The development of robust nonviral vectors could facilitate clinical gene therapy

The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. applied as a tool for functional genomic studies, genetic modification of somatic FTY720 manufacturer and embryonic cells and generation of induced pluripotent stem cells.9,10 However, there are only few studies that focus on direct gene transfer with PB and most of these relied on reporter genes.11,12,13,14,15 In this study, we explored the use of PB transposons for liver-directed gene therapy of hemophilia B, which has not yet been investigated. Hemophilia B is a hereditary bleeding disorder caused by a defective factor IX (FIX) gene. It is currently treated FTY720 manufacturer by repeated clotting factor infusions but EIF2Bdelta this treatment is not curative.16 Moreover, some patients develop neutralizing antibodies against the administered recombinant FIX protein that renders the therapy ineffective and bleeding episodes difficult to manage. Hence, hemophilia B is an attractive target disease to validate PB transposon-based gene therapy approaches, which has implications for other hereditary disorders,3 including liver-borne diseases. The main objective of this study therefore consisted of establishing proof-of-concept that PB transposons encoding FIX can be used for liver-directed gene delivery to cure hemophilia B and to assess their overall efficacy and safety in appropriate mouse models. Maximizing the therapeutic index of a given gene transfer vector is a crucial step toward implementation of successful clinical trials. Hence, we wanted to augment the efficiency of transposon-mediated gene therapy using a multilayered strategy by optimizing each one of its components including the PB transposase and the transposon, the liver-specific promoter used to drive FIX and the FIX transgene itself. Our results demonstrated that PB transposons in conjunction with a mouse codon-optimized PB transposase (mPB) resulted in prolonged FIX expression and cure hemophilia B in FIX-deficient mice, which had not been shown previously. Moreover, we showed that the efficiency of PB-mediated gene therapy could be enhanced by using the latest generation hyperactive PB transposase (hyPB) and by modifying the transposon terminal repeats.17,18,19 In addition, the use of a liver-specific promoter coupled to designed (provides long-lasting therapeutic hFIX expression levels and phenotypic correction in hemophilia B mice The codon usage optimized transposase (mPB)25 was evaluated in combination with (Figure 1d) that carried a wild-type ((Figure 1e) with a codon-optimized hFIX (transposon contained a small intron upstream of the to boost FIX expression.2,26 Both the and were driven from a novel and potent chimeric liver-specific promoter that contained an designed hepatocyte-specific transposon (10 g) along with 2 g transposon and resulted in a significant 12-fold higher ( 0.001) FTY720 manufacturer hFIX protein and activity level that stabilized in the supraphysiologic range (Figure 2b). In contrast, hFIX expression and activity gradually declined to basal levels in control mice that received either PBS or only the transposons in the absence of transposase (Figure 2a,?bb). Anti-hFIX antibodies could not be detected (data not shown). Transposition is therefore necessary for sustained expression. The initial high peak of FIX expression is likely due to the presence of nonintegrated or episomes due to the fact that we used an excess amount of transposon plasmid (10 g). These episomes are gradually lost and are also epigenetically silenced (Figure 2), consistent with previous observations.2,28,29 Open in a separate window Figure 1 Schematic representation of PB transposon and transposase constructs. (a) Transposase constructs encoding for the native transposase mouse codon-optimized (mPB) driven by the CMV promoter cloned upstream of a -globin intron (GI). (b) The hyperactive transposase mouse codon-optimized (hyPB). The hyperactivating mutations are indicated. (c) The empty control plasmid contains a multiple cloning site (MCS) between the promotor and polyadenylation signal. (d) The transposon is flanked by wild-type inverted repeats (transposon, the hFIXIA minigene was replaced by the synthetic codon-optimized hFIX (hFIXco) including a partial 3 untranslated region. In addition, the minute virus of mice small intron (MVM) was introduced downstream of the chimeric HS-CRM8/TTRmin promoter. The PB transposons were flanked by three different inverted repeats: (e) wild-type minimal inverted repeats (transposon is flanked by and contains a codon-optimized hFIX sequence with an R338L substitution. (i) The contains a codon-optimized B-domain deleted FVIII sequence (driven by the chimeric HS-CRM8/TTRmin promoter coupled to a minute virus of mice small intron (MVM). (l) is flanked by the and contains the c-Myc oncogene under the control of the liver specific chimeric promoter. All the PB transposons were flanked with.