Delivery of CRISPR/Cas9 Ribonucleoprotein using Carrier-Free Protein Engineering or Porous Silicon Nanoparticles

Abstract

Gene editing unlocks the source code of life—DNA—for human engineering. This could enable treating myriad diseases, with huge academic and industrial efforts ongoing towards treatments for numerous cancers, neurodegenerative diseases (like Alzheimer’s or Huntington’s), hypercholesteremia, osteoarthritis, and, of course, genetic diseases like Duchenne muscular dystrophy. The biggest technical challenge remaining is delivery—getting the treatment to the right cells. Against this challenge, we engineered two very different strategies, which approached the challenge from very different angles, and demonstrated efficacy in mouse models of post-traumatic osteoarthritis (PTOA) and Duchenne’s muscular dystrophy. The first strategy is the first to ever apply porous silicon nanoparticles (PSiNPs) to Cas9 delivery. Optimizing the formulation enabled gene editing twice as efficient as a commercial standard in vitro. High levels of gene editing were also achieved in mouse models of osteoarthritis and muscular dystrophy. The second strategy is a contrarian, paradigm-shifting approach to delivery. Instead of viral or nanoparticle encapsulation, we engineer the Cas9 protein to not need encapsulation (i.e. “carrier-free” delivery) by incorporating cell-penetrating peptides, chemical RNA stabilization, and targeting peptides. In a mouse model of Duchenne’s muscular Dystrophy, we demonstrate liver-avoidance, therapeutic levels of editing upon local administration, and significant levels of editing upon intravenous administration.