Genetics of cystogenesis in base edited human organoids reveal therapeutic strategies for polycystic kidney disease

Courtney E Vishy; Chardai Thomas; Thomas Vincent; Daniel K Crawford; Matthew M Goddeeris; Benjamin S Freedman

doi:10.1016/j.stem.2024.03.005

. Author manuscript; available in PMC: 2025 Apr 4.

Published in final edited form as: Cell Stem Cell. 2024 Apr 4;31(4):537–553.e5. doi: 10.1016/j.stem.2024.03.005

Genetics of cystogenesis in base edited human organoids reveal therapeutic strategies for polycystic kidney disease

Courtney E Vishy ¹, Chardai Thomas ¹, Thomas Vincent ¹, Daniel K Crawford ², Matthew M Goddeeris ², Benjamin S Freedman ^1,^3,^*,^†

PMCID: PMC11325856 NIHMSID: NIHMS2010691 PMID: 38579684

SUMMARY

In polycystic kidney disease (PKD), microscopic tubules expand into macroscopic cysts. Among the world’s most common genetic disorders, PKD is inherited via heterozygous loss-of-function mutations, but theorized to require additional loss-of-function. To test this, we establish human pluripotent stem cells in allelic series representing four common nonsense mutations, using CRISPR base editing. When differentiated into kidney organoids, homozygous mutants spontaneously form cysts, whereas heterozygous mutants (original or base-corrected) express no phenotype. Using these, we identify eukaryotic ribosomal selective glycosides (ERSGs) as PKD therapeutics enabling ribosomal read-through of these same nonsense mutations. Two different ERSGs not only prevent cyst initiation, but also limit growth of pre-formed cysts, by partially restoring polycystin expression. Glycosides also accumulate in cyst epithelia in organoids and mice. Our findings define the human polycystin threshold as a surmountable drug target for pharmacological or gene therapy interventions, with relevance for understanding disease mechanism and future clinical trials.

INTRODUCTION

Human organoids and organ-on-chip devices are being developed to better understand disease mechanism and enable investigational new drug (IND) applications¹. For genetic disorders, in which the human genome is a major therapeutic target, animal models are low throughput and may not fully recapitulate human conditions due to differences between species². Simpler human cell cultures, such as monolayers, remain immature compared to tissues and lack heterocellularity and 3D architecture³. Human organoids and related systems provide a natural solution to these challenges, for a wide variety of disorders.

One such disorder is polycystic kidney disease (PKD), a severe, life-threatening genetic disease featuring progressive expansion of microscopic tubules into numerous fluid-filled cysts. Affecting approximately 1 in 1000 individuals worldwide, PKD is the leading monogenic cause of renal failure^4,5. Tolvaptan, a vasopressin antagonist, can modestly slow cyst growth, but is associated with side effects of frequent thirst and risk of severe hepatotoxicity, preventing widespread usage in PKD patients⁶. Vasopressin activity in PKD is also primarily limited to the collecting ducts, whereas cysts also arise in other nephron segments⁷. Further, no treatments are available prior to cyst formation. Thus, there remains a large unmet clinical need for the development of new therapeutics for PKD.

PKD commonly results from loss-of-function mutations in PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively^8,9. Mutations are inherited in a germline heterozygous fashion, and somatic ‘second hit’ mutations are theorized to contribute to disease^10–13. Thus, at the cellular level, PKD is proposed to involve a loss of polycystin function below heterozygosity, although this has been difficult to prove. Recent studies in mice suggest that restoring gene levels can be therapeutic in PKD, even after cysts form, but the relevance of this approach to humans is not clear, as mice are much less susceptible to PKD as heterozygotes^14,15.

Nephron-like kidney organoids derived from human pluripotent stem cells (hPSCs) contain renal epithelial segments^16–19, with great relevance for studying PKD. Organoids with knockout mutations in either PKD1 or PKD2 express a robust cystic phenotype not observed in isogenic controls^17,20–22. Determining whether heterozygous organoids also show a cystic phenotype, similar to heterozygous patients, is important to provide insight into the fundamental mechanisms of PKD, and guide therapeutic strategies, such as gene correction. If repairing a single allele were sufficient to rescue the cystic phenotype, it would support future efforts towards monoallelic gene therapy. If, however, the cystic phenotype could not be rescued with a monoallelic edit, then it would indicate that both alleles need to be corrected for a gene therapy to take effect.

Another therapeutic strategy, which has yet to be tested, is to restore PC1 or PC2 expression using pharmacological agents that enable ribosomal read-through of in-frame nonsense mutations. Such mutations, which result in truncated proteins, constitute ~40% of PKD1 and ~50% of PKD2 cases^23–28, and produce more severe disease, compared to non-truncating mutations²⁶. Eukaryotic ribosomal selective glycosides (ERSGs) are aminoglycoside analogues, including the investigational drug ELX-02 (NB124) and the similarly active preclinical molecule ELX-10 (NB157)^29,30. ERSGs permit eukaryotic ribosomal read-through of premature stop codons by stabilizing the “exo” conformation^31–33, which allows a near-cognate tRNA to displace releasing factors³⁴. ERSG activity is specific to premature stop codons as compared to nascent stop codons³⁵. In preclinical evaluations for treatment of cystic fibrosis and cystinosis, ERSGs have demonstrated read-through and functional protein restoration both in vitro and in vivo^36–38. In Phase 1 clinical trials, ELX-02 has been well tolerated with appropriate pharmacokinetics^39,40. ELX-02 is preferentially absorbed by kidney tubular epithelial cells, which results in high exposure, but is optimized to avoid nephrotoxic side effects that arise with other aminoglycosides³⁸.

The therapeutic potential of ERSGs for PKD has not previously been tested, because mouse and organoid models with in-frame nonsense mutations do not exist. CRISPR base editing is a newer technology that confers the potential to specifically introduce a single nucleotide change, instead of indels^41,42, and can be more efficient and safer than homologous recombination following a double-strand break^43,44. Here, we establish CRISPR base edited organoid models of PKD, with clinically relevant nonsense mutations amenable to targeting with ERSGs, to evaluate the potential of restoring polycystin dosage as a therapeutic approach.

RESULTS

Base Editing Generates PKD hPSCs with Nonsense Mutations

Four clinically documented PKD nonsense mutations were selected for targeting by CRISPR base editing in hPSCs: PKD1-R2430X, PKD1-Q3838X, PKD2-R186X, and PKD2-R872X (Fig. 1A and Fig. S1A)²³. PKD1-R2430X and PKD2-R872X are the most common PKD patient nonsense truncating mutations in PKD1 and PKD2, respectively. The PKD1-R2430X site lies within a difficult-to-target region where six pseudogenes align, but we identified primers that enabled us to detect targeted editing events (Fig. S1B–C). The other two sites (PKD1-Q3838X and PKD2-R186X) were chosen in regions close in proximity to those for which we have previously established indel mutants modeling PKD^17,20. PKD1-R2430 and PKD2-R872 sites were not conserved between mouse and human (Fig. S1D).

To generate multiple allelic series, we employed the Base-Edited Isogenic hPSC Line Generation Using a Transient Reporter for Editing Enrichment (BIG-TREE) method (Fig. 1B and Fig. S1E–F)^45,46. Unedited C/C, heterozygous edited C/T, and homozygous edited T/T clones were identified for all four targeted PKD mutations (Fig. 1C–D)⁴⁷. In total, 280 clones were screened for editing and multiple clones of each genotype were obtained (Fig. 1C–D and S1F–G). 31 null mutant clones and 77 heterozygous clones were generated in this process, in addition to non-mutant controls. Representative clones of each genotype were confirmed to exhibit features of normal hPSC morphology (Fig. S2A).

