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 hardto-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.

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Duchenne muscular dystrophy (DMD) is an intractable genetic muscular disorder characterized by the loss of DYSTROPHIN. The restoration of DYSTROPHIN is expected to be a curative therapy for DMD. Because muscle stem cells (MuSCs) can regenerate damaged myofibers with full-length DYSTROPHIN in vivo, their transplantation is being explored as such a therapy. As for the transplanted cells, primary satellite cells have been considered, but donor shortage limits their clinical application. We previously developed a protocol that differentiates induced pluripotent stem cells (iPSCs) to MuSCs (iMuSCs). To ameliorate the respiratory function of DMD patients, cell transplantation to the diaphragm is necessary but difficult, because the diaphragm is thin and rapidly moves. In the present study, we explored the transplantation of iMuSCs into the diaphragm. First, we show direct cell injection into the diaphragm of mouse was feasible. Then, to enhance the engraftment of the transplanted cells in a rapidly moving diaphragm, we mixed polymer solutions of hyaluronic acid, alginate and gelatin to the cell suspension, finding a solution of 20% dissolved hyaluronic acid and 80% dissolved gelatin improved the engraftment. Thus, we established a method for cell transplantation into mouse diaphragm and show that an injectable hyaluronic acid-gelatin solution enables the engraftment of iMuSCs in the diaphragm.