Ultrasound-Assisted AQP1 Gene Therapy for Functional Restoration of Salivary Glan

描述(由申请人提供)：我们最近报道了一种在唾液腺中进行超声辅助基因转移(UAGT)的方法。这项技术可以使用FDA批准的微泡和临床超声设备将瞬时、非侵入性、非病毒的基因转移到唾液腺。我们认为，考虑到病毒载体系统的局限性，这项技术可能被证明能够应用于口干症的基因治疗，包括涎腺炎和随之而来的唾液腺功能减退。考虑到已经在小型猪身上进行了大量的前期工作，作为唾液腺功能的接近人的模型，我们的首要目标将是在小型猪上升级我们的uAGT技术，并将结果基因表达索引到病毒载体上。这第一个目标的总体目标将是 展示了与病毒载体的等价性，从而为它们的替代提供了理由。我们还将在我们的系统中测试一种高级一代(微环)质粒，该质粒在非病毒基因转移后可能具有更持久的基因表达能力。我们的下一步将是利用水通道蛋白-1(AQP1)对我们的猪受试者进行恢复性基因治疗，这种治疗性转基因在正在进行的人类基因治疗临床试验中显示出唾液修复的前景。UAGT和病毒方法将并行使用，主要的实验指标是功能性指标，即唾液流量。动物将按照之前公布的方法进行照射。使用病毒载体(腺病毒和腺相关病毒)进行AQP1治疗将如前所述，并将作为衡量我们基于uAGT的AQP1基因治疗效果的一个指标。与以前的研究不同，当唾液流量下降到对侧(对照)腺体的20%时，我们将努力再次治疗，我们的目标是保持12个月的功能改善。因此，我们将检验我们的假设，即uAGT将通过再次治疗实现恢复性基因治疗的慢性维持，而病毒载体不会。在我们的最终目标中，我们将探索一个重要但迄今尚未解决的问题，即AQP1基因治疗产生的唾液蛋白质组质量。为此，我们将对使用AQP1基因治疗的动物受试者产生的唾液进行全蛋白质组谱分析，并与未受辐射的猪和受辐射的猪的唾液进行比较，在这些唾液中，唾液腺发生了部分保留。扫描将使用二维差示凝胶电泳法，辅以多维色谱和质谱学方法。

DESCRIPTION (provided by applicant): We have recently reported the development of an ultrasound-assisted gene transfer (UAGT) method in the salivary gland. This technique enables transient, non-invasive, non-viral gene transfer to the salivary gland using "off the shelf" FDA-approved microbubbles and clinical ultrasonography equipment. We submit that this technology may prove enabling for applications in gene therapy for Xerostomia given the limitations of viral vector systems, which include sialoadenitis and consequent hyposalivation. Considering the substantial prior work that has been performed in miniature swine as a close-to- man model of salivary gland function, our first aim will be to upscale our UAGT technique in miniature swine and index resultant gene expression to viral vectors. The overall goal of this first aim will be to show equivalence to viral vectors, thereby providing a rationale for their replacement. We will also test in our system an advanced-generation (minicircle) plasmid with the putative capability of longer-lasting gene expression following non-viral gene transfer. Our next step will be to undertake a restorative gene therapy treatment in our swine subjects utilizing Aquaporin-1 (AQP1), the therapeutic transgene that has shown salivary restorative promise in an ongoing human gene therapy clinical trial. UAGT and viral methods will be utilized in parallel, with primary experimental metrics being functional, namely salivary flow. Animals will be irradiated according to previously published methodologies. AQP1 treatment with viral vectors (Adenovirus and Adeno- associated virus) will be undertaken as previously described and will serve as an index by which to measure the efficacy of our UAGT-based AQP1 gene therapy. In contrast to earlier studies, we will endeavor retreatment when salivary flow declines to <20% of that in the contralateral (control) gland, with our goal being maintenance of functional improvement for 12 months. We will thereby test our hypothesis that UAGT will enable chronic maintenance of restorative gene therapy through retreatment, whereas viral vectors will not. In our final aim, we will explore an important but heretofore unaddressed issue, that of the proteomic quality of saliva produced by AQP1 gene therapy. To do this, we will undertake full- proteome profiling of the saliva produced in our animal subjects with AQP1 gene therapy, compared with saliva from non-irradiated swine and irradiated swine wherein partial sparing of the salivary gland has occurred. Scans will be performed using 2-dimensional difference gel electrophoresis, supplemented by multi- dimensional chromatography and mass spectrometry methods.