IMPROVED ADENOVIRAL VECTORS FOR HEPATIC GENE THERAPY

这里提出的研究目标是开发改良腺病毒。 可用于将基因转移到肝脏进行治疗的载体 肝脏缺乏症。虽然腺病毒介导的基因转移 活体肝细胞在动物模型中非常有效，改进是 因为基因表达的暂时性和不能 进行重复转导。具体目标是：1)开发一种 E4基因功能缺陷的重组腺病毒载体。E4 缺陷载体除了为克隆外源基因提供了更大的空间外， 应导致内源性腺病毒基因在转导的 细胞。因此，对宿主细胞基因的干扰可能较少。 调节和降低了载体产生抗原的可能性 最终将限制体内转导细胞的寿命。2) 确定抗原特异性免疫反应在a)丢失中的作用 腺病毒介导的基因在肝细胞中的表达和b)不能 腺病毒介导的肝细胞二次转导 体内基因转移。对这些免疫反应的描述将允许 合理设计非免疫原性载体。3)调查 腺病毒E3区编码的单个蛋白在人类免疫缺陷病毒中的意义 体内肝细胞基因转导后的重组腺病毒载体。 一些E3基因产物的结构性高水平基因表达可能 保护被转导的细胞免受免疫介导的破坏。我们建议 在活体内直接解决这些问题。4)研究腺病毒介导的 将基因转移到胆管上皮细胞中，将其作为一种方法 治疗一些内科疾病。5)评估胆道 在体内将基因运送到肝细胞的途径。这种模式 与血管输送相比，基因输送具有潜在的优势 并将作为当前方法的替代方法进行探索。 该项目的成功完成将对以下方面产生普遍的应用 目前在腺病毒作为载体的所有细胞类型 用于基因转移。以肝脏为靶器官将具有普遍意义 基因治疗在大量遗传性疾病中的应用 由肝脏不足引起的。

The goal of the research proposed here is to develop improved adenoviral vectors that can be used for gene transfer to the liver for the treatment of hepatic deficiencies. Although adenoviral-mediated gene transfer to hepatocytes in vivo is very efficient in animal models, improvements are needed because of the transient nature of gene expression and the inability to perform repeat transduction. The specific aims are to: 1) Develop a recombinant adenoviral vector deficient in E4 gene function. The E4 deficient vectors besides allowing more room for cloning exogenous genes, should lead to less endogenous adenoviral gene expression in transduced cells. As a result, there may be less interference with host cell gene regulation and less probability that the vector will produce antigens that ultimately will limit the life-span of transduced cells in vivo. 2) Establish the role of the antigen-specific immune response in a) the loss of adenoviral-mediated gene expression in hepatocytes and b) the inability to achieve secondary transduction of hepatocytes after adenoviral-mediated gene transfer in vivo. Delineation of these immune responses will allow for rational design of vectors which are non-immunogenic. 3) Investigate the significance of individual proteins encoded in the adenoviral E3 region in recombinant adenoviral vectors after in vivo hepatocyte gene transduction. Constitutive high level gene expression of some of the E3 gene products may protect transduced cells from immune-mediated destruction. We propose to directly address these issues in vivo. 4) Investigate adenoviral-mediated gene transfer into biliary epithelial cells to develop this as a method for the treatment of a number of medical disorders. 5) Evaluate the biliary tract as a means of delivering genes into hepatocytes in vivo. This mode of gene delivery has potential advantages over the vascular routes of delivery and will be explored as an alternative to current methods. The successful completion of this project will have general applications to all cell types that are currently being targeted by adenovirus as a vehicle for gene transfer. Using the liver as a target organ will have general application for gene therapy for a large number of genetic disorders resulting from hepatic deficiencies.