Integration-deficient lentiviral vectors expressing codon-optimized R338L human FIX restore normal hemostasis in Hemophilia B mice.
Integration-deficient lentiviral vectors (IDLVs) have been shown to transduce a wide spectrum of target cells and organs in vitro and in vivo and to maintain long-term transgene expression in nondividing cells. However, epigenetic silencing of episomal vector genomes reduces IDLV transgene expression levels and renders these safe vectors less efficient. In this article, we describe for the first time a complete correction of factor IX (FIX) deficiency in hemophilia B mice by IDLVs carrying a novel, highly potent human FIX cDNA. A 50-fold increase in human FIX cDNA potency was achieved by combining two mechanistically independent yet synergistic strategies: (i) optimization of the human FIX cDNA codon usage to increase human FIX protein production per vector genome and (ii) generation of a highly catalytic mutant human FIX protein in which the arginine residue at position 338 was substituted with leucine. The enhanced human FIX activity was not associated with liver damage or with the formation of human FIX-directed inhibitory antibodies and rendered IDLV-treated FIX-knockout mice resistant to a challenging tail-clipping assay. A novel S1 nuclease-based B1-quantitative polymerase chain reaction assay showed low levels of IDLV integration in mouse liver. Overall, this study demonstrates that IDLVs carrying an improved human FIX cDNA safely and efficiently cure hemophilia B in a mouse model.
Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA.
Thipparat
T
Suwanmanee
Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA.
Genlin
G
Hu
Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA.
Tong
T
Gui
Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
Cynthia C
CC
Bartholomae
Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
Ina
I
Kutschera
Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
Christof
C
von Kalle
Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
Manfred
M
Schmidt
Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA; Department of Pediatrics, Division of Hematology/Oncology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Paul E
PE
Monahan
Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA; Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA. Electronic address: kafri@med.unc.edu.
Tal
T
Kafri
D000818:Animals; D001120:Arginine / Q000378:metabolism; D003062:Codon; D004195:Disease Models, Animal; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000627:therapeutic use; D002836:Hemophilia B / Q000473:pathology / Q000628:therapy*; D056945:Hep G2 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout
D003062:Codon; D005164:Factor IX; D001120:Arginine; D007930:Leucine