PHENOTYPIC AND IMMUNOLOGICAL FACTORS AFFECTING PLASMID-MEDIATED IN-VIVO GENE-TRANSFER TO RAT DIAPHRAGM

Little is known about the molecular mechanisms governing adaptive resp onses of the diaphragm in the setting of lung disease. By permitting t he study of regulatory elements and the effects of overexpressing gene s of interest, direct in vivo gene transfer to the diaphragm could be used as a tool to address such questions. Therefore, we evaluated para meters affecting transfection efficiency and duration of foreign gene expression in the diaphragm after plasmid-mediated gene transfer. Repo rter gene constructs were injected into adult rat diaphragm and hindli mb muscles. Transfection efficiency at 8-10 days postinjection was dec reased in large caliber (>1,000 mu m(2)) and type II myosin heavy chai n (MHC)-expressing fibers. There were also strong trends toward augmen ted transfection efficiency in type I MHC- and embryonic MHC-expressin g fibers. All diaphragms demonstrated evidence of muscle injury and in flammatory cell infiltrates at this early time point. By 30 days posti njection, however, neither inflammation nor reporter gene expression w as detectable in diaphragm or hind-limb muscles of immunocompetent ani mals. By contrast, immunosuppressed rats (given cyclosporine; 15 mg . kg(-1). day(-1)) showed high levels of foreign gene expression at 30 d ays postinjection, which remained stable up to 60 days. Therefore, exp loitation of plasmid-mediated in vivo gene transfer as a tool for stud ying regulated gene expression in the diaphragm may be facilitated by the use of immunodeficient animal models.