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High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot

European Journal of Human Genetics volume 28, pages 1231–1242 (2020)Cite this article

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A Correction to this article was published on 03 February 2023

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Abstract

Rett syndrome is a progressive neurodevelopmental disorder which affects almost exclusively girls, caused by variants in MECP2 gene. Effective therapies for this devastating disorder are not yet available and the need for tight regulation of MECP2 expression for brain to properly function makes gene replacement therapy risky. For this reason, gene editing with CRISPR/Cas9 technology appears as a preferable option for the development of new therapies. To study the disease, we developed and characterized a human neuronal model obtained by genetic reprogramming of patient-derived primary fibroblasts into induced Pluripotent Stem Cells. This cellular model represents an important source for our studies, aiming to correct MECP2 variants in neurons which represent the primarily affected cell type. We engineered a gene editing toolkit composed by a two-plasmid system to correct a hotspot missense variant in MECP2, c.473 C > T (p.(Thr158Met)). The first construct expresses the variant-specific sgRNA and the Donor DNA along with a fluorescent reporter system. The second construct brings Cas9 and targets for auto-cleaving, to avoid long-term Cas9 expression. NGS analysis on sorted cells from four independent patients demonstrated an exceptionally high editing efficiency, with up to 80% of HDR and less than 1% of indels in all patients, outlining the relevant potentiality of the approach for Rett syndrome therapy.

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Fig. 1: Design of sgRNA and Donor for MECP2 variant c.473 C > T (p.(Thr158Met)).
Fig. 2: Plasmids strategy.
Fig. 3: sgRNA specificity and plasmid functionality in HEK293 cells and MECP2 neurons.
Fig. 4: Efficient editing in MECP2 mutated cells.

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References

  1. Operto FF, Mazza R, Pastorino GMG, Verrotti A, Coppola G. Epilepsy and genetic in Rett syndrome: a review. Brain and Behav. 2019;9:e01250.

    Article  Google Scholar 

  2. Chahrour M, Zoghbi HY. The story of Rett syndrome: from clinic to neurobiology. Neuron. 2007;56:422–37.

    Article  CAS  Google Scholar 

  3. Bienvenu T, Chelly J. Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized. Nat Rev Genet. 2006;7:415.

    Article  CAS  Google Scholar 

  4. Ehrhart F, Sangani NB, Curfs LM. Current developments in the genetics of Rett and Rett-like syndrome. Curr Opin Psychiatry. 2018;31:103–8.

    Article  Google Scholar 

  5. Kyle SM, Vashi N, Justice MJ. Rett syndrome: a neurological disorder with metabolic components. Open Biol. 2018;8:170216.

    Article  Google Scholar 

  6. Archer H, Evans J, Leonard H, Colvin L, Ravine D, Christodoulou J, et al. Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X‐chromosome inactivation. J Med Genet 2007;44:148–52.

    Article  CAS  Google Scholar 

  7. Nissenkorn A, Levy-Drummer RS, Bondi O, Renieri A, Villard L, Mari F, et al. Epilepsy in Rett syndrome-lessons from the Rett networked database. Epilepsia 2015;56:569–76.

    Article  CAS  Google Scholar 

  8. Cuddapah VA, Nwaobi SE, Percy AK, Olsen ML. MeCP2 in the regulation of neural activity: Rett syndrome pathophysiological perspectives. Degener Neurol Neuromuscul Dis. 2015;5:103.

    Google Scholar 

  9. Cohen DR, Matarazzo V, Palmer AM, Tu Y, Jeon OH, Pevsner J, et al. Expression of MeCP2 in olfactory receptor neurons is developmentally regulated and occurs before synaptogenesis. Mol Cell Neurosci. 2003;22:417–29.

    Article  CAS  Google Scholar 

  10. Chahrour M, Jung SY, Shaw C, Zhou X, Wong ST, Qin J, et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science. 2008;320:1224–9.

    Article  CAS  Google Scholar 

  11. Guy J, Gan J, Selfridge J, Cobb S, Bird A. Reversal of neurological defects in a mouse model of Rett syndrome. Science. 2007;315:1143–7.

