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.