Development of a Pkd1 mRNA Stabilizing Oligonucleotide for the Treatment of ADPKD

Background: Nearly 80% of individuals with ADPKD harbor a pathogenic mutation in one copy of the PKD1 gene. We have identified a miR-17 binding motif in the 3’ untranslated region (3’UTR) of PKD1 that represses mRNA translation emanating from the other remaining allele. Here we have engineered an oligonucleotide directly targeting the PKD1 3’UTR to sterically hinder miR-17 interaction with PKD1 mRNA and enhance its translation. Methods: We designed a 16-base pair oligonucleotide that specifically binds to the miR-17 binding motif in the Pkd13’-UTR (Pkd1 oligo). Next, we tested Pkd1 oligo in Pkd1+/+ and Pkd1RC/- kidney epithelial cells. We used CRISPR-based live-cell imaging and qRT-PCR of Pkd1 mRNA to quantify the abundance and transcript degradation rates to determine the Pkd1 mRNA half-life. We performed 3D cystogenesis assays and western blot to assess for phenotypic benefit. Finally, we characterized the effect of our oligo in multiple human ADPKD kidney cell lines. In each experiment, a non-targeting scramble oligonucleotide was used as a control. Results: Live-cell imaging of endogenous Pkd1 mRNA and qRT-PCR revealed that Pkd1 oligo treatment increased Pkd1 mRNA abundance in both Pkd1+/+ and Pkd1RC/- cells. In addition, Pkd1 oligo prolonged Pkd1 mRNA half-life compared to control oligo-treated cells. Moreover, we find that enhancement of Pkd1 mRNA by Pkd1 oligo translates to increased PC1 protein in both Pkd1+/+ and Pkd1RC/- cells. Phenotypically we find that Pkd1 oligo-treatment leads to reduced cyst size, enhanced mitochondrial membrane potential, and reduced pCREB expression in Pkd1RC/- cells. Finally, we find that treatment of multiple human ADPKD kidney cell lines with Pkd1 oligo enhances PKD1/PC1, improves mitochondrial metabolism, reduces cyst size and pathogenic marker expression. Conclusion: Our studies are the first to demonstrate the feasibility of oligonucleotides to raise endogenous PKD1/PC1expression. More broadly, our work suggests a novel 3’-UTR-based therapeutic approach for treating other haploinsufficient disorders by amplifying the healthy gene copy. Funding: NIDDK Support