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控技术具有使下一代工程T细胞和淋巴细胞用于肿瘤学适应症的潜力，通过展示高效和 将嵌合抗原受体(CAR)基因可扩展地导入αβT细胞，以及其他淋巴细胞载体，如γδT、NK和NKT淋巴细胞。CAR-mRNA-T细胞具有靶向作用 同时，由于汽车的瞬时表情，呈现出更好的安全配置。γδT、NK和NKT淋巴细胞具有先天免疫特性，可以使普遍的同种异体产品成为可能。 在这一阶段，我们提出了一系列研究，以优化αβT细胞和至少一个其他商业感兴趣的淋巴细胞群体的mrna转导，使用执行细胞的微流控设备 对流输送的体积交换(Vect)。在目标1中，我们想要确定Vect的最佳电池起始材料。我们将通过评估转基因和细胞适合性来表征商业细胞来源 在Vect之后，选择一个选项，为我们提供多个淋巴细胞群来处理(例如，新鲜的外周血单核细胞)。我们将挑选细胞活性最好的细胞起始材料， 在流经我们的微流控通道后恢复。在目标2中，我们将通过观察以下变量来优化GFP mRNA在这些细胞中的传递：流速、压缩间隙大小和有效载荷浓度。在……里面 目的3，我们将利用我们的工艺制造大量抗CD19的CAR淋巴细胞，并通过释放细胞因子来证明其治疗效果。我们将把我们的生产效率作为基准 电穿孔，目前最先进的信使核糖核酸转导技术。

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.