Method for treating diseases associated with fibrosis

Abstract

Disclosed are methods of treating fibropathy-associated diseases comprising administering to a subject in need thereof an effective amount of a compound targeting at least one G protein coupled receptor. There is provided a method for identifying a therapeutic agent for treating a disease with fiberopathy, comprising providing an animal model system of the fiberopathy for testing the estimated therapeutic agent; in one embodiment, the method includes administering a disrupting agent to the animal, treating the administered animal with the presumed therapeutic agent, comparing a measurable phenotype of the treated animal to a measurable phenotype of an untreated animal, and when the measurable phenotype of the treated animal is reduced as compared to the measurable phenotype of the untreated animal, determining that the animal is not treated with the presumed therapeutic agent, and if the measurable phenotype of the treated animal is reduced as compared to the measurable phenotype of the untreated animal. Therapeutic targets are identified for use in the treatment of fibralopathy.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880068999.X, application date October 12, 2018, and invention name “Method for treating diseases associated with ciliary diseases”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This is an international application under the Patent Cooperation Treaty, which claims the benefit of U.S. Provisional Application No. 62 / 572,051, filed on October 13, 2017. The contents of the foregoing application are incorporated herein by reference in their entirety. Background Art

[0004] Cilia are microtubule-based cell surface protrusions that originate from the basal body, the membrane-docking centriole. The primary cilium is a non-motile sensory organelle present in a single copy on the surface of most growth-arrested or differentiated mammalian cells. Cilia sensory flow modifies and mediates essential signaling pathways during development and tissue homeostasis, such as Hedgehog, Wnt / PCP, and cAMP / PKA signaling. Intraflagellar transport (IFT) selects cargo at the base of the cilium and transports axonemal components required for ciliary assembly as well as proteins involved in ciliary signaling. Once cilia are formed, control of ciliary membrane composition relies on discrete molecular machines, including a barrier that prevents membrane proteins from 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) on the cilium, which is called the BBSome (a complex of Bardet–Biedl syndrome (BBS) proteins with other proteins that are components of the basal body and involved in transporting cargo to the primary cilium). Ciliogenesis requires the coordination of many processes. Complex cell cycle regulation, coordination of vesicle transport, and ciliary extension must occur in a precise timing to generate cilia. The importance of generating and maintaining properly differentiated cilia during embryonic development and in adult physiology is best illustrated by a number of human diseases associated with ciliopathies.

[0005] Ciliopathy is a group of human disorders directly caused by defects in ciliary formation or function. Defective primary cilia lead to multidirectional and highly variable abnormalities, which are consistent with the widespread tissue distribution of primary cilia and their wide range of functions. Individuals suffering from primary ciliopathies show a combination of the following: kidney and retinal abnormalities, central nervous system defects that may lead to mental retardation, liver defects (including cysts), obesity, and various skeletal defects (including abnormalities in limb length, abnormalities in the number of toes (polydactyly), abnormalities in left / right axis organization (situs inversus and craniofacial abnormalities). Abnormalities specific to the photoreceptors connecting cilia may also lead to retinal degeneration and blindness. Examples of primary ciliopathies include renal wasting (NPHP), Senior Loken syndrome (SLS), Joubert syndrome (JBTS), BardetBiedl syndrome (BBS), Meckel Gruber syndrome (MKS), oral-facial-digital syndrome (OFD) and Jeune syndrome (Jeunesyndrome, JATD).

[0006] Nephrotic syndrome (NPHP) is an autosomal recessive renal disease characterized by massive interstitial fibrosis, tubular basement membrane thickening and cyst formation, leading to end-stage renal disease (ESRD) in childhood. NPHP can be isolated or associated with different extrarenal manifestations {e.g., retinal dysplasia, hepatic fibrosis, skeletal dysplasia, etc.) in a syndromic form referred to below as NPHP-RC.

[0007] NPHP is driven by 21 NPHP genes, currently known to account for 60% of cases. It remains clear that given the high genetic heterogeneity of NPHP and the many mechanistic pathways discussed, there is no one unifying pathology leading to NPHP. Renal histology of NPHP points to a common endpoint of tubular injury and fibrosis that may have multiple triggers. With each new gene discovery paper published, there seems to be a better understanding of the molecular diagnosis, but more confusion regarding the signaling pathways that the disease is based on.

[0008] There remains a great need to characterize the poorly understood molecular basis of diseases with ciliopathies, including NPHP, and to improve the diagnosis and treatment of these diseases. Summary of the invention

[0009] In one embodiment, the present disclosure relates to a method for treating at least one ciliopathy-associated disease in a subject, comprising administering to the subject a therapeutically effective amount of at least one agent targeting at least one G protein-coupled receptor (GPCR). In embodiments, the ciliopathy-associated disease is caused by a homozygous deletion of the NPHP1 locus. In embodiments, the ciliopathy-associated disease is caused by a heterozygous deletion of the NPHP1 locus and a heterozygous or homozygous loss of function (LOF) at a second locus. In embodiments, the ciliopathy-associated disease is caused by a heterozygous deletion in one allele of NPHP1 and a LOF mutation in the second allele. In embodiments, the ciliopathy-associated disease is caused by a loss-of-function mutation in one allele of NPHP1 and a different loss-of-function mutation in the second allele.

[0010] In a specific embodiment, the at least one agent is an agonist of at least one GPCR. In a specific embodiment, the at least one agent is a prostaglandin. In a specific embodiment, 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-210669, 18a, AGN-210961, DE-117, CP-533536 and combinations thereof. In a specific embodiment, the at least one GPCR is selected from the group consisting of: EP1, EP2, EP3 and EP4. In a specific embodiment, the at least one disease is selected from the group consisting of: renal wasting (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders disease (JSRD) (which may include all variant forms of JBTS with additional features such as polydactyly, coloboma, retinal dystrophy, renal cysts, oral frenulae, and liver fibrosis), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS), orofacial digital syndrome (OFD), end-stage renal disease driven by large homozygous deletions of NPHP1, and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations, and any ciliopathies driven by NPHP genes. In a specific embodiment, the at least one agent is CP-544326, and the at least one GPCR is EP2. In a specific embodiment, the effective amount is between 100 pM and 5 μM. In a specific embodiment, the at least one disease is renal wasting.

