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

患者来源的调节性T(Treg)细胞的体外工程有望成为一种安全有效的方法 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 多发性硬化症。然而，目前用于设计患者来源细胞以进行Thera-TERA的方法。 常规使用(病毒传递和电穿孔)尚未优化，以增加PER-1的广泛可用性。 为有需要的患者提供声学免疫疗法。这项提议的重点是满足这种跨- -板式Treg细胞工程方法，从实验室的开发和优化到通用的Treg细胞工程方法。 用于处方使用的个性化Treg细胞免疫疗法的社会生产。微流控旋涡脱落 (µvs)是对患者来源的CD3+T细胞进行基因修饰的一种安全快速的方法。µVS技术需要 利用自然产生的流体动力学特性来温和和暂时地使细胞膜穿孔， 从而实现了一种快速而安全的T细胞转染法，这是目前的AP无法实现的。 行尸走肉。这项工作的目标是将µVS的用途扩展到独特的Treg细胞群，从而 论证了µVS开发和制造用于细胞免疫系统的工程树的可行性- 类人猿。研究和开发目标是(1)展示µVS-Me的技术性能- 以嵌合抗原受体(CAR)为靶点的人Treg细胞表达载体的构建 压低人类白细胞抗原A2，从而表明作为一种治疗方法对预防 移植物抗宿主病，以及(2)证明所产生的转基因Treg细胞的功能性和安全性 在基于细胞的检测和体内检测中，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细胞进行工程设计，并进行商业规模的加工和浓缩 将展示足够的可用于临床应用的转基因活细胞。

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.