Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS

TY - JOUR

T1 - Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS

AU - Østergaard, Michael E

AU - Southwell, Amber L

AU - Kordasiewicz, Holly

AU - Watt, Andrew T

AU - Skotte, Niels H

AU - Doty, Crystal N

AU - Vaid, Kuljeet

AU - Villanueva, Erika B

AU - Swayze, Eric E

AU - Bennett, C Frank

AU - Hayden, Michael R

AU - Seth, Punit P

PY - 2013/11

Y1 - 2013/11

N2 - 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.

AB - 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.

KW - Alleles

KW - Animals

KW - Base Pairing

KW - Brain

KW - Cells, Cultured

KW - Down-Regulation

KW - Fluorine

KW - Humans

KW - Huntington Disease

KW - Mice

KW - Mice, Transgenic

KW - Mutation

KW - Nerve Tissue Proteins

KW - Oligonucleotides, Antisense

KW - Polymorphism, Single Nucleotide

KW - Rats

KW - Rats, Sprague-Dawley

KW - Ribonuclease H

U2 - 10.1093/nar/gkt725

DO - 10.1093/nar/gkt725

M3 - Journal article

C2 - 23963702

SN - 0305-1048

VL - 41

SP - 9634

EP - 9650

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 21

ER -