TOPIC 397 - MICROFLUIDIC TRANSFECTION PLATFORM FOR RAPID MANUFACTURING OF MRNA CAR LYMPHOCYTE PRODUCTS.

该项目的目标是证明我们的微流体技术有潜力使下一代工程化T细胞和淋巴细胞用于肿瘤适应症，通过展示高效和 将嵌合抗原受体（CAR）mRNA可规模化地转染到αβ T细胞中;以及转染到其他淋巴细胞载体如γδ T、NK和NKT淋巴细胞中。CAR mRNA T细胞具有靶向 肿瘤，同时由于CAR的瞬时表达而呈现出更好的安全性特征。γδ T、NK和NKT淋巴细胞具有先天免疫特性，能够产生通用的同种异体产物。 在第一阶段的资助中，我们提出了一系列研究，以优化αβ T细胞和至少一种其他具有商业价值的淋巴细胞群体的mRNA转染，使用一种微流体装置， 对流传输体积交换（VECT）。在目标1中，我们希望确定什么是VECT的最佳细胞起始材料。我们将通过评估转染和细胞适应性来表征商业细胞来源 在VECT之后，同时选择为我们提供多个淋巴细胞群的选项（例如新鲜外周血单核细胞）。我们将挑选具有最佳细胞活力的细胞起始材料， 在流过我们的微流体通道后恢复。在目标2中，我们将通过观察以下变量来优化GFP mRNA向这些细胞中的递送：流速、压缩间隙大小和有效载荷浓度。在 目的3，我们将使用我们的工艺生产研究批次的抗CD 19 CAR淋巴细胞，并通过细胞因子释放证明治疗效力。我们将把我们的制造产量与 电穿孔是目前mRNA转染的最新技术。

The goal of this project is to demonstrate that our microfluidic technology has the potential to enable the next generation of engineered T cells and lymphocytes for oncologic indications, by demonstrating efficient and scalable transfection of chimeric antigen receptor (CAR) mRNA into αβ T cells; as well as into other lymphocytic carriers such as γδ T, NK and NKT lymphocytes. CAR mRNA T cells have the potential to target tumours while presenting a better safety profile, due to the transient expression of the CAR. γδ T, NK and NKT lymphocytes present innate immune properties that could enable universal allogeneic products. In this phase I grant, we propose a series of studies to optimize mRNA transfection of αβ T cells and at least one other lymphocyte population of commercial interest, using a microfluidic device that performs cell volume exchange for convective transfer (VECT). In Aim 1, we want to establish what is the best cell starting material for VECT. We will characterize commercial cell sources by assessing transfection and cell fitness after VECT, while select an option that provides us with multiple lymphocyte populations to work with (e.g. fresh peripheral blood mononuclear cells). We will pick the cell starting material with best cell viability and recovery after flowing through our microfluidic channel. In Aim 2, we will optimize the delivery of GFP mRNA into these cells, by looking at the variables: flow rate, compression gap size and payload concentration. In Aim 3, we will use our process to manufacture an investigational lot of anti-CD19 CAR lymphocytes and demonstrate therapeutic potency by cytokine release. We will benchmark our manufacturing yield against electroporation, the current state-of-the-art for mRNA transfection.