Abstract
Major challenges such as nuclease degradation, rapid renal clearance, non-specific delivery, poor cellular uptake and inflammatory response have limited the clinical application of small RNA-mediated gene silencing. To overcome these challenges, we designed a novel targeting small RNA delivery platform comprising of three oligonucleotides: (1) a guide RNA sequence, (2) part of a passenger sequence linked to a DNA aptamer via a PEG linker, and (3) another passenger sequence conjugated to cholesterol, which assemble through complementary base pair annealing. Remarkably, in the presence of magnesium, this molecule self-assembled into a nanoparticle with a hydrophobic cholesterol core, hydrophilic RNA oligonucleotide shell and PEG-linked DNA aptamer flare. The nanoparticles conferred protection to the RNA oligonucleotides against nuclease degradation, which increased bioavailability, and reduced systemic inflammatory responses. The aptamer allowed targeted delivery of RNA therapeutics through cell-specific surface markers, and once inside the cell, the nanoparticles induced lysosomal leakage that released the RNA oligonucleotides into the cytosol to achieve gene silencing. We created a c-Kit-targeting miR-26a delivery particle that specifically accumulated in c-Kit+ breast cancer, significantly increased T cell recruitment, and inhibited tumor growth. Regression of large established tumors were achieved when the nanoparticle was used in combination with anti-CTLA-4 monoclonal antibody.