Abstract Background The clinical use of cytokines like IL-10, IL-22, and IL-2 for Inflammatory Bowel Disease (IBD) is hindered by their pleiotropy, which causes off-target toxicity, and by their poor pharmacokinetics. We aimed to overcome these limitations by developing a synergistic platform combining structure-based protein engineering with an advanced nucleic acid delivery system. Methods We engineered biased mutant cytokines (mIL-10, mIL-22, mIL-2) designed to selectively activate specific pathways: mIL-10 (D25A/E96A) for macrophage-specific suppression, mIL-22 (S45E, Q116A, R124A, R128A) for STAT3-biased mucosal repair, and mIL-2 (L18R, Q22E, Q126H) for Treg-specific expansion. We encoded these mutants into highly stable, low-immunogenicity circular RNAs (circRNAs) generated via an in vitro self-splicing permuted intron-exon (PIE) system. These circRNAs were co-encapsulated in a four-component lipid nanoparticle (LNP) formulated using a dendrimer-based (e.g., PAMAM) cationic lipid, DSPC, cholesterol, and a PEG-lipid, prepared via microfluidics. The formulation was administered intravenously to C57BL/6 mice with DSS-induced colitis. Results In vitro assays confirmed the variants’ biased signaling (e.g., selective pSTAT activation) in target cells (macrophages, HT-29, and Tregs). In the DSS colitis model, mice treated with the combined LNP-circRNA therapy showed significantly reduced body weight loss, lower Disease Activity Index (DAI) scores, and preserved colon length compared to controls. Histological analysis revealed attenuated inflammatory cell infiltration and enhanced epithelial repair (Ki-67). Mechanistically, the therapy simultaneously suppressed inflammatory macrophages (mIL-10), promoted mucosal healing (mIL-22), and expanded colonic Foxp3+ Tregs (mIL-2). Unlike treatment with wild-type cytokine payloads, this formulation showed no signs of systemic IFN-gamma elevation or hepatotoxicity. Conclusion A dendrimer-LNP system delivering a circRNA payload encoding three biased, complementary cytokines (IL-10, IL-22, IL-2) offers a safe and highly effective, multi-pronged therapeutic strategy for IBD. This platform simultaneously drives anti-inflammation, tissue repair, and immune regulation, successfully overcoming the primary limitations of traditional cytokine and linear mRNA therapies. Conflict of interest: Dr. Lin, Lang: No conflict of interest Tang, Jian: No conflict of interest Chao, Kang: No conflict of interest Gao, Xiang: No conflict of interest
Lin et al. (Thu,) studied this question.