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细胞免疫缺陷方面取得了显著成功。 恶性肿瘤;然而，很少有研究评估CAR T细胞疗法治疗T细胞 恶性肿瘤在将这种疗法转化为T细胞疾病方面存在两个主要的制造挑战。第一、 由于恶性T细胞上缺乏癌症特异性抗原，靶向T细胞抗原的CAR T细胞经历了 这是一种自相残杀，从而使得CAR T细胞产物的有效扩增变得困难。第二，隔离的困难 白细胞去除术期间的健康T细胞导致产物污染，其中恶性T细胞无意中 被转导以表达CAR的细胞变得具有治疗抗性。因此，靶向T细胞疾病理想地需要 同种异体“现成的”抗杀兄弟药CAR T细胞产物。这可以通过多重基因组来实现 在用CAR表达载体转导之前编辑T细胞。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，同时敲低T细胞受体（TCR） 通过T细胞受体α链（TRAC）基因座编辑表达将防止危及生命的移植物， 抗宿主病然而，需要新的递送技术来促进T细胞疗法的生产 需要多次基因组编辑。低效率的转染和组合随机性可以产生最终的 含有不安全或无效的细胞亚群的产品，降低了产量和产品效力。 目前的目标标准是通过使用非病毒递送系统进行敲除编辑， 电穿孔当连续进行多个基因组编辑时，电穿孔导致大量的细胞分裂。 细胞增殖减少和产量低。或者，当作为分批方法进行时，电穿孔 可能会导致CRISPR编辑的干扰，或者更糟的是，过多的双链断裂，最终导致 基因组不稳定性和体内低增殖。在这个合作的多个主要研究者（mPI） 我们计划测试一种新的微流体转染技术，以产生有效的CAR T细胞。 用于T细胞恶性肿瘤的产品。我们的微流体平台，称为对流转移的体积交换 （VECT）机械穿孔是一种非病毒的生物力学方法，其能够有效地递送基因组 编辑产品到细胞内部。它有可能允许在高转染率下进行多次CRISPR编辑 效率和活力，同时足够温和以避免对治疗细胞的有害脱靶损伤。 VECT机械穿孔已经显示出对T细胞核的低损伤，因此，提供了一种途径， 产生更多增殖性治疗性T细胞。在目标1中，我们将建立微流控装置和工艺 这些参数用于以连续和连续两种方式将CD 5和TRAC CRISPR-Cas9编辑分子最佳地递送至T细胞， 多重方法。在目标2中，编辑的T细胞将用编码CD 5-CAR的慢病毒载体转导 和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.