Structural optimization and additional targets identification of antisense oligonucleotide G3139 encapsulated in a neutral cytidinyl-lipid combined with a cationic lipid in vitro and in vivo

Antisense oligonucleotides (ASOs) usually contain a fully phosphorothioate (PS) backbone, which possibly interact with many genes and proteins under intracellular conditions. G3139 is an ASO that targets Bcl-2 mRNA and induces cell apoptosis. Here, we report a kind of cytidinyl-lipid combined with a cationic lipid (DNCA/CLD, molar ration, 28:3, named mix), which may interact with oligonucleotides via H-bond formation, pi-stacking and electrostatic interaction, accompanied by low zeta potentials. The IC50 value of G3139 delivered by mix-lipid reduced from above 20 μM to 0.158 μM for MCF-7/ADR, and exhibited stronger antiproliferation upon other cancer cell lines. In addition, PS modification in the 3′-half of G3139 (especially at positions 13-16) enhanced serum stability, target specificity and anticancer activity. Also, a locked nucleic acid (LNA) gapmer G3139 (LNA-G3139) showed superior antiproliferation (78.5%) and Bcl-2 mRNA suppression effects (85.5%) at 200 nM, mainly due to its high complementary RNA affinity. More apoptosis-associated targets were identified, and a lower level of non-specific protein binding (HSA) revealed that both antisense and aptamer mechanisms might simultaneously exist. A combination of a new delivery system and chemical modifications, such as in LNA-G3139, may have potential clinical application prospects in the future.