Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system.

Annalisa Lattanzi, Stephanie Duguez, Arianna Moiani, Araksya Izmiryan, Elena Barbon, Samia Martin, Kamel Mamchaoui, Vincent Mouly, Francesco Bernardi, Fulvio Mavilio, Matteo Bovolenta

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Exonic duplications account for 10-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one gRNA directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.
LanguageEnglish
Pages11-19
Number of pages8
JournalMolecular Therapy - Nucleic Acids
Volume7
Early online date10 Feb 2017
DOIs
Publication statusPublished - 16 Jun 2017

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Duchenne Muscular Dystrophy
Myoblasts
Exons
Guide RNA
Dystrophin
Skeletal Muscle Fibers
Gene Deletion
Genome
Mutation
Proteins
Therapeutics

Keywords

  • Correction of the DMD exon 2
  • CRISPR/Cas9

Cite this

Lattanzi, Annalisa ; Duguez, Stephanie ; Moiani, Arianna ; Izmiryan, Araksya ; Barbon, Elena ; Martin, Samia ; Mamchaoui, Kamel ; Mouly, Vincent ; Bernardi, Francesco ; Mavilio, Fulvio ; Bovolenta, Matteo. / Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system. In: Molecular Therapy - Nucleic Acids. 2017 ; Vol. 7. pp. 11-19.
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title = "Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system.",
abstract = "Exonic duplications account for 10-15{\%} of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one gRNA directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.",
keywords = "Correction of the DMD exon 2, CRISPR/Cas9",
author = "Annalisa Lattanzi and Stephanie Duguez and Arianna Moiani and Araksya Izmiryan and Elena Barbon and Samia Martin and Kamel Mamchaoui and Vincent Mouly and Francesco Bernardi and Fulvio Mavilio and Matteo Bovolenta",
note = "Reference text: 1. Mendell, JR, Shilling, C, Leslie, ND, Flanigan, KM, al-Dahhak, R, Gastier-Foster, J, et al. (2012). Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann Neurol 71:304-313. 2. Kinali, M, Arechavala-Gomeza, V, Cirak, S, Glover, A, Guglieri, M, Feng, L et al. (2011). Muscle histology vs MRI in Duchenne muscular dystrophy. Neurology 76: 346–353. 3. Aartsma-Rus, A, Van Deutekom, JC, Fokkema, IF, Van Ommen, GJ, Den Dunnen, JT (2006). Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 34:135–144. 4. Wilton, SD, Veedu, RN and Fletcher, S (2015). The emperor's new dystrophin: finding sense in the noise Trends Mol Med 21:417-26. 5. Lu, QL, Cirak, S and Partridge, T (2014). What Can We Learn From Clinical Trials of Exon Skipping for DMD? Mol Ther Nucleic Acids 11:3:e152 6. Aoki, Y, Nakamura, A, Yokota, T, Saito, T, Okazawa, H, Nagata T, et al. (2010). In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Mol Ther 18:1995-2005. 7. Arechavala-Gomeza, V, Graham, IR, Popplewell, LJ, Adams, AM, Aartsma-Rus, A, Kinali, M et al. (2007). Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin premRNA splicing in human muscle. Hum Gene Ther 18:798-810. 8. Aartsma-Rus, A, Janson, AA, van Ommen, GJ, van Deutekom, JC (2007). Antisenseinduced exon skipping for duplications in Duchenne muscular dystrophy. BMC Med Genet 8:43. 9. Greer, KL, Lochm{\"u}ller, H, Flanigan, K, Fletcher, S, Wilton, SD (2014). Targeted exon skipping to correct exon duplications in the dystrophin gene. Mol Ther Nucleic Acids 18:3:e155. 10. van Vliet, L, de Winter, CL, van Deutekom, JC, van Ommen, GJ, Aartsma-Rus, A (2008). Assessment of the feasibility of exon 45-55 multiexon skipping for Duchenne muscular dystrophy. BMC Med Genet 9:105. 