Use of Lentiviral vector for improved system of protein expression in mammalian cells

Abstract

Lentiviral vectors can efficiently deliver genetic payloads, making it possible to deliver expression cassettes that direct long term expression of recombinant transgene products, in broad range of cell types. The present dissertation reports development and efficacy validation of HIV-2 derived multiple differently configured transfer vectors with expanded utility for in vitro and in vivo transgenesis. Among the features imparted, the new ones include a blue/white colony screening platform, a reduced vector backbone and availability of default dual tags for functional proteomics studies. Simultaneously, panels with different utilities were also made these include neomycin or puromycin selection markers, with options of default promoter and availability of dual multiple cloning site (MCS). Each transfer vector format was tested by appropriate transgene expression function by transduction of target cells. During lentiviral transgene delivery, only the cells that are transduced by the vector receive the effect of the transgene coded recombinant protein. To bypass this limitation, we report a novel strategy to amplify the effect of the lentivirally delivered gene product in bystander cells. In this vector system the transgene coded protein is secreted with a cell penetrating peptide (CPP) allowing entry of the same to nearby untransduced bystander cells resulting effectively in an increased biodistribution of the delivered gene product. The efficacy of the enhanced biodistribution system was tested in vitro and in vivo using GFP as a transgene product and protein transfer to neighboring untransduced cells was observed when green fluorescent protein (GFP) was secreted with a CPP tag. This novel lentiviral vector platform can thus be used to effectively deliver recombinant proteins with enhanced bioavailability into the target organ for desired effect. Furthermore, LV platform was also effectively used for the development of novel reporter cell based antiviral screening assay for rapid evaluation of TatTAR interaction.