title: Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.
abstract: Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.
authors.affiliation: Bioinformatics Group Leader, Johnson & Johnson Pharmaceutical Research & Development, Welsh and McKean Roads, Spring House, PA 19477, USA.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Darrow
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Conway
authors.forename: Anil H
authors.initials: AH
authors.lastname: Vaidya
authors.forename: Daniel
authors.initials: D
authors.lastname: Rosenthal
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Wildey
authors.forename: Lisa
authors.initials: L
authors.lastname: Minor
authors.forename: Zina
authors.initials: Z
authors.lastname: Itkin
authors.forename: Yan
authors.initials: Y
authors.lastname: Kong
authors.forename: Jennifer
authors.initials: J
authors.lastname: Piesvaux
authors.forename: Jenson
authors.initials: J
authors.lastname: Qi
authors.forename: Marc
authors.initials: M
authors.lastname: Mercken
authors.forename: Patricia
authors.initials: P
authors.lastname: Andrade-Gordon
authors.forename: Carlos
authors.initials: C
authors.lastname: Plata-Salamán
authors.forename: Sergey E
authors.initials: SE
authors.lastname: Ilyin
mesh_terms: D000256:Adenoviridae; D019295:Computational Biology; D005822:Genetic Vectors; D011994:Recombinant Proteins / Q000032:analysis*
chemical_list: D011994:Recombinant Proteins