Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

Figure 1. Characterisation of allele-selective siRNAs targeted to three SNPs in heterozygous human HD patient cells. Monocyte-derived macrophages were isolated from SNP-genotyped HD patients and treated with GeRPs containing either nonsense, anti-total HTT or allele-selective siRNA targeted to (a) rs362331 in exon 50, (b) rs362273 in exon 57 or (c) rs362307 in the 3′-UTR of the HTT transcript. Expression of each SNP allele was then measured 72 h later by qPCR. Data show mean expression of each SNP allele ± SEM (n = 3 for rs362331, n = 4 for rs362273 and rs362307), statistical analysis carried out using two-way ANOVA with Bonferroni post-tests. Asterisks above the horizontal line at the top of each graph show significance on ANOVA across the whole experiment before post-hoc testing. Asterisks above individual bars represent post-test significance for the mRNA expression of that allele compared to the same allele in the nonsense-treated samples. Asterisks above horizontal lines between two bars denote allele expression differences within the same treatment on post-hoc testing. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N = individual biological repeats.

Figure 2. Selective suppression of mHTT protein in human HD patient cells using siRNA targeted to rs362331. Monocyte-derived macrophages were isolated from HD patients heterozygous for rs362331 with linkage of the T allele to mHTT, before treating with GeRPs containing either nonsense, anti-total HTT or allele-selective siRNA targeted to each allele of rs362331. Expression of (a) total and (b) mutant HTT protein was measured after 72 h using MSD assays, before normalisation to total cellular protein content as measured by BCA assays. Data show mean protein levels ± SEM (n = 5), statistical analysis carried out using one-way ANOVA with Tukey post-hoc testing. Asterisks above the horizontal line at the top of each graph show significance on ANOVA across the whole experiment before post-hoc testing. Asterisks above bars show post-test significance for each siRNA compared to the nonsense-treated samples. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N = individual biological repeats.

Figure 3. Allele-selective suppression of wild-type and mHTT reverses the hyper-reactive phenotype of human HD myeloid cells. Monocyte-derived macrophages were isolated from SNP-genotyped HD patients and treated with GeRPs containing either nonsense, anti-total HTT or allele-selective siRNA targeted to each allele of rs362331. Linkage of each SNP allele to either wild-type or mHTT was determined using the SNP linkage by circularization protocol. After 72 h the macrophages were stimulated with LPS and IFNγ, before the culture supernatants were collected after a further 24 h and analysed using MSD assays. Cytokine values were normalised to total protein content as measured by BCA assays. Data show mean cytokine levels ± SEM (n = 5 for IL-6 and TNFα, n = 4 for IL-8), statistical analysis carried out using one-way ANOVA with Tukey post-hoc testing. Asterisks above the horizontal line at the top of each graph show significance on ANOVA across the whole experiment before post-hoc testing. Asterisks above bars show post-test significance for each siRNA compared to the nonsense-treated samples. *P < 0.05, **P < 0.01, ***P < 0.001. N = individual biological repeats.