The Characterization Of Modified Adenovirus Vector

Object The replication deficient first generation adenovirus vector (FGAd) was widely used in gene therapy for its safty, highly efficient transgene expression and a broad natural tropism. There are, however, some limitations associated with the use of FGAd in oncotherapy. One such disadvantage is that FGAd is ineffcient in gene transfer to cells such as dendritic cells (DCs) and many cancer cells lacking the primary Ad receptors of coxsackievirus and adenovirus receptor (CAR). Several studies have showed that FGAd containing arginine-glycine-aspartate (RGD) peptide in the HI loop of adenoviral fiber knob can enhance gene transfer efficient in cancer cells. So far the methods of constructing such FGAd were very complicated to manipulate, no praticalbe ones have been reported. RD and T24 cancer cells are deficient in CAR and refractory to convetional FGAd. In this study, we developed a simple way to construct FGAd-RGD-EGFP and FGAd-RGD-Fsyn containing a peptide-RGD in the HI loop of the fiber knob, respectively. RD and T24 cells were infected by FGAd-RGD-EGFP to investigate its infection efficiency using fluorescent microscope and flow cytometry. BALB/c mice were vaccinated by FGAd-RGD-Fsyn via intramuscular and imtranasal routes, respectively, to evaluate the ability to induce humoral responses against transgenes in vivo.Methods pAdEasy-1 was cut into two fragment by restriction enzyme EcoRⅠ. The 23 kb fragment was preserved for future use, whereas the 9 kb fragment, which contained fiber gene and other genes, was self-ligated to form a plasmid with low molecular weight, named pY. Using PCR method, RGD was inserted into pY to construct the plasmid of pY1Y2-RGD. After pY1Y2-RGD was digested with EcoRⅠ, dephosphorized, and ligated with the above 23 kb fragment of pAdEasy-1, we generated pAdEasy-RGD-1. Then, the pShuttle-CMV?EGFP and pShuttle-CMV?Fsyn were used to produce recombinant adenoviral plasmids by homologous recombination in E.coli BJ5183 with pAdEasy-RGD-1. The resulting plasmids pAd-RGD-EGFP and pAd-RGD-Fsyn were digested with PacⅠand transfected into HEK293 cells to produce the recombinant adenovirus vectors of FGAd-RGD-EGFP and FGAd-RGD-Fsyn. The RD and T24 cells were infected with 1000 vp per cell of FGAd-EGFP and FGAd-RGD-EGFP respectively and analyzed by flow cytometry. The BALB/c mice were vaccinated with 1×108 vp per mouse of FGAd-Fsyn and FGAd-RGD-Fsyn via intramuscular and imtranasal routes, respectively, and analyzed by ELISA.Result FGAd-RGD-EGFP and FGAd-RGD-Fsyn were successfully constructed in this method. The infection efficiency on RD and T24 cells was higher with FGAd-RGD-EGFP than FGAd-EGFP, and immunization with FGAd-RGD-Fsyn generated similar serum IgG against Fsyn with FGAd-Fsyn.Conclusion PCR is a simple and practical method to construct adenovirus vectors containing a peptide-RGD in the HI loop of the fiber knob. The resultant adenovirus vectors display improved infection efficiency on RD and T24 cells and similar humoral immune responses to transgene as compared to original adenovirus vector.