Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Center for Fetal Research, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.
Lab Chip. 2024 Aug 6;24(16):3790-3801. doi: 10.1039/d4lc00283k.
mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized , these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing and facilitated 3-fold greater reduction of a therapeutically-relevant protein relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.
mRNA 为基础的基因编辑平台在治疗遗传疾病方面具有巨大的潜力。然而，为了实现这一潜力，这些核酸载体必须安全有效地递送到靶细胞。可离子化脂质纳米粒（LNPs）是最先进的非病毒 RNA 递药系统，已被广泛研究用于 mRNA 的递送，但尚未针对基于 mRNA 的 CRISPR-Cas9 平台的递送进行系统优化。在这项研究中，我们研究了微流控和脂质辅料参数对 LNP 基因编辑效果的影响。通过在肝细胞中的筛选，我们发现 LNP 制剂中的磷脂、胆固醇和脂质-PEG 结构在递送方面存在明显的趋势。最佳脂质辅料的组合产生了一种 LNP 制剂，与未经优化的 LNP 制剂相比，其基因编辑效率提高了 3 倍，并且能够降低 3 倍的治疗相关蛋白。因此，对 LNP 制剂参数的系统优化揭示了一种新型 LNP 制剂，它具有将基因编辑物递送到肝脏以治疗代谢疾病的强大潜力。