[0011] In one embodiment, the present disclosure relates to a method for identifying a therapeutic agent for treating at least one ciliopathy-related disease, the method comprising: (a) administering a test agent to an animal or cell model of a ciliopathy-related disease, wherein the animal or cell model exhibits a measurable phenotype of the ciliopathy-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 a ciliopathy-related disease when the measurable phenotype of the treated animal or cell model is improved compared to the measurable phenotype of the untreated animal or cell model. In a specific embodiment, the animal model can be a Danio rerio (zebrafish) or nphp1 knockout (KO) mouse model (nphp1- / -). In a specific embodiment, the animal model is generated by administering one or more disruptive agents. In a specific embodiment, the one or more disruptive agents include morpholinos. In a specific embodiment, the morpholinos inhibit the expression of at least one nephrocystin (NPHP), such as NPHP4. In a specific embodiment, the measurable phenotype is selected from the group consisting of: body curvature, pronephric cysts, lateralized heart defects, and cloacal dilation. In a specific embodiment, the at least one disease is selected from the group consisting of: renal wasting (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 driven by large homozygous deletions of NPHP1, and renal and retinal ciliopathies associated with mutations in NPHP1, NPHP4, NPHP6 / CEP290.

[0012] In one embodiment, the disclosure relates to a GPCR agonist for use in the treatment of at least one ciliary disease-related disease. In a specific embodiment, 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-210669, 18a, AGN-210961, DE-117, CP-533536 and combinations thereof. In a specific embodiment, the GPCR is selected from the group consisting of: EP1, EP2, EP3 and EP4. In a specific embodiment, the at least one disease is selected from the group consisting of: renal wasting disease (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 driven by large homozygous deletions of NPHP1, and renal and retinal ciliopathies associated with mutations in NPHP1, NPHP4, NPHP6 / CEP290.

[0013] In a specific embodiment, the animal model is produced by administering one or more destructive agents. In a specific embodiment, the one or more destructive agents include a CRISPR / Cas9 system that mediates sgRNA-directed genetic deletion. In a specific embodiment, the CRISPR / Cas9 system inhibits the expression of at least one renal cysteine ​​(NPHP), such as NPHP1. In a specific embodiment, the measurable phenotype is selected from the group consisting of: retinal photoreceptor layer thickness, electroretinogram, and rhodopsin accumulation in photoreceptor cell bodies. In a specific embodiment, the at least one disease is selected from the group consisting of: renal depletion (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS), oral-facial-finger syndrome (OFD), end-stage renal disease driven by large homozygous deletion of NPHP1, and renal and retinal cilia disease associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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, wherein like reference numerals represent like elements.

[0015] Figures 1A-1DUrine-derived renal epithelial cells are shown; 1A: normal control; 1B: NPHP patient carrying NPHP1 deletion (Pt1); 1C: RT-PCR comparison; 1D: immunoblot comparison.

[0016] Figure 2 An automated in vitro assay for quantifying ciliogenesis in cells of interest is shown.

[0017] Figure 3 It was shown that the percentage of ciliated cells (PT1) from NPHP patients was significantly lower than that of control cells (CTRL).

[0018] Figure 4 A schematic diagram showing the steps of the novel cilium-based assay.

[0019] Figure 5 Shown are the effects of the following on ciliogenesis compared to DMSO: (A) fluticasone, (B) pheniramine, (C) verapamil, (D) ML-141, (E) mitoxantrone, (F) tropisetron, (G) epropamine, (H) cyproheptadine, (I) paclitaxel, and (J) simvastatin.

[0020] Figure 6 The effect of alprostadil on ciliogenesis compared to DMSO is shown.

[0021] Fig. 7A Shown is the alprostadil dose response compared to DMSO on ciliogenesis.

[0022] Figure 7B The corresponding semi-logarithmic representation used to determine the IC50 is shown.

[0023] Figures 8A-8C Shown are meta-analyses of results obtained from multiple ciliogenesis experiments following treatment with alprostadil.

[0024] Fig. 9 Shown (AD) is a meta-analysis of results obtained from multiple ciliogenesis experiments after treatment with alprostadil, distinguishing the data from each experiment.

[0025] Fig.10 The stability of PGE1 under the experimental conditions is shown.

[0026] Fig.11A Shown are the effects of alprostadil (PGE1), dinoprostone (PGE2), and 16,16-dimethyl-PGE2 (dmPGE2) on ciliogenesis.

[0027] Fig. 11BShown is the effect of alprostadil (PGE1) on NPHP1 -deficient patient-derived cell lines.

[0028] Fig. 11C The ciliary meta-analysis is shown.

[0029] Fig.12 The effect of PGE2 on ciliogenesis is shown.

[0030] Fig.13 Shown are the expression profiles of EP1-4 in human kidney tissue obtained by Western blotting and in human retina obtained by immunohistochemistry.

[0031] Fig.14A It was shown that EP2 and EP4 mRNA were expressed in control and Pt1-derived renal epithelial cells.

[0032] Fig. 14B It was shown that EP2 was expressed at the protein level in control and Pt1-derived renal epithelial cells.

[0033] Fig. 14C Shown is the mRNA expression of EP1-4 receptor encoding genes in various control cell lines and various NPHP patient-derived renal epithelial cell lines.

[0034] Fig.15 Prostaglandin (PG) modulators (agonists and antagonists) tested for effects on ciliogenesis are shown.

[0035] Fig.16A The ciliary meta-analysis is shown.

[0036] Fig. 16B Shown are NPHP patient-derived cells treated with CP-544326.

[0037] Fig. 16C The corresponding semi-logarithmic representation is shown.

[0038] Fig.17A Shown is the effect of L-902.688 on ciliogenesis.

[0039] Fig. 17B The effects of CP-544326 and alprostadil on ciliogenesis are shown.

[0040] Fig. 17C Shown are the effects of CP-544326 on patient-derived cells.

[0041] Fig.17D The ciliary meta-analysis is shown.

[0042] Fig.18RNA extracted by RLT or Qiazol method for microarray analysis is shown.

[0043] Fig.19 Microarray data of samples analyzed by hierarchical clustering are shown.

[0044] Fig. 20 Microarray data of samples analyzed by hierarchical clustering are shown.

[0045] Fig.21 Microarray data of samples analyzed by hierarchical clustering are shown.

[0046] Fig. 22 Microarray data obtained from RLT extraction samples are summarized.

[0047] Fig.23 Microarray data obtained from Qiazol-extracted samples are summarized.

[0048] Fig.24A and 24B It showed that there were no significant differences between the microarray data obtained from various doses.

[0049] Fig.25 The process of multi-omic analysis of drug effects on ciliogenesis is shown.

[0050] Fig.26 Shown are (AE) phenotypic analyses of the effects of alprostadil on ciliogenesis.

[0051] Fig. 27 Differential expression of imRNAs of drug-targeted and drug-targetable genes is shown.

[0052] Fig.28A -C shows pathway analysis based on multi-omics data and relevant target opportunities for (A) prostaglandin E1 (alprostadil) downstream interactions, (B) NPHP1 upstream interactions, and (C) NPHP1-20 gene-related direct interactions.

[0053] Fig.29 The zebrafish NPHP4 MO model is shown.

