Abstract：
The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.
摘要：
现有的CAR-T细胞疗法制造方案带来了显著的挑战。特别是在获得持续显著持续时间的瞬时转染时。为了解决这个差距，本研究旨在制定转染方案,利用多种脂质纳米颗粒(LNPs)给药,将转染效率(TE)提高到临床相关水平.通过一系列迭代改进，系统地微调和优化我们的转染方案，我们已经在永生化的T淋巴细胞Jurkat细胞系中实现了显著的TE增加一个数量级。这种增强在2周内一直观察到，而且重要的是,已经实现了对细胞活力没有任何有害影响。在我们研究的后续阶段,我们的目的是通过使用我们改进的方案评估为DNA封装量身定制的三种基于脂质的制剂来优化基因递送系统.这些制剂包括由可电离脂质构建的两种LNP,其特征在于脂质组成(iLNP)和阳离子脂质复合物(cLNP)的系统变化。我们的研究结果表明，在三种配方中，随着cLNP成为进一步完善和整合到CAR-T疗法生产管道的领跑者。因此,仔细检查cLNP将CAR编码质粒DNA递送至HEK-293细胞系的潜力。共聚焦显微镜实验证明了它的效率，与iLNP相比，揭示了实质性的内在化。通过采用最近开发的共焦图像分析方法，我们证实cLNP的细胞进入主要通过巨噬细胞增多发生。该机制导致增强的细胞内体逃逸并减轻溶酶体积累。抗CD19-CD28-CD3z的成功表达,一种针对CD19的CAR，CD19是一种经常在B细胞表面表达的蛋白质，使用基于荧光的测定法证实。总的来说,我们的结果表明cLNP在基因传递中的有效性,并提示多次给药转染作为一种完善CAR治疗中T细胞工程方案的实用方法的潜力.未来的研究可能集中在通过调整转染参数如核酸浓度来改善结果。脂质与DNA的比率，和孵育时间以实现改善的TE和增加的基因表达水平。
