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.
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References
Mann K G. Biochemistry and physiology of blood coagulation. Thromb Haemost, 1999, 82: 165–174
Dooriss K L, Denning G, Gangadharan B, et al. Comparison of factor VIII Transgenes bioengineered for improved expression in gene therapy of hemophilia A. Hum Gene Ther, 2009, 20: 465–478
Miao H Z, Sirachainan N, Palme L, et al. Bioengineering of coagulation factor VIII for improved secretion. Blood, 2004, 103: 3412–3419
Swaroop M, Moussalli M, Pipe S W, et al. Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII. J Biol Chem, 1997, 272: 24121–24124
Burton M, Nakai H, Colosi P, et al. Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein. Proc Natl Acad Sci USA, 1999, 96: 12725–12730
Scallan C D, Liu T, Parker A E, et al. Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII. Blood, 2003, 102: 3919–3926
Perler F B. The ins and outs of gene expression control. Nat Biotechnol, 2004; 22: 824–826
Chen L X, Zhu F X, Li J, et al. The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene. Mol Ther, 2007, 15: 1856–1862
Zhu F X, Liu Z L, Chi X Y, et al. Protein trans-splicing based dual-vector delivery of the coagulation factor VIII gene. Sci China Life Sci, 2010, 53: 683–689
Gnatenko D V, Saenko E L, Jesty J, et al. Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy. Br J Haematol, 1999, 104: 27–36
Sarkar R, Tetreault R, Gao G, et al. Total correction of hemophiliaAmice with canine FVIII using anAAV 8 serotype. Blood, 2004, 103: 1253–1260
Zhang K, Shen X, Wu J, et al. Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response. Cell, 2006, 124: 587–599
Kaufman R J, Pipe S W, Tagliavacca L, et al. Biosynthesis, assembly and secretion of coagulation factor VIII. Blood Coagul Fibrinolysis, 1997, 8(Suppl 2): S3–S14
Pipe S W. Coagulation factors with improved properties for hemophilia gene therapy. Semin Thromb Hemost, 2004, 30: 227–237
Cunningham M A, Pipe S W, Zhang B, et al. LMAN1 is a molecular chaperone for the secretion of coagulation factor VIII. J Thromb Haemost, 2003, 1: 2360–2367
Dorner A J, Wasley L C, Kaufman R J. Protein dissociation from GRP78 and secretion are blocked by depletion of cellular ATP levels. Proc Natl Acad Sci USA, 1990, 87: 7429–7432
Pittman D D, Tomkinson K N, Kaufman R J. Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells. J Biol Chem, 1994, 269: 17329–17337
Gimble F S. Putting protein splicing to work. Chem Biol, 1998, 5: R251–R256
Cheriyan M, Perler F B. Protein splicing: A versatile tool for drug discovery. Adv Drug Deliv Rev, 2009, 61: 899–907
Zhu F X, Yang S D, Liu Z L, et al. vWF improves secretion and activity of intein spliced BDD-FVIII (in Chinese). Acta Pharm Sin, 2010, 45: 595–600
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Zhu, F., Yang, S., Liu, Z. et al. The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor VIII gene. Chin. Sci. Bull. 56, 158–163 (2011). https://doi.org/10.1007/s11434-010-4244-7
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DOI: https://doi.org/10.1007/s11434-010-4244-7