title: In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumor cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo.
abstract: BACKGROUND
  Hybrid adeno-retroviral vector systems utilize the high efficiency of adenovirus transduction to direct the in situ production of retroviral progeny. In this study, we show that a single-step transduction of glioma cells with trans-complementing hybrid adeno-retroviral vectors effectively turns these cells into retrovirus vector-producing cells, which in turn facilitates the transduction of adjacent cells.
  
  
  METHODS
  We have adapted the adeno-retroviral hybrid viral vector system to enhance the ganciclovir (GCV) killing of glioma cells following transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene. To assess the effect of the in situ production of retroviral vectors on the transduction efficiency of glioma cells, 9L cells were transduced with adeno-retroviral hybrid vectors that separately express a retroviral genome (AVC2.GCEGFP or AVC2.GCTK) and retroviral packaging proteins (AxTetGP and AxTetVSVG). The generation of an integrated HSV-tk provirus by trans-complementation of the adeno-retroviral vectors was verified by analysis of the flanking retroviral LTR sequences. Tumors established on nu/nu mice were injected with the viruses followed by intraperitoneal injections of either PBS or GCV. We also estimated the copy numbers of the HSV-tk transgene present in the tumors of the treated mice. To determine the expression pattern of the HSV-tk transcripts within a tumor, in situ hybridization analysis was performed using an RNA probe specific for HSV-tk.
  
  
  RESULTS
  The co-transduction of rat 9L glioma cells with AVC2.GCEGFP together with vectors expressing packaging proteins of retroviruses increased the transduction efficiency. Transduction with AVC2.GCTK together with packaging vectors increased the in vitro sensitivity of cells to the pro-drug GCV by one log compared with control cells that were incapable of generating retrovirus. In vivo, the injection of established subcutaneous 9L tumors on athymic mice with a combination of AVC2.GCTK and packaging vectors followed by GCV treatment resulted in complete tumor regression in 50% of tumors at day 22, while no tumor regression was observed in control animals. Retroviral sequences diagnostic of 3' LTR reduplication in vivo were detected in genomic DNA extracted from the transduced tumors, indicating pro-viral integration of the retroviral genome derived from the adeno-retroviral hybrid vector. Furthermore, the relative copy number of the HSV-tk gene in tumors treated with the adeno-retroviral vectors was up to approximately 250-fold higher than in control tumors. In situ hybridization suggested dispersion of the HSV-tk product across a wider area of the tumor than in control tumors, which indicates the spread of the in situ generated retroviruses.
  
  
  CONCLUSIONS
  Although the efficacy of this system has to be evaluated in orthotopic models, our observations suggest that this hybrid adeno-retroviral vector system could improve the suicide gene therapy of tumors.
authors.affiliation: Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan. tokada@jichi.ac.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Okada
authors.forename: Natasha J
authors.initials: NJ
authors.lastname: Caplen
authors.forename: W Jay
authors.initials: WJ
authors.lastname: Ramsey
authors.forename: Masafumi
authors.initials: M
authors.lastname: Onodera
authors.forename: Kuniko
authors.initials: K
authors.lastname: Shimazaki
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Nomoto
authors.forename: Rahim
authors.initials: R
authors.lastname: Ajalli
authors.forename: Oliver
authors.initials: O
authors.lastname: Wildner
authors.forename: John
authors.initials: J
authors.lastname: Morris
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kume
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Hamada
authors.forename: R Michael
authors.initials: RM
authors.lastname: Blaese
authors.forename: Keiya
authors.initials: K
authors.lastname: Ozawa
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology; D018628:Gene Dosage; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D005910:Glioma / Q000628:therapy*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014161:Transduction, Genetic; D016662:Virus Integration / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D013937:Thymidine Kinase; D015774:Ganciclovir