A20 mRNA treatment may improve systemic sclerosis by blocking inflammation signals and reducing tissue scarring

The A20 coding sequence was optimized using codon optimization services provided by GenScript (Piscataway, NJ, USA). The optimized sequence was synthesized by Cosmogenetech (Seoul, Korea) and subsequently cloned into the CUK3.1 mRNA expression platform (11). To generate A20 mRNA, the gene was inserted into a capped mRNA expression construct incorporating a loop structure at the poly(A) tail (Figure 1A). The DNA template was linearized using NotI (R001H, Enzynomics, Daejeon, Korea) and transcribed using the EZ T7 High Yield In Vitro Transcription Kit (EZ027S, Enzynomics) according to the manufacturer's protocol. Capping was performed with SC101 (STPharm, Siheung, South Korea), and uridine triphosphate was substituted with N1-methyl-pseudouridine (N-1081, Trilink, San Diego, CA, USA) to enhance mRNA stability and translation. Double-stranded RNA contaminants were removed using a cellulose-containing column, as previously described (12).

Human dermal fibroblasts (HDFs) and human embryonic kidney 293 (HEK293) cells were cultured to 70–80% confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (#12800017 for HDFs and #16000044 for HEK293, Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL penicillin–streptomycin (#15140-122, Gibco, Carlsbad, CA, USA). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. Transfections were performed using Lipofectamine™ MessengerMAX™ Transfection Reagent (LMRNA003, Thermo Fisher Scientific) according to the manufacturer's instructions. For transfection, the culture medium was replaced with fresh Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and 2 µg of plasmid DNA was added. Transfected cells were then treated with recombinant human TGF-β (20 ng/mL; #100-21C-10, Thermo Fisher Scientific) for 48 h. For transcriptional inhibition experiments, cells were pretreated with actinomycin D (5 µg/mL; #A1410, Sigma-Aldrich, St. Louis, MO, USA) for 30 min prior to A20 mRNA-LNP transfection. ActD treatment was maintained during transfection, and cells were subsequently cultured for 24 h.

The lipid components were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, consisting of SM-102, distearoylphosphatidylcholine, cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. The mRNA was dissolved in a 50 mM sodium citrate buffer. Lipid nanoparticles (LNPs) were formulated using an Encell Master (Enparticle, Busan, South Korea) by mixing the aqueous and organic solutions at a 3:1 ratio. The resulting LNP solution was then concentrated via ultrafiltration using an Amicon ultracentrifugal filter (UFC9030, Merck Millipore, Billerica, MA, USA), following the manufacturer's instructions. The size and zeta potential of the LNPs were assessed using a Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK), with the mRNA diluted in water.

Antibodies targeting A20 (ab13597, Abcam, Cambridge, UK), TRAF6 (SC-8409, Santa Cruz Technologies, Santa Cruz, CA, USA), NF-κB (ab16502, Abcam), DREAM (SC-166916, Santa Cruz Technologies), SMAD2 (ab33875, Abcam), Col1a1 (PA5-29569, Invitrogen, CA, USA) αSMA (ab7817, Abcam), c-Myc (SC-40, Santa Cruz), p53 (SC-6243, Santa Cruz) and GAPDH (ab181602, Abcam) were used to measure the corresponding protein levels by Western blotting (SNAP i.d. Protein Detection System, SNAP2BHMB050, Merck Millipore). Protein concentrations were determined using the bicinchoninic acid assay (23235, Thermo Fisher Scientific). Samples were separated on 4–12% sodium dodecyl sulfate–polyacrylamide gels and transferred to nitrocellulose membranes (Amersham Pharmacia, Uppsala, Sweden). Band densities were quantified using image capture densitometry.

The mRNA expression levels of COL1A1 (5'-CATAAAAGGCCCACTACCCAAC-3'/5'-ACCTTGCTCTCCTCTTACTGC-3') and αSMA (5'-TGGGTGACGAAGCACAGAGC-3'/5'-CTTCAGGGGCAACACGAAGC-3') were normalized to the expression of β-actin. Total mRNA was extracted using TRIzol (Molecular Research Center, Cincinnati, OH, USA). cDNA was synthesized using a reverse transcription system (TaKaRa, Shiga, Japan). qPCR was performed using LightCycler FastStart DNA Master SYBR Green I (TaKaRa), following the manufacturer's instructions.