11. Taylor, PJ, Maroulis, S, Mullan, GL, Pedersen, RL, Baumli, A, Elakis, G et al. (2007). Measurement of the clinical utility of a combined mutation detection protocol in carriers of Duchenne and Becker muscular dystrophy. J Med Genet 44:368–372. 12. Wein, N, Vulin, A, Falzarano, MS, Szigyarto, CA, Maiti, B, Findlay, A et al. (2014). Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nat Med 20:992-1000. 13. Ryan, NJ. (2014). Ataluren: first global approval. Drugs 74:1709-1714. 14. Traynor, K. (2016) Eteplirsen approved for Duchenne muscular dystrophy. Am J Health Syst Pharm 73:1719. 15. Gaj, T, Gersbach, CA, Barbas, CF 3rd (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397-405. 16. Joung, JK, Sander, JD (2013). TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14:49-55. 17. Silva, G, Poirot, L, Galetto, R, Smith, J, Montoya, G, Duchateau, P et al. (2011). Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther 11:11-27. 18. Mali, P, Yang, L, Esvelt, KM, Aach, J, Guell, M, DiCarlo, JE et al. (2013). RNA-guided human genome engineering via Cas9. Science 339:823-826. 19. Nakamura, K, Fujii, W, Tsuboi, M, Tanihata, J, Teramoto, N, Takeuchi, S et al. (2014). Generation of muscular dystrophy model rats with a CRISPR/Cas system. Sci Rep 4:5635. 20. Chen, Y, Zheng, Y, Kang, Y, Yang, W, Niu, Y, Guo, X et al. (2015). Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Hum Mol Genet 24:3764- 3774. 21. Larcher, T, Lafoux, A, Tesson, L, Remy, S, Thepenier, V, Fran{\cc}ois, V et al. (2014). Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy. PLoS One 9:e110371. 22. Ousterout, DG, Perez-Pinera, P, Thakore, PI, Kabadi, AM, Brown, MT, Qin, X et al. (2013). Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Mol Ther 21:1718-1726. 23. Ousterout, DG, Kabadi, AM, Thakore, PI, Perez-Pinera, P, Brown, MT, Majoros, WH et al. (2015). Correction of dystrophin expression in cells from duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases. Mol Ther 23:523- 532. 24. Li, HL, Fujimoto, N, Sasakawa, N, Shirai, S, Ohkame, T, Sakuma, T et al. (2015). Precise correction of the dystrophin gene in Duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Reports 4:143-154. 25. Long, C, McAnally, JR, Shelton, JM, Mireault, AA, Bassel-Duby, R, Olson, EN. (2014). Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345:1184-1188. 26. Goyenvalle, A, Griffith, G, Babbs, A, El Andaloussi, S, Ezzat, K, Avril, A et al. (2015). Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo- DNA oligomers. Nat Med 21:270–275. 27. Vila, MC, Klimek, MB, Novak, JS, Rayavarapu, S, Uaesoontrachoon, K, Boehler, JF et al. (2015). Elusive sources of variability of dystrophin rescue by exon skipping. Skeletal Muscle 5:44. 28. Wojtal, D, Kemaladewi, DU, Malam, Z, Abdullah, S, Wong, TW, Hyatt, E et al. (2016). Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders. Am J Hum Genet 98:90-101. 29. Bovolenta, M, Neri, M, Fini, S, Fabris, M, Trabanelli, C, Venturoli, A et al. (2008). A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies. BMC Genomics 9:572. 30. Bovolenta, M, Scotton, C, Falzarano, MS, Gualandi, F, Ferlini, A (2012). Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array. Hum Mutat 33:572-581. 31. del Gaudio, D, Yang, Y, Boggs, BA, Schmitt, ES, Lee, JA, Sahoo, T et al. (2008). Molecular diagnosis of Duchenne/Becker muscular dystrophy: enhanced detection of dystrophin gene rearrangements by oligonucleotide array-comparative genomic hybridization. Hum Mutat 29:1100-1107. 