Non-viral ex vivo transduction of human hepatocyte cells to express factor VIII using a human ribosomal DNA-targeting vector.
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.


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)).


CONCLUSIONS
It has been indicated that the targeting vector pHrneo-BDDAK39 can be used in gene therapy for hemophilia A.
Liu
X
X
National Laboratory of Medical Genetics of China, Xiangya Hospital, Central South University, Changsha, China.
Liu
M
M
Xue
Z
Z
Pan
Q
Q
Wu
L
L
Long
Z
Z
Xia
K
K
Liang
D
D
Xia
J
J
D002460:Cell Line; D004275:DNA, Ribosomal / Q000235:genetics*; D018274:Electroporation; D005169:Factor VIII / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014161:Transduction, Genetic / Q000379:methods*
D004275:DNA, Ribosomal; D005169:Factor VIII