Seven-week-old male SKG mice were purchased from Orient Bio Inc. (Seongnam, Korea) and housed under specific-pathogen-free conditions at the Institute of Medical Sciences, Catholic University of Korea, with ad libitum access to water and standard mouse chow (Ralston Purina, St. Louis, MO, USA). To induce SSc, bleomycin (HY-17565A, MedChemExpress, USA) was dissolved in phosphate-buffered saline and administered subcutaneously at a dose of 50 µg in 100 µL daily for 3 weeks. All in vivo studies were conducted in three independent experiments, each comprising five mice per group. Animal experiments were performed in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments of the Institutional Animal Care and Use Committee of the Catholic University of Korea.

A20 mRNA-LNP was administered via intramuscular injection beginning 3 days after the initiation of bleomycin treatment. The treatment regimen involved weekly injections of 10 µg of A20 mRNA-LNP for 6 weeks. The control group received LNP alone, following the same dosing schedule and route of administration.

Th2 and Th17 cells were stained with PC5.5-CD4 (45-0042-82, eBioscience, San Diego, CA, USA), PE-IL-4 (554435, eBioscience), and APC-IL17 (17-7177-81, eBioscience). Spleen cells were stained with antibodies against CD4, IL-4, and IL-17 to identify Th2 and Th17 cell populations, and analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

After euthanasia, skin and lung tissues were collected, fixed in 10% formalin (#HT501320, Sigma), and embedded in paraffin. Sections (5 µm) were prepared using a microtome (Leica Biosystems) and stained with hematoxylin and eosin (H&E), Masson's trichrome, and Sirius Red. Dermal thickness was defined as the distance between the epidermal–dermal and dermal–adipose junctions. The severity of lung fibrosis was semi-quantitatively assessed, as described by Ashcroft et al. (13). Briefly, lung fibrosis was scored on a scale of 0–8: grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening without structural damage; grade 5, definite fibrosis with fibrous bands or small masses; grade 7, severe distortion with large fibrotic areas; and grade 8, complete fibrous obliteration. Grades 2, 4, and 6 represented intermediate changes. Fibrotic areas in Masson's trichrome- and Sirius Red–stained sections were quantified using ImageJ. In skin tissues, the collagen-positive area was expressed as the percentage of blue (Masson) or red (Sirius Red) area within the dermis. In lung tissues, the collagen-positive area was calculated in the same manner and expressed as the percentage of fibrotic tissue per field. All histological analyses were performed under blinded conditions.

For determination of Skin dermal and Lung lobes collagen content, colorimetric quantification of hydroxyproline was performed in a small skin and lung biopsy (5 mm diameter) taken from every animal. Briefly, frozen skin tissues were dehydrated, weighed, and hydrolyzed in 12 N HCl at 120°C for 3 h (BM-HYP-100, Biomax). After hydrolyed, the samples were mixed with chloramine T reagent and incubated for 5 min at room temperature. Next, a reagent mixed with perchloric acid/isopropanol solution and 2X DMAB was added, and the sample was incubated at 60°C for an additional 90 minutes. The absorbance was measured at 560 nm in duplicate with a microplate spectrophotometer.

The skin and lung tissues from the mice undergoing immunofluorescence analysis were embedded in paraffin and then sliced into 5 μm sections. After blocking with 10% NGS (Normal goat serum with PBS), the sections were placed in a humid chamber and incubated with the corresponding primary antibodies overnight at 4°C. The following primary antibodies were used: anti-CD4 (14-9766-82, Invitrogen), anti-IL-4 (PA5-25165, Invitrogen), and anti-IL-17 (PA5-114455, Invitrogen). Subsequently, the sections were incubated with fluorescence-labeled secondary antibodies (Mouse anti-rat FITC, Mouse anti-rabbit APC) at room temperature in the dark for 2 hours. Finally, the sections were stained with DAPI solution (Servicebio G1012) for 30 min to visualize the cell nuclei. After quenching the autofluorescence of the tissue, the sections were mounted with anti-fade mounting medium for fluorescence (CS70330-2, Dako). Fluorescence images were captured using an Confocal Laser Scanning Microscope(LSM700, ZEISS), and at least three representative fields were selected for analysis using Image J software.

