AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepat...AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepatocellular carcinoma （HCC）. METHODS： CNHK500-p53 was constructed by using human telomerase reverse transcriptase （hTERT） promoter to drive adenovirus E1a gene and hypoxia response element （HRE） promoter to drive adenovirus E1b gene. p53 gene expressing cassette was inserted into the genome of replicative virus. Viral replication experiments, cytopathic effect （CPE） and methyl thiazolyl tetrazolium （MTT） assay were performed to test the selective replication and oncolytic efficacy of CNHK500-p53. RESULTS： Immunohistochemistry verified that infection with CNHK500-p53 was associated with selective replication of adenovirus and production of p53 protein in telomerase-positive and hypoxia-inducible factordependent HCC cells, p53 protein secreted from HepG2, infected with CNHK500-p53 was significantly higher than that infected with nonreplicative adenovirus Ad-p53 in vitro （388 ± 34.6 μg/L vs 76.3 ± 13.17 μg/L）. Viral replication experiments showed that replication of CNHK500-p53 and CNHK500 or WtAd5, was much stronger than that of Ad-p53 in tested HCC cell lines. CPE and H1-F assay indicated that CNHK500-p53 selectively replicated in and killed HCC cells while leaving normal cells unaffected. CONCLUSION： A more efficient gene-viral system is developed by combining selective oncolysis with exogenous expression of p53 against HCC cells.展开更多
Objective: To evaluate the therapeutic efficacy of replicative adenovirus CNHK500 in the treatment of hepatocellular carcinoma. Methods: Virus proliferation assay, cell viability assay and Western blot were performed ...Objective: To evaluate the therapeutic efficacy of replicative adenovirus CNHK500 in the treatment of hepatocellular carcinoma. Methods: Virus proliferation assay, cell viability assay and Western blot were performed to assess the selective replication and cytolysis of CNHK500 in telomerase positive liver cancer cells Hep3B, HepGII, SMMC7721 and in normal cells. Results: The replicative multiples of CNHK500 in HepGII, Hep3B and SMMC7221 after 96 h of virus proliferation were 52 000, 396 984.9 and 632 911.3 fold respectively, similar to those of wtAd5. However, CNHK500 demonstrated more significant attenuated replicative ability in normal cell lines than wtAd5. CNHK500 replicated only 3.1-100 fold at 96 h, while the wtAd5 still reached 3160-17 357 fold. CNHK500 could cause half of HepGII cells death within 7 days at MOI 2, in Hep3B cell lines the IC50 was as low as MOI 0.01, whereas the IC50 in BJ cell was as high as MOI 1000. CNHK500 E1A protein could only be detected in hepatocellular cancer cells but not in normal cells under normoxia. E1B protein could only be detected under hypoxia condition at a MOI of 1. Conclusion: CNHK500 can efficiently replicate in and kill liver cancer cells as well as wtAd5 do while it is severely attenuated in proliferation and cytolysis among normal cells. It would be a prominsing strategy for liver cancer tratment.展开更多
Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various...Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various breast cancer and normal fibroblast cell lines. Virus proliferation assay, cell viability assay and Western blot were applied to evaluate the proliferation and cytolysis selectivity of CNHK300. Results： The telomerase activity of MCF-7, BT-549 and SK-BR-3 was positive, while telomerase in MRC-5 and BJ was negative. The progeny virus titers in MCF-7, BT-549 and SK-BR-3 after 48 h of CNHK300 exposure was 40 625, 1 265 and 20 000 fold higher than those of 0 h, even slightly higher than those of wtAd5 （except in SK-BR-3）. ONYX-015 virus proliferation ability was weaker than that of CNHK300 in cancer cells. However, CNHK300 exhibited attenuated replicative ability as compared with wtAd5 in MRC-5 and BJ. The CNHK300 replicatative multiple was 63 and 192 fold at 48 h respectively, while the wtAd5 still multiplied 3 160-4 846 fold. CNHK300 could cause about half of breast cancer cells to die within 7 days at MOI 10 pfu/cell and below, whereas the IC50 in BJ and MRC-5 was as high as MOI 100 pfu/cell. CNHK300 E1A protein could be detected in breast cancer cells and 293 cells but not in normal fibroblast cells. Conclusion： hTERT promoter can successfully modulate the CNHK300 to be selectively replicated in breast cancer cells positive for telomerase, which may be a potential treatment strategy in breast cancer.展开更多
