ABSTRACT
Objective: To establish a quality control method for Gene-Viral Therapy system CNHK200-hEndostatin. Methods: According to "The Guideline on Quality Control Methods of Human Gene Therapy Products", a quality control method for adenovirus was set up, which consisted of adenovirus protein identification, genomic identification, hEndostatin gene identification, hEndostatin protein quantitation, adenovirus particle quantitation, virus titer quantitation, determination of particle to PFU ratio, purity detection, AdWT detection, host cell DNA residue detectiong bovine serum protein residue detection, etc. The purified adenovirus product of CNHK200-hEndostatin was subjected to the above process. Results: We set up a SDS-PAGE method to identify the adenovirus protein and identified adenovirus genomic DNA by restriction endonuclease enzyme analysis. Human endostatin gene was identified by PCR method and its expression product was quantitated by ELISA. The number of adenovirus particle was quantitated by D260 method and HPLC method. The purity of adenovirus was determined by D260/D280 method and HPLC method. A PCR reaction was introduced to detect AdWT and hybridization method was used in host cell DNA residue detection. Bovine serum protein residue was detected by reverse indirect hemagglutination assay. All these methods were confirmed feasible in adenovirus quality control and our purified adenovirus sample was eligible in all test items. Conclusion: A series of basic methods have been successfully established for quality control of gene-viral therapy system CNHK200-hEndostatin. Our modified method for adenovirus particle quantitation by HPLC is more rapid and accurate than traditional method. The established methods have been approved in testing the purified adenovirus samples.
ABSTRACT
<p><b>OBJECTIVE</b>To develop a novel vector system, which combines the advantages of the gene therapy, antiangiogenic therapy and virus therapy, and to observe its effect on lung cancer.</p><p><b>METHODS</b>Human angiostatin gene hA(k1-5) was inserted into the genome of the replicative virus specific for the tumor cells by virus recombination technology. The expression of hA(k1-5), its effect on tumor growth in vitro and in vivo were studied.</p><p><b>RESULTS</b>A new kind of gene-viral vector system, designated as CNHK200-hA(k1-5), in which the E1b55 000 gene was deleted but the E1a gene of adenovirus preserved, was constructed. The novel vector system possessed the same property as the replicative virus ONYX-015, which replicates in p53- tumor cells but not in normal cells, thus specifically kills tumor cells. In vitro, CNHK200-hA and Ad-hA both could kill A549 tumor cells but the latter needed 100 times more MOI to achieve the same amplitude of cell killing. In vivo, the therapeutic effect of CNHK200-hA on human lung cancer A549 xenograft in nude mice was significantly better than that of Ad-hA and that of tumor-replicative virus ONYX-015.</p><p><b>CONCLUSION</b>CNHK200-hA(k1-5), a novel vector is constructed in which the angiostatin gene is inserted into the genome of the replicative adenovirus cytotoxic to p53-negative tumor cells. It has the advantages of specific tumor targeting, high level gene expression in tumor cells, and potent tumoricidal activity.</p>