Paraffin-embedded tissue sections were incubated at 4°C with the following primary monoclonal antibodies: anti-A20 (ab13597, Abcam), anti-TRAF6 (SC-8409, Santa Cruz Technologies), anti-NF-κB (3033S, Cell Signaling), anti-DREAM (SC-166916, Santa Cruz Technologies), anti-SMAD2 (ab33875, Abcam), anti-TGF-β (BS-0086R; Fisher Scientific), anti-procollagen (ab64409, Abcam), and anti-αSMA (ab7817, Abcam). The sections were then incubated with biotinylated secondary antibodies and subsequently with streptavidin–peroxidase complex for 30 min. Reaction products were developed using 3,3-diaminobenzidine chromogen (K3468, Dako Corp., Carpinteria, CA, USA).

Results are presented as means ± standard error of mean (SEM). Data were analyzed using Student's t-test, the Mann–Whitney U test, or one-way ANOVA, performed with Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). P values < 0.05 (two-tailed) were considered statistically significant.

To generate A20 mRNA, the A20 gene was inserted into a capped mRNA expression construct containing a loop structure at the poly(A) tail (Figure 1A). The A20 mRNA was encapsulated in LNPs composed of four lipid components, as described in the Methods section (Figure 1B). The resulting A20 mRNA-LNPs displayed a Z-average particle size of approximately 110.88 ± 0.7 nm and a zeta potential of 10.4 ± 0.523 (Figure 1C). These results demonstrate the successful development of an A20 mRNA-LNP formulation with stable physicochemical properties.

To evaluate the inhibitory effect of A20 mRNA on TRAF6/NF-κB signaling-mediated fibrosis, HDFs were transfected with A20 mRNA to induce A20 expression. The cells were then stimulated with TGF-β to activate TRAF6/NF-κB signaling and induce a fibrotic response. Western blot analysis showed that A20 mRNA transfection increased A20 protein levels while reducing the expression of TRAF6, pNF-κB, αSMA, and Col1 (Figures 1D, E). Consistently, qPCR analysis in HDFs demonstrated that A20 mRNA transfection significantly reduced TGF-β (20 ng/mL)-induced upregulation of COL1A1 and αSMA, 24 hours after stimulation (Figure 1F). These data confirm that A20 overexpression suppresses fibrotic gene transcription downstream of TGF-β signaling, complementing the observed inhibition of pro-fibrotic proteins. Together, these findings demonstrate that A20 effectively inhibits both upstream TGF-β/NF-κB signaling and downstream fibrotic gene expression, highlighting its potential as a negative regulator of fibroblast activation. To evaluate whether A20 delivery restores protein expression independently of host transcription, fibroblasts were pretreated with actinomycin D (ActD, 5 µg/mL) prior to A20 mRNA-LNP administration. ActD treatment markedly reduced endogenous A20 and c-Myc protein levels, while inducing p53 accumulation, consistent with its role as a marker of transcriptional stress (Figure 1G). Notably, subsequent A20 mRNA-LNP delivery significantly restored A20 expression despite transcriptional inhibition. These findings demonstrate that exogenous A20 mRNA-LNP enables protein restoration under transcriptionally repressive conditions (Figure 1H).

