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| Propeptide-deleted von Willebrand Factor Improves Heavy Chain Secretion and Bioactivity by Split FVIII Gene Co-transfected Cells |
| ZHU Fu-xiang, LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan |
| Life Science College of Ludong University, Yantai 264025, China |
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Abstract Dual-vector based FVIII gene split delivery has been developped as an altenative strategy to overcome the packaging limitation of adeno-associated virus (AAV) vectors in hemophilia A gene therapy but the efficacy is undesired for the inefficient secretion of heavy chain. A propeptide-deleted mutant of von Willebrand factor(vWF-ΔPro), which functions as a carrier of FVIII was tested for its effect on dual-vector FVIII gene delivery. 48 hours post-transfection of HEK293 cell with vWF-ΔPro, heavy and light chain genes of a B-domain deleted FVIII (BDD-FVIII), a chain-specific ELISA showed high levels of heavy chain secretion of 142?29ng/ml in the culture supernatant, greater than that of cell without vWF-ΔPro co-transfection(87?15ng/ml). The heavy chain transfected cells showed a very low levels of heavy chain secretion in absence of vWF-ΔPro although improved in presence of vWF-ΔPro, but lower than that of heavy and light chain co-transfected cells indicating a pro-secretion effect of light chain on heavy chain in trans. The light chain showed higher efficient secretion in light chain gene transfection alone or co-transfection with heavy chain, which was not affected by vWF-ΔPro. The Coatest assay showed an obviously higher bioactivity (0.80?0.15IU/ml) in supernatant of vWF-ΔPro, heavy and light chain genes co-transfected cell, compared to vWF-ΔPro-free co-transfection cell (0.41?0.08IU/ml). The supernatant from combined cells separately transfected with heavy and light chain displayed FVIII bioactivity of 0.23?0.09IU/ml in presence of vWF-ΔPro, suggesting a function of vWF-ΔPro in assembly of secreted heavy and light chain into a functional hetero-dimer. The data demonstrated that vWF-ΔPro transfection could improve dual-vector mediated FVIII gene delivery forming a basis for ongoing in vivo study.
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Received: 05 December 2011
Published: 25 July 2012
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