[0054] Fig.30 A scheme of drug treatment in the zebrafish NPHP4 MO model is shown.

[0055] Fig.31 (AC) Summarizes the effects of morpholino injections in zebrafish.

[0056] Fig.32Shown are (A) representative body axis bending of zebrafish; and (B, C) the effects of alprostadil on body axis bending of zebrafish.

[0057] Fig.33 Shown are (A) a representative pronephric cyst of zebrafish; and (B, C) the effects of alprostadil on pronephric cyst of zebrafish.

[0058] Fig.34 Shown are (A, B) the effect of dinoprostone on the body axis bending of zebrafish; and (C) the effect of dinoprostone on pronephric cysts in zebrafish.

[0059] Fig.35 Shown is the effect of CP-544326 on pronephric cysts in zebrafish.

[0060] Fig.36 The pharmacokinetic study design is shown.

[0061] Figures 37A-37E Results of pharmacokinetic studies are shown.

[0062] Fig.38A Figure 2 shows the relationship between wt mice and Nphp1 - / - Periodic acid-Schiff staining of retina in mouse.

[0063] Fig.38B A semi-automated quantification method for retinal layer thickness is shown.

[0064] Fig.38C Compared with wt mice, Nphp1 - / - Quantification of retinal layer thickness in mice.

[0065] Fig.39A and 39B Figure 3 shows the expression of Cep290 as a ciliary marker and rhodopsin and PNA (peanut agglutinin) as photoreceptor markers for the outer segment (OS) and inner / outer segments, respectively, in wt mice and Nphp1 mice. - / - Immunohistochemical staining of mouse retina.

[0066] Fig.40A , Fig.40B and Fig.40C Compared with wt mice, Nphp1 - / - Electroretinogram in mice.

[0067] Fig.41 The EP2 receptor is expressed in wt mice and Nphp1 - / - Expression in mice.

[0068] Fig.42 A study design according to one embodiment of the present disclosure is shown.

[0069] Fig.43 The effect of CP-544326 on Nphp1 - / - Effect of ONL / OPL retinal layer thickness ratio in mice.

[0070] Fig.44 The effect of CP-544326 on Nphp1 - / - Effects of mislocalization of green-labeled rhodopsin in the ONL of mice.

[0071] Fig.45 The effect of CP-544326 on Nphp1 - / - Effects on the electroretinogram in mice. DETAILED DESCRIPTION

[0072] It should be understood that the present disclosure is not limited to the specific embodiments described below, as changes may be made to the specific embodiments and these changes still fall within the scope of the appended claims. It should also be understood that the terminology employed is for the purpose of describing specific embodiments and is not intended to be limiting.

[0073] In this specification and the appended claims, unless the context clearly indicates otherwise, "a / an" and "the" include plural referents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0074] NPHP patients

[0075] Renal wasting (NPHP) is a hidden tubulointerstitial ciliary disease characterized by progressive destruction of the kidney, leading to end-stage renal disease (ESRD). The range of onset of ESRD driven by NPHP is from the first month of birth (infant NPHP) to >60 years old (adult NPHP), of which >17% cases suffer from ESRD after 20 years old. Pathogenic mutations have been identified in more than 20 NPHP-related genes (e.g., NPHP1–20, IFT140, TRAF3IP1 / IFT54), accounting for about 60% of all cases with NPHP. The full locus deletion of NPHP1 (NPHP1 (del)) accounts for more than 20% of NPHP cases. Traditionally, the global incidence frequency reported by the rare disease portal Orphanet is approximately 1 / 100,000 (1 / 50,000 in Canada, 1 / 900,000 in the United States, 1 / 100,000 in Finland, and 1 / 50,000 in France). There is currently no treatment for NPHP.

[0076] Ciliopathies are often caused by mutations in genes encoding transition zone (TZ) proteins or intraflagellar 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 that controls the entry and exit 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, the TZ consists of different multiprotein complexes, namely 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.

[0077] Mutations and / or inactivation in one or more genes encoding NPHP module proteins may adversely affect ciliogenesis and / or epithelialization, leading to fibrosis and cyst development in NPHP patients. The IFT machine selects cargo at the base of the cilia and transports the axoneme components required for ciliary assembly and proteins involved in ciliary signaling. The IFT-B complex, composed of 16 different proteins, mediates anterograde transport by associating with kinesin II. Retrograde transport is mediated by 6 subunits of dynein 2 and IFT-A complexes. Mutations in 6 genes encoding IFT-A subunits have been identified in NPHP-related ciliopathies, and only 3 IFT-B subunits are associated with renal depletion (IFT172, IFT54) (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, appendage proteins and GPCRs are also essential factors for ciliary function and maintenance.

[0078] Regarding the NPHP module, Nphp4 mutant mice develop retinal degeneration, but do not develop renal cysts or severe ciliogenesis defects; males are infertile and present sperm with reduced motility (Won, J. et al., Hum. Mol. Genet., 20:482-96, 2011). Similarly, targeted disruption of Nphp1 in mice (deletion of the last C-terminal exon 20) does not produce renal depletion, but exhibits rapid retinal degeneration starting at P14-P21 (Jiang, S. et al., Hum. Mol. Genet., 17:3368-79, 2008) and causes male infertility (Jiang, S. et al., Hum. Mol. Genet., 18:1566-77, 2009). Cep290 knockout mice lack connecting cilia in photoreceptors and fail to mature motor ependymal cilia, consistent with their retinal degeneration and hydrocephalus phenotype (Rachel, R. et al., Hum. Mol. Genet., 24:3775-91, 2015).

[0079] Mutations in NPHP1 are the most common cause of NPHP. In a large group of adult-onset ESRD patients (not selected for etiology), NPHP caused by homozygous whole gene deletion of NPHP1 (NPHP (del)) has a prevalence of 1 / 200 patients (0.5%) in all adult-onset ESRD (Snoek, R. et al., J. Am. Soc. Nephrol., 29: 772-9, 2018). Although the incidence is significantly higher in ESRD-onset patients between the ages of 18 and 50 (prevalence of 0.9%), the age of onset of NPHP can be as high as 61 years old. 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 that may not be fully diagnosed in current daily practice.

[0080] In the cohort of renal transplant recipients and (corresponding donors) controls from the International Genetics and Translational Research in Transplantation Network (iGenTRAiN) Alliance in the transplantation network, homozygous NPHP1 deletion patients with a relative frequency of about 0.5% (26 in 5606) were identified in ESRD (18 to 50 years old) adults. Among these, only 13% (3 in 26) were correctly diagnosed as NPHP, and about half (11 in 26%) were diagnosed as CKD patients of unknown etiology. These results show that up to 1 / 200 (0.5%) of ESRD adults are NPHP1del genotypes; when the age of ESRD onset is between 18 and 50 years old, this number increases to 0.9% (Abstract.ASN2017&Nephr Dial Trans, Vol. 32, 2017).

