LARGE DELETION ADENOVIRAL VECTORS FOR CYSTIC FIBROSIS

本申请寻求开发实质上改进的载体， 用于囊性纤维化（CF）基因治疗的临床试验。CF是一 一种常见的、严重的遗传性疾病，基因治疗是一种潜力 挽救生命的治疗CF在以下方面表现出其临床意义： 肺，这是与CF基因治疗最相关的组织， 这次使用腺病毒载体和脂质体治疗CF的临床试验 正在进行中，结果表明，新载体的开发是 needed.本提案将解决三个具体问题： 目前与之相关的毒性和表达持续时间短 利用肺中的载体，2）载体进入完整的 肺上皮，以及3）不能监测肺中的基因转移 在CF患者中，无侵入性手术。新一代腺病毒 矢量，称为增量矢量，将用于解决这些问题， 这些载体将有可能用于肺的想法， CF患者δ载体与其他腺病毒载体的不同之处在于， 它们不含病毒编码序列。这些载体含有 在目的表达盒中，腺病毒反向末端重复序列 和包装信号，以及任何必要的填充DNA， 分子到合适的大小。它们是在存在 一种辅助病毒，它提供所有必要的蛋白质载体DNA 包装.最近使用静脉内递送的结果提供了强有力的 这表明这些媒介具有提高安全性巨大潜力 和改善治疗基因表达的持续时间。这项建议 将通过评估三种特定的 目的：1）比较含有CF基因的δ载体与第一个 代腺病毒载体的毒性和持续时间的差异， 递送至肺后的基因表达; 2）比较δ-载体 将含有CF基因和修饰的纤维蛋白的纤维蛋白转化为不含有CF基因和修饰的纤维蛋白的纤维蛋白 修饰的纤维蛋白，用于病毒进入 气道上皮细胞; 3）评估含有血清和细胞因子的A载体， 报告基因甲胎蛋白（AFP）和CF，以确定 AFP作为肺中的报告基因和使用AFP追踪CF的能力 表达，希望使基因转移的监测更多 有效且侵入性更低。为了实现这些目标，质粒可以 用于产生携带CF和/或AFP的多种δ载体 通过分子克隆制备表达盒。这些质粒 将用于在辅助病毒存在下产生载体。在 此外，辅助病毒的纤维基因被修改为含有多聚赖氨酸 将产生残基并将其用于繁殖载体。后 载体繁殖，将在适当的体内试验载体 和体外模型来确定它们在人类中的潜在价值。

This application seeks to develop substantially improved vectors that can be used in clinical trials for cystic fibrosis (CF) gene therapy. CF is a common, serious genetic disease for which gene therapy is a potential life-saving treatment. CF manifests much of its clinical significance in the lung, which is the tissue of most relevance to CF gene therapy at this time. Clinical trials using adenovirus vectors and liposomes for CF are ongoing, and results indicate that the development of new vectors is needed. Three specific problems will be addressed in this proposal 1) the toxicity and short duration of expression associated with presently utilize vectors in the lung, 2) the inefficiency of vector entry to intact lung epithelium, and 3) the inability to monitor gene transfer in the lung in CF patients without invasive procedures. A new generation of adenovirus vectors, termed delta-vectors, will be used to address these issues, with the idea that these vectors will have potential to be used in the lung of CF patients. Delta-vectors differ from other adenovirus vectors in that they contain no viral coding sequences. These vectors contain the expression cassette of interest, the adenovirus inverted terminal repeats and packaging signal, and any stuffer DNA necessary to bring the DNA molecular to the appropriate size. They are propagated in the presence of a helper virus, which supplies all necessary proteins vector DNA packaging. Recent results using intravenous delivery provide a strong indication that these vectors have great potential for increasing safety and improving duration of expression of therapeutic genes. This proposal will address their potential in the lung by evaluating three specific aims: 1) to compare delta-vectors containing the CF gene to first generation adenoviral vectors for differences in toxicity and duration of gene expression after delivery to the lung; 2) to compare delta-vectors containing the CF gene and modified fiber proteins to those not containing modified fiber proteins for differences in efficiency of viral entry into airway epithelial cells; 3) to evaluate A-vectors containing both a serum reporter gene alpha-fetoprotein (AFP) and CF to determine the utility of AFP as a reporter gene in the lung and the ability to use AFP to follow CF expression, hopefully making the monitoring of gene transfer more effective and less invasive. To acheive these goals, plasmids that can be used to generate a variety of delta-vectors carrying CF and/or AFP expression cassettes will be made by molecular cloning. These plasmids will be used to generate vectors in the presence of helper virus. In addition, helper virus with a fiber gene modified to contain polylysine residues will be made and will be used to propagate the vectors. After vector propagation, the vectors will be tested in the appropriate in vivo and in vitro models to determine their potential value in humans.