The Construction Of Tumor-targeting Recombinant Adenovirus Vector And Its Applications In Cancer Gene Therapy

Cancer is one of the major threats to public health in modern society. It is the second leading cause of death world wide. As traditional therapies such as surgery, radiotherapy and chemotherapy show little effectiveness in many cases, gene therapy instead has been developing so rapidly and will bring about promise in conquering this severely life-threatening disease.The gene delivery system is the most important part of the overall gene therapy system. While in the study of cancer gene therapy, adenovirus-based vector is the most widely used vector in that it has a broad spectrum of hosts, can efficiently infect either dividing or non-dividing cells, can embrace larger exotic DNA fragments, is not integrated into host genome, and in addition, it is easy to prepare high-titre stocks of purified virus. However, the very benefit of adenovector's broad spectrum of hosts is on the other hand an impediment for its application in cancer gene therapy because of the existence of coxsackievirus and adenovirus receptor (CAR) on the surface of most normal cells in the body. Also the expression of CAR on the cell surface of some kinds of cancers is lower when compared with their normal tissues, which prevents adenovirus vectors from infecting the targeted cells efficiently. Therefore, it has become the spotlight to enhance recombinant adenvector's infection efficiency on cancer cells as well as minimizing normal tissue lesions in the study of cancer gene therapy.The abnormal over-expression of EGFR is normally seen in various solid tumors, and have close correlates with their initiation, progress and prognostics. For this matter, EGFR has become a significant target for cancer therapy research. And our research is intended to establish a targeted EGFR gene transferring system by piloting adenovirus to the EGFR-overexpressed surface of cancer cells with the help of a bifunctional crosslinkers, thus promote adenovector's infection efficiency on cancer cells as well as minimizing normal tissue lesions, provide reliable theoretical evidence as well as practical direction for cancer gene therapy.SECTION I. Relationship Between Adenovirus Infection and the expression of receptors on Cell Surface[Objective] To study relationship between adenovirus infection and expression of CAR and integrin in different cell lines, and offer basis for further study of targeted gene therapy by adenovirus[Methods] We examined expression levels of CAR and integrin in different cell lines by using RT-PCR, Western blot, immunohistochemistry. We infected cells with Ad-CMV-GFP and Ad-CMV-luc carrying report genes, tested their infection efficiency and expression levels by using flow cytometry and Luciferase Assay System. Evaluated and compared the variation of adenovirus infection efficiency when enhanced the CAR expression on cell surface by transfecting eukaryotic with plasmid expressing CAR, or blocked with the help of antibodies of CAR and integrin.[Result] We examined the expression levels of CAR and integrin on the cancer cell surface by using real-time PCR, Western blot, flow cytometry and immunohistochemistry respectively, and found that the final results tended to be consistent with one another. More specifically, the highest CAR expression occurred in SMMC-7721 and A549 cell lines, while in K562 cell lines the lowest one occurred. With respect to the expression level of integrin, we found that nearly all examined cancer cell lines expressed it to some extent, among which SK-OV-3 and A549 cell lines showed the highest expression level, and K562 showed the lowest.The results attained with the help of fluorescence microscope and flow cytometry suggested that Ad5-CMV-GFP infected SMMC-7721 and A549 cell lines with the highest efficiency, and when it came to HXO-RB₄₄ and K562 cell lines, the infection efficiency was the lowest. We also successfully constructed the recombinant plasmid pCDNA3.1-FCAR that expressed CAR, and found that after transfecting various cancer cell lines in conjunction with liposome, the efficiency of adenovirus-mediated infection would improve rapidly. In detail, the infection efficiency in A549 improved with 10 folds, SK-OV-3 with 4.4 folds, K562 with 3.4 folds, HXO-RB₄₄ with 1.8 folds and SMMC-7721 with 1.2 folds, respectively.On the other hand, after blocking of CAR and knob sites by their corresponding antibodies, the infection efficiency in SMMC-7721 and SK-OV-3 cell lines declined, and within the same antibody concentration, infection efficiency decline was more dramatic in SMMC-7721 cell lines than in Sk-OV-3. While in the case of integrin's antibody blocking, infection efficiency in either SMMC-7721 or SK-OV-C declined remarkably, and the blocking effect in SK-OV-3 was more evident.[conclusion] The infection efficiency of adenovirus was determined by the expression level of CAR and integrin on cancer cell surface, lack of CAR exression is associated with poor infectivity. Transfecting pCDNA3.1-fCAR into cancer cells can artificially enhance the CAR expression level on cell surface and therefore make the efficiency of adenovirus-mediated infection dramatically highlighted. No matter whether CAR's, knob's or integrin's antibody is used to block adenovirus infection, the efficiency always declines to some extent, while the efficiency decline caused by blocking of integrin's antibody is the most evident, showing that integrin plays a more important role in adenovirus infection.SECTION II Role of bifunctional fusion protein in improving adenovirus-mediated infection[Objective] In order to improve the efficiency of adenovirus-mediated infection on cancer cells, on the basis that the EGFR expression is often upregulated in cells of most solid tumors, we constructed the adenovector that could express fusion protein sCAR-EGF, and then studied the role of that fusion molecule in improving the infection efficiency of adenovirus on cancer cells, providing a new approach and tool for enhancing the delivery efficiency of therapeutic genes into certain targets in cancer cells.[Methods] We constructed a fused sCAR and EGF gene by PCR and in vitro ligation. After that, the fused gene was inserted into the shuttle plasmid pDC315. Then, we used Ad-MAX adenovirus package system to transfect the recombinant shuttle plasmid pDC315-sCAR-EGF into 293 cells with the cooperation of pBHGloxΔE13cre which was a large plasmid carrying major genes of adenovirus and producing the adenovirus that expressed fusion protein sCAR-EGF. Finally we used report-gene-contained adenoviruses, Ad5-CMV-GFP and Ad5-CMV-luc, to evaluate the role of bifunctional fusion protein in enhancing the efficiency of adenovirus-mediated infection on cancer cells as well as expression levels of exotic genes. [Result] We successfully packaged a replicative-deficiency adenovirusAd-CMV-sCAR-EGF. The sCAR-EGF gene fragment contained in the virus was identified by PCR assay and sCAR-EGF fusion protein was proved by Western blotting. In vivo test demonstrated that the virus infection and the fusion protein could help Ad5-CMV-luc infect cancer cells efficiently. The infection efficiency was positively-related with the EGFR expression on the surface of cancer cells, and also has a cause-and-effect relation with the dosage of bifunctional crosslinkers.[conclusion] The fusion protein sCAR-EGF can enhance the infection efficiency of adenovirus on cancer cells. After binding the fusion protein sCAR-EGF, the adenovirus then successfully attaches to the cell surface and is then internalized into the cell by using EGFR recptor which is located on the cell surface. EGFR antibody can effectively block the binding site and avoid infection by adenovirus, while CAR antibody does not show this characteristic evidently. These phenomena aptly illustrate that the sCAR-EGF-mediated adenovirus attachment and internalization is dependent on EGFR, no longer on its natural receptor CAR. By using fusion protein sCAR-EGF, we changed adenovirus's natural tropism, retargeted the adenovirus via CAR-independ pathway, and enhanced the infection efficiency, providing new approach to the gene therapy for those tumors that over express EGFR.