Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Removal of the GAA repeat in the heart of a Friedreich’s ataxia mouse model using CjCas9

Gene Therapy volume 30, pages 612–619 (2023)Cite this article

Subjects

Abstract

Most Friedreich ataxia (FRDA) cases are caused by the elongation of the GAA repeat (GAAr) sequence in the first intron of the FXN gene, leading to a decrease of the frataxin protein expression. Deletion of this GAAr with CRISPR/Cas9 technology leads to an increase in frataxin expression in vitro. We are therefore aiming to develop FRDA treatment based on the deletion of GAAr with CRISPR/Cas9 technology using a single AAV expressing a small Cas9 (CjCas9) and two single guide RNAs (sgRNAs) targeting the FXN gene. This AAV was intraperitoneally administrated to YG8sR (250–300 GAAr) and to YG8-800 (800 GAAr) mice. DNA and RNA were extracted from different organs a month later. PCR amplification of part of intron 1 of the FXN gene detected some GAAr deletion in some cells in heart and liver of both mouse models, but the editing rate was not sufficient to cause an increase in frataxin mRNA in the heart. However, the correlation observed between the editing rate and the distribution of AAV suggests a possible therapy based on the removal of the GAAr with a better delivery tool of the CRISPR/Cas9 system.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: GAAr deletion induced by CjCas9/sgRNAs.
Fig. 2: Frataxin expression by CRISPR-edited YG8sR fibroblasts.
Fig. 3: Detection of AAV in mouse tissues.
Fig. 4: CjCas9 + 2 sgRNAs delivered by an AAV9 induced GAAr deletion in YG8sR and YG8-800 mouse heart and liver.
Fig. 5: Expression of the frataxin mRNA in mice treated with AAV9-CjCas9-2sgRNA.

Similar content being viewed by others

Data availability

All data will be made available upon request.

References

  1. Pandolfo M. Friedreich ataxia. In: PJ Vinken and GW Bruyn, editors. Handbook of clinical neurology. 2012;103:275–94.

  2. Vankan P. Prevalence gradients of Friedreich’s ataxia and R1b haplotype in Europe co-localize, suggesting a common Palaeolithic origin in the Franco-Cantabrian ice age refuge. J Neurochem. 2013;126:11–20.

    Article  CAS  PubMed  Google Scholar 

  3. Pandolfo M. Friedreich ataxia: new pathways. J Child Neurol. 2012;27:1204–11.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Tsou AY, Paulsen EK, Lagedrost SJ, Perlman SL, Mathews KD, Wilmot GR, et al. Mortality in Friedreich ataxia. J Neurol Sci. 2011;307:46–9.

    Article  PubMed  Google Scholar 

  5. Pandolfo M. Friedreich ataxia: the clinical picture. J Neurol. 2009;256:3–8.

    Article  PubMed  Google Scholar 

  6. Galea CA, Huq A, Lockhart PJ, Tai G, Corben LA, Yiu EM, et al. Compound heterozygous FXN mutations and clinical outcome in friedreich ataxia. Ann Neurol. 2016;79:485–95.

    Article  CAS  PubMed  Google Scholar 

  7. Soragni E, Herman D, Dent SY, Gottesfeld JM, Wells RD, Napierala M. Long intronic GAA*TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia. Nucleic Acids Res. 2008;36:6056–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Anzovino A, Lane DJ, Huang ML, Richardson DR. Fixing frataxin: ‘ironing out’ the metabolic defect in Friedreich’s ataxia. Br J Pharmacol. 2014;171:2174–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lill R. Function and biogenesis of iron-sulphur proteins. Nature. 2009;460:831–8.

    Article  CAS  PubMed  Google Scholar 

  10. Perdomini M, Hick A, Puccio H, Pook MA. Animal and cellular models of Friedreich ataxia. J Neurochem. 2013;126:65–79.

