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.

Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide

Abstract

Expansions of a G4C2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72 BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G4C2 repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72 with the G4C2 repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72 gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72 gene mutations.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: G4C2-targeting LNA- and MOE-modified ASOs reduce the C9ORF72 repeat-containing transcripts in patient-derived fibroblasts, C9BAC mouse-derived neurons and C9BAC mice.
Fig. 2: Analogues of ASO5 with reduced PS content maintain robust and durable biological activity after CNS administration in heterozygous C9BAC mice.
Fig. 3: Clinical summary and afinersen (ASO5-2) dosing.

Data availability

The data supporting the findings of this study are available within the main text and the Supplementary Information. The full clinical trial protocol for this study is available upon reasonable request to the corresponding authors. C9BAC mice and PS-targeted polyclonal antibody are available by contacting the investigators.

References

  1. Dejesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Renton, A. E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cook, C. & Petrucelli, L. Genetic convergence brings clarity to the enigmatic red line in ALS. Neuron 101, 1057–1069 (2019).

    Article  CAS  PubMed  Google Scholar 

  4. Amick, J. & Ferguson, S. M. C9orf72: at the intersection of lysosome cell biology and neurodegenerative disease. Traffic 18, 267–276 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Belzil, V. V. et al. Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood. Acta Neuropathol. 126, 895–905 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhu, Q. et al. Reduced C9ORF72 function exacerbates gain of toxicity from ALS/FTD-causing repeat expansion in C9orf72. Nat. Neurosci. 23, 615–624 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. O’Rourke, J. G. et al. C9orf72 is required for proper macrophage and microglial function in mice. Science 351, 1324–1329 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Burberry, A. et al. Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Sci. Transl. Med. 8, 347ra393 (2016).

    Article  CAS  Google Scholar 

  9. Shi, Y. et al. Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat. Med. 24, 313–325 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ash, P. E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mori, K. et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345, 1192–1194 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tao, Z. et al. Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity. Hum. Mol. Genet. 24, 2426–2441 (2015).

    Article  CAS  PubMed  Google Scholar 

  14. Freibaum, B. D. et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525, 129–133 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Loveland, A.B. et al. Ribosome inhibition by C9orf72-ALS/FTD poly-PR and poly-GR proteins revealed by cryo-EM. Preprint at https://www.biorxiv.org/content/10.1101/2020.08.30.274597v1 (2020).

  16. Khvorova, A. & Watts, J. K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 35, 238–248 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jafar-Nejad, P. et al. The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration. Nucleic Acids Res. 49, 657–673 (2020).

    Article  PubMed Central  CAS  Google Scholar 

  18. Peters, O. M. et al. Human C9ORF72 hexanucleotide expansion reproduces RNA foci and dipeptide repeat proteins but not neurodegeneration in BAC transgenic mice. Neuron 88, 902–909 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. O’Rourke, J. G. et al. C9orf72 BAC transgenic mice display typical pathologic features of ALS/FTD. Neuron 88, 892–901 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Donnelly, C. J. et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415–428 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sareen, D. et al. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci. Transl. Med. 5, 208ra149 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Lagier-Tourenne, C. et al. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc. Natl Acad. Sci. USA 110, E4530–E4539 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jiang, J. et al. Gain of toxicity from ALS/FTD-linked repeat expansions in C9ORF72 is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs. Neuron 90, 535–550 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gendron, T. F. et al. Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis. Sci. Transl. Med. 9, eaai7866 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Cook, C. N. et al. C9orf72 poly(GR) aggregation induces TDP-43 proteinopathy. Sci. Transl. Med. 12, eabb3774 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhang, K. et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56–61 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Almeida, S. et al. Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons. Acta Neuropathol. 126, 385–399 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zu, T. et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl Acad. Sci. USA 110, E4968–E4977 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rigo, F. et al. Pharmacology of a central nervous system delivered 2′-O-methoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates. J. Pharmacol. Exp. Ther. 350, 46–55 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 24, 374–387 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. Sztainberg, Y. et al. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides. Nature 528, 123–126 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Becker, L. A. et al. Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice. Nature 544, 367–371 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tabrizi, S. J. et al. Targeting Huntingtin expression in patients with Huntington’s disease. N. Engl. J. Med. 380, 2307–2316 (2019).

    Article  CAS  PubMed  Google Scholar 

  34. Moazami, M.P. et al. Quantifying and mitigating motor phenotypes induced by antisense oligonucleotides in the central nervous system. Preprint at https://www.biorxiv.org/content/10.1101/2021.02.14.431096v1 (2021).

  35. Waite, A. J. et al. Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion. Neurobiol. Aging 35, 1779.e5–1779.e13 (2014).

