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 ammonia is generated and it then diffuses into the portal blood. If the liver is unable to convert ammonia to urea due to an enzymatic defect or severe liver disease, the ammonia enters the systemic circulation intoxicating the brain. The goal of this project is to research a novel biological gene therapy for hyperammonemia aimed at the bowel. This approach seeks to deliver and maintain overexpression of genes encoding the first and second urea cycle enzymes within the bacterial flora of the gut. The hypothesis is that the expressed enzymes residing in an ammonia rich environment, will trap ammonia and convert it to citrulline rendering it non toxic. The gene encoding the first two enzymes of the urea cycle will be incorporated into a single plasmid expression vector overexpressing these genes simultaneously. E. coli carbamyl phosphate synthetase (CPS) large subunit carB, capable of converting ammonia to carbamyl phosphate with or without the gene encoding the small subunit, carA capable of hydrolyzing glutamine to ammonia and the human ornithine transcarbamylase (OTC), converting carbamyl phosphate to citrulline will be used to construct this expression vector. Various regulated promoters will be tested for optimal expression of these enzymes. Strains of E. coli and B.fragilis obtained from human colon will then be transformed with these plasmids and the ability of the genetically engineered bacteria to convert free ammonia and glutamine derived ammonia to citrulline will be tested initially in vitro studying bacterial cultures and then in vivo by intestinal colonization of the spfASH mouse. The expression of the transformed bacteria in culture will be investigated for enzymatic activity, ability to convert ammonia to citrulline, the effect of pH (availability of bicarbonate) and the requirement of exogenous ornithine. Subsequently, the transformed bacteria will be fed yo OTC deficient spfASH mice. The survival of the transformed bacteria in the mouse colon and the metabolic effects of this therapy will be tested with respect to ammonia metabolism and ureagenesis. 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.
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