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

Abstract

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.

Keywords

hemophilia A / adeno-associated virus (AAV) / human/rat hybrid factor VIII / gene therapy / dual chain strategy

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Acknowledgements

We thank all the laboratory members for the helpful discussion. We thank Karl Ernest Helwig from the Huazhong University of Science and Technology for the manuscript revision. This work was financially supported by the National Key Basic Research Program of China (No. 2013CB966800), National Natural Science Foundation of China (Nos. 81970112, 81670127, and 81101721), the Novo Nordisk Hemophilia Foundation, grants from the Shanghai Health Commission in China (No. 201940342), grants from the Science and Technology Commission of Shanghai Municipality in China (Nos. 16PJ1406100 and 16ZR1421000), and Zhejiang Provincial Natural Science Foundation of China (No. LY17H080004).

Compliance with ethics guidelines

Jianhua Mao, Yun Wang, Wei Zhang, Yan Shen, Guowei Zhang, Wenda Xi, Qiang Wang, Zheng Ruan, Jin Wang, and Xiaodong Xi declare that they have no conflicts of interest. All experimental procedures were approved by the Studies Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. All institutional and national guidelines for the care and use of laboratory animals were followed.

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