Expression Of Human Clotting Factor Ⅸ And Utero Gene Therapy Mediated By Recombinant Lentiviral Vector

(一)Hemophilia B is an X chromsome-linked recessive factor IX (hFIX)deficiency bleeding disorder occurring 1 in 30,000 males. Up to now, Treatments mainly focus on transfusing clotting factor IX concentrates prepared from human blood or recombinant sources. Gene therapy may provide a safe and effective treatment for patients. In this study, we constructed the recombinant lentiviral vectors of human clotting factor IX gene.The recombinant FUXW vector was regulated by ubiquitin promoter and the recombinant FAXW vector was regulated by liver specific promoter (ABP) respectively. High titer of recombinant lentivirus was prepared from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids, and purified by ultracentrifugation . The viral titer was measured by Southern Dot Blot. Expression of hFIX in vitro and in hemophilia B mice mediated by recombinant lentiviral vectors was reported.Those data offered a promising result for further study. The major findings are as follows.High level of hFIX expression was detected in supernatant of 293T,BHK and L-02 cells which were infected with recombinant FUXW viruses .The results showed that the expression levels were 465ng/106cell/72h(BHK), 613ng/106cell/72h(293T), 300ng/106cell/72h (L-02) respectively. High level of hFIX expression was detected only in supernatant of L-02 cells which were infected with recombinant FAXW viruses, less expression of hFIX in BHK and 293T cells. High level of GFP expression was observed in 72 hours after being infected with recombinant FUGW viruses, and the rate of fluorescent cells was 80%. Recombinant FUXW viruses and FAXW viruses were transferred to hemophilia B mice by injection and hydrodynamics-based administration via tail vein. Serum hFIX antigen was detected in all treated mice, and the levels of FIX antigen showdose-dependent. The highest levels of hFIX were 45ng/ml. The expression lasted more than 60 d.The exprssion level of FAXW group was higher than that of FUXW group.There was no significant difference of expression level between injection and hydrodynamics-based administration via tail vein.(二) To evaluate utero gene therapy feasibility of utero gene therapy for hemophilia B mediated by recombinant lentivirus,ICR fetuses at 17-19 days' gestation were received lentiviral vectors carrying the transgene hFIX under the control of the liver specific promoter (ABP) by intrahepatic injection. The results showed that serum hFIX antigen were detected in all newborn mice, The highest level of hFIX was 50ng/ml. The expression lasted more than 30d. Anti-hFIX antibody was not found. PCR and RT-PCR analysis showed that hFIX cDNA was detected in liver,spleen and heart.The transcriptional copy of hFIX cDNA was only detected in liver. Besides, no germ line transmission was found at DNA and RNA levels, and no side effects associated with gene transfer were detected.