The anti-fibrotic effects of A20 mRNA-LNP were evaluated to assess its therapeutic potential in an SSc mouse model. SSc was induced by bleomycin, which was administered daily for 3 weeks, and A20 mRNA-LNP was given over seven doses during a 6-week period (Figure 2A). Treatment with A20 mRNA-LNP resulted in a reduction in the activity of Th2 (Vehicle: 3.49%, A20 mRNA: 2.94%) and Th17 cells (Vehicle: 3.81%, A20 mRNA: 2.73%), which are known to promote fibrotic responses in SSc (Figure 2B). Although a decreasing trend in Th2 cells was observed, this difference did not reach statistical significance. In bleomycin-induced fibrotic mice, A20 mRNA-LNP treatment significantly reduced CD4⁺IL-4⁺ and CD4⁺IL-17⁺ T cell infiltration in the skin and lung compared to vehicle, indicating suppression of Th2- and Th17-mediated responses (Figure 2C). Histological analyses were performed to assess fibrosis in the SSc model. In skin sections, H&E staining demonstrated dermal thickening, Masson's Trichrome (MT) staining revealed collagen deposition, and Sirius Red staining quantified the fibrotic area (Figures 2D, E). In lung sections, H&E staining showed fibrotic changes with alveolar wall thickening, MT highlighted collagen accumulation, and Sirius Red staining measured the extent of fibrosis (Figures 2F, G). Hydroxyproline levels were measured in skin and lung tissues to assess collagen deposition (Figure 2H). Administration of A20 mRNA-LNP significantly decreased hydroxyproline levels in both skin and lung, demonstrating its anti-fibrotic effect. Collectively, these findings indicate that A20 mRNA-LNP effectively suppresses fibrosis in the SSc model.

To investigate the inhibitory effect of A20 overexpression on TRAF6/NF-κB signaling-mediated fibrosis, immunohistochemistry analyses were performed in both skin and lung tissues. In skin sections, administration of A20 mRNA-LNP increased A20 expression, accompanied by a relative decrease in TRAF6 and NF-κB levels compared to the vehicle group (Figure 3A). Expression of the fibrotic factor TGF-β was also reduced in A20-overexpressing skin. In lung sections, A20 mRNA-LNP treatment similarly enhanced A20 expression and reduced the levels of TRAF6, NF-κB, and TGF-β compared with the vehicle group (Figure 3B). These findings demonstrate that A20 overexpression via A20 mRNA-LNP suppresses the TRAF6/NF-κB pathway in both skin and lung tissues in the SSc model.

Recent studies have suggested an inverse relationship between A20 and the DREAM complex, with the latter promoting fibrosis by suppressing A20 expression. This study investigated the hypothesis that A20 mRNA-LNP regulates the DREAM–SMAD2 pathway in SSc. In the SSc mouse model, the vehicle group exhibited increased DREAM expression in skin tissues compared to wild-type controls, which is consistent with observations in SSc patients. Treatment with A20 mRNA-LNP effectively suppressed bleomycin-induced DREAM upregulation (Figure 4A). Inhibition of DREAM also suppressed SMAD2, a downstream target of DREAM, and SMAD2-mediated fibrotic factors, such as Procollagen and α-SMA. To further validate this mechanism, the effect of A20 overexpression in HDFs was examined. The results demonstrated significant inhibition of DREAM–SMAD2 activity in A20-overexpressing HDFs (Figure 4B), confirming the in vivo findings.

In the SSc mouse models, the vehicle group exhibited increased DREAM expression in lung tissue compared to wild-type controls, consistent with the pattern observed in skin tissue. In the A20 mRNA-LNP treatment group, lung fibrosis was effectively inhibited by modulating the DREAM–SMAD2 pathway in the bleomycin-induced SSc model (Figure 5). These results suggest that A20 mRNA-LNP not only inhibits fibrosis mechanisms in skin tissues but also in lung tissues, with the regulation of the DREAM–SMAD2 pathway playing a crucial role in this process.

To provide a conceptual overview of the therapeutic mechanism, we illustrated a schematic comparison between the fibrotic signaling pathways observed in systemic sclerosis and those modulated by A20 mRNA treatment (Figure 6). In the systemic sclerosis condition, activation of TLR4–TRAF6–NF-κB signaling and subsequent induction of TGF-β promote fibroblast activation and extracellular matrix (ECM) deposition, leading to excessive collagen and α-SMA expression. In contrast, delivery of A20 mRNA via lipid nanoparticles restored A20 protein expression in fibroblasts, suppressing TRAF6-dependent NF-κB activation and downregulating TGF-β/SMAD and DREAM signaling. These changes collectively attenuated pro-fibrotic gene expression and ameliorated skin and lung fibrosis in the systemic sclerosis model.