All four PKD targeted nonsense mutants lacked detectable expression of the targeted PC1 or PC2 full-length protein, respectively (Fig. 1E). PKD1-R2430X and PKD2-R872X clones expressed truncated forms of PC1 and PC2, respectively (Fig. 1E and S1H). For the carboxy-terminal Q3838X PC1 mutant, the truncated form could not be resolved using our antibody (7e12) against the amino terminus of PC1, which is cleaved from the carboxy terminus during PC1 maturation (Fig. 1E)⁴⁸. However, levels of the PC1 amino terminus were reduced in the Q3838X mutant, suggesting degradation or nonsense-mediated decay (Fig. 1E–F). Another antibody suggested to recognize the C-terminus of human and mouse PC1 (E8–8C3C10) was unsuccessful in demonstrating specificity (Fig. S1H)⁴⁸. In each of our allelic series, PC1 or PC2 protein levels reflected their genetic dosage, with no detectable expression in homozygous mutants, and ~50% expression in heterozygotes (Fig. 1E–F).

Kidney Organoids with Homozygous Nonsense Mutations Model PKD

To evaluate the capacity of nonsense mutant hPSCs to model PKD, we first differentiated them into nephron-like kidney organoids (Fig. 2A)^17,49. Control and nonsense mutant kidney organoids expressed markers for podocytes, proximal tubules, and distal tubules in a similar fashion (Fig. 2B). Upon reaching maturity at day 18 of differentiation, kidney organoids were manually dissected out of adherent culture and transferred to suspension culture, a condition in which PKD organoids form cysts with high efficiency (Fig. 2A)²⁰. 75–95% of PKD nonsense mutant kidney organoids formed cysts when maintained over fourteen days in suspension culture, > 15-fold higher compared to isogenic unedited controls that largely failed to produce cysts in side-by-side experiments (Fig. 2C–G and Fig. S2A). Rates of cystogenesis and cyst growth were similar across all four targeted PKD mutations (Fig. 2D–G). To account for potential clonal variability, we utilized multiple independent clones per genotype in all experiments. PKD kidney organoid cysts expressed diverse markers of proximal (LTL, CUBN, OAT1, SLC3A1) and distal (ECAD, MUC1, NKCC2, DBA) tubular regions of the nephron, which co-existed in cyst-lining epithelia while remaining partially compartmentalized (Fig. 2H–I). These findings supported the observation that PKD organoid cysts arise rapidly from large stretches of tubular epithelium comprising multiple nephron segments of origin^20,22,50,51. Thus, base edited organoids with a spectrum of clinically-relevant mutations robustly recapitulated the PKD phenotype.

Heterozygosity Rescues PKD Cystogenesis

We assessed the phenotype of PKD heterozygous compared to homozygous mutant organoids, as well as isogenic controls, generated from our cohort of cytosine base edited hPSCs (Fig. 3A). In two allelic series, organoids heterozygous for either PKD1 or PKD2 nonsense truncations did not produce cysts at a significant rate, when compared with homozygous mutants, even after many weeks in culture (Fig. 3B–E and S3A). These findings indicated that the human gene dosage threshold to express PKD phenotype was below heterozygosity, i.e. ~50% of functional PC1 or PC2 relative to isogenic controls (above Fig. 1E–F).

To determine if a genetic approach for correcting nonsense mutations could be a viable option for PKD gene therapy, we employed CRISPR adenine base editing to revert a mutated base to the consensus (non-mutant) sequence (Fig. 3F–G). We selected the PKD2-R186X site as it was the only mutation in our cohort that lied within an editing window of the DNA sequence amenable to adenine base editing without bystander effects (Fig. S3B). We utilized the Cas9-Mediated Adenosine Transient Reporter for Editing Enrichment (XMAS-TREE) method to edit hPSCs (Fig. 3G and Fig. S3C–D)⁵². By Sanger sequencing, unedited PKD2-R186X homozygous mutants (A/A) and heterozygous gene corrected (A/G) clones were identified (Fig. 3H)⁴⁷. 76 clones were screened for editing and multiple heterozygous clones were obtained (Fig. S3E). Clones were maintained for many passages in culture and confirmed to exhibit features of normal hPSCs (Fig. S3F). Protein level changes were analyzed by immunoblot, where PKD2-R186X reversion rescued PC2 abundance to ~50% of non-mutant levels (Fig. 3I–J).

To evaluate the capacity of heterozygous corrected hPSCs to rescue PKD cystogenesis, we differentiated them into kidney organoids (Fig. 3K). To account for potential clonal variability, we utilized multiple independent clones per genotype. In side-by-side experiments, PKD2-R186X reversion reduced rates of cystogenesis from greater than 70% for unedited homozygous mutant organoids to ~ 10% for heterozygous gene corrected organoids, accompanied by a reduction in cross-sectional cyst area (Fig. 3L–N). In comparison to the original PKD2-R186X heterozygous organoids, the gene corrected PKD2-R186X heterozygous organoids appeared to form cysts at a slightly higher rate (Fig. 3B–C and Fig. 3L–N). This may reflect a batch effect between different experiments, or an off-target effect of repeated editing in these clones. Taken together, heterozygous organoids generated either from control (non-mutant) hPSCs by cytosine base editing, or from homozygous mutant hPSCs corrected by adenine base editing, demonstrated that mutations in a single copy of PKD1 or PKD2 were not sufficient to induce a cystic phenotype in human organoids.

We have previously shown that PC1 expression is dramatically reduced in hPSCs and organoids derived from PKD2^−/− mutants with indel mutations²⁰. Similarly, in our homozygous PKD2-R186X mutants generated using base editing, PC1 expression was greatly reduced (Fig. S3G–H). In gene-corrected heterozygous (PKD2^+/−) cells, PC1 abundance was restored to nearly that of non-mutant levels (Fig. S3G–H). Thus the molecular phenotype of PC1 loss was a specific consequence of PC2 loss in human cells, and could be prevented by restoring a single copy of PC2.

Read-Through Treatments Safely Reduce PKD Cyst Formation

The innovation of nonsense mutation PKD organoids opened the potential to testing ERSG therapeutics that promote read-through. Two unique ERSGs, ELX-02 and ELX-10, were first tested in this model system using a ‘prophylactic treatment’ approach. Following terminal differentiation into nephron-like structures, but prior to PKD cyst formation, organoids were placed in individual wells to allow for consistent tracking over a 14-day treatment time course (Fig. 4A). We tested a dose range of 10 – 90 uM, which approximates systemic doses of ELX-02 that were previously shown to be well tolerated in humans subjects in vivo, and which correspond to the human equivalent doses from experiments conducted in cystinotic mice^38–40. A dose-dependent decrease was observed in the percentage of organoids forming cysts across all four targeted PKD mutations in response to treatment with either ERSG compound (Fig. 4B–C and Fig. S4A). When treated with ERSGs, the percentage of organoids forming cysts dropped to lower than 25%, significantly lower than the 75% observed in untreated organoids (Fig. 4C). Moreover, we observed a dose-dependent decrease in cyst size, as measured by cross-sectional area, with both of these treatments and in all of our mutants (Fig. 4D). At low doses, a reduction in cyst area could still be detected even in cases where effect on the percentage of organoids was more modest (Fig. 4C–D).

