A Chemopreventive Strategy Based on Edible MicroRNAs Produced in Plants

Plants and plant-derived products have long been popular complementary health interventions inAmerica and throughout the world. Recently, there is increased evidence that plant RNAs ingested in thecourse of eating ordinary plant-based foods are taken up by the mammalian digestive tract and remainfunctional. These observations raise the possibility of incorporating delivery of modern molecular RNA-basedtherapeutics into what has traditionally been a complementary health intervention modality. MicroRNAs(miRNAs) are a class of small non-coding RNAs that play a critical role in gene expression in eukaryotes,controlling virtually every physiological process in the body. Reduction in certain miRNAs is associated withmany diseases, and restoration of the missing miRNA blocks disease progression. Although the therapeuticpotential of these miRNAs is clear, delivery of the needed miRNA to the diseased cells is a critical barrier toimplementation of the therapy. This R21 proposal is focused on developing a safe, effective, and economicallyfeasible strategy for delivering therapeutic miRNAs in vivo. The strategy is based on the scientific premise thatedible plants can be used as biofactories to produce therapeutic mammalian miRNAs that can be administeredvia ingestion. This strategy represents a fundamental shift from the current mainstream approach in the field,which has focused on synthesizing miRNAs as double-stranded ?miRNA mimics? and delivering themintravenously, formulated with lipids or liposomes. The idea of using plants to produce therapeutic miRNAs isparticularly attractive for several reasons. Plants can be bioengineered to make miRNAs of any desiredsequence and plant-made miRNAs are biologically natural - not double-stranded or chemically modified.Furthermore, plants package miRNAs into nanoparticles that resemble exosomes. These plant exosomesstabilize miRNAs in the GI tract and are taken up via intestinal stem cells and macrophages, providing aneffective delivery mechanism. Finally, the plant exosomal membranes themselves have been shown to have anti-inflammatory properties. The proposed experiments build on our preliminary results from feeding experimentsusing plant-produced mammalian tumor suppressor miRNAs to reduce tumor burden in the well-establishedApcmin/+ mouse model of colon cancer. The goals of the proposal are three-fold: 1) Characterize the miRNAcontent of exosomes isolated from our transgenic plants that have been bioengineered to produce validatedmammalian tumor suppressor miRNAs; 2) establish methods to enhance packaging of bioengineeredmammalian miRNAs into the plant exosomal fraction; and 3) test the efficacy of orally administered tumorsuppressor miRNA-containing plant exosomes in chemoprevention, using the APCmin/+ mouse model of coloncancer. To our knowledge, we are the only group exploring this potentially transformative approach to enablingthe use of miRNA therapeutics.