The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highes...The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest liters up to 8.5×108cfu .mL-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P【0.001). Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P【0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5+6.1) ng- mL11. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4±1.5)% and APTT (activated partial thromboplastin time) reduced to (43.2±7.2) s. The anti-hFIX antibody was展开更多
The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection...The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.展开更多
Hematopoietic stem cells(HSCs)are an attractive target for gene therapy,especially for inherited blood diseases.Moreover,recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for the hig...Hematopoietic stem cells(HSCs)are an attractive target for gene therapy,especially for inherited blood diseases.Moreover,recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for the high efficiency of infection.In this study,murine mononuclear cells(MNCs)were isolated from bone marrow and cultured in suspension,and then Lin−CD117+HSCs were isolated by immunomagnetic beads.During culturing,cells and colonies increased in HSCs supplied with cytokines while no change was observed in the control group without cytokines.FUXW recombinant lentiviral vectors were produced by calcium phosphate-mediated transient cotransfection infected MNCs from ICR and C57 mice.The hFIX expressions were 41.7±4.2 ng/mL and 34.5±6.6 ng/mL in supernatant on 7d.The hFIX expressions of HSCs infected by FUXW recombinant lentiviral vectors were 46.6±5.7 ng/mL(with cytokines)and 33.3±4.8 ng/mL(without cytokines)in supernatant on 7d.Results indicate that recombinant lentiviral vectors can infect murine MNCs and Lin−CD117+HSCs efficiently,and expression of the transgene can be improved when supplied with cytokines.展开更多
Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level in hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene...Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level in hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene was constructed in forwarded configuration, but the intron 1 was found spliced in virus particles by RT_PCR detection. So the inverted configuration vector G1NaPAi′IX was suggested and constructed on the basis of SNMBAIXm and transfected into PA317. Then C2C12 cells were transfected using the above virus supernatant and the G418_resistant clones were selected. PCR and RT_PCR detection found that intron 1 structure existed in C2C12 clones and retroviral particles. And the expression level of inverted vector was 3 times higher than that of forwarded vector. These results showed that the inverted configuration vector was in deed able to avoid splicing of intron 1 during the process of retroviral packaging and improved the expression level of hFIX protein.展开更多
基金This work was supported by the National "863" High-Tech Program (Grant No. Z20-02-02) the National Natural Science Foundation of China (Grant No. 39880019)+1 种基金 Shanghai Scientific Venus Program (Grant No. 98QMA1409) Huo Yingdong Foundation (Grant No. 7
文摘The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest liters up to 8.5×108cfu .mL-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P【0.001). Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P【0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5+6.1) ng- mL11. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4±1.5)% and APTT (activated partial thromboplastin time) reduced to (43.2±7.2) s. The anti-hFIX antibody was
文摘The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.
基金supported by the National Natural Science Foundation of China (No.30100102).
文摘Hematopoietic stem cells(HSCs)are an attractive target for gene therapy,especially for inherited blood diseases.Moreover,recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for the high efficiency of infection.In this study,murine mononuclear cells(MNCs)were isolated from bone marrow and cultured in suspension,and then Lin−CD117+HSCs were isolated by immunomagnetic beads.During culturing,cells and colonies increased in HSCs supplied with cytokines while no change was observed in the control group without cytokines.FUXW recombinant lentiviral vectors were produced by calcium phosphate-mediated transient cotransfection infected MNCs from ICR and C57 mice.The hFIX expressions were 41.7±4.2 ng/mL and 34.5±6.6 ng/mL in supernatant on 7d.The hFIX expressions of HSCs infected by FUXW recombinant lentiviral vectors were 46.6±5.7 ng/mL(with cytokines)and 33.3±4.8 ng/mL(without cytokines)in supernatant on 7d.Results indicate that recombinant lentiviral vectors can infect murine MNCs and Lin−CD117+HSCs efficiently,and expression of the transgene can be improved when supplied with cytokines.
文摘Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level in hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene was constructed in forwarded configuration, but the intron 1 was found spliced in virus particles by RT_PCR detection. So the inverted configuration vector G1NaPAi′IX was suggested and constructed on the basis of SNMBAIXm and transfected into PA317. Then C2C12 cells were transfected using the above virus supernatant and the G418_resistant clones were selected. PCR and RT_PCR detection found that intron 1 structure existed in C2C12 clones and retroviral particles. And the expression level of inverted vector was 3 times higher than that of forwarded vector. These results showed that the inverted configuration vector was in deed able to avoid splicing of intron 1 during the process of retroviral packaging and improved the expression level of hFIX protein.
