%0 Journal Article
%T The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor VIII gene<br>
%A FuXiang Zhu
%A ShuDe Yang
%A ZeLong Liu
%A Jing Miao
%A HuiGe Qu
%A XiaoYan Chi
%A <br>
%J 科学通报(英文版)
%D 2011
%I 
%X 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) vectors. Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain. Previously, 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 BDD-FVIII. However, its level of secretion was affected by inefficient secretion the heavy chain. Here, 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 gene. Eukaryotic 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 analysis. The 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 chain. The 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 cells. Spliced 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 transfections. This 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 dual-vector. These results encourage the transfer of this technology to an animal model using a dual-AAV vector.
%K coagulation factor VIII
%K secretion
%K mutant heavy chain
%K intein
%K protein splicing<br>
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=01BA20E8BA813E1908F3698710BBFEFEE816345F465FEBA5&cid=96E6E851B5104576C2DD9FC1FBCB69EF&jid=DD6615BC9D2CFCE0B6F945E8D5314523&aid=1C2E6D9CA08B27C23A4D12DE2B6BD263&yid=9377ED8094509821&vid=014B591DF029732F&iid=0B39A22176CE99FB&sid=4C100B7696CE9E24&eid=D5C9DC4EF2F78008&journal_id=1001-6538&journal_name=科学通报(英文版)&referenced_num=0&reference_num=20