Investigating the Biophysical Properties and <i>In Vivo</i> Activity of 7′,5′-Alpha-Bicyclo-DNA Phosphodiester Backbone Gapmer Antisense Oligonucleotides

Advances in backbone modifications are driving the development of nucleic acid therapeutics, yet there is still a need to establish compounds that avoid the potential dose-limiting toxicity of phosphorothioate internucleosidic linkages, particularly outside the central nervous system. We have developed a novel 7',5'-α-bc-DNA (abcDNA) scaffold, and here we benchmark the biophysical and <i>in vivo</i> gene knockdown efficacy of gapmer antisense oligonucleotides (ASOs) containing abcDNA nucleotides. Melting curve analyses show gapmers with abcDNA bases in both wings maintain a good affinity for complementary RNA and demonstrate stable mismatch discrimination, in addition to a high degree of serum biostability. To assess <i>in vivo</i> on-target activity and tolerability, mice were dosed systemically with abcDNA ASOs targeting <i>Malat-1</i>, with or without conjugation to palmitic acid. Multiple tissues were assessed for on-target knockdown efficiency by RT-PCR and <i>in situ</i> hybridization alongside biodistribution analysis by immunohistochemistry. abcDNA ASOs were well tolerated and performed at a comparable level to an equivalent 2'-<i>O</i>-methoxyethylribose gapmer. We also present the first <i>in vivo</i> pharmacokinetic data generated using an enzyme-free nucleic acid nanorobotics method. Together, these data support the utility of abcDNA as a valuable addition to ASO technology that maintains a natural phosphodiester backbone, providing a combination of preferred biostability and on-target affinity with acceptable <i>in vivo</i> tolerability.