The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'- O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs

Abstract

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

Keywords: 2′-O-methyl; RNase H1; antisense oligonucleotide; mesyl-phosphoramidate.

FIG. 1.

MsPA backbone improves activity, whereas gap2 OMe reduces toxicity. (A) Different sequences and chemical modifications of CXCL12 PS-ASOs indicate MsPA modifications improved potency than gap 2 OMe ASO in vitro and in vivo, also reduced toxicity at certain levels. Blue letters indicate cEt, black PS DNA, subscript pink μ = MsPA, red indicates 2′-OMe. For in vitro studies, activity was done in NIH3T3 cells and toxicity was done in Hepa1–6 cells by electroporation. For in vivo studies, BALB/c mice (N = 3 animals per group) were subcutaneously administered a single dose of parental, gap2 OMe, and MsPA PS-ASOs at indicated doses for 72 h. CXCL12 mRNA by 50% (ED₅₀), Levels of ALT, AST, Cdkn1a mRNA, Tnfrsf10b mRNA, and RNase H1 in the liver. (B) Percentage of CXCl12 mRNA in livers of mice treated subcutaneously with indicated ASO relative to saline control. n = 3. PS, phosphorothioate; ASO, antisense oligonucleotide; 2′-OMe, 2′-O-methyl; MsPA, mesyl-phosphoramidate.

FIG. 2.

The combination of MsPA linkages with the gap2 OMe modification reduces toxicity and enhances potency. (A) Summary of caspase activity (red indicates high; green indicates low) and potency as indicated by IC₅₀ values (green indicates high potency; red indicates low). Blue letters indicate cEt, black PS DNA, subscript pink μ MsPA, red 2′-OMe. Red arrow indicates parental ASO and blue arrow the ASO modified with gap2 OMe but not MsPA. Green dots indicate the ASOs with the lowest toxicity and highest activity. n = 4. (B) Dose–response curves for targeting CXCl12 mRNA in NIH3T3 cells by electroporation in Fig. 2A, n = 3.

FIG. 3.

MsPA linkage modifications enhance RNase H1 cleavage and off-rate. (A) Analysis of RNase H1 cleavage of FITC-labeled RNA, n = 3. In the carton, the circles indicate residues with the blue circles denoting the wings and the open circles the gap; the green circle indicates the gap2 OMe. The lines show the positions of MsPA linkages. (B) Percentage cleaved product as a function of time in RNase H1 cleavage assay, n = 3. (C) Gel analysis of affinity selection with parent and ASOs modified with gap2 OMe or with gap2 OMe and MsPA. (D) Western blot showed the ASOs with modifications of MsPA and gap2 OMe bind less RNase H1 proteins, Ku70 as control.

FIG. 4.

The combination of gap2 OMe and MsPA linkages enhances results in high potency without toxicity. (A) The carton indicated the four kinds of 3-10-3 ASOs for each group tested in cells. (B) The IC₅₀s of ASO activities for different groups that target different genes. ASO activities targeting FXI were done in primary hepatocyte MHT cells and targeting PABPN1, Dynamin2, and YAP were done in mouse NIH3T3 cell line, n = 3. (C) The toxicities of the different ASOs groups were detected by caspase activity in Hepa 1–6 cells, n = 4.

FIG. 5.

The combinations of double MsPA linkage modifications with gap2 OMe on ASOs enhance activity and maintain safety in mice. (A) The GalNac-ASOs with different modifications targeting CXCL12 are listed. (B) ALT and AST indicate the combination of MsPA linkages and gap2 OMe modification reduce the toxicity similar to gap 2′ OMe, n = 3. (C) The dose–response of CXCL12 mRNA levels by different modifications, n = 3. (D) The combination of gap2 OMe and MsPA linkages improved the activity compared with gap2 OMe ASO, n = 3.

FIG. 6.

Analyses of ASOs targeting FXI for activity and safety in mice. (A) The dose–response of FXI mRNA levels by different modifications, n = 3. (B) The combination of gap2 OMe and MsPA linkages improved the activity compared with gap2 OMe ASO, n = 3. (C) ALT and AST indicate the combination of MsPA and 2′ OME modifications have the similar ability to gap 2′ OMe to reduce the toxicity compared with parental ASO (1599499), n = 3. (D) The double MsPA linkage and gap2 OME modifications maintain the safety shown the similar levels of P21 mRNA as gap2 OMe ASO, compared with parental ASO (1599499).