SYSTEMATIC ASSESSMENT OF MINIMALLY-INVASIVE STRATEGIES FOR LUNG-TARGETED DELIVERY OF MICRORNA MIMICS IN RATS

Abstract

MicroRNAs (miRNA) are key regulators of gene expression that have been causally implicated in the pathobiology of many different diseases. There is increasing interest in using synthetic miRNA mimics in vivo as potential therapeutic agents for pulmonary vascular disease; however, the optimal delivery method to achieve high efficiency, selective lung targeting has not been determined. To assess the relative merits of four commonly used minimally-invasive strategies for lung-targeted administration of miRNA mimics in rats. A synthetic miRNA mimic with no mammalian homologue (cel-miR-39-3p; 0.5 nmol in 50 μL invivofectamine/PBS vehicle) was delivered to male Spraque-Dawley rats using 5 different strategies (n=3 rats/method). These included intratracheal liquid instillation (IT-L), intratracheal aerosolization (IT-A), intranasal liquid instillation (IN-L), intranasal aerosolization (IN-A) and intravenous (IV; via jugular vein) delivery. Relative levels of cel-miR-39 were quantified by RT-qPCR to assess pulmonary yield and distribution, persistence over time, and tissue-specificity. At 2 h post delivery, the highest levels of cel-miR-39 were achieved in the lungs after delivery by IT-L (mean ± SD; 9.4 ± 4.5, 2-ΔCq expression units). Expression levels progressively fell with delivery by IN-L (0.9 ± 1.6), IV (0.07 ± 0.02), IN-A (0.04 ± 0.04), and IT-A (0.0008 ± 0.0007). The pulmonary distribution of cel-miR-39 was generally comparable between delivery methods, with similar levels in both right and left lung lobes, and evidence of penetration to distal regions. Cel-miR-39 remained detectable in lung tissue 24 h post delivery, but levels were 10-100 fold lower. All methods, with the exception of IV delivery, showed lung-selective targeting, with cel-miR-39 levels 10-100 fold lower in the heart and 100-10000 fold lower in the liver. MicroRNA uptake in the lungs differed markedly by up to 4 orders of magnitude depending of the method of administration, suggesting that the choice of delivery strategy could have a significant impact on potential therapeutic outcomes in preclinical investigations of miRNA-based drug candidates.