Investigating the Biophysical Properties and in Vivo 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 in vivo 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 in vivo on-target activity and tolerability, mice were dosed systemically with abcDNA ASOs targeting Malat-1, with or without conjugation to palmitic acid. Multiple tissues were assessed for on-target knockdown efficiency by RT-PCR and in situ hybridization alongside biodistribution analysis by immunohistochemistry. abcDNA ASOs were well tolerated and performed at a comparable level to an equivalent 2'-O-methoxyethylribose gapmer. We also present the first in vivo 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 in vivo tolerability.