    Article  CAS  PubMed  Google Scholar 

  11. Anjomani Virmouni S, Ezzatizadeh V, Sandi C, Sandi M, Al-Mahdawi S, Chutake Y, et al. A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia. Dis Model Mech. 2015;8:225–35.

    PubMed  PubMed Central  Google Scholar 

  12. Ocana-Santero G, Diaz-Nido J, Herranz-Martin S. Future prospects of gene therapy for Friedreich’s ataxia. Int J Mol Sci. 2021;22:1815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gerard C, Archambault AF, Bouchard C, Tremblay JP. A promising mouse model for Friedreich ataxia progressing like human patients. Behav Brain Res. 2022;436:114107.

    Article  PubMed  Google Scholar 

  14. Ouellet DL, Cherif K, Rousseau J, Tremblay JP. Deletion of the GAA repeats from the human frataxin gene using the CRISPR-Cas9 system in YG8R-derived cells and mouse models of Friedreich ataxia. Gene Ther. 2017;24:265–74.

    Article  CAS  PubMed  Google Scholar 

  15. Kotterman MA, Schaffer DV. Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet. 2014;15:445–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. He X, Urip BA, Zhang Z, Ngan CC, Feng B. Evolving AAV-delivered therapeutics towards ultimate cures. J Mol Med (Berl). 2021;99:593–617.

    Article  PubMed  Google Scholar 

  17. Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat commun. 2017;8:14500.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ding X, Seebeck T, Feng Y, Jiang Y, Davis GD, Chen F. Improving CRISPR-Cas9 genome editing efficiency by fusion with chromatin-modulating peptides. CRISPR J. 2019;2:51–63.

    Article  CAS  PubMed  Google Scholar 

  20. Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014;42:e168.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Mazzara PG, Muggeo S, Luoni M, Massimino L, Zaghi M, Valverde PT, et al. Frataxin gene editing rescues Friedreich’s ataxia pathology in dorsal root ganglia organoid-derived sensory neurons. Nat Commun. 2020;11:4178.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Li Y, Polak U, Bhalla AD, Rozwadowska N, Butler JS, Lynch DR, et al. Excision of Expanded GAA Repeats Alleviates the Molecular Phenotype of Friedreich’s Ataxia. Mol Ther. 2015;23:1055–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li J, Rozwadowska N, Clark A, Fil D, Napierala JS, Napierala M. Excision of the expanded GAA repeats corrects cardiomyopathy phenotypes of iPSC-derived Friedreich’s ataxia cardiomyocytes. Stem Cell Res. 2019;40:101529.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li Y, Li J, Wang J, Zhang S, Giles K, Prakash TP, et al. Premature transcription termination at the expanded GAA repeats and aberrant alternative polyadenylation contributes to the Frataxin transcriptional deficit in Friedreich’s ataxia. Hum Mol Genet. 2022;31:3539–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sivakumar A, Cherqui S. Advantages and limitations of gene therapy and gene editing for Friedreich’s ataxia. Front Genome Ed. 2022;4:903139.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Rocca CJ, Rainaldi JN, Sharma J, Shi Y, Haquang JH, Luebeck J, et al. CRISPR-Cas9 gene editing of hematopoietic stem cells from patients with Friedreich’s ataxia. Mol Ther Methods Clin Dev. 2020;17:1026–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Belbellaa B, Reutenauer L, Monassier L, Puccio H. Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms. Hum Mol Genet. 2019;28:1274–85.

