Development of a sensitive trial-ready poly(GP) CSF biomarker assay for <i>C9orf72</i>-associated frontotemporal dementia and amyotrophic lateral sclerosis

Abstract

Data availability statement: Data are available upon reasonable request.Supplementary Data: This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content. Data supplement 1 available at: https://jnnp.bmj.com/highwire/filestream/214878/field_highwire_adjunct_files/0/jnnp-2021-328710supp001_data_supplement.pdf .Copyright © Author(s) (or their employer(s)) 2022. Objective: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. Methods: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. Results and conclusions: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze–thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.This work was funded by Wave Life Sciences, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (648716 - C9ND) (AMI), the UK Dementia Research Institute, which receives its funding from UK DRI, funded by the UK Medical Research Council, Alzheimer's Society and Alzheimer's Research UK. The Dementia Research Centre is supported by Alzheimer's Research UK, Alzheimer's Society, Brain Research UK and The Wolfson Foundation. This work was supported by the NIHR UCL/H Biomedical Research Centre, the Leonard Wolfson Experimental Neurology Centre (LWENC) Clinical Research Facility and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. AK is supported by a Weston Brain Institute and Selfridges Group Foundation award (UB170045). JMS is supported by Engineering and Physical Sciences Research Council (EP/J020990/1), British Heart Foundation (PG/17/90/33415), EU’s Horizon 2020 research and innovation programme (666992). HZ is a Wallenberg Scholar. Simoa instruments used were funded by Wellcome Trust, Fidelity International Foundation and UK DRI. JDR is supported by the Miriam Marks Brain Research UK Senior Fellowship and has received funding from an MRC Clinician Scientist Fellowship (MR/M008525/1) and the NIHR Rare Disease Translational Research Collaboration (BRC149/NS/MH). This work was also supported by the MRC UK GENFI grant (MR/M023664/1), the Bluefield Project and the JPND GENFI-PROX grant (2019-02248). Several authors of this publication are members of the European Reference Network for Rare Neurological Diseases - Project ID No 739510