Methods for treating diseases associated with ciliopathies

Abstract

Disclosed is a method for treating ciliopathies-related diseases, comprising administering to a subject in need thereof an effective amount of a compound that targets at least one G protein-coupled receptor.Provided is a method for identifying therapeutic agents for treating diseases with ciliopathies, comprising providing an animal model system of ciliopathies to test putative therapeutic agents, administering a disrupting agent to the animal, treating the administered animal with the putative therapeutic agent, comparing the measurable phenotype of the treated animal with that of untreated animals, and identifying the therapeutic target as a treatment for ciliopathies if the measurable phenotype of the treated animal is reduced compared to that of the untreated animal. [Selection diagram] Figure 32

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international application under the Patent Cooperation Treaty, which claims the benefit of U.S. Provisional Application No. 62 / 572,051, filed October 13, 2017, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Cilia are microtubule-based cell surface protrusions that arise from basal bodies, i.e., membrane-bound centrioles. Primary cilia are nonmotile sensory organelles present in single copies on the surface of most growth-arrested or differentiated mammalian cells. Cilia sense changes in flow and mediate signaling pathways essential for development and tissue homeostasis, such as Hedgehog, Wnt / PCP, and cAMP / PKA signaling. Intraciliary transport (IFT) selects cargo at the base of the cilium and transports axonemal components required for ciliary assembly and proteins involved in ciliary signaling. Once cilium is formed, regulation of ciliary membrane composition relies on distinct molecular machinery, including a barrier to membrane proteins entering the cilium at a specialized region at the base of the cilium called the transition zone, and a transport adaptor that controls the localization of G protein-coupled receptors (GPCRs) to the cilium, called the BBSome (a complex of Bardet-Biedl syndrome (BBS) proteins and other proteins that are components of the basal body and are involved in transporting cargo to the primary cilium). Ciliogenesis requires the coordination of many processes. To generate cilia, a complex of cell cycle regulation, vesicle trafficking, and ciliary elongation must occur in precise timing. The importance of producing and maintaining properly differentiated cilia during embryonic development and adult physiology is best highlighted by the numerous human diseases associated with ciliopathy.

[0003] Ciliopathy is a group of human disorders directly caused by defects in cilia formation or function. Primary cilia defects cause multiple and highly diverse abnormalities, consistent with the widespread tissue distribution of primary cilia and their widespread functions. Patients with primary cilia exhibit a combination of kidney and retinal abnormalities, central nervous system defects that can lead to mental retardation, liver defects (including cysts), obesity, and various skeletal defects, including abnormal limb length, abnormal digit numbers (polydactyly), abnormal left / right axis organization (situs inversus), and abnormal craniofacial patterning. Specific abnormalities in the cilia connecting photoreceptors can also lead to retinal degeneration and blindness. Examples of primary ciliopathies include nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS), Orofacial-Digital syndrome (OFD), and Jeune syndrome (JATD).

[0004] Nephronophthisis (NPHP) is an autosomal recessive nephropathy characterized by massive interstitial fibrosis, tubular basement membrane thickening, and cyst formation, leading to end-stage renal disease (ESRD) in childhood. NPHP can occur alone or in association with different extrarenal manifestations (e.g., retinal dystrophy, hepatic fibrosis, skeletal dysplasia, etc.) in a syndrome hereafter referred to as nephronophthisis-related ciliopathies (NPHP-RC).

[0005] NPHP is caused by 21 NPHP genes, which are currently known to be the primary cause of 60% of cases. Given the high genetic heterogeneity of NPHP and the numerous mechanistic pathways discussed, it is clear that no single pathology leads to NPHP. Renal histology in NPHP points to a common endpoint of tubular damage and fibrosis, which may have multiple triggers. With each new gene discovery, there seems to be more clarity regarding molecular diagnosis, but more confusion regarding the underlying signaling pathways of the disease.

[0006] There remains a great need to characterize the molecular basis of ciliopathies, including NPHP, which are poorly understood, and to improve their diagnosis and treatment. Summary of the Invention

[0007] In one embodiment, the present disclosure relates to a method for treating at least one ciliopathic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one agent that targets at least one G protein-coupled receptor (GPCR). In an embodiment, the ciliopathic disease is caused by a homozygous deletion at the NPHP1 locus. In an embodiment, the ciliopathic disease is caused by a heterozygous deletion at the NPHP1 locus and a heterozygous or homozygous loss-of-function (LOF) at a second locus. In an embodiment, the ciliopathic disease is caused by a heterozygous deletion in one allele of NPHP1 and a LOF mutation in the other allele. In an embodiment, the ciliopathic disease is caused by a loss-of-function mutation in one allele of NPHP1 and a different loss-of-function mutation in the other allele.

[0008] In certain embodiments, the at least one agent is an agonist of the at least one GPCR. In certain embodiments, the at least one agent is a prostaglandin. In certain embodiments, the at least one agent is selected from the group consisting of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), 16,16-dimethyl-PGE2 (dmPGE2), L902,688, CP-544326, AGN-21 0669, 18a, AGN-21 0961, ED-1 17, CP-533536, and combinations thereof. In certain embodiments, the at least one GPCR is selected from the group consisting of EP1, EP2, EP3, and EP4. In certain embodiments, the at least one disease is selected from the group consisting of nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), including all variant forms of JBTS with additional characteristics, such as polydactyly, ocular colobomas, retinal dystrophy, renal cysts, oral frenulum, and hepatic fibrosis; Bardet-Biedl syndrome (BBS); Meckel-Gruber syndrome (MKS); orofacial-digital syndrome (OFD); end-stage renal disease caused by NPHP1 large homozygous deletion; and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations, and any ciliopathies caused by NPHP genes. In certain embodiments, the at least one agent is CP-544326 and the at least one GPCR is EP2. In certain embodiments, the effective amount is between 100 pM and 5 μM. In certain embodiments, the at least one disease is nephronophthisis.

[0009] In one embodiment, the present disclosure relates to a method for identifying a therapeutic agent for treating at least one ciliopathies-related disease, the method comprising: (a) administering a test agent to an animal or cell model of the ciliopathies-related disease, wherein the animal or cell model exhibits a measurable phenotype of the ciliopathies-related disease; (b) comparing the measurable phenotype of the treated animal or cell model with the measurable phenotype of an untreated animal or cell model; and (c) identifying the test agent as a therapeutic agent for treating the ciliopathies-related disease if the measurable phenotype of the treated animal or cell model is improved compared to that of the untreated animal or cell model. In certain embodiments, the animal model may be Danio rerio (zebrafish) or an nphpl knockout (KO) mouse model (nphpl- / -). In certain embodiments, the animal model is generated by administering one or more disrupting agents. In certain embodiments, the one or more disrupting agents include morpholinos. In certain embodiments, the morpholino inhibits expression of at least one nephrocystin (NPHP), e.g., NPHP4. In certain embodiments, the measurable phenotype is selected from the group consisting of body surface curvature, pronephric cysts, lateralized heart defects, and luminal dilation. In certain embodiments, the at least one disease is selected from the group consisting of nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS), orofacial-digital syndrome (OFD), end-stage renal disease caused by NPHP1 large homozygous deletion, and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations.

