title: Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo.
abstract: BACKGROUND
  Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEX GM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV.
  
  
  METHODS
  To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting.
  
  
  RESULTS
  Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective.
  
  
  CONCLUSIONS
  This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials.
authors.affiliation: Oncology Group, Postgraduate Medical School, University of Surrey, Surrey, GU2 5XH, UK.
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Anesti
authors.forename: Guy R
authors.initials: GR
authors.lastname: Simpson
authors.forename: Toby
authors.initials: T
authors.lastname: Price
authors.forename: Hardev S
authors.initials: HS
authors.lastname: Pandha
authors.forename: Robert S
authors.initials: RS
authors.lastname: Coffin
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D020868:Gene Silencing; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage; D018316:Gliosarcoma / Q000235:genetics / Q000628:therapy* / Q000821:virology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006561:Herpes Simplex / Q000235:genetics / Q000628:therapy / Q000821:virology; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D035683:MicroRNAs / Q000502:physiology*; D050130:Oncolytic Virotherapy; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000502:physiology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018139:Simplexvirus / Q000502:physiology*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase