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

Abstract

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.

von Willebrand factor deficiency does not influence angiotensin II-induced abdominal aortic aneurysm formation in mice

“…VWF antigen (VWF:Ag) levels in plasma were determined using an in-house developed enzyme-linked immunosorbent assay as described 42 . Briefly, a microtiter plate was coated with polyclonal anti-human VWF antibody (Dako, Glostrup, Denmark), known to cross-react with mVWF.…”

How I manage severe von Willebrand disease

“…Gene therapy has only been investigated in a preclinical setting for VWD (De Meyer et al , ; Wang et al , ; Portier et al , ). In patients with haemophilia, gene therapy has shown very good results in clinical trials (Perrin et al , ).…”

“…In addition, VWF is synthesized mainly in the endothelium, which is difficult to target with gene therapy. So far, VWF liver‐specific gene transfer has been used in mice models leading to higher plasma VWF levels (Wang et al , ; Portier et al , ). More preclinical studies are needed before gene therapy can be used in clinical trials in patients suffering from severe VWD.…”

The Evolution of Gene Therapy in the Treatment of Metabolic Liver Diseases

“…It has been used for the in vivo overexpression of low-density lipoprotein and very low-density lipoprotein receptors, resulting in moderate reductions in plasma cholesterol and atherosclerosis in a murine model of familial hypercholesterolemia [ 111 ]. A study using a murine von Willebrand disease model found that SB transposon-mediated von Willebrand factor ( VWF ) gene delivery resulted in the long-term expression of supraphysiologic VWF levels [ 112 ]. While SB has been approved for certain clinical trials, delivery mechanisms to efficiently target livers in large animals are still under investigation.…”