[0010] In one embodiment, the present disclosure relates to a GPCR agonist for use in treating at least one ciliopathies-related disorder. In certain embodiments, the GPCR agonist is selected from the group consisting of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), 16,16-dimethyl-PGE2 (dmPGE2), CP-544326, L902,688, AGN-21 0669, 18a, AGN-21 0961, ED-1 17, CP-533536, and combinations thereof. In certain embodiments, the GPCR is selected from the group consisting of EP1, EP2, EP3, and EP4. In certain embodiments, the at least one disease is selected from the group consisting of nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS), Orofacial-Digital syndrome (OFD), end-stage renal disease caused by NPHP1 large homozygous deletion, and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations.

[0011] In certain embodiments, the animal model is produced by administering one or more disrupting agents. In certain embodiments, the one or more disrupting agents comprise a CRISPR / Cas9 system that mediates sgRNA-directed gene deletion. In certain embodiments, the CRISPR / Cas9 system inhibits the expression of at least one nephrocystin (NPHP), such as NPHP1. In certain embodiments, the measurable phenotype is selected from the group consisting of retinal photoreceptor layer thickness, electroretinogram, and rhodopsin accumulation in the photoreceptor cell body. In certain embodiments, the at least one disease is selected from the group consisting of nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS), Orofacial-Digital syndrome (OFD), end-stage renal disease caused by NPHP1 large homozygous deletion, and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations.

[0012] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be made to the following detailed description read in conjunction with the following drawings, in which like reference numerals indicate like elements and in which: [Brief explanation of the drawings]

[0013] [Figure 1] 1A-1D show renal epithelial cells derived from urine. 1A: normal control, 1B: NPHP patient (Pt1) with deletion of NPHP1, 1C: RT-PCR comparison, 1D: immunoblot comparison. [Figure 2] 1 shows an automated in vitro assay for quantifying ciliogenesis in cells of interest. [Figure 3] The percentage of ciliated cells from NPHP patients (PT1) is significantly lower than that of control cells (CTRL). [Figure 4] FIG. 1 is a schematic diagram showing the steps of the novel ciliary-based assay. [Figure 5]The effects of (A) fluticasone, (B) pheniramine, (C) verapamil, (D) ML-141, (E) mitoxantrone, (F) tropisetron, (G) ethopropazine, (H) cyproheptadine, (I) paclitaxel, and (J) simvastatin on ciliogenesis compared to DMSO. [Figure 6] The effect of alprostadil on ciliogenesis is shown in comparison with DMSO. [Figure 7A] 1 shows the dose response of alprostadil compared to DMSO on ciliogenesis. [Figure 7B] The corresponding semi-logarithmic expressions for IC50 determination are shown. [Figure 8A] A meta-analysis of the results obtained from multiple ciliogenesis experiments with alprostadil treatment is shown. [Figure 8B] A meta-analysis of the results obtained from multiple ciliogenesis experiments with alprostadil treatment is shown. [Figure 8C] A meta-analysis of the results obtained from multiple ciliogenesis experiments with alprostadil treatment is shown. [Figure 9] Meta-analysis of the results obtained from multiple ciliogenesis experiments with alprostadil treatment is shown, with data for each experiment separated (A–D). [Figure 10] 1 shows the stability of PGE1 under experimental conditions. [Figure 11A] 1 shows the effects of alprostadil (PGE1), dinoprostone (PGE2), and 16,16-dimethyl-PGE2 (dmPGE2) on ciliogenesis. [Figure 11B] 1 shows the effect of alprostadil (PGE1) on cell lines derived from NPHP1-deficient patients. [Figure 11C] A meta-analysis of cilia is presented. [Figure 12] The effect of PGE2 on ciliogenesis is shown. [Figure 13] 1 shows the expression profile of EP1-4 in human kidney tissue by Western blot and in human retina by immunohistochemistry. [Figure 14]Figure 14A shows that EP2 and EP4 mRNAs are expressed in control and Pt1-derived renal epithelial cells. Figure 14B shows that EP2 is expressed at the protein level in control and Pt1-derived renal epithelial cells. Figure 14C shows the mRNA expression of genes encoding EP1-4 receptors in multiple control cell lines and multiple NPHP patient-derived renal epithelial cell lines. [Figure 15] The figures show prostaglandin (PG) modulators (agonists and antagonists) whose effects on ciliogenesis were examined. [Figure 16A] A meta-analysis of cilia is presented. [Figure 16B] Cells from an NPHP patient treated with CP-544326 are shown. [Figure 16C] The corresponding semi-logarithmic representation is shown below. [Figure 17A] The effect of L-902.688 on ciliogenesis is shown. [Figure 17B] 1 shows the effects of CP-544326 and alprostadil on ciliogenesis. [Figure 17C] The effect of CP-544326 on patient-derived cells is shown. [Figure 17D] A meta-analysis of cilia is presented. [Figure 18] RNA extracted by the RLT or Qiazol method for microarray analysis is shown. [Figure 19] 1 shows microarray data of samples analyzed by hierarchical clustering. [Figure 20] 1 shows microarray data of samples analyzed by hierarchical clustering. [Figure 19] 1 shows microarray data of samples analyzed by hierarchical clustering. [Figure 20] 1 shows microarray data of samples analyzed by hierarchical clustering. [Figure 21] 1 shows microarray data of samples analyzed by hierarchical clustering. [Figure 22]Summarize the microarray data obtained from the RLT-extracted samples. [Figure 23] Microarray data obtained from Qiazol-extracted samples are summarized. [Figure 24A] Shows no significant differences between microarray data from different doses. [Figure 24B] Shows no significant differences between microarray data from different doses. [Figure 25] The process of multi-omics analysis of drug effects on ciliogenesis is shown. [Figure 26] Phenotypic analysis of the effect of alprostadil on ciliogenesis is shown (A-E). [Figure 27] Figure 1 shows the imRNA differential expression of drugged and druggable genes. [Figure 28A] Pathway analysis from multi-omics data on downstream interactions of prostaglandin E1 (alprostadil) and associated targeting opportunities are presented. [Figure 28B] Pathway analysis from multi-omics data on upstream interactions of NPHP1 and associated targeting opportunities are shown. [Figure 28C] Pathway analysis from multi-omics data on NPHP1-20 gene-related direct interactions and associated targeting opportunities are presented. [Figure 29] Figure 1 shows the zebrafish NPHP4 MO model. [Figure 30] We present a protocol for drug treatment of the zebrafish NPHP4 MO model. [Figure 31] Summarize the effects of morpholino injection on zebrafish (A–C). [Figure 32] (A) Representative axial curvature of zebrafish, and (B, C) the effect of alprostadil on axial curvature of zebrafish. [Figure 33] (A) A representative pronephric cyst in zebrafish, and (B, C) the effect of alprostadil on pronephric cysts in zebrafish. [Figure 34] (A, B) The effect of dinoprostone on axial curvature in zebrafish, and (C) the effect of dinoprostone on pronephric cysts in zebrafish. [Figure 35] The effect of CP-544326 on pronephric cysts in zebrafish is shown. [Figure 36] Pharmacokinetic study design is shown. [Figure 37A] The results of the pharmacokinetic study are shown. [Figure 37B] The results of the pharmacokinetic study are shown. [Figure 37C] The results of the pharmacokinetic study are shown. [Figure 37D] The results of the pharmacokinetic study are shown. [Figure 37E] The results of the pharmacokinetic study are shown. [Figure 38A] Periodic acid-Schiff staining of the retina in wt and Nphp1 − / − mice is shown. [Figure 38B] 1 shows a semi-automated method for quantification of retinal layer thickness. [Figure 38C] Quantification of retinal layer thickness in HphpV' mice compared to wt mice is shown. [Figure 39A] Immunohistochemical staining of wt and Nphp1 − / − mouse retinas for Cep290 as a cilia marker and rhodopsin and PNA (peanut agglutinin lectin) as photoreceptor markers for the outer segment (OS) and inner / outer segments, respectively. [Figure 39B] Immunohistochemical staining of wt and Nphp1 − / − mouse retinas for Cep290 as a cilia marker and rhodopsin and PNA (peanut agglutinin lectin) as photoreceptor markers for the outer segment (OS) and inner / outer segments, respectively. [Figure 40] Figures 40A, 40B, and 40C show electroretinograms of Nphp1- / - mice compared to wt mice. [Figure 41] EP2 receptor expression in wt and Nphp1 − / − mice is shown. [Figure 42] 1 shows a study design according to one embodiment of the present disclosure. [Figure 43] 1 shows the effect of CP-544326 on the ONL / OPL retinal layer thickness ratio in Nphp1 − / − mice. [Figure 44] 1 shows the effect of CP-544326 on mislocalization of green-labeled rhodopsin in the ONL in NphpV′ mice. [Figure 45] 1 shows the effect of CP-544326 on the electroretinogram of Nphp1 − / − mice. DETAILED DESCRIPTION OF THE INVENTION

