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 常见的、严重的遗传病，基因治疗是有潜力的 救命的治疗。Cf的临床意义主要体现在 肺，这是与CF基因治疗最相关的组织 这一次。用腺病毒载体和脂质体治疗慢性萎缩性胃炎的临床研究 正在进行中，结果表明新载体的开发是 需要的。这项提案将解决三个具体问题1) 与目前相关的毒性和表达持续时间短 利用肺部的病媒，2)病媒进入原状的效率低下 肺上皮，以及3)无法监测肺内的基因转移 在无侵入性手术的CF患者中。新一代腺病毒 将使用称为增量向量的向量来解决这些问题， 认为这些载体将有潜力用于肺脏的想法 Cf患者。Delta载体与其他腺病毒载体的不同之处在于 它们不包含病毒编码序列。这些向量包含 感兴趣的表达盒，腺病毒反向末端重复 和包装信号，以及携带DNA所需的任何填充DNA 分子到合适的大小。它们在存在的情况下被传播 一种辅助病毒，它提供所有必要的蛋白质载体DNA 包装。最近使用静脉给药的结果提供了一个强有力的 表明这些载体具有提高安全性的巨大潜力 以及提高治疗基因表达的持续时间。这项建议 将通过评估三个特定的基因来解决它们在肺中的潜力 目的：1)比较含有Cf基因的Delta载体 构建不同毒性和持续时间的腺病毒载体 基因在肺中的表达；2)比较Delta载体 包含CF基因和修饰的纤维蛋白的那些不包含 修饰纤维蛋白对病毒侵入效率的影响 3)评估包含两种血清的A载体 报告基因甲胎蛋白(AFP)与Cf测定的实用性 甲胎蛋白作为报告基因在肺中的表达及应用甲胎蛋白追踪CFs的能力 表达，希望能使对基因转移的监测 有效且侵入性较小。为了实现这些目标，可以 用于生成携带CF和/或AFP的各种增量载体 表达盒将通过分子克隆来制作。这些质粒 将用于在辅助病毒存在的情况下生成载体。在……里面 此外，带有纤维基因的辅助病毒被修改为含有多赖氨酸 残基将被制造并将被用于传播媒介。之后 载体的传播，载体将在适当的体内进行测试 以及体外模型，以确定它们在人类身上的潜在价值。

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.