Abstract 6375: Tardigrade Dsup mRNA nanoparticles reduce chemotherapy-induced normal tissue toxicity.

Abstract Bleomycin is a commonly used chemotherapeutic in the treatment of lymphomas and germ-cell tumors, yet its utility can be restricted by dose-limiting pulmonary toxicity. Bleomycin-induced generation of reactive oxygen species (ROS) causes significant damage to the alveolar epithelium. This leads to alveolar cell death, senescence, and maladaptive crosstalk with stromal cells, ultimately driving irreversible pulmonary fibrosis. Despite the clinical need, no approved strategy directly protects the lung epithelium from bleomycin toxicity. Certain natural organisms, namely tardigrades, can tolerate high levels of ROS-driven cellular damage. This tolerance is partially attributed to the tardigrade-specific Damage suppressor (Dsup) protein which interacts with chromatin and shields DNA from ROS. Our laboratory has recently shown that Dsup expression can protect other types of epithelia from radiation-induced injury. Because bleomycin and radiation share a common cytotoxic mechanism, we believe that Dsup could similarly protect alveolar epithelium. To this end, we developed a KC2/cholesterol/PEG/DOPE lipid nanoparticle (LNP) formulation optimized for Dsup mRNA delivery to human airway epithelium. This formulation was optimized to achieve >95% transfection efficiency in human small airway epithelial cells (HSAECs) and A549 cells with minimal impact on viability or clonogenicity. Transient Dsup expression significantly attenuated bleomycin-induced DNA damage in A549 and HSAECs, reducing γ-H2AX foci by ∼50% and decreasing alkaline comet % tail DNA by ∼25% relative to controls. Dsup expression also mitigated epithelial cell senescence (detected via β-galactosidase assay) after bleomycin treatment. In A549 and HSAECs, β-gal-positive cells were reduced by ∼40% at 16 days post-bleomycin treatment. To support translational feasibility, we evaluated the compatibility of our LNP formulation with nebulization and demonstrated that the LNPs retained particle size, polydispersity, and transfection efficiency after nebulization. We further assessed delivery in physiologically relevant models that typically resist nonviral mRNA transfection. In human and pig airway air-liquid-interface (ALI) cultures, multiple LNP formulations generated robust reporter (luciferase) expression. Additionally, dilution of LNPs in 4.5% NaCl further enhanced reporter expression by ∼100-fold in our ALI models compared with isotonic conditions, highlighting an additional strategy to improve airway transfection. Collectively, these data demonstrate that Dsup expression substantially reduces bleomycin-induced DNA damage and senescence in airway epithelial cells and establishes that our LNP formulation can effectively deliver mRNA to human airway epithelium in vitro. This work provides a mechanistic foundation for the development of a novel approach for preventing bleomycin-induced pulmonary fibrosis. Citation Format: Ian C. Sutton, Pascal Cuhat, Ashley Cooney, James D. Byrne, . Tardigrade Dsup mRNA nanoparticles reduce chemotherapy-induced normal tissue toxicity [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 6375.