[0014] It is to be understood that the present disclosure is not limited to the specific embodiments described below, and that variations of the specific embodiments may be made and may fall within the scope of the appended claims. It is also to be understood that the terminology used is for the purpose of describing particular embodiments and is not intended to be limiting.

[0015] As used herein and in the appended claims, "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0016] NPHP patient Nephronophthisis (NPHP) is a recessive tubulointerstitial ciliopathies characterized by progressive destruction of the kidney, leading to end-stage renal disease (ESRD). Onset of ESRD caused by NPHP ranges from a few months after birth (pediatric NPHP) to >60 years of age (adult NPHP), with >17% presenting with ESRD after age 20. Disease-causing mutations have been identified in over 20 NPHP-related genes (e.g., NPHP1-20, IFT140, TRAF3IP1 / IFT54), accounting for approximately 60% of all NPHP cases. Whole-locus deletions of NPHP1 (NPHP1(del)) account for >20% of NPHP cases. Historically, the rare disease portal Orphanet reports a global frequency of approximately 1 in 100,000 (Canada 1 / 50,000, USA 1 / 900,000, Finland 1 / 100,000, France 1 / 50,000). Currently, there is no cure for NPHP.

[0017] Ciliopathies are often caused by mutations in genes encoding transition zone (TZ) proteins or intraciliary transport (IFT) components (Reiter, J. & Leroux M., Nat. Rev. Mol. Cell Biol., 18:533-47, 2017; Hildebrandt, F. et al., N. Engl. J. Med., 364:1533-43, 2011; Czarnecki, P. & Shah, J., Trends Cell Biol., 22:201-10, 2012). Functionally, the TZ represents a compartment at the base of the primary cilium at the proximal end of the axoneme and regulates the trafficking of ciliary proteins (Betleja, E. & Cole, D., Curr. Biol., 20:R928-31, 2010; Craige, B. et al., J. Cell Biol., 190:927-40, 2010; Omran, H., J. Cell Biol., 190:715-7, 2010; Benzing, T. & Schermer, B., Nat. Genet., 43:723-4, 2011). Molecularly, TZ consists of different multiprotein complexes, the NPHP1-4-8 module, the NPHP5-6 (Cep290) module, the MKS / B9 module, and the Inversin (INVS, NPHP2) compartment (Sang, L. et al., Cell, 145:513-28, 2011). The NPHP1-4-8 module, the NPHP5-Cep290 module, and the Inversin compartment are sometimes collectively referred to as the NPHP module.

[0018] Mutations and / or inactivation of one or more genes encoding NPHP module proteins can adversely affect ciliogenesis and / or epithelialization, resulting in the development of fibrosis and cysts in NPHP patients. The IFT apparatus selects cargo at the base of the cilium and transports axonemal components required for ciliary assembly and proteins involved in ciliary signaling. The IFT-B complex, consisting of 16 distinct proteins, mediates anterograde transport by binding to kinesin II. Retrograde transport is mediated by dynein 2 and six subunits of the IFT-A complex. Mutations in six genes encoding IFT-A subunits have been identified in NPHP-related ciliopathies, and only three IFT-B subunits (IFT172, IFT54) are associated with nephronophthisis (Halbritter, J. et al., Am. J. Hum. Genet., 93:915-25, 2013; Bizet, A. et al., Nat. Commun., 6:8666, 2015). In addition to IFT and TZ, accessory proteins and GPCRs are also essential factors for cilia function and maintenance.

[0019] Regarding the NPHP module, Nphp4 mutant mice developed retinal degeneration but no renal cysts or severe ciliogenesis defects, and males were infertile and showed reduced sperm motility (Won, J. et al., Hum. Mol. Genet., 20:482-96, 2011). Similarly, targeted disruption of Nphp1 in these mice (deletion of the final C-terminal exon 20) did not result in nephronophthisis, but showed rapid retinal degeneration beginning at P14-P21 (Jiang, S. et al., Hum. Mol. Genet., 17:3368-79, 2008) and male infertility (Jiang, S. et al., Hum. Mol. Genet., 18:1566-77, 2009). Cep290 knockout mice lack connecting cilia in photoreceptors and are unable to mature motile ependymal cilia, consistent with their retinal degeneration and hydrocephalus phenotype (Rachel, R. et al., Hum. Mol. Genet., 24:3775-91, 2015).

[0020] Mutations in NPHP1 are the most common cause of NPHP. In a large cohort of patients with adult-onset ESRD (randomized for etiology), NPHP due to NPHP1 homozygous whole-gene deletion (NPHP(del)) had a prevalence of 1 in 200 (0.5%) of all adult-onset ESRD patients (Snoek, R. et al., J. Am. Soc. Nephrol., 29:772-9, 2018). While the incidence was significantly higher in patients aged 18 to 50 years at onset of ESRD (prevalence 0.9%), NPHP can develop up to age 61. Because the method they used underestimated the total number of causative mutations, they concluded that NPHP is a relatively frequent monogenic cause of adult-onset ESRD and may be underdiagnosed in current routine clinical practice.

[0021] In a cohort of kidney transplant recipients and matched donor controls from the International Genetics and Translational Research in Transplantation Network (iGenTRAiN), we identified approximately 0.5% (26 of 5606) patients homozygous for the NPHP1 deletion among adults with ESRD (aged 18-50 years). Of these, only 13% (3 of 26) were correctly diagnosed as NPHP, and approximately half (11 of 26) were diagnosed as CKD of unknown etiology. These results indicated that fewer than 1 in 200 adults with ESRD (0.5%) had the NPHP1del genotype, and this figure increased to 0.9% when ESRD onset occurred within the 18-50 age range (Abstract. ASN2017 & Nephr Dial Trans, Vol 32, 2017).

