THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2008; 10: 734–743. Published online 3 April 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1196 Secretion of mouse growth hormone by transduced primary human keratinocytes: prospects for an animal model of cutaneous gene therapy Cibele Nunes Peroni 1 Cl´ audia Regina Cecchi 1 Cristiane Wanderley Rosauro 1 Suely Nonogaki 2 Enrique Boccardo 3 Paolo Bartolini 1 * 1 Biotechnology Department, National Nuclear Energy Commission (IPEN-CNEN), Cidade Universit´ aria, S˜ ao Paulo, Brazil 2 Pathology Division, Immunohistochemistry Laboratory, Adolfo Lutz Institute, S˜ ao Paulo, Brazil 3 Laboratory of Virology, Ludwig Institute for Cancer Research, S˜ ao Paulo, Brazil *Correspondence to: Paolo Bartolini, Biotechnology Department, IPEN-CNEN, Avenida Professor Lineu Prestes 2242, Cidade Universit´ aria, 05508-900, S˜ ao Paulo, SP, Brazil. E-mail: pbartoli@ipen.br Received: 11 June 2007 Revised: 29 February 2008 Accepted: 3 March 2008 Abstract Background Keratinocytes are a very attractive vehicle for ex vivo gene transfer and systemic delivery because proteins secreted by these cells may reach the circulation via a mechanism that mimics the natural process. Methods An efficient retroviral vector (LXSN) encoding the mouse growth hormone gene (mGH) was used to transduce primary human keratinocytes. Organotypic raft cultures were prepared with these genetically modified keratinocytes and were grafted onto immunodeficient dwarf mice (lit/scid). Results Transduced keratinocytes presented a high and stable in vitro secretion level of up to 11 μg mGH/10 6 cells/day. Conventional epidermal sheets made with these genetically modified keratinocytes, however, showed a drop in secretion rates of >80% due to detachment of the epithelium from its substratum. Substitution of conventional grafting methodologies with organotypic raft cultures completely overcame this problem. The stable long-term grafting of such cultures onto lit/scid mice could be followed for more than 4 months, and a significant weight increase over the control group was observed in the first 40 days. Circulating mGH levels revealed a peak of 21 ng/ml just 1 h after grafting but, unfortunately, these levels rapidly fell to baseline values. Conclusions mGH-secreting primary human keratinocytes presented the highest in vitro expression and peak circulatory levels reported to date for a form of GH with this type of cells. Together with previous data showing that excised implants can recover a remarkable fraction of their original in vitro mGH secretion efficiency in culture, the factors that might still hamper the success of this promising model of cutaneous gene therapy are discussed. Copyright  2008 John Wiley & Sons, Ltd. Keywords gene therapy; immunodeficient dwarf mice; mouse growth hormone; organotypic raft cultures; primary human keratinocytes; retroviral vector Introduction Cutaneous gene therapy is a potentially attractive method for correct- ing protein deficiencies because genetically modified human keratinocytes have recently been demonstrated to comprise a powerful tool for suc- cessfully treating severe skin disorders [1,2] and, moreover, are able to produce and secrete gene products with systemic action [3–7]. Compared to other tissues, skin offers the advantage of being easily accessible for Copyright  2008 John Wiley & Sons, Ltd.