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. Scott McIvor, Perry B. Hackett, David A. Largaespada

Research output: Contribution to journalArticle

117 Citations (Scopus)

Abstract

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 FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by tail-vein injection, to integrate B-domain-deleted FVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since FVIII protein is a neoantigen to these mice, sustaining therapeutic plasma FVIII 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 FVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10%-100% of normal) FVIII expression and phenotypic correction required co-injection of an SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated polymerase chain reaction was used to clone FVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained FVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented.

Original languageEnglish (US)
Pages (from-to)2691-2698
Number of pages8
JournalBlood
Volume105
Issue number7
DOIs
StatePublished - Apr 1 2005
Externally publishedYes

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Gene transfer
Beauty
Factor VIII
Hemophilia A
Genes
Injections
Veins
Plasmids
Transposases
Gene therapy
Polymerase chain reaction
DNA
Chromosomes
Transgenes
Genetic Therapy
Liver
Antibody Formation
Tail
Animals
Proteins

ASJC Scopus subject areas

  • Hematology

Cite this

Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system. / Ohlfest, John R.; Frandsen, Joel L.; Fritz, Sabine; Lobitz, Paul D.; Perkinson, Scott G.; Clark, Karl J; Nelsestuen, Gary; Key, Nigel S.; McIvor, R. Scott; Hackett, Perry B.; Largaespada, David A.

In: Blood, Vol. 105, No. 7, 01.04.2005, p. 2691-2698.

Research output: Contribution to journalArticle

Ohlfest, JR, Frandsen, JL, Fritz, S, Lobitz, PD, Perkinson, SG, Clark, KJ, Nelsestuen, G, Key, NS, McIvor, RS, Hackett, PB & Largaespada, DA 2005, 'Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system', Blood, vol. 105, no. 7, pp. 2691-2698. https://doi.org/10.1182/blood-2004-09-3496
Ohlfest, John R. ; Frandsen, Joel L. ; Fritz, Sabine ; Lobitz, Paul D. ; Perkinson, Scott G. ; Clark, Karl J ; Nelsestuen, Gary ; Key, Nigel S. ; McIvor, R. Scott ; Hackett, Perry B. ; Largaespada, David A. / Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system. In: Blood. 2005 ; Vol. 105, No. 7. pp. 2691-2698.
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