[0022] This paper describes findings generated using Genomics England's Research Environment, a secure workspace for approved researchers to conduct research on the 100,000 Genomes Project dataset, which aims to identify novel disease- and patient-related insights, thereby enabling scientific discovery and facilitating its translation into patient management. The 100,000 Genomes Project dataset includes patients (and their relatives) with rare diseases and cancer. Within this dataset, patients homozygous for NPHP1(del) were identified at a relative frequency of approximately 1 in 6,000 (10 of 61,554), none of whom had a previous diagnosis of NPHP. Of the 10 identified patients, seven had overt clinical signs / symptoms of NPHP, e.g., signs / symptoms of renal or ciliopathies, or were recruited as patients with congenital anomalies of the kidney and urinary tract (CAKUT). The remaining three patients presented with a more complex clinical picture, likely involving multiple rare diseases. In addition to homozygotes, 193 NPHP1(del) heterozygous patients were identified in the entire dataset (approximately 1 in 200 frequency within this dataset). These patients may be heterozygous carriers, but may also include NPHP1 compound heterozygotes (NPHP1(del) and NPHP1 loss-of-function (LOF) mutations) and / or epistasis (combination of NPHP1(del) and LOF mutations at other loci). Furthermore, patients may have additional NPHP1-LOF variants, such as splice variants, frameshift, and nonsense mutations, which may also contribute to the clinical NPHP findings.

[0023] The findings of NPHP(del) described herein were obtained from research conducted using the Genomics England database. This research was made possible through access to the data and findings generated by Genomics England's Research Environment, as well as by patients who consented to the use of their data for research purposes and NHS clinicians and medical teams who contributed data and results covered by this research. Genomics England's Research Environment is managed by Genomics England Limited (a wholly owned company of the Department of Health) and funded by the National Institute for Health Research and NHS England, The Wellcome Trust, Cancer Research UK and the Medical Research Council.

[0024] Millions of patients worldwide suffer from ESRD and congenital conditions, for which transplantation is the only cure. In the United States alone, over 600,000 transplants have been performed in the past 50 years, and today's demand is higher than ever. Unfortunately, the availability of donor organs has not kept up with the demand for transplants. Embodiments of the present disclosure include identifying and / or treating patients who are homozygous or heterozygous for NPHP (e.g., NPHP-caused ESRD) and / or patients with NPHP-associated ciliopathies (e.g., NPHP1).

[0025] Cells from NPHP patients Described herein are materials and methods for identifying therapeutic agents useful for treating ciliopathies-related diseases or disorders, such as NPHP or NPHP1(del)-related diseases or disorders. Such methods may involve the use of patient-derived cell lines. Such developed cell lines can also be used in other related methods, including, for example, monitoring the effectiveness of a given treatment for ciliopathies-related diseases or disorders or NPHP1(del)-related diseases or disorders.

[0026] To identify compounds for treating ciliopathies, such as NPHP, we obtained cells from NPHP patients and established cell lines. Briefly, exfoliated renal epithelial cells, mostly proximal tubule cells (tbcs), recovered from the urine of NPHP1-deficient patients were immortalized by retroviral gene transfer of SV40 T antigen. Cells were fixed and fluorescently labeled with Hoechst (for nuclear staining), anti-γ-tubulin (for basal body staining), and anti-ARL13B (for cilia staining) antibodies for detection using immunofluorescence microscopy. In contrast to the majority of normal urinary renal epithelial cells (URECs), which have a single cilium per cell (Figure 1A), most cells from NPHP patients lack cilia (Figure 1B). The lack of NPHP expression in these NPHP patient-derived cells was further confirmed by RT-PCR (Figure 1C) and immunoblotting (Figure 1D), demonstrating no detectable levels of NPHP RNA or protein expression, respectively.

[0027] Figure 2 shows an automated in vitro assay that can be used to quantify ciliogenesis in cells of interest. Briefly, NPHP patient-derived and control cells were cultured in complete medium at 39°C (a non-permissive temperature for SV40 expression), followed by automated cilia analysis using immunofluorescence microscopy to measure ciliogenesis, e.g., as percent cilia. The spinning wheel platform can also be used for drug screening (Figure 5, A-J) and ciliogenesis analysis of the G3 multi-omics dataset (Figure 29, A-E). The Opera Phenix platform can also be used for other phenotypic analyses (e.g., ciliogenesis titration of alprostadil and CP-544326, screening of other EP agonists based on ciliogenesis, ciliogenesis using other NPHP1 patient-derived cell lines, and α-tubulin acetylation analysis).

[0028] Figure 3 shows that the percentage of ciliated cells from NPHP patients (PT1) was significantly lower than that seen in control cells (CTRL) (p=0.0065).

[0029] Drug screening The ciliary system assay described above can be used to identify compounds that restore ciliogenesis. Figure 4 illustrates the process of the ciliary system assay. For example, cells may be seeded into cell culture (e.g., a 96-well plate) on day 0, incubated with a candidate drug on day 3, and fixed on day 5, then fluorescently labeled with Hoechst, anti-γ-tubulin, and anti-ARL13B antibodies. For example, automated random collection of 35 images per well may be performed. Each image may have a z-stack of 10 images taken at intervals of <1 μm. Sequential images of the nucleus (Hoechst, 461 nm), basal body (γ-tubulin, 555 nm), and cilium (ARL13b, 647 nm) may be obtained.

[0030] Using the process shown in Figure 4, we treated cells derived from NPHP patients with several candidate drugs to identify those capable of restoring ciliogenesis. Figure 5, panels A–J, show that fluticasone, pheniramine, verapamil, ML-141, mitoxantrone, tropisetron, ethopropazine, cyproheptadine, paclitaxel, and simvastatin, respectively, had no significant effect on ciliogenesis compared to DMSO at various test concentrations. Surprisingly, Figure 6 shows that alprostadil significantly restored ciliogenesis in cells derived from NPHP patients compared to DMSO, as shown by the increased percentage of ciliated cells.

[0031] Alprostadil, or prostaglandin E1 (PGE1), has the chemical structure [ka] Alprostadil has the following properties: it exhibits vasodilation for the treatment of cardiac disease and erectile dysfunction, inhibits platelet aggregation, and stimulates intestinal and uterine smooth muscle. Alprostadil may act as an agonist by binding to the E-type prostaglandin (EP) receptor, a G protein-coupled receptor (GPCR), with IC50 values ​​of 36, 10, 1.1, and 2.1 nM for EP1, EP2, EP3, and EP4, respectively. GPCR stimulates adenylate cyclase, which subsequently elevates intracellular cAMP.

[0032] As used herein, a "GPCR agonist" includes a composition that activates a GPCR in a manner that mimics the action of endogenous signaling molecules specific for that receptor. A "GPCR antagonist" includes a composition that inhibits GPCR activity. GPCR activity can be measured by its ability to bind to effector signaling molecules, such as G proteins. An "activated GPCR" is one that is able to interact with and activate G proteins. An inhibited receptor may have a reduced ability to bind extracellular ligands and / or a reduced ability to productively interact with and activate G proteins.

[0033] For example, GPCR agonist treatment with taprenepagisopropyl may be performed at concentrations of, for example, about 0.1 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 1 mg / kg to about 20 mg / kg, about 2 mg / kg to about 20 mg / kg, about 3 mg / kg to about 20 mg / kg, about 4 mg / kg to about 20 mg / kg, about 5 mg / kg to about 20 mg / kg, about 6 mg / kg to about 20 mg / kg, about 7 mg / kg to about 20 mg / kg, or about 8 mg / kg to about 20 mg / kg. The dose may be 20 mg / kg, about 9 mg / kg to about 20 mg / kg, about 10 mg / kg to about 20 mg / kg, about 12 mg / kg to about 20 mg / kg, about 14 mg / kg to about 20 mg / kg, about 16 mg / kg to about 20 mg / kg, or about 18 mg / kg to about 20 mg / kg, and may be administered at a frequency of, for example, every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, once a week, every 2 weeks, every 3 weeks, or once a month.

