Co‐expression of HSV ‐1 ICP34 .5 enhances the expression of gene delivered by self‐amplifying RNA and mitigates its immunogenicity

Figures

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  Fig. 1. saRNA construction and validation of synthetic saRNA. (A) The structural schematic diagram of wild‐type saRNA, 5mC‐modified saRNA‐encoding delete‐mutated or intact ICP34.5. (B) Representative fluorescence microscopy images of 293T cells transfected with three types of saRNAs at 24 h. (C) The expression of ICP34.5 was identified by qRT‐PCR. Statistical analysis was performed by Student's t‐test. Data are means ± SD. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

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  Fig. 2. saRNA‐encoding ICP34.5 mediates eIF2α dephosphorylation in UC‐MSCs. (A) Western blot analysis showing the expression levels of eIF2α and phosphorylated eIF2α (p‐eIF2α) in UC‐MSCs transfected with three distinct saRNAs. GAPDH was used as the loading control. (B) After obtaining the western blot bands, grayscale values were analyzed using ImageJ. The intensity of each band was normalized to GAPDH, which served as the internal loading control, to correct for variability in protein loading. The normalized grayscale values of the experimental groups were expressed relative to the wild‐type group, which was set as the baseline (100%). The reduction in signal intensity for the modified groups, calculated relative to the wild‐type, was represented as a percentage decrease. Statistical analysis was performed by Student's t‐test. Data are means ± SD. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 3. saRNA‐encoding intact ICP34.5 enhances EGFP expression in 293T. (A) Flow cytometry analysis of EGFP expression in 293T cells transfected with three distinct saRNAs for 24 h. (B) Quantification of mean fluorescence intensity (MFI) of EGFP expression in 293T transfected with three distinct saRNAs, based on the flow cytometry analysis shown in (A). Statistical analysis was performed by Student's t‐test. Data are means ± SD. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 4. Intact ICP34.5 incorporating saRNA efficiently enhances EGFP expression in UC‐MSCs. (A) Representative fluorescence microscopy images of UC‐MSCs transfected with three types of saRNA at 24, 48, and 72 h. (B) Flow cytometry analysis of EGFP expression in UC‐MSCs transfected with saRNAs encoding either deletion‐mutated or intact ICP34.5 at 96 h. (C) Quantification of mean fluorescence intensity (MFI) of EGFP expression in UC‐MSCs transfected with saRNAs encoding either deletion‐mutated or intact ICP34.5, based on the flow cytometry analysis shown in (B). Statistical analysis was performed by Mann–Whitney U test. Data are means ± SD. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 5. saRNA‐encoding ICP34.5 reduced innate immune responses in 293T and UC‐MSCs. (A) The structural schematic diagram of wild‐type saRNA and 5mC‐modified saRNA with or without encoding ICP34.5. (B, C) The secretion of IFNβ by saRNA‐transfected 293T cells or UC‐MSCs was assessed using ELISA. Statistical analysis was performed by Student's t‐test. Data are means ± SD. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.