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.