Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.

递送核糖核蛋白 （RNP） 用于体内基因组编辑比使用编码 Cas9 及其各自向导 RNA 的病毒更安全。然而，瞬时 RNP 活性通常不会导致最佳编辑结果。在这里，我们表明，细胞穿透肽（共价融合到蛋白质或作为赋形剂）可以提高递送 RNP 的效率，并且可以优化封装 RNP 的脂质纳米颗粒 （LNP） 以增强 RNP 稳定性、递送效率和编辑效力。具体来说，在筛选合适的可电离阳离子脂质并优化合成脂质 DMG-PEG 2000 的浓度后，我们表明，使用可电离脂质 SM102 通过微流体混合将腺嘌呤碱基编辑器和引物编辑器 RNP 封装在 LNP 中，可以导致体内编辑效率提高超过 300 倍（相对于裸 RNP 的递送），而不会检测到脱靶编辑。我们相信，针对 RNP 封装稳定性和递送效率优化的化学成分确定的 LNP 制剂将导致更安全的基因组编辑。