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Browsing by Subject "Decarboxylase"

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    Gene Therapy: a new approach for preventing calcium oxalate stones
    (2014-03) Figueiredo, Marina Cruvinel
    Calcium oxalate (CaOx) urolithiasis is an important disease in companion animals and remains challenging for veterinarians to manage, because the precise etiological cascade of events leading to urolith formation is unknown. Over the past three decades, prevalence of CaOx stones has increased in dogs and cats. Medical therapy to dissolve calcium oxalate uroliths is currently unavailable. As a result, therapies are unsuccessful and uroliths are commonly associated with lower urinary tract discomfort and potential life-threatening urethral obstruction, as well as ureteral obstruction and kidney failure. Hyperoxaluria, due to increased urine CaOx saturation, is an important risk factor for calcium oxalate stone formation. Although the majority of urinary oxalate is derived from endogenous metabolic pathways, increased urinary oxalate appears to be sustained by an increased dietary load of oxalate and increased intestinal absorption, which is then secreted through the urine, since mammals are incapable of metabolizing oxalate. Our research goal is to develop safe and effective treatments to prevent stone recurrence, evaluating various approaches for gene therapy and their feasibility in a cell culture model system. The specific goal is to evaluate oxalate-degrading enzyme gene expression and activity in a feline kidney cell line, in order to identify potential candidates for future gene therapy applications in dogs and cats. Our hypothesis is that kidney cells (Crandell-Rees Feline Kidney-CRFK cell line) will stably degrade oxalate in vitro by expressing and secreting a functional oxalate-degrading enzyme into the media of transfected cells. Our objectives are: 1) to molecularly clone oxalate-degrading enzymes from available gene sequences for gene expression analysis; 2) to evaluate plasmid transfection into feline kidney cells, in order to optimize and characterize transfection of this cell line; and 3), to test the ability of oxalate-degrading activity of clones to decrease the oxalate content of media (measured by ion chromatography). Lastly, the most active protein will be tested for functionality. Results showed that oxalate-decarboxylase from different microorganisms sources were cloned, expressed and secreted in an eukaryotic cell system, and that these enzymes retained their oxalate-degradation activity.