Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A

Gene Ther. 2013 Jun;20(6):607-15. doi: 10.1038/gt.2012.76. Epub 2012 Sep 20.


We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Factor VIII / administration & dosage
  • Factor VIII / genetics*
  • Gene Transfer Techniques
  • Genetic Therapy*
  • Genetic Vectors
  • HEK293 Cells
  • Hemophilia A* / genetics
  • Hemophilia A* / therapy
  • Humans
  • Lentivirus / genetics*
  • Mice
  • Promoter Regions, Genetic
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / genetics*
  • Swine
  • Transduction, Genetic


  • Recombinant Proteins
  • Factor VIII