Abstract 275: Bioluminescent tools for quantification of <i>in vivo</i> CAR-T delivery systems.

Abstract In vivo CAR-T engineering using mRNA-LNP, lentiviral (LV), or AAV vectors requires analytical methods that are rapid, scalable, and mechanistically informative across CMC and bioassay workflows. We assembled an integrated luminescent toolkit to quantify vector input, functional potency, and innate-immune risk. For mRNA-LNP, a Lumit® dsRNA detection assay uses a split luciferase system to quantify double-stranded RNA impurities that can trigger innate sensing and diminish transfection. For broader assessment of immunogenicity, cell-based TLR reporter assays profile vector- or formulation-induced pathway activation. For quantification of LV vectors, the homogeneous, no-wash Lumit® p24 Immunoassay measures the LV p24 capsid protein as a surrogate for viral particle count with a wide linear range in ∼60 minutes. Functional potency can be measured using a cell-based reporter bioassay in which Jurkat T cells stably expressing an NFAT-Luc2 reporter are transduced with CAR LV and co-cultured with antigen-positive targets to generate an antigen-dependent luminescent signal that reflects LV identity and potency. Together, these assays streamline development, release, and comparability for in vivo CAR-T delivery systems. Citation Format: Julia K. Gilden, Pete Stecha, Rich Moravec, Jun Wang, Rod Flemming, Jim Harnett, Steven Edenson, Kristin Riching, Jamison Grailer, Mei Cong. Bioluminescent tools for quantification of in vivo CAR-T delivery systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 275.