CRISPR activation of endogenous PKD1 increases polycystin-1 levels and suppresses cellular features of ADPKD

Most cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by mutations in PKD1, which reduce polycystin-1 (PC1) levels below a critical functional threshold. Normalizing PC1 dosage mitigates disease progression; therefore, we sought to develop a CRISPR activation (CRISPRa) strategy to transcriptionally upregulate endogenous PKD1. We systematically screened multiple single-guide RNAs using an EGFP-reporter platform and identified potent candidates targeting the proximal PKD1 promoter in mouse and human cell models. Our results demonstrate that CRISPRa effectively increased endogenous Pkd1 mRNA in the mouse collecting duct-derived Pkd1RC/-cell model and in the primary renal epithelial cells from PKD mice. In Pkd1RC/- cells, CRISPRa of Pkd1 increased PC1 protein levels and significantly reduced cell proliferation and in vitro cyst formation in 3D cultures. Mechanistically, Pkd1 activation improved mitochondrial membrane potential, reduced dependency on aerobic glycolysis, and corrected signaling pathways involved in cystogenesis, specifically reducing intracellular cAMP, cMyc, pCreb, and pErk levels, while increasing pYap1 levels. We confirmed the translational potential of this platform by successfully activating PKD1 in primary renal epithelial cells from human kidneys. We observed a heterogeneous response across both normal and ADPKD patient-derived donor lines, with significant upregulation achieved in two of the tested cell preparations. These findings provide a compelling proof-of-concept that CRISPRa-mediated gene augmentation can increase PC1 levels, establishing a foundation for promising gene therapies aimed at successfully suppressing the pathogenic features of ADPKD. New and noteworthyThis study provides proof-of-concept for a CRISPR activation (CRISPRa)-based approach to ADPKD. CRISPRa targeting the PKD1 promoter, increased polycystin-1 levels in mouse and human cells. Upregulation of a hypomorphic Pkd1 allele increased functional polycystin-1, corrected dysregulated signaling pathways, suppressed cell proliferation and in vitro cyst formation. These results establish CRISPRa as a promising therapeutic approach to restore polycystin levels above a critical threshold and suppress cystogenesis in ADPKD.