MODIFICATION OF THE ADENOVIRUS FIBER TO ALTER THE TARGET RECEPTOR SPECIFICITY

腺病毒作为基因治疗载体治疗囊性疾病的研究现状 纤维化(CF)是基于其高亲和力结合和随后的 有效地摄取呼吸道上皮细胞。腺病毒结合是 由一种未定义的细胞表面受体(ADV-R)介导，据信 结合腺病毒纤维多肽的C末端部分。一个 纤维基因组织与纤维蛋白结构的结合 元素使得对纤维多肽的遗传操作成为一种特别的 有趣的和潜在的非常有用的科学努力 基因治疗的目的。使用一种诱变方案 对于纤维基因，我们将进行i)。识别一般受体 纤维的配基结合域和II)。在细胞中靶向腺病毒 通过经修饰的纤维配体通过 指定的细胞表面受体。我们将采用的策略始于 纤维的插入突变，使用一种基础插入 在整个基因中都有六肽盒。盒式磁带的插入将 提供了一些关于功能域的信息，但它的主要目的 不是为了扰乱纤维功能。多肽盒中含有一种蛋白酶 凝血酶识别的裂解部位。使用在此 之后，我们将能够研究突变的影响。 已经产生了完整的病毒。这一战略将使我们能够专注于 影响病毒结构域的突变，这些病毒结构域负责与 受体。开发的系统将允许我们使用指定的 蛋白水解酶取代配体结合域，导致病毒 对于单元附着，该值为空。使用纤维缺陷病毒，我们 将为截然不同的共价偶联开发合适的化学 配体部分与有缺陷的病毒结合，产生结合病毒 通过改变配体受体的相互作用。对于某些定义明确的对象 受体配体，我们将通过基因修饰纤维多肽 设计并创造一种具有改变受体亲和力的嵌合纤维。 对嵌合纤维进行适当的生物学表征 病毒。在最基本的层面上，这些研究将扩展我们的知识 纤维配基结合域及其与细胞受体的结合 ADV-R拟议的研究还应产生一种 更安全有效的腺病毒载体将用于囊性纤维化 体细胞基因治疗，最后，但可能是最重要的， 这些研究应该创造一种新的腺病毒载体系统，它可以 用于组织特异性的基因靶向，目的是 纠正特定组织的病理。

Current use of adenovirus as a gene therapy vector for treatment of cystic fibrosis (CF) is based on its high affinity binding and subsequent efficient uptake into airway epithelial cells. Adenovirus binding is mediated by an undefined cell surface receptor (Adv-R) which is believed to bind the C terminal portion of the adenovirus fiber polypeptide. A combination of fiber gene organization and fiber protein structural elements make genetic manipulation of the fiber polypeptide a particularly interesting and potentially very useful scientific endeavor for the purpose of gene therapy. Using a mutagenesis protocol which is directed towards the fiber gene, we are going to i). identify the general receptor ligand binding domain of fiber and ii). Target adenovirus in a cell specific manner through a modified fiber ligand which binds via a designated cell surface receptor. The strategy we will employ begins with insertional mutagenesis of fiber, using an inframe insertion of a hexapeptide cassette throughout the gene. Insertion of the cassette will give some information about functional domains, but it's primary purpose is not to disrupt fiber function. The peptide cassette contains a protease cleavage site recognized by thrombin. Using constructs generated in this manner, we will be able to study the effects of the mutations after we have generated the intact virus. This strategy will allow us to focus on mutations which affect the virus domains responsible for attachment to the receptor. The system as it is developed will allow us to use the specified protease to displace the ligand binding domain , resulting in a virus which is null for cell attachment. Using the fiber defective virus, we will develop suitable chemistry for the covalent coupling of distinct ligand moieties to the defective virus creating a virus which will bind through an altered ligand receptor interaction. For certain well defined receptor ligands, we will modify the fiber polypeptide by genetic engineering and create a chimeric fiber with altered receptor affinity. Carry out appropriate biological characterization of the chimeric fiber viruses. At the most basic level, these studies will expand our knowledge of the fiberligand binding domain and it's attachment to the cell receptor Adv-R. The proposed studies should also result in the production of a safer and more effective adenovirus vector to be used in cystic fibrosis somatic cell gene therapy and finally but possibly of greatest importance, these studies should create a new adenovirus vector system which can be used for tissue specific targeting of genes with the intention of correcting tissue specific pathology.