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

• 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
• 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

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；然而，较少的研究评估了T细胞治疗T细胞的CAR T细胞疗法 恶性肿瘤。将这种疗法转化为T细胞疾病的主要制造挑战。第一的， 鉴于缺乏恶性T细胞上的癌症特异性抗原，靶向T细胞抗原的CAR T细胞经历 杂化，从而使CAR T细胞产品的有效扩展变得困难。第二，隔离的困难 白细胞术期间健康的T细胞会导致产物污染，其中恶性T细胞无意间 转导以表达汽车变得耐药。理想情况下，靶向T细胞疾病需要 同种异体“现成的”耐药性汽车T细胞产品。这可以通过多重基因组来实现 在用表达汽车的载体转移之前，T细胞的编辑。目标T细胞的基因组编辑 通过CRISPR/CAS9技术抗原可以防止杂化，同时击倒T细胞受体（TCR） 通过T细胞受体α链（TRAC）的表达表达将防止威胁生命的移植物 与宿主疾病。但是，需要新的交付技术来促进T细胞疗法的生产 需要多个基因组编辑。效率低下的转染和组合随机性可以产生最终 包含不安全或无效的细胞子集，产量降低以及产品效力。 当前的目标标准是使用非病毒输送系统执行淘汰赛编辑 电穿孔。当进行多个基因组编辑串行进行电穿孔导致实质性 细胞增殖和低产量的降低。替代地作为批处理过程进行电穿孔 可能导致CRISPR编辑的干扰，或更糟糕的是，大量的双链断裂了 基因组不稳定性和体内较低的增殖。在此合作多重主要研究者（MPI）中 提案，我们计划测试一种新型的微流体转染技术，以生成有效的汽车T细胞 T细胞恶性肿瘤的产品。我们的微流体平台，称为对流传输的音量交换 （vect）机理围绕是一种非病毒，生物力学方法，可有效地递送基因组 将产品编辑到细胞内部。它有可能允许高转染的多次CRISPR编辑 效率和生存能力，同时足够温和以避免探测器对治疗细胞的损害。 VECT机械配饰显示出对T细胞核的损害较低，因此提供了通往的途径 产生更多增殖的治疗性T细胞。在AIM 1中，我们将建立微流体设备和过程 在串行和 多路复用方法。在AIM 2中，编辑的T细胞将用CD5卡车编码慢病毒载体翻译 细胞毒性将在体外和体内实验中进行测试。