Fig. 4 — A) Schematic of prophylactic read-through treatment workflow. Each independent trial consists of 10 organoids per condition as indicated in plate layouts. B) Representative images of organoids in each condition at treatment day 0 and 14 (brightfield and Calcein AM/propidium iodide stain). Scale bars 100 um. C) Quantification of the percentage of organoids forming cysts for each targeted PKD mutation in each treatment condition over 14 day treatment course. n=3 independent trials each with 10 organoids per condition. Mean±SEM. Some error bars too small to appear on graphs. Nonlinear regression extra-sum-of-squares F test p-values comparing all conditions indicated in upper left. For pairwise comparisons (Water vs 10uM, Water vs 30uM, Water vs 90uM), significance p<.05 indicated by orange asterisk next to respective dose at day 14. D) Quantification of cyst area for each targeted PKD mutation in each treatment condition over 14 day treatment course. n=30 organoids pooled from 3 independent trials each with 10 organoids per condition. Mean±SEM. Some error bars too small to appear on graphs. Linear regression extra-sum-of-squares F test p-values comparing all conditions indicated in upper left. For pairwise comparisons (Water vs 10uM, Water vs 30uM, Water vs 90uM), significance p<.05 indicated by orange asterisk next to respective dose at day 14. E) Quantification (violin plots) of propidium iodide fluorescence (dead) relative to Calcein AM fluorescence (live) for each targeted PKD mutation. n=30 organoids pooled from 3 independent trials each with 10 organoids per condition. F) Relative cytotoxicity compared to vehicle control as determined by LDH release assay following 7 days of treatment for each targeted PKD mutation. n=30 organoids pooled from 3 independent trials each with 10 organoids per condition. Welch’s ANOVA with Dunnett’s T3 multiple comparisons test. ns=not significant, *p<.05, **p<.01, ***p<.001, ****p<.0001. For all violin plots, solid line indicates median and dashed lines indicate quartiles. Curve fits and all p-values are provided in supplemental materials. See also Figure S4 and Methods S1.

To evaluate the safety of these compounds, we first tested a broad range of doses in organoids derived from one of our mutant cell lines (PKD1-R2430X) using a lactate dehydrogenase (LDH) release assay at day 7 of treatment and a live/dead (Calcein AM/propidium iodide) staining assay at the day 14 experimental end-point. Concentrations up to 100 uM of either ERSG did not induce significant toxicity, whereas toxicity was observed with 300 uM of either ERSG in both assays (Fig. S4B–D). The spread of LDH levels was higher for 100 μM ELX-02 than lower concentrations, suggesting a minor population of injured cells (Fig. S4D). By day 14, the LDH release assay no longer detected actively dying cells at toxic concentrations, reflecting the fact that these structures had died (Fig. S4E). Based on these findings, we then further analyzed toxicity within the 10–90 uM treatment window during our prophylactic efficacy testing with both ERSGs in all of the mutants. A slight increase in toxicity was detected at the 90 uM dose (Fig. 4B,E–F and Fig. S4F). However, this was subtle in comparison to the toxicity observed at the 300 uM dose in the initial screening we performed in the PKD1-R2430X line (Fig. S4B–E).

We next tested whether ERSGs could reduce the rate of growth for pre-established cysts using a ‘therapeutic treatment’ approach, in which organoids were allowed to develop cysts prior to treatment initiation (Fig. 5A). A dose-dependent decrease in cyst area fold change was observed across all four targeted PKD mutations in response to treatment with either ERSG compound (Fig. 5B–C and Fig. S4G). At the highest doses, on average, cyst growth plateaued following ERSG treatment (Fig. 5C). Interestingly, individual organoids occasionally showed a reduction in cyst size (reversal), compared to untreated organoids (Fig. 5D and Fig. S4H). For the therapeutic treatment assay, no substantial toxicity was detectable by either live/dead staining or LDH release assay (Fig. 5B,E–F and Fig. S4I). The reduced toxicity observed in these pre-established cysts, compared to pre-cystic structures (above Fig. 4), may reflect changes in physiology or metabolism arising during the process of cystogenesis. Collectively, these studies suggested a broadly beneficial effect of ERSGs on PKD cystogenesis, both at pre-cystic and post-cystic stages, without inducing significant cytotoxicity.

Fig. 5 — A) Schematic of therapeutic read-through treatment workflow. B) Representative images of organoids in each condition at treatment day 0 and 14 (brightfield and Calcein AM/propidium iodide stain). Scale bars 100 um. C) Quantification of the fold-change in cyst area relative to starting cyst area at day 0 for each targeted PKD mutation in each treatment condition over 14 day treatment course. n=30 organoids pooled from 3 independent trials each with 10 organoids per condition. Mean±SEM. Some error bars too small to appear on graphs. Nonlinear regression extra-sum-of-squares F test p-values comparing all conditions indicated in upper left. For pairwise comparisons (Water vs 10uM, Water vs 30uM, Water vs 90uM), significance p<.05 indicated by orange asterisk next to respective dose at day 14. D) Percentage of cysts with fold change less than 1 for each targeted PKD mutation in each treatment condition over 14 day treatment course. n=3 independent trials each with 10 organoids per condition. Mean±SEM. Some error bars too small to appear on graphs. Nonlinear regression extra-sum-of-squares F test p-values comparing all conditions indicated in upper left. For pairwise comparisons (Water vs 10uM, Water vs 30uM, Water vs 90uM), significance p<.05 indicated by orange asterisk next to respective dose at day 14. E) Quantification of propidium iodide fluorescence (dead) relative to Calcein AM fluorescence (live) for each targeted PKD mutation. n=30 organoids pooled from 3 independent trials each with 10 organoids per condition. F) Relative cytotoxicity compared to vehicle control as determined by LDH release assay following 7 days of treatment for each targeted PKD mutation. n=30 organoids pooled from 3 independent trials each with 10 organoids per condition. Welch’s ANOVA p-values as indicated in upper left. For Welch’s ANOVA where p<.05, Dunnett’s T3 multiple comparisons test pairwise significance is shown. ns=not significant, **p<.01. For all violin plots, solid line indicates median and dashed lines indicate quartiles. Curve fits and all p-values are provided in supplemental materials. See also Figure S4 and Methods S1.

Read-through Treatments are Specific for Nonsense Mutants

We sought to confirm that our efficacy findings were the result of on-target ERSG induced read-through by monitoring expression of full-length PC1 and PC2 in nonsense mutant kidney organoids following treatment with ERSGs. For these experiments, we focused only on doses up to 60 uM, since partial toxicity was observed at 90 uM in our system. PC1 levels were dramatically reduced in PKD2 mutants, compared to isogenic controls, reflecting a requirement for PC2 to stabilize PC1 (Fig. 6A–D)²⁰. Across all PKD1 or PKD2 target mutation sites, there was a dose dependent increase in PC1 expression with ERSG treatment (Fig. 6A–D and S5A). At the 30 μM ELX-02 treatment concentration, this increase was ~50% of isogenic control levels (Fig. 6A–B).