32. White, SJ, Aartsma-Rus, A, Flanigan KM, Weiss, RB, Kneppers, AL, Lalic, T et al. (2006). Duplications in the DMD gene. Hum Mutat 27:938-945. 33. Mamchaoui, K, Trollet, C, Bigot, A, Negroni, E, Chaouch, S, Wolff, A et al. (2011). Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders. Skelet Muscle 1:34. 34. Kraft, K, Geuer, S, Will, AJ, Chan, WL, Paliou, C, Borschiwer, M et al. (2015). Deletions, Inversions, Duplications: Engineering of Structural Variants using CRISPR/Cas in Mice. Cell Reports 10: 833–839. 35. Sanjana, NE, Shalem, O, Zhang, F (2014). Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11:783–784. 36. Ousterout, DG, Kabadi, AM, Thakore, PI, Majoros, WH, Reddy, TE et al. (2015). Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun 6: 6244. 37. Tabebordbar, M, Zhu, K, Cheng, JK, Chew, WL, Widrick, JJ, Yan, WX et al. (2016). In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351:407-411. 38. Nelson, CE, Hakim, CH, Ousterout, DG, Thakore, PI, Moreb, EA, Castellanos Rivera, RM et al. (2016). In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351:403-407. 39. Long, C, Amoasii, L, Mireault, AA, McAnally, JR, Li, H, Sanchez-Ortiz, E et al. (2016). Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351:400-403. 40. Xu, L, Park, KH, Zhao, L, Xu, J, El Refaey, M, Gao, Y et al. (2016) CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice. Mol Ther. 24:564-569. 41. Lu, QL, Mann, CJ, Lou, F, Bou-Gharios, G, Morris, GE, Xue, SA et al. (2003). Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 9:1009–1014 42. Doench, JG, Fusi, N, Sullender, M, Hegde, M, Vaimberg, EW, Donovan, KF et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPRCas9. Nat Biotechnol 34: 184-91. 43. Kleinstiver, BP, Pattanayak, V, Prew, MS, Tsai, SQ, Nguyen, NT, Zheng, Z et al. (2016). High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490-495. 44. Ran, FA, Cong, L, Yan, WX, Scott, DA, Gootenberg, JS, Kriz, AJ et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186-91. 45. Zetsche, B, Gootenberg, JS, Abudayyeh, OO, Slaymaker, IM, Makarova, KS, Essletzbichler, P et al. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771. 46. Le Hir, M, Goyenvalle, A, Peccate, C, Pr{\'e}cigout, G, Davies, KE, Voit, T et al. (2013). AAV Genome Loss From Dystrophic Mouse Muscles During AAV-U7 snRNA-mediated Exonskipping Therapy. Mol Ther 21:1551–1558. 47. Fu, Y, Reyon, D, Joung, JK (2014). Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs. Methods Enzymol 546:21-45. 48. Wang, T, Wei, JJ, Sabatini, DM, Lander, ES (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80-84. 49. Cantore, A, Ranzani, M, Bartholomae, CC, Volpin, M, Valle, PD, Sanvito, F et al. (2015). Liver-directed lentiviral gene therapy in a dog model of hemophilia B. Sci Transl Med 4:277ra28. 50. Zanta-Boussif, MA, Charrier, S, Brice-Ouzet, A, Martin, S, Opolon, P, Thrasher, AJ et al. (2009). Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS. Gene Ther 16:605-619.",
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Lattanzi, A, Duguez, S, Moiani, A, Izmiryan, A, Barbon, E, Martin, S, Mamchaoui, K, Mouly, V, Bernardi, F, Mavilio, F & Bovolenta, M 2017, 'Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system.', Molecular Therapy - Nucleic Acids, vol. 7, pp. 11-19. https://doi.org/10.1016/j.omtn.2017.02.004

Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system. / Lattanzi, Annalisa; Duguez, Stephanie; Moiani, Arianna; Izmiryan, Araksya; Barbon, Elena; Martin, Samia; Mamchaoui, Kamel; Mouly, Vincent; Bernardi, Francesco; Mavilio, Fulvio; Bovolenta, Matteo.

In: Molecular Therapy - Nucleic Acids, Vol. 7, 16.06.2017, p. 11-19.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system.

AU - Lattanzi, Annalisa

AU - Duguez, Stephanie

AU - Moiani, Arianna

AU - Izmiryan, Araksya

AU - Barbon, Elena

AU - Martin, Samia

AU - Mamchaoui, Kamel

AU - Mouly, Vincent

AU - Bernardi, Francesco

AU - Mavilio, Fulvio

AU - Bovolenta, Matteo

N1 - Reference text: 1. Mendell, JR, Shilling, C, Leslie, ND, Flanigan, KM, al-Dahhak, R, Gastier-Foster, J, et al. (2012). Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann Neurol 71:304-313. 2. Kinali, M, Arechavala-Gomeza, V, Cirak, S, Glover, A, Guglieri, M, Feng, L et al. (2011). Muscle histology vs MRI in Duchenne muscular dystrophy. Neurology 76: 346–353. 3. Aartsma-Rus, A, Van Deutekom, JC, Fokkema, IF, Van Ommen, GJ, Den Dunnen, JT (2006). Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 34:135–144. 4. Wilton, SD, Veedu, RN and Fletcher, S (2015). The emperor's new dystrophin: finding sense in the noise Trends Mol Med 21:417-26. 5. Lu, QL, Cirak, S and Partridge, T (2014). What Can We Learn From Clinical Trials of Exon Skipping for DMD? Mol Ther Nucleic Acids 11:3:e152 6. Aoki, Y, Nakamura, A, Yokota, T, Saito, T, Okazawa, H, Nagata T, et al. (2010). In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Mol Ther 18:1995-2005. 7. Arechavala-Gomeza, V, Graham, IR, Popplewell, LJ, Adams, AM, Aartsma-Rus, A, Kinali, M et al. (2007). Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin premRNA splicing in human muscle. Hum Gene Ther 18:798-810. 8. Aartsma-Rus, A, Janson, AA, van Ommen, GJ, van Deutekom, JC (2007). Antisenseinduced exon skipping for duplications in Duchenne muscular dystrophy. BMC Med Genet 8:43. 9. Greer, KL, Lochmüller, H, Flanigan, K, Fletcher, S, Wilton, SD (2014). Targeted exon skipping to correct exon duplications in the dystrophin gene. Mol Ther Nucleic Acids 18:3:e155. 10. van Vliet, L, de Winter, CL, van Deutekom, JC, van Ommen, GJ, Aartsma-Rus, A (2008). Assessment of the feasibility of exon 45-55 multiexon skipping for Duchenne muscular dystrophy. BMC Med Genet 9:105. 11. Taylor, PJ, Maroulis, S, Mullan, GL, Pedersen, RL, Baumli, A, Elakis, G et al. (2007). Measurement of the clinical utility of a combined mutation detection protocol in carriers of Duchenne and Becker muscular dystrophy. J Med Genet 44:368–372. 12. Wein, N, Vulin, A, Falzarano, MS, Szigyarto, CA, Maiti, B, Findlay, A et al. (2014). Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nat Med 20:992-1000. 13. Ryan, NJ. (2014). Ataluren: first global approval. Drugs 74:1709-1714. 14. Traynor, K. (2016) Eteplirsen approved for Duchenne muscular dystrophy. Am J Health Syst Pharm 73:1719. 15. Gaj, T, Gersbach, CA, Barbas, CF 3rd (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397-405. 16. Joung, JK, Sander, JD (2013). TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14:49-55. 17. Silva, G, Poirot, L, Galetto, R, Smith, J, Montoya, G, Duchateau, P et al. (2011). Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther 11:11-27. 18. Mali, P, Yang, L, Esvelt, KM, Aach, J, Guell, M, DiCarlo, JE et al. (2013). RNA-guided human genome engineering via Cas9. Science 339:823-826. 19. Nakamura, K, Fujii, W, Tsuboi, M, Tanihata, J, Teramoto, N, Takeuchi, S et al. (2014). Generation of muscular dystrophy model rats with a CRISPR/Cas system. Sci Rep 4:5635. 20. Chen, Y, Zheng, Y, Kang, Y, Yang, W, Niu, Y, Guo, X et al. (2015). Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Hum Mol Genet 24:3764- 3774. 21. Larcher, T, Lafoux, A, Tesson, L, Remy, S, Thepenier, V, François, V et al. (2014). Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy. PLoS One 9:e110371. 22. Ousterout, DG, Perez-Pinera, P, Thakore, PI, Kabadi, AM, Brown, MT, Qin, X et al. (2013). Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Mol Ther 21:1718-1726. 23. Ousterout, DG, Kabadi, AM, Thakore, PI, Perez-Pinera, P, Brown, MT, Majoros, WH et al. (2015). Correction of dystrophin expression in cells from duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases. Mol Ther 23:523- 532. 24. Li, HL, Fujimoto, N, Sasakawa, N, Shirai, S, Ohkame, T, Sakuma, T et al. (2015). Precise correction of the dystrophin gene in Duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Reports 4:143-154. 25. Long, C, McAnally, JR, Shelton, JM, Mireault, AA, Bassel-Duby, R, Olson, EN. (2014). Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345:1184-1188. 