Examples of subjects for treatment and/or prevention of the disease include mammals such as mice, rats, hamsters, guinea pigs, cattle, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, and humans, preferably primates, and more preferably humans.

Method for Treatment

The present application also provides a method for treating renal cystic ciliopathy, which involves administering the pharmaceutical composition of the present application to a subject in need thereof. The present application also provides the use of an RAR agonist for the manufacture of the pharmaceutical composition for treating renal cystic ciliopathy. The present application further provides an RAR agonist for use in the treatment of renal cystic ciliopathy. Examples of renal cystic ciliopathy and the RAR agonist are as described above.

Method for Prevention

The present application also provides a method for preventing renal cystic ciliopathy, which involves administering the pharmaceutical composition of the present application to a subject in need thereof. The present application also provides the use of the RAR agonist for the manufacture of the pharmaceutical composition for preventing renal cystic ciliopathy. The present application further provides the RAR agonist for use in the prevention of renal cystic ciliopathy. Examples of the renal cystic ciliopathy and the RAR agonist are as described above.

EXAMPLES

The following examples are provided for further description, but the present invention is not limited to these examples in any way.

Materials and Methods
Preparation of PKD1 Knockout Induced Pluripotent Stem (iPS) Cells

Experiments using human induced pluripotent stem cells were approved by the Ethics Committee of the Faculty of Medicine and Graduate School of Medicine, Kyoto University. The 1383D2 cell line was used as a human iPS cell line. PKD1 knockout iPS cells were prepared using the CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system based on a previously reported DNA transposon (Ishida K. et al, Sci Rep. 8: 310 (2018); Shimizu T. et al., Biochem Biophys Res Commun. 529, 1186-1194 (2020)). Cells were continuously transfected with a CRONUS-Puro vector (pPV-TetO-SphcCas9-GR-iC-A-EF1α-rtTA-iP, Addgene ID 100596) and a piggyBac vector for cloning sgRNA (pPV-H1-ccdB-mEF1α-RiH, Addgene ID 100598) together with a piggyBac transposase expression plasmid (pHL-EF1α-hcPBase-A, Addgene ID 100599) through lipofection using FuGENE6 (Promega). The CRONUS-Puro vector and the piggyBac vector for cloning sgRNA were compatible with puromycin selection and hygromycin selection, respectively. The following sequence: 5′-GAGACCACTTGGATCCGGGATCAGGTCTTCATCTAGGTTTTAGAGCTAGAAATAGCA-3′ (SEQ ID NO: 1, the target site is underlined) was used to clone the gRNA sequence targeting the PKD1 exon 34 splicing acceptor site into the pPV-H1-ccdB-mEF1α-RiH vector. RFP⁺ colonies were manually harvested by drug selection of transfected cells and treated with doxycycline and dexamethasone to induce genome editing. Individual colonies were screened for in terms of genome editing efficiency by Sanger sequencing of bulk genomic DNA. Single cells were then isolated by flow cytometry, clonally grown in 24-well culture plates (Corning) coated with iMatrix-511 (Nippi), and then genotyped by Sanger sequencing. Genome editing resulted in the generation of frameshifts and immature termination codons. Two bases (AG) at different sites within exon 34 functioned as splicing acceptor sequences, resulting in the generation of two types of PKD1 mutant mRNA. The primer sequences used in this example are listed in Table 1.

TABLE 1

Primers
Sequence

PKD1
CGTCCATTGTGGGTAGC
TCTCTGTCTTCTCCCCA

exon34
AAT SEQ ID NO: 2
GGA SEQ ID NO: 3

Induction of PKD1 Knockout iPS Cells Into Ureteric Bud Organoids

PKD1 knockout iPS cells were induced to differentiate into ureteric bud organoids as previously described (Mae, S I. & Ryosaka, M. et al. Cell Reports 32, 107963, 2020).

Preparation of Ureteric Bud Tip Cells

Ureteric bud organoids derived from PKD1 knockout iPS cells were treated with Accutase (Innovative Cell Technologies) at 37° C. for 5 minutes and then dissociated into single cells by pipetting. Cells were suspended in DMEM/F12 medium (Gibco) containing B27 supplement (vitamin A-free) (Gibco), 3 μM CHIR99021 (StemRD), 0.1 μM TTNPB (Santa Cruz), 200 ng/ml FGF1 (R&D systems), 100 ng/ml GDNF (R&D systems), 10 μM Thiazovivin (Santa Cruz Biotechnology), and 1 μM A83-01 (Wako). Single cells were seeded at 1.0×10⁵cells/well into each well of a 48-well plate coated with 150 μL of hydrogel. The hydrogel was composed of DMEM/F12 medium containing 50% Matrigel (BD Biosciences) and was allowed to solidify for 1 hour at 37° C. before use. Single cells established ureteric bud tip cell colonies after 7 days. The medium was exchanged every 2 to 3 days.

