Polycystic kidney disease (PKD) is an inherited disorder that affects more than 600,000 people in the United States. The disease causes fluid-filled cysts which can affect kidney functioning and lead to kidney failure, an outcome requiring dialysis or transplant. Currently, there is no cure for PKD.
PKD is typically passed on to the next generation as a heterozygous genotype – a child inherits a different version of a gene (or allele) from each parent, and a mutation in just one of these genes is enough to cause the disease. However, scientists have theorized that PKD onset may require a second-hit mutation – an abnormality that occurs in the course of an individual’s life.
Benjamin Freedman, PhD, is an Associate Professor of Medicine/Nephrology and faculty member in the UW Institute for Stem Cell and Regenerative Medicine (ISCRM). His lab has pioneered the use of stem cell-derived organoids to study kidney disease, including how PKD originates and potential treatments. In the course of their ongoing research, Freedman and his team became curious whether kidney organoids with a heterozygous genotype would develop PKD.
To explore this question, the Freedman Lab turned to CRISPR base editing, a technology that allows scientists to change a single nucleotide without actually cutting the DNA strand, making it an especially precise approach to explore the genetics underpinning a complex inherited disease like PKD.
To explore this question, the Freedman Lab turned to CRISPR base editing, a technology that allows scientists to change a single nucleotide without actually cutting the DNA strand, making it an especially precise approach to explore the genetics underpinning a complex inherited disease like PKD. Targeting four clinically relevant mutations in PKD1 and PKD2 (encoding polycystin-1 and polycystin-2), the researchers turned normal versions of the genes into what are known as nonsense mutants. These abnormalities, which include early stop codons that prematurely halt protein synthesis, account for approximately one third of PKD mutations. The 3D organoid models revealed new insights about the genetic mechanisms of PKD and allowed Freedman and his team to test potential therapeutics.
The results of the investigation appear in the journal Cell Stem Cell. Dr. Freedman is the study’s primary investigator. The first author of the paper is Courtney E. Vishy, an MD/PhD student who did her thesis work in the Freedman Lab.
“The first observation that Courtney made in the organoids was that while the heterozygotes did have reduced expression of the gene that we were mutating, they don’t form cysts at any detectable rate compared to the controls,” explains Freedman. “This is a good support for the idea that a single mutation is not enough to cause the disease and that you really do need a second hit.”
In an important, related outcome, Freedman says the findings suggest that is possible for a person to lose 50% of the protein associated with PKD and yet not develop the cysts that cause harm to the kidneys. Establishing an approximate threshold led the researchers to ask another question. If that less-than-complete level of gene expression was enough to inhibit the disease, could it be possible to use existing, already approved drugs to treat patients with certain PKD profiles?
“It was an encouraging early sign that heterozygous organoids were not displaying PKD traits,” says Freedman. “If we had found that even the heterozygotes get cysts, then there would really be no hope for this drug to work because the drugs will not give you 50% expression back. On the other hand, if you could sustain the levels of the necessary proteins above that red line, then you could slow down the disease to a point where people might not need to get a transplant.”
The true test, then, was to determine the feasibility of restoring sufficient proteins levels in homozygous organoids, to prevent cyst formation. The researchers focused on a class of drugs called eukaryotic ribosomal selective glycosides, which have been designed to read through the type of stop codons that were impeding the construction of PKD proteins. Drugs administered to the homozygous organoids read through enough of the mutations to substantially reduce cyst formation, a finding Freedman says may be especially beneficial for certain PKD patients.
“It’s known from clinical studies that patients who have truncating mutations have a more severe and progressive form of PKD and are at greater risk for needing a kidney transplant. This technology could be used in a dish to profile different mutations and their likelihood to respond to a specific treatment.”
For now, Freedman emphasizes the significance of the recent findings. “In the investigation, we were able to create our first experimental model of PKD with these types of mutations. That gave us new insights into the genetics of the disease. From there, we were able to demonstrate that drugs known to be safe for humans can potentially be used to help prevent the formation of PKD cysts.”
Acknowledgements:
We thank Vijay Modur, Vasudeo Badarinarayana, Megan Cox, and Ali Hariri (Eloxx Pharmaceuticals), Jonathan Himmelfarb (JH) Isabella Jennings (UW), and Scott Medina (SM, University of Pittsburgh) for technical support and helpful discussions. Illustrations were created with BioRender.com. Studies were supported by an Eloxx Pharmaceuticals Award, NIH Awards R01DK117914 (BSF), UH3TR002158 (JH), UH3TR003288 (JH), U01DK127553 (BSF), U01AI176460 (BSF), U2CTR004867 (BSF), UC2DK126006 (BSF), P30DK089507 (pilot to BSF), R21DK128638 (SM), R35GM142902 (SM), the Lara Nowak Macklin Research Fund, and a Washington Research Foundation fellowship (CT).