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bioRxiv · 10.1101/2024.09.25.614766

Challenges and advances for huntingtin detection in cerebrospinal fluid: in support of relative quantification

Harding, R. J.·Xie, Y.·Caron, N. S. J.·Findlay-Black, H.·Lyu, C.·Potluri, N.·Chandrasekaran, R.·Hayden, M. R.·Leavitt, B. R.·Langbehn, D.·Southwell, A.
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Abstract

Huntington disease (HD) is a progressive and devastating neurodegenerative disease caused by expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene above a critical threshold of [~]35 repeats resulting in expression of mutant HTT (mHTT). A promising treatment approach being tested in clinical trials is HTT lowering, which aims to reduce levels of the mHTT protein. Target engagement of these therapies in the brain are inferred using antibody-based assays to measure mHTT levels in the cerebrospinal fluid (CSF), which is frequently reported as absolute mHTT concentration based on a monomeric protein standard used to generate a standard curve. However, patient biofluids are a complex milieu of different mHTT protein species, suggesting that absolute quantitation is challenging, and a single, recombinant protein standard may not be sufficient to interpret assay signal as molar mHTT concentration. In this study, we used immunoprecipitation and flow cytometry (IP-FCM) to investigate different factors that influence mHTT detection assay signal. Our results show that HTT protein fragmentation, protein-protein interactions, affinity tag positioning, oligomerization and polyglutamine tract length affect assay signal intensity, indicating that absolute HTT quantitation in heterogeneous biological samples is not possible with current technologies using a single standard protein. We also explore the binding specificity of the MW1 anti-polyglutamine antibody, commonly used in these assays as a mHTT-selective reagent and demonstrate that mHTT binding is preferred but not specific. Furthermore, we find that MW1 depletion is not only incomplete, leaving residual mHTT, but also non-specific, resulting in pull down of some wildtype HTT protein. Based on these observations, we recommend that mHTT detection assays report only relative mHTT quantitation using normalized arbitrary units of assay signal intensity, rather than molar concentrations, in the assessment of central nervous system HTT lowering in ongoing clinical and preclinical studies, and that MW1-depletion not be used a method for quantifying wildtype HTT protein.

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Harding, R. J., Xie, Y., Caron, N. S. J., Findlay-Black, H., Lyu, C., Potluri, N., Chandrasekaran, R., Hayden, M. R., Leavitt, B. R., Langbehn, D., Southwell, A.. 2024-09-27. Challenges and advances for huntingtin detection in cerebrospinal fluid: in support of relative quantification. https://doi.org/10.1101/2024.09.25.614766

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