CLAUDIA REGINA CECCHI

CLAUDIA REGINA CECCHI

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Sustained secretion of human growth hormone from TheraCyte devices encapsulated with PiggyBac-engineered retinal pigment epithelium cells
2023 - CECCHI, CLAUDIA R.; ALSING, SIDSEL; JESUS, GUSTAVO P.P.; ZACARIAS, ENIO A.; KJAER, LISBETH; CLEMENT, MICHELLE S.; KUMAGAI-BRAESCH, MAKIKO; CORYDON, THOMAS J.; BARTOLINI, PAOLO; PERONI, CIBELE N.; AAGAARD, LARS
Growth hormone (GH) deficiency is characterized by impaired growth and development, and is currently treated by repeated administration of recombinant human GH (hGH). Encapsulated cell therapy (ECT) may offer a less demanding treatment-strategy for long-term production and release of GH into circulation. We used PiggyBac-based (PB) transposon delivery for engineering retinal pigment epithelial cells (ARPE-19), and tested a series of viral and non-viral promoters as well as codon-optimization to enhance transgene expression. Engineered cells were loaded into TheraCyte macrocapsules and secretion was followed in vitro and in vivo. The cytomegalovirus (CMV) promoter supports strong and persistent transgene expression, and we achieved clonal cell lines secreting over 6 µg hGH/106 cells/day. Codon-optimization of the hGH gene did not improve secretion. ARPE-19 cells endured encapsulation in TheraCyte devices, and resulted in steady hormone release for at least 60 days in vitro. A short-term pilot experiment in immunodeficient SCID mice demonstrated low systemic levels of hGH from a single 40 µL capsule implanted subcutaneously. No significant increase in weight increase or systemic hGH was detected after 23 days in the GH-deficient lit/SCID mouse model using 4.5 µL capsules loaded with the highest secreting clone of ARPE-19 cells. Our results demonstrate that PB-mediated engineering of ARPE-19 is an efficient way to generate hormone secreting cell lines compatible with macroencapsulation, and our CMV-driven expression cassette allows for identification of clones with high level and long-term secretory activity without addition of insulator elements. Our results pave the way for further in vivo studies of encapsulated cell therapy.
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,
Efficient and non-invasive plasmid DNA administration into the tibialis cranialis muscle of dwarf mice
2014 - HIGUTI, ELIZA; CECCHI, CLAUDIA R.; LIMA, ELIANA R.; AZEVEDO, MAYARA L. de; AAGAARD, LARS; BARTOLINI, PAOLO; PERONI, CIBELE N.
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.
Modelos de terapia gênica baseados na expressão de hormônio de crescimento em células humanas e na administração de DNA plasmidial em camundongos anões
2015 - PERONI, CIBELE N.; CECCHI, CLAUDIA R.; HIGUTI, ELIZA; BARTOLINI, PAOLO
Early treatment for growth hormone deficiency based on naked DNA administration in dwart mice allows efficient catch-up growth
2015 - HIGUTI, ELIZA; CECCHI, CLAUDIA R.; LIMA, ELIANA R.; AAGAARD, LARS; JENSEN, THOMAS G.; BARTOLINI, PAOLO; PERONI, CIBELE N.
Different ex vivo and direct in vivo DNA administration strategies for growth hormone gene therapy in dwarf animals
2011 - PERONI, CIBELE N.; OLIVEIRA, NELIO A. de J.; CECCHI, CLAUDIA R.; HIGUTI, ELIZA; BARTOLINI, PAOLO
In vitro, ex vivo and in vivo recombinant growth hormone synthesis, for gene therapy applications
2011 - PERONI, CIBELE N.; OLIVEIRA, NELIO A. de J.; CECCHI, CLAUDIA R.; HIGUTI, ELIZA; SILVA, JULIANA T. da; JORGE, ALEXANDER A. de L.; JENSEN, THOMAS G.; BARTOLINI, PAOLO
Growth parameters after intramuscular hGH plasmid administration compared to recombinant hGH injections in LIT/SCID mice
2011 - HIGUTI, E.; CECCHI, C.R.; OLIVEIRA, N.A.J.; SILVA, J.T.; JORGE, A.A.L.; BARTOLINI, P.; PERONI, C.N.