Objective:Oncolytic adenovirus,also called conditionally replicating adenovirus(CRAD),has been developed for the treatment of cancer.However,there is a tremendous need to enhance their antitumor efficacy.Here we wish ...Objective:Oncolytic adenovirus,also called conditionally replicating adenovirus(CRAD),has been developed for the treatment of cancer.However,there is a tremendous need to enhance their antitumor efficacy.Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage.Methods:A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase(hTERT),the coding sequence of E1A gene,an internal ribosome entry site sequence(IRES)and the coding sequence of herpes simplex virus-thymidine kinase(HSV-TK)was constructed.Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK.The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured.The infection efficiency was determined by flow cytometry.Results:Ad-ETK deliverys E1A and HSV-TK gene,which selectively replicates in hTERT-positive tumor cells,and the progeny virus can reach up to 150 IU/cell.Our in vitro study showed that Ad-ETK plus ganciclovir(GCV)induced an obvious cell death.Conclusion:An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.展开更多
Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good antitumor and antimetastasis effect. However, clinically applying M4 faces ...Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good antitumor and antimetastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in Ⅰ–Ⅲ phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.展开更多
Objective： To investigate the effect of RNAj-mediated Survivin gene with conditionally replicating adenovirus silencing on Survivin gene in colon carcinoma cell lines HT-29 lastingly. Methods： We transfected Ad-delE...Objective： To investigate the effect of RNAj-mediated Survivin gene with conditionally replicating adenovirus silencing on Survivin gene in colon carcinoma cell lines HT-29 lastingly. Methods： We transfected Ad-delE1655KD-shRNA / Survivin-EGFP to HT-29 （control was replication defective adenovirus and liposome vector which was contained the same shRNA as Ad-delE1b55KD-shRNA / Survivin-EGFP）. The expressions of EGFP, Survivin mRNA and Survivin protein in HT- 29 were detected at the 1st, 7th, 14th and 28th days alter transfection. Results： The expression of EGFP, the inhibition of Survivin mRNA and Survivin protein in HT-29 were high in each group at the 7th day alter transfection, among the total, the effect of Ad-delE1655KD-shRNA/Survivin-EGFP group was the highest; at the 14th day, the effects of replication defective adenovirus group and liposome vector group were decreased obviously, and it was still high in Ad-delE1655KD-shRNA / Survivin-EGFP group; at the 28th day, the effects of control groups were disappeared, and it was still high in Ad-delE1b55KD- shRNA/Survivin-EGFP group like before （P 〈 0.05）. Conclusion： RNAi-mediated Survivin gene with conditionally replicating adenovirus can silence Survivin gene in colon carcinoma cell lines HT-29 lastingly.展开更多
AIM： To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatoceUular carcinoma.METHODS： CNHK300, ONYX-015 （55 kDa protein deleted adenovirus） and wtAd5 （wild...AIM： To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatoceUular carcinoma.METHODS： CNHK300, ONYX-015 （55 kDa protein deleted adenovirus） and wtAd5 （wild type adenovirus 5） were compared, and virus proliferation assay, cell viability assay, Western blot and fluorescence microscopy were used to evaluate the proliferation and cytolysis selectivity of CNHK300.RESULTS： The replicative multiples in Hep3B and HepG Ⅰ after 48 Ⅱ of CNHK300 proliferation were 40625 and 65326 fold, respectively, similar to that of wtAd5.. However, CNHK300 exhibited attenuated replicative ability in normal fibroblast cell line BJ. CNHK300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection （MOI）, but could not affect growth of normal cells even at a high MOI.CONCLUSION： CNHK300 is a cancer-selective replication-competent adenovirus which can cause oncolysis of liver cancer cells as well as wtAd5 （wild type adenovirus 5）, but had severely attenuated replicative and cytolytic ability in normal cells. This novel strategy of cancer treatment offers a promising treatment platform.展开更多
