CE17 - Recherche translationnelle en santé

Innovative strategy to treat Charcot-Marie-Tooth patients based on translational readthrough molecules by in vitro screening using CRISPR-Cas9 and iPSc technologies – NeurIT

Innovative strategy to treat patients with Charcot-Marie-Tooth disease: Readthrough molecules, CRISPR-Cas9 Molecular Scissors and iPSc Induced Pluripotent Stem Cells

Innovative strategy to treat patients with Charcot-Marie-Tooth disease based on an in vitro study of readthrough molecules in induced pluripotent stem cell-derived motor neuron models modified by CRISPR-Cas9 molecular scissors technology

Charcot-Marie-Tooth disease, the most common peripheral neuropathy in humans: Creation of innovative cellular models and testing of therapeutic molecules

Charcot-Marie-Tooth disease (CMT) is the most common hereditary peripheral neuropathy in humans (1 in 2500 people). More than 90 CMT genes are known to date. For more than 40% of them, premature stop codon (PTC) mutations are responsible for the disease. These PTC mutations lead to the production of truncated proteins and/or a decrease in RNA levels due to the unmediated mRNA degradation mechanism (NMD). NMD is a surveillance mechanism that should prevent the synthesis of truncated proteins by degrading mRNAs containing premature termination codons. Our main objective is to develop effective therapeutic approaches to treat CMT patients carrying PTC mutations. <br />Our research hypothesis is that PTC mutations could be hidden by some therapeutic molecules in neurons, allowing ribosomes to fully translate the mutated mRNAs. Such molecules are already known (Gentamycin or Ataluren for example), but they cause side effects on patients and/or have low efficacy. In 2017, Dr. F. Lejeune, our partner from Lille, started the identification of new molecules that seem to be very effective on PTC. We believe that these molecules could be used to treat CMT patients with PTC mutations. In order to reach our therapeutic goal, we must first achieve several intermediate objectives: <br />- Objective 1: First, we need to test these molecules on cell models, easily cultivated, in order to evaluate the optimal conditions to use them (concentration, optimization of molecule combinations, etc ...). <br />- Objective 2: In addition, from iPSc, we must create and characterize in vitro motor neurons mimicking the motor neurons of affected patients using direct skin biopsies or the CRISPR-Cas9 strategy in collaboration with our partners in Bordeaux. <br />- Objective 3: Next, we will study whether the new molecules, identified by our partners at Pasteur Lille, are effective on these generated motoneurons.<br />We are currently focusing our efforts on two genes involved in CMT: GDAP1 and SH3TC2.

Concerning therapeutic molecules, our collaborators in Lille of the INSERM U1277 - Institut Pasteur team have access to a chemical library of 10,000 molecules. The tests will be performed on easily cultivable Hela-type cell models transfected with reporter plasmids containing a cDNA encoding the firefly luciferase interrupted by an intron and a PTC codon to target NMD-subjected RNAs. Concerning cell models, the coordinating team UR20218-NeurIT (Limoges) masters the creation of human induced pluripotent stem cells (iPSc), from dermal fibroblasts, and their differentiation into motor neurons (MN) (cells affected in CMT patients), thus providing an excellent in vitro model to test therapeutic molecules. To date, we have performed this protocol in three control individuals and one individual carrying homozygous PTC mutations in the GDAP1 gene. In order to extend our study, we wanted to generate new models by creating new PTC mutations on the GDAP1 gene, but also by creating PTC mutations on another gene responsible for CMT. We chose the SH3TC2 gene, responsible for a severe and early form of CMT, which presents at least one allele with a PTC mutation in 88% of patients. In collaboration with the INSERM1218 laboratory (Bordeaux), an expert in CRISPR-Cas9 technology, clean and durable mutations of the GDAP1 and SH3TC2 gene will be created in iPS cells from our control individuals and then differentiated into the neuronal lineage. These models will then be used to test therapeutic molecules identified by our collaborators in Lille.

From a chemical library of 10,000 molecules, the Lille team has already tested 6,000 molecules and identified 30 interesting molecules presenting either PTC-readthrough activator or NMD inhibitor activities. Additional studies, such as toxicity evaluation, allowed retaining two molecules harboring NMD inhibitor activity. The other 4,000 additional molecules are currently under investigation.
We obtained iPSc and motoneurons from one patient harboring GDAP1 homozygous nonsense mutations (p.Ser194*), but also from three healthy patients. We characterized this model and published our results in Biomedicines. To summarize our results, we described the first GDAP1 functional study on human induced-pluripotent stem cells (hiPSCs)-derived motor neurons, obtained from normal subjects and from a CMT2H patient, carrying the GDAP1 homozygous c.581C>G (p.Ser194*) mutation. At mRNA level, we observed that, in normal subjects, GDAP1 is mainly expressed in motor neurons, while it is drastically reduced in the patient's cells containing a premature termination codon (PTC), probably degraded by the nonsense-mediated mRNA decay (NMD) system. Morphological and functional investigations revealed in the CMT patient's motor neurons a decrease of cell viability associated to lipid dysfunction and oxidative stress development. Mitochondrion is a key organelle in oxidative stress generation, but it is also mainly involved in energetic metabolism. Thus, in the CMT patient's motor neurons, mitochondrial cristae defects were observed, even if no deficit in ATP production emerged. This cellular model of hiPSCs-derived motor neurons underlines the role of mitochondrion and oxidative stress in CMT disease and paves the way for new treatment evaluation.
It is important to have additional models carrying a PTC mutation in GDAP1, but also in additional genes such as SH3TC2. We planned to generate these mutations in iPSc from one of our healthy patients. The Bordeaux Team performed the first step of these creations by testing a recent CRIPR-Cas9 strategy named Base-Editing and was successful in generating the mutation GDAP1-p.Arg191* on the easy HEK-293 cultivable cell line, but also the SH3TC2-p.Arg954*. We now test this strategy on our control iPSc to create these new models. This part of this project is in progress.

