CORRECTION OF HEPATIC DEFICIENCIES IN ANIMAL MODELS

两种人类遗传性疾病动物模型的体细胞基因治疗 肝脏缺陷的治疗 苯丙酮尿症（PKU）是一种 继发于肝酶缺乏的代谢紊乱 苯丙氨酸羟化酶，并导致严重的精神发育迟滞， 未经治疗的儿童。 该疾病是作为常染色体隐性遗传 这是一个特征，频率约为1/10，000高加索人。 血友病B是 由于缺乏血浆酶凝血因子IX而引起的凝血障碍。 这是一种X连锁疾病，影响十万分之一的男性。 本实验室 先前已经报道了重组逆转录病毒的构建 携带编码这两种人类酶的全长cDNA， 它们感染肝癌和原代肝细胞的能力， 各自基因的功能表达。 最近我们 开发了将原代肝细胞移植到活体 结果表明，移植的细胞不仅能长期存活， 在接受者中，而且在接受者中无限期地表达肝功能。 vivo. 将这些技术应用于体细胞基因治疗的尝试， 在最近创建的PKU小鼠模型中进行，以及因子IX- 缺陷犬模型。 将从受影响的动物中分离肝细胞 动物通过部分肝切除术和转导的重组 逆转录病毒 病毒感染细胞的自体移植将 进行，并分析动物的表型。 可选择地， 我们将探索针对逆转录病毒感染的可能性， 通过改变病毒对宿主细胞的嗜性而感染活体动物的肝脏。 病毒包膜基因将被工程化以编码高效表达的表位。 与肝脱唾液酸糖蛋白受体或B型肝炎病毒结合 受体的 具有这种嵌合包膜蛋白的重组逆转录病毒 在动物体内接种后可能对肝细胞具有趋向性。 最后，我们还将探索基因表达的非病毒基础方法。 转移到培养的肝细胞以及活体肝细胞中， 动物 这些实验旨在提供实验性的 PKU和因子IX缺乏患者肝脏基因治疗的基础 在未来 相应开发的技术将适用于 许多其他已知的遗传性疾病由于肝脏缺陷。

Somatic gene therapy for two animals models of human genetic disorders due to hepatic deficiencies will be attempted. Phenylketonuria (PKU) is a metabolic disorder secondary to a deficiency of the hepatic enzyme phenaylalanine hydroxylase and causes severe mental retardation in untreated children. The disorder is transmitted as an autosomal recessive trait and has a frequency of about 1 in 10,000 Caucasians. Hemophilia B is a clotting disorder secondary to a deficiency of plasma enzyme, Factor IX. It is an X-linked disorder that affects 1 in 100,000 males. Our laboratory has previously reported the construction of recombinant retroviruses bearing full-length cDNAs encoding the two human enzymes and demonstrated their ability to infect hepatoma and primary hepatocytes that resulted in the functional expression of the respective genes. Recently, we have developed technologies to transplant primary hepatocytes into living animals and showed that the transplanted cells not only survived long-term in the recipients, but also expressed hepatic functions indefinitely in vivo. Application of these technologies to attempt somatic gene therapy will be carried out in a recently created PKU mouse model, as ell as a Factor IX- deficient canine model. Hepatocytes will be isolated from the affected animals by partial hepatectomy and transduced with the recombinant retroviruses. Autologous transplantation of the virus infected cells will be performed, and the animals' phenotypes will be analyzed. Alternatively, we will explore the possibility of targeting retroviral infection of the liver in living animals by altering the host cell tropism of the virus. The viral envelope gene will be engineered to encode epitopes for efficient binding to the hepatic asialoglycoprotein receptor or the hepatitis B virus receptor. Recombinant retroviruses with such chimeric envelope proteins may have tropism for hepatic cells upon in vivo innoculation of animals. Finally, we shall also explore nonviral-base methodologies for gene transfer into hepatocytes in culture as well as the livers of living animals. These experiments are designed to provide the experimental groundwork for hepatic gene therapy of PKU and Factor IX deficient patients in the future. Technologies developed accordingly will be applicable to a multitude of other known genetic disorders due to liver deficiencies.