? DESCRIPTION (provided by applicant): Hepatocyte transplantation has been proposed as a cellular therapy for patients with inherited metabolic disorders of the liver. However, hepatocyte transplantation has several shortcomings that limit its clinical application. These limitations include a shortage and inconsistent supply of transplantable hepatocytes and a life-long dependence on immunosuppression after transplantation. We hypothesize that screenable inborn errors of metabolism, such as Hereditary Tyrosinemia Type 1 (HT1), can be treated by an individualized approach involving ex vivo gene therapy and in vivo cell transplantation. Our approach is individualized since the patient's own cells may be used in the treatment. In our initial proof of concept studies, mature human hepatocytes and less mature hepatocyte-like cells derived from human induced pluripotent stem (iPS) cells will be evaluated. These human cells will be expanded in a novel, genetically engineered large animal (pig) possessing homozygote mutations of two genes: 1) fumarylacetoacetate hydrolase (FAH-/-), the enzyme that catalyzes the final step in tyrosine metabolism; 2) recombination activating gene 2 (RAG2-/-) to produce severe immunodeficiency. FAH deficiency causes functional instability in effected hepatocytes and results in a selective milieu for robust expansion of normal (FAH+) donor hepatocytes. Rejection of FAH+ human cells in immunodeficient FAH-/- RAG2-/- pigs will be further prevented by fetal tolerization, transplanting donor cells in utero prior to immune development. We postulate that in utero cell transplantation will also provide a more natural fetal environment for differentiation of iPS-derived hepatocyte-like cells. One or more injection(s) of donor cells wll be performed postnatally to enhance engraftment. Tapered use of the protective drug, 2-nitro-4-trifluoromethylbenzoyl-1,3- cyclohexanedione (NTBC), will allow robust expansion of transplanted cells in FAH- deficient pigs. These proof-of-concept studies performed in a large animal model of HT1 will provide a foundation for hepatocyte transplantation as an individualized treatment of screenable inborn errors of liver metabolism.
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