Experimental Study On Gene Therapy Of Nasopharyngeal Carcinoma By Selectively Replicate Adenovirus Expressing Interleukin 12

In this study, we used the selective replicate adenovirus in p53 mutated tumor cells to express mouse interleukin 12 (IL12) in order to enhance the therapeutic effects on the nasopharyngeal carcinoma (NPC) with p53 mutation.We constructed the fused mIL12 gene with two subunits of mIL12 gene by PCR and cloned the fused mIL12 gene into the adenovirus vector CNHK200 that depleted ElB-55Kda gene. Then, we cotransfected CNHK200-mIL12 and the adenovirus vector PBGHE3 into 293 cell line and recombined the selective replicate adenovirus CNHK200-mIL12 containing the fused mIL12 gene. After 72 hours from CNHK200-mIL12 infected the human NPC cell lines CNE3 and 915, we assayed the concentration of mIL12 in nutrient fluid by ELISA. The results showed that the expression levels of mIL12 were as high as 84ng/105PFU/104 cells in CNE3 and 84ng/105PFU/104 cells in 915 cell line.We observed CNHK200-mIL12 replicate in CNE3 by electron microscope. By derect immunohistochemistry and flow cytometry (FCM), we found that denovirus hexon protein in CNE3 and 915 was positive after CNHK200-mIL12 infected them. Positive rates of CNE3 and 915 after 0 hour from CNHK200-mIL12 infected them were 17% and 19% respetively. They rose to 42% and 23% respetively after 24 hours. It indicated that CNHK200-mIL12 replicated in CNE3 and 915. We assayed the replicate rates in CNE3 and 915 by TCID50 and it replicated more than 1000 times in CNE3and 915 in 3 days. By cell count with trypan dyed, dead cell rates of CNE3 and 915 were 85% and 64% respectively after 7 days from CNHK200-mIL12 infected them. The results displayed its abilities of infection, replication and cytopathic effect on NPC cells had not marked difference compared to the ElB-55Kda gene depleted selective replicate adenovirus dl!520 and proved cloning mIL12 into the selective replicate adenovirus had no effect on its abilities of infection, replication and cytopathic effect.We intratumor injected CNHK200-mIL12 and dl!520 into the NPC of nude mice in subcutaneum to treat the tumor in vivo. Injecting with CNHK200-mIL12 into the NPC in nude mice resulted in marked regression of the established tumors compared to injecting with dl!520 (P<0.05).This study proved that CNHK200-mIL12 not only had the same abilities of infection, replication and cytopathic effect on tumor as dl!520, but also enhanced the therapeutic effect on tumor by expressing mIL12. The selective replicate adenovirus can target the tumor cells, not replicate in normal cells but only replicate in tumor cells, and selectively express IL12 only in tumor cells. By this way, it can decrease the side effect of system used IL12. These data suggest that CNHK200-IL12, which augments the antitumor effect of replicate adenovirus, may be a powerful tool for treating NPC. This study gave a base of using the selective replicate adenovirus expressing any other cytokines to treat tumor.