Accelerated, scalable same-day microfluidic sequential gene editing for allogeneic cell therapy

Project Summary Allogeneic therapies hold significant promise as "off-the-shelf" treatments, offering faster and more accessible alternatives to autologous therapies. However, current allogeneic CAR-T manufacturing faces challenges in preventing immune rejection and alloreactivity. These challenges are particularly pronounced in gene-editing processes, where multiple gene edits are required to modify donor T cells for immune tolerance, but existing gene delivery tools present limitations like slow recovery times, high cytotoxicity, and scaling difficulties for large- scale production. This project aims to optimize the CellFE platform, a novel microfluidic transfection technology, to enable same-day sequential multiplexed gene editing of T cells for allogeneic therapies. By enhancing the speed, precision, and scalability of CRISPR-based gene editing, the proposed approach will streamline T cell manufacturing, improving therapeutic potency while minimizing the risk of chromosomal instability or genomic translocations, which are common concerns in gene-edited therapies. The innovation of this work lies in its ability to perform sequential gene editing within a single day, ensuring efficient gene editing with minimal cytotoxicity and genomic instability. This approach will accelerate clinical translation by addressing the critical bottleneck of delayed manufacturing and enabling rapid scaling of allogeneic CAR-T cell production. In this Phase I SBIR grant, we will conduct a series of proof-of-concept studies to demonstrate the feasibility of the CellFE platform for high efficiency, same-day multiplexed gene editing. By validating the CellFE platform for same-day multiplexed gene editing, this project will significantly improve the efficiency and scalability of allogeneic CAR-T therapies, advancing the field of off-the-shelf cell therapies for a variety of cancers and other diseases.