Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro.
The construction of the high titer and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCIX, LIXSN and LCIXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporation. Human clotting factor IX was detected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNCIX was 800,000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3 micrograms per 10(6) cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5 micrograms per 10(6) cells in 24 h in hemophilia B patients' skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral titer and expression of human FIX were increased, and the construction of retroviral vector backbone was improved and the safety was guaranteed as compared to those vectors used previously. These vectors may produce a sufficient quantity of factor IX proteins to cause the phenotypic modification for hemophilia B patients.
Lu
D
D
Institute of Genetics, Fudan University, Shanghai.
Qiu
X
X
Zheng
B
B
Qiu
X
X
Xue
J
J
D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D005164:Factor IX / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics*; D012867:Skin / Q000166:cytology
D018076:DNA, Complementary; D005164:Factor IX