Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retro...Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retrovirus vectors expressing B7-1/B7-2 were constructed by gene cloning technology to produce retrovirus-infected PE501 and PA317 cell lines and murine hepatoma Hepal-6. The expression of R7-l/B7-2 was detected by fluorescence activated cell soning analysis (FACS). B7-l/B7-2 positive Hepal-6 Cell lines were used in inducing anti-hepatoma immunity in ho and in the. Results: In contrast to the excessive growth of parental Hemal-6 tumor, the growth of B7-l/B7-2-positive Hepal-6 inoculated into syngenic mice regressed. B7-1/R7-2-positive or cytokine-treated Hepal-6 alone could only induce mild cytototicity; in contrast, B7-1/B7-2-positive Hemal-6 treated with cytokine-stimulated spleen cells and activated the cytotoxicity effectively. Immunity in mice with R7-1/B7-2-positive tumor cells or cytokine-beated Hepal-6 only provided partial protection against parental Hepa1-6 tumor, whereas pretreatment of the transfected tumor cells with IFN-r and TNF-a induced complete immunity protection in vivo. Mice receiving inoculation of cytokine-treated B7-l/R7-2-positive Hemal-6 cells presented regression of the establoshed pental tUmor and survived for more than l00 d, while those untreated mice died within 40 d. Conclu sions: B7-l/R7-2 expression is necessary but not sufficient in inducing anti-hepatoma immune response, whereas it is efficient when combined with the beatment of IFN-γ and TNF-a.展开更多
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In the past few decades, many efforts have been made to improve the prognosis of GBM, however, with limited success. Many ...Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In the past few decades, many efforts have been made to improve the prognosis of GBM, however, with limited success. Many gene therapy strategies for GBM have been developed and a few have progressed to clinical trials. Retroviral vectors have superior features for gene therapy in brain cancers, including tumor specificity, immunogenicity, and longer half-life. Early gene therapy trials in GBM patients based on transplantation of retrovirus-producing cells into the brain failed to prove efficacious. Adenoviral vectors, which can be prepared as high-titer virus solutions and undergo efficient transduction in tumor cells, failed in clinical trials, likely due to immunogenicity and instability of gene expression. Alternative therapeutics such as oncolytic viruses that specifically target and destroy cancer cells are currently under investigation. In addition to novel vectors, retroviral vectors are still attractive candidates for use in gene therapy against brain tumors. Since yields of properly-packaged viral particles from virus-producing cells have been very limited so far, gene therapy by direct injection of hightiter retroviral vectors into the patients’ brains was not possible. To overcome these disadvantages, a packaging cell line that yields high-titer retroviral solutions was established by our group, enabling the direct injection of massive retroviral vector stocks directly into the brain. Mouse glioma models were effectively cured with a combination of a suicide gene/ prodrug system and a highly-concentrated retrovirus solution. Preclinical assessments, including that of replication-competent retroviruses and tumorigenicity of the combination method, have confirmed the safety of the highly-concentrated retrovirus solution. Addi tional studies are needed to address the clinical utility of such combination gene therapies. Taken together, these data suggest that retroviral vectors are still good candidates for development in gene therapy applications.展开更多
Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (Ne...Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.展开更多
Objective： To construct recombinant Apoptin gene （vp3） retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods...Objective： To construct recombinant Apoptin gene （vp3） retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods： vp3 gene was cloned and recombinated into retrovirus vector pLP-LNCX-VP3 （pLVP3） at loxP site, which was transformed into package cell line PT67 and then into NIH3T3 cells for titer assay. The human breast cancer cell line 435 was infected with retrovirus pLVP3, and then MTT assay and Western blotting were adopted to detect cellular proliferation and Apoptin protein expression. Forty-eight hours after infection flow cytometry （FCM） was used for apoptosis detection and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry （SELDI-TOF-MS） was used for protein profile assay. Nude mice model of human breast cancer cells 435 was set up to observe the tumor inhibiting rates of pLVP3, and TUNEL assay was used to detect tumor apoptosis as well as real-time PCR for vp3 gene expression. Results： Recombinant plasmid pLVP3 was successfully constructed. Virus titer reached to 5×10^8 pfu/ml in the PT67 culture supernatant. Forty-eight hours after infection, cellular inhibition rate was 65.1% in MTT assay, higher than that in blank control （P〈0.05） and Apoptin protein expressed more in test group in Western blotting. FCM assay showed apoptotic peaks with a percentage of 15.42%. SELDI-TOF-MS findings suggested that two protein peaks, M_2544.1＋H and M_3712.4＋H, were statistically different between infection group and control group （P〈0.05）. The tumor inhibition rates in pLVP3 group were 65.52% and 68.23%, much higher than that of control group （t=4.06, P〈0.01）. TUNEL assay findings showed that positive yellow stains were seen in pLVP3 retrovirus group and 5-FU positive control group without difference （t1=1.05, t2=0.84, P〉0.05）. Conclusion： The experiment demonstrated that vp3 could induce apoptosis in tumor cells in vivo and in vitro, which laid a basis for further study on molecular mechanism of tumor cell apoptosis induced by Apoptin and provided valuable reference for tumor gene therapy.展开更多
Tumor necrosis factor (TNF) has certain radioprotective effect on host tissue and is capable of enhancing the antitumor effect of radiotherapy. In addition, the transcriptional regulation of the promoter region of Eg...Tumor necrosis factor (TNF) has certain radioprotective effect on host tissue and is capable of enhancing the antitumor effect of radiotherapy. In addition, the transcriptional regulation of the promoter region of Egr 1 gene is activated by ionizing radiation. So we fused Egr 1 promoter with hTNF α cDNA, and resultantly constructed a double copy and radiationin ducible retroviral vector named as pETDC. After packaged with Psi 2 and Crip cells in vitro, the hTNF recombinant retroviruses were in the titers of 4×10 5 CFU/ml. By infection of murine fibroblast cell line NIH3T3 and murine melanoma cell line B16.F10 with the recombinant retroviruses and followed by G418 resistant selection, two positive clones secreting TNF at the levels of 2.1 ng/ml and 1.1 ng/ml respectively were generated. After exposure to 20 Gy ionizing radiation, TNF secre tions from the two positive clones were elevated to 13.8 ng/ml (6.6 fold) and 5.7 ng/ml (5.2 fold) respectively. Furthermore, hTNF α expression in pETDC transfected cells was confirmed by RT PCR. These data provide an experimental bases for the application of TNF gene therapy combined with local radiotherapy in cancer patients.展开更多
Objective: To establish a retroviral mediated suicide gene therapy system for experimental glioma and test its efficacy. Methods: C6 rat glioma cells were infected with recombinant retrovirus containing HSV tk gene...Objective: To establish a retroviral mediated suicide gene therapy system for experimental glioma and test its efficacy. Methods: C6 rat glioma cells were infected with recombinant retrovirus containing HSV tk gene. The C6/tk cell line which stably expressed tk was selected and cloned. The sensitivities of C6/tk cells to several nucleoside analogues, such as GCV, BVdU, ACV were compared by the growth inhibition studies. Antitumor effects were also observed after GCV treatment in nude mice bearing tumors derived from C6/tk cells. Results: The growth inhibition studies showed that GCV was the most efficient prodrug in this system. C6/tk cells were highly sensitive to GCV, with an IC 50 <0.2 μmol/L, being 500 fold less than that in tk negative C6 cells. In vivo studies showed significant tumor inhibition in the treatment group. Conclusion: Glioma cells can be eradicated by using retroviral mediated suicide gene system in vitro as well as in vivo .展开更多
