Could Cas9 mRNA gene editing finally offer a breakthrough for muscular dystrophy?

Quantitative confocal imaging makes it easier for scientists to verify muscle protein restoration following delivery of Cas9 mRNA via targeted lipid nanoparticles.

Limb‑girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited neuromuscular conditions characterized by progressive weakness of the muscles of the shoulder and pelvic girdles. Caused by mutations in over 30 unique muscle‑associated genes, LGMDs typically result in loss of independent mobility and significantly lower quality of life.¹

Despite progress in molecular diagnostics, disease‑modifying therapies are limited for the majority of LGMD subtypes, underscoring the critical need for new treatment approaches.²

Among upcoming strategies, CRISPR‑Cas9 genome editing has generated particular interest. By directly correcting mutations that cause disease in skeletal and cardiac muscles, gene editing could enable long-lasting, potentially curative approaches.³

Despite this, translating CRISPR‑based technologies into treatment approaches for skeletal muscle conditions has faced significant obstacles, particularly regarding the safe, efficient, and tissue‑specific delivery of gene‑editing tools.

Going beyond viral delivery: Selective organ targeting LNPs

Adeno‑associated viral (AAV) vectors have typically been the primary means of delivering in vivo gene editing to muscles. Although effective in preclinical models, AAV‑based strategies are limited by elevated production costs, cargo-size constraints, immunogenicity, and the risk of persistent nuclease expression, which can increase the likelihood of off‑target editing.^4-6

Recent research aims to overcome these constraints using selective organ targeting (SORT) lipid nanoparticles (LNPs) encapsulating Cas9 mRNA. LNPs constitute a non‑viral, transient delivery system that makes repeated dosing possible while avoiding long‑term nuclease expression.

By fine‑tuning lipid composition, SORT LNPs can preferentially accumulate in specific tissues, including skeletal muscle.⁷ Scientists assessed whether SORT LNPs carrying a Cas9 cargo could efficiently alter muscle tissue in vivo and restore the expression of Telethonin, a sarcomeric protein critical for muscle stability and force transmission.

Proof‑of‑concept: Restoring telethonin expression in a dystrophic mouse model

To illustrate therapeutic potential, the researchers focused on limb‑girdle muscular dystrophy R7 (LGMDR7), which is caused by mutations in the TCAP gene, which encodes Telethonin. A mouse model with a pathogenic microduplication in TCAP recapitulated the loss of Telethonin expression seen in patients.

The scientists evaluated SORT LNPs delivering Cas9 as either ribonucleoprotein (RNP) complexes or mRNA. Ultimately, Cas9 mRNA‑loaded LNPs were considerably more efficient at editing in skeletal muscle than their RNP counterparts. After intramuscular injection, Cas9 mRNA SORT LNPs restored Telethonin expression to around 40 % of wild‑type levels in muscles that underwent treatment.⁷

Crucially, editing was not constrained to the injection site alone. Significant levels of gene editing and protein restoration were observed in adjacent muscle groups as well, indicating local distribution within muscle tissue. These results indicate that Cas9 mRNA delivery via SORT LNPs may overcome some of the anatomical and physiological hurdles that have typically hindered non‑viral muscle targeting.

Quantitative confocal imaging confirms functional protein restoration

One of the study’s key strengths lies in its robust validation of protein restoration in tissues. Whole‑slide confocal imaging of murine limb sections makes the direct comparison of muscle architecture and protein localization between wild‑type, untreated dystrophic, and Cas9 mRNA‑treated animals possible.

Using immunofluorescence staining for Telethonin alongside its binding partner Titin, the scientists confirmed that restored Telethonin localized correctly to the sarcomeric Z‑disc. This spatial accuracy is vital given adequate subcellular localization underscores both mechanical stability and signaling inside muscle fibers.⁸

High‑content, quantitative imaging was carried out with TissueGnostics’s TissueFAXS SL Q slide scanner, which provided automated confocal whole‑slide acquisition of tissues.

The system’s motorized filter wheel, with 10 positions and high‑resolution optics, enabled reliable imaging of thick muscle sections stained for multiple markers, ultimately ensuring consistency across samples.

Subsequent analysis was conducted in StrataQuest image analysis software. This made it possible to automate tissue detection and muscle fiber segmentation, and to quantify marker-positive cells. Such a mix of robust imaging hardware and flexible analysis software was crucial for converting complicated datasets into statistically meaningful outcomes.

What this means for future muscle gene‑editing therapies

Beyond illustrating a proof‑of‑concept for LGMDR7, this research provides more insights for the development of non‑viral gene‑editing therapies targeting skeletal muscle. The superiority of Cas9 mRNA over RNP delivery in large, multinucleated myofibers highlights the importance of cargo format, particle size, and intracellular kinetics for successful editing.

