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.