[0034] To identify the effective concentration of alprostadil for restoring ciliogenesis, automated cilia analysis was performed with alprostadil titrations of 1 nM to 2 μM (Figure 7A) and 100 pM to 2 μM (Figure 7B). Effective concentrations of GPCR agonists, such as alprostadil, can be about 1 pM to about 10 μM, about 10 pM to about 5 μM, about 50 pM to about 5 μM, about 100 pM to about 5 μM, about 1 nM to about 5 μM, about 1 nM to about 4 μM, about 1 nM to about 3 μM, about 1 nM to about 2.5 μM, about 1 nM to about 2 μM, about 10 nM to about 2 μM, about 100 nM to about 2 μM, about 500 nM to about 2 μM, or about 1 μM to about 2 μM. FIG. 7C shows the corresponding semi-logarithmic representation for IC50 determination, demonstrating that alprostadil significantly increases the % ciliated cells in a dose-dependent manner in cells from NPHP patients.

[0035] Figures 8A-8C and 9 (Panels A-D) show a meta-analysis demonstrating that alprostadil treatment (2 μM) does not significantly affect ciliogenesis in control normal epithelial cells (CTRL) compared to control (DMSO 0.04%) (Figure 8A). In contrast, alprostadil treatment significantly increases ciliogenesis in cells derived from NPHP patients (PT1) compared to control (DMSO 0.04%) (Figure 8B). Figure 8C shows that the effect of alprostadil on ciliogenesis in cells derived from NPHP patients is approximately two-fold increased compared to control cells not treated with alprostadil, i.e., control (DMSO 0.04%).

[0036] The meta-analysis also shows a nearly linear effect of alprostadil dose on ciliogenesis. Figure 9 (Panels A and B), for example, shows the R 2 Similarly, Figure 9 (Panels C and D) shows the R 2 The value is 0.8489.

[0037] To characterize the stability of alprostadil (PGE1), supernatants were collected from urine-derived renal epithelial cells (URECs) exposed to different concentrations of alprostadil after 24 and 48 hours of exposure. Samples were then extracted and aliquoted for analysis on LC / MS / MS and Polar LC platforms. Figure 10 shows that PGE1 was stable under the experimental conditions.

[0038] In addition to PGE1, other EP agonists, e.g., [ka] Prostaglandin E2 (PGE2 or dinoprostone) and its long-acting derivatives, having the chemical structure [ka] 16,16-dimethyl-PGE2 (dmPGE2), which has the formula (I), was also tested for its ability to restore ciliogenesis. Figure 11A shows that PGE2 and dmPGE2 had a similar effect on restoring ciliogenesis to that of alprostadil in cells derived from NPHP patients, whereas no significant effect was observed in control normal cells. In cells derived from NPHP patients, a slight decrease in ciliogenesis restoration was observed at the highest concentrations (40 μM dinoprostone and 20 μM dmPGE2), which may be due to cytotoxicity.

[0039] To examine the effects of alprostadil (PGE1) on NPHP1-deficient cells, cell lines derived from NPHP1(del) patients, e.g., PT1, 1-03-P, 1-06-P1, 1-06-P2, 1-09-P, 1-10-P, and 1-12-P, were treated with alprostadil (2 μM) or DMSO. Figure 11B shows that alprostadil significantly increases the ciliogenesis rate in NPHP1-deficient cells, whereas alprostadil has no significant effect on the ciliogenesis rate in normal control cells, suggesting that alprostadil is effective in restoring ciliogenesis in NPHP1-deficient patients.

[0040] The meta-analysis in Figure 11C shows linear regression analysis of previous data. The slope reflects the effect of alprostadil on ciliogenesis in control cells and cell lines derived from multiple NPHP patients. Each symbol represents an independent experiment, and each color represents a patient cell line (designated 1-09-PL4, 1-06-P1, 1-06-P2, and PT-1). Linear regressions on control normal epithelial cell data showed slope values ​​ranging from 0.7665 to 0.9974, suggesting a lack of effect of alprostadil on ciliogenesis. In contrast, linear regressions on cells derived from multiple NPHP patients showed a combined slope value of 1.414 or a range of slope values ​​from 1.333 to 1.506, indicating a stimulatory effect of alprostadil on ciliogenesis.

[0041] Prostaglandins are found in most human tissues and are synthesized from essential fatty acids. Structural differences among various prostaglandins account for the variations in their biological activities. Prostanoids, including prostaglandins, are abundantly produced in the kidney. These prostanoids are generated by the release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2. Arachidonic acid undergoes the bisoxygenase and peroxidase activities of cyclooxygenase (or prostaglandin G / H synthase) to form prostaglandin G2 (PGG2) and then prostaglandin H2 (PGH2). PGH2 is a substrate for synthases, including PGE2 synthase, PGD2 synthase, prostacyclin synthase, PGF2α synthase (PGF2α can also be synthesized directly from PGE2), and thromboxane synthase, which synthesize individual classes of prostanoids, including PGE2. All of these classes have different receptor subtypes, including EP1-4, through which they initiate their actions. Cyclooxygenase 1 and 2 (COX1 and COX2) are the primary targets of nonsteroidal anti-inflammatory drugs (NSAIDs), which can be specific, i.e., selective, for one isoform or nonselective. Blocking PGH2 production through inhibition of COX can reduce levels of all downstream prostanoids.

[0042] PGE in ciliogenesis PGE2 is the most well-characterized prostanoid in renal pathophysiology. PGE2 is synthesized by COX1 and COX2 and transported to the plasma membrane via the Lkt / ABCC4 transporter. Released PGE2 binds to the EP4 receptor in cilia, activating GPCR (Gs) and adenylate cyclase (AC) to increase cAMP, thereby increasing anterograde IFT and enhancing ciliogenesis.

[0043] The COX-Lkt / ABCC4-EP4 signaling cascade is required for ciliogenesis and elongation (in mouse kidney collecting duct cells IMCD3 and zebrafish models). cAMP-dependent kinase signaling is known to increase anterograde IFT during ciliogenesis. Lkt / ABCC4-mediated PGE2 signaling affects cAMP levels and promotes ciliogenesis through an increase in the anterograde velocity of IFT. PGE2 treatment increases intracellular cAMP during ciliogenesis in IMCD3 cells, but not Ca. 2+ PGE2 acts in an autocrine and / or paracrine manner, and cells can respond to PGE2 released by themselves or by their neighbors. In human cancer cells, the interaction of PGE2 with the EP4 receptor induces Wnt / p-catenin signaling, leading to COX2 expression, thereby setting up a positive feedback loop that leads to further PGE2 synthesis.

[0044] Figure 12 shows that addition of exogenous PGE2 increased both cilium length and the percentage of ciliated cells in control cells but not in EP4-depleted cells, indicating that EP4 acts downstream of PGE2 signaling during ciliogenesis.

[0045] PGE2 is produced by PGE synthase (PGES) and signals by binding to its GPCRs, i.e., EP1-4. qActivation of intracellular Ca2+ via PLC + Activation of EP3 (coupled to Gi) increases intracellular Ca2 + and / or inhibit cAMP production via adenylate cyclase (AC). s Activation of ATP (coupled with ATP) promotes cAMP production via AC.

