uORF-targeting steric block antisense oligonucleotides do not reproducibly increase RNASEH1 expression.

Upstream open reading frames (uORFs) are -regulatory motifs that are predicted to occur in the 5' UTRs of the majority of human protein-coding transcripts and are typically associated with translational repression of the downstream primary open reading frame (pORF). Interference with uORF activity provides a potential mechanism for targeted upregulation of the expression of specific transcripts. It was previously reported that steric block antisense oligonucleotides (ASOs) can bind to and mask uORF start codons to inhibit translation initiation, and thereby disrupt uORF-mediated gene regulation.

An extracellular vesicle delivery platform based on the PTTG1IP protein.

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues.

Nanoscale photobiotinylation, pulldown and sequencing of region-specific DNA from intact cells.

Femto-seq is a novel nanoscale optical method that can be used to obtain DNA sequence information from targeted regions around a specific locus or other nuclear regions of interest. Two-photon excitation is used to photobiotinylate femtoliter volumes of chromatin within the nucleus, allowing for subsequent isolation and sequencing of DNA, and bioinformatic mapping of any nuclear region of interest in a select set of cells from a heterogenous population.

Therapeutic approaches for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disorder and a priority candidate for molecular and cellular therapeutics. Although rare, it is the most common inherited myopathy affecting children and so has been the focus of intense research activity. It is caused by mutations that disrupt production of the dystrophin protein, and a plethora of drug development approaches are under way that aim to restore dystrophin function, including exon skipping, stop codon readthrough, gene replacement, cell therapy and gene editing.

EV-mediated promotion of myogenic differentiation is dependent on dose, collection medium, and isolation method.

Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. C2C12 murine myoblast differentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respectively). Conversely, treatment with C2C12 myotube-conditioned medium enhanced myogenic differentiation.

Investigating Eukaryotic Elongation Factor 2 Kinase/Eukaryotic Translation Elongation Factor 2 Pathway Regulation and Its Role in Protein Synthesis Impairment during Disuse-Induced Skeletal Muscle Atrophy.

The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. Eukaryotic elongation factor 2 kinase (eEF2k) impairs the ability of eukaryotic translation elongation factor 2 (eEF2) to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hind limb suspension (HS) model.

Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD.

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise exon 23, with flanking intronic regions repaired by non-homologous end joining.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties.

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mice were treated with a PPMO (Pip9b2-PMO) designed to induce exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later.

Nuclear microRNA-466c regulates Vegfa expression in response to hypoxia.

MicroRNAs are well characterized in their role in silencing gene expression by targeting 3´-UTR of mRNAs in cytoplasm. However, recent studies have shown that miRNAs have a role in the regulation of genes in the nucleus, where they are abundantly located. We show here that in mouse endothelial cell line (C166), nuclear microRNA miR-466c participates in the regulation of vascular endothelial growth factor a (Vegfa) gene expression in hypoxia.

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering.

Enhancing the Therapeutic Potential of Extracellular Vesicles Using Peptide Technology.

Extracellular vesicles are lipid-bilayer-enclosed nanoparticles present in the majority of biological fluids that mediate intercellular communication. EVs are able to transfer their contents (including nucleic acids, proteins, lipids, and small molecules) to recipient cells, and thus hold great promise as drug delivery vehicles. However, their therapeutic application is limited by lack of efficient cargo loading strategies, a need to improve EV tissue-targeting capabilities and a requirement to improve escape from the endolysosomal system.

Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation.

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood.

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties.

Molecular correction of Duchenne muscular dystrophy by splice modulation and gene editing.

Duchenne muscular dystrophy (DMD) is a currently incurable X-linked neuromuscular disorder, characterized by progressive muscle wasting and premature death, typically as a consequence of cardiac failure. DMD-causing mutations in the dystrophin gene are highly diverse, meaning that the development of a universally-applicable therapy to treat all patients is very challenging. The leading therapeutic strategy for DMD is antisense oligonucleotide-mediated splice modulation, whereby one or more specific exons are excluded from the mature dystrophin mRNA in order to correct the translation reading frame.

Application of CRISPR-Cas9-Mediated Genome Editing for the Treatment of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder, caused by expansion of a CTG microsatellite repeat in the 3' untranslated region of the DMPK (dystrophia myotonica protein kinase) gene. To date, novel therapeutic approaches have focused on transient suppression of the mutant, repeat-expanded RNA. However, recent developments in the field of genome editing have raised the exciting possibility of inducing permanent correction of the DM1 genetic defect.

Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle.

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention.

Advances in oligonucleotide drug delivery.

Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation.

Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology.

Background: Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.

Potential Therapies Using Myogenic Stem Cells Combined with Bio-Engineering Approaches for Treatment of Muscular Dystrophies.

Muscular dystrophies (MDs) are a group of heterogeneous genetic disorders caused by mutations in the genes encoding the structural components of myofibres. The current state-of-the-art treatment is oligonucleotide-based gene therapy that restores disease-related protein. However, this therapeutic approach has limited efficacy and is unlikely to be curative.

Changes in nuclear and cytoplasmic microRNA distribution in response to hypoxic stress.

MicroRNAs (miRNAs) are small non-coding RNAs that have well-characterized roles in cytoplasmic gene regulation, where they act by binding to mRNA transcripts and inhibiting their translation (i.e. post-transcriptional gene silencing, PTGS).