Ultrasound-Mediated Gene Therapy for Hemophilia B

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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.