Nasr Sarah S, Paul Pascal, Loretz Brigitta, Lehr Claus-Michael
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Campus E8 1, 66123, Saarbrücken, Germany.
Department of Pharmacy, Saarland University, 66123, Saarbrücken, Germany.
Drug Deliv Transl Res. 2024 Dec;14(12):3339-3353. doi: 10.1007/s13346-024-01668-w. Epub 2024 Jul 15.
Co-delivery of different protein-encoding polynucleotide species with varying expression kinetics of their therapeutic product will become a prominent requirement in the realm of combined nucleic acid(NA)-based therapies in the upcoming years. The current study explores the capacity for time-staggered expression of encoded proteins by simultaneous delivery of plasmid DNA (pDNA) in the core and mRNA on the shell of the same nanocarrier. The core is based on a Gelatin Type A-pDNA coacervate, thermally stabilized to form an irreversible nanogel stable enough for the deposition of cationic coats namely, protamine sulfate or LNP-related lipid mixtures. Only the protamine-coated nanocarriers remained colloidally stable following mRNA loading and could successfully co-transfect murine dendritic cell line DC2.4 with fluorescent reporter mRNA(mCherry) and pDNA (pAmCyan1). Further investigation of the protamine-coated nanosystem only, the transfection efficiency (percentage of transfected cells) and level of protein expression (mean fluorescence intensity, MFI) of mRNA and pDNA, simultaneously delivered by the same nanocarrier, were compared and kinetically assessed over 48 h in DC2.4 using flow cytometry. The onset of transfection for both nucleotides was initially delayed, with levels < 5% at 6 h. Thereafter, mRNA transfection reached 90% after 24 h and continued to slightly increase until 48 h. In contrast, pDNA transfection was clearly slower, reaching approximately 40% after 24 h, but continuing to increase to reach 94% at 48 h. The time course of protein expression (represented by MFI) for both NAs essentially followed that of transfection. Model-independent as well as model-dependent kinetic parameters applied to the data further confirmed such time-staggered expression of the two NA's where mRNA's rate of transfection and protein expression initially exceeded those of pDNA in the first 24 h of the experiment whereas the opposite was true during the second 24 h of the experiment where pDNA displayed the higher response rates. We expect that innovative nanocarriers capable of time-staggered co-delivery of different nucleotides could open new perspectives for multi-dosing, pulsatile or sustained expression of nucleic acid-based therapeutics in protein replacement, vaccination, and CRISPR-mediated gene editing scenarios.
在未来几年，共同递送不同的蛋白质编码多核苷酸种类，且其治疗产物具有不同的表达动力学，将成为基于核酸（NA）的联合疗法领域的一项突出要求。当前的研究探索了通过在同一纳米载体的核心同时递送质粒DNA（pDNA）和在外壳上递送mRNA，实现编码蛋白的时间交错表达的能力。核心基于A型明胶-pDNA凝聚层，经热稳定形成不可逆的纳米凝胶，其稳定性足以用于沉积阳离子涂层，即硫酸鱼精蛋白或与脂质纳米颗粒（LNP）相关的脂质混合物。只有用鱼精蛋白包被的纳米载体在加载mRNA后仍保持胶体稳定性，并且能够成功地将荧光报告mRNA（mCherry）和pDNA（pAmCyan1）共转染小鼠树突状细胞系DC2.4。仅对用鱼精蛋白包被的纳米系统进行进一步研究，比较了由同一纳米载体同时递送的mRNA和pDNA的转染效率（转染细胞百分比）和蛋白表达水平（平均荧光强度，MFI），并在48小时内使用流式细胞术对DC2.4进行动力学评估。两种核苷酸的转染起始最初都有延迟，在6小时时水平低于5%。此后，mRNA转染在24小时后达到90%，并持续略有增加直至48小时。相比之下，pDNA转染明显较慢，在24小时后达到约40%，但持续增加，在48小时时达到94%。两种NA的蛋白表达时间进程（以MFI表示）基本遵循转染时间进程。应用于数据的独立模型和依赖模型的动力学参数进一步证实了两种NA的这种时间交错表达，即在实验的前24小时，mRNA的转染率和蛋白表达率最初超过pDNA，而在实验的第二个24小时则相反，此时pDNA显示出更高的反应率。我们预计，能够时间交错共同递送不同核苷酸的创新纳米载体，可能会为基于核酸的治疗在蛋白质替代、疫苗接种和CRISPR介导的基因编辑场景中的多剂量、脉冲或持续表达开辟新的前景。
