Related Experiment Video
Updated: Apr 30, 2026
Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy
Published on: June 27, 2018
Quantification assays for total and polyglutamine-expanded huntingtin proteins
Douglas Macdonald1, Michela A Tessari2, Ivette Boogaard3
1CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.
Insights
Researchers developed new assays to accurately measure huntingtin protein (HTT) levels in Huntington's disease (HD) models and patient samples. These sensitive electrochemiluminescence assays are crucial for developing and testing new HD therapies targeting mutant HTT.
Area of Science:
- Neuroscience
- Genetics
- Biochemistry
Background:
- Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the huntingtin gene, leading to mutant huntingtin protein (mutant HTT) with an expanded polyglutamine tract.
- RNAi suppression of mutant HTT shows promise in ameliorating HD phenotypes in animal models, highlighting the need for precise HTT measurement methods.
Purpose of the Study:
- To develop sensitive and selective assays for quantifying total and mutant huntingtin protein (HTT) levels.
- To establish methods for measuring endogenous mouse and rat HTT in preclinical HD models.
- To validate these assays in various HD in vitro, in vivo, and patient sample systems.
Main Methods:
- Development of electrochemiluminescence assays using the Meso Scale Discovery (MSD) platform.
- Creation of assays to detect total human HTT, polyglutamine-expanded human HTT, and endogenous mouse/rat HTT.
- Utilized purified recombinant HTT proteins as standards for absolute quantification.
Main Results:
- Developed assays with increased dynamic range compared to existing methods.
- Successfully measured HTT protein levels in diverse HD models (cellular, animal) and human patient biosamples.
- Demonstrated the utility of assays for monitoring both mutant and wild-type HTT in response to interventions.
Conclusions:
- The developed MSD-based assays provide a robust and sensitive platform for HTT quantification in HD research.
- These assays are essential tools for preclinical and clinical studies evaluating the efficacy of mutant HTT-lowering therapies.
- Accurate HTT measurement facilitates the assessment of therapeutic effects on both mutant and wild-type huntingtin protein.
Abstract:
The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.
