Safe and Efficient CRISPR Genome Editing of Primary Human T Cells Using a Droplet-Based Cell Mechanoporation Platform

초록

T cell engineering is a transformative strategy for adoptive cell therapy, holding the key to treating a wide array of human diseases. However, clinical translation is limited by current intracellular delivery methods that compromise viability, induce stress responses, and restrict scalability. This study presents a microfluidic droplet mechanoporation system tailored for primary human T cells, enabling efficient, stable, and clinically scalable gene delivery. Delivery of 2000 kDa fluorescein isothiocyanate (FITC)-dextran achieves similar to 98% efficiency and >90% post-treatment viability, even at high cell densities, supporting the rapid production of therapeutically relevant cell numbers. The platform efficiently delivers mRNA, achieving transfection efficiencies approaching 99%; further, chimeric antigen receptor (CAR)-encoding mRNA is successfully delivered to generate CAR-expressing T cells with tunable surface expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoproteins are effectively delivered for both single and multiplex knockouts (TRAC and PDCD-1), achieving up to a 2.35-fold higher efficiency than electroporation. Longitudinal analyses confirm preserved viability, proliferation, genome integrity, and T cell phenotypic stability. Collectively, these results establish microfluidic droplet mechanoporation as a safe, efficient, and scalable platform for the clinical manufacturing of engineered T cell therapies.

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