CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2.

A single-nucleotide deletion in the stop codon of the nuclear import receptor transportin-3 (TNPO3), also involved in human immunodeficiency virus type 1 (HIV-1) infection, causes the ultrarare autosomal dominant disease limb-girdle muscular dystrophy D2 (LGMDD2) by extending the wild-type protein. Here, we generated a patient-derived in vitro model of LGMDD2 as an immortalized myoblast cell line carrying the TNP O 3 mutation. The cell model reproduced critical molecular alterations seen in patients, such as TNP O 3 overexpression, defects in terminal muscle markers, and autophagy overactivation. Correction of the TNP O 3 mutation via CRISPR-Cas9 editing caused a significant reversion of the pathological phenotypes in edited cells, including a complete absence of the mutant TNPO3 protein, as detected with a polyclonal antibody specific against the abnormal 15-aa peptide. Transcriptomic analyses found that 15% of the transcriptome was differentially expressed in model myotubes. CRISPR-Cas9-corrected cells showed that 44% of the alterations were rescued toward normal levels. MicroRNAs (miRNAs) analyses showed that around 50% of miRNAs with impaired expression because of the disease were recovered on the mutation edition. In summary, this work provides proof of concept of the potential of CRISPR-Cas9-mediated gene editing of TNP O 3 as a therapeutic approach and describes critical reagents in LGMDD2 research.

Dystrophinopathy Phenotypes and Modifying Factors in DMD Exon 45-55 Deletion.

OBJECTIVE: Duchenne muscular dystrophy (DMD) exon 45-55 deletion (del45-55) has been postulated as a model that could treat up to 60% of DMD patients, but the associated clinical variability and complications require clarification. We aimed to understand the phenotypes and potential modifying factors of this dystrophinopathy subset. METHODS: This cross-sectional, multicenter cohort study applied clinical and functional evaluation. Next generation sequencing was employed to identify intronic breakpoints and their impact on the Dp140 promotor, intronic long noncoding RNA, and regulatory splicing sequences. DMD modifiers (SPP1, LTBP4, ACTN3) and concomitant mutations were also assessed. Haplotypes were built using DMD single nucleotide polymorphisms. Dystrophin expression was evaluated via immunostaining, Western blotting, reverse transcription polymerase chain reaction (PCR), and droplet digital PCR in 9 muscle biopsies. RESULTS: The series comprised 57 subjects (23 index) expressing Becker phenotype (28%), isolated cardiopathy (19%), and asymptomatic features (53%). Cognitive impairment occurred in 90% of children. Patients were classified according to 10 distinct index-case breakpoints; 4 of them were recurrent due to founder events. A specific breakpoint (D5) was associated with severity, but no significant effect was appreciated due to the changes in intronic sequences. All biopsies showed dystrophin expression of >67% and traces of alternative del45-57 transcript that were not deemed pathogenically relevant. Only the LTBP4 haplotype appeared associated the presence of cardiopathy among the explored extragenic factors. INTERPRETATION: We confirmed that del45-55 segregates a high proportion of benign phenotypes, severe cases, and isolated cardiac and cognitive presentations. Although some influence of the intronic breakpoint position and the LTBP4 modifier may exist, the pathomechanisms responsible for the phenotypic variability remain largely unresolved. ANN NEUROL 2022;92:793-806.

Preclinical characterization of antagomiR-218 as a potential treatment for myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the DMPK gene. CUG expansions in mutant DMPK transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an antagomiR-218 molecule using the HSALR mouse model and patient-derived myotubes. In HSALR, antagomiR-218 reached 40-60 pM 2 weeks after injection, rescued molecular and functional phenotypes in a dose- and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target.

Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening.

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient's immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and ß-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

Musashi-2 contributes to myotonic dystrophy muscle dysfunction by promoting excessive autophagy through miR-7 biogenesis repression.

Skeletal muscle symptoms strongly contribute to mortality of myotonic dystrophy type 1 (DM1) patients. DM1 is a neuromuscular genetic disease caused by CTG repeat expansions that, upon transcription, sequester the Muscleblind-like family of proteins and dysregulate alternative splicing of hundreds of genes. However, mis-splicing does not satisfactorily explain muscle atrophy and wasting, and several other contributing factors have been suggested, including hyperactivated autophagy leading to excessive catabolism. MicroRNA (miR)-7 has been demonstrated to be necessary and sufficient to repress the autophagy pathway in cell models of the disease, but the origin of its low levels in DM1 was unknown. We have found that the RNA-binding protein Musashi-2 (MSI2) is upregulated in patient-derived myoblasts and biopsy samples. Because it has been previously reported that MSI2 controls miR-7 biogenesis, we tested the hypothesis that excessive MSI2 was repressing miR-7 maturation. Using gene-silencing strategies (small interfering RNAs [siRNAs] and gapmers) and the small molecule MSI2-inhibitor Ro 08-2750, we demonstrate that reducing MSI2 levels or activity boosts miR-7 expression, represses excessive autophagy, and downregulates atrophy-related genes of the UPS system. We also detect a significant upregulation of MBNL1 upon MSI2 silencing. Taken together, we propose MSI2 as a new therapeutic target to treat muscle dysfunction in DM1.

A study of the phenotypic variability and disease progression in Laing myopathy through the evaluation of muscle imaging.

BACKGROUND: Laing myopathy is characterized by broad clinical and pathological variability. They are limited in number and protocol of study. We aimed to delineate muscle imaging profiles and validate imaging analysis as an outcome measure. METHODS: This was a cross-sectional and longitudinal cohort study. Data from clinical, functional and semi-quantitative muscle imaging (60 magnetic resonance imaging [MRI] and six computed tomography scans) were studied. Hierarchical analysis, graphic heatmap representation and correlation between imaging and clinical data using Bayesian statistics were carried out. RESULTS: The study cohort comprised 42 patients from 13 families harbouring five MYH7 mutations. The cohort had a wide range of ages, age at onset, disease duration, and myopathy extension and Gardner-Medwin and Walton (GMW) functional scores. Intramuscular fat was evident in all but two asymptomatic/pauci-symptomatic patients. Anterior leg compartment muscles were the only affected muscles in 12% of the patients. Widespread extension to the thigh, hip, paravertebral and calf muscles and, less frequently, the scapulohumeral muscles was commonly observed, depicting distinct patterns and rates of progression. Foot muscles were involved in 40% of patients, evolving in parallel to other regions with absence of a disto-proximal gradient. Whole cumulative imaging score, ranging from 0 to 2.9 out of 4, was associated with disease duration and with myopathy extension and GMW scales. Follow-up MRI studies in 24 patients showed significant score progression at a variable rate. CONCLUSIONS: We confirmed that the anterior leg compartment is systematically affected in Laing myopathy and may represent the only manifestation of this disorder. However, widespread muscle involvement in preferential but variable and not distance-dependent patterns was frequently observed. Imaging score analysis is useful to categorize patients and to follow disease progression over time.