Aptamer-directed, nanovesicle-mediated targeting of undruggable molecules in preclinical cholangiocarcinoma models

Background and Aims: Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the biliary epithelium with limited therapeutic options and poor long-term survival rates. To address the limitations in CCA treatment, we investigated cell-targeted nanovesicles as a delivery platform for transcriptome-targeting therapeutics. Approach & Results: Milk-derived nanovesicles (MNVs) were loaded with short interfering RNAs targeting YAP, the downstream effector of the Hippo pathway; LCK, an upstream regulator of YAP; and tafazzin, a protein critical for the integrity of the inner mitochondrial membrane. These transcriptome-targeting nanovesicles (tMNVs) were decorated with a lipid-coupled RNA aptamer to epithelial cell adhesion molecule (EpCAM), including a tracking fluorophore. In vitro studies were conducted using multiple CCA cell lines. In vivo studies were performed using C57BL/6 and NOD/SCID mice to evaluate delivery and efficacy in both an immunocompetent syngeneic murine and a patient-derived xenograft (PDX) model. We demonstrated that tMNVs were selectively taken up by liver tumor cells, which was augmented by the incorporation of a targeting aptamer, and that MNVs loaded with siRNA effectively downregulated target gene expression, both in vitro and in vivo. Downstream effects of target gene inhibition were observed, including downregulation of YAP-TEAD target genes and an increase in reactive oxygen species production at the mitochondrial level. Administration of tMNVs targeting YAP, LCK, and tafazzin inhibited CCA growth and further synergized with chemotherapy in two preclinical CCA models. Conclusions: Herein, we show that aptamer-directed, nanovesicle-mediated targeting of YAP, LCK, and tafazzin potentiates chemosensitivity in two CCA models when delivered using aptamer-guided milk-derived nanovesicles.