Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto, 606-8507, Japan.
Nat Commun. 2020 Mar 13;11(1):1334. doi: 10.1038/s41467-020-14957-y.
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.
病毒载体中 CRISPR-Cas9 核酸酶和 gRNA 的长时间表达可能导致脱靶突变和免疫原性。因此，对于治疗性基因组编辑应用，需要一种瞬时的递送系统。在这里，我们通过利用两种不同的归巢机制，开发了一种名为 NanoMEDIC 的基于细胞外纳米囊泡的核糖核蛋白递送系统。化学诱导二聚化将 Cas9 蛋白募集到细胞外纳米囊泡中，然后病毒 RNA 包装信号和两个自我切割的核酶将 sgRNA 连接并释放到纳米囊泡中。我们证明了在各种难以转染的细胞类型中，包括人诱导多能干细胞 (iPS) 细胞、神经元和肌母细胞，都能有效进行基因组编辑。NanoMEDIC 还能在源自杜氏肌营养不良症 (DMD) 患者 iPS 细胞的骨骼肌细胞中实现超过 90%的外显子跳跃效率。最后，单次肌肉内注射 NanoMEDIC 可在荧光素酶报告小鼠和 mdx 小鼠中诱导永久性基因组外显子跳跃，表明其可用于 DMD 及其他疾病的体内基因组编辑治疗。
