Therapeutic rescue of Polycystin-1 protein expression by targeting PKD1 upstream open reading frames

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Project Summary/Abstract Autosomal dominant polycystic kidney disease (ADPKD) affects over 12 million people worldwide. Available therapies provide only a slight delay in ongoing growth of fluid-filled cysts in the kidney and liver that progress to kidney failure and in some cases devastating pain and abdominal distension. Approximately one third of ADPKD patients have non-truncating mutations in the primary disease gene PKD1/Polycystin-1(PC1). A significant subset of these encode a version of PC1 that fails to mature to its site of action at the cell surface yet may be an at least partially functional PC1 protein. PC1 “dosage”—the functional amount of PC1 protein at its site of action—correlates with disease severity. Nonetheless, feasible approaches to increase PC1 dosage had not previously been identified to evaluate therapeutically. We have contributed to the identification and characterization of several disease genes that encode proteins in the endoplasmic reticulum (ER) that are necessary for PC1 maturation. Patients with mutations in these genes also get kidney and liver cyst due to insufficient PC1 dosage, and many are in desperate need of treatmentsIn mouse models for these genes, increasing PC1 production by increasing a mouse’s genomic copy number of Pkd1 provides a striking rescue of cyst formation. We hypothesize that increasing PC1 protein expression in patients with mutations in these ER genes and in a substantial subset of patients with PKD1 non- truncating mutations will dramatically reduce cyst burden. We have identified, with supportive preliminary data, that the 5’ untranslated region of human PKD1 contains likely highly relevant upstream open reading frames (uORFs). uORF translation distracts ribosomes away from translating the intended protein. Our data suggests that blocking PKD1 uORF translation would produce a many- fold increase in translation of PC1. Blocking uORF translation is achievable as a clinical therapy using antisense oligonucleotides (ASO). ASOs are approved therapies for other diseases. For this proposal we will test and characterize the effect of abolishing human PKD1 uORF translation to increase PC1 expression and generate in vivo models for preclinical evaluation of this treatment on cystic disease severity. For our first aim we will evaluate two independent approaches in vitro: (1) edit uORF initiation codon sequence in the human PKD1 5’UTR using CRISPR to test the effect of uORF translation on PC1 expression, and (2) test and optimize ASOs to characterize the effect of steric inhibition of uORF translation and RNA secondary structure on PC1 expression. For aim 2 we will use CRISPR to humanize the 5’UTR of our epitope-tagged Pkd1-V5 mouse with or without uORF initiation codon edits. We will test the benefit of abolished uORFs to have a clinically meaningful effect on cyst formation and severity in our PC1 dosage-dependent mouse models and optimize models to evaluate for preclinical therapies.