title: Construction of a novel oncolytic adenoviral vector and its biological             characteristics.
abstract: In this study, we aimed to construct an effective and safe oncolytic adenoviral vector for cancer treatment with gene therapy. First, the promoter of the catalytic subunit of human telomerase (hTERTp), adenovirus early region 1a gene (E1A) and thymidine kinase gene of human herpes virus type 1 (HSV-1-TK) were amplified by using PCR from genomic DNA of 293A cells and wild-type HSV-1 (wHSV-1). These specially-prepared elements were inserted into an adenoviral shuttle vector in the opposite and the same directions of left inverted terminal repeat (L-ITR), respectively, to construct pENTR-E1A-IRES-TK-hTERTp (pEITH) and pENTR-hTERTp-E1A-IRES-TK (pHEIT). LR reaction between adenoviral shuttle vectors (pEITH and pHEIT) and the backbone vector DEST was carried out to establish adenoviral expression vectors pAd-E1A-IRES-TK-hTERTp (pAd-EITH) and pAd-hTERTp-E1A-IRES-TK (pAd-HEIT). Recombinant adenovirus Ad-EITH and Ad-HEIT were produced by transfecting 293A cells and purified for the subsequent studies of titer measurement, replication capability with and without acyclovir (ACV) and antitumor ability with and without ganciclovir (GCV) to evaluate the biological characteristics. Adenoviral shuttle vectors pEITH and pHEIT and expression vectors pAd-EITH and pAd-HEIT were successfully constructed, and recombinant adenoviruses Ad-EITH and Ad-HEIT with high titer were produced. The results of replication and cytotoxicity assays showed that Ad-EITH and Ad-HEIT replicated in the hTERTp (+) human nasopharyngeal carcinoma cell line CNE and expressed the TK gene effectively leading to the death of tumor cells. In addition, there were still some Ad-HEIT particles replicating in the hTERTp (-) human osteosarcoma U-2OS cells and human lung HFL-1 fibroblasts compared to Ad-EITH which was hardly able to replicate in U-2OS and HFL-1 cells. In addition, we also observed an interesting phenomenon, that the replication of Ad-EITH could be inhibited by antiviral drug ACV on account of the expression of HSV-1-TK gene making Ad-EITH sensitive to ACV. In conclusion, a novel oncolytic adenoviral vector Ad-EITH was produced which can be used for cancer-specific and efficient viral replication, and its safety is potentially improved as replication can be inhibited by ACV in vitro.
authors.affiliation: Oncology Department of the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, PR China. mingzhi_zhang1@163.com
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Zhang
authors.forename: Xudong
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Han
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Yuqiao
authors.initials: Y
authors.lastname: Sheng
authors.forename: Jianguo
authors.initials: J
authors.lastname: Wen
mesh_terms: D000212:Acyclovir / Q000494:pharmacology; D000256:Adenoviridae / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D017500:Adenovirus E1A Proteins / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology; D001859:Bone Neoplasms / Q000628:therapy*; D002277:Carcinoma / Q000628:therapy*; D045744:Cell Line, Tumor; D005347:Fibroblasts; D015774:Ganciclovir / Q000494:pharmacology; D005784:Gene Amplification; D015870:Gene Expression / Q000187:drug effects*; D015316:Genetic Therapy; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics; D006801:Humans; D008168:Lung; D009303:Nasopharyngeal Neoplasms / Q000628:therapy*; D012516:Osteosarcoma / Q000628:therapy*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D019098:Telomerase / Q000235:genetics; D020079:Terminal Repeat Sequences; D013937:Thymidine Kinase / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
chemical_list: D017500:Adenovirus E1A Proteins; D000998:Antiviral Agents; D011994:Recombinant Proteins; D013937:Thymidine Kinase; D019098:Telomerase; D015774:Ganciclovir; D000212:Acyclovir