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

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.

Figure 1.

(A) Structures of R- and S-5′-alkyl DNA monomers evaluated in this work. (B) Structural model of an ASO showing the 5′-cEt-DNA gap junction. The 5′-methyl group at position 3 in the DNA gap occupies the same chemical space as the 2′-OMe group at position 2.

Figure 2.

Synthesis of R- and S-5′-Me DNA nucleoside phosphoramidites

Figure 3.

(A) 5′-S-Me DNA (C) 5′-R-Me DNA monomers were walked across the gap region and effect on duplex stability versus complementary RNA, antisense activity in NIH3T3 cells and cytotoxicity as measured by caspase activation in Hepa1-6 cells were determined. P54nrb mislocalization was detected by immunofluorescence staining in Hela cells. The percentage of cells containing mis-localized P54nrb protein was calculated based on manual counting of ∼100 cells. Dose response curves for reducing CXCl12 mRNA in NIH3T3 following delivery of (B) 5′-S-Me DNA and (D) 5′-R-Me DNA ASOs cells by electroporation. Blue letters indicate constrained Ethyl (cEt), black indicate DNA and red indicate 5′-alkyl DNA nucleotides. All ASOs were fully phosphorothioate (PS) modified.

Figure 4.

Characterizing the effect of inserting R- and S-5′-Me DNA and 5′-allyl DNA monomers at (A and B) positions 3 and 4 and (C and D) position 2 in the DNA gap on potency as determined by the dose required to reduce CXCL12 mRNA by 50% in the liver relative to untreated control animals (ED₅₀) and hepatotoxicity as measured by increases in plasma ALT (IU/l) in mice. (E and F) Characterizing the effect of introducing R- and S-5′-ethyl DNA monomers at positions 3 and 4 in the DNA gap on potency and hepatotoxicity in mice. For ED₅₀ determination, mice (Balb/c, n = 3/group) were injected subcutaneously with ASO (50, 16.7, 5.6 and 1.9 mg/kg) and euthanized after 72 hours. Livers were harvested and reduction in CXCl12 mRNA was measured using qRT-PCR. For plasma ALT, mice (Balb/c, n = 3/group) were injected subcutaneously with 150 mg/kg of ASO and euthanized after 72 h and ALT values were measured on a clinical analyzer. Blue letters indicate constrained Ethyl (cEt), black indicate DNA and red indicate 5′-alkyl DNA nucleotides. All ASOs were fully phosphorothioate (PS) modified.

Figure 5.

(A) Comparing the effect of introducing 2′-OMe, MOP, R-5′-methyl and R-5′-ethyl DNA modifications on potency and hepatotoxicity of ASOs targeting HDAC2, FXI and Dynamin 2 mRNA (Average ALT = 27 ± 5 IU/l for the PBS group). (B–F) Dose response curves for reducing the target mRNA in the liver in mice. Mice (Balb/c, n = 3/group) were injected subcutaneously with ASO (150, 50, 16.7, 5.6 and 1.9 mg/kg) and euthanized after 72 h. Livers were harvested and reduction in the levels of target mRNAs was measured using qRT-PCR. Plasma ALT values were measured on a clinical analyzer at study termination. Data indicated with asterix were collected from a different experiment using the same study design as indicated above. Blue letters indicate constrained Ethyl (cEt), black indicate DNA and red indicate 5′-alkyl DNA nucleotides. All ASOs were fully phosphorothioate (PS) modified.

Figure 6.

(A) Characterizing the effect of introducing R- and S-5′-Me DNA monomers in the gap on RNaseH1 cleavage patterns of ASO/RNA duplexes. (B) Structural model showing the 7-base footprint of the catalytic domain of recombinant human RNaseH1 for cleavage sites a-f on the ASO/RNA duplex. (C) Incorporation of S-5′-Me DNA at position 8 in the DNA gap ablates cleavage site a while incorporation of R-5′-Me DNA at position 8 and 9 ablates cleavage site a on the ASO/RNA duplex. (D) Structural model showing how 5′-Me substituents can modulates important backbone contacts between Arg 179, Ile239 and Trp225 on human RNAseH1 and the sugar-phosphate backbone of the ASO. (E) Newmann projections depicting how configuration of the 5′-methyl group can change the rotational preference around torsion angle γ of the sugar phosphate backbone. Blue letters indicate constrained Ethyl (cEt), black indicate DNA and red indicate 5′-alkyl DNA nucleotides. All ASOs were fully phosphorothioate (PS) modified.