Background and Challenges

Acute inflammation leads to massive release of ATP and ADP from damaged cells, causing these pro-inflammatory nucleotides to bind to P2X and P2Y receptors, resulting in excessive activation of neutrophils, monocytes, and macrophages, and increased vascular permeability. To inhibit these excessive pro-inflammatory signals, an enzyme that rapidly degrades ATP into ADP and AMP, and ultimately into adenosine, is required. A representative enzyme is CD39 (ectonucleoside triphosphate diphosphohydrolase-1), which consumes ATP-ADP at the site of inflammation, thereby suppressing the immune response. Direct administration of pure CD39 protein has been attempted, but it has a short half-life in the blood (only a few minutes), and it is difficult to achieve tissue-specific delivery, leading to frequent reports of systemic side effects and reduced efficacy, which has been a major barrier to clinical application. Recently, the ability to design mRNA therapeutics to temporarily produce target proteins within cells has been highlighted, and a strategy to directly synthesize CD39 locally in the inflammatory site is attracting attention as a new solution.

Research Methods and Findings

The research team efficiently packaged CD39 mRNA using cationic nanoliposomes (NLps) and directly injected them into a matrigel to establish a local inflammation model. The positive charge of the NLps promotes affinity with cell membranes, significantly increasing the efficiency of intracellular mRNA delivery. After 24 hours, tissue analysis showed that the NLps were strongly fixed within the matrigel matrix and were not significantly released into the systemic circulation. qPCR measurements showed that CD39 mRNA levels remained high in the inflammatory site for at least 5 days, demonstrating sustained protein expression. Flow cytometry showed that monocytes and macrophages extracted from the matrigel strongly expressed CD39 protein, and phosphate release assays showed that these cells exhibited efficient ectonucleotidase activity, hydrolyzing ATP, which confirmed functional success. Histological analysis showed that the CD39 mRNA-NLp treatment group had significantly reduced monocyte and macrophage infiltration on day 5, based on HE staining. ELISA analysis showed that the concentration of the pro-inflammatory cytokine IL-6 in the matrigel extract was approximately 20% lower than in the control group, objectively demonstrating the anti-inflammatory effect. In addition, blood compatibility tests showed that the NLps did not cause blood coagulation factors or red blood cell lysis, indicating that the risk of systemic hematological side effects is minimized during in situ injection, and safety data was also obtained.

Future Significance or Prospects

This study presents a new therapeutic paradigm by combining mRNA and cationic nanoparticles to enable direct production of CD39 enzyme in the inflammatory site, which can selectively calm the target tissue while avoiding side effects such as systemic steroid administration. In the future, if CD39 mRNA-NLp is applied to various chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and myocarditis, where ATP-P2X signaling is overactivated, it is expected to have a significantly lower risk of side effects and can exhibit therapeutic efficacy at the site of action. Currently, only preclinical data have been obtained in a mouse model, but a phase 1 clinical trial is being designed to evaluate mRNA delivery efficiency and long-term safety in human tissues, and a GMP-grade NLp manufacturing process is also being optimized to lower the barrier to clinical entry. If successful in clinical trials, a multi-mRNA-nanoparticle therapeutic strategy that simultaneously delivers other anti-inflammatory enzymes or immune-modulating proteins based on this platform may become a reality, ushering in a new era of personalized inflammation therapy.