Immune profiling revealed unique yet manageable innate and adaptive immune responses to repeated LNP dosing, without compromising editing efficiency. This finding supports the feasibility of redosing approaches, which is a significant advantage over viral vectors for chronic conditions like muscular dystrophy.

Conclusion

Combined, these findings show that Cas9 mRNA‑based gene editing delivered via SORT lipid nanoparticles can partially restore disease‑relevant protein expression in dystrophic skeletal muscle. Of equal importance, the research underscores the crucial role of advanced quantitative imaging in validating potential therapeutic outputs.

By using core facility-ready solutions, such as TissueGnostics’ TissueFAXS SL Q slide scanner, which combines rapid confocal imaging with a high capacity of 120 slides, and the StrataQuest analysis platform, the researchers were able to confirm their hypothesis with a high level of confidence and spatial precision.

This combination of technologies enabled reproducible, whole‑tissue assessment of protein localization and expression levels over sample sets, supporting statistically robust outcomes.

As non‑viral genome editing continues to advance, integrated strategies that combine innovative delivery technologies with robust quantitative imaging will be critical to transitioning promising therapies from the lab to the clinic.

References and further reading

1. Bouchard, C. and Tremblay, J.P. (2023). Limb–Girdle Muscular Dystrophies Classification and Therapies. Journal of Clinical Medicine, (online) 12(14), p.4769. DOI: 10.3390/jcm12144769. https://www.mdpi.com/2077-0383/12/14/4769.
2. Liewluck, T. and Milone, M. (2018). Untangling the complexity of limb‐girdle muscular dystrophies. Muscle & Nerve, 58(2), pp.167–177. DOI: 10.1002/mus.26077. https://onlinelibrary.wiley.com/doi/abs/10.1002/mus.26077.
3. Benarroch, L. et al. (2024) The 2024 version of the gene table of neuromuscular disorders (nuclear genome), Neuromuscular Disorders, 34, pp. 126–170. DOI: 10.1016/j.nmd.2023.12.007. https://www.nmd-journal.com/article/S0960-8966(23)00838-6/abstract.
4. Wang, D., Tai, P. W. L., and Gao, G. (2019). Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews Drug Discovery, 18(5), 358–378. DOI: 10.1038/s41573-019-0012-9. https://www.nature.com/articles/s41573-019-0012-9.
5. Made Harumi Padmaswari, et al. (2023). Delivery challenges for CRISPR - Cas9 genome editing for Duchenne muscular dystrophy. Biophysics reviews, (online) 4(1). DOI: 10.1063/5.0131452. https://pubs.aip.org/aip/bpr/article-abstract/4/1/011307/2879046/Delivery-challenges-for-CRISPR-Cas9-genome-editing?redirectedFrom=fulltext.
6. Nelson, C.E., et al. (2019). Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nature Medicine, 25(3), pp.427–432. DOI: 10.1038/s41591-019-0344-3. https://www.nature.com/articles/s41591-019-0344-3.
7. Iyer, S., et al. (2026). SORT LNPs encapsulating Cas9 mRNA achieve efficient editing in skeletal muscle in a dystrophic mouse model. Molecular Therapy. DOI: 10.1016/j.ymthe.2026.03.010. https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(26)00195-4.
8. Zou, P., et al. (2006). Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature, 439(7073), pp.229–233. DOI: 10.1038/nature04343. https://www.nature.com/articles/nature04343.

About TissueGnostics

TissueGnostics (TG) is an Austrian company focusing on integrated solutions for high content and/or high throughput scanning and analysis of biomedical, veterinary, natural sciences, and technical microscopy samples.

TG has been founded by scientists from the Vienna University Hospital (AKH) in 2003. It is now a globally active company with subsidiaries in the EU, the USA, and China, and customers in 30 countries.

TissueGnostics portfolio

TG scanning systems are currently based on versatile automated microscopy systems with or without image analysis capabilities. We strive to provide cutting-edge technology solutions, such as multispectral imaging and context-based image analysis as well as established features like Z-Stacking and Extended Focus. This is combined with a strong emphasis on automation, ease of use of all solutions, and the production of publication-ready data.

The TG systems offer integrated workflows, i.e. scan and analysis, for digital slides or images of tissue sections, Tissue Microarrays (TMA), cell culture monolayers, smears, and other samples on slides and oversized slides, in Microtiter plates, Petri dishes and specialized sample containers. TG also provides dedicated workflows for FISH, CISH, and other dot structures.

TG analysis software apart from being integrated into full systems is fully standalone capable and supports a wide variety of scanner image formats as well as digital images taken with any microscope.

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