(W1030-07-43) Immunogenicity and Safety Assessment of Engineered mRNA Lipid Nanoparticles for Pulmonary Delivery

Purpose: Pulmonary administration of nonviral carriers delivering gene therapies is a promising strategy to achieve local nucleic acid delivery at high therapeutic concentrations without the concern for systemic circulation and off-target toxicity. However, to advance nonviral carriers in drug development, it is necessary to ensure that the carriers can be dosed repeatedly and do not possess undesired immunogenicity [1]. To address these challenges, we tested the safety and immunogenicity of B-1, a lipid nanoparticle (LNP) that we have previously developed in our lab. B-1 maintained stability upon aerosolization, achieved high mRNA expression in air-liquid interface human lung models and mouse lungs, and selectively transfected lung epithelia [2]. Here, we performed immunogenicity studies of B-1 and B-1 with a mucus-penetrating, cell-targeting peptide C that we previously identified (B-1 Peptide C) [3]. After intratracheal administration of these LNPs in BALB/c mice, we evaluated serum chemistry, cytokine expression from bronchoalveolar lavage fluid (BALF), and histopathology of the lung, liver, and spleen. These findings validate that B-1 and B-1 Peptide C are safe and can be further developed and translated into the clinic towards gene therapy of diseases that affect the pulmonary tract, such as cystic fibrosis.

Methods: BALB/c mice were intratracheally administered with two doses of 10 µg GFP mRNA (0.5 mg/kg) on Days 0 and 2, with groups receiving either PBS, B-1, or B-1 with a targeting peptide ligand (B-1 Peptide C). After the second dose, mice were euthanized at 4 hours (n=4 per group) and 24 hours (n=7 per group). Whole blood was collected via intra-cardiac puncture. Serum was then isolated using SST-MINI Gold Top Tubes and analyzed at a 2-fold dilution using the Research Liver Panel (#60405) from IDEXX BioAnalytics. Afterward, BALF was collected by inserting a catheter into the exposed trachea of the euthanized mice. A syringe connected to the catheter was used to introduce 1 mL of 1x PBS into the lungs, followed by gentle repeated flushing to sample the lung lumen. The collected BALF was then processed undiluted using the Mouse Cytokine/Chemokine 32-Plex Discovery Assay® Array by Eve Technologies. Following BALF collection, the lungs, liver, and spleen were harvested, rinsed in 1x PBS, placed in tissue embedding cassettes, fixed in 10% formalin overnight at room temperature, and then transferred to 70% ethanol. Tissues were paraffin embedded, sectioned, mounted on slides, stained with hematoxylin and eosin, and coverslipped by the South Texas Reference Laboratory at UT Health San Antonio. Histopathology was evaluated by a licensed pathologist at the Veterinary Medicine & Biomedical Sciences Core Histology Lab using the International Harmonization of Nomenclature and Diagnostic (INHAND) criteria, with microscopic changes graded as to severity (0 = no significant change, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe). Total lesion scores were calculated to determine the prevalence and severity of tissue changes within and between groups.

Results: Serum chemistry analysis demonstrated that LNP-treated groups exhibited results comparable to those of the PBS-treated group, supporting the safety of intratracheal administration of B-1 and B-1 Peptide C. Total protein concentration and AST/ALT levels were not statistically different between LNP- and PBS-treated mice, with mean values for all groups remaining within the normal range of BALB/c mice (Figure 1A-B). BALF cytokine analysis revealed time-dependent differences between LNP- and PBS-treated groups (Figure 2A-D). At the 4 hour timepoint, key pro-inflammatory cytokine levels (e.g., IL-6) and anti-inflammatory cytokines (e.g., IL-10) were not significantly different between groups. By 24 hours, LNP treatment resulted in significantly higher levels of several pro-inflammatory cytokines, including IL-6, IL-1β, TNFα, IL-1α, and IFNγ, as compared to PBS. In contrast, levels of anti-inflammatory cytokines such as IL-10 and IL-13 at 24 hours did not differ significantly between groups. Potential acute lung damage was evaluated by histopathology (Figure 3A-C). No significant lesions were observed in the spleen or liver. In the lungs of LNP-treated mice, treatment-related changes were detected but were graded as minimal to mild in severity.

Conclusion: B-1 and B-1 Peptide C LNPs are well-tolerated following intratracheal administration, as they did not induce any significant changes in serum chemistry, cytokine expression from BALF, or histopathology. These results confirm that our LNPs exhibit a favorable safety and immunogenicity profile for clinical translation.

References: 1. Heiser, B. J.*, Veyssi, A.*, & Ghosh, D. (2025). Recent strategies for enhanced delivery of mRNA to the lungs. Nanomedicine, 1-27. *Authors contributed equally
2. Lewis, M. M., Soto, M. R., Maier, E. Y., Wulfe, S. D., Bakheet, S., Obregon, H., & Ghosh, D. (2023). Optimization of ionizable lipids for aerosolizable mRNA lipid nanoparticles. Bioengineering & translational medicine, 8(6), e10580.
3. Soto, M. R., Lewis, M. M., Leal, J., Pan, Y., Mohanty, R. P., Veyssi, A., ... & Ghosh, D. (2024). Discovery of peptides for ligand-mediated delivery of mRNA lipid nanoparticles to cystic fibrosis lung epithelia. Molecular Therapy Nucleic Acids, 35(4).


Figure 1: Clinical chemistry test of mouse serum harvested at (A) 4 hr (n=4; mean [SD]), and (B) 24 hr after intratracheal administration of PBS, B-1, or B-1 Peptide C (n=7; mean [SD]); one-way ANOVA (Tukey’s multiple comparisons test), *p < 0.05.


Figure 2: Cytokine expression of mouse bronchoalveolar lavage fluid harvested at 4 and 24 hrs. (A and B) Pro-inflammatory (A) and anti-inflammatory (B) cytokines at 4 hr (n=3-4; mean [SD]). (C and D) Pro-inflammatory (C) and anti-inflammatory (D) cytokines at 24 hr (n=7; mean [SD]); one-way ANOVA (Tukey’s multiple comparisons test), *p < 0.05. ****p < 0.0001.


Figure 3: (A) Summary of histopathological findings at 4 hr (n=4) and 24 hr (n=4) with microscopic changes graded as to severity using INHAND criteria, (B) Representative histology analysis of murine lungs harvested at 4 hr and (C) 24 hr.