Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping
Peter Gee, Mandy Siu Yu Lung, Yuya Okuzaki, Noriko Sasakawa, Takahiro Iguchi, Yukimasa Makita, Hiroyuki Hozumi, Yasutomo Miura, Lucy Yang, Mio Iwasaki, Xiou H. Wang, Matthew A. Waller, Nanako Shirai, Yasuko Abe, Yoko Fujita, Kei Watanabe, Akihiro Kagita, Kumiko A. Iwabuchi, Masahiko Yasuda, Huaigeng Xu, Takeshi Noda, Jun Komano, Hidetoshi Sakurai, Naoto Inukai, Akitsu Hotta
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.