Abstract LB362: Optimizing non-viral genome editing workflow for primary activated T cells with advanced electroporation technology

Abstract CAR-T therapy has shown remarkable success in treating hematologic cancers such as leukemia and lymphoma. The Neon™ NxT Electroporation System with 8-Channel Pipette is a powerful tool for streamlined optimization of electroporation programs and other electroporation conditions for primary cells and cell lines. Here, we showcase the system's performance in efficiently optimizing CRISPR/Cas9 based non-viral genome editing of primary activated T cells.Using the Neon™ NxT Electroporation System with 8-Channel Pipette, we generate CAR-T cells by delivering CD19 donor DNA, a TRAC gRNA, and CTS™ HiFi™ Cas9 to activated T cells. We measure editing efficiency as well as T cell viability and phenotype using flow cytometry through 10 days post-T cell isolation.First, we demonstrate the capacity to simultaneously test multiple electroporation conditions, significantly reducing the time required for optimization experiments. Through systematic screening of voltage, pulse duration, and pulse number, we optimize the delivery of CRISPR/Cas9 RNP based gene editing while maintaining high cell viability. We show that this system is easily adapted to DoE analysis.Furthermore, we present data on the evaluation of electroporation buffers. By comparing the performance of Genome Editing, T, and R buffers, we demonstrate that Genome Editing Buffer is the most effective buffer for CAR-T cell editing across multiple donors, resulting in up to 99% TCR knockout with 84% of those CAR positive 10 days post-isolation. Our results highlight the impact of buffer components on transfection efficiency and cell viability.Importantly, we demonstrate the clinical relevance of our findings by culturing the edited T cells in a 96-well plate format out to 10 days post-isolation and show that these results are scalable to the larger Xenon™ Electroporation System. Throughout the culture process, we measured CD4 and CD8 markers as well as markers of T cell central memory. This not only enabled determination of how the electroporation parameters impact T cell editing, but also the effect on the cellular phenotypes. This analysis provides valuable insights for developing customer-relevant protocols that result in defined cell populations.The Neon™ NxT Electroporation System enables rapid optimization of electroporation parameters for editing of activated T cell cultures and is scalable to larger manufacturing systems. Citation Format: Shahan Molla, Lauren Gentles, Bindhu Hosuru, Namritha Ravinder, Arseny Smirnov. Optimizing non-viral genome editing workflow for primary activated T cells with advanced electroporation technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB362.