Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitros...Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT- PCR method from total RNA of fresh human liven the fragment was cloned into PGEM-T vector and further subcloned into GINa retrovirus vector. Then the GINaMGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8x105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT- cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.展开更多
This work reports the investigation of the effect of airway IL-4RA gene transfer by a recombinant retroviral vector on airway inflammation and airway responsiveness in asthmatic mice. The retrovirus-mediated delivery ...This work reports the investigation of the effect of airway IL-4RA gene transfer by a recombinant retroviral vector on airway inflammation and airway responsiveness in asthmatic mice. The retrovirus-mediated delivery of IL-4RA to the airways of mice inhibited elevations of airway responsiveness and the development of allergic inflammation in asthmatic mice, and regulated the Th1/Th2 balance in OVA-sensitized and -challenged mouse models. This suggests that gene therapy is a therapeutic option for treating and controlling chronic airway inflammation and asthma symptoms.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 explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT- PCR method from total RNA of fresh human liven the fragment was cloned into PGEM-T vector and further subcloned into GINa retrovirus vector. Then the GINaMGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8x105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT- cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.
文摘This work reports the investigation of the effect of airway IL-4RA gene transfer by a recombinant retroviral vector on airway inflammation and airway responsiveness in asthmatic mice. The retrovirus-mediated delivery of IL-4RA to the airways of mice inhibited elevations of airway responsiveness and the development of allergic inflammation in asthmatic mice, and regulated the Th1/Th2 balance in OVA-sensitized and -challenged mouse models. This suggests that gene therapy is a therapeutic option for treating and controlling chronic airway inflammation and asthma symptoms.
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.
