- Email: [email protected]
Restoration of dystrophin expression and motor function in using exosomes-based non-immunogenic genetic therapy
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Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212
early phase of the disease reduces efficiently Dnm2 protein level and prevents the myopathy progression while rescuing both lifespan and body weight, and correcting muscular mass, histology and force. Importantly, systemic injections of ASO into strongly affected mice revert different myopathy signs after only 2 injections and prolonged lifespan. Noteworthy, these data validate the efficacy of such ASO for a non-dystrophic myopathy. Thus, ASO-mediated Dnm2 knockdown could revert and correct CNM features after disease onset which provides an attractive therapeutic strategy that may be applied to patients with myotubular myopathy. http://dx.doi.org/10.1016/j.nmd.2016.06.448
T.O.15 Inhibition of PIK3C2B as a treatment strategy for myotubular myopathy N. Sabha 1, J. Volpatti 1, H. Gonorazky 1, A. Reifler 2, A. Davidson 1, A. Buj-Bello 3, E. Feldman 2, J. Dowling 1 1 Hospital for Sick Children, Toronto, Canada; 2 University of Michigan, Ann Arbor, USA; 3 Genethon, Evry, France Myotubular myopathy (MTM) is an X-linked form of centronuclear myopathy associated with severe disabilities, including wheelchair and ventilator dependence, and early mortality. It results from a mutation in the PIP phosphatase MTM1, and as such is the prototypical disorder of phosphoinositide (PIP) metabolism. At present there is no cure nor any disease modifying therapies for this devastating disorder. Our goal is to develop new treatment approaches for MTM. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in MTM animal models, and hypothesized that lowering PI3P levels may be an effective therapeutic strategy in this condition. To test this, we genetically targeted class II and III PI3 kinases (PI3Ks) in an MTM mouse model. We found that germline elimination of Pik3c2b, but not Pik3c3, results in complete prevention of the MTM phenotype, and that post-symptomatic targeting of Pik3c2b also promotes a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that PI3K inhibitors with activity against PIK3C2B can significantly prevent development of the zebrafish mtm phenotype. Lastly, we examined one PI3 kinase inhibitor (wortmannin) for its ability to impact the mouse MTM phenotype, and found that, consistent with our genetic results and zebrafish studies, it significantly improves muscle function and prolongs survival. In all, we identify Pik3c2b as the first genetic modifier of Mtm1 mutation, and demonstrate that PIK3C2B inhibition is a potentially remarkable treatment strategy for MTM, either as monotherapy or in combination with ongoing efforts at rescue therapies such as gene replacement and enzyme replacement therapy. http://dx.doi.org/10.1016/j.nmd.2016.06.449
T.O.16 Phase 2 study design of antisense oligonucleotide nusinersen in presymptomatic infants with spinal muscular atrophy E. Bertini 1, P. Hwu 2, S. Reyna 3, W. Farwell 3, D. De 4 1 Post-Graduate Bambino Gesù Children’s Research Hospital, Rome, Italy; 2 National Taiwan University Hospital, Taipei, Taiwan; 3 Biogen, Cambridge, USA; 4 Columbia University Medical Center, New York, USA Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by a defect in the survival motor neuron 1 (SMN1) gene, with no effective disease-modifying therapies currently approved. Nusinersen is an antisense oligonucleotide drug that increases SMN protein levels dose dependently. Presymptomatic treatment of infants with SMA may be possible because penetrance of SMA is ~100% with a genetic diagnosis, while SMN2 copy number can predict disease severity. In a pilot study, intrathecal administration of nusinersen was well tolerated with no safety concerns, and a phase 2 study (NURTURE) has been initiated. NURTURE is a phase 2 openlabel, multicenter, multinational, single-arm study across 10 countries
