High level expression of human factor VIII in mammalian cells after retroviral-mediated gene transfer.
OBJECTIVE
To develop a retroviral-mediated high efficient expression system of human coagulation factor VIII.


METHODS
The LNC-FVIIIBD retroviral vector was generated by cloning a human B-domain-deleted (760aa-1639aa) Factor VIII (FVIII) cDNA (FVIII cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FVIII in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FVIIIBD mRNA.


RESULTS
Human FVIII was expressed in all four target cells, with the highest FVIII expression observed in NIH3T3. The coagulant activity of secreted FVIII was up to 1.6 U/10(6) cells.24 hrs-1, and the FVIII antigen was 500 ng/10(6) cells.24 hrs-1. FVIII coagulant activity and antigen expressed by transduced CHO cells were 0.12 U/10(6) cells.24 hrs-1 and 62.4 ng/10(6) cells.24 hrs-1, respectively. Human FVIII expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FVIII cDNA BD in the target cells.


CONCLUSIONS
The constructed retroviral vector was able to direct high level expression of human FVIII in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.
Guo
X
X
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Wang
H
H
Chu
H
H
Wang
X
X
Qu
B
B
Li
Z
Z
Qi
Z
Z
Wang
Z
Z
D000818:Animals; D002460:Cell Line; D005169:Factor VIII / Q000235:genetics*; D022761:Gene Transfer, Horizontal; D006801:Humans; D012190:Retroviridae / Q000235:genetics*; D014158:Transcription, Genetic
D005169:Factor VIII