    Article  CAS  Google Scholar 

  12. Ramocki MB, Tavyev YJ, Peters SU. The MECP2 duplication syndrome. Am J Med Genet Part A. 2010;152:1079–88.

    Article  Google Scholar 

  13. Koerner MV, FitzPatrick L, Selfridge J, Guy J, De Sousa D, Tillotson R, et al. Toxicity of overexpressed MeCP2 is independent of HDAC3 activity. Genes Dev. 2018;32:1514–24.

    Article  CAS  Google Scholar 

  14. Livide G, Patriarchi T, Amenduni M, Amabile S, Yasui D, Calcagno E, et al. GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells. Eur J Hum Genet. 2015;23:195.

    Article  CAS  Google Scholar 

  15. Patriarchi T, Amabile S, Frullanti E, Landucci E, Rizzo CL, Ariani F, et al. Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1+/− patients and in foxg1+/− mice. Eur J Hum Genet. 2016;24:871.

    Article  CAS  Google Scholar 

  16. Landucci E, Brindisi M, Bianciardi L, Catania LM, Daga S, Croci S, et al. iPSC-derived neurons profiling reveals GABAergic circuit disruption and acetylated α-tubulin defect which improves after iHDAC6 treatment in Rett syndrome. Exp Cell Res. 2018;368:225–35.

    Article  CAS  Google Scholar 

  17. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci. 2012;109:E2579–E2586.

    Article  CAS  Google Scholar 

  18. Khan FA, Pandupuspitasari NS, Chun-Jie H, Ao Z, Jamal M, Zohaib A, et al. CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases. Oncotarget. 2016;7:52541.

    Article  Google Scholar 

  19. Pellagatti A, Dolatshad H, Valletta S, Boultwood J. Application of CRISPR/Cas9 genome editing to the study and treatment of disease. Arch Toxicol. 2015;89:1023–34.

    Article  CAS  Google Scholar 

  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;96:755–68.

    Article  CAS  Google Scholar 

  21. Gadalla KK, Vudhironarit T, Hector RD, Sinnett S, Bahey NG, Bailey ME, et al. Development of a novel AAV gene therapy cassette with improved safety features and efficacy in a mouse model of Rett syndrome. Mol Ther-Methods Clin Dev. 2017;5:180–90.

    Article  CAS  Google Scholar 

  22. Cobb S, Guy J, Bird A. Reversibility of functional deficits in experimental models of Rett syndrome. Biochem Soc Trans. 2010;38:498–506.

    Article  CAS  Google Scholar 

  23. Auricchio A, Hildinger M, O’Connor E, Gao GP, Wilson JM. Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column. Hum Gene Ther. 2001;12:71–6.

    Article  CAS  Google Scholar 

  24. Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33:102.

    Article  CAS  Google Scholar 

  25. Dracopoli NC, Haines JL, Korf BR, Moir DT, Morton CC, Seidman CE et al. (eds). Current Protocols in Molecular Biology. John Wiley & Son: Hoboken, NJ, USA, 2000.

  26. Park J, Lim K, Kim SJ, Bae S. Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics. 2017;33:286–8.

    Article  CAS  Google Scholar 

  27. Brown K, Selfridge J, Lagger S, Connelly J, De Sousa D, Kerr A, et al. The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome. Hum Mol Genet. 2016;25:558–70.

    Article  CAS  Google Scholar 

  28. Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018;24:927.

    Article  CAS  Google Scholar 

  29. Thomas M, Kalita A, Labrecque S, Pim D, Banks L, Matlashewski G. Two polymorphic variants of wild-type p53 differ biochemically and biologically. Mol Cell Biol. 1999;19:1092–100.

    Article  CAS  Google Scholar 

  30. Le TTH, Tran NT, Dao TML, Nguyen DD, Do DH, Ha LT, et al. Efficient and precise CRISPR/Cas9-mediated MECP2 modifications in human induced pluripotent stem cells. Front Genet. 2019. 10.10.3389/fgene.2019.00625.

  31. Martufi M, Good RB, Rapiteanu R, Schmidt T, Patili E, Tvermosegaard K, et al. Single-step, high-efficiency CRISPR-Cas9 genome editing in primary human disease-derived fibroblasts. CRISPR J. 2018;2:31–40.