We have confirmed efficient anti-tumor activities of the peripheral lymphocytes transduced with a p185HEH2-specific chimeric T-cell receptor gene both in murine and in human in our previous studies. To further test th...We have confirmed efficient anti-tumor activities of the peripheral lymphocytes transduced with a p185HEH2-specific chimeric T-cell receptor gene both in murine and in human in our previous studies. To further test the feasibility of chimeric T-cell receptor in a bone marrow transplantation model, we first, made two routine tumor cell lines： MT901 and MCA-205, to express human p185HER2 by retroviral gene transduction. Murine bone marrow cells were retrovirally transduced to express the chimeric T-cell receptor and gene-modified bone marrow cells were transplanted into lethally irradiated mouse. Six months post transplantation, p185HER2-positive tumor ceils： MT-901/HER2 or MCA-205/ HER2 was subcutaneously or intravenously injected to make mouse models simulating primary breast cancer or pulmonary metastasis. The in vivo anti-tumor effects were monitored by the size of the subcutaneous tumor or counting the tumor nodules in the lungs after India ink staining. The size of the subcutaneous tumor was significantly inhibited and the number of pulmonary nodules were significantly decreased in mouse recipients transplanted with chimeric T-cell receptor modified bone marrow cells compared with the control group. Our results suggest the efficient in vivo anti-tumor activities of chimeric T-cell receptor gene modified bone marrow cells.展开更多
Mounting evidence has demonstrated that CD4^(+)T cells play an important role in anti-tumor immune responses.Thus,adoptive transfer of these cells may have great potential for anti-cancer therapy.However,due to the di...Mounting evidence has demonstrated that CD4^(+)T cells play an important role in anti-tumor immune responses.Thus,adoptive transfer of these cells may have great potential for anti-cancer therapy.However,due to the difficulty to generate sufficient tumor-specific CD4^(+)T cells,the use of CD4^(+)T cells in tumor therapy is limited.It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8+Tcells,but the effect of IL-15 transfection on CD4^(+)T cells remains unknown.Here,the effects of retrovirusmediated IL-15 expression in Ova-specific CD4^(+)T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4^(+)T cells expressed both soluble and membrane-bound IL-15.Retrovirusmediated IL-15 expression led to a selective expansion of antigen-specific CD4^(+)T cells by inhibiting their apoptosis.In vivo IL-15 transfected CD4^(+)T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones.To ensure the safety of the method,the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4^(+)T cells following ganciclovir treatment.Together,we show that IL-15 transfection induced a selective expansion of antigen-specific CD4^(+)T cells ex vivo and enhanced their tumor-suppression effects in vivo.This has an important significance for improving the efficacy of adoptive T cell therapy.展开更多
文摘Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retrovirus vectors expressing B7-1/B7-2 were constructed by gene cloning technology to produce retrovirus-infected PE501 and PA317 cell lines and murine hepatoma Hepal-6. The expression of R7-l/B7-2 was detected by fluorescence activated cell soning analysis (FACS). B7-l/B7-2 positive Hepal-6 Cell lines were used in inducing anti-hepatoma immunity in ho and in the. Results: In contrast to the excessive growth of parental Hemal-6 tumor, the growth of B7-l/B7-2-positive Hepal-6 inoculated into syngenic mice regressed. B7-1/R7-2-positive or cytokine-treated Hepal-6 alone could only induce mild cytototicity; in contrast, B7-1/B7-2-positive Hemal-6 treated with cytokine-stimulated spleen cells and activated the cytotoxicity effectively. Immunity in mice with R7-1/B7-2-positive tumor cells or cytokine-beated Hepal-6 only provided partial protection against parental Hepa1-6 tumor, whereas pretreatment of the transfected tumor cells with IFN-r and TNF-a induced complete immunity protection in vivo. Mice receiving inoculation of cytokine-treated B7-l/R7-2-positive Hemal-6 cells presented regression of the establoshed pental tUmor and survived for more than l00 d, while those untreated mice died within 40 d. Conclu sions: B7-l/R7-2 expression is necessary but not sufficient in inducing anti-hepatoma immune response, whereas it is efficient when combined with the beatment of IFN-γ and TNF-a.
