Journal: bioRxiv

Article Title: Telomerase modRNA offers a novel RNA-based approach to treat human pulmonary fibrosis

doi: 10.1101/2025.02.19.639041

Figure Lengend Snippet: A Schematic presentation of the modRNA hTERT template. B The modRNA production is divided into three main steps: the sequence design, then in vitro transcription (IVT) where the modRNA is generated and lastly the purification step. C Schematic describing experimental setup used to validate transfection efficiency of modRNA GFP and functionality of modRNA hTERT. D Transmission electron microscopy of ATII cells. Arrowheads indicate lamellar body like structures. Nucleus is depicted as n. E Fold change of hTERT mRNA expression in ATII cells transfected with modRNA hTERT in a concentration range of 0.25 – 2 µg/mL compared to modRNA GFP 2 µg/mL control (modRNA control) after 24 h and 48 h (n = 3). F TRAP assay revealed reactivation of telomerase activity in ATII cells after 24 h and 48 h in dose-dependent effect. Positive (pos.) control = human induced pluripotent stem cells, negative (neg.) control = HUVEC cells, LB = lysis buffer, 1 = cell lysate, Δ = heat inactivated lysate. G Quantification of TRAP assays indicates a dose-dependent increase of telomerase activity compared to modRNA control (n = 3). H No prolonged immune reaction was detected after 48 h modRNA treatment in ATII cells compared to untreated control (n = 3). *p<0.05; ***p<0.001; Two-way ANOVA, Dunnett’s multiple comparisons test.

Article Snippet: Human primary ATII cells (EP71AL Epithelix) were obtained from non-smoking donors with no reported pathology (Epithelix Sarl, Geneva, Switzerland).

Techniques: Sequencing, In Vitro, Generated, Purification, Transfection, Transmission Assay, Electron Microscopy, Expressing, Concentration Assay, Control, TRAP Assay, Activity Assay, Lysis

Journal: bioRxiv

Article Title: Telomerase modRNA offers a novel RNA-based approach to treat human pulmonary fibrosis

doi: 10.1101/2025.02.19.639041

Figure Lengend Snippet: A Images showing successful transfection of modRNA GFP 2 µg/mL in ATII cells as observed by GFP expression in 24 h an 48 h post transfection. B Telomere qFISH analysis demonstrated no telomere elongation after 48 h and 96 h modRNA hTERT treatment in ATII cells (n ≥ 84 nuclei per group out of 3 biological replicates were imaged) modRNA GFP is used as the modRNA control. C Representative brightfield images on the day of passaging in ATII cells of modRNA control compared to 2x treatment modRNA hTERT. D A decreasing trend of senescence-related markers (CDKN2A, CDKN1A, TP53) was detected after two doses of modRNA hTERT treatment (n=4). *p<0.05; One-way ANOVA or Two-way ANOVA, Dunnett’s or Sidak’s multiple comparisons test.

Article Snippet: Human primary ATII cells (EP71AL Epithelix) were obtained from non-smoking donors with no reported pathology (Epithelix Sarl, Geneva, Switzerland).

Techniques: Transfection, Expressing, Control, Passaging

Journal: bioRxiv

Article Title: Telomerase modRNA offers a novel RNA-based approach to treat human pulmonary fibrosis

doi: 10.1101/2025.02.19.639041

Figure Lengend Snippet: A Population doubling level (PDL) demonstrated an increase after single modRNA hTERT treatment, which was further increased upon double modRNA hTERT treatment. Arrowheads indicate the time point of modRNA hTERT treatment performed in ATII cells (n = 3). Geisser-Greenhouse’s epsilon; Two-way ANOVA; Dunnett’s multiple comparison test. B BrdU assay indicated towards an increase in proliferation in ATII cells after modRNA hTERT treatment (n = 5). C Immunofluorescence staining of Ki67, pH3 and γH2AX, (green) revealed an increase in proliferation and a decrease in DNA damage after modRNA hTERT transfection in ATII cells (n = 3). D Representative images of qFISH staining in ATII cells after modRNA hTERT treatment. Brighter telomere spots (yellow, Tel Cy3), representing longer telomeres, were visible after 2x treatment with modRNA hTERT compared to 1x treatment and modRNA control group. Nucleus is depicted in blue (Hoechst). E Telomeric spots analysis revealed an increase of telomere length compared to modRNA control group (n ≥ 95 nuclei per group out of 3 biological replicates were imaged). Welch ANOVA; Games-Howell’s multiple comparisons test. *p<0.05; **p<0.01; ***p<0.001; One-way ANOVA or Two-way ANOVA, Dunnett’s multiple comparisons test.

Article Snippet: Human primary ATII cells (EP71AL Epithelix) were obtained from non-smoking donors with no reported pathology (Epithelix Sarl, Geneva, Switzerland).

Techniques: Comparison, BrdU Staining, Immunofluorescence, Staining, Transfection, Control