Complete encapsulation of nucleic acids by lipid‐based nanoparticles (LNPs) is often thought to be one of the main prerequisites for successful nucleic acid delivery, as the lipid environment protects mRNA from degradation by external nucleases and assists in initiating delivery processes. However, delivery of mRNA via a preformed vesicle approach (PFV‐LNPs) defies this precondition. Unlike traditional LNPs, PFV‐LNPs are formed via a solvent‐free mixing process, leading to a superficial mRNA localization. While demonstrating low encapsulation efficiency in the RiboGreen assay, PFV‐LNPs improved delivery of mRNA to the retina by up to 50% compared to the LNP analogs across several benchmark formulations, suggesting the utility of this approach regardless of the lipid composition. Successful mRNA and gene editors’ delivery is observed in the retinal pigment epithelium and photoreceptors and validated in mice, non‐human primates, and human retinal organoids. Deploying PFV‐LNPs in gene editing experiments result in a similar extent of gene editing compared to analogous LNP (up to 3% on genomic level) in the Ai9 reporter mouse model; but, remarkably, retinal tolerability is significantly improved for PFV‐LNP treatment. The study findings indicate that the LNP formulation process can greatly influence mRNA transfection and gene editing outcomes, improving LNP treatment safety without sacrificing efficacy.

中文翻译：

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LNP 制造的预成型囊泡方法增强视网膜 mRNA 递送

基于脂质的纳米粒子（LNP）对核酸的完全封装通常被认为是成功核酸递送的主要先决条件之一，因为脂质环境可以保护 mRNA 免受外部核酸酶的降解并有助于启动递送过程。然而，通过预先形成的囊泡方法（PFV-LNP）传递 mRNA 违背了这一前提条件。与传统的 LNP 不同，PFV-LNP 是通过无溶剂混合过程形成的，从而导致表面 mRNA 定位。虽然在 RiboGreen 测定中表现出较低的封装效率，但与几种基准配方中的 LNP 类似物相比，PFV-LNP 将 mRNA 向视网膜的递送提高了高达 50%，这表明该方法的实用性与脂质成分无关。在视网膜色素上皮和光感受器中观察到成功的 mRNA 和基因编辑器传递，并在小鼠、非人类灵长类动物和人类视网膜类器官中得到验证。在 Ai9 报告小鼠模型中，在基因编辑实验中部署 PFV-LNP 会产生与类似 LNP 相似的基因编辑程度（基因组水平高达 3%）；但值得注意的是，PFV-LNP 治疗的视网膜耐受性显着改善。研究结果表明，LNP 配制过程可以极大地影响 mRNA 转染和基因编辑结果，在不牺牲疗效的情况下提高 LNP 治疗的安全性。