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Codon optimization of human factor VIII cDNAs leads to high-level expression.

Ward, NJ, Buckley, SM, Waddington, SN, Vandendriessche, T, Chuah, MK, Nathwani, AC, McIntosh, J, Tuddenham, EG, Kinnon, C, Thrasher, AJ and McVey, JH (2011) Codon optimization of human factor VIII cDNAs leads to high-level expression. Blood, 117 (3). pp. 798-807.

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Abstract

Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Item Type: Article
Authors :
NameEmailORCID
Ward, NJUNSPECIFIEDUNSPECIFIED
Buckley, SMUNSPECIFIEDUNSPECIFIED
Waddington, SNUNSPECIFIEDUNSPECIFIED
Vandendriessche, TUNSPECIFIEDUNSPECIFIED
Chuah, MKUNSPECIFIEDUNSPECIFIED
Nathwani, ACUNSPECIFIEDUNSPECIFIED
McIntosh, JUNSPECIFIEDUNSPECIFIED
Tuddenham, EGUNSPECIFIEDUNSPECIFIED
Kinnon, CUNSPECIFIEDUNSPECIFIED
Thrasher, AJUNSPECIFIEDUNSPECIFIED
McVey, JHj.mcvey@surrey.ac.ukUNSPECIFIED
Date : 20 January 2011
Identification Number : https://doi.org/10.1182/blood-2010-05-282707
Uncontrolled Keywords : Amino Acid Sequence, Animals, Animals, Newborn, Codon, Enzyme-Linked Immunosorbent Assay, Factor VIII, Female, Gene Expression, Genetic Therapy, Genetic Vectors, HEK293 Cells, Hemophilia A, Humans, Injections, Intravenous, Lentivirus, Male, Mice, Mice, 129 Strain, Mice, Knockout, Molecular Sequence Data, Mutation, Promoter Regions, Genetic, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Spleen Focus-Forming Viruses
Related URLs :
Depositing User : Symplectic Elements
Date Deposited : 17 May 2017 09:56
Last Modified : 17 May 2017 14:46
URI: http://epubs.surrey.ac.uk/id/eprint/825747

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