including Italy, Germany, and the UK. The efficacy, safety, tolerability, and pharmacokinetics of intrathecal nusinersen (12-mg scaled equivalent dose) will be evaluated in infants with presymptomatic SMA. Key inclusion criteria include ≤6 weeks of age at first dose, genetic diagnosis of 5q SMA homozygous gene deletion/mutation or compound heterozygous mutation, 2 or 3 copies of SMN2 and compound muscle action potential ≥1 mV. Key exclusion criteria include clinical symptoms strongly suggestive of SMA. The primary objective is to examine the efficacy of nusinersen in preventing or delaying respiratory intervention or death. Secondary objectives will assess the safety, tolerability, and pharmacokinetics of nusinersen and its effect on the development of clinically-manifested SMA through a composite of clinical features, growth and function. NURTURE has a planned study period of May 2015–April 2020, and will enroll ≤25 infants with presymptomatic SMA, aged ≤6 weeks. To date (10 February 2016), 16 infants have been screened, with 13 of these infants enrolled into the study. NURTURE will investigate the feasibility, efficacy, safety and pharmacokinetics of nusinersen in infants with pre-symptomatic SMA. http://dx.doi.org/10.1016/j.nmd.2016.06.450
ORAL PRESENTATIONS – GENERAL G.O.17 MELAS, a first-ever tRNA modification disorder is alleviated by taurine supplementation therapy Y. Sunada 1, Y. Ohsawa 1, Y. Fukai 1, M. Fujino 2, S. Nishimatsu 1, S. Ohta 3 1 Kawasaki Medical School, Kurashiki, Japan; 2 Kawasaki University of Medical Welfare, Kurashiki, Japan; 3 Nippon Medical School, Kawasaki, Japan Francis Crick postulated certain chemical modifications at the first anticodon nucleotide in each tRNA, because the first anticodon nucleotide interacts with the corresponding third codon nucleotide in mRNA through non-canonical Watson-Crick geometry. So far, several post-transcriptional chemical modifications have been discovered in the first anticodon nucleotides in tRNAs. We found a modification of taurine at the first anticodon nucleotide in the normal mitochondrial (mt) tRNALeu(UUR). Surprisingly, the taurine modification is completely deficient in the mt tRNALeu(UUR) derived from tissues from MELAS patients harboring the A3243G transition. Because the taurine modification defect in the mutant mt tRNALeu(UUR) causes a deficiency in deciphering codons, we regard MELAS as a first-ever tRNA-modification disorder. Indeed, high-dose taurine supplementation ameliorates impaired mt dysfunction in patient-derived cells and prevents stroke-like episodes in two MELAS patients for more than nine years. Here we performed a multi-center, open, phase 3 trial to approve the clinical efficiencies of oral taurine supplementation on preventing stroke-like episodes in patients with MELAS for 2 years. We enrolled 10 patients suffering from repeated stroke-like episodes in the trial. Initial two-year oral administration of taurine completely prevents stroke-like episodes in 6 patients and significantly decreased its annual relapsing rates in the other patients. During the trial, taurine modification ratio in the mt tRNALeu(UUR) in peripheral white blood cells were significantly increased in 5 out of 9 patients. Taurine could prevent stroke-like episodes in MELAS by reversing impaired the taurine modification in mt tRNALeu(UUR). http://dx.doi.org/10.1016/j.nmd.2016.06.451
G.O.18 Restoration of dystrophin expression and motor function in using exosomes-based non-immunogenic genetic therapy A. Saleem, A. Safdar, M. Khan, P. Azzopardi, D. Rusiecki, A. Saleem, M. Tarnopolsky McMaster University, Hamilton, ON, Canada Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder caused by a recessive X-linked genetic mutation in the gene encoding
Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212 dystrophin. Currently, there is no cure for DMD, and recent efforts have focused on viral-based modes of gene delivery to express truncated forms of dystrophin, or rely on exon skipping strategies to skip the locus of mutation. Here we utilized bioengineered exosomes, 40–100 nm sized extracellular vesicles, to deliver full-length dystrophin mRNA (dmd) to mdx mice allogenically. C57BL/10ScSn-Dmdmdx (mdx) mice (~14–15 weeks old) were randomly divided into prednisolone (PRED, 2 mg/kg BW/day), vehicle (VEH, i.v. injections of 0.9% sterile saline), exosomes only (EXO, i.v. injections of empty exosomes), and exosomes + dmd mRNA (EXO + mRNA, i.v. injections of exosomes containing 150 ng of dmd mRNA) groups. C57BL/10ScSn wild type (WT) mice were also included as control animals. Treatment of mdx mice with exosomes + dmd mRNA rescued the absence of dystrophin protein expression in skeletal muscle (EDL, SOL, TA, diaphragm), and heart as assessed by Western blotting and immunohistochemistry. This occurred in tandem with a complete attenuation in elevated serum creatine kinase levels, muscle hypertrophy, and grip strength deficits. Furthermore, necrosis and % fibers with centralized myonuclei were reduced in EXO + mRNA to levels observed in WT mice. Moreover we treated dermal fibroblasts isolated from control and DMD patients with dmd mRNA only, exosomes only, and exosomes + dmd mRNA. Similar to our in vivo observations, we restored dystrophin protein expression in DMD patients treated with our bioengineered exosomes. Our data clearly establish treatment with non-immunogenic bioengineered exosomes as efficient delivery vehicles for treating diseases of genetic origin such as DMD. http://dx.doi.org/10.1016/j.nmd.2016.06.452
G.O.19 ORAI1 mutations cause abnormal channel gating in tubular aggregate myopathy J. Böhm 1, M. Bulla 2, J. Urquhart 3, C. Koch 1, W. Newman 3, M. Mora 4, M. Moggio 5, N. Romero 6, N. Demaurex 2, J. Laporte 1 1 IGBMC, Strasbourg, France; 2 University of Geneva, Geneva, Switzerland; 3 St. Mary’s Hospital, Manchester, UK; 4 Istituto Neurologico C. Besta, Milan, Italy; 5 University of Milan, Milan, Italy; 6 Institut de Myologie, Paris, France Skeletal muscle physiology relies on changes in free cytosolic Ca2+ levels, and small disturbances can severely impact on fundamental cellular processes as muscle contraction or signalling pathways. A major mechanism controlling Ca2+ homeostasis is store-operated Ca2+ entry (SOCE): upon Ca2+ store depletion of the endoplasmic/sarcoplasmic reticulum, the Ca2+ sensor STIM1 oligomerizes and activates the plasma membrane channel ORAI1 to trigger extracellular Ca2+ influx. We have previously shown the implication of STIM1 in tubular aggregate myopathy (TAM), and here we report the identification of three ORAI1 mutations causing autosomal dominant TAM. This slowly progressive muscle disorder predominantly affects the proximal muscles of the lower limbs, and is characterized by the presence of densely packed membrane tubules on muscle biopsies. Immunohistofluorescence on muscle sections revealed the presence of various sarcoplasmic reticulum proteins in the tubular aggregates, including large amounts of STIM1. ORAI1 channels are hexamers forming concentric rings around a central pore. Two ORAI1 mutations (G98S and V107M) affect amino acids of ion conduction pathway, and the third mutation (T184M) resides in a concentric channel ring. We analyzed ORAI1 gating in presence and absence of STIM1, and we demonstrate that the G98S and V107M mutations produce a permeable ORAI1 channel with extended maximal activity, whereas the T184M mutation requires STIM1 to generate an excessive short-run Ca2+ influx. These different pathomechanisms correlate with the clinical presentation, as the patients harboring the G98S and V107M mutations have a more severe disease pattern. Our results suggest that overactivation of SOCE resulting from STIM1 or ORAI1 mutations represents the primary pathomechanism underlying tubular aggregate myopathy. http://dx.doi.org/10.1016/j.nmd.2016.06.453
