title: Transgene expression in mononuclear muscle cells not infiltrating inflammatory cells following intramuscular plasmid gene electrotransfer.
abstract: BACKGROUND
  In situ electroporation-assisted intramuscular plasmid DNA delivery offers high efficiency for therapeutic protein replacement. Expression may be impaired by an immune response against the plasmid or transgenic protein. Expression of the transgene in non-muscle cells may increase the immune response. Gene transfer efficiency and phenotypic identification of intramuscular transgene-expressing mononuclear cells was studied following electroporation-mediated plasmid delivery.
  
  
  METHODS
  Plasmids expressing beta-galactosidase (pVR1012-betagal) or enhanced green fluorescent protein (eGFP) (pVR1012-eGFP) were electrotransferred into rat tibialis anterior muscles. Both transfection efficiency and the inflammatory response were determined in pVR1012-betagal-injected muscles by beta-galactosidase and haematoxylin and eosin staining of muscles 7 days post-plasmid injection. Muscles injected with pVR1012-eGFP were stained for CD3, CD68 and desmin at 24 and 48 h post-injection to determine whether mononuclear cells expressing eGFP were of immune or myogenic origin.
  
  
  RESULTS
  With electroporation, beta-galactosidase expression was significantly enhanced by up to ten-fold compared to plasmid injection without electroporation. A large area of regenerating muscle fibres and inflammatory cell infiltration was found in electroporated plasmid-injected muscle. No eGFP expression was found in CD3- or CD68-positive cells. Small mononuclear cells expressing eGFP showed negative staining for CD3 and CD68, but all stained positive for desmin.
  
  
  CONCLUSIONS
  In situ electroporation enhanced transfection efficiency of plasmid DNA delivery into muscle. Alongside its advantage for improving gene transfer, electroporation led to an increased inflammatory response and muscle damage. Mononuclear cells in muscle were transfected with plasmid and expressed the transgene. These cells were of myogenic origin with no evidence of transgene expression in infiltrating inflammatory cells.
authors.affiliation: Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, UK.
authors.forename: Jarupa
authors.initials: J
authors.lastname: Ratanamart
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Huggins
authors.forename: James A M
authors.initials: JA
authors.lastname: Shaw
mesh_terms: D000818:Animals; D003893:Desmin; D018274:Electroporation / Q000379:methods*; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D032342:Muscle Cells / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D019076:Transgenes / Q000235:genetics*; D001616:beta-Galactosidase
chemical_list: D003893:Desmin; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase