Mechanism and approach to inactivate mutant KRAS of lung metastatic colon cancer by RNA-ligand-displaying exosome to co-deliver dCas9--gRNA ribonucleoprotein complex and KRAS siRNA

PROJECT SUMMARY Targeted nanotechnologies have shown great promise in overcoming roadblocks in cancer therapeutics. Tumor metastasis has a limited response or resistance to chemotherapeutics and a moderate response to new antibody therapies. Our RNA nanotechnology has shown great promise in targeting metastatic disease to deliver both gene silencing and chemical drug therapies. Our long goal is to overcome colon cancer, the second most common cause of cancer death primarily due to the mutations to KRAS and subsequent lung metastasis with expected survival of months. KRAS, the gene that codes for the K-Ras protein, is considered “undruggable”. KRAS mutations are found in up to 45% of colorectal cancers. We have designed an epigenetic repressor to silence mutant K-Ras through epigenome editing. We created a fusion protein consisting of nuclease-inactive dCas9 and the histone deacetylase HDAC1 and targeted dCas9-HDAC1 to the promoter of mutant KRAS. We can load the recombinant dCas9-HDAC1- gRNA ribonucleoprotein (RNP) complex into exosomes and silence K-Ras; we designed RNA nanoparticles carrying mutant K-Ras siRNA to inhibit KRAS mutant lung cancer; we also successfully constructed RNA 4WJ carrying SN-38 to inhibit colon cancer lung metastasis. The goal of this proposal is to identify mechanisms that govern the high delivery platform to silence K-Ras in colon cancer. We intend to deactivate mutant KRAS via RNA-ligand displaying exosomes loaded with dCas9- HDAC1-gRNA ribonucleoprotein, siRNAs, and chemical drugs individually or in combination in colorectal cancer primary and metastasis tumors, using orthotopic and PDX xenograft models. We will investigate the mechanism of action in K-Ras inhibition, including the conditions for the integration and assembly of dCas9-HDAC1 and gRNA or crRNA such as sequence and length requirement for silencing mutant KRAS and suppressing colon cancer cells. We will apply RNA nanoparticle orientation to display targeting ligands on the surface of exosomes. Instead of delivering dCas9 plasmids, we will deliver a ribonucleoprotein complex of dCas9-recombinant protein and gRNA. Exosomes will display RNA nanoparticles with an aptamer to bind colon cancer cells specifically. We will engineer RNA nanoparticles and increase the surface display density of the negatively charged RNA ligands to enhance the negative zeta potential of exosomes for preventing binding to the vital organs and healthy cells that normally have negatively charged lipid membranes. We will try to enhance therapeutic efficacy and reduce toxicity by overcoming endosome trapping and non-specific cell entry through RNA ligand manipulation. Zonal and density gradient ultracentrifugation or size exclusion columns will select exosomes smaller than 100 nm to escape macrophage engulfment and improve biodistribution. This project with a multidisciplinary approach will build a strong foundation from which researchers can deploy large protein complexes to treat cancer by tumor- specific delivery and effectively targeting those previously difficult targets like K-Ras.