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.
Highlights
The measurement of disease-causing mutant and/or misfolded proteins is essential for the successful development of diseasemodifying therapies that target such pathogenic or pathologic proteins
We demonstrate that using our standard reference proteins, we are able to quantitatively compare the amount of huntingtin protein (HTT) protein in a variety of samples which will provide guidance for dose selection and provide an ability to monitor the pharmacodynamic effects of molecular therapies and other approaches that modulation the levels of HTT protein, an important and critical step in the development of such targeted therapies
Anti-huntingtin antibody characterization We characterized a panel of anti-HTT antibodies raised to different epitopes of the HTT protein (Figure 1A and Table S1 in Text S1) by immunoblot using purified large fragment (HTT 1– 573) recombinant HTT proteins with different CAG repeat lengths (Figure 1B) as well as brain homogenates obtained from 3 month-old BAC Huntington’s disease (HD) mice or wild type littermates (Figure 1C)
Summary
The measurement of disease-causing mutant and/or misfolded proteins is essential for the successful development of diseasemodifying therapies that target such pathogenic or pathologic proteins. Development of assays to detect these types of proteins is dependent on the availability of selective antibodies as well as a sensitive and robust platform for detection. To this end, we have characterized a set of novel antibodies and employed a unique assay platform for the detection of the huntingtin protein (HTT), the causative agent in Huntington’s disease (HD). The gene product is a ubiquitously-expressed 350 kDa HTT protein, with the greatest expression found in the central nervous system [2]. The mutant polyglutamine expanded form of HTT is cytotoxic leading to the hallmark pathology of HD, pronounced atrophy of the striatum as well as other brain regions [3]
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