Piggybac Mediated Gene Transfer To Correct Hemophilia A

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder, affecting about 1 out of 5,000 males; those affected suffer disabling joint and muscle hemorrhages. Hemophilia A is an attractive gene therapy candidate, because even small increases in FVIII levels (5-10%) will alter the phenotype. Non-viral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize that a PB transposon vector carrying a codon-optimized human FVIII cDNA along with a hyperactive transposase (iPB7) will confer persistent gene expression and correction of the hemophilia A bleeding phenotype. We engineered PB transposon to carry a codon-optimized human FVIII B-domain deleted cDNA (coFVIII-BDD). We evaluated the in vivo gene transfer efficiency in hemophilia A mice by hydrodynamic tail-vein injection using PB coFVIII-BDD driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Factor VIII null mice received 25 micrograms each of the PB coFVIII-BDD transposon and iPB7 to determine long term expression and phenotypic correction. FVIII activity and antigen levels were measured prior to injection and then every 4 weeks for 24 weeks. Results revealed therapeutic levels (50-225%) of factor VIII activity and antigen post gene transfer with stable expression for 24 weeks in most mice. A goal of gene transfer based therapies is to develop the most efficacious expression vectors with the least toxicity. To assess endoplasmic reticulum stress in the livers of treated and untreated mice, we evaluated BiP, CHOP, and EDEM levels via q-PCR. All experimental mice, null mice, and transposon treated mice without the coFVIII-BDD cassette revealed no evidence of cell stress. These data indicate codon-optimized FVIII and the piggyBac transposon vector system may provide a safe long term gene transfer strategy. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. More than 75% of mice receiving PB coFVIII-BDD transposon and iPB7 demonstrated functional correction via tail clip. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression and phenotypic correction.