The generally accepted view of the pathology of Duchenne muscular dystrophy is that absence of dy... more The generally accepted view of the pathology of Duchenne muscular dystrophy is that absence of dystrophin renders myofibres abnormally susceptible to work-induced trauma, precipitating recurrent bouts of segmental myofibre necrosis. Necrosis stimulates inflammation and a regenerative response by myoblasts; the latter gradually becoming ineffective due to exhaustion of cells and accumulation of connective tissue driven by chronic inflammation. However, while searching for biomarkers in tissue cultures of dystrophic mouse muscle, we found phenomena that cause us to revise this picture. Using stable isotope labeled amino acids (SILAC) combined with liquid chromatography tandem mass spectrometry, we found that undamaged dystrophic myotubes are hypersecretive, excessively effluxing 57 intracellular proteins by mechanisms that cannot be identified as cell death, or generalized increase in passive leakiness. In addition dystrophic myotubes were smaller than wild-type and contain less protein and RNA per nucleus. We hypothesize that lack of dystrophin, as well as predisposing the myofibre to necrosis during physiological stress, has more profound general effects on its metabolic balance and its relationship with the extracellular environment, these potentially contributing to progressive muscle pathology. We identify myosin light chain 1 as a biomarker for in vitro drug screening.
Objective and DesignThe objective of this study was to assess the effect of vamorolone, a first-i... more Objective and DesignThe objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis.Animals84 DBA1/J mice were used in this study (n=12 per treatment group).TreatmentVamorolone or prednisolone was administered orally after disease onset for a duration of seven days.MethodsDisease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice.ResultsWe found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone sparing property was not observed with prednisolone treatment at any of the tested doses.ConclusionsWhile many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.
Background: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used... more Background: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. Objective: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. Methods: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. Results: A chronic, monthly regimen of high d...
Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in ... more Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin‐specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum‐circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin‐specif...
Cold Spring Harbor perspectives in medicine, Jan 3, 2014
Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with ske... more Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with skeletal muscle differentiation. One major subgroup of RMS tumors (so-called "fusion-positive" tumors) carries exclusive chromosomal translocations that join the DNA-binding domain of the PAX3 or PAX7 gene to the transactivation domain of the FOXO1 (previously known as FKHR) gene. Fusion-negative RMS represents a heterogeneous spectrum of tumors with frequent RAS pathway activation. Overtly metastatic disease at diagnosis is more frequently found in individuals with fusion-positive than in those with fusion-negative tumors. RMS is the most common pediatric soft-tissue sarcoma, and approximately 60% of all children and adolescents diagnosed with RMS are cured by currently available multimodal therapies. However, a curative outcome is achieved in <30% of high-risk individuals…
In his Correspondence, Terence Par-tridge raises two issues regarding our recent study investigat... more In his Correspondence, Terence Par-tridge raises two issues regarding our recent study investigating the stem cell properties of FACS-purified skeletal muscle precursors (SMPs) (Cerletti et al., 2008). First, he challenges our conclu-sion that SMPs represent a distinct myo-genic cell population, and second, he questions our physiological assessment of the SMP-engrafted muscles of dystro-phic
... CLARKE R. SLATER1 AND STEFANO SCHIAFFINO2 1 Institute of Neuroscience, Faculty of Medical Sci... more ... CLARKE R. SLATER1 AND STEFANO SCHIAFFINO2 1 Institute of Neuroscience, Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington ... denervated skeletal muscle (Butler-Browne et al., 1982) and is in part controlled by thyroid hormones (d&amp;#x27;Albis et al ...
