Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model.
Becker muscular dystrophy
CRISPR-Cas9
Cell model
Duchenne muscular dystrophy
Dystrophin
Gene therapy
Journal
Skeletal muscle
ISSN: 2044-5040
Titre abrégé: Skelet Muscle
Pays: England
ID NLM: 101561193
Informations de publication
Date de publication:
01 Oct 2024
01 Oct 2024
Historique:
received:
07
01
2024
accepted:
13
09
2024
medline:
2
10
2024
pubmed:
2
10
2024
entrez:
1
10
2024
Statut:
epublish
Résumé
Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.
Sections du résumé
BACKGROUND
BACKGROUND
Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies.
METHODS
METHODS
We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status.
RESULTS
RESULTS
Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55.
CONCLUSIONS
CONCLUSIONS
Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.
Identifiants
pubmed: 39354597
doi: 10.1186/s13395-024-00353-3
pii: 10.1186/s13395-024-00353-3
doi:
Substances chimiques
Dystrophin
0
DMD protein, human
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
21Subventions
Organisme : Fundación Isabel Gemio
ID : 2018/0200
Organisme : Fundación Isabel Gemio
ID : 2018/0200
Organisme : Instituto de Salud Carlos III
ID : CM19/00104
Organisme : Instituto de Salud Carlos III
ID : PI20/00114
Organisme : Instituto de Salud Carlos III
ID : CP22/00028
Organisme : Instituto de Salud Carlos III
ID : PI15/00333
Organisme : Instituto de Salud Carlos III
ID : CM19/00104
Organisme : Conselleria de Cultura, Educación y Ciencia, Generalitat Valenciana
ID : APOSTD/2021/212
Organisme : Ministerio de Ciencia e Innovación
ID : FPU21/00912
Organisme : Biobizkaia Health Research Institute
ID : BC/I/DIV/19/001
Organisme : Basque Government, Spain
ID : 2016111029
Informations de copyright
© 2024. The Author(s).
Références
Davies KE, Nowak KJ. Molecular mechanisms of muscular dystrophies: old and new players. Nat Rev Mol Cell Biol. 2006;7:762–73.
pubmed: 16971897
doi: 10.1038/nrm2024
Rando TA. The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve. 2001;24:1575–94.
Emery AEH. Population frequencies of inherited neuromuscular diseases-A world survey. Neuromuscul Disord. 1991;1:19–29.
pubmed: 1822774
doi: 10.1016/0960-8966(91)90039-U
Emery AEH, Muntoni F, Quinlivan RCM. Duchenne muscular dystrophy. OUP Oxford; 2015.
Darras BT, Urion DK, Ghosh PS. Dystrophinopathies. GeneReviews
Bushby KMD, Thambyayah M, Gardner-Medwin D. Prevalence and incidence of Becker muscular dystrophy. Lancet. 1991;337:1022–4.
pubmed: 1673177
doi: 10.1016/0140-6736(91)92671-N
Ferreiro V, Giliberto F, Muñiz GMN, Francipane L, Marzese DM, Mampel A, et al. Asymptomatic Becker muscular dystrophy in a family with a multiexon deletion. Muscle Nerve. 2009;39:239–43.
pubmed: 19012301
doi: 10.1002/mus.21193
Sanchez-Arjona MB. Spanish family with myalgia and cramps syndrome. J Neurol Neurosurg Psychiatry. 2005;76:286–9.
pubmed: 15654055
pmcid: 1739523
doi: 10.1136/jnnp.2004.037325
Nakamura A. X-Linked dilated cardiomyopathy: a cardiospecific phenotype of Dystrophinopathy. Pharmaceuticals. 2015;8:303–20.
pubmed: 26066469
pmcid: 4491663
doi: 10.3390/ph8020303
North KN, Miller G, Iannaccone ST, Clemens PR, Chad DA, Bella I, et al. Cognitive dysfunction as the major presenting feature of Becker’s muscular dystrophy. Neurology. 1996;46:461–4.
pubmed: 8614513
doi: 10.1212/WNL.46.2.461
Nicolas A, Raguénès-Nicol C, Ben Yaou R, Ameziane-Le Hir S, Chéron A, Vié V, et al. Becker muscular dystrophy severity is linked to the structure of dystrophin. Hum Mol Genet. 2015;24:1267–79.
pubmed: 25348330
doi: 10.1093/hmg/ddu537
Bello L, Pegoraro E. The Usual suspects: genes for inflammation, fibrosis, regeneration, and muscle strength modify Duchenne muscular dystrophy. J Clin Med. 2019;8:649.
pubmed: 31083420
pmcid: 6571893
doi: 10.3390/jcm8050649
Verhaart IEC, Aartsma-Rus A. Therapeutic developments for Duchenne muscular dystrophy. Nature Reviews Neurology. 2019;15:373–86.
