CRISTIANE WANDERLEY ROSAURO
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Resumo IPEN-doc 08538 High-level expression of mouse growth hormone (mGH) by primary human keratinocytes using a retroviral vector2002 - ROSAURO, C.W.; PERONI, C.N.; POIATO, E.E.; SOUSA, J.M.; BARTOLINI, P.The skin is considered a potential target for the development of gene therapy protocols for cutaneous or systemic diseases. The high proliferative capacity of cultured keratinocytes makes these epidermal colls idoal candidates for genetie manipulation. We have utilized primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding a mouse growth hormone gene (mGH). The main goals are the conetruction of an homologous ox vivo transfer protocol and the comparison with a similar however heterologous system based on the human growth hormone (NGH) gene. After transfection of the vector LmGHSN into GP + E-86 ecotropic packaging cell lins, tho exprassion of mGH (10ng/mL) was detected in the culture medium by a specific radioimmonuassay. This supernatant was then used to infect the amphotropic packaging cell lins GP + env Am12 and 36 clones were isolated by G418 selection. Clones presenting a secretion lovel ranging from 22-50ng mGH/10(sup)6 cells.day (44 to 150ng mGH/mL) and a viral titer of 10(sup)5 - 10(sup)6 CFU/mL were chosen to infect the keratinocytes. The modified keratinocytes presented a stable in vitro secretion level up to 3ug mGH/10(sup)6cells.day (approximately 4ug mGH/mL), during serial propagation. These results are analogous to those previously obtained for the HGH model, which was already utilized for grafting into immunodeficient dwarf mice (little/scid), Halt-ifo dotorminations for hGH and mGH are now being carried out trying to verify a possible difference of plasma clearance rate in mice, in order to correlats these data with the lvels of GH socreted in vivo by the gonotically modified keratinocytes.Resumo IPEN-doc 07732 Retrovirus-mediated transfer of growth hormone gene into primary human keratinocytes produces measurable circulatory levels of the hormone in immunodeficient-dwarf mice2001 - BELLINI, M.H.; ROSAURO, C.W.; PERONI, C.N.; ARKATEN, R.R.; BARTOLINI, P.Artigo IPEN-doc 13178 Secretion of mouse growth hormone by transduced primary human keratinocytes: prospects for an animal model of cutaneous gene therapy2008 - PERONI, CIBELE N.; CECCHI, CLAUDIA R.; ROSAURO, CRISTIANE W.; NONOGAKI, SUELY; BOCCARDO, ENRIQUE; BARTOLINI, PAOLOBackground: 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/106cells/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.