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An Approach to Achieve Long-Term Expression in Skin Gene Therapy

¹ Dermatology Branch, National Cancer Institute, National Institute of Health, Bethesda, Maryland, USA
² Department of Dermatology and Allergology, Philipps-University, Marburg, Germany

Correspondence: Address correspondence to: Jean-Philippe Therrien, PhD, Dermatology Branch, National Cancer Institute, NIH, Bldg. 10/Room 12N254, 10 Center Drive MSC 1908, Bethesda, MD 20892-1908; e-mail: therriej{at}mail.nih.gov.

For gene therapy purposes, the skin is an attractive organ to target for systemic delivery of therapeutic proteins to treat systemic diseases, skin diseases, or skin cancer. To achieve long-term stable expression of a therapeutic gene in keratinocytes (KC), we have developed an approach using a bicistronic retroviral vector expressing the desired therapeutic gene linked to a selectable marker (multidrug resistant gene, MDR) that is then introduced into KC and fibroblasts (FB) to create genetically modified human skin equivalent (HSE). After grafting the HSE onto immunocompromised mice, topical colchicine treatment is used to select and enrich for genetically modified keratinocyte stem cells (KSC) that express MDR and are resistant to colchicine’s antimitotic effects. Both the apparatus for topical colchicine delivery and the colchicine doses have been optimized for application to human skin. This approach can be validated by systemic delivery of therapeutic factors such as erythropoietin and the antihypertensive atrial natriuretic peptide.

Key Words: Human skin • gene therapy • multidrug resistance gene • bicistronic retroviral vectors • systemic delivery • topical selection

Abbreviations: ANP, atrial natriuretic peptide • DNA, deoxyribonucleic acid • FACS, flow activated cell sorting • FB, fibroblast • H&E, hematoxylin and eosin • HSE, human skin equivalent • JEB, junctional epidermolysis bullosa • KC, keratinocyte • KSC, keratinocyte stem cell • MDR, multi-drug resistance gene • MFI, mean fluorescence intensity • TA, transient amplifying cell • TNF-, tumor necrosis factor-alpha

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