blot for canonical EV markers. The DROSHA null HEK293Ts EVs were effectively depleted in their miRNA cargos as showed by RT-PCR analysis for HEK293T EV miRNAs. Subsequently the EVs were efficiently loaded with miRNAs (miR146b and miRN212) and exhibited robust uptake in both 2D and 3D skeletal muscle models and in mice hindlimb muscles. In vitro terminally differentiated myotubes and 3D skeletal muscle spheroids were treated with custom-engineered EVs. Treated cells have shown an increased nuclei count and enhanced the expression of some myogenic markers. In βSGC-null models, EVs partially normalized the fiber diameter, aligning more closely with control values. In vivo, we perform a series of tests every five days following these steps: weight measurement, grip strength test, gait analysis, and treadmill test. At the end of all physiological tests, we injected custom-engineered EVs into the three major muscles of the hindlimb. Day 0 was used as the baseline. On day 30, after completing the final functional tests, the mice were sacrificed, and their organs were collected for further analysis. EV-treated mice exhibited improved grip strength and significantly enhanced treadmill performance by day 25, compared to scramble treated controls. Conclusions This project demonstrates that custom-engineered EVs carrying pro-myogenic miR-146b and miR-212 can be efficiently internalized by both 2D and 3D muscle cells and lead to therapeutic effects. Preliminary in vivo data show functional improvements in muscle performance, highlighting the therapeutic promise of EV-based miRNA delivery. Future work will focus on proteomic profiling expanding in vivo studies. These findings demonstrate that custom-engineered EVs carrying pro-myogenic miRNAs can exert therapeutic effects in models of muscular dystrophy, offering a promising, cell-free strategy to promote muscle regeneration. 1. Yedigaryan L, et al. Front Immunol. 2022, 13: 977617 doi:10.3389/fimmu.2022.977617 2. Gilbert G, et al. Front Cell Dev Biol. 2021, 9: 737840 doi: 10.3389/fcell.2021.737840 3. Vainzof M, et al. Neuromuscul Disord. 2021, 31: 1021-1027 doi:10.1016/j.nmd.2021.07.014 4. Yedigaryan L, Sampaolesi M. Cells. 2021,1, 10(11): 3035 doi: 10.3390/cells10113035 5. Giacomazzi G, et al. Nat Commun 2017, 8(1): 1249 doi: 10.1038/s41467-017-01359-w 6. Yedigaryan L, Sampaolesi M. Front Physiol. 2023,14:1130063. doi: 10.3389/fphys.2023.1130063 7. Caron L, et al. Stem Cells Transl Med. 2016, 5(9): 1145-61 doi: 10.5966/sctm.2015-0224 8. Marini V, et al. 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