Fully 2‘-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA

Abstract

We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.

Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2‘-O-Ribonucleic Acid Modifications,

“…RNA interference (RNAi) experiments using chemically modified siRNAs have only been conducted to a limited extent, and relatively few modifications have been evaluated in vitro or in vivo to date (reviewed in ref 34). Given the fact that the 2′-fluoro (35) and, more recently, the 2′-O-methyl modification in combination with cholesterol conjugation in an in vivo application (36) have shown increased activity in silencing of genes via RNAi, 2′-O-modifications with promising properties regarding RNA affinity and nuclease protection that we have analyzed here may well proceed to more detailed trials in the context of RNAi.…”

“…Compound 11 (11.67 g, 85% yield) was synthesized from 3 (7.00 g, 22.27 mmol), 4,4′-dimethoxytrityl chloride (9.05 g, 26.72 mmol), and 150 mL of anhydrous pyridine with stirring of the reaction mixture for 8 h using a procedure similar to that used for the synthesis of 14. MS (AP-ES): m/z calculated for C 35 (12). Compound 4 (5 g, 16.43 mmol) was coevaporated with pyridine (2 × 20 mL), and the residue obtained was dissolved in anhydrous pyridine (40 mL).…”

Therapeutic targeting in the silent era: advances in non-viral siRNA delivery

“…Certain chemical modifications can be introduced almost all bases of both RNA strands, whereas other specific modifications must be placed only at certain positions within the siRNA strands [36] . For example, stability against nuclease degradation can be achieved via the introduction of a phosphorothioate (P=S) backbone linkage at the 3' end for exonuclease resistance and a 2' modifications (2'-O-methyl and 2'-fluoro) for endonuclease resistance [37][38][39][40] . In relation to maintaining RNAi silencing activity, exonuclease-stabilizing modifications are well tolerated.…”

Using non-coding small RNAs to develop therapies for Huntington's disease

“…Chemical changes to the sugars, backbone, or bases of nucleic acid make them more like conventional drugs with significantly increased stability, in-vivo potency and less immunostimulation. [69][70][71][72][73][74][75] After comparison of 10 types of chemical modification-including peptide nucleic acid, locked nucleic acids, (S)-cET bridged-nucleic acid, carba-locked nucleic acid, ethylene nucleic acid, altritol nucleic acid, 2¢-O-methoxyethyl, 2¢-fluoro and 2¢-fluoroarabino nucleic acid oligomers-for allele-selective inhibition, bridged-nucleic acids (includes locked nucleic acid) were highly successful and were considered a lead modification of oligomer for allele-specific silencing mutant HD. 24 To apply siRNA or other synthetic nucleic acid to inhibit mutant HD, the most prudent and robust strategy is to synthesize and screen a substantial library of siRNA duplexes (or oligomers) for multiple sites in mutant HD mRNA to identify the most promising one.…”