Objective： To develop a novel adenoviral vector system, which combines the advantages of the antiangiogenic gene therapy and virus therapy, and to investigate its antitumor activity on lung cancer. Methods： A new ki...Objective： To develop a novel adenoviral vector system, which combines the advantages of the antiangiogenic gene therapy and virus therapy, and to investigate its antitumor activity on lung cancer. Methods： A new kind of viral vector CNHK200, in which the Elb55kDa gene was deleted and the whole Ela gene was preserved, was constructed. Human angiostatin gene Kringle 1-5 （hA） was amplified and inserted into the genome of the replicative virus CNHK200, generating CNHK200-hA. The expression of hA and its effect on lung cancer cell growth in vitro and in vivo were studied. Results： The novel vector system CNHK200-hA, just like the replicative virus ONYX-015, replicated in p53-deficient tumor cells but not in normal cells, and thus specifically killed tumor cells. In in vitro experiment, both CNHK200-hA and the non-replicative virus Ad-hA could kill tumor cells but the latter needed 100 times more MOI to achieve the same level of cell killing. In in vivo experiment, the therapeutic effect of CNHK200-hA on human lung cancer A549 xenografts in nude mice was significantly better than that of Ad-hA or that of ONYX-015. Conclusion： CNHK200-hA, which carries the angiostatin gene, has the advantages of specific tumor targeting, high expression of transgene in tumor cells and potent antitumor activity.展开更多
Trans-neuronal viral tracing is becoming one of the most important methods for mapping neuronal circuit connections,but an anterograde trans-synaptic tracer compatible with functional assays is still lacking.Adeno-ass...Trans-neuronal viral tracing is becoming one of the most important methods for mapping neuronal circuit connections,but an anterograde trans-synaptic tracer compatible with functional assays is still lacking.Adeno-associated virus（AAV）is replication-defective,and its production of offspring needs the help of adenovirus（AV）[1]or herpesvirus co-infection[2].The replication deficiency,non-pathogenicity,easy manipulability,展开更多
基金Supported by the Major State Basic Research Development Program (973 Program) of China, No. 2003CB515507
文摘AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepatocellular carcinoma （HCC）. METHODS： CNHK500-p53 was constructed by using human telomerase reverse transcriptase （hTERT） promoter to drive adenovirus E1a gene and hypoxia response element （HRE） promoter to drive adenovirus E1b gene. p53 gene expressing cassette was inserted into the genome of replicative virus. Viral replication experiments, cytopathic effect （CPE） and methyl thiazolyl tetrazolium （MTT） assay were performed to test the selective replication and oncolytic efficacy of CNHK500-p53. RESULTS： Immunohistochemistry verified that infection with CNHK500-p53 was associated with selective replication of adenovirus and production of p53 protein in telomerase-positive and hypoxia-inducible factordependent HCC cells, p53 protein secreted from HepG2, infected with CNHK500-p53 was significantly higher than that infected with nonreplicative adenovirus Ad-p53 in vitro （388 ± 34.6 μg/L vs 76.3 ± 13.17 μg/L）. Viral replication experiments showed that replication of CNHK500-p53 and CNHK500 or WtAd5, was much stronger than that of Ad-p53 in tested HCC cell lines. CPE and H1-F assay indicated that CNHK500-p53 selectively replicated in and killed HCC cells while leaving normal cells unaffected. CONCLUSION： A more efficient gene-viral system is developed by combining selective oncolysis with exogenous expression of p53 against HCC cells.
基金This work is supported by International Cooperation Important Project of National Natural Science Foundation of China(No.30120160824)the State 863 High Technology R&D Project of China(No.2001AA217031).
