Genomic Deletion of P-Glycoprotein from Gliomas Via the Focused Ultrasound-Delivery of a Duplex CRISPR Cas9 Ribonucleoprotein System.

P-glycoprotein (P-gp), an efflux transporter expressed in endothelium and tumor cells, restricts drug delivery to gliomas. To address this, we engineered a CRISPR Cas9 ribonucleoprotein (RNP) system that targets indels to exons 4 and 10 of the gene encoding for P-gp. Convection-enhanced delivery of this duplexed RNP system to murine gliomas inhibited efflux function in tumor cells and endothelium and improved tumor response to paclitaxel, a P-gp substrate. We then encapsulated RNPs in a lipid nanoparticle (LNP) formulation that naturally traffics to the lungs, an organ devoid of P-gp expression, and delivered them to murine gliomas with focused ultrasound (FUS). FUS-targeted LNP-RNP delivery to gliomas inhibited efflux function in tumor cells and endothelium and enhanced paclitaxel therapy. Our results conceptually illustrate a strategy wherein LNP-encapsulated CRISPR Cas9 cargo in the bloodstream is targeted to the brain with FUS, while off-target LNPs are biochemically trafficked to an innocuous sink organ.