    Article  CAS  PubMed  Google Scholar 

  28. Belbellaa B, Reutenauer L, Messaddeq N, Monassier L, Puccio H. High levels of Frataxin overexpression lead to mitochondrial and cardiac toxicity in mouse models. Mol Ther Methods Clin Dev. 2020;19:120–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li L, Matsui M, Corey DR. Activating frataxin expression by repeat-targeted nucleic acids. Nat commun. 2016;7:10606.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Greene E, Mahishi L, Entezam A, Kumari D, Usdin K. Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia. Nucleic Acids Res. 2007;35:3383–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Li K, Singh A, Crooks DR, Dai X, Cong Z, Pan L, et al. Expression of human frataxin is regulated by transcription factors SRF and TFAP2. PLoS One. 2010;5:e12286.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Yameogo P, Duchene BL, Majeau N, Tremblay JP. CRISPR-SCReT (CRISPR-Stop Codon Read Through) method to control Cas9 expression for gene editing. Gene Ther. 2022;29:171–7.

  33. Yamada M, Watanabe Y, Gootenberg JS, Hirano H, Ran FA, Nakane T, et al. Crystal structure of the minimal Cas9 from campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 Systems. Mol Cell. 2017;65:1109–21.

    Article  CAS  PubMed  Google Scholar 

  34. Silva-Pinheiro P, Cerutti R, Luna-Sanchez M, Zeviani M, Viscomi C. A single intravenous injection of AAV-PHP.B-hNDUFS4 ameliorates the phenotype of Ndufs4 (−/−) Mice. Mol Ther Methods Clin Dev. 2020;17:1071–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chapdelaine P, Gerard C, Sanchez N, Cherif K, Rousseau J, Ouellet DL, et al. Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice. Gene Ther. 2016;23:606–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell. 2021;184:4919–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rocca CJ, Goodman SM, Dulin JN, Haquang JH, Gertsman I, Blondelle J, et al. Transplantation of wild-type mouse hematopoietic stem and progenitor cells ameliorates deficits in a mouse model of Friedreich’s ataxia. Sci Transl Med. 2017;9:eaaj2347.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Park H, Shin J, Choi H, Cho B, Kim J. Valproic acid significantly improves CRISPR/Cas9-mediated gene editing. Cells. 2020;9:1447.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chan PK, Torres R, Yandim C, Law PP, Khadayate S, Mauri M, et al. Heterochromatinization induced by GAA-repeat hyperexpansion in Friedreich’s ataxia can be reduced upon HDAC inhibition by vitamin B3. Hum Mol Genet. 2013;22:2662–75.

    Article  CAS  PubMed  Google Scholar 

  40. Rodden LN, Gilliam KM, Lam C, Rojsajjakul T, Mesaros C, Dionisi C, et al. DNA methylation in Friedreich ataxia silences expression of frataxin isoform E. Sci Rep. 2022;12:5031.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dre Chantal Guillemette for ddPCR equipment and Julie Carbonneau for her technical assistance. PY has been supported by a fellowship from the Canadian Francophonie Scholarship Program (CFSP).

Funding

This research project was supported by grants from the Canadian Institute of Health Research, ThéCel network and Ataxia Canada.

Author information

Authors and Affiliations

  1. Centre de Recherche du CHU de Québec-Université Laval, Québec city, QC, Canada

    Pouiré Yaméogo, Catherine Gérard, Nathalie Majeau & Jacques P. Tremblay

  2. Département de Médecine Moléculaire, Université Laval, Québec city, QC, Canada

    Pouiré Yaméogo, Catherine Gérard, Nathalie Majeau & Jacques P. Tremblay

Authors
  1. Pouiré Yaméogo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Catherine Gérard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nathalie Majeau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jacques P. Tremblay
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

PY designed and performed the experiments and wrote the manuscript. CG provided technical assistance for the experiments and corrected the manuscript. NM assisted with the design of the experiments and corrected the manuscript. JPT conceived the experiments and corrected the manuscript.

Corresponding author

Correspondence to Jacques P. Tremblay.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yaméogo, P., Gérard, C., Majeau, N. et al. Removal of the GAA repeat in the heart of a Friedreich’s ataxia mouse model using CjCas9. Gene Ther 30, 612–619 (2023). https://doi.org/10.1038/s41434-023-00387-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-023-00387-0

This article is cited by

Search

Advanced search

Quick links