文摘Objective: To evaluate the therapeutic efficacy of replicative adenovirus CNHK500 in the treatment of hepatocellular carcinoma. Methods: Virus proliferation assay, cell viability assay and Western blot were performed to assess the selective replication and cytolysis of CNHK500 in telomerase positive liver cancer cells Hep3B, HepGII, SMMC7721 and in normal cells. Results: The replicative multiples of CNHK500 in HepGII, Hep3B and SMMC7221 after 96 h of virus proliferation were 52 000, 396 984.9 and 632 911.3 fold respectively, similar to those of wtAd5. However, CNHK500 demonstrated more significant attenuated replicative ability in normal cell lines than wtAd5. CNHK500 replicated only 3.1-100 fold at 96 h, while the wtAd5 still reached 3160-17 357 fold. CNHK500 could cause half of HepGII cells death within 7 days at MOI 2, in Hep3B cell lines the IC50 was as low as MOI 0.01, whereas the IC50 in BJ cell was as high as MOI 1000. CNHK500 E1A protein could only be detected in hepatocellular cancer cells but not in normal cells under normoxia. E1B protein could only be detected under hypoxia condition at a MOI of 1. Conclusion: CNHK500 can efficiently replicate in and kill liver cancer cells as well as wtAd5 do while it is severely attenuated in proliferation and cytolysis among normal cells. It would be a prominsing strategy for liver cancer tratment.
基金This work was supported by International Cooperation Important Project of National Natural Sciences Foundation of China(No. 30120160824) and the State 863 High Technology R&D Project of China (No. 2001AA217031)
文摘Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various breast cancer and normal fibroblast cell lines. Virus proliferation assay, cell viability assay and Western blot were applied to evaluate the proliferation and cytolysis selectivity of CNHK300. Results： The telomerase activity of MCF-7, BT-549 and SK-BR-3 was positive, while telomerase in MRC-5 and BJ was negative. The progeny virus titers in MCF-7, BT-549 and SK-BR-3 after 48 h of CNHK300 exposure was 40 625, 1 265 and 20 000 fold higher than those of 0 h, even slightly higher than those of wtAd5 （except in SK-BR-3）. ONYX-015 virus proliferation ability was weaker than that of CNHK300 in cancer cells. However, CNHK300 exhibited attenuated replicative ability as compared with wtAd5 in MRC-5 and BJ. The CNHK300 replicatative multiple was 63 and 192 fold at 48 h respectively, while the wtAd5 still multiplied 3 160-4 846 fold. CNHK300 could cause about half of breast cancer cells to die within 7 days at MOI 10 pfu/cell and below, whereas the IC50 in BJ and MRC-5 was as high as MOI 100 pfu/cell. CNHK300 E1A protein could be detected in breast cancer cells and 293 cells but not in normal fibroblast cells. Conclusion： hTERT promoter can successfully modulate the CNHK300 to be selectively replicated in breast cancer cells positive for telomerase, which may be a potential treatment strategy in breast cancer.
文摘Objective:Oncolytic adenovirus,also called conditionally replicating adenovirus(CRAD),has been developed for the treatment of cancer.However,there is a tremendous need to enhance their antitumor efficacy.Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage.Methods:A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase(hTERT),the coding sequence of E1A gene,an internal ribosome entry site sequence(IRES)and the coding sequence of herpes simplex virus-thymidine kinase(HSV-TK)was constructed.Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK.The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured.The infection efficiency was determined by flow cytometry.Results:Ad-ETK deliverys E1A and HSV-TK gene,which selectively replicates in hTERT-positive tumor cells,and the progeny virus can reach up to 150 IU/cell.Our in vitro study showed that Ad-ETK plus ganciclovir(GCV)induced an obvious cell death.Conclusion:An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.
基金supported by grants from National Natural Science Foundation of China(No.81072135)the "973" Program of China (No.2009CB521800)
文摘Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good antitumor and antimetastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in Ⅰ–Ⅲ phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.
