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.