Abstract 3531: Development of a mRNA-LNP vaccine for Lynch syndrome

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Abstract Lynch syndrome (LS) is a genetic disorder in which colorectal cancer is caused by mutations in DNA mismatch repair enzymes. Prophylactic mRNA vaccines have become the subjects of increasing interest as platforms for the potential prevention of such disorders. Several neoantigen sequences caused by defective mismatch repair were identified as expressed in a GEMM model of Lynch Syndrome, VCMsh2, and were also confirmed to be immunogenic for T and B cell responses in the background strain C57BL/6, as measured by IFN-g ELISPOT and ELISA in peptide/adjuvant-vaccinated mice. Vaccine mRNA constructs coding for 3 neoantigen peptides were designed with the HA signal peptide sequence and BioNTech 3’ and 5’ UTR regions and synthesized on the BioXp 3250 platform using pseudouridines. Monovalent mRNA vaccines were generated by encapsulating LS neoantigen mRNA into lipid nanoparticles (LNPs) on the NanoAssemblr Spark platform. High mRNA encapsulation efficiency was verified, and LNP size, polydispersity index, concentration, and zeta potential passed vaccine specifications. In vitro translation of vaccine mRNAs in transfected HEK-293 and RAW 264.7 cells were confirmed by FLAG Western Blot and mass spectrometry. The mRNA vaccines were administered 2X monthly via IM route to C57BL/6 mice. Substantial splenic CD4 T cell responses to the LS neoantigen peptides Nacad, Maz, and Senp6 were detected although lower than T cell responses to the corresponding peptide/adjuvant vaccine. In an attempt to adjuvant the mRNA vaccine, the TLR9 agonist CpG-2395 was encapsulated into LNPs via methods identical to mRNA packaging. In vitro testing on cell lines revealed that the CpG-LNPs enhanced Luciferase mRNA expression in RAW264.7 macrophages, triggered TLR9 signaling in mTLR9-HEK Blue reporter cells, and promoted IL-12 production by transfected RAW264.7. When LS neoantigen mRNA-LNPs were co-vaccinated with CpG-LNPs in C57BL/6 mice, T cell responses were augmented to be comparable to peptide-vaccinated T cell responses. Our current efforts involve optimizing the LNP lipid composition for uptake efficiency, optimizing the in vivo trafficking of the LNPs, and determining a suitable method for adjuvanting mRNA vaccines to boost T and B cell responses before evaluation in the VCMsh2 Lynch Syndrome GEMM model. Citation Format: Yuri Koboziev, Ligia A. Pinto, Robert H. Shoemaker, Jason D. Marshall. Development of a mRNA-LNP vaccine for Lynch syndrome [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 3531.

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