A large preclinical animal model to assess ex vivo skin gene therapy applications.
Because of its easy accessibility, the skin is a very attractive target for gene therapy purposes. To study potential clinical applications in a preclinical setting, appropriate animal models are needed. Pig skin is very similar to human skin, and a variety of human diseases that are potentially amenable to gene therapy applications also occur in pigs. Only a few studies have analyzed the engraftment of transduced keratinocytes (KC) in pigs, however, with limited success. We describe a porcine model in which pig KC were transduced ex vivo with a retroviral vector encoding a marker gene and subsequently grafted onto the autologous host, utilizing a relatively simple grafting technique. Enhanced transduction efficiency was achieved by an optimized transduction protocol including centrifugation of the retroviral vector at a temperature of 32 degrees C. Transduced KC were then seeded onto acellular dermis, forming a stratified epidermis. Grafting was performed by creating full thickness wounds and placing the skin graft onto the muscle fascia, covered by a protective skin flap for several days. Successful engraftment of transduced KC was demonstrated by immunohistochemistry of biopsies taken at different time points, showing transgene expression in 40-50% of grafted KC. After 4 weeks, KC expressing a foreign marker gene was lost, suggesting a transgene-specific immune response in the immunocompetent pigs and highlighting the potential problems for clinical gene therapy studies when transferring new genetic material into a patient. The model presented here may be used to examine applications of skin gene therapy, where retroviral vectors encoding endogenous pig genes will be expressed in the skin.
Dermatologische Klinik, Universität München, Frauenlobstrasse 9-11, 80337 Munich, Germany. Wolfgang_Pfutzner@web.de
Wolfgang
W
Pfützner
Mohammed R
MR
Joari
Ruth-Ann
RA
Foster
Jonathan C
JC
Vogel
D000818:Animals; D002478:Cells, Cultured; D062109:Dermatologic Surgical Procedures; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D015603:Keratinocytes / Q000637:transplantation; D023421:Models, Animal; D012190:Retroviridae / Q000235:genetics; D016038:Skin Transplantation / Q000379:methods*; D013552:Swine; D013556:Swine, Miniature; D014161:Transduction, Genetic