Intramuscular Evaluation of Chimeric Locked Nucleic Acid/2'OMethyl-Modified Antisense Oligonucleotides for Targeted Exon 23 Skipping in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2'O-Methyl (2'OMe)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2'OMe AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2'OMe AON and LNA/2'OMe chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2'OMe AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.

Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts.

Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionarily conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing ER structures. We demonstrate that the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process that does not depend on autophagosome-lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.This article has an associated First Person interview with the first author of the paper.

Systemic Evaluation of Chimeric LNA/2'-O-Methyl Steric Blockers for Myotonic Dystrophy Type 1 Therapy.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited, multisystemic disorder characterized clinically by delayed muscle relaxation and weakness. The disease is caused by a CTG repeat expansion in the 3' untranslated region (3' UTR) of the DMPK gene, which leads to the expression of a toxic gain-of-function mRNA. The expanded CUG repeat mRNA sequesters the MBNL1 splicing regulator in nuclear-retained foci structures, resulting in loss of protein function and disruption of alternative splicing homeostasis. In this study, we used CAG repeat antisense oligonucleotides (ASOs), composed of locked nucleic acid (LNA)- and 2'-O-methyl (2'OMe)-modified bases in a chimeric design, to alleviate CUGexpanded-mediated toxicity. Chimeric 14-18mer LNA/2'OMe oligonucleotides, exhibiting an LNA incorporation of ∼33%, significantly ameliorated the misregulated alternative splicing of Mbnl1-dependent exons in primary DM1 mouse myoblasts and tibialis anterior muscles of DM1 mice. Subcutaneous delivery of 14mer and 18mer LNA/2'OMe chimeras in DM1 mice resulted in high levels of accumulation in all tested skeletal muscles, as well as in the diaphragm and heart tissue. Despite the efficient delivery, chimeric LNA/2'OMe oligonucleotides were not able, even at a high-dosage regimen (400 mg/kg/week), to correct the misregulated splicing of Serca1 exon 22 in skeletal muscles. Nevertheless, oligonucleotide doses were well-tolerated as determined by histological and plasma biochemistry analyses. Our results provide proof of concept that inhibition of MBNL1 sequestration by systemic delivery of a steric-blocking ASO is extremely challenging, considering the large number of target sites that need to be occupied per RNA molecule. Although not suitable for DM1 therapy, chimeric LNA/2'OMe oligonucleotides could prove to be highly beneficial for other diseases, such as Duchenne muscular dystrophy, that require inhibition of a single target site per RNA molecule.

Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) reduces neonatal hypoxic-ischaemic brain damage.

Hypoxic-ischaemic encephalopathy is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy and cognitive disabilities. Hypoxia-ischaemia (HI) strongly up-regulates Signal Transducer and Activator of Transcription 3 (STAT3) in the immature brain. Our aim was to establish whether STAT3 up-regulation is associated with neonatal HI-brain damage and evaluate the phosphorylated STAT3-contribution from different cell types in eliciting damage. We subjected postnatal day seven mice to unilateral carotid artery ligation followed by 60 min hypoxia. Neuronal STAT3-deletion reduced cell death, tissue loss, microglial and astroglial activation in all brain regions. Astroglia-specific STAT3-deletion also reduced cell death, tissue loss and microglial activation, although not as strongly as the deletion in neurons. Systemic pre-insult STAT3-blockade at tyrosine 705 (Y705) with JAK2-inhibitor WP1066 reduced microglial and astroglial activation to a more moderate degree, but in a pattern similar to the one produced by the cell-specific deletions. Our results suggest that STAT3 is a crucial factor in neonatal HI-brain damage and its removal in neurons or astrocytes, and, to some extent, inhibition of its phosphorylation via JAK2-blockade reduces inflammation and tissue loss. Overall, the protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal HI. Current data show that neuronal and astroglial STAT3 molecules are involved in the pathways underlying cell death, tissue loss and gliosis following neonatal hypoxia-ischaemia, but differ with respect to the target of their effect. Y705-phosphorylation contributes to hypoxic-ischaemic histopathology. Protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal hypoxia-ischaemia.