Successful quantification of mHTT in PBMCs from 8ml whole blood in HD.

<p>These data confirm that mHTT levels in leukocytes are higher at more advanced disease stages. Assay A (mHTT N-terminus assay) was used to selectively quantify mHTT: S/B, signal over background. A) Sample set 1 (mean of 2 replicates); (B) Sample set 2 (mean of 3 replicates).</p

Schematic diagram showing the capture and detection antibody pairs for each assay.

<p>The huntingtin amino acid residue epitope to which the antibodies bind are shown (not to scale). Poly-Q, polyglutamine region; Poly-P, proline rich region. Details of the antibodies are given in the text below.</p

Box plots showing average polyglutamine independent HTT levels across patient subgroups.

<p>(S/B: Signal / Background) No significant differences are seen between HTT species levels and disease stage, other than a difference in C-terminal HTT between Moderate and Early HD patients. (A) Distribution of Mid Region HTT (Assay C) in sample group 1. (B) Distribution of Mid Region HTT (Assay C) in sample group 2. (C) Distribution of polyglutamine-independent N terminal HTT (Assay B) in sample group 2. (D) Distribution of C terminal HTT (polyglutamine-independent Assay D) in sample group 2.</p

Association between HTT species level (S/B: Signal/Background, mean replicated samples) and disease burden score; adjusted for age, sex and CAG.

<p>(A) HTT mid region in sample set 1, (B) N-terminal HTT in sample set 2, (C) HTT mid region in sample set 2, (D) C-terminal HTT in sample set 2). Circles = premanifest, triangles = Early, squares = Moderate.</p

Results for each sample set and disease stage.

<p>Results for each sample set and disease stage.</p

Association between DBS and mHTT (S/B: Signal/Background, mean of replicated samples) in sample set 1 (A) and 2 (B).

<p>P values adjusted for age, sex and CAG.</p

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

<div><p>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 <i>in vitro</i> cellular and <i>in vivo</i> 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.</p></div

Specificity of human mutant and mouse HTT detection.

<p>The adeno-associated AAV-shRNA expression vector AAV-SEWB-sh4 was transduced into heterozygous zQ175 mouse primary neurons and humanized mutant (A) or endogenous mouse (B) HTT proteins were evaluated using the expanded polyglutamine human HTT MSD assay (antibody pair pAb146/MW1) or the mouse/rat HTT MSD assay (antibody pair pAb147/MAB2166), respectively. sh4, <i>HTT</i> targeting shRNA. scr6, scramble control shRNA. (C) Neuronal total tau protein levels measured using a commercially available MSD ELISA-based assay kit were monitored as loading control. Data are averages of n = 3 independent samples with correspondent standard deviations. ***, P<0.001. (D) Immunoblot confirming the AAV-mediated knockdown of humanized mutant (mut) and endogenous mouse HTT in transduced heterozygous zQ175 mouse primary neurons (AAV-SEWB-sh4: <i>HTT</i> targeting shRNA; AAV-SEWB-scr6: scrambled control shRNA). Immunoblot was probed for HTT (MAB2166, 1∶1,000; Millipore) or ATP5B as loading control.</p

HTT MSD assay sensitivity and dynamic range.

<p><i>Note</i>. Lower detection limit was determined from the calibration curve using the calculation of background mean +3× Standard deviation. Log units were defined by visual interpretation.</p><p>Antibody pairs are indicated as capture/detection antibody.</p

Anti-HTT antibody epitopes and analysis by immunoblot.

<p>(A) Diagram representing antibody epitopes on human HTT protein (relative to GenBank accession CAD38447.1). A stretch of glutamine (Q) residues near the N-terminus is expanded in individuals affected by Huntington's disease. Amino-acid 1-92 encoded by exon-1 are shown. Blue, pAb147 antibody epitope on mouse HTT protein (GenBank accession NP_034544). (B) Immunoblot analysis of human large fragment recombinant HTT proteins detected by the indicated anti-huntingtin antibodies. 5 ng of both HTT (1–573) Q23 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) and HTT (1–573) Q73 (lanes 2, 4, 6, 8, 10, 12, 14, and 16) purified large fragment proteins were analyzed by SDS-PAGE. M, molecular weight marker (kDa). (C) Immunoblot analysis of wild type littermate and transgenic BAC HD mouse whole brain extracts. Mouse endogenous wild type and human transgenic polyglutamine-expanded HTT proteins were detected using the indicated anti-huntingtin antibodies. 25 µg of normal (wt) and transgenic (tg) mouse brain extracts were analyzed by SDS-PAGE as indicated. M, molecular weight marker (kDa). Tg mHTT, transgenic human polyglutamine-expanded HTT. WT HTT, endogenous mouse wild type HTT.</p