Differential Effects on Allele Selective Silencing of Mutant Huntingtin by Two Stereoisomers of α,β-Constrained Nucleic Acid

We describe the effects of introducing two epimers of neutral backbone alpha,beta-constrained nucleic acid (CNA) on the activity and allele selectivity profile of RNase H active antisense oligonudeotides (ASOs) targeting a single nucleotide polymorphism (SNP) for the treatment of Huntington's disease (HD). ASOs modified with both isomers of alpha,beta-CNA in the gap region showed good activity versus the mutant allele, but one isomer showed improved selectivity versus the wild-type allele. Analysis of the human RNase H cleavage patterns of alpha,beta-CNA modified ASOs versus matched and mismatched RNA revealed that both isomers support RNase H cleavage on the RNA strand across from the site of incorporation in the ASO an unusual observation for a neutral linkage oligonucleotide modification. Interestingly, ASOs modified with (R)and (S)-5'-hydroxyethyl DNA (RFIE and SHE respectively) formed by partial hydrolysis of the dioxaphosphorinane ring system in alpha,beta-CNA also showed good activity versus the mutant allele but an improved selectivity profile was observed for the RUE modified ASO. Our observations further support the profiling of neutral and 5'-modified nucleic acid analogs as tools for gene silencing applications.