Recombinant adenoviruses are widely used in gene therapy clinical trials. Since these vectors have a marked tropism for the adrenal gland, they could be suitable for gene therapy of adrenocortical carcinoma (ACC). However, adrenal tropism has safety implications, highlighted by the fact that adenoviral infection of adrenal cells can impair steroidogenesis. Aim of this study was the construction of E1/E3 deleted adenoviral vectors carrying different combinations of therapeutic genes and evaluation of their efficacy against ACC and their effect on adrenal gene expression profile. Recombinant adenoviral vectors were generated by cloning therapeutic genes (HSV-TK and mutant HSV-TK30, mouse GM-CSF, mouse IL-12) and reporter genes (LacZ, eGFP) into shuttle vectors followed by homologous recombination with adenoviral genome in bacteria. Viral stocks were prepared by transfection and expansion in 293 cells. Human ACC cell lines NCI-H295R and SW13 were demonstrated to be efficiently trasduced by adenoviral vectors at MOI 10 and 100 and to express high levels of the coxsackie and adenovirus receptor CAR. Cytotoxicity assay showed that ACC cells transduced with vectors carrying mutant HSV-TK30 had higher sensitivity to ganciclovir than cells infected with adenoviral vectors carrying wild-type HSV-TK, either in a monocistronic sequence or in a polycistronic combination. In vivo experiments in nude mice s.c. inoculated with ACC cells demonstrated significant inhibition of tumor growth by intratumor injection with vectors carrying cytokine genes and a complete regression of tumor masses injected with adenoviral vectors carrying combinations of suicide and cytokine genes after ganciclovir treatment. DNA microarray analysis of gene expression profile of ACC cells infected with reporter adenoviral vectors showed overexpression of genes involved in cortisol synthesis, including CYP21, and of the |[szlig]|-adrenergic receptor ADRA1B, and down-regulation of CYP11B2, ACE, and the adenovirus E1A-binding protein gene BS69. In conclusion, adenoviral vectors efficiently transduce ACC cells in vitro and in vivo. Combined adenoviral vector-mediated delivery of suicide and cytokine genes eradicates tumors in mice. Adenoviral vector infection of ACC cells modulates gene expression profile, upregulating genes involved in cortisol production and stress response.

Adenoviral Vector-Mediated Gene Therapy for Adenocortical Carcinoma: Therapeutic Strategies and Safety Considerations

BARZON, LUISA;PACENTI, MONIA;STEFANI, ANNA-LISA;TREVISAN, MARTA;MASI, GIULIA;FINCATI, KARINA;PALU', GIORGIO
2005

Abstract

Recombinant adenoviruses are widely used in gene therapy clinical trials. Since these vectors have a marked tropism for the adrenal gland, they could be suitable for gene therapy of adrenocortical carcinoma (ACC). However, adrenal tropism has safety implications, highlighted by the fact that adenoviral infection of adrenal cells can impair steroidogenesis. Aim of this study was the construction of E1/E3 deleted adenoviral vectors carrying different combinations of therapeutic genes and evaluation of their efficacy against ACC and their effect on adrenal gene expression profile. Recombinant adenoviral vectors were generated by cloning therapeutic genes (HSV-TK and mutant HSV-TK30, mouse GM-CSF, mouse IL-12) and reporter genes (LacZ, eGFP) into shuttle vectors followed by homologous recombination with adenoviral genome in bacteria. Viral stocks were prepared by transfection and expansion in 293 cells. Human ACC cell lines NCI-H295R and SW13 were demonstrated to be efficiently trasduced by adenoviral vectors at MOI 10 and 100 and to express high levels of the coxsackie and adenovirus receptor CAR. Cytotoxicity assay showed that ACC cells transduced with vectors carrying mutant HSV-TK30 had higher sensitivity to ganciclovir than cells infected with adenoviral vectors carrying wild-type HSV-TK, either in a monocistronic sequence or in a polycistronic combination. In vivo experiments in nude mice s.c. inoculated with ACC cells demonstrated significant inhibition of tumor growth by intratumor injection with vectors carrying cytokine genes and a complete regression of tumor masses injected with adenoviral vectors carrying combinations of suicide and cytokine genes after ganciclovir treatment. DNA microarray analysis of gene expression profile of ACC cells infected with reporter adenoviral vectors showed overexpression of genes involved in cortisol synthesis, including CYP21, and of the |[szlig]|-adrenergic receptor ADRA1B, and down-regulation of CYP11B2, ACE, and the adenovirus E1A-binding protein gene BS69. In conclusion, adenoviral vectors efficiently transduce ACC cells in vitro and in vivo. Combined adenoviral vector-mediated delivery of suicide and cytokine genes eradicates tumors in mice. Adenoviral vector infection of ACC cells modulates gene expression profile, upregulating genes involved in cortisol production and stress response.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/2517943
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