In vivo localization of miR-34 delivered via liposomal MRX34: chromogenic in situ hybridization (CISH) results in a preclinical model and liver biopsies from phase I patients.

e14074 Background: Each microRNA (miRNA) modulates the expression of hundreds of genes across distinct cellular pathways. This broad activity gives miRNA-based therapy the potential to simultaneously repress multiple oncogenic processes in the tumor microenvironment, including growth and proliferation, resistance, cancer stem cells, metastasis, and immune evasion. The naturally occurring tumor suppressor miR-34a has been shown to down-regulate expression of > 30 oncogenes (eg, MET, MEK1, PDGFR-α, CDK4/6, BCL2, WNT 1/3, NOTCH1, CD44), as well as genes involved in tumor immune evasion (eg, PD-L1, DGKζ). MRX34, a liposome-encapsulated miR-34a mimic, is a potential first-in-class miRNA therapy for cancer. Methods: We developed a CISH staining method to visualize cellular and sub-cellular localization of miR-34a following MRX34 treatment. Tissue concentrations of miR-34a were determined on corresponding tissue slides using micro-dissection, extraction-free RNA isolation, and qRT-PCR. The combined protocol was applied to tissues (tumor, normal liver, bone marrow) extracted post-treatment with MRX34 or dilution buffer control from an orthotopic (Huh-7) mouse HCC model (n = 3 each treatment and control per dose level). Pre- and post-treatment liver biopsies from patients (n = 4) with advanced HCC who received IV infusions of MRX34 in the MRX34-101 phase I trial (NCT01829971) were subjected to CISH only (due to limited available tissue). Results: In the orthotopic mouse HCC model (Huh-7), miR-34a levels in tumor, normal liver, and bone marrow increased by > 2 orders of magnitude as early as 2 min after tail vein injection of MRX34, with localization to the cellular cytoplasm. CISH showed similarly increased miR-34a in tumor and normal liver in the post-treatment biopsies from phase I HCC patients receiving MRX34 infusions, also localized to the cellular cytoplasm. Conclusions: Our results demonstrate MRX34-mediated delivery of miR-34a to tumor and normal tissue, both in a preclinical model of HCC and in liver biopsies from patients with advanced HCC treated systemically with MRX34 in a first-in-human trial of miRNA cancer therapy.