BACKGROUND: In gene therapy, one of the most important issues is the choice of the vectors. pHrneo is a human-derived vector previously constructed by our group, which can target a foreign gene into a human ribosomal DNA (hrDNA) locus. METHODS AND RESULTS: In this study, we inserted an expression cassette of reconstructive hFVIII (hFVIII-BDDAK39) to pHrneo to construct a targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HL7702 cells (human hepatocyte), we identified the homologous recombinants using polymerase chain reaction, and tested the expression of hFVIII-BDDAK39 located at the hrDNA locus. The hFVIII-BDDAK39 was successfully targeted into the hrDNA locus of HL7702 by pHrneo-BDDAK39, and the efficiency of site-specific integration was 1.1 x 10(-5). The hFVIII-BDDAK39 at the hrDNA locus of HL7702 was found to be able to express efficiently (4.3 +/- 0.9 ng 10(-6) cells 24 h(-1)). CONCLUSION: It has been indicated that the targeting vector pHrneo-BDDAK39 can be used in gene therapy for hemophilia A.
BACKGROUND: In gene therapy, one of the most important issues is the choice of the vectors. pHrneo is a human-derived vector previously constructed by our group, which can target a foreign gene into a human ribosomal DNA (hrDNA) locus. METHODS AND RESULTS: In this study, we inserted an expression cassette of reconstructive hFVIII (hFVIII-BDDAK39) to pHrneo to construct a targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HL7702 cells (human hepatocyte), we identified the homologous recombinants using polymerase chain reaction, and tested the expression of hFVIII-BDDAK39 located at the hrDNA locus. The hFVIII-BDDAK39 was successfully targeted into the hrDNA locus of HL7702 by pHrneo-BDDAK39, and the efficiency of site-specific integration was 1.1 x 10(-5). The hFVIII-BDDAK39 at the hrDNA locus of HL7702 was found to be able to express efficiently (4.3 +/- 0.9 ng 10(-6) cells 24 h(-1)). CONCLUSION: It has been indicated that the targeting vector pHrneo-BDDAK39 can be used in gene therapy for hemophilia A.