Residual PMO concentration by mouse and muscle group. Residual morpholino concentration was measu... more Residual PMO concentration by mouse and muscle group. Residual morpholino concentration was measured in diaphragm, heart, gastrocnemius, quadriceps, tibialis anterior, and triceps muscle extracts at 30 days after PMO administration (n = 6). We observed variable retention levels of PMO between muscle groups and animals after 30 days of delivery. (PDF 119 kb)
Dystrophin protein expression detected by IF and WB after 7Â days of PMO delivery. Four mice were... more Dystrophin protein expression detected by IF and WB after 7Â days of PMO delivery. Four mice were treated with one high dose of PMO (800Â mg/kg) and sacrificed at 7Â days. Tibialis anterior muscles were dissected and analyzed by IF and WB. We observed low levels of dystrophin protein by both quantification methods as compared to saline-treated mice. (PDF 265 kb)
The generally accepted view of the pathology of Duchenne muscular dystrophy is that absence of dy... more The generally accepted view of the pathology of Duchenne muscular dystrophy is that absence of dystrophin renders myofibres abnormally susceptible to work-induced trauma, precipitating recurrent bouts of segmental myofibre necrosis. Necrosis stimulates inflammation and a regenerative response by myoblasts; the latter gradually becoming ineffective due to exhaustion of cells and accumulation of connective tissue driven by chronic inflammation. However, while searching for biomarkers in tissue cultures of dystrophic mouse muscle, we found phenomena that cause us to revise this picture. Using stable isotope labeled amino acids (SILAC) combined with liquid chromatography tandem mass spectrometry, we found that undamaged dystrophic myotubes are hypersecretive, excessively effluxing 57 intracellular proteins by mechanisms that cannot be identified as cell death, or generalized increase in passive leakiness. In addition dystrophic myotubes were smaller than wild-type and contain less protein and RNA per nucleus. We hypothesize that lack of dystrophin, as well as predisposing the myofibre to necrosis during physiological stress, has more profound general effects on its metabolic balance and its relationship with the extracellular environment, these potentially contributing to progressive muscle pathology. We identify myosin light chain 1 as a biomarker for in vitro drug screening.
Objective and DesignThe objective of this study was to assess the effect of vamorolone, a first-i... more Objective and DesignThe objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis.Animals84 DBA1/J mice were used in this study (n=12 per treatment group).TreatmentVamorolone or prednisolone was administered orally after disease onset for a duration of seven days.MethodsDisease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice.ResultsWe found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone sparing property was not observed with prednisolone treatment at any of the tested doses.ConclusionsWhile many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.
Background: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used... more Background: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. Objective: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. Methods: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. Results: A chronic, monthly regimen of high d...
Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in ... more Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin‐specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum‐circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin‐specif...
Cold Spring Harbor perspectives in medicine, Jan 3, 2014
Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with ske... more Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with skeletal muscle differentiation. One major subgroup of RMS tumors (so-called "fusion-positive" tumors) carries exclusive chromosomal translocations that join the DNA-binding domain of the PAX3 or PAX7 gene to the transactivation domain of the FOXO1 (previously known as FKHR) gene. Fusion-negative RMS represents a heterogeneous spectrum of tumors with frequent RAS pathway activation. Overtly metastatic disease at diagnosis is more frequently found in individuals with fusion-positive than in those with fusion-negative tumors. RMS is the most common pediatric soft-tissue sarcoma, and approximately 60% of all children and adolescents diagnosed with RMS are cured by currently available multimodal therapies. However, a curative outcome is achieved in <30% of high-risk individuals…
In his Correspondence, Terence Par-tridge raises two issues regarding our recent study investigat... more In his Correspondence, Terence Par-tridge raises two issues regarding our recent study investigating the stem cell properties of FACS-purified skeletal muscle precursors (SMPs) (Cerletti et al., 2008). First, he challenges our conclu-sion that SMPs represent a distinct myo-genic cell population, and second, he questions our physiological assessment of the SMP-engrafted muscles of dystro-phic
... CLARKE R. SLATER1 AND STEFANO SCHIAFFINO2 1 Institute of Neuroscience, Faculty of Medical Sci... more ... CLARKE R. SLATER1 AND STEFANO SCHIAFFINO2 1 Institute of Neuroscience, Faculty of Medical Sciences, University of Newcastle upon Tyne, Framlington ... denervated skeletal muscle (Butler-Browne et al., 1982) and is in part controlled by thyroid hormones (d&amp;#x27;Albis et al ...
Residual PMO concentration by mouse and muscle group. Residual morpholino concentration was measu... more Residual PMO concentration by mouse and muscle group. Residual morpholino concentration was measured in diaphragm, heart, gastrocnemius, quadriceps, tibialis anterior, and triceps muscle extracts at 30 days after PMO administration (n = 6). We observed variable retention levels of PMO between muscle groups and animals after 30 days of delivery. (PDF 119 kb)
Dystrophin protein expression detected by IF and WB after 7Â days of PMO delivery. Four mice were... more Dystrophin protein expression detected by IF and WB after 7Â days of PMO delivery. Four mice were treated with one high dose of PMO (800Â mg/kg) and sacrificed at 7Â days. Tibialis anterior muscles were dissected and analyzed by IF and WB. We observed low levels of dystrophin protein by both quantification methods as compared to saline-treated mice. (PDF 265 kb)
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