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Blood, 1 April 2005, Vol. 105, No. 7, pp. 2691-2698. Prepublished online as a Blood First Edition Paper on December 2, 2004; DOI 10.1182/blood-2004-09-3496. This Article Full Text (PDF) All Versions of this Article: 2004-09-3496v1 105/7/2691    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ohlfest, J. R Articles by Largaespada, D. A Search for Related Content PubMed PubMed Citation Articles by Ohlfest, J. R Articles by Largaespada, D. A Related Collections Related Article in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Next Article  Submitted September 9, 2004 Accepted November 17, 2004 Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system John R Ohlfest, Joel L Frandsen, Sabine Fritz, Paul D Lobitz, Scott G Perkinson, Karl J Clark, Gary Nelsestuen, Nigel S Key, R S McIvor, Perry B Hackett, and David A Largaespada* Department of Genetics, Cell Biology and Development, University of Minnesota Cancer Center, Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, Minneapolis, MN, USA Discovery Genomics Inc., Minneapolis, MN, USA Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, MN, USA Department of Medicine, University of Minnesota, Minneapolis, MN, USA Department of Genetics, Cell Biology and Development, University of Minnesota Cancer Center, Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, Minneapolis, MN, USA; Discovery Genomics Inc., Minneapolis, MN, USA * Corresponding author; email: larga002{at}umn.edu. Hemophilia A is a lead candidate for treatment by gene therapy because small increments in the missing secreted protein product, coagulation factor VIII (FVIII), would result in substantial clinical amelioration. Clinically relevant therapy might be achieved by stably delivering a human(h) FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by high-pressure tail vein injection, to integrate B-domain-deleted hFVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since hFVIII protein is a neoantigen to these mice, sustaining therapeutic plasma hFVIII levels was problematic due to inhibitory antibody production. We circumvented this problem by tolerizing 82% of neonates by a single facial vein injection of recombinant hFVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10-100% of normal) hFVIII expression and phenotypic correction required co-injection of a SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated PCR was used to clone hFVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained hFVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? Related Article in Blood Online: An ounce of prevention potentiates a pound of cure for hemophilia A Katherine Parker Ponder Blood 2005 105: 2620. [Full Text] [PDF] This article has been cited by other articles: L. Liu, H. Liu, G. Visner, and B. S. Fletcher Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats FASEB J, December 1, 2006; 20(14): 2594 - 2596. [Abstract] [Full Text] [PDF] H. Liu, L. Liu, B. S. Fletcher, and G. A. Visner Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis FASEB J, November 1, 2006; 20(13): 2384 - 2386. [Abstract] [Full Text] [PDF]

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