Microfluidic platforms to generate 'off-the-shelf' fratricide-resistant CAR T cells for T-cell malignancies

项目摘要 嵌合抗原受体(CAR)T细胞疗法在治疗B细胞方面非常成功 恶性肿瘤；然而，评价CAR T细胞疗法治疗T细胞的研究较少 恶性肿瘤。在将这种疗法转化为T细胞疾病时，存在两个主要的制造挑战。第一, 鉴于恶性T细胞上缺乏癌症特异性抗原，针对T细胞抗原的CAR T细胞经历了 自相残杀，从而使汽车T细胞产品的有效扩张变得困难。第二，隔离的难度 白细胞分离过程中的健康T细胞会导致产品污染，其中恶性T细胞在不经意间 换能器表示汽车变得对治疗产生抗药性。因此，理想情况下，针对T细胞疾病需要 同种异体“现成”抗杀人剂汽车T细胞产品。这可以通过多重基因组来实现。 利用CAR表达载体对转导前的T细胞进行编辑。靶T细胞的基因组编辑 通过CRISPR/Cas9技术的抗原将防止自相残杀，同时击倒T细胞受体(TCR) 通过编辑T细胞受体阿尔法链(TRAC)基因座表达将防止危及生命的移植物- 抗宿主疾病。然而，需要新的递送技术来促进T细胞疗法的生产 需要进行多次基因组编辑。低效的转染性和组合随机性会产生最终的 含有不安全或无效的细胞子集的产品，降低产量和产品效力。 当前的目标标准是使用非病毒传递系统通过 电穿孔。当连续进行多基因组编辑时，电穿孔导致大量的 细胞增殖减少，产量低。或者，当作为批处理过程执行时，电穿孔 可能会导致CRISPR编辑的干扰，或者更糟糕的是，过多的双链断裂最终导致 基因组不稳定，体内低增殖。在这个协作的多主体调查者(MPI)中 建议，我们计划测试一种新的微流控技术，以产生有效的CAR T细胞 治疗T细胞恶性肿瘤的产品。我们的微流控平台，称为对流换热 (Vect)机械操作是一种非病毒的生物力学方法，能够有效地传递基因组 将产品编辑到单元格内部。它有可能允许高转染率的多个CRISPR编辑 效率和生存能力，同时足够温和，以避免对治疗细胞的有害偏离目标的损害。 Vect机械修复术对T细胞核的损伤很小，因此，提供了一条途径 产生更具增殖性的治疗性T细胞。在目标1中，我们将建立微流控装置和流程 以最佳方式将CD5和TRAC CRISPR-Cas9编辑分子以串联和 多路复用方法。在目标2中，编辑的T细胞将被编码慢病毒载体的CD5-CAR转导 细胞毒性将在体外和体内实验中进行测试。

Project Summary Chimeric antigen receptor (CAR) T-cell therapy has been remarkably successful in treating B-cell malignancies; however, fewer studies have evaluated CAR T-cell therapy for the treatment of T-cell malignancies. Two main manufacturing challenges exist in translating this therapy for T-cell disease. First, given the lack of a cancer-specific antigen on malignant T cells, CAR T cells targeting T-cell antigens undergo fratricide, thus making effective expansion of a CAR T-cell product difficult. Second, the difficulty in isolating healthy T cells during leukapheresis results in product contamination, wherein malignant T cells inadvertently transduced to express the CAR become treatment-resistant. Thus targeting T-cell disease ideally requires an allogeneic “off-the-shelf” fratricide-resistant CAR T-cell product. This can be achieved by multiplex genome editing of T cells prior to transduction with the CAR-expressing vector. Genome editing of the target T-cell antigen via CRISPR/Cas9 technology would prevent fratricide, while knocking down T-cell receptor (TCR) expression through T-cell receptor alpha chain (TRAC) locus editing would prevent life-threatening graft- versus-host disease. However, new delivery technologies are needed to facilitate production of T-cell therapies requiring multiple genome edits. Inefficient transfection and combinatorial stochasticity can produce a final product that contain subsets of cells that are unsafe or ineffective, decreasing yield as well as product potency. The current goal standard is to perform knockout edits using a non-viral delivery system through electroporation. Electroporation when conducted serially for multiple genome edits results in a substantial decrease in cell proliferation and low yield. Alternatively, when performed as a batch process, electroporation can result in the interference of CRISPR edits, or worse, a plethora of double strand breaks that culminate in genomic instability and low proliferation in vivo. In this collaborative multiple principle investigator (mPI) proposal, we plan to test a novel microfluidic transfection technology to generate an effective CAR T-cell product for T-cell malignancies. Our microfluidic platform, called volume exchange for convective transfer (VECT) mechanoporation, is a non-viral, biomechanical approach that enables efficient delivery of genome editing products into the cell interior. It has the potential to permit multiple CRISPR edits with high transfection efficiency and viability, while being gentle enough to avoid detrimental off-target damage to therapeutic cells. VECT mechanoporation has shown low damage to the nucleus of T cells and therefore, offers a route to produce more proliferative therapeutic T cells. In Aim 1, we will establish the microfluidic device and process parameters to optimally deliver CD5 and TRAC CRISPR-Cas9 editing molecules to T cells, in both serial and multiplexed approaches. In Aim 2, edited T cells will be transduced with CD5-CAR encoding lentiviral vector and cytotoxicity will be tested in in vitro and in vivo experiments.