Dissertation

Development of Gene Therapy by CRISPR/Cas9 for RYR1-Related Myopathies

Beaufils, Mathilde

2023

Overview

The contraction of skeletal muscle cells consists of a succession of steps dependent of the calcium homeostasis and that need to be finely regulated. The plasma membrane of muscle cells is formed of invaginations called T-tubules. Each T-tubule is surrounded by two terminal cisternae of the sarcoplasmic reticulum, which constitute the calcium stores of the cell. Embedded in the membrane of the T-tubules are the dihydropyridine receptors (DHPR). They are directly linked to type 1 ryanodine receptors (RyR1). These calcium channels are located in the membrane of the sarcoplasmic reticulum. The excitation of the muscle cell by the motoneuron induces a depolarization of the plasma membrane. This depolarization spreads to the T-tubules and activates the DHPRs that are voltage sensors. Their conformation thus changes, allowing RyR1 to open and release calcium into the cytoplasm, generating the contraction of the contractile units of the cell, the sarcomeres. All these steps from depolarization of the membrane to contraction of the muscle cells is call the excitation contraction coupling. Calcium homeostasis is therefore crucial to contraction and the RyR1 channel has a key role in the release of calcium that regulates it. RYR1-related myopathies (RYR1-RM) are a group of genetic pathologies, all due to at least one mutation in the RYR1 gene coding for the calcium channel of the same name. These pathologies all have in common muscular weakness. The severity can be moderate to severe depending on the case.There is currently no treatment for these pathologies.In a first study, our team demonstrated an exon skipping gene therapy approach using antisense oligonucleotides, applicable to a family RYR1-RM. Despite very satisfactory results, this approach could not be developed towards the clinic because of the ultra personalized aspect of the approach. This thesis work is a continuation of this study, with the objective of developing a less personalized approach where one treatment would be applicable to more patients at the same time. The objective of this thesis was to develop a proof of concept of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated protein 9) gene therapy for RYR1-RM.Firstly, we developed an intronic deletion strategy applicable to the same family as the first study conducted by the team. Through this project, we were able to perform the expected deletion in immortalized patient’s cells. Furthermore, we demonstrated the system used to control the production of the SpCas9 nuclease in the cells in order to limit the off-target activity as much as possible.Secondly, we used CRISPR/Cas9 to develop new cellular models from immortalized muscle cells from a healthy subject. These new lines have a gene of interest knockout and constitute new tools for the study of excitation-contraction coupling, both in a physiological and pathological context.Thirdly, we have developed a therapeutic approach independent of the patient's mutations. This approach targets the mutated allele, and could thus be applicable to all patients with RYR1-RM due to a dominantly inherited mutation. Moreover, this approach could also be applicable, under certain conditions, to other pathologies and even more patients.

• Publisher: ProQuest Dissertations & Theses
• Subject: Bioinformatics / CRISPR / Gene therapy / Genetics / Homeostasis / Patients / Physiology