Abstract 5862: Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12

Abstract Engineered immune cell therapies have great potential to revolutionize the treatment of many pathologies such as cancer, autoimmunity and infectious diseases. However, limitations remain in some areas including long manufacturing times, inability to engineer critical cell functions, potential for cell exhaustion, and limited access to the disease site as in the case for solid tumors. Portal has developed a novel implementation of mechanoporation technology using a silicon membrane with pores which enables deformation of the cell and poration of the cell membrane, allowing for diffusion of any cargo, charged or uncharged, into the cell. Mechanoporation is a gentle intracellular delivery technique that preserves cell viability and function (DiTommaso, et al., PNAS 2018). It can shorten manufacturing time and enable the introduction of key therapy-enhancing factors via transient modifications shortly before administration. Portal has established methodology for cytoplasmic introduction of many diverse cargo including mRNA, circular RNA, siRNA, proteins, peptides, and CRISPR RNPs to primary immune cells, including both unstimulated and activated T cells, B cells, NK cells, monocytes and iPSCs. In this work, we demonstrated simultaneous expression of a functional CD19 CAR, membrane bound IL-2 and membrane bound IL-12 through delivery of three circRNAs into activated T cells. They are highly expressed for multiple days while maintaining high viability and ensuring robust in vitro killing. The expression of mbIL2 and mbIL12 not only promotes T cell proliferation but also upregulates CD62L and enhances the cytotoxic function of CAR T cells. At clinical scale, we have demonstrated delivery of RNA to over 1 billion T cells while achieving over 90% expression. Portal’s technology and approach has the potential to further advance immune cell therapy developments, particularly in multiplexed engineering approaches involving simultaneous delivery of multiple cargos. Coupled with Portal’s compatibility with diverse immune cell types and next generation cargos. We believe this simple approach to intracellular delivery can dramatically reduce manufacturing time and cost, easily integrate into existing clinical equipment and enable unique enhancements that underpin a new generation of therapeutics. Citation Format: Zhihui Song, Andrew Larocque, Sophia Hirsch, Eleni Rogers, Darby Kreienberg, Jacquelyn Hanson, Alec Barclay, Armon Sharei. Combining circular RNA and a silicon membrane-based cell engineering approach to create CAR-Ts with membrane bound IL-2 and IL-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5862.