Lipid nanoparticles (LNPs) have recently emerged as successful gene delivery platforms for a diverse array of disease treatments. Efforts to optimize their design for common administration methods such as intravenous injection, intramuscular injection, or inhalation, revolve primarily around the addition of targeting ligands or the choice of ionizable lipid. Here, we employed a multi-step screening method to optimize the type of helper lipid and component ratios in a plasmid DNA (pDNA) LNP library to efficiently deliver pDNA through intraduodenal delivery as an indicative route for oral administration. By addressing different physiological barriers in a stepwise manner, we down-selected effective LNP candidates from a library of over 1000 formulations. Beyond reporter protein expression, we assessed the efficiency in non-viral gene editing in mouse liver mediated by LNPs to knockdown and expression, thereby lowering low-density lipoprotein (LDL) cholesterol levels. Utilizing an all-in-one pDNA construct with Cas9 and gRNAs, our results showcased that intraduodenal administration of selected LNPs facilitated targeted gene knockdown in the liver, resulting in a 27% reduction in the serum LDL cholesterol level. This LNP-based all-in-one pDNA-mediated gene editing strategy highlights its potential as an oral therapeutic approach for hypercholesterolemia, opening up new possibilities for DNA-based gene medicine applications.

中文翻译：

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通过十二指肠内递送优化脂质纳米粒子用于肝脏基因编辑

脂质纳米颗粒（LNP）最近已成为用于多种疾病治疗的成功基因传递平台。优化其常见给药方法（例如静脉注射、肌内注射或吸入）设计的努力主要围绕添加靶向配体或选择可电离脂质。在这里，我们采用多步筛选方法来优化质粒 DNA (pDNA) LNP 库中辅助脂质的类型和成分比例，以通过十二指肠内递送有效地递送 pDNA，作为口服给药的指示性途径。通过逐步解决不同的生理障碍，我们从超过 1000 种制剂的库中筛选出有效的 LNP 候选药物。除了报告蛋白表达之外，我们还评估了 LNP 介导的小鼠肝脏非病毒基因编辑敲低和表达的效率，从而降低低密度脂蛋白 (LDL) 胆固醇水平。利用 Cas9 和 gRNA 的一体化 pDNA 构建体，我们的结果表明，十二指肠内施用选定的 LNP 促进了肝脏中的靶向基因敲除，导致血清 LDL 胆固醇水平降低 27%。这种基于 LNP 的一体化 pDNA 介导的基因编辑策略凸显了其作为高胆固醇血症口服治疗方法的潜力，为基于 DNA 的基因医学应用开辟了新的可能性。