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

Peter Gee, Mandy S. Y. Lung, Yuya Okuzaki, Noriko Sasakawa, Takahiro Iguchi, Yukimasa Makita, Hiroyuki Hozumi, Yasutomo Miura, Lucy F. Yang, Mio Iwasaki, Xiou H. Wang, Matthew A. Waller, Nanako Shirai, Yasuko O. Abe, Yoko Fujita, Kei Watanabe, Akihiro Kagita, Kumiko A. Iwabuchi, Masahiko Yasuda, Huaigeng Xu, Takeshi Noda, Jun Komano, Hidetoshi Sakurai, Naoto Inukai and Akitsu Hotta ()
Additional contact information
Peter Gee: Kyoto University
Mandy S. Y. Lung: Kyoto University
Yuya Okuzaki: Kyoto University
Noriko Sasakawa: Kyoto University
Takahiro Iguchi: Kyoto University
Yukimasa Makita: Takeda-CiRA Joint Program (T-CiRA)
Hiroyuki Hozumi: Takeda-CiRA Joint Program (T-CiRA)
Yasutomo Miura: Kyoto University
Lucy F. Yang: Kyoto University
Mio Iwasaki: Kyoto University
Xiou H. Wang: Kyoto University
Matthew A. Waller: Kyoto University
Nanako Shirai: Kyoto University
Yasuko O. Abe: Kyoto University
Yoko Fujita: Kyoto University
Kei Watanabe: Kyoto University
Akihiro Kagita: Kyoto University
Kumiko A. Iwabuchi: Kyoto University
Masahiko Yasuda: Central Institute for Experimental Animals
Huaigeng Xu: Kyoto University
Takeshi Noda: Kyoto University
Jun Komano: Nagoya Medical Center
Hidetoshi Sakurai: Kyoto University
Naoto Inukai: Takeda-CiRA Joint Program (T-CiRA)
Akitsu Hotta: Kyoto University

Nature Communications, 2020, vol. 11, issue 1, 1-18

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.

Date: 2020
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DOI: 10.1038/s41467-020-14957-y

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