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.
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