Ultrasound-Mediated Gene Therapy for Hemophilia B

描述（由申请人提供）：该提案旨在开发超声介导的非病毒基因递送至肝脏，用于治疗血友病和其他遗传疾病。我们已经证明，在微泡[MB]存在下的超声（US）增强了肝脏特异性高表达因子IX（FIX）质粒向小鼠的基因递送。通过转导一个肝叶实现了显著增强，相对于单独使用裸DNA增加了66倍。小鼠肝脏也可以用GFP质粒转导。因此，治疗性US与MB一起有可能促进hFIX的安全和有效的非病毒基因转移，用于治疗血友病。为了便于将这种方法转化为人类应用，我们建议进一步优化US参数以获得更高的疗效。将在血友病B小鼠模型中测试优化的方案以及优化的pDNA/MB复合物、改进的FIX基因转移载体，以实现长期治疗效果。将设计合适的US系统，用于在正常大鼠、正常和血友病B犬中测试US方案。我们将检验以下假设：1）通过结合最佳US方案和优化的FIX基因表达盒，可在血友病B小鼠中实现治疗水平的FIX表达，并可长期纠正其表型。2)可以优化较大动物模型中的US参数和探针，以在正常狗模型中实现高水平的报告基因表达。3)通过结合最佳US方案和优化的cFIX质粒，可以实现血友病B犬的表型校正。4)聚焦超声或冲击波治疗将适用于将超声方案转化为临床应用。 公共卫生相关性：我们的目标是开发一种超声介导的基因治疗方案，可以很容易地转化为人类应用，以治疗血友病和其他遗传疾病。我们之前已经表明，高表达的肝脏特异性hFIX质粒的递送可以通过超声（US）与微泡（MB）的组合来增强，并且与没有US和MB的基因转移相比，表达水平增加了66倍。该提案将继续优化我们在小鼠和大鼠模型中的US方案，并将开发扩展到正常和血友病B犬模型。

DESCRIPTION (provided by applicant): This proposal aims at developing ultrasound-mediated nonviral gene delivery to the liver for treatment of hemophilia and other genetic diseases. We have shown that ultrasound (US) in the presence of microbubbles [MBs] enhances gene delivery of a liver-specific, high-expressing factor IX (FIX) plasmid into mice. Significant enhancement was achieved by transducing one liver lobe, a 66-fold increment relative to use of naked DNA alone. Mouse livers could also be transduced with a GFP plasmid. Therapeutic US together with MBs therefore has the potential to promote safe and efficient nonviral gene transfer of hFIX for the treatment of hemophilia. To facilitate translation of this method to human applications, we propose to further optimize the US parameters for higher efficacy. The optimized protocols, together with optimized pDNA/MBs complexes, improved FIX gene transfer vectors, will be tested in a hemophilia B mouse model to achieve a long-term therapeutic effect. Suitable US systems will be designed for testing the US protocols in normal rats, normal and hemophilia B dogs. We will test the hypotheses that: 1) By combining the best US protocol and optimized FIX gene expression cassette, therapeutic levels of FIX expression can be achieved in hemophilia B mice and can correct their phenotype long-term. 2) US parameters and probes in larger animal models can be optimized to achieve high-level reporter gene expression in normal dog models. 3) By combining the best US protocol and optimized cFIX plasmids, phenotypic correction of hemophilia B dogs can be achieved. 4) Focused US or shockwave therapy will be suitable for translation of the US protocol into clinical application. Public Health Relevance: Our goal is to develop an Ultrasound-mediated gene therapy protocol that can be easily translated into human applications to treat hemophilia and other genetic diseases. We have previously shown that delivery of a high-expressing, liver-specific hFIX plasmid can be enhanced by ultrasound (US) in combination with microbubbles (MBs) and achieved 66 fold increment in expression levels compared with gene transfer without US and MBs. This proposal will continue optimizing our US protocol in mouse and rat models and extend the development to normal and hemophilia B dog models.