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

Nature Medicine volume 28, pages 117–124 (2022)Cite this article

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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.

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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.

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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.

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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.

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Author notes

  1. These authors contributed equally: Hélène Tran, Michael P. Moazami.

Authors and Affiliations

  1. Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA

    Hélène Tran, Huiya Yang, Diane McKenna-Yasek, Catherine L. Douthwright, Courtney Pinto, Jake Metterville, Alexandra Weiss, Nicholas Wightman & Robert H. Brown Jr

  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA

    Michael P. Moazami, Minwook Shin & Jonathan K. Watts

  3. Research Pharmacy, University of Massachusetts Medical School, Worcester, MA, USA

    Nitasha Sanil & Craig Dooley

  4. Department of Radiology, University of Massachusetts Medical School, Worcester, MA, USA

    Ajit Puri, Heather Gray-Edwards, Miklos Marosfoi & Robert M. King

  5. Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA

    Robert M. King

  6. Department of Chemistry, University of Connecticut, Storrs, CT, USA

    Thomas Kenderdine & Daniele Fabris

  7. Departments of Neurology and Neurobiology, Barrow Neurological Institute, Phoenix, AZ, USA

    Robert Bowser

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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.

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Competing interests

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

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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.

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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

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