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IL-10 mRNA Engineered MSCs Demonstrate Enhanced Anti-Inflammation in an Acute GvHD Model.

ABSTRACT: Mesenchymal stem cells (MSCs) are used in various studies to induce immunomodulatory effects in clinical conditions associated with immune dysregulation such as graft versus host disease (GvHD). However, most of these clinical trials failed to go beyond early phase 2 studies because of limited efficacy. Various methods have been assessed to increase the potency of MSCs. IL-10 is an anti-inflammatory cytokine that is known to modulate immune responses in GvHD. In this study, we evaluated the feasibility of transfecting IL-10 mRNA to enhance MSC therapeutic potential. IL-10 mRNA engineered MSCs (eMSCs-IL10) maintained high levels of IL-10 expression even after freezing and thawing. IL-10 mRNA transfection did not appear to alter MSC intrinsic characteristics. eMSCs-IL10 significantly suppressed T cell proliferation relative to naïve MSCs in vitro. In a mouse model for GvHD, eMSCs-IL10 induced a decrease in plasma level of potent pro-inflammatory cytokines and inhibited CD4+ and CD8+ T cell proliferation in the spleen. In summary, our studies demonstrate the feasibility of potentiating MSCs to enhance their immunomodulatory effects by IL-10 mRNA transfection. The use of non-viral transfection may generate a safe and potent MSC product for treatment of clinical conditions associated with immune dysregulation such as GvHD.

SUBMITTER: Zhang C

PROVIDER: S-EPMC8621791 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rationale: Tailored inflammation control is badly needed for the treatment of kinds of inflammatory diseases, such as atherosclerosis. IL-10 is a potent anti-inflammatory cytokine, while systemic and repeated delivery could cause detrimental side-effects due to immune repression. In this study, we have developed a nano-system to deliver inflammation-responsive Il-10 mRNA preferentially into macrophages for tailored inflammation control. Methods: Il-10 was engineered to harbor a modified HCV-IRES (hepatitis C virus internal ribosome entry site), in which the two miR-122 recognition sites were replaced by two miR-155 recognition sites. The translational responsiveness of the engineered mRNA to miR-155 was tested by Western blot or ELISA. Moreover, the engineered Il-10 mRNA was passively encapsulated into exosomes by forced expression in donor cells. Therapeutic effects on atherosclerosis and the systemic leaky expression effects in vivo of the functionalized exosomes were analyzed in ApoE-/- (Apolipoprotein E-deficient) mice. Results: The engineered IRES-Il-10 mRNA could be translationally activated in cells when miR-155 was forced expressed or in M1 polarized macrophages with endogenous miR-155 induced. In addition, the engineered IRES-Il-10 mRNA, when encapsulated into the exosomes, could be efficiently delivered into macrophages and some other cell types in the plaque in ApoE-/- mice. In the recipient cells of the plaque, the encapsulated Il-10 mRNA was functionally translated into protein, with relatively low leaky in other tissues/organs without obvious inflammation. Consistent with the robust Il-10 induction in the plaque, exosome-based delivery of the engineered Il-10 could alleviate the atherosclerosis in ApoE-/- mice. Conclusion: Our study established a potent platform for controlled inflammation control via exosome-based systemic and repeated delivery of engineered Il-10 mRNA, which could be a promising strategy for atherosclerosis treatment.

Project description:BackgroundMesenchymal stem cells (MSCs) are useful agents in the treatment of various inflammatory diseases. The immunomodulatory effects of MSCs are largely related to their secretory properties. mRNA engineering emerged as a safe alternative to enhance the secretory function of MSCs. Optimization of the untranslated region (UTR) sequence is important for enhancing the translational efficiency of exogenous mRNAs. However, research on the optimization of UTR in canine MSCs has not yet been conducted.ObjectivesWe aimed to identify the UTR sequence related to the expression efficiency of in vitro transcription (IVT) mRNA in canine MSCs and investigate whether mRNA-engineered MSCs that overexpress TSG-6 exhibit enhanced anti-inflammatory effects.MethodsCanine adipose tissue-derived (cAT)-MSCs were transfected with green fluorescence protein (GFP) mRNA with three different UTRs: canine hemoglobin subunit alpha-like 1 (HBA1), HBA2, and hemoglobin subunit beta-like (HBB). The translation efficacy of each mRNA was evaluated using relative fluorescence. TSG-6 mRNA was produced with the UTR optimized according to relative fluorescence results. cAT-MSCs were transfected with TSG-6 mRNA (MSCTSG-6), and TSG-6 expression was analyzed using real-time quantitative PCR, ELISA, and western blotting. To evaluate the anti-inflammatory effects of MSCsTSG-6, DH82 cells were co-cultured with MSCsTSG-6 or treated with dexamethasone, and changes in the expression of inflammatory cytokines were analyzed using qPCR.ResultsThe highest fluorescence level was observed in the HBA1 UTR at 24 h post-transfection. TSG-6 mRNA transfection yielded high levels of TSG-6 in the cAT-MSCs. In DH82 cells co-cultured with MSCsTSG-6, the expression of inflammatory cytokines decreased compared to that in co-culturing with naïve MSCs and dexamethasone treatment.ConclusionsOptimization of the HBA1 UTR improved the translation efficiency of IVT mRNA in canine MSCs. cAT-MSCs engineered with TSG-6 mRNA effectively enhanced the anti-inflammatory effects of the MSCs when co-cultured with LPS-activated DH82 cells.

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IL-10 mRNA Engineered MSCs Demonstrate Enhanced Anti-Inflammation in an Acute GvHD Model.

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