The next steps of this project will be 1) the creation of new adapted cell models of motor neurons from iPSc cells using the CRISPR-Cas9 Base-Editing strategy tested by our colleagues from Bordeaux, 2) the testing of readthrough activator molecules and/or NMD inhibitors identified by our colleagues from Lille, on our adapted cell models. These molecules will be tested either alone or in combination. At the end of this project, the molecules having shown a good efficacy on our cellular models and not presenting any toxicity will be tested on in vivo models and in fine these molecules will be used for clinical trials in humans. This project gives a new hope for therapy. This project will be the proof of concept that these readthrough activator and/or NMD inhibitor molecules are effective on «Premature Termination Codon (PTC)« mutations in our models of CMT neuropathies. This approach could also be applied to other genetic neurological diseases, but also to all genetic diseases due to a PTC mutation (cystic fibrosis, myopathy, etc.).

Miressi F, Benslimane N, Favreau F, Rassat M, Richard L, Bourthoumieu S, Laroche C, Magy L, Magdelaine C, Sturtz F, Lia AS, Faye PA. GDAP1 Involvement in Mitochondrial Function and Oxidative Stress, Investigated in a Charcot-Marie-Tooth Model of hiPSCs-Derived Motor Neurons. Biomedicines. 2021 Aug 2;9(8):945.

Palma M, Lejeune F. Deciphering the molecular mechanism of stop codon readthrough. Biol Rev Camb Philos Soc. 2021 Feb;96(1):310-329. Review

Lejeune F. Nonsense-Mediated mRNA Decay, a Finely Regulated Mechanism. Biomedicines. 2022 Jan 10;10(1):141. Review

Loret C, Pauset A, Prouzet-Mauleon V, Faye PA, Miressi F, Benslimane N, Sturtz F, Favreau F, Turcq B and Lia AS. CRISPR-Cas9 and iPSCs technologies to create Charcot-Marie-Tooth cellular models. First CRISPR and translational medicine Congress” Bordeaux, March-April 2022. Oral presentation

Miressi F, Benslimane N, Favreau F, Rassat M, Richard L, Bourthoumieu S, Laroche C, Magy L, Magdelaine C, Sturtz F, Lia AS and Faye PA. A hiPS-derived cellular model of motor neurons to investigate impaired mechanisms in GDAP1-associated Charcot-Marie-Tooth disease. Peripheral Nerve Society Annual Meeting, Miami, Mai 2022. Oral presentation

Benslimane N, Miressi F, Favreau F, Loret C, Rassat M, Richard L, Bourthoumieu S, Laroche C, Magy L, Magdelaine C, Sturtz F, Lia AS and Faye PA. GDAP1 involvement in mitochondrial function and the oxidative stress development in a hiPSCs-derived motor neurons model originating from a patient carrying the Charcot-Marie-Tooth disease. French Society for Stem Cell Research, Montpellier, 9-10 sept, 2021. Poster

Benslimane N, Miressi F, Richard L, Bourthoumieu S, Rassat M, Laroche C, Magy L, Magdelaine C, Favreau F, Sturtz F, Faye PA and Lia AS. Modelling Charcot-Marie-Tooth disease with hiPSCs-derived motor neurons model. Peripheral Nerve Society Annual Meeting, Miami, Mai 2022. Poster

This project focuses on the most common inherited peripheral neuropathy in humans: Charcot-Marie-Tooth disease (CMT). More than 90 altered genes may be responsible for CMT. Around 10% of the CMT mutations are Premature STOP Codon (PTC). Our main objective is to develop effective therapeutic approaches to treat CMT patients harboring PTC mutations, thanks to the work of three complementary teams. The Limoges coordinator team managed the creation of human induced pluripotent stem cells (iPSc) and their differentiation into motoneurons (affected cells in CMT patients), providing then an excellent in vitro model to test drugs. The Bordeaux team is an expert in CRISPR-Cas9 technology and will be able to generate PTC mutations in CMT genes on Limoges’ iPSc. We will then investigate if the new readthrough molecules, identified by Lille team, are efficient on these generated motoneurons. This project could also be the proof of concept to treat other inherited diseases due to PTC mutations.

Project coordination

Anne-Sophie LIA (Maintenance myélinique et neuropathies périphériques)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


ACTION Actions for onCogenesis understanding and Target identification in ONcology
CANTHER Cancer Heterogeneity, Plasticity and Therapy Resistance
MMNP Maintenance myélinique et neuropathies périphériques

Help of the ANR 413,828 euros
Beginning and duration of the scientific project: December 2020 - 42 Months

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