High efficient transfer and expression of human clotting factor IX cDNA in cultured human primary skin fibroblasts from hemophilia B patient by retroviral vectors.
To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor IX-deficient skin fibroblasts, we constructed four retroviral vectors containing factor IX cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human fibrosarcoma cell line, HT1080 cells, was used to assay the factor IX-virus titers of these four virus-producing PA317 cells, which ranged from 2 x 10(4) to 5 x 10(5) cfu/ml. The factor IX proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor IX at the rate of 584 ng/10(6) cells/24 h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor IX than LTR promoter. The highest expressed retroviral vector XL-IX was used to infect a line of factor IX-deficient human primary skin fibroblasts, FDIX cells. The factor IX secretion rate of the infected FDIX cells was about 549 ng/10(6) cells/24 h and over 75% of secreted factor IX was biologically active. We are convinced that this factor IX-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.
Dai
Y F
YF
Institute of Genetics, Fudan University, Shanghai, PRC.
Qiu
X F
XF
Xue
J L
JL
Liu
Z D
ZD
D000328:Adult; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004560:Electricity; D005164:Factor IX / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000473:pathology; D005822:Genetic Vectors; D002836:Hemophilia B / Q000473:pathology*; D006801:Humans; D012190:Retroviridae / Q000235:genetics; D012867:Skin / Q000473:pathology*; D014162:Transfection
D004274:DNA, Recombinant; D005164:Factor IX; D004247:DNA