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

Targeted delivery and controlled expression of mRNA encapsulated by lipid nanoparticles (mRNA-LNPs) are critical for the development of safe and effective mRNA medicines. However, efficient post-delivery regulation of mRNA-LNP expression remains challenging, and conventional approaches largely rely on modifying the 3' UTR at the expense of other regulatory elements. In this study, we engineered segmented poly(A) tail variants that function as gene-specific regulatory elements for synthetic mRNAs, providing an alternative regulatory module that preserves UTR integrity. Insertion of miR-122 or miR-142 target sites (MTSs) at various positions within the poly(A) tail suppressed luciferase expression in non-target cells in vitro and in vivo in a position-dependent manner. 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 specific arrangements that simultaneously reduced luciferase activity in three non-target hepatic cell types while preserving robust expression in hepatocytes. MTS insertion order proved critical for optimal silencing, highlighting a design parameter not observed in dual-MTS constructs. These findings establish the poly(A) tail as a programmable platform for cell-type-selective regulation, complementing tissue-tropic LNPs and expanding the toolbox for mRNA therapeutic design.