Differential effects of moderate and strong dsRNA-sensing pathway inhibition on saRNA transgene expression and cell loss.

a, Schematic of the native saRNA, E3, and E3-NSs-L* constructs designed to inhibit dsRNA-sensing pathways and report saRNA transgene expression. The native saRNA construct lacks dsRNA-sensing inhibitors. The E3 construct expresses vaccinia virus E3, a pleiotropic inhibitor of dsRNA sensing, expected to provide moderate inhibition. The E3-NSs-L* construct expresses vaccinia virus E3, and additionally includes Toscana virus NSs and Theiler’s virus L*, which target the PKR and OAS/RNase L pathways, expected to provide strong inhibition. EGFP is expressed via an IRES to report cap-independent translation, while a subgenomic promoter (depicted with an angled arrow) enables transcription of an RNA transcript that expresses mScarlet3 via cap-dependent translation. saRNA constructs were transfected into primary mouse FLS, which were labeled with BioTracker to monitor cell number.

b, Representative images of EGFP (green) and mScarlet3 (red) expression in FLS transfected with native saRNA, E3, or E3-NSs-L* over 3 weeks. Scale bar = 5 mm.

c, Representative images of FLS transfected with the same constructs, showing BioTracker intensity over time. Scale bar = 5 mm.

d, Quantification of EGFP fluorescence over time (n = 11 biological replicates). The E3 construct provided the greatest EGFP expression, while the E3-NSs-L* construct showed intermediate levels. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test.

e, Quantification of mScarlet3 fluorescence over time (n = 11 biological replicates). The E3 construct provided the greatest mScarlet3 expression, while the E3-NSs-L* showed intermediate levels. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test.

f, Quantification of BioTracker fluorescence over time (n = 11 biological replicates). The native saRNA and E3 constructs reduced BioTracker fluorescence, indicating cell loss. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test.

g, AUC analysis of BioTracker fluorescence data shown in (f), summarizing cumulative effects over the time course (n = 11 biological replicates). Increasing dsRNA-sensing pathway inhibition prevents saRNA-induced reductions in integrated BioTracker signal. Statistical analysis was performed using one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test to compare all groups. Mock transfection data is also presented in Fig. S5a.

h, Representative images of FLS stained with the cell number normalization dye, CellTag 700. Columns represent cells plated from the same biological replicate, while rows show different treatments. Scale bar = 2.5 mm.

i, Quantification of mock transfection normalized CellTag signal (n = 24 biological replicates). Increasing dsRNA-sensing pathway inhibition mitigates saRNA-induced reductions in CellTag signal. Statistical analysis was performed using one-way RM ANOVA with Greenhouse–Geisser correction and Holm-Šídák’s multiple comparisons test to compare all groups. Assays were performed 2 days post-transfection. Data in this panel were pooled from in-cell western assays, incorporating data presented in Figs. 3 and 6 as well as additional data not shown elsewhere.

For all statistical reporting, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data are presented as mean ± SEM.

For panels (d-f): Data were normalized to starting cell number, indicated by BioTracker intensity on day 0, prior to transfection. The mock transfection control data is also presented in Figs. 5c-e.

Acronyms: saRNA, self-amplifying RNA; dsRNA, double-stranded RNA; nsP, Venezuelan equine encephalitis virus non-structural protein; IRES, encephalomyocarditis virus internal ribosome entry site; moxBFP, monomeric oxidizing environment-optimized blue fluorescent protein; E3, Vaccinia virus E3 protein; NSs, Toscana virus non-structural NSs protein; L*, Theiler’s murine encephalomyelitis virus L* protein; T2A, Thosea asigna virus 2A peptide; P2A, porcine teschovirus-1 2A peptide; PKR, protein kinase R; OAS, oligoadenylate synthase; AUC, area under the curve.

saRNA induces increased phosphatidylserine staining and reduced viability, which is prevented by E3-NSs-L*.

