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Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b -/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases., (© 2024. The Author(s).)

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 HSA LR 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., Competing Interests: B.L. and R.A. are founders, and CEO and scientific consultant of Arthex Biotech, respectively. Arthex Biotech is developing antimiRs for use in the treatment of DM1., (© 2023 The Authors.)

Myotonic dystrophy type 1 is a debilitating neuromuscular disease causing muscle weakness, myotonia, and cardiac dysfunction. The phenotypes are caused by muscleblind-like (MBNL) protein sequestration by toxic RNA in the DM1 protein kinase ( DMPK ) gene. DM1 patients exhibit a pathogenic number of repetitions in DMPK , which leads to downstream symptoms. Another disease characteristic is altered microRNA (miRNA) expression. It was previously shown that miR-23b regulates the translation of MBNL1 into protein. Antisense oligonucleotide (AON) treatment targeting this miRNA can improve disease symptoms. Here, we present a refinement of this strategy targeting a miR-23b binding site on the MBNL1 3' UTR in DM1 model cells and mice by using AONs called blockmiRs. BlockmiRs linked to novel cell-penetrating peptide chemistry showed an increase in MBNL1 protein in DM1 model cells and HSA LR mice. They also showed an increase in muscle strength and significant rescue of downstream splicing and histological phenotypes in mice without disturbing the endogenous levels of other miR-23b target transcripts., Competing Interests: We declare that R.A., B.L., and E.C.-H. are co-inventors in patent PCT/EP2017/073685, currently licensed to Arthex Biotech, of which R.A. and B.L. are co-founders and R.A. is scientific consultant., (© 2022 The Author(s).)

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 HSA LR mouse model and patient-derived myotubes. In HSA LR , 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., Competing Interests: B.L., R.A., J.M.F.-C., and E.C.-H. are inventors in patent PCT/EP2017/073685. This patent is currently licensed to Arthex Biotech, of which B.L. and R.A. are founders and CEO and scientific consultant, respectively. B.L. and R.A. each hold more than 5% ownership of Arthex, a company with related interests., (© 2021 The Author(s).)