High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease
High and long-term von Willebrand factor expression after Sleeping Beauty transposon-mediated gene therapy in a mouse model of severe von Willebrand disease
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DOI：
10.1111/jth.13938
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发表时间：
2018-03-01
影响因子：
10.4
通讯作者：
De Meyer, S. F.
中科院分区：
文献类型：
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作者：
Portier, I.;Vanhoorelbeke, K.;De Meyer, S. F.
背景：3型von Willebrand病(VWD)的特点是完全缺乏von Willebrand因子(VWF)。目前的治疗方法仅限于使用外源性VWF/FVIII产品进行治疗，这些产品只能提供短期解决方案。基因疗法为VWD的长期治疗提供了可能性。目的：建立一种整合睡美人(SB)转座子介导的VWF基因转移方法，用于临床前重症VWD小鼠模型。方法：通过将肝脏特异性启动子与三明治转座子设计和SB100X转座酶通过流体动力基因传递相结合的方法，建立了转基因小鼠vWF(MVWF)在VWF(-/-)小鼠肝脏持续表达的可靠平台。结果：夹心SB转座子适合携带全长mVWF基因(8.4kb)，并支持超生理性表达，基因转移后可稳定表达长达1.5年。在没有或存在转座酶的情况下，夹心载体分别保持上体(类似于60周)或整合到宿主基因组中。利用四氯化碳诱导的肝再生证实了转基因整合。对整合位点的高通量分析揭示了夹心载体的随机整合。尽管SB载体支持超生理VWF水平的长期表达，但所有小鼠的出血表型都没有得到纠正。肝细胞长期表达VWF导致相对较少的高分子量多聚体，潜在地限制了其止血效果。结论：虽然这个整合的VWF基因转移平台是VWD基因治疗的一个重要里程碑，但细胞类型特异性靶向仍有待实现。
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 (similar to 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.