Acoustic microvortices instrumentation for dosage controlled, high efficiency cell engineering

联系PD/PI：LEE，ABRAHAM 用于剂量控制、高效细胞工程的声学微涡旋仪器 摘要-在2018年和2020年，诺贝尔奖被授予癌症免疫治疗的先驱者 和基因编辑CRISPR-Cas9方法。这开创了细胞工程学的新时代， 需要技术创新来产生基因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细胞疗法，已经在免疫治疗中取得了成功。 治疗癌症的诊所圣杯是生产通用的CAR-T细胞， 以及来自健康供体的工程化同种异体T细胞，其避免免疫排斥。这 需要基因工程技术，如CRISPR-Cas9， 以及用受控剂量敲除以增强工程化细胞的活力和功效。 尽管病毒载体是细胞工程的首选方法，但其在细胞工程中的应用仍存在重大问题。 安全性以及复杂、昂贵的制备过程。非病毒转染方法是 避免有害副产物如免疫介导的毒性的病毒载体的替代物 和致癌转基因问题。本文介绍声-电剪切轨道孔 （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， 吞吐量AESOP是基于一个微流体平台，称为“横向腔声换能器 （LCAT）”，其可以形成由声能激活的微涡旋阵列。主要创新 的AESOP平台是捕获悬浮的细胞群体在可调的3-D 微涡旋，并结合两步膜破坏策略， 使细胞膜透化。通过捕获细胞在水池状的微漩涡中翻滚，这个平台 通过均匀的细胞膜穿孔优化预期货物大小的递送， 机械剪切，然后通过电场调节这些纳米孔的扩大。使用 AESOP，我们将专注于生产通用CAR T细胞的技术开发。通过 通过控制剂量，细胞中CRISPR试剂的量减少可以减少脱靶效应。 此外，统一交付使得能够交付大型货物， 免疫治疗相关基因转染和基因编辑。这个为期四年的项目有四个具体的 目的：1-产生用于均匀细胞膜纳米孔的声学微流涡旋， 2-证明用于货物的纳米孔的均匀电场扩大 交付; 3-设计原型AESOP仪器和4-AESOP的定量基准 用于大尺寸货物的细胞内递送，具有剂量控制，用于开发 通用CAR T细胞。

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.