[en] During the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.
Disciplines :
Pediatrics Neurology
Author, co-author :
Scaglioni, Dominic
Catapano, Francesco
Ellis, Matthew
Torelli, Silvia
Chambers, Darren
Feng, Lucy
Beck, Matthew
Sewry, Caroline
Monforte, Mauro
Harriman, Shawn
Koenig, Erica
Malhotra, Jyoti
Popplewell, Linda
Guglieri, Michela
Straub, Volker
Mercuri, Eugenio
Servais, Laurent ; Centre Hospitalier Universitaire de Liège - CHU > Département de Pédiatrie > Service de pédiatrie
Aartsma-Rus A, Fokkema I, Verschuuren J, Ginjaar I, van Deutekom J, van Ommen G-J, den Dunnen JT (2009) Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Hum Mutat 30:293–299. 10.1002/humu.20918 DOI: 10.1002/humu.20918
Aartsma-Rus A, Straub V, Hemmings R, Haas M, Schlosser-Weber G, Stoyanova-Beninska V, Mercuri E, Muntoni F, Sepodes B, Vroom E, Balabanov P (2017) Development of exon skipping therapies for duchenne muscular dystrophy: a critical review and a perspective on the outstanding issues. Nucleic Acid Ther 27:251–259. 10.1089/nat.2017.0682 DOI: 10.1089/nat.2017.0682
Anthony K, Arechavala-Gomeza V, Ricotti V, Torelli S, Feng L, Janghra N, Tasca G, Guglieri M, Barresi R, Armaroli A, Ferlini A, Bushby K, Straub V, Ricci E, Sewry C, Morgan J, Muntoni F (2014) Biochemical characterization of patients with in-frame or out-of-frame DMD deletions pertinent to exon 44 or 45 skipping. JAMA Neurol 71:32–40. 10.1001/jamaneurol.2013.4908 DOI: 10.1001/jamaneurol.2013.4908
Anthony K, Cirak S, Torelli S, Tasca G, Feng L, Arechavala-Gomeza V, Armaroli A, Guglieri M, Straathof CS, Verschuuren JJ, Aartsma-Rus A, Helderman-van den Enden P, Bushby K, Straub V, Sewry C, Ferlini A, Ricci E, Morgan JE, Muntoni F (2011) Dystrophin quantification and clinical correlations in Becker muscular dystrophy: implications for clinical trials. Brain 134:3547–3559. 10.1093/brain/awr291 DOI: 10.1093/brain/awr291
Bello L, Morgenroth LP, Gordish-Dressman H, Hoffman EP, McDonald CM, Cirak S (2016) DMD genotypes and loss of ambulation in the CINRG Duchenne natural history study. Neurology 87:401–409. 10.1212/wnl.0000000000002891 DOI: 10.1212/wnl.0000000000002891
Brogna C, Coratti G, Pane M, Ricotti V, Messina S, D’Amico A, Bruno C, Vita G, Berardinelli A, Mazzone E, Magri F, Ricci F, Mongini T, Battini R, Bello L, Pegoraro E, Baranello G, Previtali SC, Politano L, Comi GP, Sansone VA, Donati A, Bertini E, Muntoni F, Goemans N, Mercuri E, on behalf on the International DMD group (2019) Long-term natural history data in Duchenne muscular dystrophy ambulant patients with mutations amenable to skip exons 44, 45, 51 and 53. PLoS ONE 14:e0218683. 10.1371/journal.pone.0218683 DOI: 10.1371/journal.pone.0218683
Charleston JS, Schnell FJ, Dworzak J, Donoghue C, Lewis S, Chen L, Young GD, Milici AJ, Voss J, DeAlwis U, Wentworth B, Rodino-Klapac LR, Sahenk Z, Frank D, Mendell JR (2018) Eteplirsen treatment for Duchenne muscular dystrophy: exon skipping and dystrophin production. Neurology 90:e2146–e2154. 10.1212/wnl.0000000000005680 DOI: 10.1212/wnl.0000000000005680
Cirak S, Arechavala-Gomeza V, Guglieri M, Feng L, Torelli S, Anthony K, Abbs S, Garralda ME, Bourke J, Wells DJ, Dickson G, Wood MJA, Wilton SD, Straub V, Kole R, Shrewsbury SB, Sewry C, Morgan JE, Bushby K, Muntoni F (2011) Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet 378:595–605. 10.1016/s0140-6736(11)60756-3 DOI: 10.1016/s0140-6736(11)60756-3