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G.O.20 Functions of the SIL1-BiP chaperone system in maintaining muscle fiber integrity A. Roos 1, S. Buchkremer 2, J. González Coraspe 2, J. Weis 2, L. Kollipara 1, R. Zahedi 1 1 Leibniz-Istitut für Analytische Wissenschaften ISAS e.V., Dortmund, Germany; 2 RWTH Aachen University Hospital, Aachen, Germany The SIL1-BiP machinery represents a major chaperone-system of the SR and is involved in protein processing and modulation of cellular stress responses. BiP functions in an ATP-dependent manner and thus depends on the co-chaperone function of SIL1 linking ATP to BiP. Prompted by the fact that the SR stress response plays a significant role in diverse muscular disorders, we hypothesized that the SIL1-BiP chaperone-system is essential in skeletal muscle cells to deal with stress and that disturbances in chaperone cycling lead to clinically relevant perturbations. Therefore, we examined level of both proteins in stressed muscle cells and tissue, diseased muscles including de-innervated mouse muscles, human sIBM specimens and muscles derived from a mouse model of caveolinopathy and detected protein increase thus indicating functional relevance of this chaperone system in muscle fiber integrity. Therefore, we addressed functional relevance of this chaperonesystem by comprehensive investigation of SIL1-deficient skeletal muscle using electron microscopy, immunoblotting, immunohistochemistry as well as unbiased proteome profiling. Additionally, targeted proteomic approaches were carried out in order to study the role of this system in protein-degradation. These examinations revealed marked dilatation of the SR, peculiar alterations of the myonuclear envelope with irregular proliferation of the nuclear lamina and build-up of autophagic vacuoles and severe mitochondrial changes. Morphological alterations could be correlated with the findings of our unbiased proteomic profiling and with the results of our targeted approaches focusing on protein degradation and thereby identifying substrates of the impaired SIL1BiP machinery. Our studies highlight involvement of the SIL1-BiP machinery in the etiopathology of (neuro)muscular disorders and demonstrate the need of its proper function in fiber maintenance especially with regard to defects in various aspects of BiP functions. http://dx.doi.org/10.1016/j.nmd.2016.06.454
G.O.21 P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye Y. Zou 1, S. Donkervoort 1, A. Salo 2, A. Barnes 3, Y. Hu 1, A. Reghan Foley 1, E. Makareeva 3, M. Leach 1, J. Dastgir 1, R. Cohn 4, W. DiNonno 5, S. Leikin 3, J. Marini 3, J. Myllyharju 2, C. Bonnemann 1 1 National Institutes of Health, NINDS, Bethesda, USA; 2 Oulu Center for Cell-Matrix Research, Biocenter Oulu, Oulu, Finland; 3 National Institutes of Health, NICHD, Bethesda, USA; 4 The Hospital for Sick Children, Toronto, Canada; 5 Eastern Virginia Medical School, Norfolk, USA Collagen prolyl 4-hydroxylases (C-P4Hs) play a central role in the formation and stabilization of the collagenous triple helical domain that is the defining structural motif of collagens. P4HA1 encodes for the catalytic α(I) subunit of the main C-P4H isoenzyme (C-P4H I), and thus far no mutation in P4HA1 has been reported to cause human disease. We report the first human P4HA1 mutations in a family with a congenital-onset disorder of connective tissue, manifesting as early-onset joint hypermobility, weakness and bone malformations as well as high myopia, with clinical improvement over time in the surviving patient. Similarly to P4ha1 null mice, which die prenatally, patients were found to have reduced collagen IV expression at the muscle basement membrane. Patients were compound heterozygous for nonsense and splice mutations leading to reduced P4HA1 protein level and C-P4H activity in dermal fibroblasts compared to age-matched control samples. Differential scanning calorimetry assay revealed reduced thermal stability of collagen with a decrease in proline 4-hydroxylation in patients versus age-matched controls. The unique P4HA1 mutations identified in our patients shed light on the normal
Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212
early phase of the disease reduces efficiently Dnm2 protein level and prevents the myopathy progression while rescuing both lifespan and body weight, and correcting muscular mass, histology and force. Importantly, systemic injections of ASO into strongly affected mice revert different myopathy signs after only 2 injections and prolonged lifespan. Noteworthy, these data validate the efficacy of such ASO for a non-dystrophic myopathy. Thus, ASO-mediated Dnm2 knockdown could revert and correct CNM features after disease onset which provides an attractive therapeutic strategy that may be applied to patients with myotubular myopathy. http://dx.doi.org/10.1016/j.nmd.2016.06.448
T.O.15 Inhibition of PIK3C2B as a treatment strategy for myotubular myopathy N. Sabha 1, J. Volpatti 1, H. Gonorazky 1, A. Reifler 2, A. Davidson 1, A. Buj-Bello 3, E. Feldman 2, J. Dowling 1 1 Hospital for Sick Children, Toronto, Canada; 2 University of Michigan, Ann Arbor, USA; 3 Genethon, Evry, France Myotubular myopathy (MTM) is an X-linked form of centronuclear myopathy associated with severe disabilities, including wheelchair and ventilator dependence, and early mortality. It results from a mutation in the PIP phosphatase MTM1, and as such is the prototypical disorder of phosphoinositide (PIP) metabolism. At present there is no cure nor any disease modifying therapies for this devastating disorder. Our goal is to develop new treatment approaches for MTM. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in MTM animal models, and hypothesized that lowering PI3P levels may be an effective therapeutic strategy in this condition. To test this, we genetically targeted class II and III PI3 kinases (PI3Ks) in an MTM mouse model. We found that germline elimination of Pik3c2b, but not Pik3c3, results in complete prevention of the MTM phenotype, and that post-symptomatic targeting of Pik3c2b also promotes a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that PI3K inhibitors with activity against PIK3C2B can significantly prevent development of the zebrafish mtm phenotype. Lastly, we examined one PI3 kinase inhibitor (wortmannin) for its ability to impact the mouse MTM phenotype, and found that, consistent with our genetic results and zebrafish studies, it significantly improves muscle function and prolongs survival. In all, we identify Pik3c2b as the first genetic modifier of Mtm1 mutation, and demonstrate that PIK3C2B inhibition is a potentially remarkable treatment strategy for MTM, either as monotherapy or in combination with ongoing efforts at rescue therapies such as gene replacement and enzyme replacement therapy. http://dx.doi.org/10.1016/j.nmd.2016.06.449
T.O.16 Phase 2 study design of antisense oligonucleotide nusinersen in presymptomatic infants with spinal muscular atrophy E. Bertini 1, P. Hwu 2, S. Reyna 3, W. Farwell 3, D. De 4 1 Post-Graduate Bambino Gesù Children’s Research Hospital, Rome, Italy; 2 National Taiwan University Hospital, Taipei, Taiwan; 3 Biogen, Cambridge, USA; 4 Columbia University Medical Center, New York, USA Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by a defect in the survival motor neuron 1 (SMN1) gene, with no effective disease-modifying therapies currently approved. Nusinersen is an antisense oligonucleotide drug that increases SMN protein levels dose dependently. Presymptomatic treatment of infants with SMA may be possible because penetrance of SMA is ~100% with a genetic diagnosis, while SMN2 copy number can predict disease severity. In a pilot study, intrathecal administration of nusinersen was well tolerated with no safety concerns, and a phase 2 study (NURTURE) has been initiated. NURTURE is a phase 2 openlabel, multicenter, multinational, single-arm study across 10 countries
