Thesis title: Custom-Engineered Extracellular Vesicles to Counteract Muscle Degeneration
Rationale
Muscular dystrophies are inherited degenerative myopathies. The most prevalent forms result from mutations in dystrophin gene or components of the dystrophin-associated protein complex (DAPC)1 including the β-sarcoglycan (βSGC) gene, leading to Limb-Girdle Muscular Dystrophy type 2E (LGMD2E) with skeletal and cardiac dysfunction2. Intercellular communication is essential for muscle homeostasis, and growing evidence highlights the involvement of extracellular vesicles (EVs) in muscle regenerative processes3. Among the cargos loaded into these vesicles are microRNAs, non-coding RNAs that play several roles in skeletal muscle pathophysiology3. Recent studies have also demonstrated that engineered EVs carrying specific miRNAs can be powerful tools in the treatment of muscle-wasting conditions4.
General Objectives
The main goal of this project is the development of custom-engineered EVs, enriched with specific pro-myogenic microRNAs (miR-146b and miR-212), and the assessment of 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 Sgcb-null C57BL/6 mice.
Experimental Design and Methods
This study involved a multi-step approach. βSGC-null and isogenic hiPSCs were differentiated into myotubes (2D) and skeletal muscle spheroids (3D) using established protocols5. EVs were isolated from DROSHA-null HEK293T cells, which lack endogenous miRNAs, through differential ultracentrifugation and characterized with nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), and Western blotting for EV markers. The EVs were loaded with miR-146b and miR-212 using ExoFect™ (SBI), and miRNA loading was confirmed by RT-qPCR. In vitro 2D and 3D muscle models were treated with engineered EVs or scramble controls and effects were assessed by immunofluorescence, morphometric analysis, and gene expression. In vivo, Sgcb-null mice received intramuscular injections of engineered EVs every five days for 30 days, with functional tests (grip strength, treadmill performance, gait analysis) conducted during treatment duration and histological analyses performed on muscles isolated post-sacrifice.
Results
βSGC-null and control hiPSCs were differentiated into myotubes and muscle spheroids. Pluripotency and skeletal muscle markers confirmed successful differentiation, with pluripotency genes decreasing and myogenesis markers increasing over time. Additionally, 2D βSGC-null myotubes exhibited increased myotube diameter and a greater number of nuclei compared to controls. Similarly, 3D βSGC-null spheroids displayed an increased number of nuclei and a larger cross-sectional area compared to controls. However, no significant differences were observed in differentiation or fusion indexes.
miRNA-depleted EVs from DROSHA-null HEK293Ts were characterized using NTA, SEM, and Western blotting to confirm EV markers. The EVs were loaded with miR-146b and miR-212 and applied to 2D and 3D models, as well as in vivo systems. In healthy models, EV treatment increased nuclei count, the differentiation and the fusion index, the number and the diameter of myotubes. In βSGC-null models, the treatment increased nuclei count, the fusion index, the number of myotubes. Interestingly, the myotube diameter is partially normalized. In vivo, Sgcb-null mice injected with EVs showed improved grip strength and treadmill performance by day 15 compared to controls.
Conclusions
This project demonstrates that custom-engineered EVs carrying pro-myogenic miR-146b and miR-212 are efficiently internalized by 2D and 3D muscle cells, leading to therapeutic effects. Preliminary in vivo data show improved muscle function, highlighting the potential of EV-based miRNA delivery. Future work will focus on proteomic profiling and expanding in vivo studies. These findings suggest that engineered EVs with pro-myogenic miRNAs are a promising cell-free strategy for muscle regeneration in muscular dystrophy.
References
1) Gilbert G, et al. Front Cell Dev Biol. 2021, 9:737840. 2) Vainzof M, et al. Neuromuscul Disord. 2021, 31:1021-1027. 3) Yedigaryan L, Sampaolesi M. Cells. 2021,1, 10(11):3035. 4) Yedigaryan L, Sampaolesi M. Front Physiol. 2023, 14:1130063. 5) Caron L, et al. Stem Cells Transl Med. 2016, 5(9): 1145-61.