Reply to: “Comment on: Polyglutamine‐Expanded Ataxin‐3: A Target Engagement Marker for Spinocerebellar Ataxia Type 3 in Peripheral Blood”
2022-05-01

We thank Al-Shaikh et al for their interest in our article on measurement of polyglutamine (polyQ)-expanded ataxin-3 in plasma and cerebrospinal fluid (CSF) as target engagement marker in spinocerebellar ataxia type 3 (SCA3) using the single molecule counting (SMC×PRO) technology.1 We agree that it is mandatory to compare our SMC×PRO immunoassay design and outcome in plasma and CSF to other ataxin-3 immunoassays such as the Mesoscale technology (MSD).2 As indicated by Al-Shaikh et al, both immunoassays share similarities, but also some differences. Methodically, both assays used the ataxin-3 antibody clone 1H9 as capture antibody, but differ in the detection antibodies that specifically bind to the expanded polyQ region (MW1 in SMC×PRO and 3B5H10 in Meso Scale Discovery). Whether both differ in their quantitative binding to different polyQ-length is controversially discussed. Miller et al3 suggest that both bind to different polyQ species, whereas others demonstrated that both have similar binding characteristics.4, 5 Although the two assays are comparable, further platform-dependent differences in antibody labeling, binding characteristics, and detection limits (the SMC×PRO is typically 30× to 1000× more sensitive) introduce additional variation. Importantly, one additional advantage of SMC×PRO technology might be the reduced assay input volume allowing measurement of limited biosamples. In Huntington's disease (HD), the MSD assay can measure mutant huntingtin (mHTT) in a picomolar range only in human mononuclear cells and post mortem brain samples from HD carriers.6 In contrast, the ultrasensitive SMC×PRO immunoassay is the gold standard for HTT protein measurements because of its ability to detect femtomolar amounts of mutant HTT. Therefore, this assay is suitable for mHTT CSF sample quantification and was recently used in a first phase 1-2a HTT antisense oligonucleotide (ASO) trial to quantify mHTT reduction by ASO treatment in CSF.6, 7 Importantly, both ataxin-3 immunoassay allow to completely discriminate between controls and SCA3 mutation carriers in CSF and plasma; however, they show significant differences to discriminate pre-ataxic and ataxic carriers (CSF: SMC×PRO 58% vs. MSD 89%; plasma: SMC×PRO 78% vs. MSD 70%).1, 2 As indicated by Al-Shaikh et al, this can be explained by different assay conditions, but also by the mode how pre-ataxic state in the SMC×PRO study or pre-symptomatic carriers in the MSD study were defined. As demonstrated by cross-sectional and longitudinal analyses using SMC×PRO, polyQ-expanded ataxin-3 protein levels are highly stable in pre-ataxic and mildly affected carriers and higher at larger disease stages. Therefore, also the disease stage of mutations carriers in each study influences outcome measures in CSF and plasma. In relation to SCA3, MSD and SMC×PRO are both able to quantify ataxin-3 protein level in CSF and plasma, but with different sensitivity, as previous discussed. Here, only a comparative analysis of the same samples using the different platforms will help to clarify if both assays are comparable. Additionally, it is unclear which immunoassay allows for analysis of target engagement under ASO therapy in biofluids. For MSD, it was shown that protein lowering can be measured at least in cell culture.2 In summary, SMC×PRO and MSD ataxin-3 immunoassays are valuable tools to detect polyQ-expanded ataxin-3 in complex human biomaterials but which immunoassay is more suitable to detect target engagement under protein lowering therapies, needs to be further evaluated. (1) Research project: A. Conception, B. Organization, C. Execution; D. Acquiring data; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the first draft, B. Review and Critique; C. Revising the manuscript J.H.S.: 1A, 1B, 1C, 2A, 2B, 3A, 3C K.K.: 1D, 2B, 2C, 3A, 3B, 3C J.P.: 1D, 2B, 2C, 3A, 3B, 3C ESMI Study group: 1A, 1B, 1C, 1D, 2C, 3B, 3C O.R.: 1A, 1B, 2C, 3B, 3C J.H.S receives funding from National Ataxia Foundation and Centre of Rare Diseases (ZSE) Tübingen Germany. O.R. received funding for the NGS Competence Center and for the role of host genetics in Corona pandemic from the German Research Foundation (DFG), as well as for the Ge-Med approach and for COVID research from Illumina. None of the funding is related to this manuscript. Data sharing is not applicable to this article as no new data were created or analyzed in this study.