UAB Childhood Cystic Kidney Disease Center (UAB-CCKDC) - Bioengineering Resource

PROJECT SUMMARY/ABSTRACT (BIOENGINEERING RESOURCE) The Bioengineering Resource (BR) will work collectively with the PKD RRC and the other PKD Centers in the consortium to advance PKD research. The BR will focus on the development and distribution of innovative new in vivo and in vitro models of PKD emphasizing cross-species comparisons (rats, mice, cell lines) along with a human IPS cell/kidney cystic organoid model system. We are developing orthologous PKD conditional mutant rat models using a tetracycline-regulated CRISPR/Cas9 strategy. This fulfills a need expressed by the PKD research base for larger models with closer physiological alignment to humans than currently exist. To overcome barriers associated with detection of the endogenous PKD proteins, we are generating mice and human IPS cell lines with self-labeling Halo/Snap tags inserted into the endogenous PKD proteins (PKD1, PKD2, PKHD1). These resources are critically needed to analyze PKD protein biochemistry and dynamics under native conditions in live cells and tissues. These Halo/Snap Tag PKD resources will enhance our understanding of PKD-related protein function, localization, proteolytic processing, transport, complex formation, and provide resources to define how patient-specific variants disrupt these proteins to contribute to cyst formation. We will establish a PKD Flp-In system in human IPS cells. This system permits very efficient integration and expression of PKD gene/cDNAs or gene regions (e.g., Pkd1 C-terminal tail) using the native PKD gene promoters. With these Flp- In resources, many PKD patient variants or protein domains can be assessed for functionality during renal cyst pathogenesis in kidney organoids. Along with the new resources and models being developed, the BR will continue to serve as a distribution hub for existing PKD and other ciliopathy resources. This includes congenital mutants, conditional nulls, reversible, and patient variant cell lines and animal models, cilia tag and pathway reporter systems, Cre recombinase mice, biosensor and bioreporters, and PKD mouse and rat biomaterials and tissues (kidney, liver, urine, blood). Collectively, the resources the BR is designing and distributing will help bridge major gaps between model systems and human clinical applications, ultimately contributing to the development of effective PKD treatments.