a, Representative cropped images of Annexin V-CF800 staining, indicating phosphatidylserine exposure or loss of membrane integrity, performed daily over 6 days using a microplate imager. Scale bar = 1.5 mm.

b, Representative cropped images of calcein AM staining, indicating viability, on day 7 post-treatment. Scale bar = 1.5 mm.

c, Annexin V staining, quantified as the area of positive pixels determined using Li thresholding (n = 6 biological replicates). Staurosporine, native saRNA, and the E3 construct significantly increased annexin V staining, while E3-NSs-L* did not. Data are normalized to the average of the mock transfection group. Statistical significance of treatment effects at each time point compared to mock transfection was determined using two-way RM ANOVA with Bonferroni’s multiple comparisons test. Data are presented as mean ± SEM.

d, Calcein AM intensity measured on day 7 post-treatment (n = 6 biological replicates). Native saRNA, and E3 significantly reduced cell viability compared to mock transfection, while E3-NSs-L* did not. Cell viability in the E3-NSs-L* group was significantly higher than the E3 group. Connecting lines indicate responses from the same biological replicate. All groups differed significantly from staurosporine; these comparisons are omitted from the figure for clarity due to the large number of statistical comparisons. Data are normalized to cell number on day 0 as determined by BioTracker staining before transfection. Statistical significance was determined by one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test comparing all groups.

saRNA constructs used are shown in Fig. 1a. For all statistical reporting, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Translational control alterations induced by saRNA are modulated by dsRNA-sensing pathway inhibitors with differing effects on RNA integrity.

a, Phosphorylated eIF2α levels examined day 2 post-transfection by in-cell western assay (n = 6 biological replicates). Both E3 and E3-NSs-L* constructs significantly reduced eIF2α phosphorylation. Data are presented as fold-change relative to mock transfected cells. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test.

b, eIF2α levels examined day 2 post-transfection by in-cell western assay (n = 5 biological replicates). E3-NSs-L* significantly increased total eIF2α levels compared to native saRNA transfection. Data are presented as fold-change relative to mock transfected cells. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test.

c, PKR levels examined day 2 post-transfection by in-cell western assay (n = 4 biological replicates). E3-NSs-L* significantly reduced PKR levels compared to both native saRNA and E3 transfection. Data are presented as fold-change relative to mock transfected cells. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test.

d, Phosphorylated eIF4E levels examined day 2 post-transfection by in-cell western assay (n = 6 biological replicates). All saRNA constructs tested significantly reduced eIF4E phosphorylation levels compared to mock transfection. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test.

e, eIF4E levels examined day 2 post-transfection by in-cell western assay (n = 6 biological replicates). One-way RM ANOVA revealed no significant differences between groups (F(3,15) = 1.207, P = 0.3410). f, rRNA integrity of FLS transfected with E3-NSs-L* is significantly higher than that of E3-transfected FLS (n = 5 biological replicates). rRNA integrity was assessed using the RNA Integrity Number (RIN) algorithm, which ranges from 1 to 10, with 10 indicating fully intact rRNA. Total RNA was extracted from FLS 1 day post-transfection. Data are shown as a Gardner-Altman comparison plot, with the right panel illustrating the mean effect size ± 95% CI. Statistical significance was assessed using a paired t-test (P = 0.0054).

saRNA constructs used are shown in Fig. 1a. Connecting lines indicate responses from the same biological replicate. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001. The mock transfection control data presented in panels (a), (b), (d) and (f) are also used in Fig. 6.

