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

微流体平台可生成用于 T 细胞恶性肿瘤的“现成”抗自相残杀 CAR T 细胞

基本信息

批准号：
10317102
负责人：
Sunil Sudhir Raikar
金额：
$ 17.32万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-10 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10317102
关键词：
Allogenic Animals Antigen Targeting Antigens B lymphoid malignancy Biological Assay Biomechanics CAR T cell therapy CD5 Antigens CRISPR interference CRISPR/Cas technology Cell Line Cell Nucleus Cell Proliferation Cell Survival Cell Therapy Cell physiology Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Convection Data Devices Disease Electroporation Engineering Evaluation Flow Cytometry Generations Genomic Instability Goals Head Heterogeneity IL2 gene Immunocompromised Host In Vitro Individual Industry Knock-out Lentivirus Vector Leukapheresis Leukemic Cell Life Luciferases Malignant - descriptor Malignant Neoplasms Methods Microfluidic Microchips Microfluidics Monitor Mus Patients Process Production Relapse Relaxation Research Research Personnel Research Project Grants Resistance Ribonucleoproteins Route Surface Antigens System T cell therapy T-Cell Leukemia T-Cell Receptor T-Lymphocyte TRA@ gene cluster Technology Testing Therapeutic Time Transfection Translating Treatment Protocols Viral Viral Vector Xenograft procedure base cancer therapy cellular engineering chimeric antigen receptor chimeric antigen receptor T cells combinatorial cytokine cytotoxicity design experimental study genetically modified cells genome editing graft vs host disease in vitro testing in vivo innovation insertion/deletion mutation knock-down mouse model novel pre-clinical prevent receptor expression success tool transcriptome sequencing vector

项目摘要

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.

项目概要嵌合抗原受体 (CAR) T 细胞疗法在治疗 B 细胞方面取得了广泛成功然而，很少有研究评估 CAR T 细胞疗法用于治疗 T 细胞将这种疗法转化为 T 细胞疾病存在两个主要的制造挑战。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细胞会经历自相残杀，导致CAR T细胞产品的有效扩增变得困难。其次，分离困难。白细胞分离过程中的健康 T 细胞会导致产品污染，从而无意中感染恶性 T 细胞转导表达 CAR 后会产生治疗耐药性，因此理想情况下，靶向 T 细胞疾病需要一种治疗方法。同种异体“现成”抗自相残杀 CAR T 细胞产品可以通过多重基因组来实现。在用 CAR 表达载体转导之前对 T 细胞进行基因组编辑。通过 CRISPR/Cas9 技术的抗原可以防止自相残杀，同时敲低 T 细胞受体 (TCR) 通过 T 细胞受体 α 链 (TRAC) 基因座编辑表达可以防止危及生命的移植物然而，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，低效的转染和组合随机性才能产生最终的结果。产品含有不安全或无效的细胞子集，从而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连续进行多个基因组编辑时，电穿孔会产生大量的结果。或者，当作为批处理进行时，电穿孔会导致细胞增殖减少和产量低。可能会导致 CRISPR 编辑的干扰，或更糟糕的是，导致大量双链断裂，最终导致在这个合作的多学科研究者（mPI）中，基因组不稳定和体内低增殖。根据提案，我们计划测试一种新型微流体转染技术来产生有效的 CAR T 细胞我们的微流体平台，称为对流转移体积交换。 (VECT) 机械穿孔是一种非病毒生物力学方法，能够有效传递基因组它有可能允许高转染的多次 CRISPR 编辑。效率和活力，同时足够温和，以避免疼痛对治疗细胞的脱靶损伤。 VECT 机械穿孔显示出对 T 细胞核的损伤较低，因此提供了一条途径产生更多的增殖性治疗性 T 细胞在目标 1 中，我们将建立微流体装置和工艺。参数以最佳方式将 CD5 和 TRAC CRISPR-Cas9 编辑分子以连续和在目标 2 中，将用编码 CD5-CAR 的慢病毒载体转导编辑后的 T 细胞。细胞毒性将在体外和体内实验中进行测试。