The group of inborn errors of hepatic metabolism continue to be a prominent cause of mental retardation because of ineffective treatment strategies. Gene replacement therapy offers the theoretic advantage of correcting the basic protein deficiency. However, progress in hepatocyte directed gene therapy has been limited by questions involving pathophysiologic processes, choice of promoter and vector delivery system, route of delivery, host immune clearance, duration of expression, availability or small and large animal disease models, and quantitative measures of clinical efficacy. The three parts of this proposal attempt to address some of these issues using the group of urea cycle defects a model system. The first goal is to better understand the pathophysiologic disturbances in patients with urea cycle defects by correlating genotype and clinical severity with in vivo measurement of nitrogen flux and ureagenesis, while also developing a quantitative measure for future in vivo gene therapeutic interventions in both animal and humans. Flux through the urea cycle pathway will be measured by quantifying the conversion of [15/N-amide]glutamine to [15/N] urea. This flux will be correlated with genetic status (homozygosity, heterozygosity, hemizygosity), nature of mutation (null versus hypomorphic), and clinical severity (neonatal versus later presentation) in affected patients, heterozygous family members, and normal controls. In the second part of the study, the relative safety and efficacy of first generation (E1a deleted) and second generation (E1a/E2a deleted and all coding sequence deleted) adenovirus vectors will be determined after intravenous (i.v.) delivery in animals. In addition, potential avenues permitting long term transgene expression will be investigated. The efficacy of transient immunosuppression for the readministration of viral vectors will be evaluated, and the potential use of mariner transposon elements in mediating transgene integration in a host mammalian genome will be studied. In the third part, the urea cycle disorders, specifically murine and bovine models of citrullinemia, will be used a model systems in applying these basic findings to a clinical setting. The efficacy of the hybrid, ubiquitously active, CAG and liver-specific human albumin promoters will be compared in vivo. These data will form the preclinical basis for designing phase I clinical trials involving gene therapy in urea cycle patients. These results together will also be more generally applicable to other inborn errors of hepatocyte metabolism and to the production of extracellular products by hepatocytes. Early and long term biochemical correction would be expected to greatly decrease the great neurologic morbidity associated with these conditions.
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