    Article  CAS  Google Scholar 

  36. Meng, L. et al. Towards a therapy for Angelman syndrome by targeting a long non-coding RNA. Nature 518, 409–412 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Zhao, H. T. et al. LRRK2 antisense oligonucleotides ameliorate α-synuclein inclusion formation in a Parkinson’s disease mouse model. Mol. Ther. Nucleic Acids 8, 508–519 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mohan, A. et al. Antisense oligonucleotides selectively suppress target RNA in nociceptive neurons of the pain system and can ameliorate mechanical pain. Pain 159, 139–149 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. McCampbell, A. et al. Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models. J. Clin. Invest. 128, 3558–3567 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Tabrizi, S. J., Smith, A. V. & Bennett, C. F. Targeting Huntingtin in patients with Huntington’s disease. Reply. N. Engl. J. Med. 381, 1181–1182 (2019).

    Article  PubMed  Google Scholar 

  41. Aditi, Aditi, Folkmann, A. W. & Wente, S. R. Cytoplasmic hGle1A regulates stress granules by modulation of translation. Mol. Biol. Cell 26, 1476–1490 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Jovicic, A. et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat. Neurosci. 18, 1226–1229 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhang, Y. J. et al. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nat. Med. 24, 1136–1142 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yuva-Aydemir, Y., Almeida, S., Krishnan, G., Gendron, T. F. & Gao, F. B. Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD. Nat. Commun. 10, 5466 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Moens, T. G. et al. C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A. Acta Neuropathol. 137, 487–500 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Coyne, A.N. et al. G4C2 repeat RNA initiates a POM121-mediated reduction in specific nucleoporins in C9orf72 ALS/FTD. Neuron 107, 1124–1140 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kumar, R. et al. The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2′-thio-LNA. Bioorg. Med. Chem. Lett. 8, 2219–2222 (1998).

    Article  CAS  PubMed  Google Scholar 

  48. DeVos, S.L. & Miller, T.M. Direct intraventricular delivery of drugs to the rodent central nervous system. J. Vis. Exp. e50326 (2013).

  49. Tran, H. et al. Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a Drosophila model of C9ORF72 FTD/ALS. Neuron 87, 1207–1214 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Shin, M., Krishnamurthy, P. M., Devi, G. & Watts, J. K. Quantification of antisense oligonucleotides by splint ligation and quantitative polymerase chain reaction. Nucleic Acid Ther. https://doi.org/10.1089/nat.2021.0040 (in the press) (2021).

Download references

Acknowledgements

The authors thank the Brown and Watts laboratories, Wave Life Sciences, the Animal Medicine and DERC Morphology Cores and F. Ladam for advice, technical support and manuscript review. Funding: This work was funded by the National Institutes of Health (R01 NS111990 to R.H.B. and J.K.W.), the Angel Fund for ALS Research and the Ono Pharmaceutical Foundation (Breakthrough Science Award to J.K.W.). R.H.B. also acknowledges funding from ALSOne, ALS Finding a Cure, the Cellucci Fund for ALS Research and the Max Rosenfeld Fund. We also acknowledge the NEALS Biorepository for providing all or part of the biofluids from the ALS, healthy controls and non-ALS neurological controls used in this study. The project described in this publication was supported, in part, by the University of Massachusetts Clinical and Translational Science Award (no. UL1TR001453) from the National Center for Advancing Translational Sciences of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

R.H.B. conceived the project. H.T., R.H.B., J.K.W. and M.P.M. designed the experimental plan. M.P.M. performed oligo synthesis. M.P.M., C.P., J.M. and A.W. supported the mouse experiments. H.T., H.Y. and N.W. processed the cell and mouse tissue experiments. M.P.M. raised the PS-targeted antibody and performed brain staining. T.K. and D.F. verified the sequence of the clinical ASO. N.S. and C.D. prepared the drug product for clinical use. R.H.B., D.M.-Y. and C.L.D. supported the clinical work, including preparing consent forms. H.G.-E., M.P.M., M.M. and R.M.K. supported the sheep studies. M.S. and N.W. evaluated ASO levels in patient CSF. H.T., R.H.B. and J.K.W. wrote manuscript. R.H.B. and J.K.W. supervised the project.

Corresponding authors

Correspondence to Jonathan K. Watts or Robert H. Brown Jr.

Ethics declarations

Competing interests

The authors have filed a patent related to this research. R.H.B. is a co-founder of Apic Bio.

Additional information

Peer review information Nature Medicine thanks Aaron Gitler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Jerome Staal was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tran, H., Moazami, M.P., Yang, H. et al. Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide. Nat Med 28, 117–124 (2022). https://doi.org/10.1038/s41591-021-01557-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41591-021-01557-6

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing