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Ignacio Perez de Castro, Study of the potential of CRISPR/Cas technology for the treatment of cardiac abnormalities associated with LMNA-CMD. Funding: 2023-2025.
An international consortium of LMNA experts, Cure CMD and the Congenital Muscle Disease International Registry (CMDIR), recently published a retrospective Natural History Study. Among many insights, the study revealed that the LMNA-R249W mutation is likely the most recurrent mutation (20%) and is associated with a severe disease course.
Although few LMNA-CMD mouse models exist, they have been key in our understanding of disease pathology and instrumental in generating treatment hypotheses. Dr. Perez de Castro’s team generated a new mouse model that carries this frequently occurring mutation. As suspected, mice with this mutant gene only die prematurely due to metabolic defects. Instead, the heterozygous mice, having one mutant copy and one wildtype (normal) copy of the LMNA gene, mimic cardiac symptoms observed in affected individuals with LMNA-CMD. These mice also have a shortened lifespan but are surviving long enough (at least 40 weeks) to test experimental therapies.
Using this novel mouse model, the primary goal of this project is to explore the potential of CRISPR/Cas9 technology to treat the cardiac pathology associated with LMNA-CMD. Researchers tested two CRISPR-based approaches showing promise in human muscle cells in vitro (in a laboratory cell culture). The first approach aims to remove the mutation (“KO” strategy), while the second involves inserting a healthy version of the gene near the mutation using CRISPR (“Replace” strategy).
So far, the “KO” strategy has produced the best results. Removing the mutant copy of the gene with the CRISPR/Cas9 tool significantly extended the survival of mice and improved their heart function. Unfortunately, the “Replace” strategy didn’t reveal any benefits, likely because this methodology still needs significant work to improve in vivo (inside a living organism) delivery and efficiency in gene expression for cardiac and skeletal muscles.
A third approach was also tested, involving overexpression of a healthy version of the LMNA gene, but this was also ineffective, even detrimental, unexpectedly shortening the mouse model’s lifespan. This result tells us that this method of gene therapy needs more tuning to control the amount of LMNA protein expressed in the tissue – enough to be effective, but not excessive to the point of causing toxicity.
To maximize learnings from this project, the team is performing molecular characterization of the effects caused by gene therapy on the status of different proteins thought to be affected by the LMNA-R249W mutation. In addition, Dr. Perez de Castro’s group had opened new lines of inquiry derived from these results. They have initiated a study on the potential for base editors (a specific “find and replace” of individual DNA bases on the mutant gene) for the treatment of LMNA-CMD. This showed very promising results in the first round of experiments on human myoblasts with the R249W mutation. Applying the latest techniques to create animal models, a new mouse model, designed to be more representative of the human condition is in development. It is expected that the current and next generation of CRISPR technology, applied to this new model, will return better results for the potential therapeutic use of this fascinating tool.
Francesco Saverio Tedesco, MAGIC consortium (Next-generation Models And Genetic TherapIes for Rare NeuromusCular Diseases).
Dr. Tedesco’s team pioneered the development of 3D artificial skeletal muscle. They are now using this technology to very closely mimic the pathology of LMNA-CMD in humans. Using skin cells from affected individuals, researchers first transformed the donated samples into LMNA-mutant iPS cells. They then differentiated those cells into inducible skeletal myogenic cells which are precursors to mature muscle cells. Finally, they turned these into terminally differentiated myotubes in vitro, or 3D muscles. This work lays the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.
This technical success has garnered significant funding support from the European Union and the UK Research and Innovation to consolidate the MAGIC consortium of multidisciplinary researchers.
The aim of MAGIC is to accelerate the development of gene therapies and genome editing strategies for muscular dystrophies by using advanced humanized muscle models and innovative gene therapy approaches. LMNA-CMD is one of four diseases selected for this study.
This project can be followed on X, Linkedin and YouTube. Cure CMD is among four patient-advocacy organizations, eight academic institutions, and four biotech companies that make up this consortium. Our role is to provide representation of the affected community and disseminate results of the project to stakeholders.
Importantly, MAGIC actively involves multiple small and medium-sized enterprises to facilitate translation and exploitation of the key project outputs, as well as national and international patient advocacy groups: Muscular Dystrophy UK, Duchenne Data Foundation, Parent Project Italy and Cure CMD, to ensure that patients’ perspective remains central to the research.
Anne Bertrand & Gisele Bonne, Optimization of a gene therapy for striated muscle laminopathy. Funding: 2021-2024.
Gene therapy aimed at correcting mutations at the RNA level (instead of the DNA level) is another therapeutic option being investigated by Bertrand team at the Bonne Laboratory. This approach has a complicated name: spliceosome-mediated RNA trans-splicing technology. To put it simply, the aim is to “trick” the messenger RNA carrying the wrong instructions provided by the mutated LMNA gene to incorporate the correct instructions and produce healthy LMNA protein. The team has shown the presence of corrected RNA and protein, reverting the phenotype in affected cells in various laboratory conditions.
Researchers have also had some success in a different type of gene therapy. This process utilizes a combination of RNA knock-down and delivery of a healthy lamin A gene. Knock-down is to reduce the expression of the mutant allele. This is done using a genetic tool called shRNA, or small hairpin RNA, which are small molecules with tight hairpins used to silence gene expression. Using this tool in combination with delivery of the wild type LMNA gene has been shown to correct LMNA mutations and promote healthy lamin A overexpression in the mouse model.
Both gene therapy approaches are being tested in vivo via AAV-vector delivery, however they have shown only limited correction at the molecular level, with little to no improvement in survival of the mouse model. These results highlight the urgent need for optimization and a more efficient delivery system of gene therapy to striated muscles. This is a crucial step, not only for LMNA-related muscle disorders, but also many other neuromuscular subtypes. Cure CMD will continue to advocate for open science and collaboration, to share learnings across all muscular dystrophy research, and accelerate the gene therapy field.
Learn more about LMNA-CMD on Cure CMD’s LMNA information page. You can also check out Cure CMD’s Research Funding Portfolio to learn about other projects we’ve funded, and our research strategy for the CMD’s.