SOMATIC GENE THERAPY FOR PHENYLKETONURIA

这项提案涉及到遗传转移方法的发展。 人苯丙氨酸羟化酶(PAH)的原代和培养克隆 细胞和整个动物。将对一系列逆转录病毒载体进行评估 作为多环芳烃基因转移载体的多环芳烃及其定向高活性 PAH表达水平，并产生高滴度的重组病毒。 我们将探索使用重组逆转录病毒来导入PAH 多种细胞类型的活动，包括小鼠NIH3T3，这是一种 用于鉴定逆转录病毒功能的标准宿主、原代和 永生化的人类细胞系，这将使我们能够表征 感染人类细胞及各种小鼠和大鼠肝癌细胞的能力 已知PAH表达不足的品系，可用于 鉴定重组逆转录病毒的重组能力 PAH全酶活性。我们将探索几种感染猪瘟的方法 活体动物和原代细胞在体外的感染 移植到宿主动物体内。特别重要的是 肝细胞体外感染方法的研究进展 将这些细胞植入宿主动物体内。为了构成多环芳烃 在肝细胞以外的细胞中的全酶活性，我们将构建 同时表达多环芳烃和二氢蝶呤还原酶的逆转录病毒载体 (Dhfr)。重组多环芳烃的表达将进行整体研究 动物和基因转移的生化效应将被评估 在高苯丙氨酸血症(PH-/PH-)小鼠中。 这些实验旨在为以下方面提供实验基础 北京大学的基因治疗。北京大学代表了一个很有吸引力的研究模式 对一种常见疾病的躯体基因治疗，这是一种公认的医学 以及对公共卫生的关注。

This proposal deals with the development of methods for genetic transfer of cloned human phenylalanine hydroxylase (PAH) into cultured and primary cells and whole animals. A series of retroviral vectors will be evaluated as vehicles for genetic transfer of PAH and their ability to direct high level of PAH expression and produce high titers of the recombinant virus. We will explore the use of recombinant retroviruses to introduce PAH activity into a variety of cell types including mouse NIH3T3, which is a standard host for characterization of retrovirus function, primary and immortalized human cell lines, which will allow characterization of the ability to infect human cells, and various mouse and rat hepatoma cell lines which are known to be deficient in PAH expression and can be used to characterize the ability of the recombinant retroviruses to reconstitute PAH holoenzyme activity. We will explore several methods for infection of live animals and infection of primary cells in vitro prior to transplantation into host animals. Of particular importance is the development of methods for infection of hepatocytes in vitro and implantation of these cells into host animals. In order to constitute PAH holoenzyme activity in cells other than hepatocytes, we will construct retroviral vectors which express both PAH and dihydropteridine reductase (DHFR). The expression of recombinant PAH will be investigated in whole animals and the biochemical effects of the gene transfer will be evaluated in the hyperphenylalaninemic (PH-/PH-) mouse. These experiments are designed to provide the experimental groundwork for genetic therapy of PKU. PKU represents an attractive model for the study of somatic gene therapy for a common disorder which is a recognized medical and public health concern.