Site-specific Incorporation of 2',5'-Linked Nucleic Acids Enhances Therapeutic Profile of Antisense Oligonucleotides

Abstract

Site-specific incorporation of 2'-modifications and neutral linkages in the deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined the effect of introducing 2',5'-linked RNA in the deoxynucleotide gap region on toxicity and potency of PS ASOs. Our results demonstrate that incorporation of 2',5'-linked RNA in the gap region dramatically improved hepatotoxicity profile of PS-ASOs without compromising potency and provide a novel alternate chemical approach for improving therapeutic index of ASO drugs.

Figure 1

(A). Structure of 3′,5′ and 2′,5′-PS-RNA. (B). Structural model of 3′,5′- and 2′,5′-RNA showing similar position of the nucleobases but different backbone geometries. (C). Chemical structure of CXCL12 cEt BNA gapmer ASO 1. (D). Structure of 3′-O-Me-2′,5′-PS-RNA

Figure 2

(A) Effect of nucleolar delocalization of p54nrb in cells treated with ASO 1 and 2, immunofluorescent staining of p54nrb in HeLa cells 4 h after transfection with 60 nM ASOs 1 and 2, toxic PS-ASO 1 induced nuclear localization of p54nrb and RNase H1 to nucleoli, RNA modified ASO 2 reduced nuclear localization of p54nrb to nucleoli. (B−C) In vitro and in vivo potency, ALT (IU/L) of mice treated with 150 mg kg⁻¹ and tissue concentration of ASO 1 and 2. *Concentration of ASOs 1−2 in livers of mice dosed at 16.7 mg kg⁻¹ via subcutaneous injection. (D) Site of metabolic cleavage in 3′,5′-DNA and 3′,5′-RNA in mouse liver. ASOs 1−2 blue letters indicate cEt BNA, black letters indicate DNA and red indicates 2′,5′-PS-RNA nucleotides.

Figure 3

(A,B) 2′,5′-PS-RNA modification was walked in the gap region (3–12) and the effect on duplex stability versus complementary RNA (T_m), potency (IC₅₀) in NIH3T3 cells, and cytotoxicity as measured by caspase activation in Hepa1–6 cells, and characterization of hepatotoxicity of ASOs 3–6 as measured by increase in plasma ALT (IU/L). (C) Characterizing the effect of 2′,5′-PS-RNA modification at positions 1–4 (3–6) in the DNA gap on potency as determined by the dose required to reduce CXCL12 mRNA by 50% in the livers relative to untreated control animal (ED₅₀). ASOs 1 and 3–12. Blue letters indicate cEt BNA, black indicates DNA, and red indicates 2′,5′-PS-RNA nucleotides. ND = not done. *ΔT_m relative to parent ASO 1.

Figure 4

(A−B) 3′-O-Me-2′,5′-PS-RNA modification was walked in the gap region (13−16) and the effect on duplex stability versus complementary RNA (T_m), potency (IC₅₀) in NIH3T3 cells, and cytotoxicity as measured by caspase activation in Hepa1-6 cells, and characterization of hepatotoxicity of ASOs 13−16 as measured by increase in plasma ALT (IU/L). (C) Effect of 3′-O-Me-2′,5′-PS-RNA in the DNA gap position 1−4 (ASOs 13−16) on activity in the livers of mouse dosed 17 mg kg⁻¹ of ASOs once via subcutaneous injection. ASOs 1, 13−16 blue letters indicate cEt, BNA, black letters indicate DNA and red indicates 3′-O-Me-2′,5′- PS-RNA nucleotides. *ΔT_m relative to parent ASO 1.