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




The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor VIII gene - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Chinese Science Bulletin

, Volume 56, Issue 2, pp 158–163

First Online: 19 January 2011Received: 13 July 2010Accepted: 22 October 2010

Abstract

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.

Keywordscoagulation factor VIII secretion mutant heavy chain intein protein splicing This article is published with open access at Springerlink.com

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Autor: FuXiang Zhu - ShuDe Yang - ZeLong Liu - Jing Miao - HuiGe Qu - XiaoYan Chi

Fuente: https://link.springer.com/article/10.1007/s11434-010-4244-7







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