CONSTRUCTION OF A RECOMBINANT ADENOVIRAL VIRUS HARBOURING THE CDNA OF THE ENZYME AROMATASE: A NOVEL TOOL FOR THE STUDY OF AROMATASE FUNCTION IN THE BRAIN.

BELLINI MJ; GOYA RG; GARCIA-SEGURA LM

TORINO, ITALIA

Congreso; 5th International Meeting STEROIDS AND NERVOUS SYSTEM; 2009

The remarkable potential for vector-mediated delivery of therapeutic genes to treat neurodegenerative diseases and injuries to the CNS is well recognized Aromatase, the enzyme that synthesises estrogens from androgen precursors, is expressed in the brain, where it has been classically associated with the regulation of neuroendocrine events and behaviours linked with reproduction. Recent findings, however, have revealed new unexpected roles for brain aromatase, indicating that the enzyme regulates synaptic activity, synaptic plasticity, neurogenesis and the response of neural tissue to injury, and may contribute to control nonreproductive behaviours, mood and cognition. Therefore, the function of brain aromatase is not restricted to the regulation of reproduction as previously thought.In this context the overall goal of the present work is to implement aromatase gene therapy in the nervous system.AIM: The aim of our study was to develop an effective gene delivery system for aromatase enzyme.METHODS:     The sequence coding for human aromatase (1840 bp) was  excised from the plasmid pUC119-human aromatase cDNA, kindly donated for Dr. Nobuhiro Harada, with Sal I and was inserted in the same restriction sites of the MCS  of Ad5 shuttle pDC516 (Microbix, Ontario, Canada). Digestion with EcoRI and Xho I was used to verify the correct orientation (5´-3´sense). The shuttle so loaded was termed pDC516-hAro, and was cotransfected with the genomic plasmid pBHGfrt-delE1-delE3-FLP, in HEK 293 cells in order to generate the desired recombinant adenoviral vector (RAd), termed RAd-h Aro. The virus was subsequently purified and titered.The gene transduction efficiency was evaluated with real time PCR using recombinant adeno virus-green fluorescent protein as a control.The activity of the enzyme was evaluated by measuring the estrogen production of cells transduced with the virus.RESULTS: In the present study, we found: (1) that recombinant adenovirus efficiently transduced primary cultures of astrocytes or neurospheres. (2) that RAd-h Aro overexpress the cDNA of the aromatase in astrocytes from the cerebral cortex of mice transduced with the virus; and (3) the estrogen in the supernatant of cells transduced with the virus is higher than in non transduced cells or cells transduced with control virus CONCLUSION: Our findings implied that RAd-h Aro may offer great potential for the study of aromatase function in the brain.