CORRECTION OF HEPATIC DEFICIENCIES IN ANIMAL MODELS

两种人类遗传病动物模型的体细胞基因治疗 对肝虚的治疗将有所尝试。苯丙酮尿症是一种 继发于肝酶缺乏的代谢紊乱 苯丙氨酸羟基酶和导致严重精神发育迟滞 未经治疗的儿童。这种疾病是以常染色体隐性遗传方式传播的 该基因的频率约为每10,000名高加索人中就有1例。血友病B是 凝血酶，凝血因子，凝血酶。 这是一种X连锁疾病，每10万名男性中就有一人受到影响。我们的实验室 曾报道过重组逆转录病毒的构建 携带编码两种人类酶的全长cDNA，并展示了 它们感染肝癌和原代肝细胞的能力导致 各自基因的功能表达。最近，我们有 开发出将原代肝细胞移植到活细胞中的技术 并表明，移植的细胞不仅能长期存活 在受体中，还无限期地表达了肝功能 活着。 应用这些技术来尝试体细胞基因治疗将是 在最近创建的北大小鼠模型中进行，以及因子IX- 虚证犬模型。将从受影响的患者体内分离出肝细胞 大鼠肝部分切除后转导重组 逆转录病毒。病毒感染细胞的自体移植将 并对动物的表型进行分析。或者， 我们将探讨针对人的逆转录病毒感染的可能性 通过改变病毒的宿主细胞嗜性，使活体动物的肝脏。 病毒包膜基因将被设计成编码表位，以有效地 与肝脏去唾液酸糖蛋白受体或乙肝病毒结合 受体。含有这种嵌合包膜蛋白的重组逆转录病毒 可能对动物体内接种的肝细胞有趋向性。 最后，我们还将探索基因的非病毒基础方法。 转移到培养中的肝细胞以及活着的肝脏 动物。这些实验旨在提供实验 PKU和FIX缺乏症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是由肝脏缺陷引起的。

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.