(102a) A Fiberless Adenovirus Vector for Delivering the Gfp Reporter Gene

Adenovirus is widely used as a vector for gene therapy and vaccine delivery due to its ability to achieve high transduction efficiency and high levels of gene expression. An adenovirus vector that lacks the virus fiber protein was previously developed. When complexed with polymers, the fiberless adenovirus produced nanoparticles capable of delivering genes with reduced immunogenicity and flexible cell targeting. In this study, a homologous recombination technique was used to produce a fiberless adenovirus that packaged a reporter gene for green fluorescent protein (GFP). The incorporated gfp gene will allow for quantitative characterization of the infectivity, as well as enable the study of cellular trafficking and nuclear entry of the vector. To produce the gfp-packaging vector, the gfp gene was first cloned into a shuttle vector. The resultant plasmid was linearized by restriction endonuclease, PmeI, and then was cotransformed into DH10B electrocompetent cells with an adenoviral backbone plasmid. The linearized recombinants that were selected for kanamycin resistance were then transfected into HEK293 cells to produce the fiberless adenovirus packaging the gfp reporter gene. The infectivity of the vector was characterized using fluorescence microscopy. Through future analysis, we hope to identify the limiting steps in the gene delivery process and gain insight to how the modified vector can be improved further.