CRAd improve gene replication efficiency and cancer killing effect of E1-deleted adenovirus including killing gene in head and neck cancer cell line


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Seon-Hui Shim, Myung-Whun Sung

Cancer Research Institute, Seoul, Republic Of Korea

Abstract

To overcome low efficiency of gene therapy, we tried to combine adenoviral vector including killing gene and CRAd (conditionally replicating adenovirus). CRAd show oncolytic action in cancer cells with abnormal Rb activity and helped replication of E1-deleted adenovirus including a killing gene. We hypothesized that conventional E1-deleted adenovirus can replicate competently when co-transfected with a CRAd to selectively supply E1 in tumors by combining with CRAd and adenovirus. As combining with CRAd and adenovirus carrying TRAIL, we expected both an increase of killing gene expression in cancer cells and a tumor killing effect. Additionally, we expected that this combination strategy could extend to multi-targeting cancer therapy. First, to identify any increase of gene expression in conventional E1-deleted adenovirus, we tested combinational application with CRAd and Adeno-luciferase in a head and neck cancer cell line and observed a significant increase of luciferase activity, 10-50 times greater than transfection with Adeno-luciferase alone. Next, we investigated the anti-cancer-effect of combination with Ad-TRAIL and CRAd, comparing with single transfection of Adeno –TRAIL, and observed that combining CRAd and Adeno-TRAIL showed the increasing sub-G1 above 30 fold by PI staining than any single application in head and neck cancer cell lines. When co-transfected with CRAd, increasing expression of TRAIL by 3-5-fold than transduction with Adeno-TRAIL alone. These results represent that this combination with CRAd and Adenovirus increased killing gene transfer rate and enhanced its anti-tumor effect. Next, we established head and neck cancer xenografts models in vivo and confirmed the same result as in vitro. CRAd and Ad-trail treated mice showed complete regression of established tumors compared with CRAd or Adeno-TRAIL alone respectively As an ongoing experiment, we investigated whether multi-targeting was also effective. We tried multi targeting gene therapy by combining with E1-deleted adenovirus and CRAd. We combined Adeno-luciferase plus Adeno-LacZ plus Adeno-GFP and CRAd. We checked the activity of each gene. All of these reporter genes were expressed well without interruption. From these results, we expect that multi-targeting gene therapy could be possible. However, to achieve multi targeting gene therapy a suitable killing gene must be found.

To overcome low efficiency of gene therapy, we tried to combine adenoviral vector including killing gene and CRAd (conditionally replicating adenovirus). CRAd show oncolytic action in cancer cells with abnormal Rb activity and helped replication of E1-deleted adenovirus including a killing gene. We hypothesized that conventional E1-deleted adenovirus can replicate competently when co-transfected with a CRAd to selectively supply E1 in tumors by combining with CRAd and adenovirus. As combining with CRAd and adenovirus carrying TRAIL, we expected both an increase of killing gene expression in cancer cells and a tumor killing effect. Additionally, we expected that this combination strategy could extend to multi-targeting cancer therapy. First, to identify any increase of gene expression in conventional E1-deleted adenovirus, we tested combinational application with CRAd and Adeno-luciferase in a head and neck cancer cell line and observed a significant increase of luciferase activity, 10-50 times greater than transfection with Adeno-luciferase alone. Next, we investigated the anti-cancer-effect of combination with Ad-TRAIL and CRAd, comparing with single transfection of Adeno –TRAIL, and observed that combining CRAd and Adeno-TRAIL showed the increasing sub-G1 above 30 fold by PI staining than any single application in head and neck cancer cell lines. When co-transfected with CRAd, increasing expression of TRAIL by 3-5-fold than transduction with Adeno-TRAIL alone. These results represent that this combination with CRAd and Adenovirus increased killing gene transfer rate and enhanced its anti-tumor effect. Next, we established head and neck cancer xenografts models in vivo and confirmed the same result as in vitro. CRAd and Ad-trail treated mice showed complete regression of established tumors compared with CRAd or Adeno-TRAIL alone respectively As an ongoing experiment, we investigated whether multi-targeting was also effective. We tried multi targeting gene therapy by combining with E1-deleted adenovirus and CRAd. We combined Adeno-luciferase plus Adeno-LacZ plus Adeno-GFP and CRAd. We checked the activity of each gene. All of these reporter genes were expressed well without interruption. From these results, we expect that multi-targeting gene therapy could be possible. However, to achieve multi targeting gene therapy a suitable killing gene must be found.
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