26. Goyenvalle, A, Griffith, G, Babbs, A, El Andaloussi, S, Ezzat, K, Avril, A et al. (2015). Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo- DNA oligomers. Nat Med 21:270–275. 27. Vila, MC, Klimek, MB, Novak, JS, Rayavarapu, S, Uaesoontrachoon, K, Boehler, JF et al. (2015). Elusive sources of variability of dystrophin rescue by exon skipping. Skeletal Muscle 5:44. 28. Wojtal, D, Kemaladewi, DU, Malam, Z, Abdullah, S, Wong, TW, Hyatt, E et al. (2016). Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders. Am J Hum Genet 98:90-101. 29. Bovolenta, M, Neri, M, Fini, S, Fabris, M, Trabanelli, C, Venturoli, A et al. (2008). A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies. BMC Genomics 9:572. 30. Bovolenta, M, Scotton, C, Falzarano, MS, Gualandi, F, Ferlini, A (2012). Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array. Hum Mutat 33:572-581. 31. del Gaudio, D, Yang, Y, Boggs, BA, Schmitt, ES, Lee, JA, Sahoo, T et al. (2008). Molecular diagnosis of Duchenne/Becker muscular dystrophy: enhanced detection of dystrophin gene rearrangements by oligonucleotide array-comparative genomic hybridization. Hum Mutat 29:1100-1107. 32. White, SJ, Aartsma-Rus, A, Flanigan KM, Weiss, RB, Kneppers, AL, Lalic, T et al. (2006). Duplications in the DMD gene. Hum Mutat 27:938-945. 33. Mamchaoui, K, Trollet, C, Bigot, A, Negroni, E, Chaouch, S, Wolff, A et al. (2011). Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders. Skelet Muscle 1:34. 34. Kraft, K, Geuer, S, Will, AJ, Chan, WL, Paliou, C, Borschiwer, M et al. (2015). Deletions, Inversions, Duplications: Engineering of Structural Variants using CRISPR/Cas in Mice. Cell Reports 10: 833–839. 35. Sanjana, NE, Shalem, O, Zhang, F (2014). Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11:783–784. 36. Ousterout, DG, Kabadi, AM, Thakore, PI, Majoros, WH, Reddy, TE et al. (2015). Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun 6: 6244. 37. Tabebordbar, M, Zhu, K, Cheng, JK, Chew, WL, Widrick, JJ, Yan, WX et al. (2016). In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351:407-411. 38. Nelson, CE, Hakim, CH, Ousterout, DG, Thakore, PI, Moreb, EA, Castellanos Rivera, RM et al. (2016). In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351:403-407. 39. Long, C, Amoasii, L, Mireault, AA, McAnally, JR, Li, H, Sanchez-Ortiz, E et al. (2016). Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351:400-403. 40. Xu, L, Park, KH, Zhao, L, Xu, J, El Refaey, M, Gao, Y et al. (2016) CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice. Mol Ther. 24:564-569. 41. Lu, QL, Mann, CJ, Lou, F, Bou-Gharios, G, Morris, GE, Xue, SA et al. (2003). Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 9:1009–1014 42. Doench, JG, Fusi, N, Sullender, M, Hegde, M, Vaimberg, EW, Donovan, KF et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPRCas9. Nat Biotechnol 34: 184-91. 43. Kleinstiver, BP, Pattanayak, V, Prew, MS, Tsai, SQ, Nguyen, NT, Zheng, Z et al. (2016). High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490-495. 44. Ran, FA, Cong, L, Yan, WX, Scott, DA, Gootenberg, JS, Kriz, AJ et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186-91. 45. Zetsche, B, Gootenberg, JS, Abudayyeh, OO, Slaymaker, IM, Makarova, KS, Essletzbichler, P et al. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771. 46. Le Hir, M, Goyenvalle, A, Peccate, C, Précigout, G, Davies, KE, Voit, T et al. (2013). AAV Genome Loss From Dystrophic Mouse Muscles During AAV-U7 snRNA-mediated Exonskipping Therapy. Mol Ther 21:1551–1558. 47. Fu, Y, Reyon, D, Joung, JK (2014). Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs. Methods Enzymol 546:21-45. 48. Wang, T, Wei, JJ, Sabatini, DM, Lander, ES (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80-84. 49. Cantore, A, Ranzani, M, Bartholomae, CC, Volpin, M, Valle, PD, Sanvito, F et al. (2015). Liver-directed lentiviral gene therapy in a dog model of hemophilia B. Sci Transl Med 4:277ra28. 50. Zanta-Boussif, MA, Charrier, S, Brice-Ouzet, A, Martin, S, Opolon, P, Thrasher, AJ et al. (2009). Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS. Gene Ther 16:605-619.

PY - 2017/6/16

Y1 - 2017/6/16

N2 - Exonic duplications account for 10-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one gRNA directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.

AB - Exonic duplications account for 10-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one gRNA directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications.

KW - Correction of the DMD exon 2

KW - CRISPR/Cas9

U2 - 10.1016/j.omtn.2017.02.004

DO - 10.1016/j.omtn.2017.02.004

M3 - Article

VL - 7

SP - 11

EP - 19

JO - Molecular Therapy - Nucleic Acids

T2 - Molecular Therapy - Nucleic Acids

JF - Molecular Therapy - Nucleic Acids

SN - 2162-2531

ER -