Adeno-associated viral (AAV) vector delivery of canine factor VIII (cFVIII) results in long-term dose dependent expression of FVIII in hemophilia A dogs for >8 years (Sabatino 2011). These data demonstrate that AAV-FVIII can significantly improve the disease phenotype. Interestingly, we found that cFVIII is inherently more stable than human FVIII (hFVIII), resulting in increased biological activity (Sabatino 2009). Thus, the AAV dose of cFVIII does not predict the efficacy of hFVIII and preclinical studies with hFVIII will be important for predicting the therapeutic dose of AAV-hFVIII. Furthermore, recent clinical trials for AAV-mediated gene transfer for hemophilia B have demonstrated sustained long-term expression of therapeutic levels of factor IX (FIX) but established that the AAV vector dose may be limiting due to anti-AAV immune responses to the AAV capsid. Therefore, our goal is to generate a novel hFVIII transgene cassette that increases hFVIII expression so that we can achieve therapeutic FVIII levels with a lower AAV dose. In our efforts to develop a hFVIII transgene cassette, we addressed this systematically by comparing different codon-optimized B-domain deleted (BDD) hFVIII genes, alternate promoters as well as hFVIII variants of the PACE-furin cleavage recognition site. We identified a novel codon-optimized hFVIII-BDD transgene (CO3) that expresses 15-fold higher than wild-type hFVIII-BDD. CO3 was introduced into a minimal expression cassette driven by the liver-specific transthyretin (TTR) promoter. At 4 weeks after delivery of AAV8-TTR-hFVIII-BDD and AAV8-TTR-hFVIII-CO3 (1x1011 vector genomes/mouse), the hFVIII expression in the peripheral blood based on ELISA was 35 ± 5 ng/ml and 492 ± 72 ng/ml, respectively. Using a modified transthyretin (TTRm) promoter (222bp), hFVIII expression further increased 2-fold compared to a wild type TTR promoter. In our previous work, we demonstrated that the R1645H substitution in the PACE-furin (P/F) cleavage recognition site (hFVIII-R1645H) confers increased stability and higher biological activity (Siner 2013). More recently, we have characterized a series of deletion variants at this P/F site in hFVIII with similar properties to R1645H. Therefore, in an effort to further enhance the efficacy of our hFVIII gene therapy approach, we introduced the R1645H or a 4 amino acid deletion (residues 1645-1648) of the P/F site (delP/F) into our hFVIII-CO3 transgene. Administration of AAV8-TTR-hFVIII-CO3, AAV8-TTR-hFVIII-CO3-R1645H and AAV8-TTR-hFVIII-CO3-delP/F in hemophilia A mice (1x1011vg/mouse) demonstrated that the hFVIII expression from R1645H (528 ± 30 ng/ml) was higher than CO3 (378 ± 75 ng/ml) at this vector dose. However, introduction of delP/F (TTR-hFVIII-CO3-delP/F) resulted in hFVIII expression that was 1.8-fold higher (685 ± 93 ng/ml)(p = 0.036) than CO3 alone. Importantly, the introduction of the deletion of the PACE-furin recognition site into the codon-optimized hFVIII resulted in an additive effect on the hFVIII expression. Delivery of AAV8-TTRm-hFVIII-CO3-delP/F using the modified TTR promoter resulted in 1018 ± 117 ng/ml hFVIII demonstrating that the TTRm promoter further enhances transgene expression. Notably, the AAV8-TTRm-hFVIII-CO3-delP/F construct results in hFVIII expression that is 30-fold higher than the wild type hFVIII-BDD. Our studies demonstrate that a combination of a liver-specific promoter, codon-optimization and modification of hFVIII at the PACE-furin recognition site (delP/F) significantly increases hFVIII expression in the setting of gene transfer. This work supports the feasibility of lowering the AAV vector dose for a gene-based therapeutic application for hemophilia A.


Elkouby:Spark Therapeutics: Research Funding. High:Bluebird Bio: Consultancy, Equity Ownership; Alnylam: Consultancy; Spark Therapeutics: Consultancy, Equity Ownership. Sabatino:Spark Therapeutics: Research Funding; Pfizer, Inc.: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.