Design And Construction Of Recombinant Ad5 Modified By Fiber

Adenovirus working as a virus vector has many outstanding advantages, for example, basic biology has been studied extensively, the viral genome can accommodate large heterologous transgene insertions, readily infect quiescent and dividing cells, amplified to high titers and have previously been shown to be relatively safe for use in humans. Adenovirus accounts for a high proportion in the clinic therapy. Recombinant adenovirus type 5(rAd5) has been widely used in therapy, because of several upwards. In order to transfer Ad5 into a more perfect therapeutic vector, the capsid protein, including Fiber, will be modified. At present, a series of modifications of Fiber basically focused on the original Fiber, most strategies are as follows:the use of other subtypes of adenovirus to replace the corresponding parts of or the entire rAd5 Fiber, inserting some ligands to Knob area which will enhance its binding to tumor cell receptors, some components of the rAd5 Fiber are replaced with other protein substances substitutions, such as replacing Fiber Knob with the trimerization motif. However, such transformations would undermine Fiber original structure more or less, reducing the ability to infect tumor cells,In order to remain the natural structure and function of Ad5, we proposed a new strategy which remained the original Fiber without any modifications and inserted another Fiber. Our idea comes from avian adenovirus which contains dual Fiber on the same Penton (a long Fiber, a short Fiber), also the short Fiber has a higher homology with Ad5 Fiber. So we simulate this avian adenovirus, short Fiber from avian adenovirus behind the original Fiber of Ad5 was inserted into Ad5, a series of genetic modification were made in the short Fiber, such as replacing Knob district with TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) which was capable of binding to CAR receptor on tumor cell surface to activate the apoptosis pathway inside tumor cells. Such transformation strategy had several advantages: Firstly, no damage to rAd5 original Fiber, which would ensure the normal function. Secondly, adding another gene modified short Fiber to exercise tumor targeting and killing functions.Such a transformation strategy was to explore two aspects:Firstly, whether the virus will affect its packaging because of an external source of Fiber. Secondly, how about the rAd5 yield after the transformation.Finally,whether rAd5 would have tumor-killing ability after transformation.Through the final experimental data we found:Firstly, although increased the size of the capsid protein, it still could be packaged successfully, indicating that such a transformation strategy would not hinder the virus packaging. Secondly, Testing the pfu and vp of rAd5, we found those values were in the normal range, indicating that the transformation strategy would not affect the yield of the virus. Finally, through the different experiments we found could be toxic to the tumor cells to some extent, indicating that addition of exogenous gene TRAIL in the short Fiber strategy is effective.