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Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology. A current challenge in genome editing is delivering Cas9 and sgRNA into target cells. Here the authors engineer a delivery system based on murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins to induce efficient genome editing in both cell culture and in vivo in mouse.

Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts—the effector cells of muscle growth and regeneration—are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmd mdx myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD ( Myog, Mymk, Mymx , epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmd mdx-βgeo myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-020-66016-7, published online 04 June 2020 The original version of this Article contained errors in the names of the authors Mathilde Sanson, Ai Vu Hong, Emmanuelle Massourides, Nathalie Bourg, Laurence Suel, Fatima Amor, Guillaume Corre, Paule Bénit, Inès Barthelemy, Stephane Blot, Anne Bigot, Christian Pinset, Pierre Rustin, Laurent Servais, Thomas Voit, Isabelle Richard & David Israeli, which were incorrectly given as M. Sanson, A. Vu Hong, E. Massourides, N. Bourg, L. Suel, F. Amor, G. Corre, P. Bénit, I. Barthélémy, S. Blot, A. Bigot, C. Pinset, P. Rustin, L. Servais, T. Voit, I. Richard & D. Israeli. The original Article and accompanying Supplementary Information file have been corrected.

Background Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise—even asymptomatically—is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. Methods We have used both human tissue‐derived myoblasts and human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis and compared their differentiation dynamics with that of healthy control cells by a comprehensive multi‐omic analysis at seven time points. Results were strengthened with the analysis of isogenic CRISPR‐edited human embryonic stem cells and through comparisons against published transcriptomic and proteomic datasets from human DMD muscles. The study was completed with DMD knockdown/rescue experiments in hiPSC‐derived skeletal muscle progenitor cells and adenosine triphosphate measurement in hiPSC‐derived myotubes. Results Transcriptome and miRnome comparisons combined with protein analyses demonstrated that hiPSC differentiation (i) leads to embryonic/foetal myotubes that mimic described DMD phenotypes at the differentiation endpoint and (ii) homogeneously and robustly recapitulates key developmental steps—mesoderm, somite, and skeletal muscle. Starting at the somite stage, DMD dysregulations concerned almost 10% of the transcriptome. These include mitochondrial genes whose dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of DMD skeletal muscle cells that begins early during myogenesis. All the omics data are available online for exploration through a graphical interface at https://muscle‐dmd.omics.ovh/. Conclusions Our data argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin roles during muscle development. This hiPSC model of skeletal muscle differentiation offers the possibility to explore these functions as well as find earlier DMD biomarkers and therapeutic targets.

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000–6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

Use of human induced pluripotent stem cells (h-iPSCs) for bone tissue engineering is most appealing, because h-iPSCs are an inexhaustible source of osteocompetent cells. The present study investigated the contribution of undifferentiated h-iPSCs and elucidated aspects of the underlying mechanism(s) of the involvement of these cells to new bone formation. Implantation of undifferentiated h-iPSCs seeded on coral particles in ectopic sites of mice resulted in expression of osteocalcin and DMP-1, and in mineral content similar to that of the murine bone. The number of the implanted h-iPSCs decreased with time and disappeared by 30 days post-implantation. In contrast, expression of the murine osteogenic genes at day 15 and 30 post-implantation provided, for the first time, evidence that the implanted h-iPSCs affected the observed outcomes via paracrine mechanisms. Supporting evidence was provided because supernatant conditioned media from h-iPSCs (h-iPSC CM), promoted the osteogenic differentiation of human mesenchymal stem cells (h-MSCs) in vitro . Specifically, h-iPSC CM induced upregulation of the BMP-2, BMP-4 and BMP-6 genes, and promoted mineralization of the extracellular matrix. Given the current interest in the use of h-iPSCs for regenerative medicine applications, our study contributes new insights into aspects of the mechanism underlying the bone promoting capability of h-iPSCs.

One of the most frequent genetic abnormalities associated with non Hodgkin lymphoma is the structural alteration of the 5' non coding/regulatory region of the BCL6 (LAZ3) protooncogene. BCL6 encodes a POZ/Zn finger protein, a structure similar to that of many Drosophila developmental regulators and to another protein involved in a human hematopoietic malignancy, PLZF. BCL6 is a sequence specific transcriptional repressor controlling germinal center formation and T cell dependent immune response. Although the expression of BCL6 negatively correlates with cellular proliferation in different cell types, the influence of BCL6 on cell growth and survival is currently unknown so that the way its deregulation may contribute to cancer remains elusive. To directly address this issue, we used a tetracycline-regulated system in human U2OS osteosarcoma cells and thus found that BCL6 mediates growth suppression associated with impaired S phase progression and apoptosis. Interestingly, overexpressed BCL6 can colocalize with sites of ongoing DNA synthesis, suggesting that it may directly interfere with S phase initiation and/or progression. In contrast, the isolated Zn finger region of BCL6, which binds BCL6 target sequence but lacks transcriptional repression activity, slows, but does not suppress, U2OS cell growth, is less efficient at delaying S phase progression, and does not trigger apoptosis. Thus, for a large part, the effects of BCL6 overexpression on cell growth and survival depend on its ability to engage protein/protein interactions with itself and/or its transcriptional corepressors. That BCL6 restricts cell growth suggests that its deregulation upon structural alterations may alleviate negative controls on the cell cycle and cell survival.

Abstract Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

Introduction and hypothesis Cell therapy for stress urinary incontinence (SUI) management has been experienced with encouraging results. Methods We conducted an open prospective study on 12 women presenting severe SUI with fixed urethra, after previous failed surgical management. Patients underwent intrasphincteric injections of autologous progenitor muscular cells isolated from a biopsy of deltoid muscle. Primary endpoint focused on safety (measurement of Q max variation after 3 months). Secondary endpoints assessed side effects and efficacy. Results No variation was diagnosed on Q max measurements. Efficacy data show that three of 12 patients are dry at 12 months, seven other patients are improved on pad test but not on voiding diary, and two patients were slightly worsened by the procedure. Quality of life was improved in half of patients. Conclusions Cell therapy for severe multioperated cases of SUI is a mini-invasive, feasible, and safe procedure that can improve urinary condition in as a second line therapy.