Subculture of Ureteric Bud Tip Cells

The hydrogel was dissolved with Cell Recovery Solution (Corning) for 30 minutes at 4° C., and then ureteric bud tip cell colonies were isolated. After further washing with Cell Recovery Solution at 4° C. for 30 minutes, ureteric bud tip cell colonies were centrifuged at 500 g for 5 minutes at room temperature. Ureteric bud tip cell colonies were treated with Accutase at 37° C. for 5 minutes and then dissociated into single cells by pipetting. Cells were suspended in DMEM/F12 medium containing B27 supplement (vitamin A-free), 3 μM CHIR99021, 0.1 μM TTNPB, 200 ng/ml FGF1, 100 ng/ml GDNF, 10 μM Thiazovivin and 1 μM A83-01. Cells were seeded at 1.0×10⁵cells/well into each well of a 48-well plate coated with 150 μL hydrogel. The hydrogel was composed of DMEM/F12 medium containing 50% Matrigel and allowed to solidify for 1 hour at 37° C. before use. Ureteric bud tip cell colonies were prepared by culturing single cells at 37° C. with 5% CO₂for 7 days. The medium was exchanged every 2 to 3 days. The prepared ureteric bud tip cell colonies were passaged every 7 days in the same manner. The tip cell colonies cultured for 4 to 6 weeks or longer were used to reconstitute the following collecting duct organoids.

Reconstitution of Collecting Duct Organoids From Ureteric Bud Tip Cells

The hydrogel was dissolved using Cell Recovery Solution at 4° C. for 30 minutes, so as to isolate ureteric bud tip cell colonies. After further washing with Cell Recovery Solution at 4° C. for 30 minutes, the ureteric bud tip cell colonies were centrifuged at 500 g for 5 minutes at room temperature. Ureteric bud tip cell colonies were treated with Accutase at 37° C. for 5 minutes and then dissociated into single cells by pipetting. Cells were suspended in Essential 6 medium (Gibco) containing 3 μM CHIR99021, 0.1 μM TTNPB, 200 ng/ml FGF1, 100 ng/ml GDNF, 10 μM Thiazovivin and 1 μM A83-01. Cells were seeded at 5.0×10³cells/well into 96-well low-adhesion plates (Sumitomo Bakelite). Single cells were cultured for 2 days at 37° C. with 5% CO₂, thereby preparing spheroids.

Spheroids were suspended in Essential 6 medium containing 10% Afamin/Wnt3a conditioned medium (MBL), 200 ng/ml R-spondin 1 (R&D systems), 0.1 μM LDN193189 (Axon medchem), 200 ng/ml FGF1, 200 ng/ml FGF8 (Peprotech), 100 ng/ml GDNF, 0.1 μM TTNPB, 50 ng/ml EGF (R&D systems), 1 μM A83-01 and 10% Matrigel, and then distributed in 35 mm low-adhesion dishes (Sumitomo Bakelite) at 2.5 to 3 mL/dish. The medium was exchanged every 3 to 4 days. Spheroids were cultured at 37° C. with 5% CO₂for 14 to 21 days, thereby preparing artificial collecting duct organoids.

RNA Sequencing Analysis

RNA sequence library preparation, sequencing, mapping and gene expression analysis were performed at DNAFORM. The quality of total RNA was evaluated using a Bioanalyzer (Agilent Technologies) and the RNA Integrity Number was confirmed to be 7.0 or greater. After enrichment of poly (A)+RNA by the use of the NEBNext Poly (A) mRNA Magnetic Isolation Module (New England BioLabs), Double-stranded cDNA libraries (RNA-seq library) were prepared according to the manufacturer's instructions using a SMARTer Stranded Total RNA Seq Kit v2 Pico Input Mammalian (Clontech). RNA-seq libraries were sequenced using paired-end reads (50 nt read 1 and 25 nt read 2) on a NextSeq 500 system (Illumina). The obtained reads were mapped to the human GRCh38 genome using STAR (version 2.7.2b) or Hisat2 (version 2.1.0). Reads of annotated genes were counted using featureCounts (version 1.6.1). FPKM and TPM values were calculated from the mapped reads by normalization with total counts. Differentially Expressed Genes (DEGs) were detected using the DESeq2 package (version 1.26.0). The list of DEGs detected by DESeq2 was used for GSEA. Heat maps and volcano plots were created using the bioinfokit package (version 2.0.1).