    Article  Google Scholar 

  32. Xie N, Gong H, Suhl JA, Chopra P, Wang T, Warren ST. Reactivation of FMR1 by CRISPR/Cas9-mediated deletion of the expanded CGG-repeat of the fragile X chromosome. PLoS ONE 2016;11:e0165499.

    Article  Google Scholar 

  33. Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med. 2017;377:1713–22.

    Article  CAS  Google Scholar 

  34. Lombardi LM, Baker SA, Zoghbi HY. MECP2 disorders: from the clinic to mice and back. J Clin Investig. 2015;125:2914–23.

    Article  Google Scholar 

  35. Bowers WJ, Breakefield XO, Sena-Esteves M. Genetic therapy for the nervous system. Hum Mol Genet. 2011;20:R28–41.

    Article  CAS  Google Scholar 

  36. Daya S, Berns KI. Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev. 2008;21:583–93.

    Article  CAS  Google Scholar 

  37. Kaplitt MG, Feigin A, Tang C, Fitzsimons HL, Mattis P, Lawlor PA, et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet. 2007;369:2097–105.

    Article  CAS  Google Scholar 

  38. Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol. 2009;27:59.

    Article  CAS  Google Scholar 

  39. Hareendran S, Balakrishnan B, Sen D, Kumar S, Srivastava A, Jayandharan GR. Adeno‐associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them. Rev Med Virol. 2013;23:399–413.

    Article  CAS  Google Scholar 

  40. Sinnett SE, Hector RD, Gadalla KKE, Heindel C, Chen D, Zaric V, et al. Improved MECP2 gene therapy extends the survival of MeCP2-null mice without apparent toxicity after intracisternal delivery. Mol Ther Methods Clin Dev. 2017;5:106–15.

    Article  CAS  Google Scholar 

  41. Senís E, Fatouros C, Große S, Wiedtke E, Niopek D, Mueller AK, et al. CRISPR/Cas9‐mediated genome engineering: an adeno‐associated viral (AAV) vector toolbox. Biotechnol J. 2014;9:1402–12.

    Article  Google Scholar 

  42. Bak RO, Porteus MH. CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep. 2017;20:750–6.

    Article  CAS  Google Scholar 

  43. Saraiva J, Nobre RJ, Pereira de Almeida L. Gene therapy for the CNS using AAVs: the impact of systemic delivery by AAV9. J Control Release. 2016;10:94–109.

    Article  Google Scholar 

  44. Gray SJ, Nagabhushan Kalburgi S, McCown TJ, Jude Samulski R. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene Ther. 2013;20:450–9.

    Article  CAS  Google Scholar 

  45. Maguire CA, Ramirez SH, Merkel SF, Sena-Esteves M, Breakefield XO. Gene therapy for the nervous system: challenges and new strategies. Neurotherapeutics 2014;11:817–39.

    Article  CAS  Google Scholar 

  46. Büning H, Srivastava A. Capsid modifications for targeting and improving the efficacy of AAV Vectors. Mol Ther-Methods Clin Dev. 2019;12:248–65.

    Article  Google Scholar 

  47. Ruan G-X, Barry E, Yu D, Lukason M, Cheng SH, Scaria A. CRISPR/Cas9-mediated genome editing as a therapeutic approach for leber congenital amaurosis 10. Mol Ther. 2017;25:331–41.

    Article  CAS  Google Scholar 

  48. Gong H, Liu M, Klomp J, Merrill BJ, Rehman J, Malik AB. Method for dual viral vector mediated CRISPR-Cas9 gene disruption in primary human endothelial cells. Sci Rep. 2017;7:42127.

    Article  CAS  Google Scholar 

  49. Chu HW, Rios C, Huang C, Wesolowska-Andersen A, Burchard EG, O’Connor BP, et al. CRISPR–Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18. Gene Ther. 2015;22:822.

    Article  CAS  Google Scholar 

  50. Voets O, Tielen F, Elstak E, Benschop J, Grimbergen M, Stallen J, et al. Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells. PloS ONE. 2017;12:e0182974.

