Data in support of selective targeting of mutant huntingtin intron-1 improves rescue provided by antisense oligonucleotides in HttQ111/+ mice
Data files
Mar 05, 2026 version files 6.77 MB
-
Bragg_data.xlsx
6.76 MB
-
README.md
16.40 KB
Abstract
Huntington’s disease (HD) arises from the toxic gain-of-function caused by a CAG expansion in the coding region of the HTT gene. HD is increasingly appreciated to emerge from multiple pathogenic processes, including somatic instability in mutant HTT’s (mHTT) CAG repeat tract, which leads to diverse deleterious consequences. These include the alternative processing of HTT pre-mRNA to generate the HTT1a transcript that encodes the very toxic, mHTT isoform referred to as HTT1a. We set out to compare the efficacy and safety of allele-selective lowering of mHTT compared to non-allele-selective lowering using antisense oligonucleotides (ASOs) in heterozygous HttQ111 (Q111) mice. We developed a mutant-specific ASO (MutASO) targeting Htt intron-1 that selectively reduced mutant full-length HTT, as well as HTT1a, in the brains of Q111 mice. Compared to the rescue provided by a pan-allele-targeting ASO (PanASO) that lowers wild-type HTT and full-length mHTT (sparing HTT1a), the MutASO essentially eliminated aggregate formation and provided marked protection from transcriptional dysregulation in HD knock-in mice. Thus, by targeting the ASO to the region upstream of the cryptic polyadenylation sites required to generate the HTT1a transcript, our allele-selective MutASO potently reduced HTT1a protein levels. Here, our findings advocate that HTT1a may have a disproportionate impact on aggregate formation and transcriptional dysregulation and that lowering the levels of HTT1a could provide benefit when designing HTT-lowering-based therapeutic strategies for HD.
https://doi.org/10.5061/dryad.6wwpzgn8j
Description of the data and file structure
The dataset and R scripts (uploaded to Zenodo) used to generate all statistics and figures for the associated manuscript are deposited here.
The 'Bragg_data.xlsx' file comprises 17 separate worksheets with a leading number (e.g., 1b, 1e, etc) which indicates where the data was used in the manuscript. Each worksheet includes a description of the data, and a description of each column and variable is defined below.
Worksheet(s): ‘fig.1b’, ‘fig.1e’
Description: Figure 1 describes the development of mutant allele-selective ASOs in the context of Q111 mice. Datasets included are for Fig 1B: Htt mRNA abundance in IPSC-derived neurons after 7 days of treatment with 0.4uM, 4uM, or 20uM ASO targeting human intron 1 sequences; Fig 1E: wtHTT and mHTT protein abundance from the cortex of Q111 mice treated with 300 µg of ASO by unilateral ICV injection. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| fig.1b | complex_name | ASO identity |
| fig.1b | dose_um | ASO dose (micromolar) |
| fig.1b | lin_estimate | Htt expression |
| fig.1b | lin_sem_lower | Expression, SEM, lower bound |
| fig.1b | lin_sem_upper | Expression, SEM, upper bound |
| fig.1e | Tissue | Tissue measured by MSD |
| fig.1e | Mouse | Animal ID |
| fig.1e | ASO | ASO ID |
| fig.1e | duration | Treatment duration (weeks) |
| fig.1e | mean.pg.ml | Mean pg/ml of Htt |
| fig.1e | allele | Huntingtin allele |
| fig.1e | normalized. y.group | HTT concentration, normalized for each input control |
Worksheet(s): ‘fig2’
Description: Figure 2 demonstrates that ASO-induced HTT lowering requires multiple injections for a longitudinal study. Datasets included are for Fig 2A, which measured wtHTT and mHTT protein abundance in the cortex of Q111 mice at 4, 10, and 16 weeks after a single unilateral ICV injection of the indicated treatment. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| fig2 | Tissue | Tissue measured by MSD |
| fig2 | Mouse | Animal ID |
| fig2 | ASO | ASO ID |
| fig2 | allele | Huntingtin allele |
| fig2 | Duration | Treatment duration (weeks) |
| fig2 | normalized.HTT | HTT concentration, normalized for each input control |
Worksheet(s): ‘fig3a’, ‘fig3b’, ‘fig3d_1’, ‘fig3d_2’
