A novel oncolytic adenovirus based on simian adenovirus serotype 24

Abstract

Among the oncolytic virotherapy, an emerging treatment for tumor, adenoviruses are widely used at present in preclinical and clinical trials. Traditionally, oncolytic adenoviruses were developed based on the human adenovirus serotype 5 (AdHu5). However, AdHu5 has the drawbacks of preexisting anti-AdHu5 immunity in most populations, and extensive sequestration of Adhu5 by the liver through hexon, blood coagulation factor X (FX), and FX receptor interactions. To tackle these problems, we explored a novel oncolytic adenovirus AdC7-SP/E1A-ΔE3 for cancer treatment. AdC7-SP/E1A-ΔE3 was constructed by replacing the E1A promoter with tumor specific promoter survivin promoter and deleting E3 region using direct cloning methods based on simian adenovirus serotype 24 (namely AdC7). We showed that AdC7-SP/E1A-ΔE3 significantly killed tumor cell lines NCI-H508 and Huh7, and inhibited tumor growth in both NCI-H508 and Huh7 xenograft tumor models. Importantly, AdC7-SP/E1A-ΔE3 exhibited the antitumor efficacy via systemic administration. Mechanistically, infected cells were killed by AdC7-SP/E1A-ΔE3 via the p53-independent mitochondrial apoptosis pathway in which phosphorylation of BAD markedly declined and the expresses of Bik significantly went up. Therefore, AdC7-SP/E1A-ΔE3 is a promising candidate for liver and colon tumor treatment.

Keywords: AdC7; chimpanzee adenoviruses; oncolytic adenoviruses; p53-independent mitochondrial apoptosis; tumor treatment.

Figure 1. Expression of survivin and E1A genes in different cell types

(A) Western blotting was performed to detect survivin proteins in 1 × 10⁶ cells. (B–F) A panel of NCI-H508 (B), Huh7 (C), A549 (D), SiHa (E), and MRC-5 (F) cells was infected with AdC7-ΔE1-ΔE3, AdC7-ΔE3, and AdC7-SP/E1A-ΔE3 at 10 MOI. E1A mRNA was quantitated by real-time PCR at 24 h post infection. Relative levels of E1A mRNA were shown in reference to GAPDH expression; relative E1 mRNAs in cells infected with AdC7-ΔE1-ΔE3 were defined as 1. Data shown are means ± standard errors of the means (SEM); statistical significance was determined by one-way ANOVA: ***P < 0.0001. Each experiment was performed three times.

Figure 2. Replication of AdC7-ΔE1-ΔE3 in a panel of cells

(A–E) A panel of NCI-H508 (A), Huh7 (B), A549 (C), SiHa (D), and MRC-5 (E) cells was infected with AdC7-ΔE1-ΔE3, AdC7-ΔE3, and AdC7-SP/E1A-ΔE3 at 10 MOI, and sampled at different times post-infection. Relative viral genome copies were determined through real-time PCR. GAPDH was used as the reference and viral copies was defined as 1 in cells infected with AdC7-ΔE1-ΔE3. Data shown are means ± standard errors of the means (SEM); statistical significance was determined by one way ANOVA: ***P < 0.0001. Each experiment was performed three times. (F, G) A panel of NCI-H508, Huh7, A549, SiHa, and MRC-5 cells was infected with AdC7-ΔE1-ΔE3, AdC7-ΔE3, and AdC7-SP/E1A-ΔE3 at 10 MOI, and was twice collected and washed with PBS, at 24 h (F) and 48 h (G) post-infection. After three freeze-thaw cycles, progeny virus was quantitated with the TCID₅₀ assay. Data shown are mean of two independent experiments.

Figure 3. AdC7-ΔE1-ΔE3 induced cytotoxicity of tumor cells

A panel of NCI-H508, Huh7, A549, SiHa, and MRC-5 cells was infected with AdC7-ΔE1-ΔE3, AdC7-ΔE3, and AdC7-SP/E1A-ΔE3 at various MOIs. At 5 d post infection (A–E), cells were stained with crystal violet. Graph represents one of three different experiments. (F–K) Infected cells were subject to MTT assay kat 5 d post infection. Data are presented as means ± SEM of triplicate samples, and are representative of three independent experiments.

