SignificanceEx vivo manipulation of primary cells is critical to the success of this emerging generation of cell-based therapies, such as chimeric antigen receptor T cells for the treatment of cancer and CRISPR for the correction of developmental diseases. However, the limitations of existing delivery approaches may dramatically restrict the impact of genetic engineering to study and treat disease. In this paper, we compared electroporation to a microfluidic membrane deformation technique termed “squeezing” and found that squeezed cells had dramatically fewer side effects than electroporation and gene expression profiles similar to those of unmanipulated cells. The significant differences in outcomes from the two techniques underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications.

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Background: Tumor-specific T cells possess unique potential for cancer therapy but are limited by T cell exhaustion and anergy induced in the tumor microenvironment. Ex vivo manipulation of these T cells to maintain their full function is critical to their success clinically. Yet, limitations of existing ex vivo delivery approaches dramatically restrict their function and thus limit their therapeutic use. Methods: Genome-wide profiling was used to identify the impact of optimized electroporation treatment and the SQZ cell therapy platform on gene expression in human T cells. The profiling was paired with a 42 key T cell cytokine-multiplex analysis comprised of to assess perturbation of cytokine secretion. We then compared the in vivo functionality of immune checkpoint deleted antigen-specific T cells, modified by either electroporation or SQZ delivery of CRISPR/Cas9, and adoptively transferred into tumor bearing mice. Finally, genomic editing of tumor infiltrating leukocyte (TIL) derived T cells was compared using either electroporation or SQZ and subsequent effector response upon re-exposure to tumor cells. Results: Impactful disruptions in transcript expression after treatment with electroporation (17% of genes mis-regulated, FDR q <0.1) we identified, whereas cells treated with SQZ had similar expression profiles to untreated control cells (0% of genes mis-regulated, FDR q <0.1). These genetic disruptions result in concomitant perturbation of cytokine secretion and effector response. Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vitro and in vivo with electroporated antigen-specific and TIL derived T cells failing to demonstrate sustained antigen-specific effector responses and tumor control with or without immune checkpoint editing. Conclusions: This work demonstrates that functional modifications to tumor-specific T cells ex vivo can restore and improve their function upon re-exposure to tumor cells but that the delivery mechanism used is critical to the desired phenotype. The significant differences in outcomes from the two techniques tested here underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications. For both research and therapeutic applications with primary T cells, the functional consequences of the selected intracellular delivery technique and its impact on cell phenotype should be carefully evaluated. Citation Format: Luke Cassereau, Julie M. Cole, Roslyn Yi, Jacquelyn L. Hanson, Josh Bugge, Tia DiTommaso, Howard Bernstein, Armon Sharei. Tumor-specific T cell engineering for enhanced effector function via microfluidic delivery of bioactive molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1445.