Abstract A55: Vector-free genome editing of immune cells for cell therapy (2018)

Abstract The ex vivo manipulation of primary cells is critical to an emerging generation of cell-based therapies, such as chimeric antigen receptor systems and CRISPR mediated genomic editing. However, the limitations of existing methods for delivering desired material to cells of interest could dramatically hinder the development and impact of these therapies. To overcome the challenges associated with conventional cell delivery and engineering systems, we have developed a microfluidic approach, CellSqueeze®, where cells are mechanically deformed as they pass through constricting channels. This process disrupts the cell membrane resulting in the diffusion of material from the surrounding buffer directly into the cytosol. The CellSqueeze® 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 for gene editing to primary human T cells via membrane deformation with little detectable perturbation in baseline gene expression, cell function, and viability. To determine the 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. We performed differential gene expression analysis and found that 6 hours post transfection, electroporation induced statistically significant changes in 33% (7944/23786) of all genes as compared to untreated control cells, whereas cell squeeze treatment significantly changed expression of 0% (0/23786) of genes (FDR q&amp;lt;0.25.) To determine the functional impact of this differential gene expression, we compared T cell homing post electroporation or CellSqueeze®. Briefly, CD45.1 mice were subjected to CellSqueeze® while T cells from CD90.1 mice were electroporated, the cells were mixed at a 1:1 ratio, and injected into mice. After 1 day the blood, spleen, and lymph nodes were harvested and FACS analysis was performed on recovered T cells. Despite being injected at a 1:1 ratio, over 80% of the T cells recovered from the target homing organs had been treated with CellSqueeze® as opposed to electroporation, indicating T cells more effectively home to tissues after CellSqueeze®. 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). Here, we show efficacious editing of several clinically relevant loci (including B2M-up to 50% editing, CCR5-up to 80% editing, and checkpoint proteins-up to 60% editing) 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. Citation Format: Luke Cassereau, Tia DiTommaso, Scott Loughhead, Jonathan Gilbert, Howard Bernstein, Armon Sharei. Vector-free genome editing of immune cells for cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A55.