Site-specific incorporation of 5′-methyl DNA enhances the therapeutic profile of gapmer ASOs

• Published in: Nucleic acids research Vol. 49; no. 4; pp. 1828 - 1839
• Main Authors: Vasquez, Guillermo, Freestone, Graeme C, Wan, W Brad, Low, Audrey, De Hoyos, Cheryl Li, Yu, Jinghua, Prakash, Thazha P, Ǿstergaard, Michael E, Liang, Xue-hai, Crooke, Stanley T, Swayze, Eric E, Migawa, Michael T, Seth, Punit P
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 26-02-2021
• Subjects: Animals; Chemical Biology and Nucleic Acid Chemistry; DNA - chemistry; Glucose - analogs & derivatives; Glucose - chemistry; HeLa Cells; Humans; Liver - drug effects; Male; Mice; Mice, Inbred BALB C; NIH 3T3 Cells; Oligonucleotides, Antisense - chemistry; Oligonucleotides, Antisense - therapeutic use; Oligonucleotides, Antisense - toxicity; Organophosphorus Compounds - chemical synthesis; Ribonuclease H
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkab047

Abstract
We recently showed that site-specific incorporation of 2′-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5′-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5′-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2′-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5′-Me and R-5′-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5′-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.