including Italy, Germany, and the UK. The efficacy, safety, tolerability, and pharmacokinetics of intrathecal nusinersen (12-mg scaled equivalent dose) will be evaluated in infants with presymptomatic SMA. Key inclusion criteria include ≤6 weeks of age at first dose, genetic diagnosis of 5q SMA homozygous gene deletion/mutation or compound heterozygous mutation, 2 or 3 copies of SMN2 and compound muscle action potential ≥1 mV. Key exclusion criteria include clinical symptoms strongly suggestive of SMA. The primary objective is to examine the efficacy of nusinersen in preventing or delaying respiratory intervention or death. Secondary objectives will assess the safety, tolerability, and pharmacokinetics of nusinersen and its effect on the development of clinically-manifested SMA through a composite of clinical features, growth and function. NURTURE has a planned study period of May 2015–April 2020, and will enroll ≤25 infants with presymptomatic SMA, aged ≤6 weeks. To date (10 February 2016), 16 infants have been screened, with 13 of these infants enrolled into the study. NURTURE will investigate the feasibility, efficacy, safety and pharmacokinetics of nusinersen in infants with pre-symptomatic SMA. http://dx.doi.org/10.1016/j.nmd.2016.06.450
ORAL PRESENTATIONS – GENERAL G.O.17 MELAS, a first-ever tRNA modification disorder is alleviated by taurine supplementation therapy Y. Sunada 1, Y. Ohsawa 1, Y. Fukai 1, M. Fujino 2, S. Nishimatsu 1, S. Ohta 3 1 Kawasaki Medical School, Kurashiki, Japan; 2 Kawasaki University of Medical Welfare, Kurashiki, Japan; 3 Nippon Medical School, Kawasaki, Japan Francis Crick postulated certain chemical modifications at the first anticodon nucleotide in each tRNA, because the first anticodon nucleotide interacts with the corresponding third codon nucleotide in mRNA through non-canonical Watson-Crick geometry. So far, several post-transcriptional chemical modifications have been discovered in the first anticodon nucleotides in tRNAs. We found a modification of taurine at the first anticodon nucleotide in the normal mitochondrial (mt) tRNALeu(UUR). Surprisingly, the taurine modification is completely deficient in the mt tRNALeu(UUR) derived from tissues from MELAS patients harboring the A3243G transition. Because the taurine modification defect in the mutant mt tRNALeu(UUR) causes a deficiency in deciphering codons, we regard MELAS as a first-ever tRNA-modification disorder. Indeed, high-dose taurine supplementation ameliorates impaired mt dysfunction in patient-derived cells and prevents stroke-like episodes in two MELAS patients for more than nine years. Here we performed a multi-center, open, phase 3 trial to approve the clinical efficiencies of oral taurine supplementation on preventing stroke-like episodes in patients with MELAS for 2 years. We enrolled 10 patients suffering from repeated stroke-like episodes in the trial. Initial two-year oral administration of taurine completely prevents stroke-like episodes in 6 patients and significantly decreased its annual relapsing rates in the other patients. During the trial, taurine modification ratio in the mt tRNALeu(UUR) in peripheral white blood cells were significantly increased in 5 out of 9 patients. Taurine could prevent stroke-like episodes in MELAS by reversing impaired the taurine modification in mt tRNALeu(UUR). http://dx.doi.org/10.1016/j.nmd.2016.06.451
G.O.18 Restoration of dystrophin expression and motor function in using exosomes-based non-immunogenic genetic therapy A. Saleem, A. Safdar, M. Khan, P. Azzopardi, D. Rusiecki, A. Saleem, M. Tarnopolsky McMaster University, Hamilton, ON, Canada Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder caused by a recessive X-linked genetic mutation in the gene encoding
Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212 dystrophin. Currently, there is no cure for DMD, and recent efforts have focused on viral-based modes of gene delivery to express truncated forms of dystrophin, or rely on exon skipping strategies to skip the locus of mutation. Here we utilized bioengineered exosomes, 40–100 nm sized extracellular vesicles, to deliver full-length dystrophin mRNA (dmd) to mdx mice allogenically. C57BL/10ScSn-Dmdmdx (mdx) mice (~14–15 weeks old) were randomly divided into prednisolone (PRED, 2 mg/kg BW/day), vehicle (VEH, i.v. injections of 0.9% sterile saline), exosomes only (EXO, i.v. injections of empty exosomes), and exosomes + dmd mRNA (EXO + mRNA, i.v. injections of exosomes containing 150 ng of dmd mRNA) groups. C57BL/10ScSn wild type (WT) mice were also included as control animals. Treatment of mdx mice with exosomes + dmd mRNA rescued the absence of dystrophin protein expression in skeletal muscle (EDL, SOL, TA, diaphragm), and heart as assessed by Western blotting and immunohistochemistry. This occurred in tandem with a complete attenuation in elevated serum creatine kinase levels, muscle hypertrophy, and grip strength deficits. Furthermore, necrosis and % fibers with centralized myonuclei were reduced in EXO + mRNA to levels observed in WT mice. Moreover we treated dermal fibroblasts isolated from control and DMD patients with dmd mRNA only, exosomes only, and exosomes + dmd mRNA. Similar to our in vivo observations, we restored dystrophin protein expression in DMD patients treated with our bioengineered exosomes. Our data clearly establish treatment with non-immunogenic bioengineered exosomes as efficient delivery vehicles for treating diseases of genetic origin such as DMD. http://dx.doi.org/10.1016/j.nmd.2016.06.452
G.O.19 ORAI1 mutations cause abnormal channel gating in tubular aggregate myopathy J. Böhm 1, M. Bulla 2, J. Urquhart 3, C. Koch 1, W. Newman 3, M. Mora 4, M. Moggio 5, N. Romero 6, N. Demaurex 2, J. Laporte 1 1 IGBMC, Strasbourg, France; 2 University of Geneva, Geneva, Switzerland; 3 St. Mary’s Hospital, Manchester, UK; 4 Istituto Neurologico C. Besta, Milan, Italy; 5 University of Milan, Milan, Italy; 6 Institut de Myologie, Paris, France Skeletal muscle physiology relies on changes in free cytosolic Ca2+ levels, and small disturbances can severely impact on fundamental cellular processes as muscle contraction or signalling pathways. A major mechanism controlling Ca2+ homeostasis is store-operated Ca2+ entry (SOCE): upon Ca2+ store depletion of the endoplasmic/sarcoplasmic reticulum, the Ca2+ sensor STIM1 oligomerizes and activates the plasma membrane channel ORAI1 to trigger extracellular Ca2+ influx. We have previously shown the implication of STIM1 in tubular aggregate myopathy (TAM), and here we report the identification of three ORAI1 mutations causing autosomal dominant TAM. This slowly progressive muscle disorder predominantly affects the proximal muscles of the lower limbs, and is characterized by the presence of densely packed membrane tubules on muscle biopsies. Immunohistofluorescence on muscle sections revealed the presence of various sarcoplasmic reticulum proteins in the tubular aggregates, including large amounts of STIM1. ORAI1 channels are hexamers forming concentric rings around a central pore. Two ORAI1 mutations (G98S and V107M) affect amino acids of ion conduction pathway, and the third mutation (T184M) resides in a concentric channel ring. We analyzed ORAI1 gating in presence and absence of STIM1, and we demonstrate that the G98S and V107M mutations produce a permeable ORAI1 channel with extended maximal activity, whereas the T184M mutation requires STIM1 to generate an excessive short-run Ca2+ influx. These different pathomechanisms correlate with the clinical presentation, as the patients harboring the G98S and V107M mutations have a more severe disease pattern. Our results suggest that overactivation of SOCE resulting from STIM1 or ORAI1 mutations represents the primary pathomechanism underlying tubular aggregate myopathy. http://dx.doi.org/10.1016/j.nmd.2016.06.453
