Hyperammonemia is a clinical problem with severe consequences to the central nervous system. It is usually caused by liver disease, inherited metabolic disorders and various toxins. The main source of ammonia production is the bowel where it is generated and then diffuses into the portal blood. If the liver is unable to convert ambiguous ammonia to urea due to liver disease or an inherited enzymatic defect, the ammonia enters the systemic circulation exerting toxicity on the brain. The goal of this project is to develop a novel form off therapy for hyperammonemia aimed to act within aimed the intestinal environment. This approach will deliver into the bowel's lumen bacterial genes over-expressing arginine biosynthesis enzymes so that large amounts of arginine can be synthesized. Our hypothesis is that the expressed enzymes, residing within an ammonia rich environment, will trap ammonia and covert it to arginine rendering it non toxic. Escherichia coli strains incorporating large amounts of nitrogen into arginine have already been successfully engineered. These bacteria will be delivered to the bowel of a hyper- ammonemic animal model, the spf-ASH mouse. We will study the viability, colonization and anatomical distribution of the engineered bacteria in the bowel. The metabolic effects of this treatment of this treatment on the host with respect to ammonia metabolism will be investigated. Various regulated promoters will be tested for optimal expression of arginine biosynthesis genes in the bowel's environment As the anaerobic bacteria Bacteroides fragilis is orders of magnitudes more numerous within the colonic bacterial flora than E. coli, it is our ultimate vehicle for bacterial therapy. We are currently completing the identification of arginine biosynthesis genes and their regulation in this organism. B. fragilis will be engineered to over-produce arginine and will then be used to trap ammonia in the intestine of the spf-ASH mouse. The expression of arginine of arginine genes in the transformed B. fragilis will be investigated and their hyperammonemic mice as with engineered E. coli. The long term goal of this project is to test this therapy in humans with hyperammonemia after its efficacy and safety have been demonstrated in laboratory animals. There are many other inherited and acquired conditions which may be amenable to bacterial therapy. This project will provide better understanding of the promise and challenges of such therapeutic approach.
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