Metabolic consequences of cystinuria and genome engineering therapeutics

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ABSTRACT Cystinuria is an inherited human renal disease with significant morbidity affecting 1 in 7000 veterans. The disease is caused by mutation of genes involved in renal cystine transport resulting in elevated urinary cystine with kidney stone formation. Historically, the primary clinical concern of cystinuria has been that cystine spills into the urine resulting in nephrolithiasis. However, cystinuria patients develop more chronic kidney disease and hypertension than other stone formers. Additionally, little is known about other consequences of loss of amino acid transport function (of cystine, ornithine, lysine, and arginine). These additional metabolic consequences of cystinuria likely contribute to the observations that cystinuria patients develop more chronic kidney and hypertension compared to other kidney stone formers. With the genetic basis of the disorder defined (mutation in Slc3a1, cystinuria type I), opportunities for targeted molecular therapies exist. Building upon our previous grant, we propose an innovative experimental design to demonstrate long-term phenotypic correction of cystinuria in an intact animal using a combination of transposon, adeno-associated virus (AAV), and CRISPR/Cas9 genome engineering technologies. In specific aim 1, we will evaluate the effects of the metabolic changes on sensitivity to and recovery from kidney injury and the development of hypertension. We will use ischemia reperfusion injury models and the unilateral ureteral obstruction to evaluate sensitivity to and recovery from kidney injury. We will also evaluate whether α- lipoic acid has any effect on these metabolic consequences other than its known ability to increase the solubility of cystine in the urine and prevent cystine stone formation. In specific aim 2, we propose to engineer a chimeric piggyBac transposase capable of rescuing of Slc3a1 expression and we will compare this to CRISPR/Cas9 mediated targeted integration in mouse proximal tubular cells lacking Slc3a1. We also propose to attempt permanent correction of cystinuria in vivo by multiple genome engineering technologies including transposon technology with concomitant immunosuppression, hybrid AAV-transposon technology and CRISPR/Cas9 mediated genome editing or targeted integration. We will attempt correction both in neonatal and adult mice lacking Slc3a1 assaying for reduction of cystine level in the urine, increase of cystine level in the plasma, and reduction of cystine stones. The proposed studies will lead to a greater understanding of the metabolic consequences of cystinuria and develop genome engineering approaches for cystinuria and potentially other kidney diseases affecting veterans.