Modelling undiagnosed muscular dystrophies using patients's stem cells

Project Information

MUMDUPSC

Grant agreement ID: 101025510

DOI

10.3030/101025510

Project closed

EC signature date 13 April 2021

Start date 15 September 2021

End date 14 September 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 196 707,84

EU contribution

€ 196 707,84

Coordinated by

UNIVERSITE D'AIX MARSEILLE
France

Periodic Reporting for period 1 - MUMDUPSC (Modelling undiagnosed muscular dystrophies using patients's stem cells)

Reporting period: 2021-09-15 to 2023-09-14

Summary of the context and overall objectives of the project

Rare diseases affect around 30 million people in Europe, of which 500,000 are affected with neuromuscular diseases (NMDs). NMDs, affecting both children and adults with a prevalence of 1 in a 1000 people, form a large and heterogeneous group of genetic diseases causing progressive degeneration of skeletal muscles. Most NMDs result in chronic long-term disability imposing a significant burden on patients, families, and public health care. In many cases, patients die prematurely from respiratory, and in some cases cardiac, muscle impairment. There are more than 200 NMDs, including over 30 types of Muscular Dystrophy (MD) which results from the mutation of genes controlling muscle functions and structures. Over 100 genes involved in MDs have already been identified; however, many are still to be discovered. The classification of MDs is not fixed but evolves with the constant discovery of new genes/mutations responsible for these diseases. In Europe, thousands of MDs patients currently have no molecular diagnosis. Late or inaccurate diagnoses delay the start of adequate treatments and may have irreversible consequences for the patients.
The goal of MUMDUPSC is to better understand the mechanisms contributing to previously undiagnosed MD (UMDs), to orient their diagnoses and facilitate their classification, in the hope that this may lead to new therapies. To reach this aim, MUMDUPSC met the three specific objectives:

1. Establishment of in vitro models recapitulating UMDs muscle phenotypes using patients hiPSC lines.
2. Identification of Genes whose expression is modulated in UMDs compared to control lines (unaffected and known MDs)
3. Identification of genetic alterations associated with UMDs in patients.

By combining disease modelling from patients’ hiPSC and state-of-the-art molecular and functional technologies, MUMPDUPSC objectives are to identify genes, biological pathways and functional properties affected in these various debilitating NMDs. Overall, through stem cells modeling, this project offers new avenues for understanding muscle pathologies and will, facilitate the development of innovative therapeutic approaches.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Our team continuously collaborates with hospital services and ten patients affected with different clinical forms of UMDs were selected for this study, based on the age at onset, the severity of the disease, and clinical data available. A bank of induced pluripotent stem cells (hiPSC) derived from this cohort of patients affected with UMDs was established. Patient’s hiPSC were subsequently pushed to form skeletal muscle cells the affected cell types in muscular dystrophies, using our previously established protocols that we further optimized1. Differentiated muscle cells were thoroughly characterized and compared to cells from healthy control (non-affected) and patients with known MDs. We observed calcium signaling defects in UMD patients skeletal muscle cells, characteristic of muscular dystrophies, which confirms their pathological status.
Our initial in-depth analysis, which focused on the first two UMD patients, led to the identifications of genes and biological pathways specifically modulated in each of these two pathologies, and highlighted potential diseases mechanisms including alteration of sodium channels activity in patient 1 and increased cell death as well as changes in extracellular matrix components in patient 2.
In parallel, we sought to identify genetic mutations associated with UMDs. Using Whole exome sequencing (WES) methodology, pathogenic variants and variants of unknown significance (VUS) were identified in the first two UMD patients. WES analysis is currently underway for the other selected UMD patients. Specifically, the analysis revealed that patient 1 carries a homozygous VUS in the PLEC gene already associated with several forms of muscular dystrophies; patient 2 carries heterozygous compound variants (one pathogenic and one VUS) in the LAMA2 gene also associated with a form of muscular dystrophy. Genetic corrections of these VUS in PLEC or LAMA2 genes are currently underway in respective patients’ hiPSC. Corrected cells will then be differentiated into skeletal muscle and thoroughly characterized to confirm that these mutations corrections rescue the disease phenotypes previously identified in patients’ hiPSC, and therefore validate the pathogenicity of the variants.

Dissemination of the results:

1 The optimization of the skeletal muscle differentiation protocol used to differentiate hiPSC in this study led to the publication of an article in the journal Cells (Ruan et al., 2022) and was presented at “Myology” conference in Nice in September 2023.

2 A review on hiPSC-based models of NMDs, on which I am first author, was also published in the Journal of Neuromuscular Diseases (Caron et al., JND, 2023) in July 2023.

3 The main publication related to MUMDUPSC and presenting the results obtained from patients’ hiPSC modeling and characterization is currently in progress and expected to be submitted in 2024.

The overall MUMDUPSC project and the results obtained from several UMD patients will be presented at the conference “Myology” in Paris in April 2024.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In the context of genetic NMDs, hiPSC-based models represent unprecedented opportunities to investigate the different aspects of a given pathology, identify new genes, validate the pathogenicity of variants or define early stages of muscle wasting. By identifying genes and pathways specifically modulated in UMDs, MUMDUPSC revealed some of the cellular mechanisms specifically contributing to the specific diseases of UMD patients. It represents a proof of concept for the classification of unlabeled muscle pathologies and, in the long term, will provide innovative leads for the diagnosis and the treatment of these debilitating diseases. HiPSC-based models also supports the development of innovative treatments for muscle conditions and may open the door for preclinical screening of a personalized panel of drug candidates to improve these debilitating disorders.

MUMDUPSC is aligned with (1) the perspective of the French National Plan for Rare Diseases (PMRIII) and more particularly the project of the Filnemus Network (French Rare Health Care for Neuromuscular Diseases Network) in close connection with the AFM Telethon project "a diagnosis for everyone" that aims to fight against lack of diagnostic; and (2) with the European directives of personalized medicine.

Project summary diagram