Identification of SNP alleles that are associated with HD chromosomes and can be used for allele-specific targeting in HD. A SNP allele was defined as a disease allele if it is significantly more common on HD than control chromosomes. In a population of 234 Caucasian patients with HD, we identify 50 SNPs with disease alleles (chi-squared, Bonferroni-corrected P value P < 0.00055) and present the heterozygosity rate for the disease allele in patients with HD (percentage heterozygosity in italics). The best targets for allele-specific therapy will be specific SNP alleles that maximize both the frequency on HD chromosomes and heterozygosity in patients with HD. HD, Huntington disease; SNP, single-nucleotide polymorphism.

Development and use of a primary cortical neuron counter screen utilizing YAC and BACHD transgenic mice. (a) Left—Development of primary neuron assay in cortical neurons from the YAC128 mice. Bath application at DIV 2 with a non-allele-specific positive control ASO leads to clear dose-dependent silencing of HTT (IC₅₀ = 27.03 nmol/l, 95% CI 22.66–32.24 nmol/l) after 6 days of treatment. Right—Treatment of primary cortical neurons from YAC18 and BACHD mice with 500 or 1,500 nmol/l of non-allele-specific positive control ASO leads to potent reduction of transgenic human HTT, detected with a human HTT-specific antibody (N = 6 YAC18 cultures, 10 BACHD cultures; 2-way ANOVA genotype F(1,42) = 23.08, P < 0.0001; treatment F(1,42) = 1170, P < 0.0001; interaction F(1,42) = 6.2, P = 0.0042). (b) Paired primary cortical neurons from YAC18 or BACHD mice were treated after 2 days in vitro with the indicated doses of ASO for 6 days and HTT detected with a human-specific HTT antibody. ASOs targeting SNP alleles selectively knockdown BACHD, but not YAC18, HTT transgene. rs7685686: N = 6 YAC18 cultures, 6 BACHD cultures; 2-way ANOVA genotype F(1,30) = 67.24, P < 0.0001; treatment F(2,30) = 24.9, P < 0.0001; interaction F(2,30) = 17.5, P <0.0001. rs4690072: N = 6 YAC18 cultures, 5 BACHD cultures; 2-way ANOVA genotype F(1,24) = 29.6, P < 0.0001; treatment F(2,24) = 18.3, P < 0.0001; interaction F(2,24) = 10.0, P <0.0001. rs2024115: N = 6 YAC18 cultures, 4 BACHD cultures; 2-way ANOVA genotype F(1,33) = 66.2, P < 0.0001; treatment F(2,33) = 7.5, P = 0.0021; interaction F(2,33) = 17.0, P <0.0001. rs363088: N = 6 YAC18 cultures, 4 BACHD cultures; 2-way ANOVA genotype F(1,24) = 62.3, P < 0.0001; treatment F(2,24) = 9.3, P = 0.0010; interaction F(2,24) = 16.2, P <0.0001.(*, **** Represent Bonferroni multiple comparison P values of P < 0.05 and P < 0.0001, respectively).

ASOs are potent and selective after acute delivery to the murine central nervous system (CNS). (a) YAC18 and BACHD mice were injected intrastriatally with PBS vehicle (sham) or 50 µg ASO (non-allele-specific positive control or allele-specific targeting rs7685686). Injected striata were examined with antibodies reactive to the ASO backbone, DARPP-32, and GFAP. ASO immunoreactivity is observed throughout the DARPP-32 positive striatum in injected animals. GFAP immunoreactivity is limited proximally to the needle tract in both sham- and ASO-injected animals. (b) Transgenic HTT levels were examined by western blot 14 days post-injection. Both YAC18 and BACHD transgenes are reduced after injection with non-allele-specific ASO, while the ASO targeting rs7685686_A is only potent in the BACHD striatum (YAC18 versus BACHD Bonferroni post-test t = 8.4, P < 0.0001). ASO, antisense oligonucleotide; GFAP, glial fibrillary acidic protein; PBS, phosphate-buffered saline.

Selectivity and potency of lead ASOs in primary human HD patient fibroblasts. (a) Forty-eight ASOs targeting SNPs reduce total HTT mRNA in a panel of on target human HD fibroblasts (black bars = range). (b) Summary of single dose cell line counter screen demonstrating selectivity of ASOs targeting SNPs in homozygous on (top panel) and off (bottom panel) target human HD fibroblasts. (c) Sample dose–response data in primary on and off target fibroblasts. Left—An ASO targeting rs7685686_A is more potent in on target cell lines versus off target. Right—A non-SNP-targeted positive control ASO shows equivalent silencing in both cell lines. F-values in c are the results of an extra sum of squares test after fitting a log(inhibitor) versus concentration curve. ASO, antisense oligonucleotide; HD, Huntington disease; SNP, single-nucleotide polymorphism.

An ASO with S-constrained ethyl motif (cET) backbone has increased potency and equivalent allele-selectivity to a traditional MOE backbone. (a) Motifs used in ASO backbones. (b) Dose–response curves for HTT silencing after electroporation of human HD fibroblasts with MOE or cEt-modified ASOs targeting rs7685686_A. ASOs were, in each case, more potent toward HTT in cell lines homozygous for the targeted SNP allele versus homozygous for the untargeted allele. ASO potency is increased with no loss of specificity by addition of cEt motifs to wing structures, relative to 5-9-5 MOE design (IC₅₀'s indicated). Error bars indicate range, and lines indicate fit curves. (c) Verification of increased potency and preserved specificity of cEt-modified ASOs targeting rs7685686_A (blue) toward HTT protein in BACHD neurons, relative to the equivalent MOE ASO (red). (Two-way ANOVA effect of ASO modification in BACHD neurons P < 0.0001. **, **** Represent Bonferroni multiple comparisons between MOE ASO and cET ASO P values of P < 0.01 and P < 0.0001, respectively). ASO, antisense oligonucleotide; MOE, 2'-O-Methoxyethyl.