Adenoviral gene delivery to primary human cutaneous cells and burn wounds.
The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has potential for clinical application.
Hirsch
Tobias
T
Department for Plastic Surgery, Burn Center, BG University Hospital Bergmannsheil, Ruhr University Bochum, Germany.
von Peter
Sebastian
S
Dubin
Grzegorz
G
Mittler
Dominik
D
Jacobsen
Frank
F
Lehnhardt
Markus
M
Eriksson
Elof
E
Steinau
Hans-Ulrich
HU
Steinstraesser
Lars
L
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002056:Burns / Q000235:genetics* / Q000473:pathology; D002452:Cell Count; D016923:Cell Death; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012867:Skin / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D014162:Transfection / Q000379:methods*; D019076:Transgenes
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins