In certain inherited metabolic disorders, the absence of a functional enzyme can result in toxic accumulation of its substrate throughout the body. Often however, toxicity is manifest only in certain organs or tissues. in one such example, an adenosine deaminase deficiency results in the accumulation of (deoxy)adenosine substrate which is toxic for T and B cells. Nonetheless, this disorder is amenable to Current gene therapy approaches because peripheral lymphocytes and marrow progenitor cells can be targeted for gene transfer with the normal gene. in contrast, in a second example, where ornithine aminotransferase is deficient, ornithine accumulates and is toxic to retinal pigment epithelial cells. However, no realistic way exists at the present time to transfer the normal allele to these cells in humans. In this proposal we will test a new method of gene therapy applicable to both kinds of disorders. This is possible by focusing on reducing the systemic accumulation of a toxic enzyme substrate. Specifically, the approach is to create an intracellular enzyme sink in a patch of autologous skin which metabolizes the circulating precursor and thus reduces the systemic load. Keratinocytes and fibroblasts cultured from an enzyme deficient donor will receive the missing gene by ex vivo transfer and be used in the construction of transplantable organotypic cultures. Two specific disease models will be tested, severe combined immunodeficiency caused by adenosine deaminase deficiency and gyrate atrophy caused by ornithine aminotransferase deficiency.
The specific aims i nclude (i) a proof of concept in athymic mice; (ii) a quantitative assessment of substrate metabolism by genetically modified skin using an organotypic culture model; and (iii) an evaluation of methods designed to circumvent the loss of gene expression that is known to occur following transplantation of genetically modified cells to animal hosts. The information gained from this research will permit an accurate assessment of cutaneous gene therapy for reducing substrate loads in inherited metabolic disorders and will provide the basis for clinical trials.