Cirak S, Feng L, Anthony K, Arechavala-Gomeza V, Torelli S, Sewry C, Morgan JE, Muntoni F (2012) Restoration of the dystrophin-associated glycoprotein complex after exon skipping therapy in duchenne muscular dystrophy. Mol Ther 20:462–467. 10.1038/mt.2011.248 DOI: 10.1038/mt.2011.248
Claflin DR, Brooks SV (2008) Direct observation of failing fibers in muscles of dystrophic mice provides mechanistic insight into muscular dystrophy. Am J Physiol Cell Physiol 294:C651–658. 10.1152/ajpcell.00244.2007 DOI: 10.1152/ajpcell.00244.2007
Clemens PR, Rao VK, Connolly AM, Harper AD, Mah JK, Smith EC, McDonald CM, Zaidman CM, Morgenroth LP, Osaki H, Satou Y, Yamashita T, Hoffman EP, Investigators CINRGDNHS (2020) Safety, tolerability, and efficacy of viltolarsen in boys with duchenne muscular dystrophy amenable to exon 53 skipping: a phase 2 randomized clinical trial. JAMA Neurol. 10.1001/jamaneurol.2020.1264 DOI: 10.1001/jamaneurol.2020.1264
Constantin B (2014) Dystrophin complex functions as a scaffold for signalling proteins. Biochimica et Biophysica Acta (BBA) Biomembranes 1838:635–642. 10.1016/j.bbamem.2013.08.023 DOI: 10.1016/j.bbamem.2013.08.023
Duan D (2018) Systemic AAV micro-dystrophin gene therapy for duchenne muscular dystrophy. Mol Ther 26:2337–2356. 10.1016/j.ymthe.2018.07.011 DOI: 10.1016/j.ymthe.2018.07.011
Dubowitz V, Oldfors A, Sewry CA (2020) Muscle biopsy: a practical approach
Ervasti JM (2003) Costameres: the Achilles’ Heel of Herculean muscle. J Biol Chem 278:13591–13594. 10.1074/jbc.r200021200 DOI: 10.1074/jbc.r200021200
Ervasti JM (2013) Structure and function of the dystrophin-glycoprotein complex. Landes Bioscience, Austin
Finkel RS, Flanigan KM, Wong B, Bönnemann C, Sampson J, Sweeney HL, Reha A, Northcutt VJ, Elfring G, Barth J, Peltz SW (2013) Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy. PLoS ONE 8:e81302. 10.1371/journal.pone.0081302 DOI: 10.1371/journal.pone.0081302
Flanigan KM (2014) Duchenne and Becker muscular dystrophies. Neurol Clin 32:671–688. 10.1016/j.ncl.2014.05.002 DOI: 10.1016/j.ncl.2014.05.002
Frank DE, Schnell FJ, Akana C, El-Husayni SH, Desjardins CA, Morgan J, Charleston JS, Sardone V, Domingos J, Dickson G, Straub V, Guglieri M, Mercuri E, Servais L, Muntoni F (2020) Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy. Neurology 10:15–20. 10.1212/wnl.0000000000009233 DOI: 10.1212/wnl.0000000000009233
Gao QQ, McNally EM (2015) The dystrophin complex: structure, function, and implications for therapy. Compr Physiol 5:1223–1239. 10.1002/cphy.c140048 DOI: 10.1002/cphy.c140048
Guiraud S, Davies KE (2019) Regenerative biomarkers for Duchenne muscular dystrophy. Neural Regener Res 14:1317. 10.4103/1673-5374.253534 DOI: 10.4103/1673-5374.253534
Guiraud S, Edwards B, Squire SE, Moir L, Berg A, Babbs A, Ramadan N, Wood MJ, Davies KE (2019) Embryonic myosin is a regeneration marker to monitor utrophin-based therapies for DMD. Hum Mol Genet 28:307–319. 10.1093/hmg/ddy353 DOI: 10.1093/hmg/ddy353
Gumerson JD, Michele DE (2011) The dystrophin-glycoprotein complex in the prevention of muscle damage. J Biomed Biotechnol 2011:210797. 10.1155/2011/210797 DOI: 10.1155/2011/210797
Hoffman EP, Brown RH, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919–928. 10.1016/0092-8674(87)90579-4 DOI: 10.1016/0092-8674(87)90579-4
