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.

联系人 PD/PI：LEE, ABRAHAM 用于剂量控制、高效细胞工程的声学微涡流仪器 摘要：2018年和2020年诺贝尔奖颁发给了癌症免疫疗法的先驱 基因编辑CRISPR-Cas9方法开创了细胞工程的新时代。 需要技术创新来生产转基因和重新编程的免疫细胞（例如， CAR T细胞免疫疗法是一种已经在多个领域取得成功的细胞疗法。 治疗癌症的诊所的圣杯是生产转基因的通用 CAR T 细胞。 以及来自健康供体的工程化同种异体 T 细胞，以避免免疫排斥。 需要基因工程技术，例如 CRISPR-Cas9，可以实现多重基因插入 以及控制剂量的敲除，以增强工程细胞的活力和功效。 尽管病毒载体是细胞工程的首选方法，但人们对它们的主要担忧 非病毒转染方法的安全性以及复杂、昂贵的制备过程都是如此。 病毒载体的替代品，避免产生免疫介导的毒性等有害副产物 在这里我们介绍声电剪切轨道穿孔。 (AESOP) 平台解决了非病毒转染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，同时实现相对较高的 AESOP 基于称为“侧腔声换能器”的微流体平台。 （LCAT）”，可以形成由声能激活的微涡流阵列。 AESOP 平台的特点是在可调谐 3-D 中捕获悬浮的细胞群 微涡流并结合两步膜破坏策略来精确地 该平台通过捕获细胞在漩涡状微涡中翻滚来透化细胞膜。 通过细胞膜的均匀穿孔优化预期货物尺寸的输送 机械剪切，然后通过电场调节这些纳米孔的扩大。 AESOP，我们将专注于生产通用CAR T细胞的技术开发。 通过控制剂量，减少细胞中 CRISPR 试剂的量可以减少脱靶效应。 此外，统一交付可以交付所需的大件货物。 免疫治疗相关的基因转染和基因编辑这个为期4年的项目有四个具体内容。 目标：1-产生声学微流涡流以形成均匀的细胞膜纳米孔和 均匀的局部混合；2-展示货物纳米孔的均匀电场放大 交付；3- 设计原型 AESOP 仪器和 4- AESOP 定量基准 用于大尺寸货物的细胞内递送，并进行剂量控制以开发 通用 CAR T 细胞。