Development of a genome editing therapeutic approach for RyR1-related diseases: from in vitro study to in vivo application

On Tuesday September 30th 2025, Margaux MELKA will defend his thesis "Development of a genome editing therapeutic approach for RyR1-related diseases: from in vitro study to in vivo application".

This thesis has been directed by Isabelle Marty and John Rendu of the "Cellular Myology and Pathologies" team of the GIN.

Jury composition

- Caroline LE GUINER, Inserm, Grand-Ouest Delegation, reviewer 
- Emiliano RICCI, Inserm, Auvergne Rhône-Alpes Delegation, reviewer 
- Bruno ALLAR, Université Lyon 1, examiner 
- Walid RACHIDI, Université Grenoble Alpes, examiner
- John RENDU, CHU, Université Grenoble Alpes, PhD co-supervisor 
- Isabelle Marty, Inserm, Auvergne Rhône-Alpes Delegation, PhD supervisor 
 

Abstract

The type 1 ryanodine receptor (RyR1) is a calcium channel essential for skeletal muscle contraction. Mutations in the RYR1 gene are responsible for rare disorders known as "RyR1-related myopathies," which cause muscle weakness in affected patients. To date, no curative treatment exists for these pathologies. One of the main challenges in developing gene therapies for these myopathies lies in the large number and wide distribution of mutations within the RYR1 gene. Furthermore, gene replacement therapy is difficult to envision due to the large size of the RYR1 gene.

To overcome these limitations, we developed an innovative, mutation-independent strategy aimed at restoring RyR1 function. This approach relies on the CRISPR/Cas9 system, which enables selective deletion of the pathogenic allele while preserving expression of the healthy allele. We first validated this concept in vitro by demonstrating efficient allele-specific deletion in immortalized myoblasts derived from a patient carrying a dominant RYR1 mutation.

To move towards in vivo application, we used a well-established mouse model carrying the dominant RYR1 R163C mutation, associated with malignant hyperthermia, a pharmacogenetic disorder characterized by an abnormal response to volatile anesthetics. Our goal is to reduce the animals’ sensitivity to anesthesia, thereby providing proof-of-concept of the therapeutic potential of allele-specific editing.

For this in vivo application, we optimized both the CRISPR/Cas9 system and its delivery method. We tested different guide RNAs (gRNAs) targeting the pathogenic allele and evaluated the most effective administration route to maximize efficiency and specificity of transduction in skeletal muscle.

Although validation of the most promising gRNAs is still ongoing, this study establishes the first steps towards developing our strategy in this model. In the longer term, this project aims to pave the way for new genome editing therapies capable of treating RyR1-related myopathies, regardless of the patient’s specific mutation.