[0081] This article describes discoveries generated using the Genomics England Research Environment, a secure workspace for approved researchers to conduct research on the 100,000 Genomes Project dataset, with the goal of identifying new diseases and patient-relevant insights to enable scientific discoveries and accelerate their translation into patient care. The 100,000 Genomes Project dataset includes patients with rare diseases (and their relatives) as well as cancer patients. Within this dataset, homozygous NPHP1(del) patients were identified at a relative frequency of approximately 1 in 6,000 (10 out of 61,554) – none of whom had been previously diagnosed with NPHP. Of the 10 identified patients, 7 had clear clinical signs / symptoms of NPHP, such as renal or ciliopathy signs / symptoms, or were recruited as patients with congenital anomalies of the kidney and urinary tract (CAKUT). The remaining 3 patients had a more complex clinical presentation – likely with multiple rare diseases. In addition to homozygotes, 193 NPHPI (del) heterozygous patients were identified in the full data set (with an approximate frequency of 1 / 200 in this data set); these patients may be heterozygous carriers, but may also include NPHP1 compound heterozygotes (NPHP1 (del) with NPHP1 loss-of-function (LOF) mutations) and / or epistasis (NPHP1 (del) combined with LOF mutations at another locus). In addition, patients may have additional NPHP1-LOF variants, like splice-variants, frameshifts, and nonsense mutations, which may also lead to clinical NPHP manifestations.

[0082] NPHP (del) described herein is found to be the result of the research conducted using the UK Genomics database. This research is achieved by accessing the data and findings produced by the UK Genomics Research Environment, the patients who agree to use their data for research purposes, and the NHS clinicians and medical teams who contribute to the data and results covered by this research. The UK Genomics Research Environment is managed by UK Genomics Ltd. (a wholly owned company of the Ministry of Health), and is funded by the National Institute for Health Research and the UK National Health Insurance System (National Institute for Health Research and NHSEngland), the Wellcome Trust (The Wellcome Trust), UK Cancer Research and the Medical Research Council (CancerResearch UK and the Medical Research Council).

[0083] Millions of individuals worldwide suffer from ESRD and congenital diseases for which the only treatment is transplantation. In the United States alone, more than 600,000 transplants have been performed in the past 50 years, and the need is now higher than ever. Unfortunately, the availability of donor organs has not kept pace 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-driven ESRD) and / or NPHP-related ciliopathies (e.g., NPHP1).

[0084] NPHP patient-derived cells

[0085] Described herein are materials and methods for identifying therapeutic agents useful for treating ciliopathy-related diseases or conditions, such as NPHP or NPHP1(del)-related diseases or conditions. Such methods may include the use of patient-derived cell lines. Such cell lines as developed may also be used in other related methods, including, for example, monitoring the efficacy of a given treatment for a ciliopathy-related disease or condition or a NPHP1(del)-related disease or condition.

[0086] In order to identify compounds for the treatment of diseases associated with ciliopathies (such as, for example, NPHP), NPHP patient-derived cells were obtained and cell lines were established. Briefly, exfoliated renal epithelial cells, primarily proximal tubular cells (tbc) recovered from urine of NPHP1-deficient patients, were immortalized by retroviral gene transfer of the SV40 T antigen. The cells were fixed and fluorescently labeled with Hoechst (for nuclear staining), anti-γ tubulin antibody (for basal body staining), and anti-ARL13B antibody (for cilia staining) for detection using immunofluorescence microscopy. Figure 1A In contrast, most NPHP patient-derived cells lack cilia ( Figure 1B ). Further RT-PCR ( Figure 1C ) and immunoblotting ( Figure 1D ) confirmed the lack of NPHP expression in these NPHP patient-derived cells, with these two methods failing to show detectable levels of NPHP RNA and NPHP protein expression in NPHP patient-derived cells, respectively.

[0087] Figure 2 An automated in vitro assay that can be used to quantify ciliogenesis in cells of interest is shown. Briefly, NPHP patient-derived cells and control cells are cultured in complete medium at 39C (a non-permissive temperature for SV40 expression), followed by automated ciliary analysis using immunofluorescence microscopy to measure ciliogenesis, e.g., as % cilia. Drug screening can be performed using a spinning disk platform ( Figure 5 , AJ) and analysis of ciliogenesis in G3 multi-omics datasets ( Fig.29 , AE). Other phenotypic analyses can be performed using the Opera Phenix platform (e.g., alprostadil and CP-544326 ciliogenesis titration, ciliogenesis-based screening of other EP agonists, ciliogenesis using other NPHP1 patient-derived cell lines, alpha-tubulin acetylation analysis).

[0088] Figure 3 It was shown that the percentage of ciliated cells from NPHP patients (PT1) was significantly lower than that found in control cells (CTRL) (p=0.0065).

[0089] Drug Screening

[0090] The ciliary-based assays described above can be used to identify compounds that restore ciliogenesis. Figure 4 The process of a cilium-based assay is shown, where cells can be plated in cell culture (e.g., 96-well plates) on day 0, incubated with drug candidates on day 3, and fixed and fluorescently labeled with Hoechst, anti-γ tubulin antibodies, and anti-ARL13B antibodies on, for example, day 5. For example, an automated random acquisition of 35 images / well can be performed. Each image can have a z-stack of 10 images taken at <1 μm intervals. Continuous imaging of nuclei (Hoechst, at 461 nm), basal bodies (γ tubulin, at 555 nm), and cilia (ARL13b, at 647 nm) can be obtained.

[0091] use Figure 4 In the process shown in , NPHP patient-derived cells were treated with several drug candidates to identify drugs that could restore ciliogenesis. Figure 5 (Panels AJ) show that fluticasone, pheniramine, verapamil, ML-141, mitoxantrone, tropisetron, epromezine, cyproheptadine, paclitaxel, and simvastatin had no significant effect on ciliogenesis at the various concentrations tested compared to DMSO. Figure 6 It was shown that alprostadil significantly restored ciliogenesis in NPHP patient-derived cells by increasing the percentage of ciliated cells compared with DMSO.

[0092] Alprostadil, also known as prostaglandin E1 (PGE1), has the chemical structure

[0093]

[0094] It exhibits vasodilation, inhibition of platelet aggregation, and stimulation of intestinal and uterine smooth muscle activity for the treatment of heart disease and erectile dysfunction. Alprostadil may act as an agonist by binding to the E-type prostaglandin (EP) receptor as 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 and subsequently increases in intracellular cAMP.

