Abstract B43: Quantitative PCR and in-situ-hybridization analysis to determine tissue concentration and localization of MRX34

Abstract MRX34, a liposomal microRNA (miRNA)-based therapy for cancer, has recently entered clinical trials as a potential first clinical candidate in its class. MRX34 is a mimic of naturally occurring microRNA-34 (miR-34) encapsulated in a liposomal nanoparticle formulation. Preclinical animal studies have shown that intravenous delivery of MRX34 can increase miR-34 levels in liver tumor cells more than 100-fold when analyzing whole-tissue RNA extracts by quantitative PCR (qPCR). MRX34-induced tumor regression has enhanced the survival of mice by inhibiting the growth of both hepatic and non-hepatic tumors. We have established a chromogenic in situ hybridization (CISH) method to track the cellular location of the miR-34 mimic in tissues after systemic MRX34 administration. In contrast to conventional biodistribution approaches that cannot distinguish between spatial differences in tissue accumulation, CISH in conjunction with microscopy allows the detection of the miRNA mimic on a cellular level and may provide new insights into cell-type specific accumulation. Here, we evaluated the localization and tissue concentrations of systemically delivered MRX34 in mice bearing orthotopic Huh7 tumors by CISH followed by image correlation to a formalin-fixed and paraffin-embedded tissue equivalent, and isolation-free qPCR analysis. Our results show that the CISH procedure is a reproducible and robust assay capable of over 2 logs of miR-34 detection when correlated to qPCR data from matching micro-dissected samples. Systemic MRX34 delivery leads to accumulation of miR-34 mimics in tumor cells with Cmax reached approximately 2 hrs post dosing. In addition, the CISH data reveal how biodistribution data generated from whole-tissue extracts can be biased due to minute impurities and suggests that these methods should be used in combination with CISH for an accurate assessment of tissue concentrations. Citation Format: Desiree Martin, Kevin Kelnar, Andreas G. Bader. Quantitative PCR and in-situ-hybridization analysis to determine tissue concentration and localization of MRX34. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B43.