| Factor IX (FIX), a crucial component of theblood-clotting cascade,is dysfunctional in 1 in 30 000 males suffering from X-linked hemophilia B. The clinical symptoms are usually spontaneous bleeding episodes into joints or internal organs and physician apply blood recombinant human factor IX (hFIX) for these patients. Most of patients can not afford the high cost and have to give up the treatment. In some patients, the development of inhibitory antibodies against the recombinant protein severely compromises therapy; neutralizing antibodies appear in 3% to 4% of treated patients. In roughly half of those patients who develop inhibitors, anaphylaxis or severe allergic reactions occur on infusion of any type of factor IX–containing product. Also, some patients will beaffected by AIDS and hepatitis. Because the development of inhibitors can be associated with dramatically increased morbidity and mortality3 and tremendous treatment costs, novel therapeutic strategies such as gene therapy have to address this dominant problem in the treatment of hemophiliacs. The term "gene therapy" refers to the transfer of genetic material to a target cell to achieve aclinical benefit. Through use of gene transfer techniques, introduced DNA sequences may interfere with a gene's function, restore a lost function, or initiate a new function. It provides great hope to cure the kind of disease. hFIX has been cloned and modulated simply and studied widely by scientists. The symptoms will be improved or disappeared if the level of exogenous FIX given is 5-10% of therapeutic concentration, respectively.Sustained FIX expression and long-term substitution of hFIX at therapeutic levels without immune response remains, therefore, one of the major tasks in hemophilia gene therapy. Therefore, we transferred the target gene into the yolk sac vessel of mouse to study the immune response and the hFIX sustained expression. The experiment was divided into three phases: phase 1, analysis of hFIX expression and persistence; phase 2, changes of hFIX antibody development after hFIX reinjected; phase3, changes of hFIX antibody development after AdFIX reinjected. MF1 mices are fecund and easily tolerate complicated in utero manipulation. Furthermore, as they are an outbred strain, they are less likely to show immunologic vagaries as observed in other strains.Results: 1. Injection into the yolk sac vessel provides a highly effective route for gene delivery to the fetal mouse circulation. The blood flows from the yolk sac vessel through the umbilical vein to the fetus where the blood stream is divided between the liver and via the ductus venosus to the heart. This circulation system may explain the relatively high infection in these organs. However, expression in the cardiac muscle was not restricted to tissue adjacent to the chambers and was absent in the endothelium. Therefore, this restricted expression pattern may not be due to physiologic constraints but instead to vector tropism or promoter selectivity. 2. The most interesting observation in this study that we want to get is the prolonged expression of hFIX, without development of significant levels of hFIX antibodies, following prenatal transgene delivery and expression by injection of hFIX into abdominal cavity and AdhFIX into tail vein. The results of phase 2 show: to determine whether an immune response to hFIX had been affected by the prenatal expression of hFIX, we applied purified hFIX protein repeatedly to the D15 and D17 fetal AdhFIX groups and to the adult AdhFIX group; In the D15 and D17 fetal AdhFIX groups, hFIX concentrations were still high in 5 mice after the seventh injection. No hFIX was ever detected in the adult AdhFIX group mice following protein injection. In the protein challenge control group, circulating hFIX fell to undetectable levels in all 6 mice after the fifth and sixth protein injections. These results also were tested when AdhFIX was reinjected into the D15 and D17 groups and no AdhFIX antibody detected in phase 3. |
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