Exploring Backbone Chemistries in Morpholino Gapmers of Oblimersen: It’s Effect on Biophysical Parameters and RNaseH Activity

The phosphorothioate (PS) linkage represents a privileged modification in oligonucleotide therapeutics. Within cells, PS ASOs interact non-specifically with cellular proteins which causes toxicity and immune response. To address these issues, we investigated the effect of replacing the 2'-OMe-PS wings of a reported sequence targeting Bcl 2 mRNA with morpholino wings. The goal is to use the safe and effective features of morpholino oligonucleotides to improve the efficacy of a known oligonucleotide sequence, Oblimersen. Considering the synthetic challenges associated with incorporating morpholino modifications into a gapmer design, this work pioneers a novel strategy highlighting the importance of the neutral Phosphorodiamidate (PMO) backbone over its charged isoforms like thiophosphoramidate (TMO) and phosphoramidate (MO). Biophysical studies revealed that the incorporation of morpholino wings does not perturb the global geometry of the tested Gapmer-RNA duplexes as compared to that of 2'-OMe-PS gapmer. However, the presence of charge neutral backbone significantly affects the morpholino-gapmer duplex stability with its complementary RNA as well as its biological efficacy. This work segregates the functional role of backbone chemistry from the morpholino nucleobases holding a potential to inform safer, more potent morpholino gapmer antisense therapeutic design.