Development of adenoviral vector for high-throughput production of recombinant adenoviruses and its applications to G protein-coupled receptor-based drug discovery and drug repositioning

Abstract

The need for efficient high-throughput gene delivery system for mammalian cells is rapidly increasing with the growing request for functional genomics studies and drug discoveries in various physiologically relevant systems. However, plasmid-based gene delivery has limitations in co-transfection efficiency and available cell types. Viral vectors have advantages in transfection efficiency, but construction of recombinant viruses remains to be a big hurdle for high-throughput applications.

In this thesis, a rapid and simple high-throughput system for constructing recombinant adenoviruses as efficient gene delivery tools in mammalian system in vitro and in vivo was developed and recombinant adenoviruses expressing GPCRs (G-protein coupled receptors) produced by this system were used for drug repositioning of marketed drugs and drug discovery of natural products.

Firstly, adenoviral vector was modified to contain sequences for site-specific recombination for simplified generation. And then, isolation of adenoviral DNA (DNA-terminal protein complex) was developed for the multiple generation of recombinant adenoviruses in multi-well plate simultaneously.

Secondly, GPCR based screening system was established by production of 106 recombinant adenoviruses containing GPCR gene. The 106 GPCRs include most of the popular drug target GPCRs such as dopaminergic, serotonergic, muscarinic, purinergic, angiotensin, calcitonin, leukotriene, prostanoid, peptide, and hormone receptors.

Thirdly, application to the drug repositioning was performed by high-throughput screening of marketed drug library. Several compounds were identified as cannabinoid1 receptor antagonists and observed in vivo as a candidate for appetite suppressant.

Fourthly, application to the drug discovery was performed by high-throughput screening and high-content analysis of natural product library. Several products were identified as β2 adrenergic receptor agonist and developed for anti-asthma drug.