Fig. 6 — A) Representative immunoblots of PC1 with alpha-tubulin loading control from kidney organoids following 14 days of treatment. WT indicates untreated isogenic C/C control organoids. Carat (>) symbol at left indicates band of truncated protein. For *PKD1*-R2430X, blue arrowhead indicates full-length PC1 above truncated band. Dashed line indicates merging of lanes from same immunoblot. B) Quantification of immunoblot PC1 fold change following treatment with ELX-02 relative to WT PC1 abundance. n=3 independent biological replicates per genotype; Welch’s ANOVA with Dunnett’s T3 multiple comparisons test where *p<.05 and **p<.01. Welch’s ANOVA p-value as indicated for graphs without pairwise significance. Mean±SEM. C) Schematic depicting proposed effects of treatment with ELX compounds. D) Quantification of PC1 fold change following 10 day treatment with 30 uM ELX-10 relative to WT PC1 abundance for each targeted PKD mutation. n=3 independent biological replicates per genotype. Unpaired t-test with Welch’s correction where *p < .05. E) Representative images of *PKD2*^−/− organoids with indicated frameshift mutation in each condition at day 0, 7, and 14 of treatment with water (vehicle), 10 uM, or 30 uM ELX-02 (blue) or ELX-10 (purple). Scale bar 100 um. F) Quantification of the percentage of organoids forming cysts for *PKD2* frameshift mutant in each treatment condition over 14 day treatment course with ELX-02. n=3 independent trials each with 10 organoids per condition. Mean±SEM. Nonlinear regression extra-sum-of-squares F test p-values as indicated. G) Quantification of cyst area for *PKD2* frameshift mutant over 14 day treatment course with ELX-02. n=30 organoids pooled from 3 independent trials each with 10 organoids per condition. Mean±SEM. Linear regression extra-sum-of-squares F test p-values as indicated. H-I) Same as F-G but for ELX-10 treatment. See also Figures S5–6.

Interestingly, in addition to an increase in full-length PC1, the PKD1-R2430X mutant also demonstrated accumulation of the truncated PC1 form (Fig. 6A and S5A). We hypothesized that this difference in truncated PC1 expression might reflect effects of ELX-02 on nonsense-mediated decay of the mRNA, which can be sequence dependent⁵³. Indeed, we observed an increase in PKD1 mRNA levels for this R2430X mutant when treated with ELX-02, but not the other mutants (Fig. S5B).

We previously showed that PC1 is dramatically reduced in PKD2^−/− hPSCs and organoids²⁰. In our ERSG-treated PKD2 nonsense mutants, we observed restoration of PC1 to wild-type levels, suggesting that PC2 was being restored (Fig. 6A–C). PC2 has also been shown to promote the maturation of PC1 from a lower molecular weight, Endoglycosidase H (EndoH)-sensitive form in the endoplasmic reticulum (ER) to a higher molecular weight, EndoH-resistant form in post-ER membrane compartments⁵⁴. Compared to untreated cells, the PC1 bands from the ERSG treatment groups in the PKD2 nonsense mutants showed an increase in both forms of PC1, but favored the lower molecular weight form, compared to wild type controls, suggesting that PC2 levels in these cells were not fully adequate to rescue PC1 trafficking and maturation. Read-through rates produced by aminoglycoside-derived compounds vary between proteins and mutation context, sometimes with low efficiency (< 5 %), which can pose a significant challenge for detection of read-through using endogenous protein levels^55,56. Consistent with this, we were unable to directly detect changes in PKD2 transcript or PC2 protein abundance in our PKD2 mutants after treatment with ERSGs (Fig. S5C–E). This may reflect reduced sensitivity of reagents for detecting PC2 compared to PC1 or lower efficiency of read-through for the PKD2 mutation sites. Indeed, in our system, PC2 abundance was not readily detectable below 10–25% of that in control cells with no mutations (Fig. S5F–G). PC2 abundance is generally higher than PC1 abundance, thus a small quantity of PC2 may be sufficient to stabilize PC1 and contribute to function⁵⁴.

To evaluate read-through capacity using an alternative method, we used a dual luciferase reporter assay in immortalized cells transfected with plasmids specific to each mutation⁵⁷. A dose dependent increase was observed in the percentage of read-through by both ERSGs, on par or higher than read-through levels achieved by treatment with positive control compounds gentamicin and G418 (Fig. S5H). Consistent with our above observations, read-through efficiency was lower in the PKD2 mutants compared to PKD1 mutants. Read-through efficiency is context dependent on the nucleotide immediately downstream of the stop codon^58,59. Both of the PKD1 mutants have a cytosine base and both of the PKD2 mutants have a guanine base at this position, likely explaining the observed differences in read-through efficiency in these cohorts. For the PKD2 mutations, read-through levels plateaued at 3–6%, lower than the detection threshold of our protein abundance assay (Fig. S5F–H). Thus, the increase in PC1, which is stabilized by PC2, in ERSG-treated PKD2 nonsense mutants was consistent with the hypothesis that low levels of PC2 were being expressed^54,60.

Certain small molecules, such as mefloquine, can function as enhancers of aminoglycoside-mediated read-through^61,62. Mefloquine is thought to potentiate the effects of read-through compounds by binding to the eukaryotic 80S ribosome, inducing a favorable conformational change^61,63. We tested whether mefloquine addition might enable us to detect direct effects of ERSGs on PKD2 transcript levels. In undifferentiated hPSCs, PKD2 levels were significantly reduced in PKD2-R186X mutant hPSCs versus isogenic controls, likely reflecting a process of nonsense-mediated decay in these cells (Fig. S5I). Treatment with ELX-02 resulted in a slight increase in transcript, which was enhanced ~40 % by supplementation of ELX-02 with mefloquine, but not mefloquine alone (Fig. S5I). Levels of PKD1 in these cells were unaffected by mefloquine, suggesting that this was effect was specific to the mutant PKD2 transcript (Fig. S5J). These findings further supported a direct effect of ERSGs on PKD2.

To test whether the ERSG treatment response in our organoid model was specific to nonsense mutations, we applied ELX-02 or ELX-10 to organoids derived from PKD2^−/− hPSCs with homozygous deletion mutations (p.Ser88SerfsTer207) generated with CRISPR-Cas9²⁰. As such mutations are not amenable to read-through, there was no effect on cystogenesis in PKD2^−/− organoids lacking nonsense mutations when treated with either ERSG at doses up to 30 uM (Fig. 6E–I and Fig. S6A–D). To further control for off-target effects of ERSGs, we added ELX-02 to organoid cultures at an earlier stage of differentiation, and observed that that renal vesicle structures maintained in 10 uM or 30 uM progressed normally to terminal differentiation (Fig. S6E–F). Taken together, the observed restoration of full-length PC1 protein and specificity of the treatment response to nonsense mutations indicate on-target activity of ERSGs in our organoid model system.

ERSGs Accumulate in Kidney Cysts

Aminoglycosides are taken up by nephron epithelia, but whether this extends to PKD cysts is unclear^38,64–67. As ELX drugs with fluorescent conjugates are not available, we instead utilized Texas red-gentamicin (GTTR), a fluorophore-conjugated aminoglycoside that closely resembles the structure of ELX-02 and ELX-10, and is known to accumulate in kidney tubules in vivo in a similar manner^38,64–66. In PKD organoid cysts, GTTR uptake into the cyst lining epithelial cells occurred over a period of hours (Fig. 7A). In fixed samples, GTTR signal was present in both proximal (LTL^hiECAD^lo) and distal (ECAD^hiLTL^lo) cells in an intracellular localization pattern (Fig. 7B and Fig. S7A).

