title: Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.
abstract: In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India ; Centre for Nanotechnology and Advanced Biomaterials, Shanmugha Arts, Science, Technology and Research Academy University, Tanjore, India.
authors.forename: Ravikanth
authors.initials: R
authors.lastname: Danda
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India ; Centre for Nanotechnology and Advanced Biomaterials, Shanmugha Arts, Science, Technology and Research Academy University, Tanjore, India.
authors.forename: Gopinath
authors.initials: G
authors.lastname: Krishnan
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Kalaivani
authors.initials: K
authors.lastname: Ganapathy
authors.affiliation: Centre for Nanotechnology and Advanced Biomaterials, Shanmugha Arts, Science, Technology and Research Academy University, Tanjore, India.
authors.forename: Uma Maheswari
authors.initials: UM
authors.lastname: Krishnan
authors.affiliation: Departments of Ocular Oncology and Vitreoretina, Medical Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Khetan
authors.initials: K
authors.lastname: Vikas
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Sailaja
authors.initials: S
authors.lastname: Elchuri
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Nivedita
authors.initials: N
authors.lastname: Chatterjee
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Krishnakumar
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy; D015815:Cell Adhesion Molecules / Q000235:genetics; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D063928:Ependymoglial Cells / Q000378:metabolism; D000071858:Epithelial Cell Adhesion Molecule; D005134:Eye Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012334:RNA, Neoplasm / Q000235:genetics; D012175:Retinoblastoma / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014018:Tissue Distribution
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D035683:MicroRNAs; D012334:RNA, Neoplasm; C487931:mirnlet7 microRNA, human; D013937:Thymidine Kinase; D015774:Ganciclovir