Boosting epidermal growth factor receptor expression by gene gun transfection stimulates epidermal growth in vivo

Expression constructs encoding a full-length cDNA encoding the human epider mal growth factor receptor, or reporter gene for green fluorescent protein or luciferase were coated onto gold particles and driven into porcine skin using a gene gun delivery system. Strategies for epidermal growth factor re ceptor boosting were tested in two types of wounds. For grafted wounds, int act porcine skin was pretreated by the introduction of the epidermal growth factor receptor expression construct 24 hours before its harvesting as a s plit-thickness skin graft. Partial-thickness excisional wound beds (donor s ites) were transfected at the time of their creation. Wound healing paramet ers were subsequently tested in the presence or absence of excess epidermal growth factor ligand. Initial distributions of gene gun delivered gold par ticles as well as luciferase expression levels suggested that optimal skin penetrations and expression levels were achieved at 500 psi for intact epid ermis and 300 psi for exposed wound beds. At 2 days after gene delivery, vi sualization of green fluorescent protein by fluorescence microscopy showed focal expression of green fluorescent protein at the advancing epithelial o utgrowths found at wound edges or surviving epithelial remnants. Green fluo rescent protein expression appeared transient since no green fluorescent pr otein was noted in specimens removed at 4 days after injury. Northern blot analysis on mRNA isolated from wounds 2 days after introduction of epiderma l growth factor receptor coated gold particles by gene gun confirmed the ex pression of the human epidermal growth factor receptor transgene in both sk in grafts and excisional wounds. Skin grafts showed subsequent biological r esponses to the introduction of excessive epidermal growth factor receptor as well as expression of the human epidermal growth factor receptor constru ct within healing epidermis. While control autografts (reporter gene treate d, epidermal growth factor alone, placebo formula, no treatment) showed few 5'-bromodeoxyuridine-labeled cells, epidermal growth factor receptor autog rafts showed 5'-bromodeoxyuridine labeling of nearly every basal cell. Favo rable wound healing outcomes were also shown within excisional wounds follo wing in vivo boosting of epidermal growth factor receptor. Four days after receiving epidermal growth factor receptor particle bombardment, resurfacin g was significantly accelerated in those wounds receiving epidermal growth factor receptor transgene. Application of topical epidermal growth factor l igand resulted in the highest percentage of resurfacing. Maximal re-epithel ialization was noted in wound beds receiving both receptor boosting and exc essive daily epidermal growth factor ligand. A modest increase in the thick ness of the granulation tissue followed gene therapy with epidermal growth factor receptor. In summary these in vivo data suggest that it is possible to boost in vivo expression of a tyrosine kinase receptor during wound repa ir, increased epidermal growth factor receptor expression has an integral i mpact on cell proliferation, rates of resurfacing and dermal components and merits consideration as a possible therapeutic agent.