Segmented poly(A) tails with microRNA target sites confer tissue-specific regulation for mRNA therapeutics

Abstract Targeted delivery and controlled expression of mRNA-LNPs are critical for the development of safe and effective mRNA medicines. However, efficient post-delivery regulation of mRNA-LNP expression remains challenging. In this study, we engineered segmented poly(A) tail variants that function as gene-specific regulatory elements for synthetic mRNAs. Specifically, we inserted microRNA target sites (MTS) for miR-122 or miR-142 at various positions of the poly(A) tail of synthesized luciferase reporter mRNA. These modifications significantly reduced luciferase expression in non-target tissues in vitro and in vivo, demonstrating position-dependent selective expression control. Furthermore, by incorporating triple-MTS sequences for miR-142, miR-126, and miR-148a in all possible combinations at the 5’ end of the poly(A) tail, we identified triple-MTS arrangements that simultaneously decrease luciferase activities in three non-target hepatic cell types, while preserving robust expression in hepatocytes. The results highlighted the importance of MTS insertion order for optimal mRNA silencing. These triple-MTS modules significantly expanded the utility of single miRNA-responsive elements, enabling cell-type selective regulation for mRNA therapeutics. By complementing tissue-tropic delivery LNPs, our mRNA cargo regulatory elements have the potential to improve tissue and cell type selectivity as a novel platform for post-delivery regulation of mRNA-LNP. Competing Interest Statement Weiguo Zhang, Yijie Dong, Rui Chen, and Rui Qi are listed as co-inventors of a patent related to this work. Rui Qi, Rui Chen, Hua Chen, Ruiwen Xu, Lu Han, Yingmei Xu, Juan Li, Na Li, Qiang Li, Hui Bao, Tingting Zhang, Yijie Dong, and Weiguo Zhang were employees of RinuaGene, Inc. at the time of the work.