Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

TLDR

Prolonged expression of CRISPR‑Cas9 and gRNA from viral vectors can cause off‑target mutagenesis and immunogenicity. A transient delivery system is required for therapeutic genome editing. We developed NanoMEDIC, an extracellular nanovesicle‑based ribonucleoprotein delivery platform that uses chemical‑induced dimerization to load Cas9 and a viral RNA packaging signal with self‑cleaving riboswitches to release sgRNA. NanoMEDIC achieves efficient genome editing in hard‑to‑transfect cells, over 90 % exon skipping in DMD patient‑derived myoblasts, and permanent exon skipping after a single intramuscular injection in reporter and mdx mice.

Abstract

Abstract Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.