Microarray Analysis

For comprehensive profiling of transcripts encoding human proteins, microarray analysis was performed at Filgen using the Human 8×60K LncRNA expression array (ArrayStar). Labeling of samples and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Data were collected using Agilent's Feature Extraction software. Raw signal intensities were normalized using the quantile normalization method of GeneSpring GX v12.1. Statistically significantly differentially expressed mRNAs between two groups were extracted by volcano plotting, followed by heat mapping and GSEA analysis.

Animal Model

Pkd1^flox/flox: Ksp-cre mice, which are ADPKD model mice, develop numerous cysts progressively after birth and die at approximately 14 days of age due to renal failure.

Mouse lineages were maintained with heterozygous Pkd1^flox/+: Ksp-Cre mice, and Pkd1^flox/flox: Ksp-cre mice were generated by mating mice of the same lineage.

ATRA Treatment

On postnatal day 3, Pkd1^flox/flox: Ksp-cre mice (Cystic) and Pkd1^flox/+: Ksp-Cre mice (Non-cystic) were intraperitoneally administered with ATRA (Sigma, #R2625) dissolved in sunflower oil containing 2% DMSO (solvent) to achieve a dose of 10 mg/kg. A group of mice administered with only the solvent intraperitoneally was served as a negative control. Mice were euthanized on postnatal day 9, and renal tissues and blood samples were collected.

AM80 Treatment

On postnatal day 3, Pkd1^flox/flox: Ksp-cre mice (Cystic) and Pkd1^flox/+: Ksp-Cre mice (Non-cystic) were intraperitoneally administered with AM80 (Tamibarotene; Tocris, #3507) dissolved in sunflower oil containing 2% DMSO (solvent) to achieve a dose of 5 or 10 mg/kg. A group of mice administered with only the solvent intraperitoneally was served as a negative control. Mice were euthanized on postnatal day 9 and renal tissue samples were collected.

Measurement of BUN (Blood Urea Nitrogen)

BUN in mouse serum was measured using an UN-L kit (Serotec, #A667-00).

Measurement of Hematoxylin-Eosin (HE) Staining and Cystic Index

Kidneys were fixed in 4% paraformaldehyde solution, and 4 μm-thick paraffin sections were prepared. After deparaffinization, HE staining was performed and renal tissues were photographed under an optical microscope. Cystic index was calculated by total cystic area/total renal tissue area×100 using image analysis and measurement software (WinROOF, Mitani Corporation).

Result
Examination of Compounds That Inhibit Cyst Enlargement

Using PKD1 knockout iPS cells of the 1383D2 cell line, artificial collecting duct organoids with cystic structures were induced by the above method. The following protocol was used to examine compounds that inhibit cyst enlargement.

- 1. Organoids in which cyst formation was occurring were collected in tubes.
- 2. Medium was removed and 2 ml of Cell Recovery Solution was added. (4° C., 30 minutes)
- 3. Gel was dissolved by gentle pipetting with a P-1000 pipetteman and then centrifuged. (500 g, 2 minutes)
- 4. After removing the supernatant from 3 and adding 5 ml of FBS-containing medium (STO medium), cysts were separated from the organoids by pipetting.
- 5. Using a stereomicroscope, cysts were collected in 15 ml tubes.
- 6. The supernatant was removed, and then 2 ml of Accutase was added to perform treatment at 37° C. for 5 minutes.
- 7. Cells were dissociated down to single cells by pipetting with a P-1000 pipetteman.
- 8. After filtering, cells were counted.
- 9. After cell counting, a cell suspension containing the required number of cells was transferred to a tube, and then 5 ml of FBS-containing medium (STO medium) was added to stop the Accutase reaction. To seed at 5×10⁴cells/well, the amount of the cell suspension required was calculated.
- 10. Pellets were prepared by centrifugation (200 g, 5 minutes).
- 11. The supernatant was thoroughly removed, and then DMEM/F12+B27 w/o V.A. medium (10% Afamin/Wnt3a CM, 200 ng/ml R-Spondin 1, 200 ng/ml FGF1, 10 μM Forskolin, 2.5 μM AVP, 10 μM Y27632) was added for suspension. To seed at 5×10