The therapeutic promise of small-RNA therapeutics is limited, not only by the lack of delivery vehicles, but also by the inability of the small RNAs to reach intracellular compartments where they can be biologically active. We previously reported successful delivery of functionally active miRNAs via receptor-mediated endocytosis. This type of targeted therapy still faces a major challenge in the delivery field: endosomal sequestration. Here, a new method has been developed to promote endosomal escape of delivered miRNA. The strategy relies on the difference in solute contents between nascent endosomes and the cytoplasm; early endosomes are rich in sodium ions, whereas the intracellular fluid is rich is potassium ions. Exploiting this difference through favoring the influx of potassium into the endosomes without the exchange of osmotically active sodium, results in an osmotic differential leading to the endosomes swelling and bursting. One molecule that is able to exchange potassium for an osmotically inactive hydrogen ion is the ionophore nigericin. Through generating an intramolecular miRNA delivery vehicle, containing a ligand, in this case folate and nigericin, we enabled the escape of folate-RNA conjugates from their entrapping endosomes into the cytoplasm where they bound the RNA-induced silencing complex and activated the RNAi response.

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Based on the knowledge that microRNAs (miRNAs) are dysregulated in diseases such as cancer, various attempts have been explored to develop miRNA-based cancer therapeutics. Although many strategies have been used to restore the levels of therapeutically relevant microRNAs, clinical delivery of the processed or mature miRNA to cancer cells still remains a challenge. To overcome this challenge, innovative methods are being explored to increase the pools of tumor-suppressive miRNAs, such as enhancing miRNA biogenesis. This is especially true for the let-7 family of tumor-suppressive miRNAs, which has an additional layer of regulation over the canonical miRNA biogenesis pathway. In particular, the DROSHA and DICER cleavage steps are blocked when unprocessed let-7 is bound by the RNA-binding protein LIN28. LIN28 interacts specifically with unprocessed let-7 via a sequence-specific motif, GGAG, contained in most let-7 family members. Because previous attempts to restore mature let-7 levels through increasing let-7 pools have been quite successful at reducing tumor burden, identifying novel and clinically relevant ways to increase the level of this tumor-suppressive miRNA represents a critical need. In this study, we hypothesize that small-molecule inhibitors that disrupt the LIN28-let-7 interaction will lead to enhanced processing and increased levels of mature, tumor-suppressive let-7. Thus, an in vitro high-throughput fluorescence polarization screen has been conducted using His-tagged LIN28 and a Cy5 fluorophore-tagged let-7 RNA probe. The nine-nucleotide RNA probe was designed through in silico modeling to include the GGAG motif, which associates with LIN28 through the zinc knuckle domain of LIN28. Due to the size difference between unbound let-7 probe and LIN28-bound let-7 probe, a wide window of polarization values was achieved, resulting in a Z′ score of 0.61, indicating that the assay was robust to proceed with the screen. The screen was performed against 23,680 compounds, which consist of FDA-approved LOPAC and blood-brain barrier-permeable CNS library compounds. Eight compounds were identified as positive hits from the screen. These compounds are currently being characterized in secondary assays both in vitro and in cell culture. The significance of this research is that small-molecule inhibitors may provide novel therapeutic strategies to increase the pool of mature, tumor-suppressive let-7 in tumors with elevated LIN28. Citation Format: Sunghyun Myoung, Sergey Savinov, Lan Chen, Gaurav Chopra, Larisa Avramova, James Welch, Bradley Loren, David Thompson, Andrea L. Kasinski. Development of an RNA-based cancer therapeutic targeting the let-7-LIN28 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4656.