Fig. 7 — A) Representative time course images of PKD organoid cysts treated with Gentamicin-Texas Red (GTTR) or water (vehicle). Red arrow indicates start of treatment. n=4 organoids from 3 independent differentiations. Scale bar 100 um. B) Representative confocal 20X and 40X images of PKD organoid cysts treated with 30 uM GTTR followed by fixation at 48 hours post treatment initiation. LTL stains proximal tubule and ECAD stains distal tubule. Scale bars 100 um. C) Schematic depicting GTTR uptake assay. D) Representative confocal images of cysts receiving PBS or GTTR perfusion. Two representative cysts are shown for GTTR, with digital zoom below. Scale bar 200 um PBS and GTTR, 50 um for zoom. E) Line scan analysis of PKD cysts after perfusion (mean ± SEM, n = 20 cysts per condition pooled from 4 male *Pkd1*^RC/RC mice of C57BL/6J background). Yellow arrows in (D) represent direction of line scans. See also Figure S7.

In mouse studies in vivo, GTTR was previously shown to persist in proximal and distal tubules for 2–3 days after administration^65,66. Similarly, in PKD organoids, GTTR administered for 2 hours and subsequently removed (washout) was still visible in cyst lining epithelium 48 hours later, and co-localized with markers of proximal and distal tubules (Fig. S7B–C). When cysts were treated with both GTTR and unlabeled gentamicin as a competitor for binding, the fluorescent signal decreased in a dose-dependent manner with increasing concentrations of the unlabeled compound, demonstrating specificity (Fig. S7D–E). Thus, aminoglycoside derivatives accumulated to a steady-state level inside organoid cyst-lining epithelial cells.

To test accumulation in vivo, we injected GTTR into the vasculature and immediately harvested the kidneys and processed them unfixed for fluorescence analysis (Fig. 7C)⁵¹. This was performed using Pkd1^RC/RC mice, which are homozygous for a hypomorphic mutation to Pkd1, and develop progressive PKD^68,69. Analysis of tissue sections revealed that a subset of cyst-lining epithelia readily accumulated GTTR (Fig. 7D–E). Immunofluorescence analysis on serial tissue sections indicated that GTTR accumulated in cysts lacking specific markers of collecting ducts (AQP2⁺) or proximal tubules (LTL⁺), whereas cysts that stained positive for AQP2 did not exhibit significant uptake in these short-term GTTR injections (Fig. S7F–G). Cysts lacking specific markers develop progressively in these PKD mice, and are thought to represent dedifferentiation events in which a subset of cysts lose their segment markers of origin^68,69. Uptake of GTTR was also observed in non-cystic proximal tubules, as expected from prior analyses, which further supported the specificity of the signal observed in these experiments (Fig. S7F–G)^38,64–67. Thus, ERSGs were capable of uptake into PKD cysts in vivo.

DISCUSSION

Nonsense Mutants Test Therapeutic Approaches

Conceptually, the major advance of the hPSC-derived PKD organoid system over other cellular models is that it demonstrates a clear genotype-phenotype relationship specific to PKD. Our comprehensive cohort of base edited allelic series, representing > 130 sequenced clonal cell lines, and gene corrected lines, robustly supports this conclusion and our foundational studies in this area.

In therapeutic ‘trials in a dish’ on pre-established cysts, we found that ERSGs significantly limited cyst expansion. This expands the potential to treat PKD patients with advanced disease in order to further delay disease progression. A recent report shows that induced expression of PKD2 from a BAC transgene reversed cysts beginning at 13 weeks of age in an in vivo mouse model of PKD¹⁵. Our own data also indicate a dose-dependent level of cyst reversal in organoids treated with read-through ERSGs, although we were only able to detect this in individual organoids and not at the level of the general population, using a compound to induce treatment effects rather than a purely genetic strategy. Notably, even a modest increase in PC1 abundance was sufficient to reduce cystogenesis.

The ERSGs used in this study were selectively optimized to reduce nephrotoxicity compared to traditional aminoglycoside antibiotics³¹. As cytotoxicity can potentially masquerade as efficacy in cystogenesis experiments, it is important to conduct experiments with multiple doses and appropriate controls. For experiments in which treatment was initiated prior to cyst formation, we observed partial toxicity at 90 μM, possibly because the tubules are more functional prior to cyst formation and accumulate higher doses of drug. Thus, to prevent cyst initiation, doses of 30 μM or below would be more suitable, to avoid off-target toxicity.

Support for the Two-Hit Hypothesis

Organoids with heterozygous nonsense mutations, lacking only one copy of either PKD1 or PKD2, failed to produce robust cysts. This was not due to compensatory upregulation of the mutant allele, as we confirmed that expression of the mutant protein was only ~50 % in our heterozygotes. Rather, this finding supports a threshold model in which additional events lower the functional dosage of PC1 or PC2 below heterozygosity, enabling cystogenesis to occur^10–13. We also demonstrate that heterozygous restoration of PKD2 expression by CRISPR base editing significantly reduces the rate of cystogenesis, establishing a ‘floor’ of ~50 % protein level that is sufficient to rescue the phenotype, and demonstration that base editing can constitute a viable therapeutic strategy for PKD.

Other groups recently described kidney organoids developed using the conventional CRISPR-Cas9 system (indel generating) or PKD patient-derived, in which an increased level of cystogenesis was observed in heterozygotes compared to wild type, although this remained much less than isogenic homozygotes^70,71. In those studies, which utilized a distinct differentiation protocol, cystogenesis in PKD organoids (heterozygous or homozygous) required the addition of forskolin, a strong artificial secretagogue that can also trigger cysts in non-PKD structures^20,22,72. In contrast, PKD organoids generated using our differentiation protocol form cysts robustly without the use of forskolin, a more natural context. Thus, in light of our findings, the data in human organoids support the conclusion that heterozygotes resemble control organoids, rather than homozygotes.

In hPSCs and organoids with indel mutations, PC2 loss results in a dramatic reduction in full-length PC1 protein²⁰. Interestingly, heterozygous PKD2^+/− hPSCs show a near-complete rescue of PC1 expression, demonstrating that full dosage of PC2 is not required. Similarly, treatment of PKD2^−/− nonsense mutants with read-through compounds greatly increases PC1, even though PC2 levels are only partially rescued. This dosage-independent effect of PC2 on PC1 levels in human cells appears to be distinct from mice, in which Pkd2 exhibits a dosage-dependent effect on PC1 maturation⁵⁴.

Limitations of the Study

The nephron-like organoids used in these studies lack collecting ducts. Distinct protocols are needed to differentiate hPSCs into the ureteric lineage that produces collecting ducts, and currently these are neither fully mature nor capable of modeling PKD cystogenesis without forskolin^70,73. Our work reveals that aminoglycoside derivatives can reach kidney cysts in vivo, but as the injections we performed were short in duration, further studies are needed to demonstrate biodistribution, safety, and efficacy in longer-term treatments of animals. Proximal tubules are a logical segment to target for therapy, given their tendency to take up aminoglycosides. Achieving efficacy in distal tubules and collecting ducts would further enhance the clinical relevance of this approach but may require higher doses of drug.

