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

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 (m)VWF 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 year 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. While the SB vector supported long-term expression of supraphysiological mVWF 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: While this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type specific targeting is yet to be solved