Development of an RNA virus-based episomal vector with artificial aptazyme for gene silencing

Abstract

RNA virus-based episomal vector (REVec), engineered from Borna disease virus, is an innovative gene delivery tool that enables sustained gene expression in transduced cells. However, the difficulty in controlling gene expression and eliminating vectors has limited the practical use of REVec. In this study, we overcome these shortcomings by inserting artificial aptazymes into the untranslated regions of foreign genes carried in vectors or downstream of the viral phosphoprotein gene, which is essential for vector replication. Non-transmissive REVec carrying GuaM8HDV or the P1-F5 aptazyme showed immediate suppression of gene expression in a guanine or theophylline concentration-dependent manner. Continuous compound administration also markedly reduced the percentage of vector-transduced cells and eventually led to the complete elimination of the vectors from the transduced cells. This new REVec is a safe gene delivery technology that allows fine-tuning of gene expression and could be a useful platform for gene therapy and gene-cell therapy, potentially contributing to the cure of many genetic disorders. KEY POINTS: • We developed a bornavirus vector capable of silencing transgene expression by insertion of aptazyme • Transgene expression was markedly suppressed in a compound concentration-dependent manner • Artificial aptazyme systems allowed complete elimination of the vector from transduced cells.

Keywords: Aptazyme; Borna virus vector; Gene regulation; Viral vector.

Fig. 1

Regulation of gene expression by insertion of aptazymes into plasmids. A Relative luciferase activity after the addition of compounds that react with aptazymes in plasmid-transfected cells. After HEK293T cells were transfected with 10 ng of plasmids encoding NanoLuc with GuaM8HDV or the P1-F5 aptazyme, guanine or theophylline was added at each concentration, and luciferase activity was measured 4 days later. The aptazyme was inserted into the N terminus (+ N) of the NanoLuc gene. B Relative luciferase activity for plasmids in which the aptazyme was inserted at the C terminus (+ C) of the NanoLuc gene. C Relative luciferase activity for plasmids in which the aptazyme was inserted at both ends (+ NC) of the NanoLuc gene. The values are expressed as the means ± SEs of the results from three biologically independent replicates. Significance was analyzed by Dunnett’s multiple-comparison test. *, p < 0.01, **, p < 0.001

Fig. 2

Construction of aptazyme-containing REVec with controllable gene expression. A Schematic showing that aptazymes enable the suppression of gene expression induced by REVec. Administration of the aptazyme ligand induces self-cleavage of the untranslated regions at both ends of the luciferase gene, resulting in the shutdown of gene expression. B Detection of antigens in REVec-transduced cells. After the transduction of REVec with Vero cells at an MOI of 0.25, the N and P proteins were detected by Western blot analysis. The results are shown for REVec-transduced cells with GuaM8HDV or P1-F5 aptazymes inserted at both ends of the NanoLuc gene. C Subcellular localization of the N and P proteins of the REVec in transduced cells. REVec with aptazymes added to both ends of the luciferase gene was transduced into Vero cells at an MOI of 0.25, and the subcellular localization of the N and P proteins was analyzed by IFA. The white arrows point to vSPOT in transduced cells. Scale bar, 10 μm. D Relative luciferase activity in cells transduced with the aptazyme-containing REVec. Each vector was transduced into Vero cells at an MOI of 0.25, after which luciferase activity was measured 3 days later. + N, + C, and + NC indicate the insertion sites of the aptazymes. E Evaluation of the effect of aptazyme insertion on the persistence of gene expression. Each vector was transduced into Vero cells at an MOI of 0.25, and luciferase activity was subsequently measured at each time point. The values are expressed as the means ± SEs of the results from three biologically independent replicates

Fig. 3

Aptazymes enable the suppression of gene expression induced by REVec. A Aptazyme enable the suppression of gene expression induced by REVec. After transduction into Vero cells with aptazyme-inserted REVec (+ NC) at an MOI of 0.25, the transduced cells were treated with guanine or theophylline at various concentrations. Relative luciferase activity was measured at 4 days post-administration. After treatment of transduced cells with the compounds, the amount of genomic RNA in the REVec (B) and the amount of mRNA in the luciferase gene (C) were quantified by qRT–PCR. Each value was normalized to the Ct value of the GAPDH gene. D Cytotoxicity of guanine and theophylline. Cell viability was measured at 4 days post-administration. The data are presented as the means + SEs of results from three independent experiments. Significance was analyzed by Dunnett’s multiple-comparison test. *, p < 0.01, **, p < 0.001

Fig. 4

Aptazymes can eliminate REVec from transduced cells. A Schematic showing that aptazymes can eliminate REVec from transduced cells. The GuaM8HDV or P1-F5 aptazyme was inserted downstream of the P gene of REVec and induced self-cleavage of the mRNA after aptazyme activation via the administration of each compound. B Detection of antigens in REVec-transduced cells. After the transduction of REVec with Vero cells at an MOI of 0.25, the N and P proteins were detected by Western blot analysis. C Subcellular localization of the N and P proteins of the REVec in transduced cells. Each vector with aptazymes inserted downstream of the P gene was transduced into Vero cells at an MOI of 0.25, and the subcellular localization of the N and P proteins was analyzed by IFA. The white arrows point to vSPOT in transduced cells. Bar, 10 μm

Fig. 5

Aptazyme enables the complete elimination of REVec from transduced cells. A Suppression of P gene expression by aptazymes leads to elimination of the genomic vector RNA. After transduction into Vero cells with aptazyme-inserted REVec at an MOI of 0.25, the transduced cells were treated with guanine or theophylline at various concentrations. The amount of genomic vector RNA of the REVec (A) and the mRNA of the P gene (B) were quantified by qRT–PCR at 4 days post-administration. Each value was normalized to the Ct value of the GAPDH gene. C Cytotoxicity of guanine and theophylline. Cell viability of A549 cells was measured at 4 days post-administration. After transduction into A549 cells with aptazyme-inserted REVec at an MOI of 0.2. The amount of genomic vector RNA of the REVec (D) and the mRNA of the P gene (E) were quantified by qRT–PCR at 4 days post-administration. Each value was normalized to the Ct value of the GAPDH gene. F Dose-dependent decrease in the percentage of vector-transduced cells. HEK293T cells were transduced with each vector at an MOI of 0.3. At 8 days post-administration, the percentage of GFP-positive cells was measured by a Tali cytometer and IFA. G Decrease in the amount of vector antigen caused by aptazyme activation. After compound administration at each concentration and aptazyme activation, N and P proteins were detected by WB at 4 days post-administration. The right lane indicates higher compound concentrations. H The amounts of N and P protein at 0, 14, and 21 days post-administration. After the administration of guanine or theophylline at 200 μM to transduced cells, the cells were passaged every 3 days. N and P proteins in transduced Vero cells were detected by Western blot analysis. The right graphs show the quantification and comparison of band intensities. The data were normalized to the band intensity of tubulin. The analysis was performed by ImageJ. (I) Relative amount of genomic vector RNA of GuaM8HDV vector and (J) P1-F5 vector after continuous compound administration. After the continued administration of guanine or theophylline at 200 μM to transduced cells for 0, 7, 14, or 21 days, the amount of the genomic vector RNA was quantified via qRT–PCR. After continuing treatment with the compound for 21 days, the cells were cultured in the absence of the compound for 6 days, after which the amount of genomic RNA was quantified in the same manner. Each value was normalized to the Ct value of the GAPDH gene. The data are presented as the means and + SEs of results from three independent experiments. Significance was analyzed by Dunnett’s multiple-comparison test. *, p < 0.01, **, p < 0.001. n.d., not detected