5'-Palmitate Lipid and Internal LNA-Piperidyl Triesters Enhance the RNA Affinity and Activity of Splice-Switching Oligonucleotides.

Therapeutic oligonucleotides represent a paradigm shift in medicine, but their modest therapeutic index remains a major obstacle. While chemical modification is essential for stability and cell uptake, only limited diversity is represented in oligonucleotides currently in the clinic. In fact, phosphorothioate and morpholino (PMO) are the only backbone modifications used clinically. Alternative phosphorus-based oligonucleotide backbones such as alkyl phosphonate and phosphoramidate have been explored, but their synthesis is challenging. In this work, we present the synthesis, physical, and biological properties of oligonucleotides containing THP and piperidyl phosphothiotriesters combined with LNA, 2'-OMe, 2'-MOE, and 2'-F ribose sugar derivatives. The LNA triester analogues increase duplex stability compared to phosphorothioate, while 2'-OMe, 2'-MOE, and 2'-F ribose phosphothiotriesters reduce it. Oligonucleotides containing LNA-piperidyl triester backbones enhance duplex stability to levels far beyond the classical phosphorothioate linkage, and their gymnotic splice-switching activity is significantly enhanced when conjugated to palmitate lipid.