Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy


Autoři: Sara Mata López aff001;  Cynthia Balog-Alvarez aff001;  Stanislav Vitha aff002;  Amanda K. Bettis aff001;  Emily H. Canessa aff003;  Joe N. Kornegay aff001;  Peter P. Nghiem aff001
Působiště autorů: Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, United States of America aff001;  Microscopy and Imaging Center, Texas A&M University, College Station, TX, United States of America aff002;  Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Johnson City, NY, United States of America aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0228072

Souhrn

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.

Klíčová slova:

Body limbs – Dogs – Muscle proteins – Mutation – Myoblasts – Polymerase chain reaction – TALENs – Dystrophin


Zdroje

1. Hoffman EP, Brown RH Jr., Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51(6):919–28. Epub 1987/12/24. doi: 10.1016/0092-8674(87)90579-4 3319190.

2. Klingler W, Jurkat-Rott K, Lehmann-Horn F, Schleip R. The role of fibrosis in Duchenne muscular dystrophy. Acta Myol. 2012;31(3):184–95. Epub 2013/04/27. 23620650; PubMed Central PMCID: PMC3631802.

3. Wang B, Li J, Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc Natl Acad Sci U S A. 2000;97(25):13714–9. Epub 2000/11/30. doi: 10.1073/pnas.240335297 11095710; PubMed Central PMCID: PMC17641.

4. Cirak S, Arechavala-Gomeza V, Guglieri M, Feng L, Torelli S, Anthony K, et al. 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. 2011;378(9791):595–605. Epub 2011/07/26. doi: 10.1016/S0140-6736(11)60756-3 21784508; PubMed Central PMCID: PMC3156980.

5. 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. 2016;351(6271):400–3. Epub 2016/01/02. doi: 10.1126/science.aad5725 26721683; PubMed Central PMCID: PMC4760628.

6. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345(6201):1184–8. Epub 2014/08/16. doi: 10.1126/science.1254445 25123483; PubMed Central PMCID: PMC4398027.

7. Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, et al. Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Reports. 2015;4(1):143–54. doi: 10.1016/j.stemcr.2014.10.013 25434822; PubMed Central PMCID: PMC4297888.

8. Bengtsson NE, Hall JK, Odom GL, Phelps MP, Andrus CR, Hawkins RD, et al. Corrigendum: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat Commun. 2017;8:16007. Epub 2017/06/24. doi: 10.1038/ncomms16007 28643790; PubMed Central PMCID: PMC5489999.

9. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403–7. doi: 10.1126/science.aad5143 26721684; PubMed Central PMCID: PMC4883596.

10. Lee Kunwoo, Conboy Michael, Hyo Min Park Fuguo Jiang, Hyun Jin Kim Mark A. Dewitt, et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nature Biomedical Engineering. 2017;1(November):889–901. doi: 10.1038/s41551-017-0137-2 29805845

11. 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. 2018;362(6410):86–91. Epub 2018/09/01. doi: 10.1126/science.aau1549 30166439; PubMed Central PMCID: PMC6205228.

12. Ousterout DG, Perez-Pinera P, Thakore PI, Kabadi AM, Brown MT, Qin X, et al. Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Mol Ther. 2013;21(9):1718–26. Epub 2013/06/05. doi: 10.1038/mt.2013.111 23732986; PubMed Central PMCID: PMC3776627.

13. Tang L, Bondareva A, Gonzalez R, Rodriguez-Sosa JR, Carlson DF, Webster D, et al. TALEN-mediated gene targeting in porcine spermatogonia. Mol Reprod Dev. 2018;85(3):250–61. Epub 2018/02/03. doi: 10.1002/mrd.22961 29393557; PubMed Central PMCID: PMC6370346.

14. Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016;351(6271):407–11. Epub 2016/01/02. doi: 10.1126/science.aad5177 26721686; PubMed Central PMCID: PMC4924477.

15. Yu HH, Zhao H, Qing YB, Pan WR, Jia BY, Zhao HY, et al. Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy. Int J Mol Sci. 2016;17(10). Epub 2016/10/14. doi: 10.3390/ijms17101668 27735844; PubMed Central PMCID: PMC5085701.

16. 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. Epub 2015/02/19. doi: 10.1038/ncomms7244 25692716; PubMed Central PMCID: PMC4335351.