    Article  Google Scholar 

  51. Chapman JR, Taylor MR, Boulton SJ. Playing the end game: DNA double-strand break repair pathway choice. Mol Cell. 2012;47:497–510.

    Article  CAS  Google Scholar 

  52. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med. 2015;21:121.

    Article  CAS  Google Scholar 

  53. Ishizu T, Higo S, Masumura Y, Kohama Y, Shiba M, Higo T, et al. Targeted genome replacement via homology-directed repair in non-dividing cardiomyocytes. Sci Rep. 2017;7:9363.

    Article  Google Scholar 

  54. Keimling M, Wiesmüller L. DNA double-strand break repair activities in mammary epithelial cells––influence of endogenous p53 variants. Carcinogenesis. 2009;30:1260–8.

    Article  CAS  Google Scholar 

  55. Moureau S, Luessing J, Harte EC, Voisin M, Lowndes NF. A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining. Open Biol. 2016;6:160225.

    Article  Google Scholar 

  56. Hustedt N, Durocher D. The control of DNA repair by the cell cycle. Nat Cell Biol. 2016;19:1–94.

    Article  Google Scholar 

  57. IHry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat Med. 2018;24:939.

    Article  CAS  Google Scholar 

  58. Conti A, Di Micco R. p53 activation: a checkpoint for precision genome editing? Genome Med. 2018;10:66.

    Article  Google Scholar 

  59. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187–97.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank MECP2 patients and their families. The “Cell lines and DNA bank of Rett Syndrome, X-linked mental retardation and other genetic diseases”, member of the Telethon Network of Genetic Biobanks (project no. GTB12001), funded by Telethon Italy, and of the EuroBioBank network, and the “Associazione Italiana Rett O.N.L.U.S.” provided us with specimens. This work is generated within the ITHACA (European Reference Network for Intellectual Disability Telehealth and Congenital Anomalies). We thank SienaGenTest srl, a Spin-off of the University of Siena (www.sienagentest.dbm.unisi.it) for gene editing efficiency analysis. Two of several authors of this publication are members of the European Reference Network for rare malformation syndromes and rare intellectual and neurodevelopmental disorders, ERN-ITHACA.

Author Contributors

CS, CML, CK, CS, RA and MI have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and have been involved in drafting the manuscript. DS, DF, FE, BE, FS, TR, GA, CA, VF and PAM has made substantial contributions to acquisition and analysis of the data. LV and LRC have made substantial contributions to interpretation of data and clinical evaluation. All authors have been involved in drafting the manuscript, have given final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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Authors and Affiliations

  1. Medical Genetics, University of Siena, Siena, Italy

    Susanna Croci, Miriam Lucia Carriero, Katia Capitani, Sergio Daga, Francesco Donati, Elisa Frullanti, Vittoria Lamacchia, Annarita Giliberti, Floriana Valentino, Elisa Benetti, Alessandra Renieri & Ilaria Meloni

  2. ISPRO, Institute for Cancer Research, Prevention and Clinical Network, Firenze, Italy

    Katia Capitani, Francesco Donati & Silvestro Giovanni Conticello

  3. Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy

    Vittoria Lamacchia, Rossella Tita, Caterina Lo Rizzo, Anna Maria Pinto & Alessandra Renieri

  4. Department of Medical Biotechnologies, University of Siena, Siena, Italy

    Elisa Benetti & Simone Furini

  5. Laboratory of Molecular Microbiology and Biotechnology, Department of Medical Biotechnologies, University of Siena, Siena, Italy

    Annalisa Ciabattini

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  1. Susanna Croci
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Correspondence to Alessandra Renieri.

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The study was approved by Azienda Ospedaliera Universitaria Senese Ethics Committee, Prot Name CRI, Prot n 12362_2018.

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Patenting The approach described in this work is covered by Italian patent application n.102018000020230.

The original online version of this article was revised: Update in the Acknowledgment section.

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Croci, S., Carriero, M.L., Capitani, K. et al. High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot. Eur J Hum Genet 28, 1231–1242 (2020). https://doi.org/10.1038/s41431-020-0624-x

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