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Duchenne muscular dystrophy, or DMD, is a severe inherited muscle disorder caused by mutations in the DMD gene. Without functional dystrophin protein, muscle fibers become progressively damaged, leading to worsening muscle weakness.
One major challenge in treating DMD is the enormous size of the dystrophin gene. Adeno-associated virus, or AAV, vectors have limited carrying capacity, so current viral approaches generally deliver shortened versions of dystrophin rather than the full-length protein.
Researchers at Peking University, along with colleagues, investigated a different strategy using engineered extracellular vesicles.
Full-length DMD EV mRNA restores muscle dystrophin protein expression in vitro
a, Schematic of the production and delivery of EVs loaded with full-length DMD mRNA encoding dystrophin. b, Number of EVs per millilitre produced by nHDFs stimulated for 24 h with PBS, bulk electroporation (BEP) or CNP (n = 3 biologically independent samples per group; ***P < 0.001 for control versus CNP and BEP versus CNP). c, Morphologic characterization of EVs before and after CNP by cryo-electron microscopy. The experiments were repeated independently three times with similar results. Scale bars, 50 nm. d, RT-qPCR of DMD mRNA revealed that EVs produced by CNP contained much larger amounts of transcribed mRNAs than EVs produced by BEP or controls at 24 h (n = 3 biologically independent samples per group; ***P < 0.001 for control versus CNP and BEP versus CNP). e, Copies of DMD mRNA per EV produced by nHDFs stimulated for 24 h with PBS, BEP or CNP (n = 6 biologically independent samples per group; ***P < 0.001 for control versus CNP and BEP versus CNP). f, Gel electrophoresis of total RNA extracted from 3 × 1011 EVs generated by CNP with DMD-GFP plasmids compared with synthetic in-vitro-transcribed DMD-GFP mRNA. The experiments were repeated independently three times with similar results. g, Myoblasts were isolated from mdx mice and treated with three different doses of DMD EVs (low, medium and high doses with 4.56 × 108, 4.56 × 109 and 9.12 × 109 copies of DMD-GFP mRNA, respectively) cultured in differentiation medium for 8 d, followed by immunofluorescence triple staining for dystrophin (red fluorescent protein (RFP)), GFP and MYHC to evaluate dystrophin expression and confirm myotube differentiation. Nuclei were counterstained blue. Scale bars, 50 µm. h, Quantification of the RFP fluorescence intensity (as a marker of dystrophin expression) in differentiated myoblasts from WT (C57BL/6) mice, untreated mdx mice (controls) or mdx mice given DMD EVs for 8 d at the three doses (n = 5 biologically independent samples per group; ***P < 0.001 for control versus low dose, medium dose and high dose). i,j, Western blot (i) and quantitative analysis (j) of dystrophin restoration after treatment with DMD EVs for 8 d in differentiated myoblasts from mdx mice (n = 3 biologically independent samples per group; ***P < 0.001 for control versus low dose, medium dose and high dose). All of the data were from three independent experiments and are presented as means ± s.e.m. Statistical significance in b, d, e, h and j was determined by two-sided Student’s t-test. DAPI, 4′,6-diamidino-2-phenylindole; kb, kilobases; MYHC, myosin heavy chain; NS, not significant; nt, nucleotides; WT, wild type.
Extracellular vesicles are small membrane-bound particles naturally released by cells. The researchers engineered these vesicles to carry full-length DMD messenger RNA, or mRNA, and to preferentially target skeletal muscle.
Once the vesicles enter muscle cells, the delivered mRNA provides instructions for producing full-length dystrophin. Because mRNA works in the cell cytoplasm, this approach does not require permanent changes to the cell’s DNA.
In a mouse model of DMD, treatment restored substantial dystrophin expression and improved muscle function. The researchers also demonstrated skeletal muscle targeting and dystrophin production in non-human primates.
Extracellular vesicles could provide a way to deliver large RNA molecules that are difficult to package into conventional viral vectors. This may be especially valuable for disorders involving very large genes such as DMD.
The approach remains preclinical, and further work is needed to evaluate repeat dosing, long-term safety, immune responses, manufacturing, and durability of protein expression.
Still, the findings show that engineered extracellular vesicles can deliver full-length DMD mRNA systemically and support production of full-length dystrophin, offering a potential non-viral strategy for treating Duchenne muscular dystrophy and other inherited muscle diseases.
Source – EIN Newswire
