Magnetic bead-sensitized optoporation coupled with antibodies-based activation for mRNA CAR-T cell manufacturing.

Immunotherapy is facing a revolution with the advent of immune cell engineering. Chimeric antigen receptor (CAR)-T cell therapy has shown unprecedented efficacy in B cell malignancies and is now being evaluated in other disease areas. Viral transduction is the most common method for immune cell genetic engineering, but presents important limitations, such as high reagent costs and regulatory concerns due to mutagenesis risk. One prevailing non-viral gene delivery strategy relies on the electroporation of non-integrating RNA. However, most modern electroporation technologies also require high reagent costs and rely on the use of proprietary software and transfection buffers. Nanoparticle-sensitized optoporation represents an alternative method for transient permeabilization of cells. Here, we introduce magnetic bead-sensitized optoporation, in which commercially available superparamagnetic beads coupled with anti-human CD3 and CD28 antibodies are used as photosensitizers for efficient genetic cargo delivery into human primary T cells and other immune cells. We show that magnetic bead-sensitized optoporation of human T cells generates functional mRNA-based CAR-T cells without affecting T cell product memory phenotype or activation potential. Importantly, optoporated T cells exhibited a greater proliferation capacity relative to electroporated T cells. In conclusion, our findings suggest that magnetic bead-sensitized optoporation holds promise as mRNA delivery strategy for immune cell therapy.