Read-through compounds are typically capable of achieving only relatively modest efficiency, which varies depending on the read-through target^55,56. Future studies may employ more advanced and sensitive techniques, such as mass spectrometry, to quantify the PC1 and PC2 polypeptide fragments generated by read-through. Given the very robust effects of these read-through compounds on PC1 levels, compared to relatively minor effects on PC2, the complete molecular mechanisms remain elusive, and it is possible that the compounds may contribute specifically to PC1 stabilization via additional mechanisms, which remain to be determined. This would be an interesting topic for future studies, which may lead to targets for PC1 restoration. For the gene therapy aspect, we have been able to achieve rescue by gene correction, but it would be valuable to establish techniques to efficiently modify mature organoids, which would be the equivalent of the kidneys in patients. Our toolkit of genetic and chemical tools might be further developed to assess such questions in future studies.

STAR★METHODS

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Benjamin Freedman (benof@uw.edu).

Materials Availability

All unique reagents generated in the study are available from the lead contact under material transfer request.

Data and Code Availability

All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Scans of full-length Western blots are available on Mendeley Data (doi:10.17632/8t4y3f8zs8.1) at the following link: https://data.mendeley.com/preview/8t4y3f8zs8?a=a9a27a05-74fc-450e-8ff6-7eeebbd4fcae

EXPERIMENTAL MODELS AND STUDY PARTICIPANT DETAILS

Animals

All animal studies were conducted in accordance with all relevant ethical regulations under protocols approved by the Institutional Animal Care and Use Committee at the University of Washington in Seattle. C57BL/6J Pkd1^RC/RC mice (MGI: 5476822), males ≥ 18 months old were utilized for the experiments. Mice were maintained on a standard diet under standard pathogen-free housing conditions, with food and water freely available.

Cell Lines

WTC11 hPSCs (Coriell 25256, female) were maintained on 1% reduced growth factor GelTrex (Life Technologies) in mTeSR1 media (Stem Cell Technologies) and dissociated with Accutase (Stem Cell Technologies). HeLa cells (CCL-2, ATCC, female) were cultured in DMEM (Thermo Fisher), supplemented with 10% of non-heat-inactivated fetal bovine serum (Sigma-Aldrich) and 1% PenStrep (Thermo Fisher).

METHOD DETAILS

Kidney Organoid Differentiation.

hPSCs were dissociated with Accutase (Stem Cell Technologies) and seeded at a density of 1500 cells per well in a 24-well plate pre-coated with GelTrex in mTeSR1 supplemented with 10 μM Rho-kinase inhibitor Y27632 (Tocris Bioscience). The media was replaced with 500 μL mTeSR1 + 1:40 growth factor reduced Matrigel (Corning) at 16 hours, 500 μL mTeSR1 at 36 hours, Advanced RPMI + Glutamax (Life Technologies) + 12 μM CHIR99021 (Tocris Bioscience) at 50 hours, and RB - Advanced RPMI + 1x Glutamax + 1x B27 Supplement (Life Technologies) at 86 hours⁴⁹. RB was changed two days later and every three days thereafter. Organoids were differentiated for 18 days from the time of plating.

For cystogenesis assays, adherent organoids were microdissected with a 23-gauge syringe needle from 24-well plates on an inverted phase-contrast microscope, and carefully transferred using a P1000 pipette into an ultra-low attachment 6-well plate (Corning) containing 2 mL RB. RB media was changed by gravity every three days. Wells were imaged using a Nikon TiE inverted widefield microscope. Cyst percentage and area were measured using FIJI software. Kidney organoid cysts were identified as having a bright, translucent thin-walled epithelium with 3D curvature surrounding a hollow, fluid-filled space. This is in contrast to the kidney organoid body that was identified as having a dull, dense multilayer architecture occasionally associated with a stromal cell layer. Cross-sectional cyst area was traced using the free-form tool in FIJI, outlining only the areas of translucency while excluding areas identified as the organoid body.

CRISPR Base Editing.

Gene alignments and amino acid alignments were performed in Benchling. Base editing guide sequences (listed in Supplemental Table 1) were generated using the Benchling CRISPR Design and Analyze Guides tool with base editing selected as the design type. Guides were optimized for highest base editing scores as predicted from sequence context and target C position⁴¹ as well as lowest number of off-target sites. BIG-TREE cytosine base editing plasmids were obtained from Addgene (pEF-AncBE4max #138270, pDT-sgRNA #138271, and pEF-BFP #138272)⁴⁵. Guide oligonucleotides with CACC (sense) and AAAC (antisense) 5’-overhangs were duplexed and cloned into the pDT-sgRNA plasmid at the BbsI cut site using a Quick Ligation Kit (New England BioLabs). All intermediate plasmid products were confirmed via Sanger sequencing (Genewiz). 500,000 WTC11 hPSCs (Coriell 25256) were seeded in a 6 well plate with mTeSR1 media supplemented with 10 μM Rho-kinase inhibitor Y27632 24 hours prior to transfection. pEF-AncBE4max (1800 ng), pDT-sgRNA with unique guide (900 ng), and pEF-BFP (900 ng) were co-transfected using 8 μL Lipofectamine Stem transfection reagent (Themo Fisher). Media was replaced with mTeSR1 24 hours post transfection. 48 hours post transfection, hPSCs were dissociated as single cells using Accutase and passed through a 35 μm cell strainer. BFP⁺ and GFP⁺ cells were isolated using a FACS Aria cell sorter and plated at low density in recovery media (mTeSR1 supplemented with 1X CloneR (Stem Cell Technologies)). Recovery media was changed 48 hours post plating and supplemented with 20% recovery media by volume 72 hours post plating without a full change. Media was changed to standard mTeSR1 96 hours post plating followed by daily changes until small colonies formed. Individual colonies were manually isolated with a 10 μl pipet and transferred to a 96-well plate mTeSR1 media supplemented with 10 μM Rho-kinase inhibitor Y27632 followed by daily changes with mTeSR1 media until confluent. Clones were then passaged with one replica plate dissociated and temporarily frozen in FreSR-S (Stem Cell Technologies) at −80°C and the other replica plate used for genotype screening. Genomic DNA was prepared from the resulting clonal cell lines using QuickExtract DNA extraction solution (Lucigen). The targeted region was PCR amplified using Platinum Hot Start 2X Master Mix (Thermo Fisher) by thermocycling at 94°C or 2 minutes followed by 35 cycles of 94°C, 57°C, and 72°C for 30 seconds each followed by 72°Cfor 5 minutes. PCR products sizes were confirmed on a 1% agarose gel and were purified using ExoSAP-IT (Applied Biosystems) prior to Sanger sequencing (Genewiz). Genomic DNA primer sequences are listed in Supplemental Table 1. Resultant sequencing files were analyzed with EditR software for chromatogram alignment and detection of editing⁴⁷. All isogenic sets for a targeted mutation site were then thawed in parallel from temporary storage at −80°C and expanded for long-term storage in liquid nitrogen. Isogenic control clones were obtained through the same CRISPR base editing procedure but were found to have no modification at the target site upon screening.

For reversion of the PKD2-R186X mutation, the guide sequence (listed in Supplemental Table 1) was generated using the CRISPR RGEN Tools BEdesigner optimized for location of target base within the editing window sequence and lowest number of off-target binding sites⁷⁴. XMAS-TREE adenine base editing plasmids were obtained from Addgene pEF-ABEmax (#164415), pDT-sgRNA-XMAS-1x (#164413), and pEF-XMAS-1xStop (#164411)⁵². The same protocol was followed as described above for adenine base editing except cell sorting was performed for mCherry⁺ and GFP⁺ cells. All oligonucleotides and PCR primers were synthesized by Integrated DNA Technologies.

