A novel oHSV-1 targeting telomerase reverse transcriptase-positive cancer cells via tumor-specific promoters regulating the expression of ICP4

Abstract

Virotherapy is a promising strategy for cancer treatment. Using the human telomerase reverse transcriptase promoter, we developed a novel tumor-selective replication oncolytic HSV-1. Here we showed that oHSV1-hTERT virus was cytopathic in telomerase-positive cancer cell lines but not in telomerase-negative cell lines. In intra-venous injection in mice, oHSV1-hTERT was safer than its parental oHSV1-17+. In human blood cell transduction assays, both viruses transduced few blood cells and the transduction rate for oHSV1-hTERT was even less than that for its parental virus. In vivo, oHSV1-hTERT inhibited growth of tumors and prolong survival in telomerase-positive xenograft tumor models. Therefore, we concluded that this virus may be a safe and effective therapeutic agent for cancer treatment, warranting clinical trials in humans.

Keywords: ICP4; hTERT; oncolytic HSV-1; oncolytic virotherapy; tumor specific.

Figure 1. Replication of oHSV1-hTERT is correlated with telomerase activity

A. Schematic construction of oHSV1-hTERT, oHSV1-GFP and oHSV1-hTERT-GFP/Luc. oHSV1-hTERT was developed from oHSV1-d34.5-d47, referred to as oHSV1-17+. The hTERT core promoter contains two E-box domains and five SP1 binding sites. The modifications included the deletion of the ICP47 and ICP34.5 genes, the replacement of the ICP4 promoter with the hTERT promoter and the insertion of the CMV promoter controlled GFP or luciferase expression cassette at the deleted ICP34.5 sites. B. Expression of TERT in different human cancer cell lines. The expression levels of TERT mRNA were measured using qRT-PCR and were normalized to the corresponding expression level of GAPDH. The bars represent the relative expression levels of mRNA. C. Relationship between TERT mRNA expression and GFP fluorescence intensity in human cancer cell lines. The expression levels of TERT mRNA were normalized to the level of BGC823, and the GFP fluorescence intensity was normalized to the level of A549.

Figure 2. In vitro oncolytic activity comparison of oHSV1-hTERT and oHSV1-17+

A. oHSV1-hTERT and oHSV1-17+ were used to infect human cancer cell lines with high telomerase activity at the indicated MOIs for the indicated times. The human cancer cell lines included BGC823, HOS, Krause, PC-3, HepG2, HuH7, LoVo and Y. B. oHSV1-hTERT and oHSV1-17+ were used to infect the human cancer cell lines lacking telomerase activity at the indicated MOIs for the indicated times. The human cancer cell lines included Saos-2, Wi-38 and M. C. oHSV1-hTERT and oHSV1-17+ were used to infect mouse cancer cell lines at the indicated MOIs for the indicated times. The mouse cancer cell lines included 4T-1 and B16R. There was a significant difference in the oncolytic activity of oHSV1-hTERT and oHSV1-17+ (p = 0.0002, 0.0005, 0.0006, 0.0007 and <0.0001) for the Saos-2, Wi38, M, 4T-1 and B16R cells, respectively. Each value represents the mean ± SED of three independent samples.

Figure 3. Comparison of oHSV1-hTERT and oHSV1-17+ replication

A. oHSV1-17+ and oHSV1-hTERT replication were assessed in the telomerase-negative Saos-2 and Wi-38 cell lines by growth curve analysis at MOI = 0.1. B. oHSV1-17+ and oHSV1-hTERT replication were assessed in the telomerase-positive BGC823 and HuH7 cell lines by growth curve analysis at MOI = 0.1. C. The expression of ICP4 in the telomerase-positive cell lines was measured after oHSV1-hTERT infection at an MOI of 1 using western blot analysis. D. The expression of ICP4 in telomerase-negative cell lines was measured after oHSV1-hTERT or oHSV1-17+ infection at an MOI of 1 using western blot analysis.

Figure 4. oHSV1-hTERT induces necrosis in telomerase-positive cancer cells

A. Flow cytometry analysis of cancer cell lines after oHSV1-hTERT infection at the indicated MOIs. P1, apoptosis; P2, necrosis. B. The necrosis and apoptosis rates of the BGC823 and HuH7 were measured after oHSV1-hTERT infection. C. The necrosis/apoptosis rates were analyzed in both the telomerase-negative and telomerase-positive cell lines. necrosis/apoptosis = P2/P1. D. The necrosis and apoptosis rates of Saos-2 and Wi-38 were measured after oHSV1-hTERT infection. Each value represents the mean ± SED of three independent samples. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. oHSV1-hTERT is tumor specific and safe

A. The rates of GFP-positive cell in WBCs after oHSV1-hTERT-GFP or oHSV1-GFP infection. Each value represents the mean ± SED of three independent samples. ***p < 0.001. B. Acute toxicity test for oHSV1-17+ and oHSV1-hTERT. oHSV1-hTERT or oHSV1-17+ (1 × 10⁶, 1 × 10⁷ or 1 × 10⁸ pfu) was injected intraveinly. The number of mice survied was calculated two weeks after injection. C. Representative BGC823 mouse model for tracer analysis. oHSV1-hTERT-Luc (5 × 10⁶ pfu) was injected into the tumor (i.t.), muscle (i.m.) or subcutaneous tissue (s.c.) of the BGC823 mouse model (n = 3). Luciferase expression was measured using the IVIS Imaging System at the indicated times. D. The fluorescence intensity at different injection sites was measured at the indicated times. The BALB/c nude mice bearing BGC823 tumors were treated with oHSV1-hTERT-Luc.

Figure 6. Therapeutic effect of oHSV1-hTERT in vivo

A. The BGC823 average tumor volume within the different groups was measured every four days following treatments. The data represent the mean ± SEM (n = 6). P = 0.0432 and 0.0354 for oHSV1-hTERT and oHSV1-17+, respectively. B. The HuH7 average tumor volume within the different groups was measured every three days following treatments. The data represent the mean ± SEM (n = 6). P = 0.01 and 0.0086 for oHSV1-hTERT and oHSV1-17+, respectively. C. BGC823 model Kaplan–Meier survival curves (n = 6) for oHSV1-hTERT vs control. Median survival time: 29 days for control, 77 days for oHSV1-17+ and 91 days for oHSV1-hTERT. (P = 0.0004, 0.0005 for oHSV1-17+ and oHSV1-hTERT, respectively; log rank test) D. HuH7 model Kaplan–Meier survival curves (n = 6) for oHSV1-hTERT vs control. Median survival time: 38 days for control, 62 days for oHSV1-17+ and 75 days for oHSV1-hTERT. (P = 0.0082, 0.006 for oHSV1-17+ and oHSV1-hTERT, respectively; log rank test).