Gene Therapy (2000) 7, 1640–1647  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt ACQUIRED DISEASES RESEARCH ARTICLE Transcriptional targeting of adenoviral gene delivery into migrating wound keratinocytes using FiRE, a growth factor-inducible regulatory element P Jaakkola 1,2 , M Ahonen 1,3 , V-M Ka ¨ha ¨ri 1,3 and M Jalkanen 1 1 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University; and 3 Departments of Medical Biochemistry, and Dermatology, University of Turku, Turku, Finland Impaired cutaneous wound healing is a common compli- cation in diabetes, ischemia and venous insufficiency of lower extremities, and in long-term treatment with cortico- steroids or other immunosuppressive agents. In develop- ment of gene therapy for wound repair, expression of thera- peutic transgenes should be precisely targeted and controlled. Here, we describe a recombinant adenovirus RAdFiRE-EGFP, in which a growth factor inducible element (FiRE) of the murine syndecan-1 gene controls the expression of enhanced green fluorescent protein (EGFP) reporter gene. Treatment of RAdFiRE-EGFP-transduced murine epidermal keratinocytes in culture with FiRE-activat- ing growth factor markedly enhanced the expression of EGFP. In ex vivo organ culture of wounded murine skin transduced with RAdFiRE-EGFP, the EGFP expression was Keywords: adenovirus; EGF; FiRE; syndecan-1; transcription; wound repair Introduction Wound healing is a dynamic process, which includes inflammation, tissue formation, and tissue remodeling. 1 Impaired cutaneous wound healing is an important medical problem and a common complication, eg in dia- betes, ischemia and venous insufficiency of lower extremities, as well as in long-term treatment with corticosteroids or other immunosuppressive agents. Chronic dermal ulcers are characterized by impaired re- epithelialization and granulation tissue formation, as well as insufficient neovascularization. 1 Recently, the possi- bility of correcting these defects in wound repair by intro- ducing polypeptide growth factors or cytokines to chronic wounds has been under intensive investigation. Accordingly, topical application of epidermal growth fac- tor (EGF), fibroblast growth factors (FGFs) and platelet- derived growth factor (PDGF) have been shown to stimu- late wound healing. 1–5 In addition, the feasibility of other therapeutic proteins, such as inducible nitric oxide Correspondence: V-M Ka ¨ha ¨ri, Turku Centre for Biotechnology, Univer- sity of Turku, Tykisto ¨katu 6B, FIN-20520 Turku, Finland 2 Current address: Wellcome Trust Centre, University of Oxford, Roose- velt Drive, Headington, Oxford OX3 7BN, UK Received 28 February 2000; accepted 5 July 2000 specifically detected in wound margin keratinocytes, but not in intact skin. Activity of EGFP was first detected 2 days after a single application of RAdFiRE-EGFP and persisted up to 10 days. Similarly, FiRE-driven EGFP expression was detected specifically in epidermal keratinocytes in the edge of incisional wounds in murine skin transduced with RAd- FiRE-EGFP. In contrast, adenovirus-mediated lacZ expression driven by CMV promoter was detected scattered in epidermal, dermal and subcutaneous layers in ex vivo and in vivo wounds, as well as in intact skin. These data demon- strate the feasibility of FiRE as a tool for transcriptional tar- geting of adenovirus-mediated transgene expression to cutaneous wound edge keratinocytes. Gene Therapy (2000) 7, 1640–1647. synthase (iNOS) in therapy of chronic ulcers has been suggested. 6 Although easily performed, topical administration of therapeutic polypeptides to wounds has several disad- vantages. First, the effect of topically applied growth fac- tors can not be targeted to specific cells in the wounds and their effects are therefore difficult to control. In addition, production of recombinant growth factors is expensive, and due to their instability in the wound environment they should be administered frequently to obtain a sufficient effect. It is expected that controlled local production of therapeutic polypeptides in the wound as a result of effective gene delivery, would over- come these problems. The feasibility of viral gene deliv- ery using adenovirus, herpes simplex virus and retro- virus vectors, as well as nonviral gene transfer by naked DNA injection, liposomes or particle-mediated gene delivery to skin cells either in vivo or ex vivo has been demonstrated. 7–11 One main goal in development of gene therapy aimed at improving cutaneous wound repair is targeting of therapeutic genes to a defined cell population in the wound to avoid undesired effects of growth fac- tors. This could be achieved by transcriptional targeting of transgene expression to a specific cell population using cell specific regulatory elements, as has been demon- strated in gene delivery to melanoma, liver, mammary gland, and smooth muscle cells. 12–15