Tang A, Yokota T. Duchenne muscular dystrophy: promising early-stage clinical trials to watch. Expert Opin Investig Drugs. 2024;33:201–17.
Arechavala-Gomeza V, Anthony K, Morgan J, Muntoni F. Antisense oligonucleotide-mediated exon skipping for Duchenne muscular dystrophy: Progress and challenges. Curr Gene Ther. 2012;12:152–60.
pubmed: 22533380
doi: 10.2174/156652312800840621
Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Rivera RMC et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science (1979). 2016;351:403–7.
Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science (1979). 2016;351:400–3.
Amoasii L, Hildyard JCW, Li H, Sanchez-Ortiz E, Mireault A, Caballero D et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science (1979). 2018;362:86–91.
Tabebordbar M, Cheng J, Wagers AJ. Therapeutic Gene Editing in Muscles and Muscle Stem Cells. Research and Perspectives in Neurosciences. 2017;103–23.
Duan D. Systemic AAV micro-dystrophin gene therapy for Duchenne muscular dystrophy. Mol Ther. 2018;26:2337–56.
pubmed: 30093306
pmcid: 6171037
doi: 10.1016/j.ymthe.2018.07.011
Hammond SM, Aartsma-Rus A, Alves S, Borgos SE, Buijsen RAM, Collin RWJ et al. Delivery of oligonucleotide-based therapeutics: challenges and opportunities. EMBO Mol Med. 2021;13:e13243.
Anthony K, Cirak S, Torelli S, Tasca G, Feng L, Arechavala-Gomeza V, et al. Dystrophin quantification and clinical correlations in Becker muscular dystrophy: implications for clinical trials. Brain. 2011;134:3544–56.
pmcid: 3235564
doi: 10.1093/brain/awr291
Anthony K, Arechavala-Gomeza V, Ricotti V, Torelli S, Feng L, Janghra N et al. Biochemical Characterization of Patients With In-Frame or Out-of-Frame DMD Deletions Pertinent to Exon 44 or 45 Skipping. JAMA Neurol. 2014;71:32.
Nakamura A, Shiba N, Miyazaki D, Nishizawa H, Inaba Y, Fueki N, et al. Comparison of the phenotypes of patients harboring in-frame deletions starting at exon 45 in the Duchenne muscular dystrophy gene indicates potential for the development of exon skipping therapy. J Hum Genet. 2017;62:459–63.
pubmed: 27974813
doi: 10.1038/jhg.2016.152
Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Gappmaier E, Howard MT, et al. Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009;30:1657–66.
pubmed: 19937601
pmcid: 3404892
doi: 10.1002/humu.21114
Béroud C, Tuffery-Giraud S, Matsuo M, Hamroun D, Humbertclaude V, Monnier N, et al. Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy. Hum Mutat. 2007;28:196–202.
pubmed: 17041910
doi: 10.1002/humu.20428
Poyatos-García J, Martí P, Liquori A, Muelas N, Pitarch I, Martinez‐Dolz L et al. Dystrophinopathy Phenotypes and Modifying Factors in DMD Exon 45–55 Deletion. Ann Neurol. 2022;92:793–806.
Aoki Y, Yokota T, Nagata T, Nakamura A, Tanihata J, Saito T et al. Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery. Proceedings of the National Academy of Sciences. 2012;109:13763–8.
Echigoya Y, Aoki Y, Miskew B, Panesar D, Touznik A, Nagata T, et al. Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45–55 with a cocktail of vivo-morpholinos in Mdx52 mice. Mol Ther Nucleic Acids. 2015;4:e225.
pubmed: 25647512
pmcid: 4345310
doi: 10.1038/mtna.2014.76
Lee J, Echigoya Y, Duddy W, Saito T, Aoki Y, Takeda ichi et al. Antisense PMO cocktails effectively skip dystrophin exons 45–55 in myotubes transdifferentiated from DMD patient fibroblasts. 2018;13(5):e0197084.
