Abstract
Huntington’s disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1–97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.
Highlights
Huntington’s disease (HD) is a neurodegenerative genetic disorder that leads to motor dysfunction and cognitive decline [1]
HD is caused by the abnormal expansion of CAG trinucleotide repeats within exon 1 of the huntingtin (HTT) gene, which leads to the expansion of the poly-glutamine stretch located in the N-terminus of the HTT protein [4]
Meso Scale Discovery (MSD) assays have been described for detection of mouse HTT, human HTT or mutant HTT [19]
Summary
Huntington’s disease (HD) is a neurodegenerative genetic disorder that leads to motor dysfunction and cognitive decline [1]. The expanded polyQ stretch increases the propensity of the HTT protein to form aggregates that accumulate within both the nucleus and cytoplasm of cells, most prominently in neurons. The rate of mHTT aggregation is proportional to the increase in CAG repeat length and age [6], and correlates with susceptibility to cell death and loss of neuronal tissue [7, 8]. Modulation of mHTT and mHTT aggregate levels represents a promising therapeutic approach for HD patients. For this reason, multiple HTT-lowering agents such as antisense oligonucleotides (ASO), small interfering RNAs (siRNA), micro RNAs (miRNA), and zinc finger repression proteins (ZFP) are in or under development for clinical testing based on promising results generated in preclinical models
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