Biopharmaceutical Development, Biopharmaceuticals R&D, AstraZeneca, Gaithersburg, MD, 20878, USA; Johns Hopkins University, Department of Chemical and Biomolecular Engineering, Baltimore, MD, 21218, USA.
Eurofins Lancaster Laboratories Professional Scientific Services, LLC, Gaithersburg, MD, 20878, USA.
Anal Biochem. 2021 Aug 15;627:114259. doi: 10.1016/j.ab.2021.114259. Epub 2021 May 23.
mRNA-based therapeutics have progressed significantly over the past decade and hold great potential to treat several diseases, including but not limited to cancer, infectious diseases, and genetic disorders. Many mRNA therapeutics are encapsulated in lipid nanoparticles (LNPs), which stabilize the mRNA and, following patient administration, transfect target cells to deliver the therapeutic mRNA. Developing reliable and robust in vitro bioassays is critical for expediting early screening of LNP-mRNA formulations, as well as identifying and developing lead candidate drug products. In this study, high-content fluorescent imaging was used to monitor LNP-mRNA transfections in both two- and three-dimensional HepG2 cell cultures. This technique allowed for continuous and simultaneous measurement of multiple LNP-mRNA transfection parameters, including cellular cytotoxicity, mRNA delivery, and mRNA translation/protein expression. Moreover, high-content imaging demonstrated acceptable accuracy and precision when quantifying the potency of mRNA delivery and subsequent translation and protein expression, thereby establishing the method as fit-for-purpose. Furthermore, concurrent evaluation of two different LNP formulations showed high-content imaging is sensitive at detecting pharmacological differences among varying LNP formulations. This study details the efficiency and reliability of LNP-mRNA characterizations that can be accomplished with high-content imaging.
mRNA 疗法在过去十年中取得了重大进展，具有治疗多种疾病的巨大潜力，包括但不限于癌症、传染病和遗传疾病。许多 mRNA 疗法被包裹在脂质纳米颗粒（LNPs）中，这些颗粒稳定了 mRNA，并且在患者给药后，转染靶细胞以递送治疗性 mRNA。开发可靠和强大的体外生物测定对于加速 LNP-mRNA 配方的早期筛选以及鉴定和开发先导候选药物产品至关重要。在这项研究中，高内涵荧光成像被用于监测二维和三维 HepG2 细胞培养物中的 LNP-mRNA 转染。该技术允许连续和同时测量多个 LNP-mRNA 转染参数，包括细胞毒性、mRNA 传递和 mRNA 翻译/蛋白质表达。此外，高内涵成像在定量 mRNA 传递及其随后的翻译和蛋白质表达的效力方面表现出可接受的准确性和精密度，从而确立了该方法的适用性。此外，同时评估两种不同的 LNP 制剂表明，高内涵成像在检测不同 LNP 制剂之间的药理学差异方面具有敏感性。本研究详细介绍了可以通过高内涵成像完成的 LNP-mRNA 特性的效率和可靠性。
