title: VP22-mediated intercellular transport correlates with enhanced biological activity of MybEngrailed but not (HSV-I) thymidine kinase fusion proteins in primary vascular cells following non-viral transfection.
abstract: BACKGROUND
  The intercellular transport properties of the herpes simplex virus (HSV) protein VP22 have been harnessed to enhance the effectiveness of viral gene transfer. We investigated the intercellular transport and biological effects of VP22 fused with the dominant negative c-Myb chimera, MybEngrailed (MybEn) and HSV-I thymidine kinase (TK), in primary vascular smooth muscle cells (VSMC) following non-viral transfection.
  
  
  METHODS
  Porcine VSMC transfected with plasmids encoding MybEn, TK and their respective N- and C-terminal VP22 fusion proteins were assayed for the extent and distribution of transgene expression (by immunohistochemistry), culture growth and apoptosis.
  
  
  RESULTS
  The N-terminal MybEn fusion with VP22 (MybEnVP22) and both TK fusions, but not VP22MybEn, exhibited intercellular spread from primary transfected to up to 200 surrounding cells. pMybEnVP22-transfected cultures exhibited growth inhibition and apoptosis rates that were 10.6 +/- 3.6 and 3.2 +/- 1.0 fold higher than in pMybEn-transfected cultures; pVP22MybEn-transfected cultures showed no difference in these parameters. pTK-transfected cultures underwent 60-70% cell death in the presence of ganciclovir despite <2% primary transfection, which was not increased in cultures transfected with plasmids encoding VP22-TK fusions.
  
  
  CONCLUSIONS
  The close correlation between immunocytochemical and biological assays suggests that intercellular transport is crucial to the enhanced biological activity of the MybEnVP22 fusion. The "intrinsic" bystander activity of TK was 4-fold greater than was "engineered" by VP22 fusion, probably reflecting the abundance of gap junctions between VSMC. VP22 fusion may enhance the efficiency of non-viral gene delivery when combined with the appropriate therapeutic transgene, target tissue and transfection method.
authors.affiliation: Cardiovascular Research Unit, Division of Clinical Sciences (North), Clinical Sciences Centre, University of Sheffield, Northern General Hospital, Sheffield S5 7AU, UK. PaulJohn.Sheridan@btinternet.com
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Sheridan
authors.forename: Allan
authors.initials: A
authors.lastname: Lawrie
authors.forename: David C
authors.initials: DC
authors.lastname: Crossman
authors.forename: Cathy M
authors.initials: CM
authors.lastname: Holt
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Newman
mesh_terms: D000818:Animals; D017209:Apoptosis; D001692:Biological Transport / Q000502:physiology; D024201:Bystander Effect; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D020626:Oncogene Proteins v-myb / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018398:Homeodomain Proteins; D020626:Oncogene Proteins v-myb; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D015678:Viral Structural Proteins; C106185:engrailed homeobox proteins; C073413:herpes simplex virus type 1 protein VP22; D013937:Thymidine Kinase