Peptide-conjugated antimiRs improve myotonic dystrophy type 1 phenotypes by promoting endogenous MBNL1 expression.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the DMPK gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In HSALR mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

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.

Deciphering the Complex Molecular Pathogenesis of Myotonic Dystrophy Type 1 through Omics Studies.

Omics studies are crucial to improve our understanding of myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults. Employing tissue samples and cell lines derived from patients and animal models, omics approaches have revealed the myriad alterations in gene and microRNA expression, alternative splicing, 3' polyadenylation, CpG methylation, and proteins levels, among others, that contribute to this complex multisystem disease. In addition, omics characterization of drug candidate treatment experiments provides crucial insight into the degree of therapeutic rescue and off-target effects that can be achieved. Finally, several innovative technologies such as single-cell sequencing and artificial intelligence will have a significant impact on future DM1 research.

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.

Increased Muscleblind levels by chloroquine treatment improve myotonic dystrophy type 1 phenotypes in in vitro and in vivo models.

Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. In this work, we demonstrate that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in Drosophila and mouse (HSALR) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSALR mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.