[0046] There are approximately 800 human GPCRs, which are divided into five major phylogenetic families: rhodopsin, secretin, adhesion, glutamate, and Frizzled / Taste2. GPCRs are attractive targets for recombinant proteins, small molecule compounds, allosteric ligands, or antibodies. 46 GPCRs serve as drug targets for hypertension, pain, ulcers, allergies, alcoholism, obesity, glaucoma, psychotic disorders, and HIV. One of the major obstacles is the overall lack of knowledge regarding the association of putative GPCRs with precise physiological functions or disease states.

[0047] Figure 13 shows that EP1-4 are expressed in the kidney and retina, and both organs are affected by NPHP and NPHP-RC. In the kidney, EP receptors are differentially expressed along the nephron, highlighting the distinct functional consequences of activation of each EP receptor subtype in the kidney. EP receptors regulate vascular tone in the afferent arteriole, where EP1 / EP3 act as vasoconstrictors and EP2 / EP4 act as vasodilators. EP1 / EP4 regulate proximal tubule transport. EP3 and EP4 regulate large ascending limb and distal tubule transport. EP4 promotes renin release from the macula densa. EP2 / EP4 dilate the vasa recta. EP receptors are expressed by EP1, which mediates Na+ transport. + EP1 inhibits the reabsorption of H2O, EP2 inhibits the reabsorption of H2O, and EP4 promotes the reabsorption of H2O, thereby regulating collecting duct transport.

[0048] Expression of components of the PG pathway, including EP receptors, in URECs was measured by qRT-PCR. Figure 14A shows that EP2 and EP4 are expressed at the mRNA level, and that EP2 is expressed primarily at the mRNA level. Figure 14B shows EP2 protein expression in URECs.

[0049] PGE2 modulator (EP2) Selective agonists and antagonists of the EP2 receptor are shown, for example, in Markovic, T. "Structural features of subtype-selective EP receptor modulators," Drug Discovery Today. 2017, 22(1):57-71, which is incorporated by reference. The first class of agonists includes ligands that are structurally similar to the endogenous ligand PGE2 but incorporate key modifications in the ω-lipophilic chain that contribute to improved potency and selectivity. The second class of agonists is non-prostanoid pyridylsulfonamide derivatives, the most potent of which is taprenepag isopropyl (PF 04217329, a prodrug of CP544326). Taprenepag has a non-prostanoid structure. [ka] It has.

[0050] A third class of agonists includes non-prostanoid N-phenyl-y-lactam derivatives, which include AGN-21 0669 and AGN-21 0961.

[0051] PF-0441 8948, an azetidine-3-carboxylic acid derivative, is the first selective EP2 antagonist, which has an IC50 of 16 nM (Kb=1.8 nM) and exhibits a >10,000-fold increase in selectivity for the EP2 receptor over other prostanoid receptors.

[0052] Figure 5 of Markovic (incorporated by reference) shows selective agonists of the EP4 receptor, namely, (a) derivatives based on a functionalized cyclopentane core, (b) derivatives bearing the lactam counterpart of the hydroxycyclopentanone core, and (c) structurally diverse EP4 agonists. The introduction of a tetrazole feature into the a-chain instead of a terminal carboxylic acid functionality with the intent of improving bioavailability led to the discovery of L902,688, a subnanomolar agonist of the EP4 receptor (EC50 = 0.2 nM). L902,688 has a prostanoid structure. [ka] It has.

[0053] Structure of KAG-308, a low nanomolar EP4 agonist [ka] is somewhat unique in the field of EP4 agonists as it is the only one based on the 7,7-difluoroprostacyclin scaffold.

[0054] Figure 6 of Markovic (incorporated by reference) shows a selective antagonist of the EP4 receptor and the switching of functional responses as a result of minimal structural changes: (a) a selective antagonist of the EP4 receptor, and (b) the switching between agonism and antagonism at the EP4 receptor. PG-1 531, a trisubstituted furan derivative, is a nanomolar EP4 antagonist with an excellent selectivity profile and improved water solubility. By introducing minor modifications to the molecule, it is possible to fine-tune the latter's intrinsic activity at the EP4 receptor (an example is shown in Figure 17). For example, it has been shown that the intrinsic activity (agonism vs. antagonism) depends solely on the substitution pattern of the trifluoromethyl substituent of the benzyl group of the compound in Figure 17 (panel b). Dramatic changes in function can be achieved with minimal changes in the ligand structure.

[0055] FIG. 15 shows PG modulators (agonists and antagonists) that were examined for their effect on ciliogenesis.

[0056] Figure 16A shows that the non-prostanoid EP2 agonist CP-544326 restores ciliogenesis to levels similar to alprostadil. Figure 16B shows that CP-544326 restores ciliogenesis in a dose-dependent manner compared to DMSO. Figure 16C is a semi-logarithmic representation of the results from Figure 16B, showing a CP-544326 titration with an EC50 of 11 nM for EP2. The restoration of ciliogenesis for the non-prostanoid CP-544326 confirms its specificity in mechanism of action. In contrast, Figure 17A shows that the prostanoid EP4 agonist L-902.688 has no significant effect on ciliogenesis. These results indicate that EP2 plays a more important role in ciliogenesis than EP4.

[0057] Figure 17C shows that, like alprostadil, CP-544326 treatment increased ciliogenesis in multiple cell lines derived from NPHP1(del) patients, e.g., 1-09-P, 1-06-P1, and 1-06-P2, compared to those treated with DMSO. Figure 17D shows a meta-analysis of the linear regression analysis in Figure 17D. The slope reflects the effect of CP-544326 on ciliogenesis in control cells and multiple NPHP patient-derived cell lines. Each symbol represents an independent experiment, and each color represents a patient cell line (designated 1-09-P L4, 1-06-P1, 1-06-P2, or PT-1). Linear regression of control normal epithelial cell data showed slope values ​​ranging from 0.6369 to 1.03, suggesting that CP-544326 does not affect ciliogenesis. In contrast, linear regression on cells from multiple NPHP patients showed an integrated slope value of 1.36 or a range of slope values ​​from 1.245 to 1.532, indicating a stimulatory effect of alprostadil on ciliogenesis.

[0058] Differential expression analysis Microarray analysis was performed to identify expressed genes involved in alprostadil-mediated restoration of ciliogenesis. URECs were cultured in 96-well plates and treated with different concentrations of alprostadil, as summarized in Figure 18, followed by RNA extraction using either the RLT or Qiazol method.

[0059] Figure 19 shows the microarray data of samples analyzed by hierarchical clustering. Data were first clustered by type of extraction (Qiazol vs. RLT). Qiazol samples were then clustered by condition, e.g., control vs. alprostadil treatment, and RLT samples were then clustered by iteration.

[0060] Figure 20 shows the microarray data for samples analyzed by hierarchical clustering. Data from Qiazol-extracted samples were clustered by condition and then by replicate, rather than by dose within treatment or medium / DMSO within control.

[0061] Figure 21 shows the microarray data of samples analyzed by hierarchical clustering. Data from RLT-extracted samples were clustered by replicate and then by condition (control vs. alprostadil treatment), rather than by dose within treatment or medium / DMSO within control.

[0062] Regarding the microarray data obtained from the RLT-extracted samples, there were no significant differences between DMSO and medium, e.g., only four differentially expressed genes with no regulated exons / patterns. Figure 22, however, shows approximately the same number of expressed and regulated genes across the three alprostadil concentrations compared, comparing control (DMSO) with alprostadil treatment (0.2 μM, 2 μM, and 10 μM). The top three regulated genes were also approximately the same, sharing the same signaling pathways, e.g., downregulation of cell adhesion and extracellular matrix.