Read-Through Compound Treatment.

For prophylactic treatment assays, organoids were micro dissected as described above. Single organoids were then transferred to individual wells of an ultra-low attachment round bottom 96 well plate (Corning) using a P200 pipette. Organoids were maintained in 100 μL RB media supplemented with water, ELX-02 (NB124), or ELX-10 (NB157) at indicated concentrations. On days 3, 5, 7, 10, and 12, RB media was changed by gravity with removal of 50 μL existing media followed by addition of 50 μL fresh media supplemented with water, ELX-02, or ELX-10 at 2x concentration. Therapeutic treatment assays were performed in the same way, but starting with cystic organoids that had been maintained in suspension for 7 days prior to transfer. For LDH release assays, 25 μL of media per well was transferred to a white 96 well plate (Cellstar). LDH-Glo cytotoxicity assay was performed according to manufacturer’s protocol (Promega). Gentamicin-Texas Red (GTTR) organoid experiments were performed in the same manner with removal of 50 μL existing media followed by addition of 50 μL fresh media supplemented with GTTR (AAT Bioquest 24300), with or without unconjugated gentamicin (Gibco 15710–064). Washout was performed by 3 successive changes of normal media.

Immunoblotting.

Undifferentiated hPSCs or derived organoids were maintained in adherent culture to obtain sufficient material for lysis. The same dosing schedule was used for organoid treatments as described above – day 0, 3, 5, 7, 10, and 12. Once hPSCs were confluent or on day 10 (ELX-10)/day 14 (ELX-02) of organoid treatment, cells were lysed using RIPA buffer (Thermo Fisher) with complete mini EDTA-free protease inhibitor (Sigma), PhosSTOP (Sigma), and benzonase nuclease (Sigma). Samples were centrifuged at 13,000 rpm for 10 minutes at 4°C to remove cell debris. Resulting lysates were quantified using a BCA protein assay kit (Thermo Fisher). Samples were prepared with 50 μg protein in Laemmli buffer (Bio-Rad) containing 2-mercaptoethanol (Sigma). Precision Plus Protein Kaleidoscope Standards (Bio-Rad) were loaded as a marker for molecular weights. Samples were separated in a 4–20% gel (Bio-Rad) and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% non-fat milk and probed with primary antibodies. The antibodies used were anti-PC1 7e12 (sc-130544; Santa Cruz), anti-PC1 E8 (E8–8C3C10, Kerafast), anti-PC2 (sc-25749; Santa Cruz), anti-αtubulin (2144s; Cell Signaling), anti-rabbit HRP (1706515; Bio-Rad), and anti-mouse HRP (176516; Bio-Rad). Signal was captured using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific) and ProSignal ECL Blotting Film (Prometheus). Band intensities were quantified with FIJI Gel Analyzer and normalized to α-tubulin loading control.

Immunostaining and Imaging.

Kidney organoids or mouse tissue slices were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 minutes at room temperature. Samples were washed with PBS, blocked in 5% donkey serum (Millipore)/0.3% Triton-X-100/PBS for 1 hour, and incubated overnight in 3% bovine serum albumin/PBS with primary antibodies. The following day, samples were washed with PBS and incubated overnight with Alexa-Fluor secondary antibodies (Invitrogen) and DAPI (Thermo Scientific). Prior to imaging, samples were washed with PBS and subsequently imaged in PBS. Primary antibodies used were biotinylated LTL (FL-1320, Vector Labs), DBA (Vector Labs RL-1032), ECAD (Abcam ab11512), PODXL (R&DAF1658), CUBN (Invitrogen PA5–83684), SLC3A1 (Sigma HPA038360), OAT1 (Invitrogen PA5–26244), MUC1 (Abcam ab15481), NKCC2 (Proteintech 18970–1-AP), and AQP2 (Abcam 199975). Calcein AM (Invitrogen) and Propidium Iodide (Thermo Fisher) stains were performed according to manufacturer’s instructions. Confocal fluorescence images were captured using a Yokogawa W1 Spinning Disk confocal head mounted on an inverted Nikon Ti widefield microscope. For cystogenesis assays, wells were imaged using a Nikon TiE inverted widefield microscope. For compound treatment assays, brightfield images were captured using the transmitted light detector on a Leica SP8 microscope. Cyst percentage and area were quantified using FIJI. Dead/live ratios were quantified as mean fluorescent intensity measurements on max intensity projections of organoids which were traced manually using FIJI. For suspension days 30 and 48, organoids were imaged overhead using an iPhone 12.

RT-PCR.

RNA was prepared using the Quick RNA MiniPrep kit (Zymo). 666 ng RNA was reverse transcribed using the SuperScriptIV Reverse Transcription kit (Invitrogen). For each independent experiment, quantitative RT–PCR reactions were run in triplicate using cDNA (diluted 1:2, 300 nM primers, and PowerUp SYBR Green Master Mix (Applied Biosystems). For read-through enhancer treatments, confluent hPSCs were treated with ELX-02 with or without mefloquine hydrochloride (Milipore Sigma M2319). For these experiments, 1000ng RNA was reverse transcribed and the cDNA was diluted 1:300. qPCR primer sequences are listed in Supplemental Table 1. mRNA fold change was calculated using 2^−μμCT with actin as the housekeeping gene.

Dual Luciferase Assay.

HeLa cells were plated in a 96 well format at a density of 7500 cells per well in DMEM (Thermo Fisher), supplemented with 10% of non-heat-inactivated fetal bovine serum (Sigma-Aldrich). Cellular transfection occurred one day later using 0.1 μL Turbofect (ThermoFisher) and 25 ng DNA (pSGDluc plasmid with insert sequences as listed in Supplemental Table 1 (GenScript)). Compound treatment was initiated 24 h post transfection. FF and RNL luciferase measurements were made 24 h after compound treatment using the Dual Luciferase kit (Promega) as per the manufacturer protocol. Briefly, cells were lysed with 25 μL of Passive Lysis Buffer. 10 μL of lysate was transferred to a secondary plate and 25 μL of Luciferase Assay Reagent II was added. FF activity was measured on an Envision plate reader (PerkinElmer). 25 μL of Stop & Glo reagent was then added to each well followed by RNL activity measurements in the Envision plate reader. For each well, raw FF were normalized to raw RNL to obtain FF/RNL ratios. FF/RNL ratios of mutant plasmids were then normalized to wild-type plasmids. The percent increase in read-through activity was derived by calculating the difference between vehicle and compound-induced read-through, then dividing that value by the read-through value for vehicle and expressing the result as a percentage.

In Vivo Perfusion Experiments.

