Lentiviral Vector Mediated Gene Therapy For Hemophilia B

Hemophilia B(HB)is an X-linked recessive disorder caused by a mutation in the FⅨ gene which leads to the dysfunction of activated coagulation factor IX(FⅨ).In coagulation cascade,FⅨ plays a role in intrinsic coagulation pathway and converts the prothrombin to thrombin.Current protein replacement therapy uses extended half-life rFⅨ 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 FⅨ 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 FⅨ 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 FⅨ gene(optFⅨ),and evaluate the expression and function of the tissue specific gene therapy approach.In this study,LV-EF1α-FⅨ was first constructed to investigate the effect of two human MSCs(FT902 and 1207-9)to express exogenous FⅨ protein.Expression results showed the effective expression and secretion of active FⅨ produced by MSC-EF1α-FⅨ.Results of post-translational modification indicated the complete glycosylation of FⅨ in MSCs but theγ-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 FⅨ expression.Endothelium-specific LVs,LV-VEC-FⅨ and LV-KDR-FⅨ were constructed by replacing the EF1αsequence with VEC and KDR,and used to transduce FT902 and 1207-9.FⅨ ELISA showed that EF1α,VEC and KDR expressed FⅨ in amounts of 1300~1500 ng/10~6/24h,200~500 ng/10~6/24h,and 500~800 ng/10~6/24h,respectively.The FⅨ 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 FⅨ driven by VEC and KDR promoters reduced the expression of FⅨ and were independent of Vitamin K.The regulation of VEC and KDR promoters improved the FⅨ 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.