Proximal Tubule Targeted Gene Therapy for Cystinuria

PROJECT SUMMARY Monogenic kidney diseases such as cystinuria are well characterized genetically, but lack safe and effective clinical treatments. Patients with cystinuria form numerous cystine-based stones in their urinary tract due to failure to reabsorb cystine in renal proximal tubule cells, leading to chronic kidney disease in up to 70% of cases. The most common subclass of cystinuria, type A, is a result of a homozygous deficiency of SLC3A1, which encodes an amino acid transporter (rBAT) that reabsorbs cystine in proximal tubules. Recent advances in the genome engineering field have allowed for potentially curative therapy for monogenic diseases including cystinuria. Current barriers to renal genome engineering include delivery and sustained expression of transgenes. However, cystinuria is an ideal model disease to investigate and potentially overcome these barriers as the proximal tubule is targetable within the kidney, a low level of rBAT is estimated to prevent stone formation, and cystinuria could be targeted at any stage of life. When designing renal gene therapy for cystinuria, previous work has shown advantageous integration efficiency of transgenes using the piggyBac transposon system. Kidney-targeted genome engineering using piggyBac transposons for in vivo models can be accomplished with a novel proximal tubule-targeted adeno-associated virus (AAV). I hypothesize that the combination of a renal specific AAV with piggyBac transposon integration of SLC3A1 will lead to stable, kidney- targeted phenotypic correction in models of cystinuria. To test this hypothesis, I will engineer renal-specific AAV vectors to contain piggyBac-SLC3A1 in AIM 1. Self-complementary AAV has shown improved kidney specificity, but its’ compact size necessitates the splitting of SLC3A1 into two AAVs. Therefore, I will design a dual AAV system that recombines in vivo to express full length SLC3A1 using homologous recombination and mRNA splicing. I will also test the recombination and functionality of the dual AAV-piggyBac-SLC3A1 system in vitro. In AIM 2, I will generate SLC3A1-/- kidney organoids derived from human inducible pluripotent stem cells (iPSCs). I will then quantify expression of rBAT and cystine transport after delivery of the proposed system. Finally, I will assess the potential of AAV-piggyBac-SLC3A1 to phenotypically correct cystinuria through prevention of cystine stone formation in a mouse model of type A cystinuria in AIM 3.