Abstract 6692: The combination of 5-methyltetrahydrofolate and nigericin enhances the cytosolic release of chemically modified miRNA-34a, significantly boosting its anti-tumoral activity

A major driver of cancer progression involves the downregulation of tumor-suppressive miRNAs like miR-34a, which regulates key cancer-promoting genes such as MYC, AXL, and BCL-2. Thus, it is not surprising that miR-34a restoration has been proposed as an anticancer strategy. miRNAs, such as miR-34a can precisely target cancer-related genes with limited toxicity, provided they are properly designed. Effective therapeutic design must address RNA specificity, stability, and endosomal escape. Our lab has made significant progress in addressing these unmet challenges, particularly in enhancing specificity and stability of miRNA therapeutics through ligand-mediated delivery and chemical modifications, respectively. Our ligand-mediated delivery strategy capitalizes on overexpression of Folate Receptor 1 (FR1) in cancer cells, and uses the high-affinity FR1 ligand, folate, to deliver miRNAs to cancer cells with high specificity. To overcome the issues with poor instability, we created a fully modified version of miR-34a (FM-34a), increasing its stability by ∼400-fold. Despite significant progress in developing FM- 34a as a therapeutic agent, efficient endosomal escape remains a major hurdle. To address this, a small molecule ionophore, nigericin that disrupts endosomal membranes was conjugated to the folate-FM-34a conjugate (Fol-N-FM-34a). However, nigericin alone achieved only modest improvements in escape efficiency, likely due to the inability of folate to dissociate from the receptor at the elevated endosomal pH levels induced by nigericin. To overcome this limitation, a lower affinity ligand of FR1, 5-methyltetrahydrofolate (5-MTHF), was used in combination with nigericin (5M-N-FM-34a). Unlike folate, 5-MTHF retains a similar binding affinity for FR1 at physiological pH but dissociates more effectively at the higher pH levels attainable in the presence of nigericin, thereby enhancing endosomal escape and cytosolic delivery. We validated that the 5-MTHF ligand binds to FR1-expressing cells in both in vitro and in vivo models and that 5-MTHF-N-FM-34a lead to a higher cytosolic abundance of miR-34a and superior downregulation of miR-34a target genes compared to Fol-N-FM-34a in vitro. To evaluate in vivo activity both single dose targeting and multi-dose efficacy studies were conducted. 5M-N-FM-34a achieved greater target downregulation and enhanced anti-tumor activity in a KRASG12D p53-/- genetically engineered mouse model of non-small cell lung cancer in comparison to other ligands. These findings highlight 5-MTHF-nigericin as a promising ligand for targeting FR1-overexpressing tumors. Citation Format: Kenan E. Ozcan, Sophia A. Matthias, Shreyas G. Iyer, Ahmed M. Abdelaal, Sudarsan R. Kasireddy, Philip S. Low, Andrea L. Kasinski. The combination of 5-methyltetrahydrofolate and nigericin enhances the cytosolic release of chemically modified miRNA-34a, significantly boosting its anti-tumoral activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6692.