Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling

Muscle satellite stem cells (MuSCs) are responsible for skeletal muscle growth and regeneration. Despite their differentiation potential, human MuSCs have limited in vitro expansion and in vivo migration capacity, limiting their use in cell therapies for diseases affecting multiple skeletal muscles. Several protocols have been developed to derive MuSC-like progenitors from human induced pluripotent stem (iPS) cells (hiPSCs) to establish a source of myogenic cells with controllable proliferation and differentiation. However, current hiPSC myogenic derivatives also suffer from limitations of cell migration, ultimately delaying their clinical translation. Here we use a multi-disciplinary approach including bioinformatics and tissue engineering to show that DLL4 and PDGF-BB improve migration of hiPSC-derived myogenic progenitors. Transcriptomic analyses demonstrate that this property is conserved across species and multiple hiPSC lines, consistent with results from single cell motility profiling. Treated cells showed enhanced trans-endothelial migration in transwell assays. Finally, increased motility was detected in a novel humanised assay to study cell migration using 3D artificial muscles, harnessing advanced tissue modelling to move hiPSCs closer to future muscle gene and cell therapies

Chris-TestProject

To understand how it work

Low-dose of peptide-conjugate antisense oligonucleotides targeting CUGexp-RNA in murine skeletal muscles normalizes Myotonic Dystrophy 1 phenotype

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including Myotonic Dystrophy type 1 (DM1). DM1 is a dominant neuromuscular disease caused by the abnormal amplification of a CTG repeated sequence in the 3’UTR of the DMPK gene. Expression of mutant transcripts containing expanded CUG repeats (CUGexp) leads to a deleterious RNA gain-of-function mechanism, in which CUGexp-transcripts are retained within the nucleus and alter the function of RNA-binding proteins such as MBNL splicing factors, leading to splicing defects and muscular dysfunction. ASO strategies can reverse the RNA toxicity induced by the expression of CUGexp-RNA. However systemic use of ASOs remains challenging for this muscular dystrophy because of the poor skeletal muscle uptake as pointed out by a recent clinical trial. Here, we show that advanced Pip6a-conjugate peptides strongly enhanced morpholino phosphorodiamidate oligomer (PMO) delivery into skeletal muscles of a DM1 mouse model (HSA-LR) after systemic administration in comparison to naked PMO. Antisense Pip6a-PMOs targeting pathologic CUGexp repeats inhibit the detrimental sequestration of MBNL1 splicing factor by nuclear CUGexp-RNA foci and consequently its functional loss. Thus low-dose of Pip6a-CAG ASOs induces a complete normalization of the myotonia in treated HSA-LR mice along with the correction of splicing defects and altered gene expression. Long-term studies reveal than the compound is still active six months after injection. In addition, the beneficial effect of the Pip6-CAG treatment was confirmed in human DM1 patient-derived muscle cells containing >2000 CTG repeats. This study demonstrates that pip6a-PMO treatment allows a lasting normalization of DM1-associated phenotypes at both molecular and functional levels supporting the use of advanced pip-conjugates for enhanced ASO systemic delivery in DM1 skeletal muscles

Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice

International audienceAntisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1

Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats

International audienc

EMERGEN-BioInfo The digital platform for the French SARS-CoV-2 genomic surveillance and research program

International audienceWe present EMERGEN-Bioinfo, the digital platform to collect, process, manage and divulgate viral sequences and non-sensitive metadata, developed in the context of EMERGEN, the French plan for COVID-19 genomic surveillance and research. The bioinformatics platform relies on different components to manage all the steps from raw sequence collection to deposition in international repositories. This includes: (1) specific storage spaces for each one of the 60 teams of the consortium; (2) a data lake gathering all sequences (raw, mapped, consensus genomes, aligned genomic and peptidic sequences); (3) system-level workflows to handle the data flow trough all the components of the platform; (4) a covid-19 specific domain of the national Galaxy server (covid19.usegalaxy.fr); (5) EMERGEN-DB, a database to store and manage non-sensitive metadata and genomic consensus sequences ; (6) data brokering services to facilitate metadata management and curation, submission to international repositories (GISAID&nbsp;and ENA) and follow-up of their acceptance status. The EMERGEN-Bioinfo platform is complemented by a high-security digital platform (EMERGEN-HDS) certified for Health Data Storage, which will enable researchers to pair EMERGEN data with patient data from different sources (national COVID-19 and healthcare databases). All the software resources developed for this projects will be accessible under an open license, and re-usable for other national projects (e.g. ABRomics multi-omics platform for surveillance and research on antimicrobial resistance) or international cooperation (e.g. sharing with partners of the European bioinformatics infrastructure ELIXIR).</p

Clonal hematopoiesis driven by chromosome 1q/MDM4 trisomy defines a canonical route toward leukemia in Fanconi anemia

International audienceFanconi anemia (FA) patients experience chromosome instability, yielding hematopoietic stem/progenitor cell (HSPC) exhaustion and predisposition to poor-prognosis myeloid leukemia. Based on a longitudinal cohort of 335 patients, we performed clinical, genomic, and functional studies in 62 patients with clonal evolution. We found a unique pattern of somatic structural variants and mutations that shares features of BRCA-related cancers, the FA-hallmark being unbalanced, microhomology-mediated translocations driving copy-number alterations. Half the patients developed chromosome 1q gain, driving clonal hematopoiesis through MDM4 trisomy downmodulating p53 signaling later followed by secondary acute myeloid lukemia genomic alterations. Functionally, MDM4 triplication conferred greater fitness to murine and human primary FA HSPCs, rescued inflammation-mediated bone marrow failure, and drove clonal dominance in FA mouse models, while targeting MDM4 impaired leukemia cells in vitro and in vivo. Our results identify a linear route toward secondary leukemogenesis whereby early MDM4-driven downregulation of basal p53 activation plays a pivotal role, opening monitoring and therapeutic prospects