[0095] As used herein, "GPCR agonists" include compositions that activate GPCRs to mimic the effects of endogenous signaling molecules specific for this receptor. "GPCR antagonists" include compositions that inhibit GPCR activity. GPCR activity can be measured by the ability to bind effector signaling molecules (such as G proteins). An "activated GPCR" is an activated GPCR that is able to interact with and activate a G protein. The ability of an inhibited receptor to bind extracellular ligands and / or to effectively interact with and activate a G protein may be reduced.

[0096] For example, GPCR agonist treatment with taperenepag isopropyl can be performed at, 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, about 8 mg / kg to about 20 mg / kg. / kg to about 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, 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, once every 2 weeks, once every 3 weeks, or once a month.

[0097] To determine the effective concentration of alprostadil for restoring ciliogenesis, 1 nM to 2 μM ( Fig. 7A ) and 100pM to 2μM ( Fig. 7A) of alprostadil titer. The effective concentration of a GPCR agonist, 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. Figure 7B The corresponding semi-logarithmic representation used to determine the IC50 is shown, indicating that alprostadil significantly increased the % ciliated cells in NPHP patient-derived cells in a dose-dependent manner.

[0098] Figures 8A-8C and Fig. 9 (Panels AD) show meta-analysis indicating that alprostadil treatment (2 μM) did not significantly affect ciliogenesis in control normal epithelial cells (CTRL) compared to control (DMSO 0.04%). Fig. 8A In contrast, alprostadil treatment significantly increased ciliogenesis in NPHP patient-derived cells (PT1) compared with control (DMSO 0.04%) ( Figure 8B ). Figure 8C It is shown that the effect of alprostadil on ciliogenesis in NPHP patient-derived cells was increased approximately two-fold relative to control cells not receiving alprostadil treatment, ie, control (DMSO 0.04%).

[0099] The meta-analysis also showed that alprostadil dose had an approximately linear effect on ciliogenesis. Fig. 9 (Panels A and B) show, for example, the R for the effect of alprostadil on ciliogenesis in control normal epithelial cells. 2 The value is 0.9194. Similarly, Fig. 9 (Panels C and D) show the R for the effect of alprostadil on ciliogenesis in NPHP patient-derived cells. 2 The value is 0.8489.

[0100] To determine the stability of alprostadil (PGE1), supernatants were obtained from urine-derived renal epithelial cells (URECs) after exposure to different concentrations of alprostadil for 24 and 48 hours. The samples were then extracted and aliquoted for analysis on LC / MS / MS and polar LC platforms. Fig.10 It was shown that PGE1 is stable under the experimental conditions.

[0101] In addition to PGE1, other EP agonists have been tested, such as prostaglandin E2 (PGE2 or dinoprostone) which has the following chemical structure:

[0102]

[0103] and its long-acting derivative, 16,16-dimethyl-PGE2 (dmPGE2)-, which has the following chemical structure:

[0104]

[0105] Restores the ability of ciliogenesis. Fig.11A PGE2 and dmPGE2 showed that PGE2 and dmPGE2 had a ciliogenesis restoration effect similar to alprostadil in NPHP patient-derived cells, while no significant effect was observed in control normal cells. A slight decrease in ciliogenesis restoration was observed in NPHP patient-derived cells at the highest concentration (40 μM dinoprostone and 20 μM dmPGE2), which may be due to cytotoxicity.

[0106] To test the effect of alprostadil (PGE1) on NPHP1-deficient cells, cell lines derived from NPHPI(del) patients, such as 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. Fig. 11B It was shown that alprostadil significantly increased the ciliogenesis rate in NPHP1-deficient cells, whereas alprostadil had no significant effect on the ciliogenesis rate in normal control cells, indicating that alprostadil is effective in restoring ciliogenesis in NPHP1-deficient patients.

[0107] Fig. 11C Meta-analysis in shows linear regression analysis of previous data, wherein slope reflects the influence of alprostadil on the ciliogenesis of control cells and cell lines derived from multiple NPHP patients, each symbol represents independent experiment and each color represents patient cell line (named as 1-09-P L4, 1-06-P1, 1-06-P2, PT-1). Linear regression of control normal epithelial cell data shows that slope value is between 0,7665 and 0,9974, indicating that alprostadil has no influence on ciliogenesis. In contrast, linear regression of cells derived from multiple NPHP patients shows that the pooled slope value is 1.414, or the slope value range is 1.333 to 1.506, indicating that alprostadil has an influence on the stimulation of ciliogenesis.

[0108] Prostaglandins are present in most human tissues, and are synthesized by essential fatty acids. The structural differences between various prostaglandins are the reasons for different biological activities. Prostanoids including prostaglandins (Prostanoid) are produced abundantly in kidney. Prostaglandins originate from the arachidonic acid (AA) released from membrane phospholipids by phospholipase A2. Arachidonic acid is subjected to the dioxygenase and peroxidase activity of cyclooxygenase (or prostaglandin G / H synthetase) to form prostaglandin G2 (PGG2), and then forms prostaglandin H2 (PGH2). PGH2 is the substrate of synthetase (comprising PGE2 synthetase, PGD2 synthetase, prostaglandin synthetase, PGF2α synthetase (PGF2α can also be directly synthesized by PGE2) and thromboxane synthetase), for the synthesis of the prostaglandins including PGE2 of each classification. These classifications all have the discrete receptor subtypes including EP1-4, and they start their effects by the receptor subtypes. Cyclooxygenases 1 and 2 (COX1 and COX2) are the primary targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but these drugs may be specific for one isoform or the other, selective or nonselective. Blocking PGH2 production by COX inhibition can reduce the levels of all downstream prostanoids.

[0109] PGE in ciliogenesis

[0110] PGE2 is the best characterized prostanoid in renal pathophysiology. PGE2 is synthesized by COX1 and COX2 and exported through the Lkt / ABCC4 transporter on the cell membrane. The released PGE2 binds to the EP4 receptor on the cilium, leading to the activation of GPCR (G) and adenylate cyclase (AC) to increase cAMP, thereby increasing anterograde IFT and enhancing ciliogenesis.

[0111] The formation and elongation of cilia require the COX-Lkt / ABCC4-EP4 signaling cascade (in mouse renal collecting duct cells IMCD3 and in 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 leads to an increase in intracellular cAMP during ciliogenesis in IMCD3 cells, but Ca 2+ Do not increase. PGE2 works in an autocrine and / or paracrine manner, because cells can respond to the PGE2 released by themselves or their surroundings. In human cancer cells, the interaction of PGE2 and the EP4 receptor induces Wnt / p-catenin signal transduction, causing COX2 to express, and thereby setting up a positive feedback loop that causes further PGE2 synthesis.

[0112] Fig.12 It was shown that addition of exogenous PGE2 increased ciliary length and the percentage of ciliated cells in control cells but not in EP4-deficient cells, indicating that EP4 functions downstream of PGE2 signaling during ciliogenesis.

