Abstract 6401: <i>In vivo</i> mRNA CAR-T cell engineering using peptide based targeted nanoparticles

Abstract The Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a ground-breaking immunotherapeutic approach in cancer treatment and demonstrated encouraging clinical results in autoimmune disease indications. However, personalized CAR T cell production is a bottleneck due to challenges in cell manufacturing, scalability, high production costs and the use of viral transduction. In vivo CAR T cell generation has been proposed to make this treatment immediately accessible to patients and applicable for broader use. However, efficient and highly selective gene delivery to target cells in vivo is challenging. To that end, we are developing a new peptide based (non-lipid) delivery platform (PEP-NP) to enable in vivo delivery of CAR mRNA to T-cells. The PEP-NP platform is based on short amphipathic peptides that form stable, scalable nanoparticles capable of efficiently packaging and delivering RNA to the target site. PEP-NPs have been designed to bypass the liver and to enhance mRNA expression in the spleen and T-cells in vivo. PEP-NPs were selected based on their efficiency to deliver functional Fluc-mRNA on Jurkat or THP-1 cells and primary human T cells from different donors. Efficiency of PEP-NP to deliver a mRNA encoding for αCD-19 protein was evaluated on human primary T cells. The cytotoxicity of CAR-PEP-NPs transfected T cells was evaluated by co-cultured with either Nalm6 (CD19+) or K651 (CD19-) cells. In-vivo efficacy of PEP-NP (0.5 and 1.0 mg/kg) to deliver mRNA in the spleen and in specific immune cells, was evaluated in C57BL/C6 mice using luciferase or eGFP encoding mRNA and analyze by flow cytometry. PEP-NPs carrying mRNA encoding luciferase showed a dose responsive transfection starting at 6 hr post treatment with more than 80% of treated cells expressing Luciferase after 72 hr. We validated transfection, translation and surface expression of a functional CD19 CAR protein by transfecting T cells with PEP-NPs mRNA encoding for αCD19 CAR protein. CAR expression was observed in more than 80% of the transfected T cells from different donors. CAR-PEP-NPs transfected T Cells showed a robust dose response specific cytotoxic effect, resulting in 65% to 92% killing of CD19+Nalm6 cells, while in contrast, no cytotoxicity was observed on K561 (CD19-) cells or with T cells transfected with PEP-NP eGFP mRNA . Then we demonstrated that IV administration of T cell targeted PEP-NPs resulted in a high Luciferase or GFP mRNA expression in the spleen. PEP-NPs carrying GFP mRNA showed a dose-dependent mRNA delivery to T cells in the blood as well as splenic T cells with 15-22% of GFP positive T cells after a single injection. No toxicity of PEP-NP was detected after single or repeat dosing. Our study provides a proof-of-concept that PEP-NP platform can be applied to directly target T cells for in vivo CAR T cell engineering. PEP-NP is a novel strategy for specific T cell reprogramming providing redosable, off the shelf T cell gene therapies for patients. Citation Format: Gilles Divita, Audrey Grunenberger, Léa Cabrera, Julien Vollaire, Veronique Josserand, Neil Desai. In vivo mRNA CAR-T cell engineering using peptide based targeted nanoparticles [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 6401.