Janghra N, Morgan JE, Sewry CA, Wilson FX, Davies KE, Muntoni F, Tinsley J (2016) Correlation of utrophin levels with the dystrophin protein complex and muscle fibre regeneration in duchenne and becker muscular dystrophy muscle biopsies. PLoS ONE 11:e0150818. 10.1371/journal.pone.0150818 DOI: 10.1371/journal.pone.0150818
Koenig M, Beggs AH, Moyer M, Scherpf S, Heindrich K, Bettecken T, Meng G, Müller CR, Lindlöf M, Kaariainen H, de la Chapelle A, Kiuru A, Savontaus M-L, Gilgenkrantz H, Récan D, Chelly J, Kaplan J-C, Covone AE, Archidiacono N, Romeo G, Liechti-Gallati S, Schneider V, Braga S, Moser H, Darras BT, Murphy P, Francke U, Chen JD, Morgan G, Denton M, Greenberg CR, Wrogemann K, Blonden LAJ, van Paassen HMB, van Ommen GJB, Kunkel LM (1989) The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 45:498–506
Li D, Yue Y, Duan D (2008) Preservation of muscle force in Mdx3cv mice correlates with low-level expression of a near full-length dystrophin protein. Am J Pathol 172:1332–1341. 10.2353/ajpath.2008.071042 DOI: 10.2353/ajpath.2008.071042
Malerba A, Sharp PS, Graham IR, Arechavala-Gomeza V, Foster K, Muntoni F, Wells DJ, Dickson G (2011) Chronic systemic therapy with low-dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice. Mol Ther 19:345–354. 10.1038/mt.2010.261 DOI: 10.1038/mt.2010.261
Mendell JR, Rodino-Klapac LR, Sahenk Z, Roush K, Bird L, Lowes LP, Alfano L, Gomez AM, Lewis S, Kota J, Malik V, Shontz K, Walker CM, Flanigan KM, Corridore M, Kean JR, Allen HD, Shilling C, Melia KR, Sazani P, Saoud JB, Kaye EM, Eteplirsen Study Group (2013) Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann Neurol 74:637–647. 10.1002/ana.23982 DOI: 10.1002/ana.23982
Mendell JR, Shilling C, Leslie ND, Flanigan KM, Al-Dahhak R, Gastier-Foster J, Kneile K, Dunn DM, Duval B, Aoyagi A, Hamil C, Mahmoud M, Roush K, Bird L, Rankin C, Lilly H, Street N, Chandrasekar R, Weiss RB (2012) Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann Neurol 71:304–313. 10.1002/ana.23528 DOI: 10.1002/ana.23528
Meng J, Counsell JR, Reza M, Laval SH, Danos O, Thrasher A, Lochmüller H, Muntoni F, Morgan JE (2016) Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne muscular dystrophy. Sci Rep 6:19750. 10.1038/srep19750 DOI: 10.1038/srep19750
Meng J, Muntoni F, Morgan J (2018) CD133 + cells derived from skeletal muscles of Duchenne muscular dystrophy patients have a compromised myogenic and muscle regenerative capability. Stem Cell Res 30:43–52. 10.1016/j.scr.2018.05.004 DOI: 10.1016/j.scr.2018.05.004
Neri M, Torelli S, Brown S, Ugo I, Sabatelli P, Merlini L, Spitali P, Rimessi P, Gualandi F, Sewry C, Ferlini A, Muntoni F (2007) Dystrophin levels as low as 30% are sufficient to avoid muscular dystrophy in the human. Neuromuscul Disord 17:913–918. 10.1016/j.nmd.2007.07.005 DOI: 10.1016/j.nmd.2007.07.005
Omairi S, Hau K-L, Collin-Hooper H, Montanaro F, Goyenvalle A, Garcia L, Patel K (2017) Link between MHC fiber type and restoration of dystrophin expression and key components of the DAPC by tricyclo-DNA-mediated exon skipping. Mol Ther Nucleic Acids 9:409–418. 10.1016/j.omtn.2017.10.014 DOI: 10.1016/j.omtn.2017.10.014
Potaczek DP, Garn H, Unger SD, Renz H (2016) Antisense molecules: a new class of drugs. J Allergy Clin Immunol 137:1334–1346. 10.1016/j.jaci.2015.12.1344 DOI: 10.1016/j.jaci.2015.12.1344
van Putten M, Hulsker M, Nadarajah VD, van Heiningen SH, van Huizen E, van Iterson M, Admiraal P, Messemaker T, den Dunnen JT, ’t Hoen PAC, Aartsma-Rus A (2012) The effects of low levels of dystrophin on mouse muscle function and pathology. PLoS ONE 7:e31937. 10.1371/journal.pone.0031937 DOI: 10.1371/journal.pone.0031937
van Putten M, Hulsker M, Young C, Nadarajah VD, Heemskerk H, van der Weerd L, ’t Hoen PAC, van Ommen GJB, Aartsma-Rus AM (2013) Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice. FASEB J 27:2484–2495. 10.1096/fj.12-224170 DOI: 10.1096/fj.12-224170
Ricotti V, Ridout DA, Pane M, Main M, Mayhew A, Mercuri E, Manzur AY, Muntoni F, UK NorthStar Clinical Network (2016) The NorthStar ambulatory assessment in Duchenne muscular dystrophy: considerations for the design of clinical trials. J Neurol Neurosurg Psychiatry 87:149–155. 10.1136/jnnp-2014-309405 DOI: 10.1136/jnnp-2014-309405
Sardone V, Zhou H, Muntoni F, Ferlini A, Falzarano MS (2017) Antisense oligonucleotide-based therapy for neuromuscular disease. Molecules. 10.3390/molecules22040563 DOI: 10.3390/molecules22040563
Scaglioni D, Ellis M, Catapano F, Torelli S, Chambers D, Feng L, Sewry C, Morgan J, Muntoni F, Phadke R (2020) A high–throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies. Acta Neuropathol Commun 8:53. 10.1186/s40478-020-00918-5 DOI: 10.1186/s40478-020-00918-5
Sharp PS, Bye-a-Jee H, Wells DJ (2011) Physiological characterization of muscle strength with variable levels of dystrophin restoration in mdx mice following local antisense therapy. Mol Ther 19:165–171. 10.1038/mt.2010.213 DOI: 10.1038/mt.2010.213
Shimizu-Motohashi Y, Murakami T, Kimura E, Komaki H, Watanabe N (2018) Exon skipping for Duchenne muscular dystrophy: a systematic review and meta-analysis. Orphanet J Rare Dis 13:93. 10.1186/s13023-018-0834-2 DOI: 10.1186/s13023-018-0834-2
Tinsley J, Muntoni F, Layton G, Faelan C, Patterson-Kane J, Hetherington A, Davies K (2018) DMD clinical therapies II: P.132 identification of developmental myosin positive fibres acts both as a clinical biomarker for muscle disease and an important component of the process to confirm ezutromid target engagement. Neuromuscul Disord 28:S69. 10.1016/j.nmd.2018.06.160 DOI: 10.1016/j.nmd.2018.06.160
Wang B, Li J, Xiao X (2000) Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. PNAS 97:13714–13719. 10.1073/pnas.240335297 DOI: 10.1073/pnas.240335297
Watanabe N, Nagata T, Satou Y, Masuda S, Saito T, Kitagawa H, Komaki H, Takagaki K, Takeda S (2018) NS-065/NCNP-01: an antisense oligonucleotide for potential treatment of exon 53 skipping in Duchenne muscular dystrophy. Mol Ther Nucleic Acids 13:442–449. 10.1016/j.omtn.2018.09.017 DOI: 10.1016/j.omtn.2018.09.017
van Westering TLE, Lomonosova Y, Coenen-Stass AML, Betts CA, Bhomra A, Hulsker M, Clark LE, McClorey G, Aartsma-Rus A, van Putten M, Wood MJA, Roberts TC (2020) Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology. J Cachexia Sarcopenia Muscle 11:578–593. 10.1002/jcsm.12506 DOI: 10.1002/jcsm.12506
Yiu EM, Kornberg AJ (2015) Duchenne muscular dystrophy. J Paediatr Child Health 51:759–764. 10.1111/jpc.12868 DOI: 10.1111/jpc.12868
Yoshimura M, Sakamoto M, Ikemoto M, Mochizuki Y, Yuasa K, Miyagoe-Suzuki Y, Takeda S (2004) AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype. Mol Ther 10:821–828. 10.1016/j.ymthe.2004.07.025 DOI: 10.1016/j.ymthe.2004.07.025
