DESCRIPTION (provided by applicant): 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 and kidney stone formation. Medical treatments to prevent the formation of cystine stones are not very effective and have unpleasant side effects. With the genetic basis of the disorder defined (mutation in SLC3A1, cystinuria type I), new opportunities for targeted molecular therapies exist. Transposons are non- viral plasmid-DNA based vectors that represent promising emerging tools for chromosomal transgene insertion and establishment of persistent gene expression. In order for transposon-based technologies to mediate effective and safe therapeutic gene transfer for cystinuria, kidney-targeted gene delivery must be combined with strategies able to target integration of therapeutic genes to unique chromosomal elements thereby limiting genotoxicity imparted by gene transfer. In specific aim 1, we have developed a novel hydrodynamic injection method for kidney-specific DNA delivery and have demonstrated that transposons are capable of achieving long-term gene expression using this technology. We propose to evaluate for the extent of long-term gene expression after kidney specific gene transfer, further elucidate the kidney cell types stably transfected, analyze transposon integration sites after gene transfer in vivo, and evaluate for the possibility of short- and long-term toxicity after transposon- mediated gene transfer to intact kidney. Thus far, long-term phenotypic correction of an inherited kidney disease has not been demonstrated using gene transfer. Mutation of a dibasic amino acid transporter encoded by SLC3A1 is the most frequent cause of cystinuria. In specific aim 2, we propose to use transposon-mediated kidney-specific gene transfer to phenotypically correct cystinuria in SLC3A1 -/- mice. We have recently demonstrated the ability to manipulate chromosomal integration site selection of the piggyBac transposon system by fusing a highly site-specific zinc finger protein (ZFP) to the piggyBac transposase, thereby directing integration into user-selected chromosomal elements. In specific aim 3, we propose an innovative method of selecting for site-directed events and will deliver SLC3A1 in a site-directed manner into cultured proximal tubular kidney cells. In additon, we propose to develop an animal model for evaluating for site-directed integration in vivo. These studies are intended to demonstrate the capability of transposon-mediated gene transfer for potential treatment renal syndromes in veterans.

PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE This project is focused on developing gene transfer technology for cystinuria, an inherited kidney disease in veterans. The proposed strategy could also be used for therapy for a variety of other human kidney diseases which plague veterans. Our proposed strategy is thefore extremely significant and relevant to the health of veterans and the overall mission of research within the VA.