Ultrasound-Mediated Gene Therapy for Hemophilia B

描述（由申请人提供）：本提案旨在开发超声介导的非病毒基因传递到肝脏，用于治疗血友病和其他遗传疾病。我们已经证明，微泡[mb]存在下的超声（US）可以增强肝脏特异性高表达因子IX （FIX）质粒向小鼠的基因传递。通过转导一个肝叶实现了显著的增强，相对于单独使用裸DNA增加66倍。小鼠肝脏也可以用GFP质粒转导。因此，治疗性US与MBs有可能促进安全有效的hFIX非病毒基因转移治疗血友病。为了便于将该方法转化为人类应用，我们建议进一步优化美国参数以获得更高的有效性。优化的方案以及优化的pDNA/ mb复合物、改进的FIX基因转移载体将在血友病B小鼠模型中进行测试，以获得长期治疗效果。将设计合适的美国系统，用于在正常大鼠、正常狗和B型血友病狗身上测试美国方案。我们将验证以下假设：1)通过结合最佳的US方案和优化的FIX基因表达盒，可以在血友病B小鼠中达到治疗水平的FIX表达，并可以长期纠正其表型。2)优化大型动物模型中的US参数和探针，在正常犬模型中实现高水平的报告基因表达。3)将最佳US方案与优化后的cFIX质粒相结合，可实现B型血友病犬的表型校正。4)聚焦超声或冲击波治疗将适合将超声治疗方案转化为临床应用。

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.