Targetable deoxycholic acid-9R self-assembly for the delivery of mRNA cancer vaccine and macrophage repolarization

다국어 초록 (Multilingual Abstract)

Messenger RNA (mRNA)-based cancer vaccines represent great potential in terms of the flexible tumor antigens-encoding capability of mRNA. However, the efficacy of vaccines is hindered by the immunosuppressive tumor-associated macrophages (TAMs) in the tumor microenvironment, indicating a hurdle in therapeutic cancer vaccines. Here, an mRNA cancer vaccine was developed, consisting of deoxycholic acid-conjugated 9R (DOCA-9R) self-assembly coated with mannosylated lipid, TLR7/8 agonist R848 as an adjuvant, and model antigen ovalbumin (OVA)-encoded mRNA (A-mL-DRO). The objective was to overcome the limitation of therapeutic cancer vaccines by targeting and repolarizing TAMs, as well as targeting dendritic cells to achieve amplified cancer immunotherapy. A- mL-DRO effectively encapsulated R848 and complexed mRNA safely, preventing its degradation from serum. Following targeting for higher internalization, A-mL- DRO enabled effective transfection, maturation, and antigen presentation in dendritic cells, resulting in an elicited antigen-specific T cell immune response against the OVA-expressing tumor. A-mL-DRO revealed improved therapeutic effects and immune responses synergized by TAM repolarization. Combination therapy with anti-PD-1 robustly enhanced therapeutic immune response in the OVA-expressing melanoma model. A-mL-DRO represented a powerful mRNA vaccine strategy for enhanced antigen-specific immunotherapy against tumors.

목차 (Table of Contents)

• List of figures
• Abstract
• 1. Introduction
• 2. Material and Methods
• 2.1 Materials
• 2.2 Preparation of A-mL-DRO
• 2.3 Agarose gel retardation and mRNA degradation protection assay
• 2.4 Characterization of A-mL-DRO
• 2.5 Drug loading, encapsulation efficiency, and releasing profiles
• 2.6 Cell culture
• 2.7 In vitro luciferase assay
• 2.8 CCK-8 assay
• 2.9 In vitro cellular uptake and microscopy imaging
• 2.10 In vitro dendritic cell maturation and cytokine secretion
• 2.11 Western blot
• 2.12 In vitro analysis of mRNA expression level
• 2.13 Biodistribution
• 2.14 In vivo bioluminescence imaging
• 2.15 Ex vivo transfection and antigen-specific killing assay
• 2.16 In vivo therapeutic effect, combination therapy effect with immune checkpoint blockade, and prophylactic effect of A-mL-DRO
• 2.17 Ex vivo toxicity of A-mL-DRO
• 2.18 Analysis of tumor infiltrating lymphocytes (TILs)
• 2.19 Ex vivo analysis of cytokines
• 2.20 Immunofluorescence analysis of tissues
• 2.21 Statistical analysis
• 3. Results and Discussion
• 3.1 Design, optimization, and physicochemical characterization of A-mL-DRO for mRNA cancer vaccine
• 3.2 Evaluation of drug encapsulation, cellular uptake, and transfection efficiency of A-mL-DRO
• 3.3 Effect of A-mL-DRO on dendritic cell maturation in vitro
• 3.4 Targeting and repolarizing effects of A-mL-DRO on M2 macrophages in vitro
• 3.5 In vivo delivery effectiveness of A-mL-DRO via mannose-mediated targeting to dendritic cells and M2 macrophages
• 3.6 Screening in vivo transfection efficiency of A-mL-DRO for inducing antigen-specific T cell response
• 3.7 Therapeutic effect of A-mL-DRO in B16-OVA melanoma mouse model
• 3.8 Enhanced efficacy of A-mL-DRO with immune checkpoint blockade for combination therapy
• 3.9 Immunoprophylactic effect of A-mL-DRO on B16-OVA melanoma mouse model and safety evaluation of A-mL-DRO
• 4. Conclusion
• 5. References
• 6. 국문요지