Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA and Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, V5Z 4H4, Canada.
Nucleic Acids Res. 2013 Nov;41(21):9634-50. doi: 10.1093/nar/gkt725. Epub 2013 Aug 19.
Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.
常染色体显性疾病，如亨廷顿病 (HD)，是由功能获得性突变蛋白和/或 RNA 引起的。这些疾病的理想治疗方法是选择性地抑制突变等位基因的表达，同时保留野生型变异体的表达。具有 RNase H 活性的反义寡核苷酸 (ASO) 或小干扰 RNA 可以通过靶向与重复扩展相关的单核苷酸多态性 (SNP) 来实现基因表达的等位基因选择性抑制。ASO 以前被证明可以通过 5 倍的选择性来区分靶向 RNA 中的单核苷酸变化。基于 RNase H 酶学，我们通过在寡核苷酸中位置整合化学修饰来增强单核苷酸的区分，从而限制 RNase H 对非靶向转录本的切割。在导致亨廷顿病的 huntingtin mRNA 中的 SNP 位点、在患者细胞中和在完全人源化的 HD 小鼠模型中，这些修饰后的 ASO 对单个核苷酸变化的区分度大于 100 倍。在野生型动物的中枢神经系统中注射修饰后的 ASO 后，耐受性也很好，这表明它们的耐受性适合作为潜在的等位基因选择性 HD 治疗药物的进一步开发。我们的研究结果为使用 ASO 进行高效的基因表达等位基因选择性下调奠定了基础——这一结果广泛适用于 HD 和其他显性遗传疾病。
