Increased Cytopathic Effect of Replicating Adenovirus Expressing Adenovirus Death Protein / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Replication-competent adenoviruses (Ads) are promising new modalities for the treatment of cancer. Selective replication of a viral agent in tumor may lead to improved efficacy over non-replicating Ads due to viral multiplication, lysis of the infected cancer cell and spread to surrounding cells. In our previous studies it was shown that the E1B 55 kD-deleted Ad (YKL-1) exhibits tumor specific replication and cell lysis, but with reduced cytolytic effects compared to the wild type adenovirus (Int J Cancer 2000;88: 454-463). Thus, improving the potency of oncolytic Ads remains an important goal for cancer gene therapy. To increase the oncolytic ability of YKL-1, an adenovirus death protein (ADP) gene was reintroduced under the control of a CMV or MLP promoter at the E3 region of the YKL-1, generating an YKL-cADP and YKL-mADP, respectively. MATERIALS AND METHODS: The in vitro cytolytic effect of ADP expressing Ads was evaluated by MTT assay, and the induction of apoptosis by ADP expressing Ads was examined by TUNEL analysis. Finally, the antitumor effect of ADP expressing Ads was demonstrated in C33A xenograft tumor model. RESULTS: The YKL-cADP exerted a markedly enhanced cytolytic effect against H460 and SK-Hep1 cancer cell lines. The TUNEL assay indicated that the ADP-mediated cytotoxicity was largely driven by apoptosis. Finally, the YKL-cADP showed a superior antitumor effect than the YKL-1 or YKL-mADP in C33A xenografts. CONCLUSION: These lines of evidence demonstrate that the YKL-cADP induces efficient cell lysis, which is critical for the addition of therapeutic value to replicating Ads in cancer gene therapy.

Development of a Conditional Replication Competent Adenovirus, Controlled by the Human Telomerase Promoter (hTERT) / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study has been planned to generate a replication-competent adenovirus which replicates in a cancer cell-specific manner, thus minimizing the side effects and toxicity of cancer gene therapy. MATERIALS AND METHODS: we have generated an E1B 19 kD attenuated recombinant adenoviruses, Ad-TERT-delta19 and Ad-mTERT-delta19, which encode E1A gene driven by the wild type hTERT and modified m-hTERT promoter containing additional c-myc and Sp1 binding sites in the backbone of Ad-deltaE1B19. The in vitro efficacy and specificity of the hTERT and m-hTERT promoter have been evaluated by the comparison of viral replication and cytopathic effect in cancer cells and normal cell lines. To assess anti-tumor effect and safety of hTERT or m-hTERT promoter driven replication competent adenoviruses, tumor regression after subcutaneous injection in subcutaneous C33A xenografts and lacZ expression after systemic injection in organs were examined. RESULTS: The activation of hTERT or m-hTERT promoter was significantly up-regulated only in hTERT-positive cells, but not in hTERT-negative cells. Moreover, the activity of m-hTERT promoter was substantially increased in hTERT-positive cancer cells, but not in hTERT-negative cells. While Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect in cancer cells only. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. CONCLUSIONS: The use of m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of m-hTERT promoter may be a new promising tool for the treatment of human malignancies.

Evaluation of E1B-mutant Replicating Adenoviruses for Cancer Gene Therapy / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. In an effort to continually improve upon cancer gene therapy, we have modified gene- attenuated replication-competent adenoviruses so as to cause them to replicate efficiently and lyse the infected cancer cells more effectively. MATERIALS AND METHODS: We modified the E1 region of the adenovirus (Ad) systematically, generating Ad-deltaE1B19, Ad-deltaE1B55, Ad-deltaE1B19/55, and Ad-WT. The cytopathic effects (CPE) and viral replication of these four gene modified adenoviruses were compared, and the morphology and DNA fragmentation of the infected cells was evaluated. RESULTS: Among the constructed adenoviruses, E1B 19kD-inactivated adenovirus (Ad-deltaE1B19) was the most potent, inducing the largest-sized plaques and markedCPE. Moreover, cells infected with Ad-deltaE1B19 showed complete cell lysis with disintegrated cellular structure whereas cells infected with Ad-WT maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL assay was also used to monitor DNA integrity, and a more profound induction of apoptosis was observed in the Ad-deltaE1B19 infected cells in comparison to wild type adenovirus infected cells. CONCLUSION: We demonstrate that the inactivation of the E1B19kD gene in a replicating adenovirus leads to increased CPE, rapid viral release, improved cell-to-cell viral spread and increased induction of apoptosis.

Construction of replication-competent adenovirus shuttle plasmids pAd-delE1b55kD / 重庆医科大学学报

Objective: To construct a replication-competent adenovirus shuttle plasmids named pAd-delE1b55kD,which can transfect the cell of tumor targeted.Methods: delete the E1B55 region of pXC1 and retain a site for Bgl II by such technique of gene recombination as nested PCR;verify the correctness of pAd-delE1b55kD by gel image analysis and sequencing the gene order.Results: The 2279~3326 region of E1B in pXC1 was deleted.The 2024 basic radical C was mutated to T,the 2252 basic radical C to T and the 2261 basic radical G to T.Stop codon TGA?TAG and TAA were inserted.A site for Bgl II was retained in downstream of the stop codon.Conclusion: The replication-competent adenovirus shuttle plasmids named pAd-delE1b55kD is constructed,which provided a carrier for the gene therapy of tumor targeted.