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

描述（由申请人提供）：我们最近报道了超声辅助基因转移（UAGT）方法在唾液腺中的发展。该技术能够使用FDA批准的“现成”微泡和临床超声设备将瞬时、非侵入性、非病毒基因转移到唾液腺。我们认为，考虑到病毒载体系统的局限性，包括涎腺炎和随之而来的唾液分泌不足，这项技术可能被证明能够应用于口腔干燥症的基因治疗。考虑到大量的先前的工作，已经在小型猪作为一个接近人的模型的唾液腺功能，我们的第一个目标将是升级我们的UAGT技术在小型猪和索引所得的基因表达的病毒载体。第一个目标的总体目标是 显示出与病毒载体的等效性，从而为它们的替代提供了理论依据。我们还将在我们的系统中测试先进的一代（小环）质粒，其具有在非病毒基因转移后更持久的基因表达的推定能力。我们的下一步将是利用水通道蛋白-1（AQP 1）在我们的猪受试者中进行恢复性基因治疗，水通道蛋白-1（AQP 1）是一种治疗性转基因，在正在进行的人类基因治疗临床试验中显示出唾液恢复的前景。将平行使用UAGT和病毒方法，主要实验指标是功能性的，即唾液流。将根据先前发表的方法对动物进行辐照。用病毒载体（腺病毒和腺相关病毒）进行的AQP 1治疗将如前所述进行，并将作为衡量我们基于UAGT的AQP 1基因疗法的功效的指标。与早期研究相反，我们将在唾液流量下降到对侧（对照）腺体的20%以下时奋进再治疗，我们的目标是维持12个月的功能改善。因此，我们将测试我们的假设，即UAGT将使恢复性基因治疗通过再治疗的长期维持，而病毒载体不会。在我们的最终目标，我们将探讨一个重要的，但迄今尚未解决的问题，即由AQP 1基因治疗产生的唾液的蛋白质组质量。为此，我们将对在我们的动物受试者中用AQP 1基因治疗产生的唾液进行全蛋白质组分析，与来自未照射的猪和其中发生唾液腺部分保留的照射的猪的唾液进行比较。将使用二维差异凝胶电泳进行扫描，并辅以多维色谱和质谱法。

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.