17. Zhang Y, Long C, Li H, McAnally JR, Baskin KK, Shelton JM, et al. CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice. Sci Adv. 2017;3(4):e1602814. Epub 2017/04/26. doi: 10.1126/sciadv.1602814 28439558; PubMed Central PMCID: PMC5389745.

18. He Z, Proudfoot C, Whitelaw CB, Lillico SG. Comparison of CRISPR/Cas9 and TALENs on editing an integrated EGFP gene in the genome of HEK293FT cells. Springerplus. 2016;5(1):814. Epub 2016/07/09. doi: 10.1186/s40064-016-2536-3 27390654; PubMed Central PMCID: PMC4916124.

19. Devkota S. The road less traveled: strategies to enhance the frequency of homology-directed repair (HDR) for increased efficiency of CRISPR/Cas-mediated transgenesis. BMB Rep. 2018;51(9):437–43. Epub 2018/08/15. doi: 10.5483/BMBRep.2018.51.9.187 30103848; PubMed Central PMCID: PMC6177507.

20. Nishiyama J, Mikuni T, Yasuda R. Virus-Mediated Genome Editing via Homology-Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain. Neuron. 2017;96(4):755–68 e5. Epub 2017/10/24. doi: 10.1016/j.neuron.2017.10.004 29056297; PubMed Central PMCID: PMC5691606.

21. Kornegay JN, Bogan JR, Bogan DJ, Childers MK, Li J, Nghiem P, et al. Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies. Mamm Genome. 2012;23(1–2):85–108. Epub 2012/01/06. doi: 10.1007/s00335-011-9382-y 22218699; PubMed Central PMCID: PMC3911884.

22. Sharp NJ, Kornegay JN, Van Camp SD, Herbstreith MH, Secore SL, Kettle S, et al. An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics. 1992;13(1):115–21. Epub 1992/05/01. doi: 10.1016/0888-7543(92)90210-j 1577476.

23. Kornegay JN. The golden retriever model of Duchenne muscular dystrophy. Skelet Muscle. 2017;7(1):9. Epub 2017/05/21. doi: 10.1186/s13395-017-0124-z 28526070; PubMed Central PMCID: PMC5438519.

24. Mata Lopez S, Hammond JJ, Rigsby MB, Balog-Alvarez CJ, Kornegay JN, Nghiem PP. A novel canine model for Duchenne muscular dystrophy (DMD): single nucleotide deletion in DMD gene exon 20. Skelet Muscle. 2018;8(1):16. Epub 2018/05/31. doi: 10.1186/s13395-018-0162-1 29843823; PubMed Central PMCID: PMC5975675.

25. Schneider SM, Sridhar V, Bettis AK, Heath-Barnett H, Balog-Alvarez CJ, Guo LJ, et al. Glucose Metabolism as a Pre-clinical Biomarker for the Golden Retriever Model of Duchenne Muscular Dystrophy. Mol Imaging Biol. 2018. Epub 2018/03/07. doi: 10.1007/s11307-018-1174-2 29508262.

26. Kornegay JN, Spurney CF, Nghiem PP, Brinkmeyer-Langford CL, Hoffman EP, Nagaraju K. Pharmacologic management of Duchenne muscular dystrophy: target identification and preclinical trials. ILAR J. 2014;55(1):119–49. doi: 10.1093/ilar/ilu011 24936034; PubMed Central PMCID: PMC4158345.

27. Li Y, Pan H, Huard J. Isolating stem cells from soft musculoskeletal tissues. J Vis Exp. 2010;(41). Epub 2010/07/21. doi: 10.3791/2011 20644509; PubMed Central PMCID: PMC3156067.

28. Pawlikowski B, Lee L, Zuo J, Kramer RH. Analysis of human muscle stem cells reveals a differentiation-resistant progenitor cell population expressing Pax7 capable of self-renewal. Dev Dyn. 2009;238(1):138–49. Epub 2008/12/20. doi: 10.1002/dvdy.21833 19097049; PubMed Central PMCID: PMC2799339.

29. Jensen ON, Wilm M, Shevchenko A, Mann M. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. Methods Mol Biol. 1999;112:513–30. Epub 1999/02/23. doi: 10.1385/1-59259-584-7:513 10027274.

30. Bartlett RJ, Winand NJ, Secore SL, Singer JT, Fletcher S, Wilton S, et al. Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs. Am J Vet Res. 1996;57(5):650–4. Epub 1996/05/01. 8723876.

31. Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014;30(10):1473–5. Epub 2014/01/28. doi: 10.1093/bioinformatics/btu048 24463181; PubMed Central PMCID: PMC4016707.

32. Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, Vandyk JK, et al. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 2012;40(Web Server issue):W117–22. Epub 2012/06/14. doi: 10.1093/nar/gks608 22693217; PubMed Central PMCID: PMC3394250.

33. Doetschman T, Georgieva T. Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease. Circ Res. 2017;120(5):876–94. Epub 2017/03/04. doi: 10.1161/CIRCRESAHA.116.309727 28254804.

34. Cotten SW, Kornegay JN, Bogan DJ, Wadosky KM, Patterson C, Willis MS. Genetic myostatin decrease in the golden retriever muscular dystrophy model does not significantly affect the ubiquitin proteasome system despite enhancing the severity of disease. Am J Transl Res. 2013;6(1):43–53. Epub 2013/12/19. 24349620; PubMed Central PMCID: PMC3853423.

35. Spitali P, van den Bergen JC, Verhaart IE, Wokke B, Janson AA, van den Eijnde R, et al. DMD transcript imbalance determines dystrophin levels. FASEB J. 2013;27(12):4909–16. Epub 2013/08/27. doi: 10.1096/fj.13-232025 23975932.

36. Cannell IG, Kong YW, Bushell M. How do microRNAs regulate gene expression? Biochem Soc Trans. 2008;36(Pt 6):1224–31. Epub 2008/11/22. doi: 10.1042/BST0361224 19021530.

37. Lukashev AN, Zamyatnin AA Jr. Viral Vectors for Gene Therapy: Current State and Clinical Perspectives. Biochemistry (Mosc). 2016;81(7):700–8. Epub 2016/07/28. doi: 10.1134/S0006297916070063 27449616.

38. Miki Y, Ono K, Hata S, Suzuki T, Kumamoto H, Sasano H. The advantages of co-culture over mono cell culture in simulating in vivo environment. J Steroid Biochem Mol Biol. 2012;131(3–5):68–75. Epub 2012/01/24. doi: 10.1016/j.jsbmb.2011.12.004 22265957.

39. FDA. Peripheral and Central Nervous System Drugs Advisory Committee Meeting: FDA; 2016. https://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/PeripheralandCentralNervousSystemDrugsAdvisoryCommittee/UCM497063.pdf].

40. Mao Z, Bozzella M, Seluanov A, Gorbunova V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst). 2008;7(10):1765–71. Epub 2008/08/05. doi: 10.1016/j.dnarep.2008.06.018 18675941; PubMed Central PMCID: PMC2695993.

41. Li G, Zhang X, Zhong C, Mo J, Quan R, Yang J, et al. Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells. Sci Rep. 2017;7(1):8943. Epub 2017/08/23. doi: 10.1038/s41598-017-09306-x 28827551; PubMed Central PMCID: PMC5566437.

42. Li D, Yue Y, Duan D. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice. PLoS One. 2010;5(12):e15286. Epub 2010/12/29. doi: 10.1371/journal.pone.0015286 21187970; PubMed Central PMCID: PMC3004926.

43. van Putten M, Hulsker M, Young C, Nadarajah VD, Heemskerk H, van der Weerd L, et al. Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice. FASEB J. 2013;27(6):2484–95. Epub 2013/03/06. doi: 10.1096/fj.12-224170 23460734; PubMed Central PMCID: PMC3659351.

44. Song Y, Morales L, Malik AS, Mead AF, Greer CD, Mitchell MA, et al. Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models. Nat Med. 2019. Epub 2019/10/09. doi: 10.1038/s41591-019-0594-0 31591596.

45. Nghiem PP, Kornegay JN. Gene therapies in canine models for Duchenne muscular dystrophy. Hum Genet. 2019;138(5):483–9. Epub 2019/02/09. doi: 10.1007/s00439-019-01976-z 30734120.

46. Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 2016;540(7631):144–9. Epub 2016/11/17. doi: 10.1038/nature20565 27851729; PubMed Central PMCID: PMC5331785.

47. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019. Epub 2019/10/22. doi: 10.1038/s41586-019-1711-4 31634902.


Článek vyšel v časopise

PLOS One


2020 Číslo 1