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

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.


Cell culture
The tumor cell lines, including LoVo, PC-3, HepG2, Vero, 4T1, B16, were purchased from ATCC and maintained in our laboratory. Krause, U2OS, Saos-2 and Wi-38 were purchased from the Cell Resource Center (IBMS, CAMS/PUMC). BGC823 cell line was a gift from BGI-Shenzhen (Shenzhen, China). The HuH7 cell line was a gift from Dr. Yuming Guo. The M and Y cell lines were a gift from Professor Kaitai Zhang (Cancer Hospital/Institute, CAMS, Beijing). Both the M and Y cell lines were noncancerous bronchial epithelial cells transformed with the SV40 virus. The Y cell line possesses high telomerase activity, whereas the M cell line lacks telomerase activity, as measured by gene chip. The BHK-ICP4 cell line, which is BHK-21 cells that stably express ICP4, was constructed by our laboratory. And the B16R cell line, which was stably transfected with an HSV receptor, was also constructed by our laboratory.

Plasmid construction
A number of plasmids were constructed to replace the virus's ICP4 promoter with the tumor-specific hTERT promoter. To construct the pICP4del plasmid, the ICP4 upstream (US) and downstream (DS) flanking regions (FLRs) were amplified using the ICP4USf/ ICP4USr (CCCTCCAGACGCACCGGAGTCGGGGG/ AAGTCGACTCTAGAGGATCGATCTCTGACCTGA GATTGGCGGCACTGAGGTA) and ICP4DSf/ICP4DSr primer pairs (AAAAGTCGACCTGCAGGCATGCT AACGAGGAACGGGCAGGGGGC/AAAAAAGCTT GCATGCCCACGTGCGCGGGGCCAGACGGGCT), respectively. The ICP4 US FLR was cut with SalI, and the DS FLR was cut with SalI/HindIII; the two fragments were mixed and ligated to the pSP73 plasmid (Promega, USA) that was digested with SalI and HindIII to create the pICP4del plasmid, which was verified by sequencing. The pICP4del-eGFP plasmid was derived from the pICP4del plasmid by inserting a CMV-GFP cassette into its EcoRV site. The CMV-GFP cassette was cut from the pcDNA3.1-EGFP plasmid (YRGENE, Beijing) with EcoRI and XhoI and was blunt-ended.
To construct pICP4del-hTERTp-ICP4, we first synthesized the hTERT core promoter and inserted it into the pUC57 plasmid to generate the phTERTp plasmid. The hTERT core promoter was cut out from the phTERTp plasmid by NruI and HindIII digestion and was inserted into the pcDNA3-NHN plasmid that was pre-cut with NruI and HindIII to generate the pcDNA3-NHN-hTERTp plasmid. The pCDNA3-NHN plasmid was constructed by inserting the selfcomplement linker (GCTAGCGTTAACGCTAGC) into pcDNA3 that was pre-cut with PvuII. The ICP4 gene was amplified in three overlapping fragments: ICP4 1st , ICP4 2nd and ICP4 3rd . These fragments were separately cloned into pSP73 to give rise to pSP73-ICP4 1st , pSP73-ICP4 2nd and pSP73-ICP4 3rd , which were verified by sequencing. The primers used to amplify the ICP4 fragments are listed in Supplementary Table S1. Then, the three ICP4 fragments were cut out of their respective plasmids using the EcoRI and BsrGI, BsrGI and PvuI and PvuI and XhoI restriction enzymes, respectively. These fragments were mixed and ligated to the pcDNA3-NHN plasmid that was digested with EcoRI and XhoI to generate pcDNA3-NHN-hTERTp-ICP4. Finally, the hTERT-ICP4 fragment was digested from pcDNA3-NHN-hTERTp-ICP4 using PmeI and HpaI and was inserted into pICP4del to generate pICP4del-hTERTp-ICP4. Additionally, to make an ICP4 complementary cell line, the pcDNA3-CMV-ICP4 plasmid was created using the following steps. The ICP4 gene was released by EcoRI and XhoI digestion from pcDNA3-NHN-hTERTp-ICP4 and was ligated into the pcDNA3 plasmid that was digested with EcoRI and XhoI to generate pcDNA3-CMV-ICP4.
Two other shuttle plasmids were constructed to insert the eGFP and luciferase genes into the ICP34.5 site of the virus genome. The pdICP34.5 plasmid was previously described (26), whereas the pGL4.17-CMVp-Luc2/Neo plasmid was kindly provided by Dr. Chen Ling (Cancer Hospital/Institute, CAMS, Beijing). The GFP and luciferase expression cassettes from pcDNA3.1-EGFP and pGL4.17_CMVp_Luc2/Neo were removed through EcoRV and BamHI digestion and were blunt ended with T4 DNA polymerase; these cassettes were then cloned into the AfeI site of pdICP34.5 to generate pd34.5-CMVp-eGFP and pd34.5-CMVp-Luc2, respectively. These two plasmids were then verified by sequencing.
The oHSV1-17+ stocks were prepared by infecting Vero cells and the oHSV1-hTERT based vectors were prepared by infecting BHK-ICP4 cells with 0.01 plaqueforming units (pfu)/cell. BHK-ICP4 is a complementary cell line for ICP4-deficient viruses. This cell line was made by transfecting the BHK-21 cells with the pcDNA3-CMV-ICP4 plasmid. The viruses were harvested 72 hours after infection, were freeze/thawed once and were purified by centrifugation at 3,000 rpm to remove cell debris, followed by a high speed centrifugation at 12,000 rpm to pellet the virus. The purified viruses were dissolved in SFM, titrated, divided into aliquots, and stored at −80°C until use.

CCK8 cell viability assay
The cells were seeded at a concentration range from 2 × 10 4 to 2 × 10 5 cells/ml and were seeded in 96-well plates at 100 μL per well. Each sample was run in triplicate. To test the effect of multiplicity of infection (MOI) on cell viability, every cell line was infected 24 h later with oHSV1-hTERT or oHSV1-17+ at MOIs of 0.1, 1 and 5. After culture for 24, 48 and 72 h, the culture medium was removed from the plates, and 100 μl of a mixture containing 10% CCK8 was added and incubated for an additional 4 h. The plates were examined using a model 550 microplate reader (BIO-RAD, Japan) at 450 nm with a reference of 655 nm.

DNA ladder
The cells and culture were harvested and lysed in the buffer. After Proteinase K treatment, DNA was gathered and washed by 75% ethanol. And the DNA ladder was analysed by 2% agarose gel electrophoresis.