Codon optimization of human factor VIII cDNAs leads to high-level expression.
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.
Molecular Immunology Unit, Institute of Child Health, University College London, London, UK.
Natalie J
NJ
Ward
Suzanne M K
SM
Buckley
Simon N
SN
Waddington
Thierry
T
Vandendriessche
Marinee K L
MK
Chuah
Amit C
AC
Nathwani
Jenny
J
McIntosh
Edward G D
EG
Tuddenham
Christine
C
Kinnon
Adrian J
AJ
Thrasher
John H
JH
McVey
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D003062:Codon / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D057809:HEK293 Cells; D006467:Hemophilia A / Q000097:blood / Q000235:genetics / Q000628:therapy*; D006801:Humans; D007275:Injections, Intravenous; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013155:Spleen Focus-Forming Viruses / Q000235:genetics
D003062:Codon; D005169:Factor VIII