[0063] Regarding the microarray data obtained from the Qiazol-extracted samples, there were no significant differences between DMSO and medium, e.g., 33 differentially expressed genes with no regulated exons / patterns. However, as shown in Figure 26, when comparing control (DMSO) with alprostadil treatment (0.2 μM, 2 μM, and 10 μM), there are approximately the same number of expressed and regulated genes across the three alprostadil concentrations. The top three regulated genes are also approximately the same, sharing the same signaling pathways, e.g., downregulation of cell adhesion and extracellular matrix, and upregulation of interferon signaling.

[0064] Furthermore, Figures 24A and 24B show that two clusters were defined, summarizing a total of 310 genes, i.e., "Cluster 1" = 120 down-regulated genes and "Cluster 2" = 190 up-regulated genes, indicating that no significant differences were detected between the microarray data obtained from the various doses.

[0065] Furthermore, pathway analysis by intersecting microarray data from patients with and without alprostadil treatment with RNAseq from controls versus patients revealed that alprostadil could reverse the gene expression changes observed in cells from NPHP patients compared to control cells.

[0066] Multi-omics analysis Figure 25 shows the process of multi-omics analysis of drug effects on ciliogenesis. Figures 26A-26E show the effects of alprostadil on ciliogenesis, e.g., phenotypic analysis of the percentage of ciliated cells, in five independent experiments. These results show that alprostadil partially restores ciliogenesis at similar folds without a dose-dependent response (n=1-5).

[0067] Figure 27 shows a summary of the drugged and druggable genes identified from the protein differential expression analysis of multi-omics data (cells from NPHP patients in DMSO 0.04% and cells from NPHP patients treated with alprostadil 2 μM), from which the drugged genes are named.

[0068] Figure 28 (A-C) shows pathway analysis (using Ingenuity Pathway Analysis) from multi-omics data for (A) prostaglandin E1 (alprostadil) downstream interactions, (B) NPHP1 upstream interactions, and (C) NPHP1-20 gene-related direct interactions, as well as associated targeting opportunities.

[0069] In vivo model Figure 29 shows results from a zebrafish NPHP4 morpholino (MO) model, in which single-cell wild-type zebrafish embryos were injected with a morpholino (e.g., NPHP4 ATG MO) that blocks the start site of NPHP4 mRNA from ribosome binding. This morpholino specifically inhibits NPHP4 mRNA translation. Zebrafish NPHP4 MO exhibits classic ciliopathies-associated phenotypes, including body surface curvature, pronephric cysts, laterality (cardiac looping) defects, and cloacal dilation (obstruction).

[0070] Figure 30 is a schematic diagram showing the drug treatment protocol (alprostadil: 0.5 μM and 5 μM) for the zebrafish NPHP4 MO model. Briefly, wild-type Tg(wt1b:GFP) transgenic zebrafish embryos were injected with morpholinos (e.g., nphp4 ATG MO) at the single-cell stage. At 8 h post-fertilization (hpf), injected embryos were treated with drug or vehicle in PTU-egg seawater (1 mL in a 12-well plate). At 24 hpf, drug treatment was renewed, and pronase was added at 36 hpf before dechorionation. At 54 hpf, zebrafish embryos were examined for phenotype, particularly for body surface curvature and glomerular pronephric cysts, using appropriate means, such as a stereoscope and a PerkinElmer Opera Phenix HCS system, respectively (labeled with the Tg(wt1b:GFP) transgene).

[0071] Figure 31, panel A, shows that DMSO (0.04%) did not induce lethality, body surface curvature, or pronephric cysts in wild-type zebrafish embryos. Furthermore, zebrafish injected with a control morpholino that does not affect NPHP4 expression also did not exhibit body surface curvature (Figure 31, panel B) or pronephric cysts (Figure 31, panel C). In contrast, zebrafish injected with NPHP4 MO exhibited classic ciliopathies-associated phenotypes, including body surface curvature (Figure 31, panel B) and pronephric cysts (Figure 31, panel C), in a dose-dependent manner.

[0072] Figure 32, panel A shows representative axial curvatures of four categories of zebrafish: normal, class I, class II, and class III. Figure 32 , Panel B shows that alprostadil treatment (0.5 μM and 5 μM) did not significantly affect the body axial curvature of zebrafish NPHP4 MO compared to that of DMSO treatment (p>0.05, Fisher's exact test). Similarly, using body surface curvature as an automated quantification parameter, Figure 32, Panel C shows that alprostadil treatment (0.5 μM and 5 μM) did not significantly affect the dorsal curvature of zebrafish NPHP4 MO compared to that of DMSO treatment.

[0073] Figure 33, panel A shows representative pronephric cysts in zebrafish: normal, mild, and severe. Figure 33, panel B shows that alprostadil treatment (0.5 μM) significantly reduced the percentage of severe pronephric cysts in nphp4 MO-injected embryos compared to those treated with DMSO (p<0.05, Fisher's exact test). Similarly, Figure 33, panel C shows that alprostadil treatment (5 μM) significantly reduced the percentage of severe pronephric cysts in nphp4 MO-injected embryos compared to those treated with DMSO.

[0074] To investigate the effect of dinoprostone (PGE2) on ciliopathies, zebrafish NPHP4 MO were treated with dinoprostone (50 μM) or DMSO. Figure 34, panel A, shows that dinoprostone treatment significantly increased the percentage of normal body curvature in zebrafish NPHP4 MO compared to DMSO-treated ones (p = 0.0066, Fisher's exact test). Figure 34, panel B, shows that dinoprostone treatment, however, did not significantly affect the dorsal curvature of zebrafish NPHP4 MO compared to DMSO-treated ones (p = 0.0577, t-test). Figure 34, panel C, shows that dinoprostone treatment significantly reduced the percentage of severe and mild pronephric cysts and significantly increased the percentage of normal pronephric cysts in zebrafish NPHP4 MO compared to DMSO-treated ones (p < 0.008, Fisher's exact test).

[0075] To investigate the effects of the selective EP2 agonist CP-544326, zebrafish NPHP4 MO were treated with CP-544326 (100 nM) or DMSO. Figure 35 shows that CP-544326 treatment significantly reduced the percentage of severe pronephric cysts and increased the percentage of mild and normal pronephric cysts in zebrafish NPHP4 MO compared with DMSO treatment (p<0.01, Fisher's exact test).

[0076] To investigate the in vivo stability of taprenepag isopropyl (PF 04217329, a prodrug of CP-544326) and taprenepag (CP-544326), a pharmacokinetic (PK) study was conducted in wild-type C57BL / 6J mice. Figure 36 shows the PK study design. After intraperitoneal injection of taprenepag isopropyl (1 mg / kg or 8 mg / kg) or taprenepag (8 mg / kg), concentrations of these compounds in various organs were measured at different time points. The results generally indicate that taprenepag is more stable than taprenepag isopropyl in plasma (Figure 37A), kidney (Figure 37B), testis (Figure 37C), retina (Figure 37D), and vitreous humor (Figure 37E).

[0077] Homozygous deletion of NPHP1 is the most common cause of juvenile nephronophthisis. Homozygous or compound heterozygous mutations in NPHP1 are also associated with, for example, Joubert syndrome (brain abnormalities) and Senior-Loken syndrome (retinopathy). We generated NPHP1 KO animals to investigate whether taprenepag could be used to treat these diseases. CRISPR / Cas9-engineered Nphp1 - / - To establish the mouse model, we injected single-guide RNA into C57BL / 6J embryos to create a 76-bp deletion encompassing the ATG of exon 1 of Nphpl. - / - To characterize the natural development of the mouse model, Nphp1 + / + and Nphpl - / - Histochemical staining of mouse kidney and retinal sections was performed. - / - In contrast, P14 Nphpl mouse models do not exhibit renal phenotypes. - / - Mice begin to show a decrease in the thickness of photoreceptor layers (e.g., inner segment (IS), outer segment (OS), and outer nuclear layer (ONL)) until sacrificed at P28, demonstrating rapid retinal degeneration in this model, corresponding to signs of ciliopathies.

