Development of high activity human muscle-specific regulatory cassettes and their

项目-3（Hauschka，Stephen D.，P.I.）我们假设DMD和其他肌肉疾病的最佳治疗 无论是细胞还是载体介导的疾病，都需要一系列肌肉特异性调节盒， 不同治疗产品在不同水平的表达。实现其中一些目标的卡萨布兰卡 已经设计和测试，但这些包含小鼠肌肉基因成分，所有的测试都已经完成， 在小鼠细胞培养物中和在全身性病毒递送后的小鼠中。由于，和间距的序列 小鼠和人类肌肉基因增强子控制元件之间的差异，由于初步研究表明， 由于小鼠表达盒在人肌肉细胞中的活性较低，因此优化调节表达盒是至关重要的 用于在成熟的人类肌肉纤维中表达。我们将设计，构建和测试人类基因版本的 基于类似试剂盒的广泛测试的小鼠版本的调节试剂盒。单独的盒 将被设计为在各种快/慢肌纤维类型中以及在不同的肌纤维类型中具有最佳功能。 解剖肌肉人体盒将以“微型”和大型形式建造，以方便他们的工作。 在AAV和慢病毒（LV）载体中用不同大小的治疗性cDNA包装（4.8和9 kb包装 限制），因为更大的盒允许包含更复杂的调节功能。一种卡匣类型为 设计用于生产治疗产品以响应外部递送的药物;从而允许分级 治疗产品的合成水平取决于特定患者的生理需求。 将在人骨骼肌培养物中检测试剂卡功能，最佳试剂卡将在 小鼠和免疫缺陷小鼠中的人肌肉异种移植物中，以模拟成熟的 人体肌肉成功的DMD基因治疗可能需要患者卫星的稳定转导 肌细胞以及肌纤维核，以确保在 与疾病相关的和肌肉纤维的自然周转。卫星细胞转导和维持 治疗产品的合成将通过LV转导的卫星细胞的克隆测定来检查， 逐步增加的增殖轮次;并且盒将被修改以保持长期功能。 相关性（参见说明）： 这些研究是将基因和细胞介导的治疗应用于肿瘤的必要前奏。 治疗几乎所有的人类肌肉疾病。此外，肌肉调节盒的优化 功能将允许用低得多的病毒载体治疗患者以及供体细胞的离体培养物 剂量这将在患者安全方面提供重大改进，并且较低的载体要求将节省 数百万美元。

Project-3 (Hauschka, Stephen D., P.I.) We hypothesize that optimal therapy for DMD and other muscle diseases, whether cell or vector mediated, will require an array of muscle-specific regulatory cassettes for expression of different therapeutic products at different levels. Cassettes meeting some of these goals have been designed & tested, but these contain mouse muscle gene components and all of their testing was done in mouse cell cultures and in mice after systemic viral delivery. Since the sequences of, and spacing between mouse & human muscle gene enhancer control elements differs, and since pilot studies indicate lower activity of mouse cassettes in human muscle cells, it is critical that regulatory cassettes be optimized for expression in mature human muscle fibers. We will design, construct, and test human gene versions of regulatory cassettes based on extensively tested mouse versions of similar cassettes. Individual cassettes will be designed for optimal function in a variety of fast/slow muscle fiber types, as well as in different anatomical muscles. Human cassettes will be built in both "miniature" and large forms to facilitate their packaging with therapeutic cDNAs of different sizes in AAV & Lentiviral (LV) vectors (4.8 & 9 kb packaging limits), as larger cassettes permit including more complex regulatory functions. One cassette type will be designed to produce therapeutic products in response to an externally delivered drug; thus permitting graded synthesis levels of the therapeutic product, depending on the physiological needs of particular patients. Cassette function will be tested in human skeletal muscle cultures, and the best cassettes will be retested in mice and in human muscle xenografts in immunodeficient mice, to mimic the in vivo properties of mature human muscle. Successful DMD gene therapy will likely require stable transduction of patient satellite muscle cells, as well as myofiber nuclei, to ensure continued therapeutic product synthesis following disease-related and natural turnover of muscle fibers. Satellite cell transduction and the maintenance of therapeutic product synthesis will be examined via clonal assays of LV-transduced satellite cells at progressively increased rounds of proliferation; and cassettes will be modified to retain long-term function. RELEVANCE (See instructions): These studies are a necessary prelude to the application of gene- and cell-mediated therapy to the treatment of virtually all human muscle diseases. Additionally, the optimization of muscle regulatory cassette function will permit treating patients as well as ex vivo cultures of donor cells with much lower viral vector doses. This will provide major improvements in patient safety, and the lower vector requirements will save millions of hfialth r.ara dollars.