Lentiviral Vector Mediated Gene Therapy For Hemophilia B

Hemophilia B(HB)is an X-linked recessive disorder caused by a mutation in the F? gene which leads to the dysfunction of activated coagulation factor IX(F?).In coagulation cascade,F? plays a role in intrinsic coagulation pathway and converts the prothrombin to thrombin.Current protein replacement therapy uses extended half-life rF? protein to treat HB patients but the product incurs high cost and requires life-time application.Stem cell transplantation is an alternative treatment for hemophilia B.Stem cells extracted from patients are transduced to express F? proteins and transplanted back to patients.Mesenchymal stem cells(MSCs)have also been extensively studied in cell therapy with wide differentiation potential and easy access.We propose to investigate F? gene therapy based on MSCs using an advanced lentiviral vector system(NHP/TYF)and tissue specific promoters to drive the expression of a codon-optimized F? gene(optF?),and evaluate the expression and function of the tissue specific gene therapy approach.In this study,LV-EF1?-F? was first constructed to investigate the effect of two human MSCs(FT902 and 1207-9)to express exogenous F? protein.Expression results showed the effective expression and secretion of active F? produced by MSC-EF1?-F?.Results of post-translational modification indicated the complete glycosylation of F? in MSCs but the?-carboxylation was inefficient,and thus,the coagulation activity was reduced.In view of this,endothelial tissue-specific promoters VEC and KDR were selected to regulate F? expression.Endothelium-specific LVs,LV-VEC-F? and LV-KDR-F? were constructed by replacing the EF1?sequence with VEC and KDR,and used to transduce FT902 and 1207-9.F? ELISA showed that EF1?,VEC and KDR expressed F? in amounts of 1300~1500 ng/10~6/24h,200~500 ng/10~6/24h,and 500~800 ng/10~6/24h,respectively.The F? functional activity assays showed the same trend,and the relative activities were~10%,~2%,and~5%of the normal blood coagulation activity,respectively.It is of interest to observe that the activities of F? driven by VEC and KDR promoters reduced the expression of F? and were independent of Vitamin K.The regulation of VEC and KDR promoters improved the F? expression stability of MSC and the specific expression in endothelial tissue could reduce the risk of inducing an immune response,which supports the potential application of MSC-mediated cell transplantation in the treatment of hemophilia B.