Structure - silencing duration relationships in RNAi medicines in rapidly dividing cells

RNA interference (RNAi)-based medicines offer precise targeting of virtually any transcript, making them an appealing new drug class for addressing unmet needs in immune-oncological applications. While RNAi therapies show exceptional duration of effect in non-dividing cells, their efficacy in rapidly dividing cells, crucial for immune-oncology, remains largely unexplored. Unlike in non-dividing cells, full chemical modification in rapidly dividing cells has not consistently extended silencing duration, according to limited data available. In this study, we investigated key factors affecting the duration of effect for three main types of RNAi-based therapeutics (siRNA, miRNA mimics, and miRNA inhibitors) in rapidly dividing cancer and immune cells. Saturation of intracellular depots by multiple loading doses, a common strategy to prolong silencing duration in non-dividing hepatocytes, had minimal impact on siRNA duration of effect in rapidly dividing cells. However, modifying the antisense strand with a 5'-(E)-vinylphosphonate (5'-VP) to protect siRNAs from exonucleases and enhance AGO2 binding significantly extended siRNA silencing duration to over 30 days both in vitro and in vivo. For miRNA mimics, extensive stabilization of the antisense strand with phosphorothioates was not effective and led to reduced potency and silencing duration. Interestingly, a shorter duplex region commonly seen in therapeutic siRNAs partially rescued duration of silencing in miRNA mimics with extended phosphorothioate modifications. On the other hand, miRNA inhibitors demonstrated robust reversal of miRNA activity for an impressive 25 days in cancer cell lines. Our findings enable the rational design of the chemical architecture and administration regimens of RNAi-based therapies in oncology and immunology.