Evaluation of the activity levels of rat FVIII and human FVIII delivered by adeno-associated viral vectors both in vitro and in vivo.

The development of a novel coagulation factor VIII (FVIII) expression cassette with an enhanced activity for gene therapy of hemophilia A (HA) is essential. The biological properties of several non-human FVIII sequences, such as porcine and canine, have been evaluated. Here, we compared the activity level of rat FVIII (rFVIII) and human FVIII (hFVIII) by using single-chain and dual-chain strategies in 293 T cells and the HA mice. In both in vitro and hydrodynamic injection studies, the activity of rFVIII detected by the activated partial thromboplastin time assay was higher than that of hFVIII both by single-chain (~2.96-fold and ~1.72-fold, respectively) and dual-chain (~7.69-fold and ~2.35-fold, respectively). Moreover, the dual chain exerted a potentially higher delivery efficacy compared with the single chain (~4.96-fold and ~2.99-fold, respectively). The blood loss of HA mice administrated with rFVIII was less than those with hFVIII. AAV-delivered rFVIII and hFVIII also exerted long-term therapeutic effects on HA mice and caused a transient ALT elevation. These data might help to the development of novel, optimized FVIII expression cassettes based on the amino acid difference between rFVIII and hFVIII. These data indicate that the dual-chain strategy would likely enhance the delivery efficiency of the AAV-mediated FVIII gene therapy.

Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FVIII composed of the human heavy chain and the rat light chain.

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC + rLC protein had a higher activity than the hHC + hLC protein at comparable expression levels. The specific activity of hHC + rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC + rLC treatment exhibited a visibly higher activity than those treated with hHC + hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC + rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.