Echigoya Y, Lim KRQ, Melo D, Bao B, Trieu N, Mizobe Y, et al. Exons 45–55 skipping using mutation-tailored cocktails of antisense morpholinos in the DMD Gene. Mol Ther. 2019;27:2005–17.
pubmed: 31416775
pmcid: 6838919
doi: 10.1016/j.ymthe.2019.07.012
Ousterout DG, Kabadi AM, Thakore PI, Majoros WH, Reddy TE, Gersbach CA. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun. 2015;6:6244.
pubmed: 25692716
doi: 10.1038/ncomms7244
Young CS, Hicks MR, Ermolova NV, Nakano H, Jan M, Younesi S, et al. A single CRISPR-Cas9 deletion strategy that targets the majority of DMD patients restores dystrophin function in hiPSC-Derived muscle cells. Cell Stem Cell. 2016;18:533–40.
pubmed: 26877224
pmcid: 4826286
doi: 10.1016/j.stem.2016.01.021
Young CS, Mokhonova E, Quinonez M, Pyle AD, Spencer MJ. Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne muscular dystrophy and application of a CRISPR/Cas9 gene editing therapy. J Neuromuscul Dis. 2017;4:139–45.
pubmed: 28505980
pmcid: 5565771
doi: 10.3233/JND-170218
Thorley M, Duguez S, Mazza EMC, Valsoni S, Bigot A, Mamchaoui K, et al. Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines. Skelet Muscle. 2016;6:1–12.
doi: 10.1186/s13395-016-0115-5
Mamchaoui K, Trollet C, Bigot A, Negroni E, Chaouch S, Wolff A, et al. Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders. Skelet Muscle. 2011;1:34.
pubmed: 22040608
pmcid: 3235972
doi: 10.1186/2044-5040-1-34
Soblechero-Martín P, Albiasu-Arteta E, Anton-Martinez A, de la Puente-Ovejero L, Garcia-Jimenez I, González-Iglesias G et al. Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening. Sci Rep. 2021;11:18188.
Sabater-Arcis M, Bargiela A, Moreno N, Poyatos-Garcia J, Vilchez JJ, Artero R. Musashi-2 contributes to myotonic dystrophy muscle dysfunction by promoting excessive autophagy through miR-7 biogenesis repression. Mol Ther Nucleic Acids. 2021;25:652–67.
pubmed: 34589284
pmcid: 8463325
doi: 10.1016/j.omtn.2021.08.010
Poyatos-García J, Blázquez-Bernal Á, Selva-Giménez M, Bargiela A, Espinosa-Espinosa J, Vázquez-Manrique RP et al. CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2. Mol Ther Nucleic Acids. 2023;31:324–38.
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protocols. 2013;8:11.
Fuster-García C, García-García G, González-Romero E, Jaijo T, Sequedo MD, Ayuso C, et al. USH2A gene editing using the CRISPR System. Mol Ther Nucleic Acids. 2017;8:529–41.
pubmed: 28918053
pmcid: 5573797
doi: 10.1016/j.omtn.2017.08.003
Bae S, Park J, Kim J-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014;30:1473–5.
pubmed: 24463181
pmcid: 4016707
doi: 10.1093/bioinformatics/btu048
López-Martínez A, Soblechero-Martín P, Arechavala-Gomeza V. Evaluation of Exon Skipping and Dystrophin Restoration in In Vitro Models of Duchenne Muscular Dystrophy. Arechavala-Gomeza V, Garanto A, editors. Methods Mol Biol. 2022;2434:217–33.
Ruiz-Del-Yerro E, Garcia-Jimenez I, Mamchaoui K, Arechavala-Gomeza V. Myoblots: dystrophin quantification by in-cell western assay for a streamlined development of Duchenne muscular dystrophy (DMD) treatments. Neuropathol Appl Neurobiol. 2018;44:463–73.
