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

Author:

Gee PeterORCID,Lung Mandy S. Y.ORCID,Okuzaki YuyaORCID,Sasakawa Noriko,Iguchi TakahiroORCID,Makita YukimasaORCID,Hozumi Hiroyuki,Miura Yasutomo,Yang Lucy F.,Iwasaki MioORCID,Wang Xiou H.ORCID,Waller Matthew A.ORCID,Shirai Nanako,Abe Yasuko O.ORCID,Fujita YokoORCID,Watanabe KeiORCID,Kagita Akihiro,Iwabuchi Kumiko A.ORCID,Yasuda Masahiko,Xu HuaigengORCID,Noda Takeshi,Komano JunORCID,Sakurai Hidetoshi,Inukai NaotoORCID,Hotta AkitsuORCID

Abstract

AbstractProlonged 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.

Funder

Takeda Pharmaceutical Company

Japan Agency for Medical Research and Development

MEXT | Japan Society for the Promotion of Science

Publisher

Springer Science and Business Media LLC

Subject

General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry

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