CLAUDIA REGINA CECCHI

CLAUDIA REGINA CECCHI

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Optimization of mouse growth hormone plasmid DNA electrotransfer into tibialis cranialis muscle of "little" mice
2020 - 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.
Efficient non-invasive plasmid-DNA administration into tibialis cranialis muscle of “little” mice
2017 - CECCHI, C.R.; HIGUTI, E.; LIMA, E.R.; VIEIRA, D.P.; SQUAIR, P.L.; PERONI, C.N.; BARTOLINI, P.
Background: An alternative treatment for growth hormone deficiency based on hGH-DNA administration, followed by electro gene transfer, was investigated by injecting the plasmid into surgically exposed or non-exposed quadriceps or tibialis muscle of immunodeficient “little” mice. Methods: An optimization of electrotransfer conditions via a new combination of high/low voltage pulses is presented. After 3 days, serum hGH was determined and in a 28-day assay, the relative growth parameters were compared. Results: Both groups exhibited similar results: 5.0 ± 2.2 (SD) and 3.5 ± 0.9 ng hGH/ml (P>0.05; n=7) for the exposed quadriceps and non-exposed tibialis treatments, respectively. The final body weight increases were 16.1% for the quadriceps and 18.9% for the tibialis group. The tail and nose-to-tail length increases were 4.5% and 7.1% for the quadriceps and 4.8 and 4.6% for the tibialis group. The right and left femur length increases, obtained from radiographic measurements, were 16.9% and 12.7% for the quadriceps and 19.4% and 12.3% for the tibialis, respectively. A non-significant difference between exposed quadriceps and non-exposed tibialis treatments (P=0.48) was confirmed via a completely integrated statistical analysis. Circulating mIGF-1 levels were 126 ± 47, 106 ± 93 (P>0.05) and 38 ± 15 ng/ml for the quadriceps, tibialis and saline treatments, respectively. Conclusion: These results show that hGH-DNA administration into non-exposed tibialis muscle followed by the new HV/LV electrotransfer protocol was an equally efficient, less traumatic treatment,
Partial correction of the dwart phenotype by non-viral transfer of the growth hormone gene in mice: treatment age is critical
2016 - HIGUTI, ELIZA; CECCHI, CLAUDIA R.; OLIVEIRA, NELIO A.J.; LIMA, ELIANA R.; VIEIRA, DANIEL P.; AAGAARD, LARS; JENSEN, THOMAS G.; JORGE, ALEXANDER A.L.; BARTOLINI, PAOLO; PERONI, CIBELE N.
A novel homologous model for gene therapy of swarfism by non-viral transfer of the mouse growth hormone gene into immunocompetent dwarf mice
2014 - CECCHI, CLAUDIA R.; HIGUTI, ELIZA; OLIVEIRA, NELIO A.J.; LIMA, ELIANA R.; JAKOBSEN, MARIA; DAGNAES-HANSEN, FREDERIK; BARTOLINI, PAOLO; PERONI, CIBELE N.
Growth responses following a single intra-muscular hGH plasmid administration compared to daily injections of hGH in dwarf mice
2012 - HIGUTI, ELIZA; CECCHI, CLAUDIA R.; OLIVEIRA, NELIO A.J.; VIEIRA, DANIEL P.; JENSEN, THOMAS G.; JORGE, ALEXANDER A.L.; BARTOLINI, PAOLO; PERONI, CIBELE N.
Long-term human growth hormone expression and partial phenotypic correction by plasmid-base gene therapy in an animal model of isolated growth hormone deficiency
2010 - OLIVEIRA, NELIO A.J.; CECCHI, CLAUDIA R.; HIGUTI, ELIZA; OLIVEIRA, JOAO E.; JENSEN, THOMAS G.; BARTOLINI, PAOLO; PERONI, CIBELE N.
High-level secretion of growth hormone by retrovirally transduced primary human keratinocytes
2006 - PERONI, CIBELE N.; CECCHI, CLAUDIA R.; DAMIANI, RENATA; SOARES, CARLOS R.J.; RIBELA, MARIA T.C.P.; ARKATEN, ROSANGELA R.; BARTOLINI, PAOLO
A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 μg/106 cells/d) and mGH (11 μg/106 cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice (lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase (p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy.
Secretion of mouse growth hormone by transduced primary human keratinocytes: prospects for an animal model of cutaneous gene therapy
2008 - PERONI, CIBELE N.; CECCHI, CLAUDIA R.; ROSAURO, CRISTIANE W.; NONOGAKI, SUELY; BOCCARDO, ENRIQUE; BARTOLINI, PAOLO
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/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.