Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.This is the publisher’s final pdf. The article is copyrighted by the American Society of Gene & Cell Therapy and published by the Nature Publishing Group. It can be found at: http://www.nature.com/mtna/index.html.Keywords: Antisense oligonucleotide, Exon skipping, Chimeric peptide conjugate, Duchenne muscular dystroph

Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies

MoultonHongVeterinaryMedicineContextDependentEffects.pdf

We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Keywords: Antisense oligonucleotide, Chimeric peptide conjugate, Duchenne muscular dystrophy, Exon skippingKeywords: Antisense oligonucleotide, Chimeric peptide conjugate, Duchenne muscular dystrophy, Exon skippin

MoultonHongVeterinaryMedicineContextDependentEffects_FigureS1.pdf

We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Keywords: Antisense oligonucleotide, Exon skipping, Chimeric peptide conjugate, Duchenne muscular dystroph

Gene Expression Networks in the Murine Pulmonary Myocardium Provide Insight into the Pathobiology of Atrial Fibrillation

The pulmonary myocardium is a muscular coat surrounding the pulmonary and caval veins. Although its definitive physiological function is unknown, it may have a pathological role as the source of ectopic beats initiating atrial fibrillation. How the pulmonary myocardium gains pacemaker function is not clearly defined, although recent evidence indicates that changed transcriptional gene expression networks are at fault. The gene expression profile of this distinct cell type in situ was examined to investigate underlying molecular events that might contribute to atrial fibrillation. Via systems genetics, a whole-lung transcriptome data set from the BXD recombinant inbred mouse resource was analyzed, uncovering a pulmonary cardiomyocyte gene network of 24 transcripts, coordinately regulated by chromosome 1 and 2 loci. Promoter enrichment analysis and interrogation of publicly available ChIP-seq data suggested that transcription of this gene network may be regulated by the concerted activity of NKX2-5, serum response factor, myocyte enhancer factor 2, and also, at a post-transcriptional level, by RNA binding protein motif 20. Gene ontology terms indicate that this gene network overlaps with molecular markers of the stressed heart. Therefore, we propose that perturbed regulation of this gene network might lead to altered calcium handling, myocyte growth, and contractile force contributing to the aberrant electrophysiological properties observed in atrial fibrillation. We reveal novel molecular interactions and pathways representing possible therapeutic targets for atrial fibrillation. In addition, we highlight the utility of recombinant inbred mouse resources in detecting and characterizing gene expression networks of relatively small populations of cells that have a pathological significance

Functional Rescue of Dystrophin-deficient mdx Mice by a Chimeric Peptide-PMO

Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy

Small-scale convection at a continental back-arc to craton transition: Application to the southern Canadian Cordillera

A step in the depth of the lithosphere base, associated with lateral variations in the upper mantle temperature structure, can trigger mantle flow that is referred to as edge-driven convection. This paper aims at outlining the implications of such edge-driven flow at a lateral temperature transition from a hot and thin to a cold and thick lithosphere of a continental back-arc. This configuration finds application in the southern Canadian Cordillera, where a hot and thin back-arc is adjacent to the cold and thick North American Craton. A series of geodynamical models tested the thermodynamical behavior of the lithosphere and upper mantle induced by a step in lithosphere thickness. The mantle flow patterns, thickness and heat flow evolution of the lithosphere, and surface topography are examined. We find that the lateral temperature transition shifts cratonward due to the vigorous edge-driven mantle flow that erodes the craton edge, unless the craton has a distinct high viscosity mantle lithosphere. The mantle lithosphere viscosity structure determines the impact of edge-driven flow on crustal deformation and surface heat flow; a dry olivine rheology for the craton prevents the edge from migrating and supports a persistent surface heat flow contrast. These phenomena are well illustrated at the transition from the hot Canadian Cordillera to craton that is supported by a rheological change and that coincides with a lateral change in surface heat flow. Fast seismic wave velocities observed in the upper mantle cratonward of the step can be explained as downwellings induced by the edge-driven flow.Geoscience & EngineeringCivil Engineering and Geoscience

Partition of aerobic and anaerobic swimming costs related to gait transitions in a labriform swimmer

Members of the family Embiotocidae exhibit a distinct gait transition from exclusively pectoral fin oscillation to combined pectoral and caudal fin propulsion with increasing swimming speed. The pectoral–caudal gait transition occurs at a threshold speed termed Up–c. The objective of this study was to partition aerobic and anaerobic swimming costs at speeds below and above the Up–c in the striped surfperch Embiotoca lateralis using swimming respirometry and video analysis to test the hypothesis that the gait transition marks the switch from aerobic to anaerobic power output. Exercise oxygen consumption rate was measured at 1.4, 1.9 and 2.3 L s–1. The presence and magnitude of excess post-exercise oxygen consumption (EPOC) were evaluated after each swimming speed. The data demonstrated that 1.4 L s–1 was below the Up–c, whereas 1.9 and 2.3 L s–1 were above the Up–c. These last two swimming speeds included caudal fin propulsion in a mostly steady and unsteady (burst-assisted) mode, respectively. There was no evidence of EPOC after swimming at 1.4 and 1.9 L s–1, indicating that the pectoral–caudal gait transition was not a threshold for anaerobic metabolism. At 2.3 L s–1, E. lateralis switched to an unsteady burst and flap gait. This swimming speed resulted in EPOC, suggesting that anaerobic metabolism constituted 25% of the total costs. Burst activity correlated positively with the magnitude of the EPOC. Collectively, these data indicate that steady axial propulsion does not lead to EPOC whereas transition to burst-assisted swimming above Up–c is associated with anaerobic metabolism in this labriform swimmer