Inhibiting inflammatory signaling reduces saRNA-induced anti-viral cytokine secretion

a, Schematic of the moxBFP, srIκBα, and srIκBα-Smad7-SOCS1 saRNA constructs designed for inhibition of inflammatory signaling. These constructs include dsRNA-sensing pathway inhibitors (vaccinia virus E3, Toscana virus NSs, and Theiler’s virus L*). The moxBFP construct, serving as a control, lacks inflammatory signaling inhibitors. The srIκBα construct co-expresses srIκBα, which blocks the NF-κB inflammatory signaling pathway and represents moderate inflammatory signaling inhibition. The srIκBα-Smad7-SOCS1 construct co-expresses srIκBα, Smad7 and SOCS1 to additionally inhibit TGF-β and IFN pathways, representing strong inflammatory signaling inhibition. The angled arrow denotes the subgenomic promotor.

b, FLS were transfected with saRNA constructs, and anti-viral cytokines were quantified by bead-based immunoassay on day 2 post-transfection (n = 6 biological replicates). dsRNA-sensing pathway inhibition and inflammatory signaling inhibition significantly reduced cytokine secretion induced by saRNA. Cytokine levels were scaled within each biological replicate, with the highest value set to 100%, and the mean value displayed in the heatmap. Detailed graphs of individual cytokines are shown in Supplementary Fig. S4. For statistical analysis, cytokine levels were normalized to pre-transfection cell number (measured using BioTracker). Statistical significance for each cytokine was assessed using one-way RM ANOVA followed by Bonferroni’s multiple comparisons test against the mock transfection control. A Bonferroni correction was applied to P values to account for multiple independent hypothesis testing. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Acronyms: nsP, Venezuelan equine encephalitis virus non-structural protein; IRES, encephalomyocarditis virus internal ribosome entry site; moxBFP, monomeric oxidizing environment-optimized blue fluorescent protein; E3, Vaccinia virus E3 protein; NSs, Toscana virus non-structural NSs protein; L*, Theiler’s murine encephalomyelitis virus L* protein; T2A, Thosea asigna virus 2A peptide; P2A, porcine teschovirus-1 2A peptide, E2A, equine rhinitis A virus 2A peptide, srIкBα, super-repressor inhibitor of κBα; smad7, mothers against decapentaplegic homolog 7; SOCS1, suppressor of cytokine signaling 1; IFN-γ, interferon-γ; CXCL1, C-X-C motif chemokine ligand 1; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein-1; IL-12p70, interleukin-12; CCL5, chemokine ligand 5; IL-1β, interleukin-1β; CXCL10, C-X-C motif chemokine ligand 10; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-10, interleukin-10; IFN-β, interferon-β; IFN-α, interferon-α; IL-6, interleukin-6.

Differential effects of srIκBα and srIκBα-Smad7-SOCS1 on cell number and transgene expression.

a, Representative images of BioTracker staining over 3 weeks in FLS transfected with different saRNA constructs. Scale bar = 5 mm.

b, Representative images of EGFP (green) and mScarlet3 (red) expression over 3 weeks in FLS transfected with different saRNA constructs. Scale bar = 5 mm.

c, Quantification of BioTracker fluorescence intensity over time (n = 11 biological replicates). srIκBα induces reduction in cell number, which is prevented by srIκBα-Smad7-SOCS1. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test.

d, Quantification of EGFP fluorescence intensity over time (n = 11 biological replicates). All constructs showed low levels of EGFP expression. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test.

e, Quantification of mScarlet3 fluorescence intensity over time (n = 11 biological replicates). The srIκBα-Smad7-SOCS1 produced 2-5 times more mScarlet3 fluorescence than either moxBFP or srIκBα constructs. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test.

saRNA constructs used are shown in Fig. 4a. For all statistical reporting, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data were normalized to cell number (BioTracker intensity) on day 0, prior to transfection. The mock transfection control data used in this figure is also presented in Fig. 1. Data are presented as mean ± SEM.

srIκBα reduces eIF2α phosphorylation and total eIF4E levels, effects reversed by co-expression of Smad7 and SOCS1 In-cell western assays were performed 2 days post-transfection. Data are presented as fold change relative to mock-transfected cells.

