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

Abstract

Self-amplifying RNA (saRNA) vectors have garnered significant attention for their potential in transient recombinant protein expression and vaccination strategies. These vectors are notable for their safety and the ability to produce high levels of protein from minimal input templates, offering a promising avenue for gene therapy applications. Despite their advantages, saRNA vectors face a critical challenge in their propensity to trigger a robust innate immune response. The presence of double-stranded RNA intermediates during saRNA replication activates pattern recognition receptors (PRRs), leading to the activation of protein kinase R (PKR) and interferon (IFN) signaling, which can result in a general translational shutdown within the host cell. To mitigate the stimulatory effects on PRRs and enhance the translation efficiency of saRNA, this study employs the saRNA-encoding HSV-1 neurovirulence protein ICP34.5, which is known for its ability to counteract the effects of PKR activation, potentially improving the translation efficiency of saRNA. It was shown that saRNA-encoding ICP34.5 clearly mediated the eukaryotic initiation factor 2 alpha subunit (eIF2α) dephosphorylation and significant suppression of innate immune responses in vitro, leading to enhanced expression of saRNA-encoded genes. The application of ICP34.5 incorporating saRNA vectors offers a more efficient and cost-effective solution for the production of proteins and the development of vaccines. This strategy could revolutionize the fields where saRNA utilization is envisioned, particularly in neurotropic disease applications where HSV-1 proteins may offer additional benefits.

Keywords: ICP34.5; gene expression; innate immune response; saRNA; vaccines.

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.

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.

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.

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.

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.