Modelling gene therapy for haemophilia

Gene therapy is a proposed new treatment for haemophilia, an X-linked disorder caused by a deficiency of either factor VIII or IX. However, many problems of safety, efficacy and long term expression need to be overcome before it becomes a reality. Muscle cells secrete biologically active clotting factors (Yao et al, 1994), and research from this laboratory previously described a muscle-specific expression system which expressed low levels of factor VIII (Steinbrecher et al, 1993). I tested whether this expression system would be more successful for human factor VII. Biologically active fVII was detected in cultured mouse muscle cells, and in adult C57/B16 mice 4-5 days after direct intramuscular injection in vivo (Miller et al. 1995). However, I could not determine whether the expression occurred over a longer time course due to an anti-human fVII antibody response. In an attempt to overcome this problem, murine fVII (mfVII) was expressed with either a C- or N-terminal tag. However, expression of C-terminal tagged mfVII could only be detected in transfected cells by immunofluorescence, and not by any other tested methods. When N-terminal tagged mfVII was used, biologically active mfVII was detected in vitro, but the N-terminal tag was cleaved off, precluding its use in vivo. Finally, as endothelial cells may be a suitable alternative to muscle for haemophilia gene therapy, expression levels of the CAT reporter gene, from muscle-derived promoter and enhancer elements, were compared to those from the viral SV40 early gene promoter, in both primary human umbilical vein cells and the ECV304 line. However, no improvement upon expression from the SV40 promoter was found with any of the non-viral promoter and enhancer combinations used. The results demonstrated that muscle-derived elements can drive expression of biologically active coagulation factor VII in muscle, and that this system may have the potential to correct the phenotype of fVII-deficient mice. This data also suggests that a non-viral promoter and/or enhancer could be used to control the expression of a biologically active coagulation factor in endothelial cells