Precise Editing of the Central Six Nucleotides in the Pkd1-3’UTR miR-17 Binding Motif Is Sufficient to Restore Pkd1 and Slow PKD Progression

Background: Nearly 80% of ESRD caused by ADPKD is due to mutations in the PKD1 gene. Large deletions encompassing the microRNA-17 binding motif within the Pkd1 3’UTR (P1MBM) can restore Pkd1 and slow cyst growth. Whether a fine-tuned approach to target the central 6 nucleotides in the miR-17 binding motif of the Pkd1 3’UTR will prevent miR-17 hybridization and rescue PKD remains unknown. Methods: We generated Pkd1RCΔ6/RC mice using CRISPR/Cas9 in conjunction with a novel donor template, where nucleotides 2-7 of the 8bp P1MBM were mutated. KspCre;Pkd1RCΔ6/RC mice were crossed with Pkd1F/F mice to generate KspCre;Pkd1RC/F and KspCre;Pkd1RCΔ6/F mice. Tissues were harvested at various time points for histological and molecular analysis, and omics studies. We also designed and tested a small antisense oligonucleotide to sterically bind P1MBM and prevent miR-17 mediated translational repression. Results: At P18, KspCre;Pkd1RCΔ6/F mice exhibited a 75% reduction in kidney-to-body weight ratio, and a 50% decline in serum creatinine compared to KspCre;Pkd1RC/F mice. Longitudinal studies with total kidney volume measured by MRI demonstrated a sustained cyst-attenuating effect in KspCre;Pkd1RCΔ6/F mice. Western blot (WB) exhibited an increase in PC1, andproteomics identified reduced proliferation and enhanced mitochondrial metabolism in KspCre;Pkd1RCΔ6/F mice kidneys. These findings were confirmed by WB and IF staining for pHH3, pCREB, and OXPHOS markers. Interestingly, cMyc, a driver of miR-17, and miR-17 itself remained increased in KspCre;Pkd1RCΔ6/F mice, suggesting that the 6bp change is sufficient to decouple the cMyc-miR-17-Pkd1 axis. Hence, we designed a 16-base pair anti-sense oligonucleotide and tested if it could hybridize to the Pkd1 3’UTR to mask P1MBM. Indeed, Pkd1 ASO stabilized Pkd1 mRNA, increased PC1 protein, slowed cyst growth, and improved metabolism in Pkd1RC/- and human ADPKD kidney cyst derived cells. Conclusion: Mutation of 6 cardinal nucleotides within the Pkd1 3’UTR is sufficient to rescue a mouse model of ADPKD, independent of miR-17 expression. Steric blockade of the P1MBM with a small ASO similarly stabilized Pkd1 mRNA, increased PC1 protein and reduced cysts in-vitro. These studies define a precise novel target within the Pkd1 3’UTR to treat ADPKD. Funding: NIDDK Support