Figure 4. AdC7-ΔE1-ΔE3 trigger tumor cell apoptosis

(A) NCI-H508 and Huh7 cells were infected at 100 MOI with AdC7-ΔE1-ΔE3, AdC7-ΔE3, and AdC7-SP/E1A-ΔE3. Two cells were collected 24 h and 48 h post infection respectively. Flow cytometry was performed to evaluate tumor cell apoptosis after staining NCI-H508 and Huh7 with propidium iodide (PI)/annexin V. Early apoptosis: AV⁺/PI⁻ (%); later stage apoptosis: AV⁺/PI⁺ (%). Data are presented as means ± SEM of triplicate samples, and are representative of three independent experiments. Statistical significance was determined by one way ANOVA: ***P < 0.0001, **< 0.001, *< 0.05. (B) NCI-H508 and Huh7 cells were infected at 100 MOI with AdC7-ΔE1-ΔE3, AdC7-ΔE3, and AdC7-SP/E1A-ΔE3. NCI-H508 cells were collected at 12 h and 24 h post infection, and Huh7 cells were collected 24 h and 48 h post infection. Western blotting was carried out to detect levels of p62, LC3, cleaved caspase3, and cleaved PARP in NCI-H508 and Huh7 cells. β-actin was used as a loading control.

Figure 5. Tumor cell apoptosis is induced by AdC7-SP/E1A-ΔE3 via a p53-independent mitochondrial pathway

NCI-H508 and Huh7 cells were infected at 100 MOI with AdC7-ΔE1-ΔE3, AdC7-ΔE3, and AdC7-SP/E1A-ΔE3. NCI-H508 cells were collected at 12 h and 24 h post infection, and Huh7 cells were collected 24 h and 48 h post-infection. Western blotting was carried out to detect levels of p53, phosphorylation of p53, MCL-1, Bcl-2, Bcl-xl, Bik and phosphorylation of Bad in NCI-H508 and Huh7 cells. β-actin was used as a loading control.

Figure 6. AdC7-SP/E1A-ΔE3 inhibit tumor growth in nude mouse NCI-H508 cell xenografts

NCI-H508 cells (10⁷ ) were inoculated into the right flanks of nude mice, and tumor was injected with 5 × 10⁸ PFU adenovirus (AdC7-ΔE1-ΔE3, AdC7-ΔE3, or AdC7-SP/E1A-ΔE3) suspended in 50 μL of PBS or 50 μL PBS alone when reaching 100–150 mm³. (A) Tumor volume (n = 6) was measured every three days. (B) In left images, TUNEL staining of tumor cells, and in right images, TUNEL positive cells (n = 5) were quantitated. Data are presented as means ± SEM. Statistical significance was determined by one way ANOVA: ***< 0.0001, **< 0.001. Data are representative of two independent experiments.

Figure 7. AdC7-SP/E1A-ΔE3 inhibit tumor growth in nude mouse Huh7 cell xenografts

Huh7 cells (5 × 10⁶) were inoculated into the right flanks of nude mice, and tumor was injected with 5 × 10⁸ PFU adenovirus (AdC7-ΔE1-ΔE3, AdC7-ΔE3, or AdC7-SP/E1A-ΔE3) suspended in 50 μL of PBS or 50 μL PBS alone when reaching 100-150 mm³. (A) Tumor volume (n = 6) was measured every two days. (B) In left images, TUNEL staining of tumor cells, and in right images, TUNEL positive cells (n = 5) were quantitated. Data are presented as means ± SEM. Statistical significance was determined by one way ANOVA: ***< 0.0001, **< 0.001. Data are representative of two independent experiments.

Figure 8. The antitumor efficacy of AdC7-SP/E1A-ΔE3 via systemic administration

Huh7 cells (5 × 10⁶) were inoculated into the right flanks of nude mice, and tumor was injected with 1 × 10⁹ PFU adenovirus (AdC7-ΔE1-ΔE3 or AdC7-SP/E1A-ΔE3) suspended in 50 μL of PBS three times at the interval of one day when reaching 100–150 mm³. (A) Tumor volume (n = 6) was measured every two days. (B) In left images, TUNEL staining of tumor cells, and in right images, TUNEL positive cells (n = 5) were quantitated. Data are presented as means ± SEM. Statistical significance was determined by student's t test: ***< 0.0001, *< 0.05. Data are representative of two independent experiments.

Figure 9. Flowchart of cloning AdC7-ΔE3 and AdC7-SP/E1A-ΔE3

(A, B) Construction of AdC7-ΔE3 and AdC7-SP/E1A-ΔE3. A fragment containing the E1 region was cut from pshoncoE1 and pshoncoSPE1, and inserted into the ponco3 region between I-Ceu / and PI-Sce /. See Methods for more detail.