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

项目3(Hauschka，Stephen D.，P.I.)我们假设对DMD和其他肌肉的最佳治疗方法 疾病，无论是细胞介导的还是媒介介导的，都需要一系列肌肉特异的调节盒来 不同的治疗产品在不同水平上的表达。满足其中一些目标的磁带有 经过设计和测试，但它们包含老鼠肌肉基因成分，并且它们的所有测试都已完成 在小鼠细胞培养和全身病毒传递后的小鼠体内。由于、和间隔的顺序 小鼠和人类肌肉基因增强子控制元件不同，因为初步研究表明 为了降低小鼠盒在人类肌肉细胞中的活性，关键是要优化调节盒 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 基于广泛测试的类似盒式磁带的小鼠版本的调节盒式磁带。单个盒式磁带 将在各种快/慢肌纤维类型以及不同类型的肌肉纤维中实现最佳功能 解剖肌肉。人体录像带将以“微型”和大型两种形式建造，以方便他们的 用不同大小的治疗性DNA包装在AAV和慢病毒(LV)载体中(4.8&9kb包装 限制)，因为较大的盒式磁带允许包括更复杂的监管功能。一种盒式磁带将是 设计用于生产治疗产品以响应外部输送的药物；因此允许分级 治疗产品的合成水平，取决于特定患者的生理需求。 卡带功能将在人类骨骼肌培养中进行测试，最好的卡带将在 在小鼠和人肌肉移植中免疫缺陷小鼠，以模拟体内成熟的特性 人类的肌肉。成功的DMD基因治疗可能需要患者卫星的稳定转导 肌肉细胞以及肌纤维核，以确保在以下情况下持续的治疗产品合成 与疾病相关的肌肉纤维的自然周转。卫星细胞转导及其维持 治疗产品的合成将通过LV转导的卫星细胞的克隆性分析在 逐渐增加的增殖轮；将对盒式磁带进行改装，以保留长期功能。 相关性(请参阅说明)： 这些研究是将基因和细胞介导性治疗应用于 治疗几乎所有人类肌肉疾病。此外，肌肉调节盒的优化 该功能将允许治疗患者以及使用低得多的病毒载体的供体细胞的体外培养 剂量。这将大大提高患者的安全性，较低的病媒要求将节省 数以百万计的巨额收入。

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.