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Kidney Week

Abstract: TH-OR39

Read-Through Therapeutics Reduce Cystogenesis in a Novel Cohort of CRISPR Base Edited ADPKD Organoids

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Vishy, Courtney E., University of Washington, Seattle, Washington, United States
  • Crawford, Daniel, Eloxx Pharmaceuticals, Waltham, Massachusetts, United States
  • Goddeeris, Matthew, Eloxx Pharmaceuticals, Waltham, Massachusetts, United States
  • Freedman, Benjamin S., University of Washington, Seattle, Washington, United States
Background

In autosomal dominant polycystic kidney disease (ADPKD), truncating nonsense mutations are responsible for 40-50% of cases, with increased disease severity and limited treatment options. Eukaryotic ribosomal selective glycosides (ERSGs) allow read-through of premature stop codons to restore full-length proteins as a novel therapeutic approach. However, existing animal and kidney organoid models of ADPKD lack mutations amenable to read-through.

Methods

Human pluripotent stem cells were CRISPR base edited to introduce four specific nonsense mutations previously documented in ADPKD patients – PKD1 R2430X and Q3838X and PKD2 R186X and R872X. Mutations were confirmed by sequencing and protein changes by immunoblot. Mutant and isogenic control stem cells were differentiated into kidney organoids to determine if nonsense mutations conferred a cystic phenotype. Premature stop codon read-through potential was evaluated for impact on cyst formation and toxicity (live/dead staining and LDH release) over a period of two weeks using two unique ERSGs.

Results

Nonsense mutant clones of each targeted genotype were obtained with the desired single base pair mutation and lacked expression of full-length protein. Fewer than 5% of isogenic control organoids formed cysts compared to > 80% in untreated mutant organoids. Treatment of mutant organoids with ERSGs reduced cystogenesis to < 20% and slowed the rate of cyst expansion in a dose-dependent manner. Treatment associated toxicity was not significantly detected at efficacious doses.

Conclusion

CRISPR base editing enabled rapid generation of an ADPKD organoid cohort with patient targeted nonsense mutations. The data suggest that read-through by ERSGs is a viable therapeutic approach for reducing cystic burden in a large subpopulation of patients with ADPKD, supporting the advancement of ERSGs in human clinical trials.

Funding

  • NIDDK Support