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The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor Ⅷ gene

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chinese Science Bulletin 2011年第2期56卷 158-163页

作者： zhu fuxiang yang shude liu zelong miao jing qu huige chi xiaoyan College of Life Sciences Ludong University Yantai 264025 China

Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) *** secretion of FVIII is caused mainly by inefficient secretion of its heavy ***,we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene,suggesting that the light chain,covalently ligated to a co-expressed heavy chain can improve the secretion of spliced ***,its level of secretion was affected by inefficient secretion the heavy ***,we studied the effect of a mutant heavy chain with L303E/F309S substitutions,which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII *** vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells.A spliced FVIII protein was seen in co-transfected cells by Western blot *** heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene,which was greater than the secretion of wild-type heavy *** amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL,with an activity of (0.61±0.08) IU/mL,which was greater than that of wild-type FVIII co-transfected *** FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain *** suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a *** result

关键词：分泌作用基因传递因子和内含肽轻链载体基础蛋白质剪接

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Enhanced plasma factor Ⅷ activity in mice via cysteine mutation using dual vectors

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Science china(Life Sciences) 2012年第6期55卷 521-526页

作者： zhu fuxiang liu zelong miao jing qu huige chi xiaoyan Life Science College of Ludong University Yantai 264025China

Hemophilia A is caused by a genetic mutation in coagulation factor VIII （FVIII） gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII （BDD-FVIII） leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately （239＋_56） ng mL-1 above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately （1.09＋_0.25） IU mL-1 above en- dogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.

关键词： coagulation factor VIII dual-vector gene delivery inter-chain disulfide bonding heavy chain secretion coagulation ac-tivity

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Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

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Science china(Life Sciences) 2013年第3期56卷 262-267页

作者： zhu fuxiang liu zelong WANG XiaoLei miao jing qu huige chi xiaoyan College of Life Science Ludong University Department of Hematology Yuhuangding Hospital

Protein trans-splicing based dual-vector factor VIII （FVIII） gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide （442± 151 ng mL i vs. 305±103 ng mL-1） and coagulation activi- ty （1.46±0.37 IU mL i vs. 0.85±0.23 IU mL-1） compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector ex- pressing FVIII （1.79_＋0.59 IU mL-l）. These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.

关键词： coagulation factor VIII protein trans-splicing dual-vector gene delivery plasma coagulation activity

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