基金a grant from the Nature Sciences Foundation of Chongqing (No. 2005BB5225)
文摘Objective： To investigate the effect of RNAj-mediated Survivin gene with conditionally replicating adenovirus silencing on Survivin gene in colon carcinoma cell lines HT-29 lastingly. Methods： We transfected Ad-delE1655KD-shRNA / Survivin-EGFP to HT-29 （control was replication defective adenovirus and liposome vector which was contained the same shRNA as Ad-delE1b55KD-shRNA / Survivin-EGFP）. The expressions of EGFP, Survivin mRNA and Survivin protein in HT- 29 were detected at the 1st, 7th, 14th and 28th days alter transfection. Results： The expression of EGFP, the inhibition of Survivin mRNA and Survivin protein in HT-29 were high in each group at the 7th day alter transfection, among the total, the effect of Ad-delE1655KD-shRNA/Survivin-EGFP group was the highest; at the 14th day, the effects of replication defective adenovirus group and liposome vector group were decreased obviously, and it was still high in Ad-delE1655KD-shRNA / Survivin-EGFP group; at the 28th day, the effects of control groups were disappeared, and it was still high in Ad-delE1b55KD- shRNA/Survivin-EGFP group like before （P 〈 0.05）. Conclusion： RNAi-mediated Survivin gene with conditionally replicating adenovirus can silence Survivin gene in colon carcinoma cell lines HT-29 lastingly.
基金International Cooperative Key Project of the National Natural Science Foundation of China, No. 30120160824the State 863 High Technology R&D Project of China, No. 2001AA217031
文摘AIM： To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatoceUular carcinoma.METHODS： CNHK300, ONYX-015 （55 kDa protein deleted adenovirus） and wtAd5 （wild type adenovirus 5） were compared, and virus proliferation assay, cell viability assay, Western blot and fluorescence microscopy were used to evaluate the proliferation and cytolysis selectivity of CNHK300.RESULTS： The replicative multiples in Hep3B and HepG Ⅰ after 48 Ⅱ of CNHK300 proliferation were 40625 and 65326 fold, respectively, similar to that of wtAd5.. However, CNHK300 exhibited attenuated replicative ability in normal fibroblast cell line BJ. CNHK300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection （MOI）, but could not affect growth of normal cells even at a high MOI.CONCLUSION： CNHK300 is a cancer-selective replication-competent adenovirus which can cause oncolysis of liver cancer cells as well as wtAd5 （wild type adenovirus 5）, but had severely attenuated replicative and cytolytic ability in normal cells. This novel strategy of cancer treatment offers a promising treatment platform.
基金This work was supported by the NationalNatural Sciences Foundation of China (No.30572149) andthe National "863" High Technology R & D Project ofChina (No. 2003AA216030).
文摘Objective： To develop a novel adenoviral vector system, which combines the advantages of the antiangiogenic gene therapy and virus therapy, and to investigate its antitumor activity on lung cancer. Methods： A new kind of viral vector CNHK200, in which the Elb55kDa gene was deleted and the whole Ela gene was preserved, was constructed. Human angiostatin gene Kringle 1-5 （hA） was amplified and inserted into the genome of the replicative virus CNHK200, generating CNHK200-hA. The expression of hA and its effect on lung cancer cell growth in vitro and in vivo were studied. Results： The novel vector system CNHK200-hA, just like the replicative virus ONYX-015, replicated in p53-deficient tumor cells but not in normal cells, and thus specifically killed tumor cells. In in vitro experiment, both CNHK200-hA and the non-replicative virus Ad-hA could kill tumor cells but the latter needed 100 times more MOI to achieve the same level of cell killing. In in vivo experiment, the therapeutic effect of CNHK200-hA on human lung cancer A549 xenografts in nude mice was significantly better than that of Ad-hA or that of ONYX-015. Conclusion： CNHK200-hA, which carries the angiostatin gene, has the advantages of specific tumor targeting, high expression of transgene in tumor cells and potent antitumor activity.
文摘Trans-neuronal viral tracing is becoming one of the most important methods for mapping neuronal circuit connections,but an anterograde trans-synaptic tracer compatible with functional assays is still lacking.Adeno-associated virus（AAV）is replication-defective,and its production of offspring needs the help of adenovirus（AV）[1]or herpesvirus co-infection[2].The replication deficiency,non-pathogenicity,easy manipulability,
