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

Fuxiang Zhu,Jing Miao,Xiaoyan Chi,Huige Qu,Shude Yang,Zelong Liu

doi:10.1007/s11434-010-4244-7

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.

Highlights

Treatment of hemophilia A by gene therapy is adversely affected by inefficient factor VIII (FVIII) secretion and the large FVIII gene, which is difficult to package in the promising adeno-associated virus (AAV) vectors
FVIII binds to immunoglobulin-binding protein (BiP), an endoplasmic reticulum (ER) chaperone protein, during secretion and the release of FVIII is dependent on a high concentration of intracellular ATP
The FVIII heavy chain-specific monoclonal antibody, ESH5, light chain-specific monoclonal antibody, ESH8, and the horseradish peroxidase (HRP)-conjugated ESH8, HRP-ESH8, were purchased from American Diagnostica (Stamford, CT, USA); HRP-conjugated rabbit anti-human FVIII polyclonal antibody was purchased from Novus (Littleton, CO, USA); HRP-conjugated sheep anti-rabbit secondary antibody and the ECL Plus Western blotting detection kit were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA)

Summary

Introduction

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. Spliced FVIII and bioactivity were 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. The data showed an obviously improved secretion of the mutant heavy chain by itself and spliced FVIII with an increased bioactivity This provides an experimental basis for transferring dual-AAV vector co-delivery of full-length FVIII gene to animal models

Methods

Results

Conclusion