Antisense oligonucleotide depletion of CCDC146 is a broad-spectrum therapeutic strategy for ALS

Abstract Amyotrophic lateral sclerosis (ALS) is a heritable and incurable disease defined by the degeneration of motor neurons (MNs), yet the genetics of ALS remain partially understood. Using a genomic deep learning-powered whole-genome analysis of 6,715 ALS patients, we identify four rare noncoding variants associated with patient survival, including chr7:76,009,472:C>T which is linked to a 70.6% reduction in survival. Genetic editing of this variant into iPSC-derived MNs increases CCDC146 expression and exacerbates ALS-specific phenotypes including TDP-43 mislocalization. We reveal that CCDC146 was located within the basal body of primary cilia in human MNs, and that cilia structure and function is impaired by CCDC146 overexpression but is restored by its depletion. Suppressing CCDC146 using an antisense oligonucleotide (ASO) completely rescues ALS-specific survival defects in neurons derived from both sporadic and familial patients, and it extends survival and reverses TDP-43 pathology in an aggressive ALS mouse model. Taken together, CCDC146 is a new modifier of ALS survival that acts via the primary cilia of MNs. ASO targeting of CCDC146 is a potential therapeutic approach for both sporadic and genetic forms of ALS, particularly because congenital absence of CCDC146 is well tolerated. Competing Interest Statement M.P.S. is a co-founder and the scientific advisory board member of Personalis, Qbio, January, SensOmics, Filtricine, Akna, Protos, Mirvie, NiMo, Onza, Oralome, Marble Therapeutics and Iollo. He is also on the scientific advisory board of Danaher, Genapsys, and Jupiter. J.K.I. is a co-founder and a scientific advisory board member of AcuraStem, Inc. and Modulo Bio, a scientific advisory board member of Synapticure and Vesalius Therapeutics. J.K.I. is also an employee of BioMarin Pharmaceutical. The remaining authors declare no competing interests. We have a patent application related to this work. Funding Statement This work was supported by NIH (CEGS 5P50HG007735 to M.P.S., R01NS131409 and R01NS097850 to J.K.I.), Wellcome Trust (216596/Z/19/Z to J.C.-K.), NIHR (NF-SI-0617-10077 to P.J.S.), Motor Neurone Disease (MND) Association (894-791, 899-792 and 913-792 to C.H., S.G., S.Z. and J.C.-K.), John Douglas French Alzheimer's Foundation (to J.K.I.), and Robert Packard Center for ALS Research (1375407 to S.Z.). We also acknowledge support from the MND Association Fellowship (2323-799) and the Kingsland Fellowship (to T.M.), the NIHR Sheffield Biomedical Research Centre (NIHR203321), and the NIHR Sheffield Clinical Research Facility. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the South Sheffield Research Ethics Committee. Also, this study followed study protocols approved by Medical Ethical Committees for each of the participating institutions. Written informed consent was obtained from all participating individuals. All methods were performed in accordance with relevant national and international guidelines and regulations. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes We added more in vitro and in vivo experiments to validate our discovery. Data Availability The epigenome and transcriptome profiling data of iPSC-derived motor neurons used in the ABC model and NeuroNet are available at encodeproject.org with the following link: https://www.encodeproject.org/publications/de19555b-a49f-471c-bfbc-be3b628fe9bf/. NeuroNet variant effect scores can be downloaded at https://doi.org/10.6084/m9.figshare.25444534. The Project MinE WGS data are accessible upon application approval (https://www.projectmine.com/research/data-sharing/). The AnswerALS WGS and RNA-seq data are available upon request approval (https://dataportal.answerals.org/home). https://www.encodeproject.org/publications/de19555b-a49f-471c-bfbc-be3b628fe9bf/ https://doi.org/10.6084/m9.figshare.25444534