Pkd1^RC/RC mice (≥ 18 months old) were sacrificed through isoflurane overdose and immediately incised through the chest and nicked at the vena cava with a 27-gauge needle. Keeping pressure on the vena cava, mice were perfused systemically through the heart with a syringe containing 10 ml of PBS, followed by a second syringe containing either 2.5 ml of PBS (control) or 2.5 ml of PBS + 83.33 μM Gentamicin-Texas Red (AAT Bioquest 24300). Kidneys were excised immediately and embedded directly in OCT without fixation or sucrose equilibration. 20 μm thick kidney sections were cut using a cryostat, mounted on a microscope slide in OCT, and imaged on a confocal microscope with 10X objective.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses were performed using GraphPad Prism Software. Information regarding specific statistical tests used, representation of data in graphs, and replicates for each experiment are included in the corresponding figure legend. For nonlinear regression, p-values were calculated with an extra sum-of-squares F test using an exponential plateau model comparing YM and k values for percentage of organoids forming cysts and percentage of cysts with fold change less than 1, a straight-line model comparing slopes for cyst area, and a logistic growth model comparing YM and k values for cyst fold change. Nonlinear regression curves for all plots are provided in Data S2-S3. At least 2 unique clones per genotype were used in each experiment. In total, over 11,500 images of individual organoids were analyzed.

Supplementary Material

Supplement

NIHMS2010691-supplement-Supplement.pdf^{(9.5MB, pdf)}

KEY RESOURCES TABLE (KRT).

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
anti-PC1 7e12	Santa Cruz	sc-130554
anti-PC1 E8	Kerafast	E8-8C3C10
anti-PC2 H280	Santa Cruz	sc-25749
anti-α-tubulin	Cell Signaling	2144
anti-ECAD	Abcam	ab11512
anti-CUBN	Invitrogen	PA5-83684
anti-OAT1	Invitrogen	PA5-26244
anti-MUC1	Abcam	ab15481
anti-NKCC2	Proteintech	18970-1-AP
anti-SLC3A1	Sigma	HPA038360
anti-PODXL	R&D	AF1658
anti-AQP2	Abcam	ab199975
Bacterial and Virus Strains
NEB Stable Compotent E. Coli	New England Biolabs	C3040H
Chemicals, Peptides, and Recombinant Proteins
Calcein AM	Invitrogen	C3099
Propodium Iodide	Invitrogen	P1304MP
CHIR99021	Tocris	4423
ELX-02	Eloxx Pharmaceuticals	NB142
ELX-10	Eloxx Pharmaceuticals	NB157
Biotinylated LTL	Vector Labs	FL-1320
DBA	Vector Labs	RL-1032
DAPI	Cayman Chemical	14285
Gentamicin	Gibco	15710-064
GTTR	AAT Bioquest	24300
Mefloquine	Milipore Sigma	M2319
Critical Commercial Assays
Quick Ligation Kit	New England BioLabs	M2200
Lipofectamine Stem Transfection Reagent	Invitrogen	STEM00001
Sanger Sequencing	Genewiz	https://www.genewiz.com
QuickExtract	Lucigen	QE09050
Platinum Hot Start PCR Master Mix	Invitrogen	13000012
ExoSAP-IT Express	Applied Biosystems	75001
LDH-Glo Cytotoxicity Assay	Promega	J2381
Quick RNA MiniPrep Kit	Zymo	R1054
SuperScript IV	Invitrogen	18091050
PowerUp SYBR Green Master Mix	Applied Biosystems	A25743
TurboFect	Thermo Fisher	R0531
Dual-Glo Luciferase Assay	Promega	E2920
Experimental Models: Cell Lines
WTC11	Coriell	25256
HeLa	ATCC	CCL-2
Experimental Models: Organisms/Strains
Mouse: C57BL/6J Pkd1^RC/RC	The Jackson Laboratory	MGI:5476822
Oligonucleotides
Cytosine Base Editing gRNAs	This Paper: Supplemental Table 1	N/A
Genomic DNA Sequencing Primers	This Paper: Supplemental Table 1	N/A
qPCR Primers	This Paper: Supplemental Table 1	N/A
Dual Luciferase Insert Sequences	This Paper: Supplemental Table 1	N/A
Recombinant DNA
pEF-AncBE4max	Addgene	#138270
pDT-sgRNA	Addgene	#138271
pEF-BFP	Addgene	#138272
pEF-ABEmax	Addgene	#164415
pDT-sgRNA-XMAS-1x	Addgene	#164413
pEF-XMAS-1xStop	Addgene	#164411
pSGDluc	Addgene	#87323
Software and Algorithms
EditR	Kluesner, M.G., Nedveck, D.A., Lahr, W.S., Garbe, J.R., Abrahante, J.E., Webber, B.R., and Moriarity, B.S. (2018). EditR: A Method to Quantify Base Editing from Sanger Sequencing. Cris. J. 1, 239–250. 10.1089/crispr.2018.0014.	https://moriaritylab.shinyapps.io/editr_v10/
CRISPR Guide RNA Design Tool	Benchling	https://www.benchling.com/crispr
Fiji (2.0.0)	Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. 10.1038/nmeth.2019	https://imagej.net/software/fiji/
Prism (9.3.1)	GraphPad	https://www.graphpad.com
BioRender	BioRender	https://www.biorender.com
CRISPR RGEN Tools BEdesigner	Hwang, GH., Park, J., Lim, K. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics 19, 542 (2018). https://doi.org/10.1186/s12859-018-2585-4	http://www.rgenome.net/be-designer/

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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).

Footnotes

DECLARATION OF INTERESTS

Eloxx Pharmaceuticals is developing ELX-02 and ELX-10 as investigational drugs, and holds patents related to their use. BSF is an inventor on patents and/or patent applications related to human kidney organoid differentiation and modeling of PKD in this system. BSF holds ownership interest in Plurexa LLC.

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Data Availability Statement

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PERMALINK

Genetics of cystogenesis in base edited human organoids reveal therapeutic strategies for polycystic kidney disease

Courtney E Vishy

Chardai Thomas

Thomas Vincent

Daniel K Crawford

Matthew M Goddeeris

Benjamin S Freedman

SUMMARY

INTRODUCTION

RESULTS

Base Editing Generates PKD hPSCs with Nonsense Mutations

Fig. 1. CRISPR base editing generates hPSCs with PKD patient nonsense mutations.

Kidney Organoids with Homozygous Nonsense Mutations Model PKD

Fig. 2. Kidney organoids with homozygous nonsense mutations model PKD.

Heterozygosity Rescues PKD Cystogenesis

Fig. 3. Heterozygosity rescues PKD cystogenesis.

Read-Through Treatments Safely Reduce PKD Cyst Formation

Fig. 4. Prophylactic read-through treatment prevents PKD cyst formation.

Fig. 5. Therapeutic read-through treatment slows PKD cyst expansion.

Read-through Treatments are Specific for Nonsense Mutants

Fig. 6. Read-through treatments are specific for nonsense mutants.

ERSGs Accumulate in Kidney Cysts

Fig. 7. Aminoglycosides localize to PKD cysts.

DISCUSSION

Nonsense Mutants Test Therapeutic Approaches

Support for the Two-Hit Hypothesis

Limitations of the Study

STAR★METHODS

RESOURCE AVAILABILITY

Lead Contact

Materials Availability

Data and Code Availability

EXPERIMENTAL MODELS AND STUDY PARTICIPANT DETAILS

Animals

Cell Lines

METHOD DETAILS

Kidney Organoid Differentiation.

CRISPR Base Editing.

Read-Through Compound Treatment.

Immunoblotting.

Immunostaining and Imaging.

RT-PCR.

Dual Luciferase Assay.

In Vivo Perfusion Experiments.

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

KEY RESOURCES TABLE (KRT).

ACKNOWLEDGEMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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