Hirose Jun, Aizawa Emi, Yamamoto Shogo, Xu Mingyao, Iwai Shigenori, Suzuki Keiichiro
Graduate School of Engineering Science, Osaka University, Toyonaka 560-8531, Osaka, Japan.
Graduate School of Frontier Bioscience, Osaka University, Suita 565-0871, Osaka, Japan.
Biomolecules. 2024 Dec 6;14(12):1558. doi: 10.3390/biom14121558.
In vivo genome editing holds great therapeutic potential for treating monogenic diseases by enabling precise gene correction or addition. However, improving the efficiency of delivery systems remains a key challenge. In this study, we investigated the use of lipid nanoparticles (LNPs) for in vivo knock-in of ectopic DNA. Our in vitro experiments demonstrated that the homology-independent targeted integration (HITI)-mediated genome-editing method achieved significantly higher knock-in efficiency at the locus in hepatic cells compared to the traditional homology-directed repair (HDR)-mediated approach. By optimizing LNP composition and administration routes, we successfully achieved HITI-mediated GFP knock-in (2.1-2.7%) in the livers of mice through intravenous delivery of LNP-loaded genome editing components. Notably, repeated intravenous dosing led to a twofold increase in liver GFP knock-in efficiency (4.3-7.0%) compared to a single dose, highlighting the potential for cumulative genome editing effects. These findings provide a solid foundation for the use of LNPs in in vivo knock-in strategies, paving the way for future genome-editing therapies.
体内基因组编辑通过实现精确的基因校正或添加，在治疗单基因疾病方面具有巨大的治疗潜力。然而，提高递送系统的效率仍然是一个关键挑战。在本研究中，我们研究了脂质纳米颗粒（LNP）用于体内异位DNA敲入的情况。我们的体外实验表明，与传统的同源定向修复（HDR）介导的方法相比，同源性非依赖性靶向整合（HITI）介导的基因组编辑方法在肝细胞的位点实现了显著更高的敲入效率。通过优化LNP组成和给药途径，我们通过静脉注射负载LNP的基因组编辑组件，成功在小鼠肝脏中实现了HITI介导的GFP敲入（2.1 - 2.7%）。值得注意的是，与单次给药相比，重复静脉给药导致肝脏GFP敲入效率提高了两倍（4.3 - 7.0%），突出了累积基因组编辑效应的潜力。这些发现为LNP在体内敲入策略中的应用提供了坚实的基础，为未来的基因组编辑疗法铺平了道路。
