Adenoviral vector-mediated gene replacement therapy for inborn errors involving hepatocyte metabolism has been hindered by toxicity, limited duration of transgene expression, and restrictions on vector cloning capacity. These obstacles are due in part to the viral sequences within the vector and to a combination of both humoral and cell mediate immune responses of the host against products of viral transcripts and the therapeutic transgene. The recent development of helper-dependent adenoviral vectors (HDV) devoid of all coding viral sequences holds great promise for circumventing these obstacles. However, much remains to be done regarding the understanding of factors including liver-specific gene expression and nature a transgene product in modifying host-vector interactions. These will ultimately impact on efforts to obtain high-level prolonged expression of a therapeutic transgene for correction of a metabolic deficiency. A model of one such enzymatic deficiency of hepatocyte metabolism is citrullinemia, a defect in human argininosuccinate synthetase (hASS). This urea cycle disorder is characterized by severe neurologic morbidity associated with hyperammonemia which may present in both neonates, children, and adults. Moreover, the development of gene replacement intervention in this model is appropriate given the inadequacy of current treatments, the availability of both bovine and murine disease models, and the development of in viva isotopic measures of clinical efficacy of treatment. This group of disorders are an important model for developing liver directed gene replacement of diseases whose pathogenesis require high level gene expression of intracellular proteins and efficient transduction of hepatocytes. We propose to 1) elucidate the effects of liver-specific vs. ubiquitous expression of a therapeutic, eukaryotic intacellular transgene (hASS) on host cell mediated immune response (specifically cytotoxic T lymphocytes), duration of expression, and liver toxicity using a HDV, 2) determine whether prolonged transgene expression mediated by liver-specific expression is due to induction of tolerance or modification of antigen presenting cell function using hASS and/or ovalbumin, and 3) develop helper-dependent adenoviral vectors expressing hASS from an optimal liver-specific promoters to achieve prolonged clinical correction in a murine models of citrullinemia. The data from these studies would be widely applicable to gene replacement therapy in a host of intrinsic disorders of liver metabolism as well as deficiencies of secretory proteins.
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