[0078] To assess retinal degeneration, a semi-automated tool was developed to detect and quantitatively measure the thickness of each retinal layer at five separate planes manually marked on retinal sections (Figure 38B). Semi-automated quantitative analysis confirmed a clear decrease in the thickness of the photoreceptor layers ONL, IS, and OS (Figure 38C).

[0079] To examine the effect of Nphpl deletion on the structural organization of photoreceptors in this model, immunohistochemistry (IH) was performed on Nphpl + / + and Nphpl - / - This was performed on mouse retinal sections (Figures 39A and B). The tissues were fixed and fluorescently labeled with DAPI (for nuclear staining), anti-rhodopsin antibody (for OS staining), and anti-Cep290 antibody (for connecting cilia staining) or PNA (for OS and IS staining), and detected using immunofluorescence microscopy. Figure 39A shows that Nphp1 - / - Mouse models show that rhodopsin localizes along the OS and exhibits well-organized photoreceptor structures surrounded by a punctate distribution of Cep290 in the connecting cilia, indicating that the connecting cilia are functional in transporting rhodopsin from the IS to the light-sensitive OS. In contrast, Nphpl - / - Mice fail to form connecting cilia and show clear rhodopsin mislocalization at the IS and OS, suggesting that rhodopsin transport requires the correct formation / maintenance of connecting cilia. Consistently, Figure 39B shows that Nphp1 + / + In contrast to mice, Nphpl - / - 1 shows that mice exhibit clear rhodopsin mislocalization in the IS / OS and ONL as well.

[0080] To assess the effect of Nphpl deletion on photoreceptor function, electroretinograms (ERGs) were recorded under different intensities of light stimulation. + / + and Nphp1 - / - This was performed in mice (Figures 40A-C). Figures 40A and 40B show the a- and b-waves of ERGs recorded from the same animal at P21 for a given light intensity. + / + In contrast to mice, Nphp1 - / -Mice exhibit dramatically lower ERG amplitudes at a given light stimulus intensity. Figure 40C shows enlarged views of the a-wave of ERGs under different intensities of light stimulation, where the a-wave reflects photoreceptor function.

[0081] Before examining the effects of CP-544326 on ciliopathies-related phenotypes, we examined the expression of its potential target, EP2, by immunohistochemistry. Fluorescence microscopy revealed that EP2 inhibited the expression of Nphp7 in P21 cells. + / + and Nphpl - / - It is well expressed at the protein level in the photoreceptor layers IS and ONL of mice, but the boundary between the OS / IS / ONL is marked by the Nphp1 - / - It was difficult to distinguish in mice.

[0082] Figure 42 shows that CP-544326 is Nphp7 - / - The experimental design for assessing the effects of CP-544326 on retinal degeneration in a mouse model is shown. Briefly, animals were injected (ip) with either vehicle or CP-544326 in vehicle (18 mg / kg) every 3 or 4 days from P6 to P21. Phenotypic measurements were performed using the Nphp1 (Nphp1) and Nphp1 / N ... - / - Include the structural and functional parameters previously described for characterization of the mouse model.

[0083] Figure 43 shows the effect of CP-544326 on the thickness of the photoreceptor layer ONL, as expressed by the ONL / OPL ratio calculated from semi-automated quantification of retinal layers in IHC sections. CP-544326 treatment (18 mg / kg) reduced the thickness of Nphp7 - / - CP-544326 treatment (18 mg / kg) significantly reduced the ONL / OPL ratio in mice compared to vehicle-treated mice (p<0.05, Mann-Whitney test). Similarly, CP-544326 treatment (18 mg / kg) significantly reduced the Nphpl, a parameter semi-automatically quantified by fluorescence microscopy in IHC sections, expressed as the mean green intensity in the ONL. - / - It significantly reduced rhodopsin mislocalization in mice (p<0.05, unpaired t-test) (FIG. 44).

[0084] To evaluate the effect of CP-544326 on photoreceptor responsiveness, electroretinograms (ERGs) were recorded under different intensities of light stimulation in Nphp1 mice treated with CP-544326 (18 mg / kg) or vehicle. + / + and Nphp1 - / - A magnified view of the ERG a-wave (Figure 45) shows that CP-544326 (18 mg / kg) produced a small improvement in the amplitude of the photoreceptor response compared to that of vehicle-treated Nphpl^ mice.

[0085] All references cited herein are incorporated by reference as if each reference were specifically and individually indicated to be incorporated by reference. It will be understood that each of the above elements, or two or more together, may find useful applications in other types of methods different from those described above. Without further analysis, the foregoing sufficiently reveals the gist of the present disclosure so that others, by applying their current knowledge, can readily adapt it to various applications without omitting features that, in view of the prior art, fairly constitute essential features generally or of the particular aspects of the present disclosure as set forth in the appended claims. The foregoing embodiments are presented by way of example only, and the scope of the present disclosure is limited only by the following claims.

Claims

1. A pharmaceutical composition for treating nephronophthisis, comprising an agonist of E-type prostaglandin receptor 2 (EP2), The pharmaceutical composition, wherein the EP2 agonist comprises CP-544326 (taprenepag), PF 04217329 (taprenepag isopropyl), or both.

2. 2. The pharmaceutical composition of claim 1, wherein the nephronophthisis is caused by a homozygous deletion of the NPHP1 locus.

3. 2. The pharmaceutical composition of claim 1, wherein the nephronophthisis results from a heterozygous deletion of the NPHP1 locus and a heterozygous or homozygous loss of function at a second locus.

4. 2. The pharmaceutical composition of claim 1, wherein the nephronophthisis results from a heterozygous deletion in one allele of NPHP1 and a loss-of-function mutation in the other allele.

5. 2. The pharmaceutical composition of claim 1, wherein the nephronophthisis is caused by a loss-of-function mutation in one allele of NPHP1 and a different loss-of-function mutation in another allele.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the EP2 agonist further comprises prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), or both.

7. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the EP2 agonist further comprises prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), 16,16-dimethyl-PGE2 (dmPGE2), AGN-210669 (simenepag isopropyl), 18a, AGN-210961 (aganepag isopropyl), DE-117 (omidenepag isopropyl), CP-533536 (evatanepag), or a combination thereof.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the at least one EP2 agonist is CP-544326 (taprenepag).

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the dosage of the EP2 agonist is 100 pM to 5 µM.

10. Use of at least one agonist of E-type prostaglandin receptor 2 (EP2) for the preparation of a medicament for use in the treatment of nephronophthisis, comprising: The at least one EP2 agonist comprises CP-544326 (taprenepag), PF 04217329 (taprenepag isopropyl), or both.

11. 11. The use of claim 10, wherein the EP2 agonist further comprises prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), 16,16-dimethyl-PGE2 (dmPGE2), AGN-210669 (simenepag isopropyl), 18a, AGN-210961 (aganepag isopropyl), DE-117 (omidenepag isopropyl), CP-533536 (evatanepag), or a combination thereof.

Images