title: Secretion of mouse growth hormone by transduced primary human keratinocytes: prospects for an animal model of cutaneous gene therapy.
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 microg 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.
authors.affiliation: Biotechnology Department, National Nuclear Energy Commission (IPEN), Cidade Universitária, São Paulo, Brazil.
authors.forename: Cibele Nunes
authors.initials: CN
authors.lastname: Peroni
authors.forename: Cláudia Regina
authors.initials: CR
authors.lastname: Cecchi
authors.forename: Cristiane Wanderley
authors.initials: CW
authors.lastname: Rosauro
authors.forename: Suely
authors.initials: S
authors.lastname: Nonogaki
authors.forename: Enrique
authors.initials: E
authors.lastname: Boccardo
authors.forename: Paolo
authors.initials: P
authors.lastname: Bartolini
mesh_terms: D000818:Animals; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D023421:Models, Animal; D012190:Retroviridae
chemical_list: D013006:Growth Hormone