Validation of Ultrasensitive Mutant Huntingtin Detection in Human Cerebrospinal Fluid by Single Molecule Counting Immunoassay

Abstract

Background:The measurement of disease-relevant biomarkers has become a major component of clinical trial design, but in the absence of rigorous clinical and analytical validation of detection methodology, interpretation of results may be misleading. In Huntington’s disease (HD), measurement of the concentration of mutant huntingtin protein (mHTT) in cerebrospinal fluid (CSF) of patients may serve as both a disease progression biomarker and a pharmacodynamic readout for HTT-lowering therapeutic approaches. We recently published the quantification of mHTT levels in HD patient CSF by a novel ultrasensitive immunoassay-based technology and here analytically validate it for use.Objective:This work aims to analytically and clinically validate our ultrasensitive assay for mHTT measurement in human HD CSF, for application as a pharmacodynamic biomarker of CNS mHTT lowering in clinical trials.Methods:The single molecule counting (SMC) assay is an ultrasensitive bead-based immunoassay where upon specific recognition, dye-labeled antibodies are excited by a confocal laser and emit fluorescent light as a readout. The detection of mHTT by this technology was clinically validated following established Food and Drug Administration and European Medicine Agency guidelines.Results:The SMC assay was demonstrated to be accurate, precise, specific, and reproducible. While no matrix influence was detected, a list of interfering substances was compiled as a guideline for proper collection and storage of patient CSF samples. In addition, a set of recommendations on result interpretation is provided.Conclusions:This SMC assay is a robust and ultrasensitive method for the relative quantification of mHTT in human CSF.

Huntingtin Lowering Strategies for Disease Modification in Huntington’s Disease

“…Additionally, this study provided evidence that mHTT is released by dying neurons, suggesting that CSF mHTT levels may reflect ongoing neurodegeneration (Southwell et al, 2015). The single-molecule counting immunoassay was recently validated according to clinical regulatory standards (Fodale et al, 2017). Together, these studies provide a basis for the use of CSF mHTT levels to assess CNS target engagement in HTT lowering studies and were critical in generating the first evidence of successful mHTT lowering in the Ionis ASO trial (Leavitt et al, 2016).…”

AAV5-miHTT Lowers Huntingtin mRNA and Protein without Off-Target Effects in Patient-Derived Neuronal Cultures and Astrocytes

“…Htt protein expression in cell pellet homogenates was analyzed by an ultrasensitive single molecular counting (SMC) immunoassay (IRBM Science Park, Pomezia, Italy), similar to the previously described analysis of mutant Htt protein 27 . The assay is based on the use of a capture antibody (2B7), coated on magnetic beads, and a detection antibody (MAB2166) labeled with a fluorescent dye.…”

Fluid and imaging biomarkers for Huntington's disease

“…This assay uses the 2B7 antibody, which binds the very N-terminus of HTT, for capture, and the MW1 antibody, which binds to polyglutamine tracts, for detection. It has since been validated to a high standard along the guidelines for regulatory approval (Fodale, Boggio et al 2017) and is in use in multiple clinical trials.…”