Anthony K, Arechavala-Gomeza V, Taylor LE, Vulin A, Kaminoh Y, Torelli S, et al. Dystrophin quantification: Biological and translational research implications. Neurology. 2014;83:2062–9.
pubmed: 25355828
pmcid: 4248450
doi: 10.1212/WNL.0000000000001025
Oliveros JC, Franch M, Tabas-Madrid D, San-León D, Montoliu L, Cubas P, et al. Breaking-Cas—interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes. Nucleic Acids Res. 2016;44:W267–71.
pubmed: 27166368
pmcid: 4987939
doi: 10.1093/nar/gkw407
Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol. 2019;37:224–6.
pubmed: 30809026
pmcid: 6533916
doi: 10.1038/s41587-019-0032-3
Chal J, Pourquié O. Making muscle: skeletal myogenesis in vivo and in vitro. Development. 2017;144:2104–22.
pubmed: 28634270
doi: 10.1242/dev.151035
Schmidt M, Schüler SC, Hüttner SS, von Eyss B, von Maltzahn J. Adult stem cells at work: regenerating skeletal muscle. Cellular and Molecular Life Sciences. 2019;76:2559–70.
Zhang H, Shang R, Bi P. Feedback regulation of Notch signaling and myogenesis connected by MyoD–Dll1 axis. PLoS Genet. 2021;17:e1009729.
Doorenweerd N, Straathof CS, Dumas EM, Spitali P, Ginjaar IB, Wokke BH, et al. Reduced cerebral gray matter and altered white matter in boys with Duchenne muscular dystrophy. Ann Neurol. 2014;76:403–11.
pubmed: 25043804
doi: 10.1002/ana.24222
Felisari G, Boneschi FM, Bardoni A, Sironi M, Comi GP, Robotti M, et al. Loss of Dp140 dystrophin isoform and intellectual impairment in Duchenne dystrophy. Neurology. 2000;55:559–64.
pubmed: 10953192
doi: 10.1212/WNL.55.4.559
Gargaun E, Falcone S, Solé G, Durigneux J, Urtizberea A, Cuisset JM, et al. The lncRNA 44s2 study applicability to the design of 45–55 exon skipping therapeutic strategy for DMD. Biomedicines. 2021;9:219.
pubmed: 33672764
pmcid: 7924625
doi: 10.3390/biomedicines9020219
Lidov HGW, Selig S, Kunkel LM. Dp140: a novel 140 kDa CNS transcript from the dystrophin locus. Hum Mol Genet. 1995;4:329–35.
pubmed: 7795584
doi: 10.1093/hmg/4.3.329
Bovolenta M, Erriquez D, Valli E, Brioschi S, Scotton C, Neri M, et al. The DMD Locus Harbours multiple long non-coding RNAs which orchestrate and Control Transcription of Muscle Dystrophin mRNA isoforms. PLoS ONE. 2012;7:e45328.
pubmed: 23028937
pmcid: 3448672
doi: 10.1371/journal.pone.0045328
Wojtal D, Kemaladewi DU, Malam Z, Abdullah S, Wong TWY, Hyatt E, et al. Spell checking Nature: versatility of CRISPR/Cas9 for developing treatments for inherited disorders. Am J Hum Genet. 2016;98:90–101.
pubmed: 26686765
doi: 10.1016/j.ajhg.2015.11.012
Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing. Theranostics. 2021;11:614–48.
pubmed: 33391496
pmcid: 7738854
doi: 10.7150/thno.47007
Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014;24:1012–9.
pubmed: 24696461
pmcid: 4032847
doi: 10.1101/gr.171322.113
Li L, Hu S, Chen X. Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities. Biomaterials. 2018;171:207–18.
pubmed: 29704747
pmcid: 5944364
doi: 10.1016/j.biomaterials.2018.04.031
Taha EA, Lee J, Hotta A. Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges. J Controlled Release. 2022;342:345–61.
doi: 10.1016/j.jconrel.2022.01.013
Mcinerney P, Adams P, Hadi MZ. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. 2014;204:287430.
Tuffery-Giraud S, Miro J, Koenig M, Claustres M. Normal and altered pre-mRNA processing in the DMD gene. Hum Genet. 2017;136:1155–72.
pubmed: 28597072
doi: 10.1007/s00439-017-1820-9
Arechavala-Gomeza V, Kinali M, Feng L, Brown SC, Sewry C, Morgan JE, et al. Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression. Neuropathol Appl Neurobiol. 2009;36:265–74.
pubmed: 20002311
doi: 10.1111/j.1365-2990.2009.01056.x
van den Bergen JC, Wokke BH, Janson AA, van Duinen SG, Hulsker MA, Ginjaar HB et al. Dystrophin levels and clinical severity in Becker muscular dystrophy patients. J Neurol Neurosurg Psychiatry. 2014;85:747–53.