Antisense Oligonucleotides (ASO) are short (12 to 25 base-pairs) chemically modified oligonucleotides which bind to their complementary mRNA in cells by Watson-Crick base-pairing and modulate gene expression via multiple pathways. Over the years, a large number of 2'-modified nucleotides have been investigated to improve the drug-like properties of ASOs. For example, introducing electron withdrawing groups at the 2'-position shifts the conformational equilibrium of the furanose ring towards the C3'-endo sugar pucker and improves ASO binding affinity for complementary RNA. Further conformational restriction of the 2'-substituent into the 4'-position of the furanose ring or completely replacing the furanose ring with a hexitol or a cyclohexenyl ring system can provide dramatic to moderate improvements in binding affinity of the modified oligonucleotides for complementary nucleic acids. We recently undertook the rational design of RNase H active antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms (SNPs) for allele selective silencing of mutant Huntingtin protein in Huntington's disease (HD) patient-derived fibroblasts and in the CNS of a completely humanized mouse model of HD. We found that position specific incorporation of 2'-modified nucleotides, which differentially bias the conformation of the furanose ring, within the DNA gap-region of the ASO can profoundly improve allele selectivity without compromising activity against the mutant allele. The modified ASOs were also well tolerated after injection into the CNS of wild-type animals suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings highlight the effect of subtle structural changes to modulate ASO behavior and lay the foundation for efficient allele-selective silencing of gene expression using ASOs � an outcome with broad application to dominant genetic disorders.

Conformational tuning of the furanose ring in antisense oligonucleotides to achieve allele selective silencing of mutant Huntingtin in the CNS

246th National Meeting (2013)

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