2025 AIChE Annual Meeting

(183ap) Harnessing miRNA Target Sites for Cell-Specific mRNA Therapeutics

Authors

Shaoyi Jiang, Cornell University
Precision delivery of mRNA constructs to target cell types is a prerequisite for myriad therapeutic applications of mRNA, ranging from vaccination to cancer treatment. Currently, the expression of mRNA in undesirable cell types or organs, resulting in off-target effects, remains a significant barrier. One method to overcome this limitation is the incorporation of microRNA (miRNA) target sites. miRNAs are short, non-coding sequences of RNA that are endogenously present in all cells; after binding to complementary target sites on mRNAs, they exert notable regulatory control over translation of that mRNA. By modifying the 3’ untranslated region (UTR) of mRNAs to include one or more of these target sites, translation can be modulated depending on inherent miRNA levels. Through strategic utilization of specific miRNAs that are upregulated or downregulated in certain cell populations, such as cancer cells, we can achieve precise expression in these cell types, enabling accurate delivery of mRNA therapeutics.

We sought to alter mRNA expression between healthy and cancerous cells by incorporating target sites for miRNAs associated with tumor suppression in breast cancer. We formulated a range of mRNA constructs including one or multiple relevant miRNA target sites in the 3’ UTR, using a luminescent reporter system to compare gene expression in healthy cells and breast cancer cells in vitro. With this technique, we were able to achieve over 2-fold increased luminescence in breast cancer cells relative to the control mRNA, while in healthy cells there was no significant change with the same mRNA. Additionally, we performed RT-qPCR to confirm and quantify the presence of miRNAs in the cell lines used, validating the connection between endogenous miRNA levels and regulatory control of mRNA expression.

These results demonstrate that incorporating miRNA target sites into the 3’ UTR of an mRNA construct has a powerful effect on the expression of that mRNA, and further, that this effect varies considerably depending on miRNA sequence and cell type. The ability to fine-tune expression levels such that translation occurs at a much higher degree in specific cell populations enables a vast array of potential therapeutic applications. In concert with delivery technologies such as lipid nanoparticles, which can incorporate additional targeting mechanisms, synergistic and incredibly precise cell targeting can be achieved. The long-term goal of this research is differentiation between various healthy and cancer cell types, such that cytotoxic agents or other oncologic treatments can be accurately delivered to tumors while minimizing damage to healthy tissue. Additional experiments will be undertaken toward this end: the inclusion of additional miRNA candidates, expansion to a wider array of cell types, and murine tumor models.

Figure 1. Average luminescence measurements across 4 mRNA constructs (3' UTR modification to include a target site for miRNA 1, 2, both, or neither) and 2 cell lines, one healthy and one cancerous, normalized to the control of the same cell type with standard error.