Dystrophin involvement in peripheral circadian SRF signalling.

Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes.

Selection of Fluorescent, Bioluminescent, and Radioactive Tracers to Accurately Reflect Extracellular Vesicle Biodistribution .

The ability to track extracellular vesicles (EVs) without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63.

Extracellular vesicles induce minimal hepatotoxicity and immunogenicity.

Extracellular vesicles (EVs) mediate cellular communication through the transfer of active biomolecules, raising interest in using them as biological delivery vehicles for therapeutic drugs. For drug delivery applications, it is important to understand the intrinsic safety and toxicity liabilities of EVs. Nanoparticles, including EVs, typically demonstrate significant accumulation in the liver after systemic administration in vivo.

2'-O-(2-Methoxyethyl) Nucleosides Are Not Phosphorylated or Incorporated Into the Genome of Human Lymphoblastoid TK6 Cells.

Nucleoside analogs with 2'-modified sugar moieties are often used to improve the RNA target affinity and nuclease resistance of therapeutic oligonucleotides in preclinical and clinical development. Despite their enhanced nuclease resistance, oligonucleotides could slowly degrade releasing nucleoside analogs that have the potential to become phosphorylated and incorporated into cellular DNA and RNA. For the first time, the phosphorylation and DNA/RNA incorporation of 2'-O-(2-methoxyethyl) (2'-O-MOE) nucleoside analogs have been investigated.

The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions.

Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions.

Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy.

The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration.

Identification of novel, therapy-responsive protein biomarkers in a mouse model of Duchenne muscular dystrophy by aptamer-based serum proteomics.

There is currently an urgent need for biomarkers that can be used to monitor the efficacy of experimental therapies for Duchenne Muscular Dystrophy (DMD) in clinical trials. Identification of novel protein biomarkers has been limited due to the massive complexity of the serum proteome and the presence of a small number of very highly abundant proteins. Here we have utilised an aptamer-based proteomics approach to profile 1,129 proteins in the serum of wild-type and mdx (dystrophin deficient) mice.

Implications for Cardiac Function Following Rescue of the Dystrophic Diaphragm in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by absence of the integral structural protein, dystrophin, which renders muscle fibres susceptible to injury and degeneration. This ultimately results in cardiorespiratory dysfunction, which is the predominant cause of death in DMD patients, and highlights the importance of therapeutic targeting of the cardiorespiratory system. While there is some evidence to suggest that restoring dystrophin in the diaphragm improves both respiratory and cardiac function, the role of the diaphragm is not well understood.

Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD).

Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis.

Prevention of exercised induced cardiomyopathy following Pip-PMO treatment in dystrophic mdx mice.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder caused by mutations in the Dmd gene. In addition to skeletal muscle wasting, DMD patients develop cardiomyopathy, which significantly contributes to mortality. Antisense oligonucleotides (AOs) are a promising DMD therapy, restoring functional dystrophin protein by exon skipping.

Re-evaluation of the Mutagenic Response to Phosphorothioate Nucleotides in Human Lymphoblastoid TK6 Cells.

The degradation of phosphorothioate oligonucleotides (PS-ONDs) and the release of potentially genotoxic modified mononucleotides raise a safety concern for OND-based therapeutics. Deoxyadenosine monophosphorothioate (dAMPαS), a PS nucleotide analog, has been reported to be a potent in vitro mutagen at the thymidine kinase (TK) locus in human TK6 lymphoblastoid cells. This led us to explore the mechanism behind the apparent positive response induced by dAMPαS in the TK gene-mutation assay in TK6 cells.

Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.

Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems.

Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy.

The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs).

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA.

Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment.

Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart.

Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy.

The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein.

Overview of alternative oligonucleotide chemistries for exon skipping.

The chemistry of the oligonucleotide backbone is crucial to obtaining high activity in vivo in exon skipping applications. Apart from the ability to bind strongly and sequence-specifically to pre-mRNA targets, the type of backbone also influences cell delivery, in vivo pharmacology, bio-distribution, toxicology, and ultimately the therapeutic use in humans. Reviewed here are classes of oligonucleotide commonly used for exon skipping applications, namely negatively charged backbones typified by RNA analogues having 2'-O-substitution and a phosphorothioate linkage and charge-neutral backbones such as PNA and PMO.

Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice.

Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials.

Diaphragm rescue alone prevents heart dysfunction in dystrophic mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function.

Splice redirection as a convenient assay to monitor CPP-ON efficiency and mechanism.

Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligonucleotides (PMO), to cell-penetrating peptides (CPP) has recently been proposed as a new delivery strategy.

Synthesis and splice-redirecting activity of branched, arginine-rich peptide dendrimer conjugates of peptide nucleic acid oligonucleotides.

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection.

In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy.

Background: Targeted splice modulation of pre-mRNA transcripts by antisense oligonucleotides (AOs) can correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) arises as a result of mutations that interrupt the open-reading frame in the DMD gene encoding dystrophin such that dystrophin protein is absent, leading to fatal muscle degeneration. AOs have been shown to correct this dystrophin defect via exon skipping to yield functional dystrophin protein in animal models of DMD and also in DMD patients via intramuscular administration.

Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the mdx mouse.

Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration.