Biology subjects

Gourdon, G.

Publications and source records attributed to Gourdon, G..

7 recordsLinked to original sources

bioRxiv2026

Cortical organoids from congenital DM1 PSCs reveal MBNL-dependent corticogenesis defects and enable preclinical testing of therapeutic compounds

Myotonic dystrophy type 1 (DM1) is caused by an expansion of a CTG repeat in the 3' untranslated region of the DMPK gene, leading to accumulation of toxic CUG-repeat RNAs, sequestration of MBNL proteins and widespread splicing dysregulation. Congenital DM1 (CDM), the most severe form of the disease, is associated with profound muscular and neurodevelopmental defects, yet the mechanisms underlying early human brain involvement remain poorly understood. Here, we generated cortical organoids from patient-derived pluripotent stem cells carrying >1000 CTG repeats, an expansion typically associated with CDM, to model early human neurodevelopment. DM1 molecular and cellular hallmarks were detected at early developmental stages, including nuclear DMPK RNA foci in neural progenitor cells and reduced proliferative capacity. As organoids matured, CDM cultures displayed altered cortical composition, with reduced CTIP2 and SATB2 neuronal populations and increased NFIA/GFAP glial cells. In parallel, 120-day-old organoids recapitulated splicing abnormalities previously identified in DM1 patient brain tissue. To assess the contribution of MBNL dysfunction, we analyzed cortical organoids derived from MBNL2 and MBNL1/2/3 knockout induced pluripotent stem cells, which reproduced key neurodevelopmental phenotypes observed in CDM organoids, supporting a central role for MBNL loss of function in impaired corticogenesis. Finally, we evaluated the translational relevance of this model using tideglusib and erythromycin, two compounds currently under clinical evaluation in DM1 patients. Both treatments reduced DMPK RNA foci and restored proliferation defects in SOX2 neural progenitors. Together, these findings establish cortical organoids as a robust human model of CDM-associated neurodevelopmental defects, uncover MBNL-dependent mechanisms underlying early corticogenesis impairment and demonstrate the utility of this platform for translational therapeutic discovery in DM1.

pathology↗

bioRxiv2025

Enhanced muscle uptake of chemically optimized miR-23b antisense oligonucleotides as lead compounds for Myotonic Dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by CTG repeat expansions in DMPK. Mutant transcripts containing expanded CUG repeats form ribonuclear foci that sequester muscleblind-like splicing regulator (MBNL) proteins, key regulators of RNA splicing and metabolism. This functional depletion leads to widespread mis-splicing and persistence of fetal transcript profiles, which underlie muscle weakness, myotonia, and muscle atrophy. In addition, miR-23b is upregulated in DM1 muscle and further represses MBNL1 translation, amplifying molecular defects. We developed chemically optimized miRNA-targeting antisense oligonucleotides (antimiRs) to inhibit miR-23b and restore functional MBNL1 levels. Using a multi-step screening process, we evaluated antimiRs with varying sequences, lengths, chemical modifications, and lipid conjugations. A key optimization was a 3-oleic acid conjugation combined with specific chemical modifications, which enhanced muscle uptake and efficacy. Lead candidates showed strong activity in preclinical models (HSALR and DMSXL mice, and human myoblasts), increasing MBNL1 levels, correcting mis-splicing, improving muscle strength, and reducing myotonia. They also exhibited efficient biodistribution to skeletal muscle, a critical DM1-affected tissue. In vitro toxicology indicated a favorable safety profile with minimal immune or renal toxicity. The antimiR mechanism was conserved in rat and pig fibroblasts. Overall, two lead antimiRs emerged as promising therapeutic candidates for DM1, with improved pharmacokinetics, tissue targeting, and safety, supporting the potential of microRNA-based approaches to correct key molecular defects in this disorder.

genetics↗

bioRxiv2024

Comparative analysis of CRISPR/Cas9-targeted nanopore sequencing approaches in repeat expansion disorders

More than 50 repeat expansion disorders have been identified, with long-read sequencing marking a new milestone in the diagnosis of these disorders. Despite these major achievements, the comprehensive characterization of short tandem repeats in a pathological context remains challenging, primarily due to their inherent characteristics such as motif complexity, high GC content, and variable length. In this study, our aim was to thoroughly characterize repeat expansions in two neuromuscular diseases: myotonic dystrophy type 1 (DM1) and oculopharyngodistal myopathy (OPDM) using CRISPR/Cas9- targeted long-read sequencing (Oxford Nanopore Technologies, ONT). We conducted precise analyses of the DM1 and OPDM loci, determining repeat size, repeat length distribution, expansion architecture and DNA methylation, using three different basecalling strategies (MinKnow software, Dorado and Bonito). We demonstrated the importance of the basecalling strategy in repeat expansion characterization. We proposed guidelines to perform CRISPR-Cas9 targeted long-read sequencing (no longer supported by ONT), from library preparation to bioinformatical analyses. Finally, we showed, for the first time, somatic mosaicism, hypermethylation of LRP12 loci in OPDM symptomatic patients and changes in the repeat tract structure of these patients. We propose a strategy based on CRISPR/Cas9-enrichment long-read sequencing for repeat expansion diseases, which could be readily applicable in research but also in diagnostic settings.