[0113] PGE2 is produced by PGE synthase (PGES) and conducts signal transduction by binding to its GPCRs: EP1-4. q Activation of the PLC increases intracellular Ca 2+ Activation of EP3 (coupled to Gi) increases intracellular Ca via PLC 2+ , and / or inhibit cAMP production via adenylate cyclase (AC). EP2 or EP4 (both with G s Activation of the mitochondria (mitochondria) stimulates cAMP production through AC.

[0114] There are approximately 800 human GPCRs, divided into five major phylogenetic families: rhodopsins, secretins, adhesions, glutamate, and Frizzled / Taste2. GPCRS are attractive targets for recombinant proteins, small molecule compounds, allosteric ligands, or antibodies. 46 GPCRs have served as drug targets for hypertension, pain, ulcers, allergies, alcoholism, obesity, glaucoma, mental disorders, and HIV. Among many obstacles, a major obstacle is the general lack of knowledge about the association of putative GPCRs with accurate physiological functions or disease conditions.

[0115] Fig.13 EP1-4 are shown to be expressed in the kidney and retina - both organs are affected by NPHP and NPHP-RC. In the kidney, EP receptors are differentially expressed along the nephron, highlighting the different functional consequences of activating each EP receptor subtype in the kidney. EP receptors regulate vascular tone in the afferent arterioles, with EP1 / EP3 acting as vasoconstrictors and EP2 / EP4 acting as vasodilators. EP1 / EP4 regulate proximal tubular transport. EP3 and EP4 regulate the thick ascending limb and distal tubular transport. EP4 stimulates the release of renin from the macula densa. EP2 / EP4 causes vasodilation of the vasodilata. EP receptors regulate collecting duct transport, whereby EP1 inhibits Na + EP3 inhibits H2O reabsorption, and EP4 stimulates H2O reabsorption.

[0116] The expression of PG pathway components including EP receptors in URECS was determined by qRT-PCR. Fig.14A It was shown that EP2 and EP4 were expressed at the mRNA level, and EP2 was mainly expressed at the imRNA level. Fig. 14BEP2 protein expression in URECS is shown.

[0117] PGE2 modulators (EP2)

[0118] Selective agonists and antagonists of EP2 receptors 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 consists of ligands that are structurally similar to the endogenous ligand PGE2, but have major modifications incorporated into the ω-lipophilic chain that promote enhanced efficacy and selectivity. The second class of agonists is a pyridylsulfonamide derivative of the non-prostanoid series, the most potent of which is taprenepag isopropyl (PF04217329, a prodrug of CP 544326). Taprenepag has the following non-prostanoid structure

[0119]

[0120] The third class of agonists includes non-prostaglandin series of N-phenyl-gamma-lactam derivatives, including AGN-210669 and AGN-210961.

[0121] PF-04418948 is an azetidine-3-carboxylic acid derivative and the first selective EP2 antagonist with an IC50 of 16 nM (Kb=1.8 nM). Its selectivity for EP2 receptors relative to other prostanoid receptors is increased by >10,000-fold.

[0122] Markovic Figure 5 (which is incorporated by reference) shows selective agonists of the EP4 receptor: (a) derivatives based on functionalized cyclopentane cores, (b) derivatives of lactam counterparts carrying hydroxycyclopentanone cores, and (c) structurally different EP4 agonists. The tetrazole feature is introduced into the a-chain to replace the terminal carboxylic acid functional group, with the intention of improving bioavailability, which led to the discovery of L902,688, a sub-nanomolar agonist of the EP4 receptor (EC50=0.2nM). L902,688 has the following prostanoid structure:

[0123]

[0124] Structure of KAG-308, a low nanomolar EP4 agonist

[0125]

[0126] It is somewhat unique in the field of EP4 agonists as it is the only one based on the 7,7-difluoroprostacyclin scaffold.

[0127] Markovic Figure 6 (which is incorporated by reference) shows selective antagonists of the EP4 receptor and switching of functional responses due to minor structural changes: (a) selective antagonists of the EP4 receptor, and (b) switching of agonism and antagonism at the EP4 receptor. PG-1531 is a trisubstituted furan derivative that is a nanomolar EP4 antagonist with an excellent selectivity spectrum and enhanced water solubility. By introducing minor modifications to the molecule, the intrinsic activity of the molecule can be fine-tuned at the EP4 receptor (an example is shown in Figure 17). For example, it has been demonstrated that the intrinsic activity (agonism versus antagonism) depends solely on the substitution pattern of the trifluoromethyl substituents on the benzyl group of the compounds of Figure 17 (group b). Great functional changes can be achieved by minor changes in the ligand structure.

[0128] Fig.15 PG modulators (agonists and antagonists) tested for effects on ciliogenesis are shown.

[0129] Fig.16A showed that CP-544326, a non-prostanoid EP2 agonist, restored ciliogenesis to levels similar to alprostadil. Fig. 16B It was shown that CP-544326 restored ciliogenesis in a dose-dependent manner compared with DMSO. Fig. 16C yes Fig. 16B The semi-logarithmic representation of the results, where CP-544326 titration indicated an EC50 = 11 nM for EP2. Restoration of ciliogenesis by non-prostanoid CP-544326 confirmed the specificity of its mechanism of action. In contrast, Fig.17A L-902.688, a prostanoid EP4 agonist, did not significantly affect ciliogenesis. These results indicate that the role of EP2 in ciliogenesis is more important than that of EP4.

[0130] Fig. 17C It was shown that, similar to alprostadil, CP-544326 treatment increased ciliogenesis in various NPHPI(del) patient-derived cell lines, such as 1-09-P, 1-06-P1, and 1-06-P2, compared to treatment with DMSO. Fig.17D The meta-analysis in Fig.17DLinear regression analysis of CP-544326, wherein the slope reflects the effect of CP-544326 on ciliogenesis of control cells and multiple NPHP patient-derived cell lines, each symbol represents an independent experiment and each color represents a patient cell line (named 1-09-P L4, 1-06-P1, 1-06-P2, PT-1). Linear regression of control normal epithelial cell data showed slope values ​​between 0.6369 and 1.03, indicating that CP-544326 does not affect ciliogenesis. In contrast, linear regression of multiple NPHP patient-derived cells showed a pooled slope value of 1.36, or a slope value range of 1.245 to 1.532, indicating the stimulatory effect of alprostadil on ciliogenesis.

[0131] Differential display analysis

[0132] Microarray analysis was performed to identify the expressed genes responsible for alprostadil-mediated restoration of ciliogenesis. URECs were cultured in 96-well plates and treated with different concentrations of alprostadil, followed by RNA extraction using either the RLT or Qiazol method, as described in Fig.18 As summarized in .