文摘Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In the past few decades, many efforts have been made to improve the prognosis of GBM, however, with limited success. Many gene therapy strategies for GBM have been developed and a few have progressed to clinical trials. Retroviral vectors have superior features for gene therapy in brain cancers, including tumor specificity, immunogenicity, and longer half-life. Early gene therapy trials in GBM patients based on transplantation of retrovirus-producing cells into the brain failed to prove efficacious. Adenoviral vectors, which can be prepared as high-titer virus solutions and undergo efficient transduction in tumor cells, failed in clinical trials, likely due to immunogenicity and instability of gene expression. Alternative therapeutics such as oncolytic viruses that specifically target and destroy cancer cells are currently under investigation. In addition to novel vectors, retroviral vectors are still attractive candidates for use in gene therapy against brain tumors. Since yields of properly-packaged viral particles from virus-producing cells have been very limited so far, gene therapy by direct injection of hightiter retroviral vectors into the patients’ brains was not possible. To overcome these disadvantages, a packaging cell line that yields high-titer retroviral solutions was established by our group, enabling the direct injection of massive retroviral vector stocks directly into the brain. Mouse glioma models were effectively cured with a combination of a suicide gene/ prodrug system and a highly-concentrated retrovirus solution. Preclinical assessments, including that of replication-competent retroviruses and tumorigenicity of the combination method, have confirmed the safety of the highly-concentrated retrovirus solution. Addi tional studies are needed to address the clinical utility of such combination gene therapies. Taken together, these data suggest that retroviral vectors are still good candidates for development in gene therapy applications.
文摘Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.
文摘Objective： To construct recombinant Apoptin gene （vp3） retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods： vp3 gene was cloned and recombinated into retrovirus vector pLP-LNCX-VP3 （pLVP3） at loxP site, which was transformed into package cell line PT67 and then into NIH3T3 cells for titer assay. The human breast cancer cell line 435 was infected with retrovirus pLVP3, and then MTT assay and Western blotting were adopted to detect cellular proliferation and Apoptin protein expression. Forty-eight hours after infection flow cytometry （FCM） was used for apoptosis detection and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry （SELDI-TOF-MS） was used for protein profile assay. Nude mice model of human breast cancer cells 435 was set up to observe the tumor inhibiting rates of pLVP3, and TUNEL assay was used to detect tumor apoptosis as well as real-time PCR for vp3 gene expression. Results： Recombinant plasmid pLVP3 was successfully constructed. Virus titer reached to 5×10^8 pfu/ml in the PT67 culture supernatant. Forty-eight hours after infection, cellular inhibition rate was 65.1% in MTT assay, higher than that in blank control （P〈0.05） and Apoptin protein expressed more in test group in Western blotting. FCM assay showed apoptotic peaks with a percentage of 15.42%. SELDI-TOF-MS findings suggested that two protein peaks, M_2544.1＋H and M_3712.4＋H, were statistically different between infection group and control group （P〈0.05）. The tumor inhibition rates in pLVP3 group were 65.52% and 68.23%, much higher than that of control group （t=4.06, P〈0.01）. TUNEL assay findings showed that positive yellow stains were seen in pLVP3 retrovirus group and 5-FU positive control group without difference （t1=1.05, t2=0.84, P〉0.05）. Conclusion： The experiment demonstrated that vp3 could induce apoptosis in tumor cells in vivo and in vitro, which laid a basis for further study on molecular mechanism of tumor cell apoptosis induced by Apoptin and provided valuable reference for tumor gene therapy.
