Abstract 6107: Tailoring mRNA expression in T-cell therapy: advancing CAR-T functional optimization

Abstract Background: T cell engineering is an emerging field in immune-oncology, and new T cell-specific modalities are required, such as gene insertion via mRNA. Cells display diverse functions and identities, requiring tailored approaches for efficient mRNA delivery. Most mRNA constructs contain standard α-globin UTRs, lacking the specificity needed for optimal T cell mRNA expression. Methods: In our study, we developed modRNA constructs by replacing the standard hemoglobin 5’-UTR with sequences derived from genes highly expressed in normal or exhausted T cells, aiming to enhance mRNA stability and protein expression. mRNA constructs containing reporter genes were electroporated into PBMC-derived T cells, with expression levels measured by flow cytometry. Results: Interestingly, we observed significant differences, with notable variability in expression arising from the replacement and directly linked to the specific gene substituted. Certain UTRs, such as Interferon gamma (IFNG), showed superior and sustained GFP expression, while others, such as TNF, exhibited reduced activity. Based on these observations, we expressed diverse CAR constructs in T cells, revealing distinct protein turnover and tonic signaling profiles. Specific constructs exhibited rapid decay, while others displayed more stable expression. These turnover patterns suggested significant differences in CAR expression levels across constructs and their impact on tonic signaling, reinforcing earlier findings on the influence of 5'-UTRs in CAR-T expression. Particularly notable were constructs that exhibited significantly lower activity and reduced expression of key activation and well-established hallmark markers of exhaustion. Cytokine secretion assays further confirmed diminished tonic signaling, especially in non-target co-cultures. Conclusions: These results underscore the critical role of T cell-specific UTRs in fine-tuning CAR-T cell activity and reducing off-target effects. Citation Format: Gilad Gibor, Shai Kilim, Neve Tzvi, Ortal Harush, Orit Itzhaki, Ronnie Shapira, Elad Jacoby, Gal Cafri, Yochai Wolf. Tailoring mRNA expression in T-cell therapy: advancing CAR-T functional optimization [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 6107.