title: VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of a VP22-HSVTk-GFP triple fusion construct.
abstract: BACKGROUND
  VP22 is a herpes simplex virus type 1 (HSV-1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22-Tk-GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo.
  
  
  METHODS
  VP22-Tk-GFP was cloned into lenti- and adenoviral vectors and used for expression studies, analyses for VP22-mediated protein spreading, and to study the effect of VP22 to thymidine kinase/ganciclovir-mediated cytotoxicity. The function of VP22-Tk-GFP was also investigated in vivo.
  
  
  RESULTS
  The triple fusion protein was expressed correctly in vitro, but intercellular trafficking was not observed in any of the studied cell lines. However, under certain conditions, VP22-Tk-GFP sensitized cells more efficiently to ganciclovir than Tk-GFP. In vivo there was a trend for increased inhibition of tumor growth with VP22-Tk-GFP when ganciclovir was present, but the difference with Tk-GFP was not statistically significant.
  
  
  CONCLUSIONS
  Based on our results, VP22 fusion proteins do not seem to traffic intercellularly at detectable levels in most tumor cell types. Even though VP22 enhanced cytotoxicity in one cell line in vitro, the effect in vivo was modest. Therefore, our results do not support the utility of VP22 as an enhancer of enzyme prodrug cancer gene therapy.
authors.affiliation: A. I. Virtanen Institute, Department of Biotechnology and Molecular Medicine, University of Kuopio, FIN-70211 Kuopio, Finland.
authors.forename: Tanja
authors.initials: T
authors.lastname: Hakkarainen
authors.forename: Tiina
authors.initials: T
authors.lastname: Wahlfors
authors.forename: Outi
authors.initials: O
authors.lastname: Meriläinen
authors.forename: Sami
authors.initials: S
authors.lastname: Loimas
authors.forename: Akseli
authors.initials: A
authors.lastname: Hemminki
authors.forename: Jarmo
authors.initials: J
authors.lastname: Wahlfors
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D005240:Feasibility Studies; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology*; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000628:therapy*; D011355:Prodrugs / Q000378:metabolism* / Q000494:pharmacology; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics*; D014161:Transduction, Genetic; D019076:Transgenes; D014407:Tumor Cells, Cultured; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir