Modulation of Pro-Inflammatory IL-6 Trans-Signaling Axis by Splice Switching Oligonucleotides as a Therapeutic Modality in Inflammation.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in maintaining normal homeostatic processes under the pathogenesis of various inflammatory and autoimmune diseases. This context-dependent effect from a cytokine is due to two distinctive forms of signaling: -signaling and . IL-6 involves binding IL-6 to the membrane-bound IL-6 receptor and Glycoprotein 130 (GP130) signal-transducing subunit.

Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype.

Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD.

Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci).

Peptide-conjugated phosphodiamidate oligomer-mediated exon skipping has benefits for cardiac function in mdx and Cmah-/-mdx mouse models of Duchenne muscular dystrophy.

Cardiac failure is a major cause of mortality in patients with Duchenne muscular dystrophy (DMD). Antisense-mediated exon skipping has the ability to correct out-of-frame mutations in DMD to produce truncated but functional dystrophin. Traditional antisense approaches have however been limited by their poor uptake into cardiac muscle.

In Vitro Assays to Assess Exon Skipping in Duchenne Muscular Dystrophy.

Cell-penetrating peptide (CPP)-mediated delivery of phosphorodiamidate morpholino oligomers (PMO) results in efficient exon skipping and has shown great promise as a potential therapy for Duchenne muscular dystrophy (DMD). However, large differences in efficiency have been observed between CPPs and in delivery to different tissues. Cellular trafficking has appeared to be an important determinant of activity.

Development and Application of an Ultrasensitive Hybridization-Based ELISA Method for the Determination of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides.

Antisense oligonucleotide (AON)-induced exon skipping is one of the most promising strategies for treating Duchenne muscular dystrophy (DMD) and other rare monogenic conditions. Phosphorodiamidate morpholino oligonucleotides (PMOs) and 2'-O-methyl phosphorothioate (2'OMe) are two of the most advanced AONs in development. The next generation of peptide-conjugated PMO (P-PMO) is also showing great promise, but to advance these therapies it is essential to determine the pharmacokinetic and biodistribution (PK/BD) profile using a suitable method to detect AON levels in blood and tissue samples.

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).

How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse.

Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage.

Delivery of therapeutic oligonucleotides with cell penetrating peptides.

Oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. Clinical translation has been often hampered by poor biodistribution, however. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids.

Parallel synthesis of cell-penetrating peptide conjugates of PMO toward exon skipping enhancement in Duchenne muscular dystrophy.

We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized.

A hydrazide-anchored dendron scaffold for chemoselective ligation strategies.

Chemoselective ligation, including "click" chemistry, has found wide utility in general synthetic strategies and the specific modification of polymers and biomolecules. This has resulted in a number of applications of such approaches, particularly in the biomedical area, including diagnostic imaging and drug delivery. However, tools to chemoselectively decorate target molecules with multiple copies of a particular drug, ligand or label are lacking.

First synthesis of an aziridinyl fused pyrrolo[1,2-a]benzimidazole and toxicity evaluation towards normal and breast cancer cell lines.

Anionic aromatic ipso-substitution has allowed an aziridine ring to be fused onto pyrrolo[1,2-a]benzimidazole. This diazole analogue of aziridinomitosene, and N-[(aziridinyl)methyl]-1H-benzimidazole are shown to be significantly more cytotoxic towards the human breast cancer cell lines MCF-7 and HCC1937 than towards a human normal fibroblast cell line (GM00637). The aziridinyl fused pyrrolo[1,2-a]benzimidazole is less cytotoxic than the non-ring fused aziridinyl analogue towards all three cell lines.

The influence of the aziridinyl substituent of benzimidazoles and benzimidazolequinones on toxicity towards normal and Fanconi anaemia cells.

Aziridinyl substituted benzimidazolequinones are more toxic than methoxy analogues towards normal human fibroblast cells (GM00637). The aziridinyl substituent is required for hypersensitive killing of Fanconi anaemia (FA) cells (PD20i) deficient in FANCD2. Despite lacking quinone functionality, 4,7-dimethoxy-N-[(aziridin-2-yl)methyl]benzimidazole also induces hypersensitivity from FA cells, similar to their response towards mitomycin C.

First synthesis of N-[(aziridin-2-yl)methyl]benzimidazolequinone and analysis of toxicity towards normal and Fanconi anemia cells.

A diazole is N-substituted with 1-trityl-2-methylaziridine and demethylated and oxidised with NBS under acidic conditions to give a benzimidazolequinone; this novel anti-tumour agent is marginally more cytotoxic than mitomycin C (MMC) towards the normal human fibroblast cell line GM00637, while the MMC-hypersensitive human Fanconi anaemia (FA) cell line, PD20i, lacking the FANCD2 protein, is also hypersensitive to the benzimidazolequinone, with expression of FANCD2 protein decreasing sensitivity to both MMC and the benzimidazolequinone.