573. Vector Free Genome Editing of Immune Cells for Cell Therapy

While the ex vivo manipulation of primary cells has signaled a new era in the application of cell-based therapies, common methods to manipulate primary cells have limitations. To overcome the limitations associated with conventional cell delivery and engineering systems, we have developed a microfluidic approach to delivery where cells are mechanically deformed as they pass through constricting channels. This deforms the cell membrane resulting in the diffusion of material from the surrounding buffer directly into the cytosol. This system has demonstrated efficacy in patient-derived cells, such as stem cells and immune cells and with a variety of target molecules that are difficult to address with alternative methods. Moreover, by eliminating the need for electrical fields or exogenous materials such as viral vectors and plasmids, it minimizes the potential for cell toxicity and off-target effects. Here, we present evidence detailing our ability to deliver functional material to primary human T cells via membrane deformation with little detectable perturbation in baseline gene expression, cell function, and viability. To determine effect of membrane deformation on gene expression and to compare to other delivery systems, human T cells were subjected to membrane deformation or electroporation and gene expression changes were compared to unmanipulated control cells using microarray analysis. Differential gene expression with respect to both methods of delivery was assessed by performing t tests on the coefficient of a linear mixed-effects model that treated delivery method as a fixed effect and donor as a random effect. Electroporation produced substantially more changes in gene expression than membrane deformation. Subsequently, we designed a series of experiments to manipulate gene expression with the CRISPR-CAS9 system using membrane deformation to deliver CAS9 ribonucleoproteins (RNPs; recombinant CAS9 protein complexed with a single-guide RNA) designed to edit a model locus, the B2 microglobulin component of MHC class 1 (B2M). Here, we show that the delivery of the CRISPR-CAS9 system via membrane deformation results in a significant reduction in B2M surface protein expression by FACS analysis. Taken together, these data suggest that membrane deformation is a viable delivery method for genetic engineering of primary human cells with little off target effects on baseline gene expression. Indeed, the ability to deliver structurally diverse materials to difficult-to-transfect primary cells indicate that this method could potentially enable many novel clinical applications.