piggyBac-mediated phenotypic correction of factor VIII deficiency

Abstract

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene

“…Codon optimization of human FVIII is a proven strategy to increase protein expression in vivo . 26,29 Here, we codon-optimized canine FVIII (CO-cFVIII) and observed enhancements to FVIII activity in vitro (Figure 2c) and in FVIII-deficient mice (Figure 2d). Wild-type cFVIII and CO-cFVIII share 100% amino acid identity, but only ~ 78% nucleotide identity.…”

“…These results are consistent with previous reports using codon-optimized human FVIII cDNAs. 26,29 On the basis of these results, we focused on the CO-cFVIII cDNA constructs for additional studies.…”

“…We hypothesized that the addition of a partial canine B-domain would decrease toxicity by increasing transport from the ER to Golgi, as described for human FVIII. 26,31,32 We compared a conventional BDD cDNA and a cDNA including a partial B-domain sequence encoding 11 predicted N-linked glycosylation sites (N11) (Figure 1). To confirm functional cDNA expression, we first delivered the expression plasmids by hydrodynamic tail vein injection in FVIII null mice.…”

“…22,25 In addition, we demonstrated improved expression of codon-optimized human FVIII in vivo following hydrodynamic injection of a DNA transposon-based vector. 26 In this study, we focus on developing the vector system for delivery and therapeutic FVIII protein expression from an optimized cFVIII cDNA to achieve safe and long-term expression from hepatocytes. This proof-of-principle study in a relevant small animal model bridges the way to pre-clinical studies in a canine model of hemophilia A.…”

Transposons: Moving Forward from Preclinical Studies to Clinical Trials

“…159,160 In addition to wild-type PBase, 162 hyPBase system mediated long-term expression of blood clotting factor VIII (FVIII) and FIX and correct coagulation defects in hemophilia A and B mice, respectively. 59,62,163 Comparative analysis in the hemophilia B murine model indicated that the hyPBase was more efficient than mPBase, resulting in higher stable FIX expression levels. 59,62 PB-mediated expression of FIX and FVIII was long lasting (>300 days) and did not induce an immunological reaction.…”