a, Phosphorylation of eIF2α is significantly reduced by srIκBα, and this reduction is reversed by co-expression of Smad7 and SOCS1 (n = 6 biological replicates). Statistical significance was determined by one-way RM ANOVA and Holm-Šídák’s multiple comparisons test to compare all groups.

b, Total eIF2α levels are not significantly affected by srIκBα or srIκBα-Smad7-SOCS1 (n = 6 biological replicates). One-way RM ANOVA revealed no significant differences among groups. F(2,8)=3.683, P=0.0735.

c, Phosphorylation of eIF4E is not significantly affected by srIκBα or srIκBα-Smad7-SOCS1 (n = 6 biological replicates). One way RM ANOVA revealed no significant difference among groups. F(2,10)=1.336, P=0.3059.

d, Total eIF4E levels are significantly reduced by srIκBα, an effect reversed by co-expression of Smad7 and SOCS1 (n = 6 biological replicates). Statistical significance was determined by one-way RM ANOVA and Holm-Šídák’s multiple comparisons test to compare all groups.

saRNA constructs are described in Fig. 4a. Connecting lines indicate responses from the same biological replicate. For all statistical reporting, *P < 0.05, **P < 0.01 and ***P < 0.001. Mock transfection data used for normalization are the same as in Fig. 3.

Prolonged transfection with srIκBα or srIκBα-Smad7-SOCS1 significantly reduces basal fibroblast activation factor-α (FAP-α) levels.

a, Representative in-cell western images showing FAP-α expression. Columns show different biological replicates, and rows show different treatments. The montage on the right shows FAP-α signal normalized to CellTag signal (FAP-α/CellTag).

b, In-cell western assay of FAP-α expression (n = 8 biological replicates). Both srIκBα and srIκBα-Smad7-SOCS1 significantly reduce FAP-α levels compared to mock transfection, while moxBFP does not differ significantly from mock. Statistical significance was determined by one-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test to compare groups to mock transfection. Connecting lines indicate responses from the same biological replicate. **P<0.01.

ML336 enables external control of transgene expression from the srIκBα-Smad7-SOCS1 construct.

a, Representative images of EGFP (green) and mScarlet3 (red) expression over 13 days in FLS transfected with srIкBα-smad7-SOCS1. Cultures were treated with vehicle or 1 μM ML336, starting 1 day post-transfection. Scale bar = 5 mm.

b, Representative images of calcein AM staining on day 13 post-transfection. Scale bar = 5 mm.

c, Quantification of EGFP fluorescence intensity in srIκBα-Smad7-SOCS1-transfected FLS (n = 6 biological replicates). Cultures were treated with vehicle or 1 μM ML336 (treatment period indicated by shading). EGFP fluorescence was significantly lower in ML336-treated cultures compared to vehicle-treated cultures on day 7. Statistical significance relative to vehicle-treated cells was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. #P<0.05, ###P<0.01.

d, Quantification of mScarlet3 fluorescence intensity in srIκBα-Smad7-SOCS1-transfected FLS (n = 6 biological replicates). Cultures were treated with vehicle or 1 μM ML336 (treatment period indicated by shading). mScarlet3 fluorescence was significantly lower in ML336-treated cultures compared to vehicle-treated cultures beginning on day 3. Statistical significance relative to vehicle-treated cells was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. #P<0.05, ##P<0.01, ###P<0.001.

e, Quantification of calcein AM staining on day 13 post-transfection, following 12 days of vehicle or ML336 treatment (n = 6 biological replicates). Cultures treated with ML336 showed no significant difference in calcein AM signal compared to mock transfection, while vehicle-treated cultures exhibited significantly lower calcein AM signals compared to both mock-transfected and ML336-treated cultures. Connecting lines indicate responses from the same biological replicate. Statistical significance was determined by one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test. **P<0.01, ***P<0.001.

Data were normalized to BioTracker intensity on day 0 and are presented as mean ± SEM.