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G.O.20 Functions of the SIL1-BiP chaperone system in maintaining muscle fiber integrity A. Roos 1, S. Buchkremer 2, J. González Coraspe 2, J. Weis 2, L. Kollipara 1, R. Zahedi 1 1 Leibniz-Istitut für Analytische Wissenschaften ISAS e.V., Dortmund, Germany; 2 RWTH Aachen University Hospital, Aachen, Germany The SIL1-BiP machinery represents a major chaperone-system of the SR and is involved in protein processing and modulation of cellular stress responses. BiP functions in an ATP-dependent manner and thus depends on the co-chaperone function of SIL1 linking ATP to BiP. Prompted by the fact that the SR stress response plays a significant role in diverse muscular disorders, we hypothesized that the SIL1-BiP chaperone-system is essential in skeletal muscle cells to deal with stress and that disturbances in chaperone cycling lead to clinically relevant perturbations. Therefore, we examined level of both proteins in stressed muscle cells and tissue, diseased muscles including de-innervated mouse muscles, human sIBM specimens and muscles derived from a mouse model of caveolinopathy and detected protein increase thus indicating functional relevance of this chaperone system in muscle fiber integrity. Therefore, we addressed functional relevance of this chaperonesystem by comprehensive investigation of SIL1-deficient skeletal muscle using electron microscopy, immunoblotting, immunohistochemistry as well as unbiased proteome profiling. Additionally, targeted proteomic approaches were carried out in order to study the role of this system in protein-degradation. These examinations revealed marked dilatation of the SR, peculiar alterations of the myonuclear envelope with irregular proliferation of the nuclear lamina and build-up of autophagic vacuoles and severe mitochondrial changes. Morphological alterations could be correlated with the findings of our unbiased proteomic profiling and with the results of our targeted approaches focusing on protein degradation and thereby identifying substrates of the impaired SIL1BiP machinery. Our studies highlight involvement of the SIL1-BiP machinery in the etiopathology of (neuro)muscular disorders and demonstrate the need of its proper function in fiber maintenance especially with regard to defects in various aspects of BiP functions. http://dx.doi.org/10.1016/j.nmd.2016.06.454
G.O.21 P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye Y. Zou 1, S. Donkervoort 1, A. Salo 2, A. Barnes 3, Y. Hu 1, A. Reghan Foley 1, E. Makareeva 3, M. Leach 1, J. Dastgir 1, R. Cohn 4, W. DiNonno 5, S. Leikin 3, J. Marini 3, J. Myllyharju 2, C. Bonnemann 1 1 National Institutes of Health, NINDS, Bethesda, USA; 2 Oulu Center for Cell-Matrix Research, Biocenter Oulu, Oulu, Finland; 3 National Institutes of Health, NICHD, Bethesda, USA; 4 The Hospital for Sick Children, Toronto, Canada; 5 Eastern Virginia Medical School, Norfolk, USA Collagen prolyl 4-hydroxylases (C-P4Hs) play a central role in the formation and stabilization of the collagenous triple helical domain that is the defining structural motif of collagens. P4HA1 encodes for the catalytic α(I) subunit of the main C-P4H isoenzyme (C-P4H I), and thus far no mutation in P4HA1 has been reported to cause human disease. We report the first human P4HA1 mutations in a family with a congenital-onset disorder of connective tissue, manifesting as early-onset joint hypermobility, weakness and bone malformations as well as high myopia, with clinical improvement over time in the surviving patient. Similarly to P4ha1 null mice, which die prenatally, patients were found to have reduced collagen IV expression at the muscle basement membrane. Patients were compound heterozygous for nonsense and splice mutations leading to reduced P4HA1 protein level and C-P4H activity in dermal fibroblasts compared to age-matched control samples. Differential scanning calorimetry assay revealed reduced thermal stability of collagen with a decrease in proline 4-hydroxylation in patients versus age-matched controls. The unique P4HA1 mutations identified in our patients shed light on the normal