CLAUDIA REGINA CECCHI
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Artigo IPEN-doc 27539 Optimization of mouse growth hormone plasmid DNA electrotransfer into tibialis cranialis muscle of "little" mice2020 - LIMA, ELIANA R.; CECCHI, CLAUDIA R.; HIGUTI, ELIZA; JESUS, GUSTAVO P.P. de; GOMES, ALISSANDRA M.; ZACARIAS, ENIO A.; BARTOLINI, PAOLO; PERONI, CIBELE N.Previous non-viral gene therapy was directed towards two animal models of dwarfism: Immunodeficient (lit/scid) and immunocompetent (lit/lit) dwarf mice. The former, based on hGH DNA administration into muscle, performed better, while the latter, a homologous model based on mGH DNA, was less efficient, though recommended as useful for pre-clinical assays. We have now improved the growth parameters aiming at a complete recovery of the lit/lit phenotype. Electrotransfer was based on three pulses of 375 V/cm of 25 ms each, after mGH-DNA administration into two sites of each non-exposed tibialis cranialis muscle. A 36-day bioassay, performed using 60-day old lit/lit mice, provided the highest GH circulatory levels we have ever obtained for GH non-viral gene therapy: 14.7 ± 3.7 ng mGH/mL. These levels, at the end of the experiment, were 8.5 ± 2.3 ng/mL, i.e., significantly higher than those of the positive control (4.5 ± 1.5 ng/mL). The catch-up growth reached 40.9% for body weight, 38.2% for body length and 82.6%–76.9% for femur length. The catch-up in terms of the mIGF-1 levels remained low, increasing from the previous value of 5.9% to the actual 8.5%. Although a complete phenotypic recovery was not obtained, it should be possible starting with much younger animals and/or increasing the number of injection sites.Artigo IPEN-doc 15564 Long-term human growth hormone expression and partial phenotypic correction by plasmid-base gene therapy in an animal model of isolated growth hormone deficiency2010 - OLIVEIRA, NELIO A.J.; CECCHI, CLAUDIA R.; HIGUTI, ELIZA; OLIVEIRA, JOAO E.; JENSEN, THOMAS G.; BARTOLINI, PAOLO; PERONI, CIBELE N.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.