High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease.

Essentials von Willebrand disease (VWD) is the most common inherited bleeding disorder. Gene therapy for VWD offers long-term therapy for VWD patients. Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice. Liver-directed transposons support sustained VWF expression with suboptimal multimerization. SUMMARY: Background Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short-term solution. Gene therapy offers the potential for a long-term treatment for VWD. Objectives To develop an integrative Sleeping Beauty (SB) transposon-mediated VWF gene transfer approach in a preclinical mouse model of severe VWD. Methods We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf(-/-) mice by combining a liver-specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery. Results The sandwich SB transposon was suitable to deliver the full-length mVWF cDNA (8.4 kb) and supported supra-physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride-induced liver regeneration. Analysis of integration sites by high-throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long-term expression of supra-physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long-term expression of VWF by hepatocytes resulted in relatively reduced amounts of high-molecular-weight multimers, potentially limiting its hemostatic efficacy. Conclusions Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type-specific targeting is yet to be achieved.