CORRECTION OF HEPATIC DEFICIENCIES IN ANIMAL MODELS

动物模型肝缺陷的校正

• 批准号: 3776724
• 负责人: SAVIO WOO
• 金额: --
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3776724
• 关键词: Retroviridae; autologous transplantation; biological models; chimeric proteins; dogs; gene therapy; genetic disorder; genetic techniques; genetic transduction; glycoproteins; hemophilia As; hepatitis B virus group; host organism interaction; laboratory mouse; laboratory rabbit; liposomes; liver cells; model design /development; phenotype; phenylketonurias; plasmids; protein engineering; receptor; tissue /cell culture; transfection; virus envelope; virus receptors; xenotransplantation

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.

针对两种人类遗传性疾病动物模型的体细胞基因治疗 将尝试治疗肝功能缺陷。 苯丙酮尿症 (PKU) 是一种 肝酶缺乏继发的代谢紊乱 苯丙氨酸羟化酶，导致严重智力低下 未经治疗的儿童。 该疾病以常染色体隐性遗传方式传播 性状，其出现频率约为万分之一。 血友病 B 是 继发于血浆酶因子 IX 缺乏的凝血障碍。 这是一种 X 连锁疾病，影响十万分之一的男性。 我们的实验室 此前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，并证明 它们感染肝癌和原代肝细胞的能力，导致 各个基因的功能表达。 最近，我们有 开发了将原代肝细胞移植到活体中的技术 动物实验表明移植的细胞不仅能长期存活 在接受者中，但也无限期地表达了肝功能 体内。 应用这些技术来尝试体细胞基因治疗将是 在最近创建的 PKU 小鼠模型（以及因子 IX）中进行 有缺陷的犬模型。 将从受影响的肝细胞中分离出来 通过部分肝切除术并用重组体转导的动物 逆转录病毒。 病毒感染细胞的自体移植 进行，并对动物的表型进行分析。 或者， 我们将探索针对逆转录病毒感染的可能性 通过改变病毒的宿主细胞向性来影响活体动物的肝脏。 病毒包膜基因将被设计为编码表位，以有效地 与肝脏脱唾液酸糖蛋白受体或乙型肝炎病毒结合 受体。 具有此类嵌合包膜蛋白的重组逆转录病毒 动物体内接种后可能对肝细胞有趋向性。 最后，我们还将探索基于非病毒的基因方法 转移到培养物中的肝细胞以及活体的肝脏中 动物。 这些实验旨在提供实验 PKU 和因子 IX 缺陷患者的肝脏基因治疗的基础 将来。 相应开发的技术将适用于 由于肝脏缺陷导致的许多其他已知遗传性疾病。