Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53715, United States.
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
ACS Appl Mater Interfaces. 2023 Mar 1;15(8):10464-10476. doi: 10.1021/acsami.2c21683. Epub 2023 Feb 17.
Genome editing mediated by the CRISPR-Cas system holds great promise for the treatment of genetic diseases. However, safe and efficient in vivo delivery of CRISPR genome editing machinery remains a challenge. Here, we report a lipopeptide-based nanoparticle (LNP) that can efficiently deliver the CRISPR Cas9/sgRNA ribonucleoprotein (RNP) and enable efficient genome editing both and . An artificial lipopeptide, GD-LP, was constructed by linking a hydrophilic guanidinium-rich head to an oleic acid-based hydrophobic tail a disulfide bond. LNP formed by the self-assembly of GD-LP can easily form a complex with RNP with a loading content of up to 20 wt %. The resulting RNP-LNP nanocomplex led to 72.6% gene editing efficiency in GFP-HEK cells with negligible cytotoxicity. The LNP also showed significantly higher transfection efficiencies than Lipofectamine 2000 for the delivery of mRNA in NIH 3T3 and RAW 264.7 and the delivery of plasmid DNA in B78 cells. In vivo studies showed that intramuscular injection of the RNP-LNP nanocomplex in Ai14 mice induced efficient gene editing in muscular tissues. Moreover, the delivery of Cas9 RNP and donor DNA by LNP (., RNP/ssODN-LNP nanocomplex) restored dystrophin expression, reduced skeletal muscle fibrosis, and significantly improved muscle strength in a Duchenne muscular dystrophy (DMD) mouse model.
基于 CRISPR-Cas 系统的基因组编辑在治疗遗传疾病方面具有巨大的潜力。然而，安全有效地将 CRISPR 基因组编辑机制递送到体内仍然是一个挑战。在这里，我们报告了一种基于脂肽的纳米颗粒 (LNP)，它可以有效地递送 CRISPR Cas9/sgRNA 核糖核蛋白 (RNP)，并能够在体内和体外实现高效的基因组编辑。人工脂肽 GD-LP 是通过将亲水的胍基丰富的头部连接到基于油酸的疏水尾部 一个二硫键来构建的。由 GD-LP 自组装形成的 LNP 可以很容易地与 RNP 形成复合物，负载量高达 20wt%。所得的 RNP-LNP 纳米复合物在 GFP-HEK 细胞中导致 72.6%的基因编辑效率，几乎没有细胞毒性。与 Lipofectamine 2000 相比，LNP 还显示出更高的转染效率，用于在 NIH 3T3 和 RAW 264.7 中递送 mRNA 以及在 B78 细胞中递送质粒 DNA。体内研究表明，在 Ai14 小鼠中肌肉内注射 RNP-LNP 纳米复合物可诱导肌肉组织中有效的基因编辑。此外，通过 LNP 递送 Cas9 RNP 和供体 DNA（即，RNP/ssODN-LNP 纳米复合物）可恢复肌营养不良蛋白的表达，减少骨骼肌纤维化，并显著改善 Duchenne 肌营养不良症 (DMD) 小鼠模型中的肌肉力量。
