Background and challenges

During acute inflammation, damaged cells release large amounts of ATP and ADP. These pro-inflammatory nucleotides bind to P2X and P2Y receptors, overactivating neutrophils, monocytes, and macrophages and increasing vascular permeability.

A key enzyme that suppresses this excessive pro-inflammatory signaling is CD39 (ectonucleoside triphosphate diphosphohydrolase-1). It hydrolyzes ATP and ADP to AMP, initiating an enzyme cascade in which CD73 subsequently generates adenosine, helping calm immune responses at sites of inflammation.

Direct administration of purified CD39 protein has faced major barriers to clinical use: its half-life in blood is only a few minutes, tissue-specific delivery is difficult, and systemic side effects or reduced efficacy have frequently been reported.

As mRNA therapies have drawn attention for their ability to be designed to make cells temporarily produce a target protein, inducing local CD39 synthesis directly at inflamed sites has emerged as a potential solution.

Methods and findings

The researchers used cationic nanoliposomes (NLp) to package CD39 mRNA efficiently and evaluated the formulation in a mouse model of localized inflammation induced by lipopolysaccharide (LPS) and Matrigel. The positive charge of NLp promoted favorable interactions with cell membranes, substantially increasing intracellular mRNA delivery efficiency.

Tissue analysis after 24 hours showed that NLp was strongly retained within the Matrigel matrix, with almost none escaping into systemic circulation. qPCR measurements showed that CD39 mRNA remained at high levels at the inflammation site through at least day 5, indicating that sustained protein expression was possible.

Flow cytometry showed strong CD39 protein expression in monocytes extracted from Matrigel. Macrophages showed a trend toward increased CD39 expression, but the increase was not statistically significant. A phosphate release assay showed that these cells exhibited ectonucleotidase activity that efficiently hydrolyzed ATP, confirming functional activity.

Histological assessment using H&E staining showed markedly reduced monocyte and macrophage infiltration on day 5 in the CD39 mRNA-NLp group. Cytometric Bead Array analysis found lower levels of the inflammatory cytokine IL-6 in Matrigel extracts from the treated group (15.30 ± 12.09) than from the LPS-only control group (89.55 ± 79.72), but the difference was not statistically significant (p=0.06).

In addition, blood compatibility tests found that NLp did not induce blood coagulation or red blood cell destruction. These safety data indicated a minimal risk of systemic hematological side effects following local injection.

Implications and outlook

By combining mRNA with positively charged nanoparticles to produce CD39 enzyme directly at sites of inflammation, this study presents a new therapeutic paradigm that showed local anti-inflammatory activity and good short-term tolerability in an LPS-Matrigel model.

Future application of CD39 mRNA-NLp to chronic inflammatory diseases involving overactive ATP-P2X signaling, such as rheumatoid arthritis, inflammatory bowel disease, and myocarditis, holds considerable promise for producing therapeutic effects promptly at the disease site.

So far, preclinical data have been obtained only in a mouse model. Further development will require manufacturing scale-up, establishment of a robust manufacturing process, and evaluation of repeat dosing and long-term safety.

If successful clinically, this platform could also enable multi-mRNA nanoparticle treatment strategies that simultaneously deliver other anti-inflammatory enzymes or immunomodulatory proteins, potentially opening a new era of personalized inflammation treatment.