[0133] Fig.19 The microarray data of the sample by hierarchical cluster analysis are shown.First, according to extraction type (Qiazol and RLT), data are clustered.Then according to condition (for example, comparison and alprostadil processing), Qiazol samples are clustered, and then by repetition, RLT samples are clustered.

[0134] Fig. 20 Microarray data for samples analyzed by hierarchical clustering are shown. Data obtained from Qiazol extracted samples were clustered by condition and then by replicate, but not by dose within treatment or media / DMSO within control.

[0135] Fig.21 Microarray data for samples analyzed by hierarchical clustering are shown. Data obtained from RLT extraction samples were clustered by replicates and then by condition (control vs. alprostadil treatment), rather than by dose within treatment or media / DMSO within control.

[0136] For the microarray data obtained from RLT extraction samples, there were no significant differences between DMSO and medium, for example, only four differentially expressed genes and no regulated exons / patterns. Fig. 22Shown is a comparison of control (DMSO) and alprostadil treatment (0.2 μM, 2 μM and 10 μM), in the comparison of three alprostadil concentrations, the number of genes expressed and regulated is almost the same. The top three regulated genes are also almost the same and share the same signaling pathways, such as cell adhesion and downregulation of the extracellular matrix.

[0137] For the microarray data obtained from Qiazol extracted samples, there were no significant differences between DMSO and medium, for example, 33 differentially expressed genes had no regulated exons / patterns. Fig.26 As shown, the number of expressed and regulated genes was almost the same in the three alprostadil concentration comparisons comparing the control (DMSO) to alprostadil treatment (0.2 μM, 2 μM and 10 μM). The top three regulated genes were also almost the same and shared the same signaling pathways, such as downregulation of cell adhesion and extracellular matrix and upregulation of interferon signaling.

[0138] also, Fig.24A and 24B It shows that two clusters were defined, bringing together a total of 310 genes, ie, "Cluster 1" = 120 down-regulated genes, and "Cluster 2" = 190 up-regulated genes. This indicates that no significant differences were detected between the microarray data obtained from the various doses.

[0139] Further, pathway analysis performed by crossing microarray data from patients treated or not with alprostadil and crossing this data with RNAseq microarray data from controls and patients revealed that alprostadil could reverse the changes in gene expression observed in NPHP patient-derived cells compared to control cells.

[0140] Multi-omics analysis

[0141] Fig.25 The process of multi-omic analysis of drug effects on ciliogenesis is shown. Fig.26 A-26E shows phenotypic analysis, for example, regarding the effect of alprostadil on ciliogenesis, for example % ciliated cells in five independent experiments. These results show that alprostadil partially restored ciliogenesis in n=1-5 with similar fold ratios and no dose-dependent response.

[0142] Fig. 27 Shown is a summary of drug-targeted and drug-targetable genes identified from protein differential expression analysis of multi-omics data (NPHP patient-derived cells in DMSO 0.04% vs. NPHP patient-derived cells treated with alprostadil 2 μM), according to which drug-targeted genes are named.

[0143] Figure 28 (AC) shows pathway analysis (using Ingenuity Pathway Analysis) from multi-omics data, and relevant target opportunities for (A) prostaglandin E1 (alprostadil) downstream interactions, (B) NPHP1 upstream interactions, and (C) NPHP1-20 gene-related direct interactions.

[0144] In vivo models

[0145] Fig.29 Results from a zebrafish NPHP4 morpholino (MO) model are shown, in which wild-type zebrafish embryos at the one-cell stage are injected with a morpholino (e.g., NPHP4 ATG MO) that blocks the start site of NPHP4 mRNA for ribosome binding. The morpholino specifically inhibits the translation of NPHP4 mRNA. The zebrafish NPHP4 MO exhibits classic ciliopathy-related phenotypes, including body curvature, pronephric cysts, laterality (cardiac ringing) defects, and cloacal dilation (blockage).

[0146] Fig.30 It is a schematic diagram showing the scheme of drug treatment (alprostadil: 0.5 μM and 5 μM) in zebrafish NPHP4 MO model. In short, morpholino (e.g., nphp4 ATG MO) is injected into wild-type Tg (wt1b: GFP) transgenic zebrafish embryos at the single-cell stage. 8 hours after fertilization (8hpf), the injected embryos are treated with PTU-egg water (1mL, in 12-well plates) containing drugs or vehicles. At 24hpf, the drug treatment agent is updated, and at 36hpf, pronase is added to remove the chorion. At 54hpf, the phenotype of zebrafish embryos, especially the pronephric cysts (marked by Tg (wt1b: GFP) transgenic markers) at body bends and glomeruli are examined using suitable means, such as stereoscopes and PerkinElmer Opera Phenix HCS systems.

[0147] Fig.31 Panel A shows that DMSO (0.04%) did not induce lethality, body bending, or pronephric cysts in wild-type zebrafish embryos. In addition, zebrafish injected with a control morpholino that does not affect NPHP4 expression also did not show body bending ( Fig.31 , Group B) or pronephric cyst ( Fig.31 , Panel C). In contrast, zebrafish injected with NPHP4 MO exhibited classic ciliopathy-associated phenotypes in a dose-dependent manner, including, for example, body bending ( Fig.31 , Group B) and pronephric cysts ( Fig.31 , Group C).

[0148] Fig.32 , Panel A shows representative body axis curvatures of four categories of zebrafish: normal, class I, class II, and class III. Fig.32 Panel B shows that alprostadil treatment (0.5 μM and 5 μM) had no significant effect on the body axis curvature of zebrafish NPHP4 MO compared with DMSO treatment (p>0.05, Fischer's exact test). Similarly, using body curvature as an automatic quantitative parameter, Fig.32 , Panel C shows that alprostadil treatment (0.5 μM and 5 μM) had no significant effect on the dorsal bending of zebrafish NPHP4 MO compared with DMSO treatment.

[0149] Fig.33 , Panel A shows representative pronephric cysts of zebrafish: normal, moderate, and severe. Fig.33 Panel B shows that alprostadil treatment (0.5 μM) significantly reduced the percentage of severe pronephric cysts in embryos injected with nphp4 MO compared with DMSO treatment (p<0.05, Fischer's exact test). Fig.33 , Panel C shows that alprostadil treatment (5 μM) significantly reduced the percentage of severe pronephric cysts in embryos injected with nphp4 MO compared with DMSO treatment.

[0150] To test the effect of dinoprostone (PGE2) on ciliopathy, zebrafish NPHP4 MO were treated with dinoprostone (50 μM) or DMSO. Fig.34 Panel A shows that dinoprostone treatment significantly increased the % normal body axis curvature of zebrafish NPHP4MO compared to DMSO treatment (p=0.0066, Fischer's exact test). Fig.34 , Panel B shows that dinoprostone treatment had no significant effect on dorsal bending of zebrafish NPHP4 MO compared with DMSO treatment (p=0.0577, t-test). Fig.34 , Panel C shows that dinoprostone treatment significantly decreased the % severe and mild pronephric cysts and significantly increased the % normal pronephric cysts in zebrafish NPHP4 MO compared with DMSO treatment (p<0.008, Fischer's exact test).

