Double-copy retroviral vector containing human factor Ⅸ cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinant virus produced in PA317 was used to transduce skin fibroblasts from a hemophilia B patient. The infected cells produced high levels of biologically active human factor Ⅸ at a rate of 3420 ng/10~6 cells/24 h. These cells were embedded in a collagen matrix and implanted into the peritoneal cavity or subcutaneous space of mice. It was demonstrated that human factor Ⅸ was produced by the implants for at least 12 days in vivo, reaching a peak of 105 ng/ml plasma. Over 90% of the protein was functionally active. This technique has the potential to be developed into a new approach for gene therapy for hemophilia B. Double-copy retroviral vector containing human factor IX cDNA driven by human cytomegalovirus enhancer-promoter was constructed. The vector was introduced into the amphotropic packaging cell line PA317. The recombinant virus produced in PA317 was used to transduce skin fibroblasts from a hemophilia B patient. The infected cells produced high levels of biologically active human factor Ⅸ at a rate of 3420 ng / 10 ~ 6 cells / 24 h. These cells were embedded in a collagen matrix and implanted into the peritoneal cavity or subcutaneous space of mice. that human factor IX was produced by the implants for at least 12 days in vivo, reaching a peak of 105 ng / ml plasma. Over 90% of the protein was functionally active. This technique has the potential to be developed into a new approach for gene therapy for hemophilia B.