Description: Figure 3 shows that both wtHTT and mHTT levels were reduced at the end of our interventional study. Datasets included are for Fig 3A: HTT protein in cortex of WT and Q111 mice treated with saline, PanASO, or MutASO harvested at 26 weeks following initial ICV injection measured by MSD; Fig 3B: additional analysis of mHTT protein levels in the striatum, cortex, and cerebellum measured by HTRF; Fig 3D: plasma NEFL levels measured by MSD assay at 4-weeks or 26-weeks after the initial ICV injection of saline or indicated ASO. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| fig3a | Mouse | Animal ID |
| fig3a | Sex | Animal sex |
| fig3a | Genotype | Animal genotype |
| fig3a | Tissue | Tissue measured by MSD |
| fig3a | ASO | ASO ID |
| fig3a | pg.ml.mean | Mean pg/ml of HTT |
| fig3a | Assay | MSD assay ID |
| fig3a | Normalized by. geno | HTT concentration, normalized for each input control |
| fig3b | ASO | ASO ID |
| fig3b | Mouse | Animal ID |
| fig3b | Tissue | Tissue measured by HTRF |
| fig3b | Signal | Raw HTRF signal |
| fig3b | WT_blank_normalized | HTRF signal with blank well subtracted |
| fig3d_1 | mouse | Animal ID |
| fig3d_1 | pg.ml.mean | Mean pg/ml of NEFL |
| fig3d_1 | ASO | ASO ID |
| fig3d_2 | Mouse | Animal ID |
| fig3d_2 | Genotype | Animal genotype |
| fig3d_2 | ASO | ASO ID |
| fig3d_2 | pg.ml.mean | Mean pg/ml of NEFL |
Worksheet(s): ‘fig4bc’
Description: Figure 4 shows that MutASO, but not PanASO, reduces soluble and aggregated HTT1a fragments as measured by HTRF. Datasets included are for Fig 4C: soluble levels of HTT1a levels detected with the 2B7 and 1B12 antibody pair; Fig 4D: aggregated HTT1a levels detected with the 4C9 and 1B12 antibody pair from striatum, cortex, and cerebellum in Q111 mice 26-weeks after initial treatment of Saline, PanASO, or MutASO. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| fig4bc | Mouse | Animal ID |
| fig4bc | ASO | ASO ID |
| fig4bc | Signal | Raw HTRF signal |
| fig4bc | Signal_blank_norm | HTRF signal with blank well subtracted |
| fig4bc | Assay | HTRF assay ID |
| fig4bc | Species | HTT allele measured |
| fig4bc | Tissue | Tissue measured |
Worksheet(s): ‘fig5a’, ‘fig5b’
Description: Figure 5 shows immunohistochemistry for mHTT neuronal nuclear aggregates in the striatum reveals the effect of MutASO, but not PanASO, treatment. The dataset included contains quantification of MW8+ positive neurons for generating Fig. 5A and S380 immunoreactive area per cell for generating Fig 5B. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| fig5a | Mouse | Animal ID |
| fig5a | Genotype | Animal genotype |
| fig5a | Sex | Animal sex |
| fig5a | ASO | ASO ID |
| fig5a | Percent | Percent of cells with HTT aggregates, per animal |
| fig5b | Pct.Area | Percent of cells with HTT positive staining |
| fig5b | image | Image ID |
| fig5b | Mouse | Animal ID |
| fig5b | Genotype | Animal genotype |
| fig5b | Sex | Animal sex |
| fig5b | ASO | ASO ID |
Worksheet(s): ‘fig6’, ‘fig6meta’, ‘fig6a’
Description: Figure 6 shows that MutASO, but not PanASO, rescues broad patterns of transcriptional dysregulation in the striatum of Q111 mice. Datasets included (fig6, fig6meta, fig6a) are used to build all figures in Fig 6 including Fig 6a: The number of up- and down-regulated striatal DEGs between saline treated WT and Q111 mice this study was compared with historical data from similar aged naïve mice (9-months) in Bragg et al., 2017; Fig 6B and 6C with volcano plots indicating the changes in gene expression induced by PanASO (6B) and MutASO (6C) in Q111 mice; Fig 6D and 6E: Heatmaps of gene expression produced by unsupervised hierarchical clustering of the 1175 Q111 induced DEGs between saline treated WT and Q111 mice from 6A. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| fig6 | Gene | Gene name |
| fig6 | CQQ2 | RNAseq counts, animal ID |
| fig6 | CQR2 | RNAseq counts, animal ID |
| fig6 | CQW1 | RNAseq counts, animal ID |
| fig6 | CQX3 | RNAseq counts, animal ID |
| fig6 | CRQ2 | RNAseq counts, animal ID |
| fig6 | CRQ6 | RNAseq counts, animal ID |
| fig6 | CRX5 | RNAseq counts, animal ID |
| fig6 | CSE1 | RNAseq counts, animal ID |
| fig6 | CSE2 | RNAseq counts, animal ID |
| fig6 | CSF3 | RNAseq counts, animal ID |
| fig6 | CSG1 | RNAseq counts, animal ID |
