The Experimental Gene Therapy on Type 2A/Iic VWD Mouse Model with AAV Vector Delivering Only VWF Propeptide Expression cassette4

Type 2A/IIC von Willebrand Disease (VWD) is caused by mutations in the propeptide of von Willebrand Factor (VWF), and characterized by the impaired multimer assembly. The structural analysis revealed that VWF propeptide (D1D2 domain) serves as a pH-sensing template for VWF multimerization and tubular storage, which implies that D1D2 expression "in trans" has the potential to restore the multimerization defect of type 2A/IIC mutatns.

The gene therapy of VWD is challenging due to the huge size of VWF gene, which cannot be packed to the virus vectors, particularly recombinant adeno-associated virus (rAAV). In the pilot studies, we had co-transfected AtT20 cell with plasmid encoding full length VWF, containing the Y87S mutation on propeptide region and causing type 2A VWD phenotype, and plasmid expressing only wild type propeptide of VWF. The co-transfection rescued the multimer formation in a concentration dependent manner (Figure 1A )and corrected the functional defects caused by the Y87S mutation (Figure 1B). Inspired by the results of in vitro studies, we thus have designed a novel gene therapy strategy for type 2A VWD utilizing the machinery of VWF multimer assembling. Instead of trying to squeeze the VWF gene into the AAV vector, we had prepared the AAV vector containing the expression cassette of propeptide region directed by endothelial cell specific promoter FLT-1. The mouse model representing natural occurring VWF Y87S mutation identified in type 2A VWD patient was established by criper9 gene editing technique. The AAV vectors was administered via tail vein injection at a dose of 5Ⅹ10¹¹ genome copies to 6 weeks old male mice to deliver the propeptide of VWF gene and the plasma of the treated mice was collected retroorbital pretreatment and at 2- ,4- and 6-weeks post vector injection. The antigen level of VWF was determined by ELISA and the function activity of VWF was measured by collagen binding assay. The status of VWF multimerization was examined by agarose gel electrophoresis. Although the Y87S mutation led to significant reduce of high molecule weight multimer in mice, unlike observed in mutation harboring patient, the mice still had medium and low molecular weight multimer, maintaining ~30% of activity. The expression of propeptide by AAV delivered vector improved the VWF activity significantly to ~70% of wild type control (Figure 1C), and restored the formation of high molecular weight multimer (Figure 1D). The experimental gene therapy conducted on VWD mouse model showed that it would be efficient to express the propeptide alone to correct the type 2A/IIC VWD defect and the preliminary results provide a novel strategy to overcome technique limits on gene therapy of VWD.

No relevant conflicts of interest to declare.