Integrating microfluidic vortex shedding-mediated gene delivery into the development and manufacture pipelines of adoptive cellular immunotherapies

患者源性调节性T（Treg）细胞的离体工程有望成为安全有效的方法 预防移植物抗宿主病和治疗一系列自身免疫性疾病，如1型糖尿病， 和多发性硬化症然而，目前用于工程化患者来源的细胞用于治疗的方法， 病毒的使用（病毒递送和电穿孔）还有待优化，以增加每种病毒的广泛可用性。 为有需要的患者提供超声波免疫治疗。这一建议的重点是满足这一需要的一个跨- Treg细胞工程方法，从实验室的开发和优化到商业化， 用于处方使用的个性化Treg细胞免疫疗法的工业化生产。微流控涡旋脱落 （µVS）是一种安全、快速的方法，可以对患者来源的CD 3 + T细胞进行遗传修饰。µVS技术需要 天然存在的流体动力学性质温和地和暂时地穿孔细胞膜的优点， 从而实现了一种快速而安全的T细胞转染方法，这是目前的方法无法实现的。 接近这项工作的目的是将µVS的效用扩展到独特的Treg细胞群，从而 证明了µVS开发和制造用于细胞免疫疗法的工程TdR的可行性， 蜜蜂。研究和开发的目标是（1）展示µVS-me的技术性能， 用嵌合抗原受体（CAR）构建体对人Treg细胞的介导转染，所述嵌合抗原受体构建体靶向来自 按压人白细胞抗原A2，从而表明作为预防性治疗的临床效用 移植物抗宿主病，和（2）证明产生的转染Treg细胞的功能性和安全性 在基于细胞的和体内试验中通过µVS。在成功完成这些目标之前，CAR-Treg 细胞将使用患者来源的Treg细胞进行工程化，商业规模的处理和富集 将证明有足够的遗传修饰的活细胞用于临床应用。

Ex vivo engineering of patient-derived regulatory T (Treg) cells holds promise as a safe and effective approach to preventing graft versus host disease and treating a range of autoimmune diseases, such as type 1 diabetes and multiple sclerosis. However, the methods currently employed to engineer patient-derived cells for thera- peutic use (viral delivery and electroporation) have yet to be optimized to increase the broad availability of per- sonalized immunotherapies to patients in need. This proposal is focused on meeting this need for an across- the-board Treg cell engineering method, from development and optimization in the laboratory to the commer- cial production of personalized Treg cell immunotherapies for prescription use. Microfluidic vortex shedding (µVS) is a safe and rapid approach to genetically modify patient-derived CD3+ T cells. µVS technology takes advantage of naturally occurring fluid dynamic properties to gently and temporarily porate cell membranes, thereby enabling a rapid, yet safe, approach to T cell transfection that cannot be achieved by current ap- proaches. The objective of this work is to expand the utility of µVS to the unique Treg cell population, thus demonstrating the feasibility of µVS to develop and manufacture engineered Tregs for cell-based immunother- apies. The research and development objectives are to (1) demonstrate the technical performance of µVS-me- diated transfection of human Treg cells with a chimeric antigen receptor (CAR) construct that targets cells ex- pressing the human leukocyte antigen A2, thus indicating clinical utility as a therapeutic treatment to prevent graft versus host disease, and (2) demonstrate the functionality and safety of transfected Treg cells generated by µVS in cell-based and in vivo assays. Pending the successful completion of these objectives, CAR-Treg cells will be engineered using patient-derived Treg cells, and commercial-scale processing and enrichment of sufficient genetically modified viable cells for clinical applications will be demonstrated.