Acoustic microvortices instrumentation for dosage controlled, high efficiency cell engineering

用于剂量控制、高效细胞工程的声学微涡流仪器

• 批准号: 10629435
• 负责人: Abraham P. Lee
• 金额: $ 29.69万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629435
• 关键词: 3-Dimensional; Acoustics; Address; Allogenic; Automation; Award; Benchmarking; CRISPR/Cas technology; Cell Membrane Permeability; Cell Nucleus; Cell Survival; Cell Therapy; Cell membrane; Cells; Cellular Phone; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoplasm; Development; Diffusion; Engineered Gene; Engineering; Exposure to; Frequencies; Genes; Genetic; Goals; Harvest; Human; Immune; Immunologics; Immunotherapy; Knock-out; Lateral; Malignant Neoplasms; Mechanics; Mediating; Membrane; Messenger RNA; Methods; Microfluidics; Molecular; Nobel Prize; Nucleic Acids; Ocular orbit; Oncogenic; Outcome; Plasmids; Population; Preparation; Process; Proteins; Pump; Reagent; Rotation; Safety; Sampling; Stream; System; T-Lymphocyte; Techniques; Toxic effect; Transducers; Transfection; Transgenes; Viral Vector; cancer immunotherapy; cell type; cellular engineering; chimeric antigen receptor T cells; cost; design; dosage; electric field; innovation; instrument; instrumentation; membrane excitation; nanopore; portability; prototype; success; technological innovation; technology development

Contact PD/PI: LEE, ABRAHAM Acoustic microvortices instrumentation for dosage controlled, high efficiency cell engineering Abstract - In years 2018 and 2020, Nobel Prizes were awarded to pioneers in cancer immunotherapy and the gene editing CRISPR-Cas9 method. This has ushered in a new era of cell engineering that demands technological innovations to produce genetic-modified and reprogrammed immune cells (e.g., T cells). CAR T cell immunotherapy is one type of cell therapy that has already achieved success in the clinic for treating cancer. The holy grail is to produce universal CAR T cells, genetically modified and engineered allogeneic T cells derived from healthy donors that avoid immunologic rejection. This requires gene engineering techniques such as CRISPR-Cas9 that can deliver multiplex gene insertions and knock-outs with controlled dosage to enhance viability and efficacy of the engineered cells. Although viral vectors are the method of choice for cell engineering, there are major concerns over their safety as well as the complex, costly preparation process. Nonviral transfection methods are alternatives to viral vectors that avoid the deleterious byproducts such as immune-mediated toxicity and oncogenic transgene concerns. Here we introduce the Acoustic-Electric Shear Orbiting Poration (AESOP) platform that addresses several of the known challenges for nonviral transfection techniques, including cell viability and dosage-controlled intracellular delivery while achieving relatively high throughput. AESOP is based on a microfluidic platform termed “lateral cavity acoustic transducers (LCATs)” that can form an array of microvortices activated by acoustic energy. The main innovation of the AESOP platform is the trapping of suspended populations of cells in tunable 3-D microvortices and incorporating a two-step membrane disruption strategy to precisely permeabilize cell membranes. By trapping cells to tumble in whirlpool-like microvortices, this platform optimizes the delivery of intended cargo sizes with uniform poration of the cell membranes via mechanical shear followed by the modulated enlargement of these nanopores via electric field. Using AESOP, we will focus on technology development for producing the universal CAR T cells. By controlling dosage, a decreased amount of CRISPR reagents in cells could reduce off-target effects. Furthermore, the uniform delivery enables the delivery of large cargo sizes necessary for immunotherapy-related gene transfection and gene editing. This 4-year project has four specific aims: 1- Generate acoustic microstreaming vortices for uniform cell membrane nanopores and uniform local mixing; 2- Demonstrate uniform electric field enlargement of nanopores for cargo delivery; 3- Design prototype AESOP instrumentation and 4- Quantitative benchmark of AESOP for intracellular delivery of large size cargos with dosage control for the development of universal CAR T cells.

联系PD/PI：Lee，Abraham 剂量控制的高效率细胞工程的声学微效应仪器 摘要 - 在2018年和2020年中，诺贝尔奖获得了癌症免疫疗法的先驱 以及基因编辑CRISPR-CAS9方法。这已经迎来了一个新的细胞工程时代 需要技术创新以产生遗传修饰和重编程的免疫细胞（例如， T细胞）。 CAR T细胞免疫疗法是一种已经成功的细胞疗法 治疗癌症的诊所。圣杯是生产通用汽车T细胞，经过基因修饰 并设计了来自健康供体的避免免疫排斥的健康供体的T型细胞。这 需要基因工程技术，例如CRISPR-CAS9，它可以输送多重基因插入 和以受控剂量的敲除，以提高工程细胞的生存力和效率。 尽管病毒矢量是细胞工程的首选方法，但对其 安全以及复杂的，昂贵的准备过程。非病毒转染方法是 避免有害副产品（例如免疫介导的毒性）的病毒载体的替代品 和致癌转化问题。在这里，我们介绍了声电剪切部分 （AESOP）平台，解决了非病毒翻译技术的几个已知挑战， 包括细胞活力和剂量控制的细胞内递送，同时实现相对高的 吞吐量。 AESOP基于称为“侧腔声传感器的微流体平台 （lcats）”可以形成通过声能激活的微构成阵列。主要创新 Aesop平台的是捕获悬浮的细胞种群在可调的3-D中 微效应并结合两步的膜破坏策略 透化细胞膜。通过将细胞捕获到漩涡状的微效应中，该平台 通过通过细胞膜统一的统一政策优化预期的货物尺寸的交付 机械剪切，然后通过电场对这些纳米孔进行调节。使用 AESOP，我们将专注于生产通用汽车T细胞的技术开发。经过 控制剂量，细胞中的CRISPR试剂量减少可能会降低脱靶效应。 此外，统一交付使得能够交付大型货物尺寸 免疫疗法相关的基因转染和基因编辑。这个4年的项目有四个特定的 目的：1-生成均匀细胞膜纳米孔和 统一的本地混合； 2-展示纳米孔的均匀电场扩展 送货; 3-设计原型Aesop仪器和4- eSop的定量基准 用于剂量控制的大尺寸大尺的细胞内输送以开发 通用汽车T细胞。