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.
Pfützner
Wolfgang
W
Dermatologische Klinik, Universität München, Frauenlobstrasse 9-11, 80337 Munich, Germany. Wolfgang_Pfutzner@web.de
Joari
Mohammed R
MR
Foster
Ruth-Ann
RA
Vogel
Jonathan C
JC
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