NF-κB promotes leaky expression of adenovirus genes in a replication-incompetent adenovirus vector

Abstract

The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes.

Figure 1. Reduction in the E2 promoter activity by deletion of an NF-κB binding site.

(A,B) HeLa cells were co-transfected with pHMCMV6-RLuc and reporter plasmids carrying the E2-promoter-driven FLuc expression cassette (pGL4, pGL4-E2, pGL4-E2-del1, pGL4-E2-del2, pGL4-E2-del2.1, or pGL4-E2-del3). After a 24-h incubation, luciferase activity was determined. The data show FLuc activity normalized by RLuc activity in the cells. Schematic diagrams of each promoter are shown at the left of the graph. (B) Nucleotide sequence around the NF-κB binding site (italic type) in the E2 promoter region. The NF-κB binding sequence is deleted in pGL4-E2-del2.1. These data are expressed as the means ± S.D. (n = 4). ***p < 0.001.

Figure 2. Promotion of Ad early gene expression and replication by TNF-α stimulation.

(A) HeLa cells were transfected with pNF-κB-Luc, followed by treatment with recombinant hTNF-α at 100 ng/ml. After 24-h incubation, luciferase activity was determined. The data show FLuc activity normalized by RLuc activity. (B,C) HeLa cells were pre-treated with hTNF-α at 100 ng/ml for 5 h, followed by infection with WT-Ad at 100 VP/cell. After 12-h incubation, the E1A, E2, E3, and E4 mRNA levels in the cells were determined by quantitative RT-PCR (B). After 24-h incubation, the E1A protein levels in the cells were determined by western blotting analysis (C). The intensity of E1A expression was quantified using Image J software. (D) HeLa cells were pre-treated with hTNF-α at the indicated concentration for 5 h, followed by infection with WT-Ad at 100 VP/cell. After 24-h incubation, Ad genome copy numbers in the cells were determined by quantitative PCR. (E) HeLa cells were pre-treated with hTNF-α at 100 ng/ml for 5 h, followed by transduction with Adv-CMVLuc at 100 VP/cell. After 12-h incubation, Ad gene mRNA levels in the cells were similarly determined. These data are expressed as the means ± S.D. (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Suppression of Ad early gene expression and replication by NF-κB inhibitors.

(A) HeLa cells were pre-treated with BAY11-7082 and MG-132 at 10 mM and 2.5 mM, respectively, for 1 h, followed by transfection with pNF-κB-Luc. After 24-h incubation, luciferase activity was determined. The data show FLuc activity normalized by RLuc activity. (B,C) HeLa cells were pre-treated with BAY11-7082 and MG-132 at 10 mM and 2.5 mM, respectively, for 1 h, followed by infection with WT-Ad at 100 VP/cell. After 12-h incubation, the E1A, E2, E3, and E4 mRNA levels in the cells were determined by quantitative RT-PCR (B). After 24-h incubation, Ad genome copy numbers in the cells were determined by quantitative PCR (C). (D) HeLa cells were pre-treated with BAY11-7082 and MG-132 at 10 mM and 2.5 mM, respectively, for 1 h, followed by infection with Adv-CMVLuc at 100 VP/cell. After 12-h incubation, Ad gene mRNA levels in the cells were similarly determined. These data are expressed as the means ± S.D. (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Suppression of Ad early gene expression and replication by NF-κB knockdown.

(A,B) HeLa and H1299 cells were transfected with siControl or sip50. After 48-h incubation, mRNA and protein levels of p50 in the cells were determined by quantitative RT-PCR (A) and western blotting analysis (B), respectively. p50 mRNA levels in the cells transfected with siControl were normalized to 1. (C,D) HeLa cells were transfected with sip50 at 50 nM, followed by infection with WT-Ad at 100 VP/cell. After 12-h incubation, the E1A, E2, E3, and E4 mRNA levels in the cells were determined by quantitative RT-PCR (C). After 24-h incubation, Ad genome copy numbers in the cells were determined by quantitative PCR (D). (E) HeLa cells were transfected with sip50 at 50 nM, followed by transduction with Adv-CMVLuc at 100 VP/cell. After 12-h incubation, Ad gene mRNA levels in the cells were similarly determined. These data are expressed as the means ± S.D. (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. NF-κB-mediated enhancement of the E2 and E3 promoter activity.

(A) HeLa cells were transfected with reporter plasmids carrying the expression cassette of Ad early gene promoter-driven FLuc (pGL4-E1A, -E1B, -E2, -E3, and -E4) for 24 h, followed by treatment with hTNF-α at 100 ng/ml. After 24-h incubation, luciferase activity was determined. (B) HeLa cells were infected with WT-Ad at 100 VP/cell. After 24-h incubation, NF-κB binding DNA levels were determined by ChIP assay. These data are expressed as the means ± S.D. (n = 3–4). **p < 0.01, ***p < 0.001.

Figure 6. Promotion of Ad early gene expression by TNF-α stimulation in mouse liver.

(A,B) C57BL/6 mice were intravenously administered 10⁹ IFU of WT-Ad (A) or Adv-CALuc (B), followed by intravenous administration of recombinant mTNF-α at 0.5 mg/mouse. Twenty-four hours after the administration, the E1A, E2, E3, and E4 mRNA levels in the mouse liver were determined by quantitative RT-PCR. These data are expressed as the means ± S.E. (n = 5–6). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7. Suppression of Ad early gene expression by expression of a dominant-negative IκBα gene.

(A) HeLa cells were transfected with pNF-κB-Luc, followed by transduction with Adv-CALuc or Adv-CADNIκBα at an MOI of 5. After 48-h incubation, luciferase activity was determined. The data show FLuc activity normalized by RLuc activity. (B) HeLa cells were transduced with Adv-CALuc or Adv-CADNIκBα at an MOI of 5. After 48-h incubation, the E2 and E4 mRNA levels in the cells were determined by quantitative RT-PCR. These data (A,B) are expressed as the means ± S.D. (n = 4). (C) C57BL/6 mice were intravenously administered 10⁹ IFU of Adv-CALuc or Adv-CADNIκBα. Forty-eight hours after the administration, the E2, E4, and pIX mRNA levels in the mouse liver were determined by quantitative RT-PCR. The data are expressed as the means ± S.E. (n = 5–6). **p < 0.01, ***p < 0.001.

Figure 8. Suppression of Ad vector-mediated hepatotoxicities by expression of a dominant-negative IκBα gene.

C57BL/6 mice were intravenously administered 10⁹ IFU of Adv-CALuc or Adv-CADNIκBα. At the indicated time points, the serum ALT and AST levels were determined. These data are expressed as the means ± S.E. (n = 5–6). (B,C) Rag2/IL2rgc double knockout mice were intravenously administered 10⁹ IFU of Adv-CALuc or Adv-CADNIκBα. (B) Forty-eight hours after the administration, the E2 and E4 mRNA levels in the mouse liver were determined by quantitative RT-PCR. These data are expressed as the means ± S.E. (n = 4). (C) At the indicated time points, the serum ALT and AST levels were determined. These data (B,C) are expressed as the means ± S.E. (n = 6–8). *p < 0.05, **p < 0.01, ***p < 0.001 (Adv-CALuc versus Adv-CADNIκBα).