Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

Tobias Hirsch,Sebastian Von Peter,Elof Eriksson,Grzegorz Dubin,Dominik Mittler,Hans-Ulrich Steinau,Lars Steinstraesser,Frank Jacobsen,Markus Lehnhardt

doi:10.2119/2006-00031.hirsch

Abstract

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.

Highlights

Gene therapy has been described as a promising approach for the treatment of skin diseases and impaired wound healing [1]
Determined by FACS cell counting system, results demonstrated an increase in expression with a peak for the HaCaT cell line of 76% ± 1.093% at day 10, followed by a decrease up to the day 30 (Figure 3a)
In this study we demonstrate that adenoviral gene delivery is highly efficient in primary human epithelial cells and in burn wounds

Summary

Introduction

Gene therapy has been described as a promising approach for the treatment of skin diseases and impaired wound healing [1]. Skin in particular has been described as an ideal target for in vivo and ex vivo gene therapy [3]. Skin therapy was beneficial in the local treatment of xeroderma pigmentosum [10], epidermolysis bullosa [11], and ichtthyoses [12]. In addition to these applications in dermatological domains, gene therapy was of use in the treatment of wounds [13] caused by decubital, vascular, and diabetic pathologies [14,15]. In particular the induction of the production of growth factors, such as PDGF-AA and IGF-1 [16], was shown to interact with and improve the molecular processes of wound healing

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