Abstract: FR-PO259
Assessing the Therapeutic Potential of Readthrough of Nonsense Mutations in Autosomal Dominant Polycystic Kidney Disease (ADPKD)
Session Information
- Genetic Diseases of the Kidneys: Cystic - II
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1101 Genetic Diseases of the Kidneys: Cystic
Authors
- Dillinger, Elizabeth K., Mayo Clinic Graduate School of Biomedical Sciences, Rochester, Minnesota, United States
- Harris, Peter C., Mayo Clinic Minnesota, Rochester, Minnesota, United States
Background
ADPKD is a common cause of ESKD, and truncating mutations are associated with more severe disease. Over 25% of catalogued PKD1 and PKD2 mutations are nonsense type, truncating mutations. Readthrough of nonsense variants resulting in full length wildtype or missense containing proteins is a promising treatment for this type of mutation. We hypothesize that readthrough therapies may result in an increase in PKD1 and PKD2 protein, polycystin-1 and -2 (PC1/PC2), and hence, decrease the severity of cystic disease.
Methods
Using a dual-fluorescent, short fragment, in vitro assay, and flow cytometry we determined the effect of various readthrough treatments on a catalogue of PKD2 nonsense mutations. After determining which treatments have the best efficacy on the PKD2 mutations we are further investigating the effects of these treatments on the level of readthrough, trafficking and localization of full length PC2 and the PC1/PC2 complex formation using a novel in vitro assay.
Results
We have assayed five PKD2 nonsense variant in detail that contain the three different stop codons and different flanking sequences and have found a dramatic difference in the level readthrough depending on the sequence. We have also added 5 mM caffeine to the culture media which increases the rate of readthrough by diminishing nonsense mediated decay, although alone did not promote readthrough. For the nonsense variant p.Trp414*, with the nonsense codon TGA C, levels of readthrough of ~28% were recorded with 1800ug/ml of gentamycin, that is promoted to ~38% with the addition of caffeine. In contrast, the nonsense variant p.Gln768*, codon TAA G, had readthrough of only ~6%, that was not further boosted with caffeine. Analysis of treated MEFs from a mouse mimicking the Gln768* variant showed minimal readthrough. The level of readthrough with the compounds G418 or ataluren for p.Trp414* were not as effective as gentamycin.
Conclusion
Our studies indicate that readthrough may be a feasible means to increase the level of functional polycystin protein in ADPKD and so a therapy worthy of consideration. However, selecting the best drug and patients based on the specific stop codon may dramatically alter the efficacy.
Funding
- NIDDK Support