| Summary: | 博士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 97 === Hemophilia B is an X-linked inherited disorder caused by the lack of a functional coagulation Factor IX (FIX) and characterized by a bleeding diathesis of variable severity. The first part of this thesis is focused on developing new factor IX for therapeutic use. In an effort to reduce factor usage and cost, we investigated the potential use of human Factor IX (FIX) variants with enhanced specific activity. We generated and purified seven recombinant FIX variants using alanine replacement and assayed their activity in vitro. One variant containing three substitutions (R86A/E277A/R338A, FIX-Triple) exhibited ~13-fold higher specific activity and a 10-fold increased affinity for human Factor VIIIa than FIX-wildtype (FIX-WT) and was thus investigated systematically in vivo. Protein infusion of FIX-Triple into hemophilia B mice resulted in greater improvement of hemostasis than FIX-WT. Additionally, liver-specific FIX-Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5-fold higher specific activity compared to FIX-WT. Moreover, tail-vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple in hemophilia B mice demonstrated a 7-fold higher specific activity of FIX-Triple than FIX-WT (35% vs. 6% of normal human plasma intrinsic activity, respectively). In conclusion, we demonstrate the generation of a novel FIX variant with substantially enhanced specific activity in vitro and in vivo, that can be utilized for protein replacement therapy as well as gene-based therapeutic strategies.
The second part of this thesis is focused on two mutant factor IX proteins. The heavy chain (amino acid residues 181-415) of human blood clotting factor IX (FIX) contains the protease function of FIX. Two hemophilia B patients with missense mutations at residues G190 (G25, chymotrypsinogen numbering system) and F192 (F27) exhibited severe and moderate bleeding phenotypes, respectively. The patient with the FIXG190V (glycine replaced by valine) variant had severe bleeding episodes with <1% clotting activity and only 36% of the normal FIX protein levels in plasma. The patient with the F192V variant had only 2% clotting activity and <6% of the normal FIX protein levels in plasma. Hemophilia B mice, which contain an engineered-deletion of FIX gene) expressing human wild type (WT) or the two mutant FIX have been created by hydrodynamic technique that directs the exogenous DNA synthesis in the liver. In this model system a significant difference in the plasma FIX clotting activity was observed (IXWT: 0.60 U/mL, G190V: 0.08 U/mL and F192V: 0.14 U/mL respectively). Since immunohistochemical staining showed similar amounts of FIX in the liver, the synthesis and secretion pathways are less likely to be the major pathogenic mechanisms onderlying the low amount of the mutant variants in the plasma. Decreased specific clotting activities were demonstrated for both the G190V (12.2% of WT FIX) and F192V (16.8% of WT FIX) variants. Purified G190V and F192V proteins were more sensitive to degradation than wild type FIX, especially after being activated by factor XIa. The vulnerable sites were mapped to the peptide bonds at Arg116-Leu117, Lys265-Tyr266, Arg327-Val328, and Arg338-Ser339, conferring the exposed loops of the FIX molecule. The zymogen form of FIX G190V was also easily hydrolyzed by plasmin. Mutations in the G190 and F192 region may result in misfolding and/or decreased stability of FIX in plasma. We conclude that the defects in both the clotting activities and stabilities of G190V and F192V variants of the FIX protein contributed to the clinical phenotypes of these two mutations in the hemophilia B patients.
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