Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping
| Autor: | Kumiko Iwabuchi, Akihiro Kagita, Lucy F. Yang, Nanako Shirai, Takahiro Iguchi, Yoko Fujita, Jun Komano, Takeshi Noda, Akitsu Hotta, Noriko Sasakawa, Yukimasa Makita, Matthew A. Waller, Naoto Inukai, Peter Gee, Mio Iwasaki, Mandy S. Y. Lung, Hidetoshi Sakurai, Yasuko O. Abe, Xiou H. Wang, Yasutomo Miura, Kei Watanabe, Masahiko Yasuda, Hiroyuki Hozumi, Huaigeng Xu, Yuya Okuzaki |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
CRISPR-Cas9 genome editing
0301 basic medicine viruses Duchenne muscular dystrophy General Physics and Astronomy Ligands 0302 clinical medicine HIV Protease Genome editing CRISPR-Associated Protein 9 CRISPR Gene delivery Guide RNA Luciferases Gene Editing Multidisciplinary Exons Tissue Donors Cell biology Ribonucleoproteins 030220 oncology & carcinogenesis tat Gene Products Human Immunodeficiency Virus Dimerization RNA Guide Kinetoplastida Cell Survival RNA Splicing Science Genetic Vectors Induced Pluripotent Stem Cells Biology Article General Biochemistry Genetics and Molecular Biology Viral vector Extracellular Vesicles 03 medical and health sciences medicine Humans RNA Catalytic Base Sequence Cas9 HEK 293 cells General Chemistry medicine.disease Exon skipping Targeted gene repair HEK293 Cells 030104 developmental biology Drug delivery Nanoparticles CRISPR-Cas Systems |
| Zdroj: | Nature Communications, Vol 11, Iss 1, Pp 1-18 (2020) Nature Communications |
| ISSN: | 2041-1723 |
| Popis: | 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. Expression of Cas9 and gRNA from viral vectors in vivo may cause off-target activity. Here the authors present NanoMEDIC, which uses nanovesicles to transiently deliver editing machinery to hard-to-transfect cells. |
| Databáze: | OpenAIRE |
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