Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / allele-specific silencing of mutant huntingtin - PubMed (original) (raw)

Figure 3

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).