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Mutations in the RYR1 gene, encoding the skeletal muscle calcium channel RyR1, lead to congenital myopathies, through expression of a channel with abnormal permeability and/or in reduced amount, but the direct functional whole organism consequences of exclusive reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Tamoxifen-induced recombination in the RYR1 gene at adult age resulted in a progressive reduction in the protein amount reaching a stable level of 50% of the initial amount, and was associated with a progressive muscle weakness and atrophy. Measurement of calcium fluxes in isolated muscle fibers demonstrated a reduction in the amplitude of RyR1-related calcium release mirroring the reduction in the protein amount. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution and membrane remodeling. An increase in the expression level of many proteins was observed, as well as an inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Dusty Core Disease patients (a subtype of Central Core Disease), pointing to common pathophysiological mechanisms related to RyR1 reduction.

BackgroundDysthyroidism (DT) is a common toxicity of immune checkpoint inhibitors (ICIs) and prior work suggests that dysthyroidism (DT) might be associated with ICI efficacy.Patients and methodsConSoRe, a new generation data mining solution, was used in this retrospective study, to extract data from electronic patient records of adult cancer patients treated with ICI at Institut Paoli-Calmettes (Marseille, France). Every DT was verified and only ICI-induced DT was retained. Survival analyses were performed by Kaplan-Meier method (log-rank test) and Cox model. To account for immortal time bias, a conditional landmark analysis was performed (2 months and 6 months), together with a time-varying Cox model.ResultsData extraction identified 1385 patients treated with ICI between 2011 and 2021. DT was associated with improved overall survival (OS) (HR 0.46, (95% CI 0.33 to 0.65), p<0.001), with a median OS of 35.3 months in DT group vs 15.4 months in non-DT group (NDT). Survival impact of DT was consistent using a 6-month landmark analysis with a median OS of 36.7 months (95% CI 29.4 to not reported) in the DT group vs 25.5 months (95% CI 22.8 to 27.8) in the NDT group. In multivariate analysis, DT was independently associated with improved OS (HR 0.49, 95% CI 0.35 to 0.69, p=0.001). After adjustment in time-varying Cox model, this association remained significant (adjusted HR 0.64, 95% CI 0.45 to 0.90, p=0.010). Moreover, patients with DT and additional immune-related adverse event had increased OS compared with patients with isolated DT, with median OS of 38.8 months vs 21.4 months, respectively.ConclusionData mining identified a large number of patients with ICI-induced DT, which was associated with improved OS accounting for immortal time bias.

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.

Some mutations in the RYR1 gene lead to congenital myopathies, through reduction in this calcium channel expression level, but the functional whole organism consequences of reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Recombination in the RYR1 gene resulted in a progressive reduction in the protein amount and was associated with a progressive muscle weakness and atrophy. Calcium fluxes in isolated muscle fibers were accordingly reduced. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution, membrane remodeling, associated with increase in the expression level of many proteins and inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Central Core Disease patients, pointing to common pathophysiological mechanisms related to RyR1 reduction. Competing Interest Statement The authors have declared no competing interest.

Longitudinal studies allow the modelling of disease progression through repeated measurement of health outcomes, such as biomarkers. Changes in measurement tools over time, due to logistical or financial constraints, may challenge the statistical modeling of outcome trajectories. This study aims to compare two methods for managing changes in blood biomarkers assays over time, in the context of modeling their longitudinal trajectories. We analyzed data from 2299 individuals in the French MEMENTO cohort, focusing on two Alzheimer's disease blood biomarkers: 181-phosphorylated tau (p-tau181) and neurofilament light chain (NfL). Baseline blood samples were quantified using an initial assay kit in 2021, while samples collected at 2- and 4-year follow-ups with updated kits in 2023. Two approaches were applied to derive conversion equations for aligning measurements from the initial to the updated assay: (i) a bridging study, requiring biomarker quantification using both the initial and the updated assay in a subsample of individuals and (ii) Latent Process Models (LPM), which established links between the two assays as measures of the same latent process over age, using biomarker measurements available at the 3 timepoints. Prediction error rates were computed, and biomarker trajectories estimated with linear mixed models according to two variables of interest (education level, cognitive impairment). Prediction error rates were slightly higher for LPM than for bridging for both NfL and p-tau181. While the two methods yielded similar predictions around the median, discrepancies were observed at the tails of the distribution of the observed values. Longitudinal trajectories showed consistent associations for the variables of interest at baseline and during follow-up for both biomarkers. LPM provide a feasible and efficient method for managing changes in biomarker quantification assays in longitudinal studies. LPM yields results comparable to traditional bridging studies without requiring additional sample analysis. This approach is particularly advantageous in studies with long-term follow-up, where changes in measurement tools cannot always be avoided, offering a straightforward and resource-efficient solution.