Fig. 2: Engineered circRNAs encoding fLuc exhibit stable and efficient expression in chondrocytes in vivo.

a Schematic diagram of ivcRNA design and synthesis in vitro based on permuted Anabaena group I intron optimized with truncated extraneous sequences (Ana_PIE_27nt system)¹⁴. ivcRNA, in vitro transcribed and circularized RNA. b Schematic diagram of ivcRNA-LNPs (lipid nanoparticles) formulation. The lipid mixture in ethanol and circRNA in aqueous solution were pumped separately into the two inlets of the microfluidic mixing device with a total flow rate of 4 mL/min. c Representative image of ivc-IRES3-fLuc is analyzed using 4% urea denaturing polyacrylamide gel electrophoresis (Urea-PAGE). Bans of RNA circles are enriched and verified with RNase R. The hollow blue circles indicate circular RNAs, single blue curves indicate nicked RNAs. d The pipeline for administering mice to characterize the expression of mRNA/circRNA encoding fLuc in vivo. Intra-articular injection of DiR labeled RNA-LNPs complexes was performed on 6-week-old C57/B6 mice (300 ng RNA per mouse). Bioluminescence imaging of fLuc activity was conducted until the signal returned to background levels. DiR, 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Iodide; PBS, phosphate-buffered saline. e Represent bioluminescence IVIS images at various time points of the live mice after intra-articular injection of DiR labeled RNA-LNPs complexes. IVIS, in vivo imaging system spectrum. f Quantitative statistical analysis of the fluorescence at the injection site for in vivo Luciferase expression at various time points (n  =  3 mice per group). The data are presented as the Mean with SD. Statistical significance was determined by multiple two-tailed unpaired t tests in fLuc mRNA group and ivc-IRES3-fLuc group. g Schematic illustration of the experimental pipeline characterizes circRNA injection in sham and DMM-treated mice. Sham (Left leg) and DMM surgery (right leg) were performed in 8-week-old wild-type mice. The dosage of circRNA injection is 500 ng. h Bioluminescent images of a representative mice. n = 4 mice per group. Left leg: Sham group; right leg: DMM group. i Quantitative statistical analysis of the fluorescence at the injection site (Sham and DMM) for in vivo Luciferase expression (n  =  4 mice per group). The data are presented as the violin plot and the number of mice is showed as points in each group. Statistical significance was determined by multiple two-tailed unpaired t test, and p-values are labelled in (i). j Representative image of ivc-IRES4-mCherry RNA is analyzed by 4% urea-PAGE. RNA circles are enriched and verified with RNase R. The hollow red circles indicate RNA circles, single red curves indicate nicked RNA. k Immunofluorescence staining of mCherry in articular cartilage of joints from mice after intra-articular injection of ivc-IRES4-mCherry-LNPs. n = 3 samples per group. Scale bars = 100 µm. l Representative image of ivc-IRES2-EGFP is analyzed using denaturing 4% urea-PAGE. RNA circles are enriched and verified with RNase R. The hollow green circles indicate RNA circles; single green curves indicate nicked RNA. m Representative microscopy immunofluorescence staining images of a cross section of day-3 cartilage pellet cultures after incubated with ivc-IRES2-EGFP-LNPs. n = 3 samples per group. Scale bars = 100 µm. Figure 2 a, b, d, g. k, m: Created in BioRender. Suo, J. (2025) https://BioRender.com/4byajfm.