Oncolytic Adenovirus Targeting Cancer Cells Through Modified Adenovirus Fiber

Adenovirus vector as a gene transfer vector has been widely used because of its wide host range,efficient transduction,insert large gene fragment, non-integrated into the genome and the higher security.Currently,the most detailed studied of adenovirus molecular biological characteristics is the human adenovirus serotype 2 (Ad2) and human adenovirus serotype5(Ad5),especially the C family adenovirus type 5 (Ad5). Adenovirus capsid protein is the most important factors to determine the tropism of adenovirus infection,especially its fiber knob CAR-binding domain and the Arg-Gly-Asp(RGD) motif of more than five peptide molecules in the penton base.To increase the targeted capacity of adenovirus infection,scientists have tried to inset the RGD motif,specific ligand sequence or polyadenylic acid and other means into the penton base and HI loop in fiber konb, which can be specifically combination with some tissue,and the receptors on cell surface.Conditionally replicative adenovirus(CRAds),also called tumor specific adeno- virus.Oncolytic Adenovirus,which are genetically programmed to replicate within tumor cells but not in normal cells.Thereby,Oncolytic Adenovirus can using their own proliferation capacity to kill tumor cells while do not produce significant toxicity,and thus have tumor targeting and drug safety.Adenovirus which using there fiber to adsorb host cells and to combine the cell surface eceptors, and then infection of host cells.Theoretically,oncolytic virus can be a large number of proliferation in tumor cells,releasing the virus also can infect surrounding tumor cells,ultimately through the cascade amplification to eliminate the whole tumor.However, in vivo experiments indicate that oncolytic virus can not completely kill the tumor cells,the international community has always been considered that it is due to the normal cells or necrotic tissue intermission in tumor.However, recent studies have shown that the main reason is in the process of adenoviral particle assembly,the fiber is overproduction.The function of excessive fiber production has been unclear,but has been suggested to be important for efficient adenoviral particle assembly.However,these over-production fiber may firstly binding with the non-infected tumor cell surface receptors, thereby to screen off the cell surface receptors, which led to the progeny adenovirus can not infect surrounding tumor cells,resistance breaking virus on tumor cells in secondary infection.Based on these considerations,we began our study with the fiber modified, However,adenovirus transformation of the traditional method is more cumbersome and difficult to operate,so we established a successful Gateway system.make the fiber transformation more easy to operate.And the fiber with red fluorescent protein behind become red observed under a fluorescence microscope,shows that we have established the Gateway recombination system successful.In this study,in order to increase the secondary infection of adenovirus,we inset the MicroRNA21, MicroRNA155 in the fiber behind to regulate the expression of fiber. Afterwards,Establish a mature Gateway recombination system,to make the adenovirus fiber-modified become easier.And using the Gateway technology to recombine the Fiber-modified into the full-length recombinant adenovirus vector which removal of the wild-type fiber.And then injecting the 293 cells and other live cancer cell lines with these modified adenovirus,hoping to select an adenoviral vector with high infectivity in target cells.we have already to establish a series of adenovirus vectors with MicroRNA and red fluorescin after the fiber,and successfully packaged the virus.Further more,to identify the effectiveness that increased the targeting to tumor cells,we have done a preliminary study.