Gene Therapy For Cancer By AD/AAV Hybrid Virus With Full-length Antibody Gene

It is fund that the cancer targeting therapy is an important strategy to cure the tumor. useing gene therapy, the virus carry the full length antibody genes into cells, the gene expression in cells can last for a long time, the cells own antibodies can kill tumor cells. In 2004, our laboratory constructed the adenovirus carrying full-length antibody gene for the first time and show its good anti-tumor effect. However, due to shortcomings of adenovirus, antibody genes can't be expressed long enough and limits the further development of clinical applications. In gene therapy there are adeno-associated virus vector (AAV), adenovirus vectors, retroviral vectors and lentiviral vectors , etc., but all of them are not perfect.Because ITR is the important for AAV copies and packagin as well as formed AAV circular intermediates, these intermediates between the ITR is the open reading frames, which characteristics of the target gene can be inserted. we insert the two AAV ITR in the adenovirus vector and then inserted full-length antibody genes sequences between the ITRs.When viral infect of target cells, antibody genes can form the ring outside the host chromosome and independent expression. This new hybrid adenovirus vector with large capacity , antibody genes also can be expresed for a long-time. It's a ideal new virus vector.Upstream AAV ITR (uITR) and downstream ITR (dITR) fusion gene fragment was geted by Overlap-PCR, and then insert the fusion gene into adenovirus vector, and then full-length antibody Cetuximab (AT2) gene fragments was inserted between the ITRs.This new virus is called A/V659-AT2.Recombinant adenovirus AD359-AT2 carrying the same antibodie can be used as the control group, the Cetuximab (AT2) antibody expressed in cell supernatant was detected by ELISA and Western blot. At the 2,4,6,8 day collecte cell supernatant, ELISA results showed that both viruses are able to successfully expression of Cetuximab (AT2) antibody in vitro, and molecular weight are the same as the commercialization one. In the first 4 days, the expression of antibodies are same, but at the first 6 day,the antibody expression in the control group began to decrease, at the 8 day, the expression of the antiboday was reduced from 138.82ng/ml(2 day) to 58.49 ng / ml ; Western Blot shown at the first 6 day, the light chain does not match to the heavy chain and in the 8th day ,the light chain is not obvious. But with AAV ITR's,the new Ad / AAV hybrid virus's express the antibodies relatively constant, around 110ng/ml, Western Blot shows the expression of its light and heavy chain are matched . It means the antibodies have biological activity.Use recombinant adenovirus vector carried fluorescent protein as a control, the new Ad / AAV hybrid vector carried fluorescent protein infect NK92 cells. Then observe EGFP's expression under a fluorescence microscope.It was found in the control group the expression of GFP is damed at the 6 day, but New vector infected cells can be long-lasting expression of GFP. Therefore, we can use the viral vectors conjunction with NK cells. As NK cells naturaly targeting tumor cells, viral vectors can be carried to tumor cells, so that a large number of antibody can be expressed in tumors for a long time, so as to kill the tumor cells.