The long-term goal of this proposal is to correct an inborn error of metabolism, citrullinemia, by hepatocyte gene therapy using the adenoviral delivery system. Citrullinemia, an autosomal recessive enzymatic deficiency of argininosuccinate synthetase (ASS), is representative of the group of urea cycle defects. They present in the neonatal period with hyperammonemic coma and result in long-term neurological and developmental deficits. Plasma, metabolites, ammonia, and citrulline can be easily quantitated to monitor the efficacy of therapeutic intervention, and both murine and bovine models are available. Protocols derived from this study are expected to be applicable to other genetic disorders characterized by primary metabolic dysfunction of hepatocytes. Transient correction of the ASS deficiency will first be attempted by in vivo transduction of hepatocytes after intravenous delivery of an E1-deleted type 5 adenovirus (Ad-ASS) into a neonatal murine model. Once the optimal dose and pattern of delivery is determined and efficacy is established by direct enzymatic assay of liver isolates and by achieving normalized growth rates and prolonged survival, the protocol will be scaled up and applied to the larger bovine model. At first, a recombinant adenovirus (Ad-AAT) expressing a serum reported gene product, alpha-antitrypsin (alpha1AT), will be delivered intravenously into normal neonatal calves. The extent, efficacy, and kinetics of heterologous gene expression and morbidity associated with infection will be determined by serial measurements of serum alpha1AT, adenovirus specific neutralizing antibodies, blood chemistries, and immuno-histologic analysis of liver isolates. Subsequently, Ad-ASS will be delivered into citrullinemic newborn calves using optimized dosage and delivery protocols. Once transient correction of the metabolic defect is demonstrated in the bovine model, human trials will be proposed. Even a transient correction may abbreviate the hyperammonemic state and reduce long-term central nervous system morbidity. Concurrent studies directed at minimizing the role of host immune response in terminating the initial period of heterologous gene expression and in limiting subsequent reinfection will be carried out in the bovine model. The ability to induce immune tolerance will be investigated by delivering the reporter construct Ad-AAT in the neonatal and in utero period. In addition, new adenoviral vectors mutated in the E2a and E4 domains and designed to minimize adenoviral protein expression and host antigen presentation will be tested.
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