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.

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides

“…In contrast to the effects on potency, the directionality of replacing PS with MsPA in the gap had a remarkable effect on cytotoxicity. As observed in our previous studies with 2′- and 5′-modifications ( 7 , 8 ), replacing PS in the vicinity of the 5′-cEt/gap junction reduced cytotoxicity whereas similar substitution near the 3′-cEt/DNA gap junction did not have a strong effect for reducing cytotoxicity.…”

“…The ASO with 4 MsPA in the gap (1462753) showed the optimal potency and mitigation of cyto- and hepatotoxicity in the experiment above and as result this design was chosen for evaluation of additional model toxic ASOs targeting HDAC2, FXI, PABN, Dynamin 2 mRNAs (Figure 5A and B ). All the parent ASOs were previously shown to be hepatotoxic in mice ( 8 , 10 ) and were not evaluated in these studies to reduce animal usage. Instead, we used ASOs with OMe at position 2 in the gap as controls to determine if MsPA substitution could maintain potency and safety relative to this design.…”

“…We recently showed that small structural changes in the ASO such as site-specific introduction of 2′- and 5′-substitutions on the 2′-deoxynucleotide sugar ( 7 , 8 ), replacing one or two PS with alkyl-phosphonate linkages ( 10 ), or replacing the natural 3′,5′-PS linkage with a 2′,5′-linkage ( 9 ) could mitigate ASO toxicities without compromising activity. However, these ASO designs still retained significant protein binding as they did not reduce the total number of PS linkages in the ASO, a major determinant of protein binding.…”

The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background

“…First, we recommend including not only libraries from untreated cells but also lipofectamine-only treated cells to evaluate the potential effects of the transfection reagent on the cell of interest [ 31 ]. Second, analysis of gap ablated ASO and ASO backbone modification data can provide further useful information on the off-target effects of the negative control ASOs and to differentiate between off-target cleavage and steric hindrance [ 34 – 36 ]. Third, a rigorous evaluation of dose-response and time-course experiments will help determine the best experimental conditions and provide direct comparisons between experiments [ 30 ].…”

Preclinical Safety Assessment of Therapeutic Oligonucleotides

“…Being more hydrophobic, the higher affinity modifications showing higher incidence of liver toxicity also show higher affinity to a number of intracellular proteins compared to the same sequence with, e.g., MOE chemistry [68][69][70][71]. Importantly, predictive in vitro models for liver and kidney toxicity have been developed [67,72,73], and design modifications reducing ASO toxicity without compromising potency have been proposed [69,74] demonstrating that highly potent ASO sequences that do not show liver toxicity can be identified and progressed to clinical trials.…”