| fig6 | CSG2 | RNAseq counts, animal ID |
| fig6 | CSG5 | RNAseq counts, animal ID |
| fig6 | CSH6 | RNAseq counts, animal ID |
| fig6 | CSI3 | RNAseq counts, animal ID |
| fig6 | CSI4 | RNAseq counts, animal ID |
| fig6 | CSI5 | RNAseq counts, animal ID |
| fig6 | CSI6 | RNAseq counts, animal ID |
| fig6 | CSJ2 | RNAseq counts, animal ID |
| fig6 | CSK5 | RNAseq counts, animal ID |
| fig6 | CSL5 | RNAseq counts, animal ID |
| fig6 | CSN5 | RNAseq counts, animal ID |
| fig6 | CSO1 | RNAseq counts, animal ID |
| fig6 | CSO2 | RNAseq counts, animal ID |
| fig6 | CSO5 | RNAseq counts, animal ID |
| fig6 | CSO6 | RNAseq counts, animal ID |
| fig6 | CSR1 | RNAseq counts, animal ID |
| fig6 | CSR5 | RNAseq counts, animal ID |
| fig6 | CSS6 | RNAseq counts, animal ID |
| fig6 | CSU6 | RNAseq counts, animal ID |
| fig6meta | Mouse | RNAseq, animal ID |
| fig6meta | Sex | RNAseq, animal sex |
| fig6meta | Genotype | RNAseq, animal genotype |
| fig6meta | Treatment | RNAseq, animal sex |
| fig6a | logFC | Log(2) fold-change, WT vs mutant |
| fig6a | logCPM | Log CPM |
| fig6a | LR | Likelihood ratio test statistic |
| fig6a | PValue | Unadjusted p-value |
| fig6a | FDR | Adjusted p-value (false discovery rate) |
| fig6a | Tissue | Tissue ID |
| fig6a | Age | Animal age |
| fig6a | Gene_Name | Gene name |
Worksheet(s): ‘fig7a’
Description: Figure 7 demonstrates that treatment with MutASO, but not PanASO, robustly protects from core HD DEG dysregulation. Datasets included are forFigureg 7A: categorization of 188 genes comprising a common HD signature into 4 bins (Full Rescue, Partial Rescue, Negligible Rescue, or Exacerbated). A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| fig7a | Ensembl | Gene (ensembl ID) from the Str266 list |
| fig7a | Gene | Gene (symbol) from the Str266 list |
Worksheet(s): ‘figS1’
Description: Figure S1 shows inferred mHTT levels across the study. The dataset approximates levels of mHTT knockdown across the study based on separate experimental data endpoint levels at week 26. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| figS1 | Week | Weeks post ASO injection |
| figS1 | ASO | ASO ID |
| figS1 | mHTT | mHTT protein |
| figS1 | Genotype | Animal genotype |
Worksheet(s): ‘figS2’
Description: Figure S2 dataset contains behavioral measures (distance traveled and time spent inthe center) and shows no differences between saline-treated WT and Q111 mice. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| figS2 | Genotype | Animal genotype |
| figS2 | Mouse | Animal ID |
| figS2 | Target | Animal treatment |
| figS2 | Distance_travelled | Total distance travelled |
| figS2 | Center_percentage | Percent of time spent in the center of the arena |
Worksheet(s): ‘figS3’
Description: Figure S3 shows that MutASO reduces Htt1a transcripts as measured by QuantSeq. A description of each variable is listed below.
| Sheet | Header | Description |
|---|---|---|
| figS3 | chr | Mouse chromosome number |
| figS3 | start | Chromosome start position |
| figS3 | end | Chromosome end position |
| figS3 | species | Huntingtin transcript species |
| figS3 | sample | Sample ID |
| figS3 | mean_CPM | Mean counts per million (CPM) |
| figS3 | Mouse | Mouse ID |
| figS3 | Genotype | Animal genotype |
| figS3 | Treatment | Animal treatment |
Corresponding R scripts for analysis are also included. R scripts pull data from the .xlsx sheet and make plots and/or run statistics used in the manuscript, and include basic annotation. R script naming follows the same convention as the data, for example,e the script 'fig1b.R' is the script that plots and analyzes the data underlying Figure 1b in the manuscript. R scripts and descriptions are listed below.
| Figure | Description |
|---|---|
| fig1.R | Script for Figure 1 |
| fig2.R | Script for Figure 2 |
| fig3.R | Script for Figure 3 |
| fig4.R | Script for Figure 4 |
| fig5.R | Script for Figure 5 |
| fig6.R | Script for figure 6 |
| fig6a.R | Script for Figure 6a |
| fig7a.R | Script for Figure 7a |
| fig7b.R | Script for Figure 7b |
| fig7c.R | Script for figure 7c |
| figS1.R | Script for figure S1 |
| figS2.R | Script for figure S2 |
| figS3.R | Script for figure S3 |