Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system

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 transposaseexpressing 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 IntroductionHemophilia A is an X-linked, recessive, genetic disorder that is caused by insufficient coagulation factor (F) VIII synthesis resulting in sustained bleeding after trauma or injury. 1 Recombinant FVIII protein is currently used to treat bleeding episodes at the cost of approximately $55 000 per person year and is not available in many parts of the world. 2 Gene therapy for hemophilia has recently been an intense area of study because even modest levels (2%-5% normal) of FVIII or FIX can improve clinical outcomes. Considerable progress has been made in developing both nonviral and viral vectors to this end. Viral vectors have been extremely effective in delivering FVIII and FIX transgenes, in some cases curing animal models of hemophilia A and B, respectively (reviewed in Nathwani et al 3 ).However, problems related to triggering of the host inflammatory response have resulted in cessation of clinical trials with both adenovirus 4 and adeno-associated virus. 5 Accordingly, improved adenoviral vectors are being developed that may be less immunogenic (reviewed in Ritter et al 6 ), and the use of alternative serotypes also has shown promise in adeno-associated virus re-administration. 7 Nevertheless, these kinds of problems, along with large-scale vector production challenges, have fueled intense interest in development of nonviral approaches for the treatment of hemophilia by gene therapy.The benefits of nonviral vectors include simplicity, ease of storage, and amenability to large-scale manufacture. 3,8 They are also potentially less immunogenic than viral vectors, which could allow for safer administration and/or re-administration. Integrating and nonintegrating plasmid-based vectors have been developed and successfully tested...

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…This procedure has been successfully applied to confer a therapeutic benefit in several animal models of human diseases, including hemophilia B after delivery of human clotting factor IX (FIX) as a therapeutic gene product in FIX-deficient mice (Yant et al, 2000) and tyrosinemia after hydrodynamic delivery of a fumarylacetoacetate hydrolase (FAH)-encoding SB transposon vector into the livers of FAH-deficient mice that showed selective outgrowth of genetically corrected hepatocytes (Montini et al, 2002). Additional, successful preclinical testing of the SB system has been established in disease models for hemophilia A (Ohlfest et al, 2005b) and mucopolysaccharidosis (Aronovich et al, , 2009.…”

“…However, with careful promoter choice, several studies have established that SB-mediated transposition provides long-term expression in vivo, as discussed previously. Notably, stable transgene expression from SB vectors was seen in mice after gene delivery in the liver (Yant et al, 2000;Ohlfest et al, 2005b;Aronovich et al, 2009;Kren et al, 2009), lung (Belur et al, 2003;L. Liu et al, 2006b), brain (Ohlfest et al, 2005a), and blood after hematopoietic reconstitution in vivo Xue et al, 2009).…”

“…There has been steadily growing interest in applying the SB system for gene therapy (Izsvák and Ivics, 2004;Essner et al, 2005;Hackett et al, 2005Hackett et al, , 2010Ivics and Izsvak, 2006;Izsvák et al, 2010) in the context of several conditions including hemophilia A and B (Yant et al, 2000;Ohlfest et al, 2005b;L. Liu et al, 2006b;Kren et al, 2009;Hausl et al, 2010) (Aronovich et al, , 2009, cancer (Ohlfest et al, 2005a;Peng et al, 2009;Jin et al, 2011), and type 1 diabetes (He et al, 2004).…”

Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection

“…SB-mediated long-term expression of clotting factors such as Factor VIII and Factor IX in the liver has cured hemophilias A and B, respectively [40][41][42] . The procedure has also been used by us and others to treat mice with genetic deficiencies for fumarylacetoacetate hydrolase deficiency 43-45 as well as by us for β-glucuronidase and iduronidase deficiencies associated with mucopolysaccharidosis Type VII and Type I, respectively 16 .…”

Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon‐mediated gene delivery: implications for non‐viral gene therapy of mucopolysaccharidoses

“…The integrating ability of SB transposons combines the advantages of plasmid-based vectors with the ability to provide long-term expression of the therapeutic gene [12]. The SB system has been used to successfully treat mice with several genetic disorders including hemophilia [13][14][15] and fumarylacetoacetate hydrolase deficiency [16]. These diseases are excellent models for gene therapy because a therapeutic benefit can be achieved by inserting a therapeutic gene into relatively few cells in liver [8,9].…”