文摘Tumor necrosis factor (TNF) has certain radioprotective effect on host tissue and is capable of enhancing the antitumor effect of radiotherapy. In addition, the transcriptional regulation of the promoter region of Egr 1 gene is activated by ionizing radiation. So we fused Egr 1 promoter with hTNF α cDNA, and resultantly constructed a double copy and radiationin ducible retroviral vector named as pETDC. After packaged with Psi 2 and Crip cells in vitro, the hTNF recombinant retroviruses were in the titers of 4×10 5 CFU/ml. By infection of murine fibroblast cell line NIH3T3 and murine melanoma cell line B16.F10 with the recombinant retroviruses and followed by G418 resistant selection, two positive clones secreting TNF at the levels of 2.1 ng/ml and 1.1 ng/ml respectively were generated. After exposure to 20 Gy ionizing radiation, TNF secre tions from the two positive clones were elevated to 13.8 ng/ml (6.6 fold) and 5.7 ng/ml (5.2 fold) respectively. Furthermore, hTNF α expression in pETDC transfected cells was confirmed by RT PCR. These data provide an experimental bases for the application of TNF gene therapy combined with local radiotherapy in cancer patients.
文摘Objective: To establish a retroviral mediated suicide gene therapy system for experimental glioma and test its efficacy. Methods: C6 rat glioma cells were infected with recombinant retrovirus containing HSV tk gene. The C6/tk cell line which stably expressed tk was selected and cloned. The sensitivities of C6/tk cells to several nucleoside analogues, such as GCV, BVdU, ACV were compared by the growth inhibition studies. Antitumor effects were also observed after GCV treatment in nude mice bearing tumors derived from C6/tk cells. Results: The growth inhibition studies showed that GCV was the most efficient prodrug in this system. C6/tk cells were highly sensitive to GCV, with an IC 50 <0.2 μmol/L, being 500 fold less than that in tk negative C6 cells. In vivo studies showed significant tumor inhibition in the treatment group. Conclusion: Glioma cells can be eradicated by using retroviral mediated suicide gene system in vitro as well as in vivo .
文摘We have confirmed efficient anti-tumor activities of the peripheral lymphocytes transduced with a p185HEH2-specific chimeric T-cell receptor gene both in murine and in human in our previous studies. To further test the feasibility of chimeric T-cell receptor in a bone marrow transplantation model, we first, made two routine tumor cell lines： MT901 and MCA-205, to express human p185HER2 by retroviral gene transduction. Murine bone marrow cells were retrovirally transduced to express the chimeric T-cell receptor and gene-modified bone marrow cells were transplanted into lethally irradiated mouse. Six months post transplantation, p185HER2-positive tumor ceils： MT-901/HER2 or MCA-205/ HER2 was subcutaneously or intravenously injected to make mouse models simulating primary breast cancer or pulmonary metastasis. The in vivo anti-tumor effects were monitored by the size of the subcutaneous tumor or counting the tumor nodules in the lungs after India ink staining. The size of the subcutaneous tumor was significantly inhibited and the number of pulmonary nodules were significantly decreased in mouse recipients transplanted with chimeric T-cell receptor modified bone marrow cells compared with the control group. Our results suggest the efficient in vivo anti-tumor activities of chimeric T-cell receptor gene modified bone marrow cells.
基金supported by the National Basic Research Program(973 Program)(No.2006CB504304)the National Programs for High Technology Research and Development Program(863 Program)(No.2006AA02Z4B9).
文摘Mounting evidence has demonstrated that CD4^(+)T cells play an important role in anti-tumor immune responses.Thus,adoptive transfer of these cells may have great potential for anti-cancer therapy.However,due to the difficulty to generate sufficient tumor-specific CD4^(+)T cells,the use of CD4^(+)T cells in tumor therapy is limited.It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8+Tcells,but the effect of IL-15 transfection on CD4^(+)T cells remains unknown.Here,the effects of retrovirusmediated IL-15 expression in Ova-specific CD4^(+)T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4^(+)T cells expressed both soluble and membrane-bound IL-15.Retrovirusmediated IL-15 expression led to a selective expansion of antigen-specific CD4^(+)T cells by inhibiting their apoptosis.In vivo IL-15 transfected CD4^(+)T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones.To ensure the safety of the method,the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4^(+)T cells following ganciclovir treatment.Together,we show that IL-15 transfection induced a selective expansion of antigen-specific CD4^(+)T cells ex vivo and enhanced their tumor-suppression effects in vivo.This has an important significance for improving the efficacy of adoptive T cell therapy.
