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 VM Methods The LNC-FVIIIBD retroviral vector was generated by cloning a human B-domain-deleted (760aa similar to 1639aa) Factor VM (FVIII) cDNA (FVIII cD NA BID) into the retroviral vector pLNCX. Several mammalian cell lines, inc luding NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduce d with viral supernatant from the highest virus-producing PA317 clone. Anti gen and coagulant activity of human FVIII in cell culture medium were measu red 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 highes t FVIII expression observed in NIH3T3. The coagulant activity of secreted F VIII was up to 1.61.1/10(6) cells . 24 hrs(-1), and the FVIII antigen was 5 00 ng/10(6) cells.24 hrs(-1). FVIII coagulant activity and antigen expresse d 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 FVIIIcDNA BID 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 potentia l utility in the future gene therapy for Hemophilia A.