Development of Adeno-Associated Viral Gene Therapy for an Inherited Kidney Stone Disorder

Abstract

While the genetic basis of monogenic kidney disorders such as cystinuria is well understood, the development of effective gene therapies remains hindered by the kidney's complex architecture and highly selective filtration barrier. This dissertation addresses these delivery challenges by establishing a comprehensive platform for renal gene therapy, using type A cystinuria — caused by mutations in the SLC3A1 gene — as a primary disease model. This work first characterizes AAV.cc47, a novel capsid variant that demonstrates robust, systemic transduction within the murine kidney. Using quantitative whole-kidney imaging, this enhanced tropism was specifically mapped to the Sglt2+ epithelial cells of the proximal convoluted tubule. Furthermore, to overcome the restrictive packaging limitations of self-complementary AAV vectors, a dual-vector hybrid reconstitution strategy was successfully developed to deliver larger transgenes in vivo. Next, rigorous comparison of AAV.cc47 alongside other purposefully evolved, kidney-targeted variants (AAV.k13 and AAV.k20) was conducted. This analysis reveals distinct functional profiles based on administration routes: AAV.cc47 emerged as the optimal candidate for systemic delivery even in diseased microenvironments, whereas AAV.k20 demonstrated unparalleled potency when administered locally via retrograde renal pelvis injection. Finally, to facilitate the translation of these therapies, a multifaceted investigative toolkit was developed. This includes adapting human kidney organoids to a scalable 96-well format for high-throughput screening, engineering a human SLC3A1-knockout iPSC disease model, optimizing a reliable colorimetric assay for urinary cystine quantification, and validating the obligate interdependence of the rBAT and b0,+AT transporter subunits. Collectively, this work provides an optimized framework of targeted capsids, advanced models, and precise assays to propel genetic medicines for inherited kidney diseases closer to clinical reality.