bioinformatics↗

bioRxiv2024

CCG interruptions are unstable and hypermethylated in DM1 patients

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The dynamics of CTG repeats and clinical variability depends on the CTG repeat number, CNG repeat interruptions, DNA methylation, somatic mosaicism, but also gene modifiers. Around 10% of the DM1 population carries triplet repeat interruptions (CCG, CGG, CTC, CAG), which differ in number and nature between DM1 families. CCG interruptions have been associated with stabilization of the CTG repeat expansions and a milder phenotype in the DM1 population. However, the dynamics and precise role of interruptions in CTG repeat instability remain relatively underexplored due to the complexities of analyzing them. In this study, we showed that the number of CCG interruptions within the expansion varies within tissues and between generations using single-molecule real-time long-read sequencing. Although the interrupted expanded alleles showed global stabilization, the CCG interruptions themselves displayed variability across generations and within somatic tissues. Importantly, our findings reveal, for the first time, CCG hypermethylation within the CTG expansion, which is linked to downstream hypermethylation of the repeat. These results support the hypothesis that methylation of CCG interruptions within the expanded DMPK alleles may contribute to the stabilization of trinucleotide repeats.

molecular biology↗

bioRxiv2024

Upregulation of FasII underlies synergistic neuropathological and behavioral defects in a Drosophila model of myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder that has been extensively studied for decades, yet our understanding of its neuropathological aspect remains rudimentary. In this study, we characterized a novel model of DM1 neuropathology by expressing untranslated expanded CUG repeats at the Drosophila larval neuromuscular junction. In this model, both pre- and postsynaptic expression of CUG repeats participate to induce reduction of synaptic boutons, increase of arbor disassembly and impairment of larval locomotor activity. We found that the expression of CUG repeats caused an upregulation of the cell adhesion molecule, FasII (NCAM1 in mammals), in both the motor neurons and the body wall muscles. Knockdown of fasII was sufficient to rescue bouton numbers and locomotor impairment in this model. Further analyses identified the upregulation of the FasII-C isoform as a major contributor of these phenotypes. Remarkably, overexpressing the FasII-A-PEST+ isoform rescued the synaptic and behavioral defects, likely by outcompeting the upregulated FasII-C. Our study provided the foundation for a basic mechanism of synapse dysregulation in DM1.

neuroscience↗

bioRxiv2023

TALEN-induced contraction of CTG trinucleotide repeats in myotonic dystrophy type 1 cells

Trinucleotide repeat expansions are the cause of two dozen neurodegenerative and developmental disorders. One of these, myotonic dystrophy type 1 (Steinert disease, or DM1) is due to the expansion of a CTG triplet in the 3 UTR of the DMPK gene. We used highly specific DNA endonucleases to induce a double-strand break in the repeat tract to contract it below pathological length. Expression of a TALE Nuclease (TALEN) in human DM1 cells induced moderate CTG repeat contractions in 27% of the clones analyzed. These clones exhibited large internal deletions within the TALEN, occurring by homologous recombination between internal TALE repeats, inactivating the nuclease, and explaining its reduced efficacy. Taking advantage of the degeneracy of the genetic code, we recoded the TALEN sequence, to decrease internal redundancy and optimize codon usage. The new recoded TALEN showed increased efficacy in DM1 cells, with 68% of clones exhibiting a moderate to large contraction of the CTG repeat tract. In contrast, Staphylococcus aureus Cas9 (SaCas9) was unable to contract the CTG repeat tract. In parallel, we completely sequenced to very high coverage the DM1 genome using the PacBio technology. Several clones in which the TALEN was induced were also totally sequenced. In some of them, length changes of other long CTG repeats were detected, possibly corresponding to off-target effects, all of them in introns or intergenic regions. Repeat contractions were never associated with recombination of flanking markers, suggesting that contractions most probably occur by an intra-allelic mechanism such as single-strand annealing. TALENs should now be considered as a promising gene therapy approach, not only for DM1 but also for many other microsatellite expansion disorders.

genomics↗

bioRxiv2018

Straightjacket/α 2δ 3 deregulation is associated with cardiac conduction defects in Myotonic Dystrophy type 1

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a complex toxic CTG repeat disorder involving misbalance between two RNA- binding factors, MBNL1 and CELF1. How this pathogenic DM1 condition translates into cardiac conduction disorders remains poorly understood. Here, we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and identified associated gene deregulations using TU-tagging based transcriptional profiling of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels and among them increased expression of straightjacket/2{delta}3 that encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heart beating, a hallmark of affected conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular 2{delta}3 expression is low in healthy mice and humans but significantly elevated in ventricular muscles from DM1 patients with conduction defects. Taken together, this suggests that reducing the straightjacket/2{delta}3 transcript levels in ventricular cardiomyocytes could represent a strategy to prevent conduction defects and in particular intraventricular conduction delay associated with DM1 pathology.

genomics↗