Secretion of mouse growth hormone by transduced primary human keratinocytes: prospects for an animal model of cutaneous gene therapy.
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.
Peroni
Cibele Nunes
CN
Biotechnology Department, National Nuclear Energy Commission (IPEN), Cidade Universitária, São Paulo, Brazil.
Cecchi
Cláudia Regina
CR
Rosauro
Cristiane Wanderley
CW
Nonogaki
Suely
S
Boccardo
Enrique
E
Bartolini
Paolo
P
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
D013006:Growth Hormone