Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10–16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%–72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%–12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.
中文翻译：
脂质纳米颗粒介导的肇事逃逸方法可在人体皮肤中实现高效、安全的原位基因编辑
尽管基因编辑取得了令人兴奋的进展，但将遗传工具有效传递到肝外组织仍然具有挑战性。对于皮肤来说尤其如此，因为皮肤构成了高度限制性的传递屏障。在这项研究中，我们对 Cas9 mRNA 或负载核糖核蛋白 (RNP) 的脂质纳米粒子 (LNP) 进行了头对头比较，以将基因编辑工具传递到人体皮肤的表皮层，旨在实现原位基因编辑。我们观察到不同的 LNP 组成和细胞特异性效应，例如 RNP 在慢循环上皮细胞中的存在时间长达 72 小时。虽然使用 Cas9 RNP 和 mRNA 与基于 MC3 的 LNP 获得了相似的基因编辑率 (10-16%)，但事实证明装载 mRNA 的 LNP 具有更强的细胞毒性。有趣的是，p K a ∼ 7.1 的可电离脂质在二维 (2D) 上皮细胞中产生了优异的基因编辑率 (55%–72%)，同时没有检测到单向导 RNA 依赖性脱靶效应。出乎意料的是，这些高 2D 编辑功效并没有转化为实际皮肤组织，无论 LNP 成分如何，单次应用后总体基因编辑率达到 5%–12% 之间。最后，我们成功地对常染色体隐性先天性鱼鳞病患者细胞中的致病突变进行了碱基校正，其疗效为~5%，展示了该策略治疗单基因皮肤病的潜力。总而言之，这项研究证明了原位纠正皮肤致病突变的可行性，可以为罕见、单基因和常见的皮肤病提供有效的治疗，甚至有可能治愈。