[0151] To test the effects of the selective EP2 agonist CP-544326, zebrafish NPHP4 MOs were treated with CP-544326 (100 nM) or DMSO. Fig.35It was shown that CP-544326 treatment significantly decreased the % severe pronephric cysts and significantly increased the % mild and normal pronephric cysts in zebrafish NPHP4 MO compared with DMSO treatment (p<0.01, Fischer's exact test).

[0152] To examine the in vivo stability of taperenepag isopropyl (PF 04217329, a prodrug of CP-544326) and taperenepag (CP-544326), a pharmacokinetic (PK) study was performed in wild-type C57BL / 6J mice. Fig.36 The PK study design is shown. After intraperitoneal injection of taperenepag isopropyl (1 mg / kg or 8 mg / kg) or taperenepag (8 mg / kg), the concentrations of these compounds in various organs were determined at different time points. The results showed that, in general, taperenepag was Fig.37A ),kidney( Fig.37B ),testis( Fig.37C ), retina( Fig.37D ) and vitreous humor ( Fig.37E ) is more stable than taperenepag isopropyl.

[0153] Homozygous deletion of NPHP1 is the most common cause of juvenile renal wasting 1. Homozygous or compound heterozygous mutations in NPHP1 are also associated with, for example, Joubert syndrome 4 (brain abnormalities) and Senior-Loken syndrome 1 (retinal disorders). NPHP1 KO animals were generated to test whether taperenepag could be used to treat these diseases. - / - Mouse model, single guide RNA was injected into C57BL / 6J embryos and a 76 bp deletion encompassing the ATG in exon 1 of Nphp1 was generated. - / - Natural history of mouse models for Nphp1 + / + and Nphp1 - / - Histochemical staining of kidney and retinal sections of mice. - / - The mouse model did not show a renal phenotype. In contrast, Nphp1 - / - Mice began to show a reduction in the thickness of photoreceptor layers (e.g., inner segment (IS), outer segment (OS), and outer nuclear layer (ONL)) until they were sacrificed at P28, indicating that the rapid retinal degeneration in this model corresponds to ciliopathy-related manifestations.

[0154] To assess retinal degeneration, a semiautomatic tool was developed to detect and quantitatively measure thickness of each retinal layer at five distant planes manually marked on retinal sections ( Fig.38B Semi-automated quantitative analysis confirmed that the thickness of the photoreceptor layers ONL, IS, and OS was significantly reduced ( Fig.38C ).

[0155] To assess the impact of Nphp1 deficiency on the structural organization of photoreceptors in this model, we isolated Nphp1-derived photoreceptors from + / + and Nphp1 - / - Immunohistochemical (IH) analysis of mouse retinal sections Fig.39A and 39B 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. Fig.39A Display, Nphp1 - / - The mouse model exhibited a well-organized photoreceptor structure, with rhodopsin localized along the OS and bounded by Cep290 punctate distribution at the connecting cilium. This suggests that the connecting cilium functions to allow rhodopsin transport from the IS to the photoreceptor OS. In contrast, Nphp1 - / - Mice fail to form connecting cilia and exhibit marked mislocalization of rhodopsin in IS and OS, suggesting that rhodopsin trafficking requires proper formation / maintenance of connecting cilia. Fig.39B Shows that compared to Nphp1 + / + Mouse, Nphp1 - / - The mice showed significant rhodopsin mislocalization in the IS / OS as well as in the ONL.

[0156] To evaluate the effect of Nphp1 deficiency on photoreceptor function, Nphp1 was expressed in the presence of light of different intensities. + / + and Nphp1 - / - Electroretinogram (ERG) examination of mice Fig.40A -C). Fig.40A and 40B ERG a- and b-waves recorded from the same animal at P21 at a given light intensity are shown. + / + Mouse, Nphp1 - / - Mice exhibited significantly lower ERG amplitudes at a given intensity of light stimulation. Fig.40C It is the amplification of the ERG a wave under light stimulation of different intensities, because the a wave reflects the function of photoreceptors.

[0157] Prior to testing the effects of CP-544326 on ciliopathy-related phenotypes, the expression of the potential target EP2 was investigated by immunohistochemistry. Fluorescence microscopy revealed that EP2 was expressed in Nphp1 cells at P21. + / + and Nphp1 - / - Nphp1 is well expressed at the protein level in the IS and ONL photoreceptor layers of mice, although the OS / IS / ONL boundary is - / - Difficult to discern in mice.

[0158] Fig.42 The results show that CP-544326 can be used to evaluate the effect of CP-544326 on Nphp1. - / - Experimental design for the effects of retinal degeneration in a mouse model. Briefly, animals were injected (ip) with vehicle or CP-544326 in vehicle (18 mg / kg) every 3 or 4 days from P6 to P21. Phenotypic readouts included those previously described for Nphp1. - / - Characterization of the mouse model described structural and functional parameters.

[0159] Fig.43 The effect of CP-544326 on the thickness of the photoreceptor layer ONL represented by the ONL / OPL ratio calculated by semi-automated quantification of retinal layers on IHC sections is shown. CP-544326 treatment (18 mg / kg) significantly prevented Nphp1 from esophageal carcinoma compared to vehicle treatment. - / - Similarly, CP-544326 treatment (18 mg / kg) significantly prevented the decrease of ONL / OPL ratio in Nphp1 mice (p<0.05, Mann-Whitney test). - / - Rhodopsin mislocalization in mice (p<0.05, unpaired t-test) expressed as the parameter “mean green intensity in ONL” ( Fig.44 ).

[0160] To assess the effect of CP-544326 on photoreceptor responsiveness, Nphp1 treated with CP-544326 (18 mg / kg) or vehicle was stimulated with light at different intensities. + / + or Nphp1 - / - The mice were examined by electroretinogram (ERG). Fig.45 ) showed that Nphp1 - / - CP-544326 (18 mg / kg) triggered a slight improvement in the amplitude of photoreceptor responses compared to mice.

[0161] All references cited in this specification are incorporated herein by reference, as if specifically and individually indicated that each reference is incorporated by reference. It should be understood that each of the elements described above or two or more together can also be applied to other types of methods different from the types described above. Without further analysis, the foregoing will fully reveal the gist of the present disclosure, so that others can easily apply the gist of the present disclosure to various applications by applying current knowledge without ignoring the features that largely constitute the necessary characteristics of the general or specific aspects of the present disclosure set forth in the attached claims based on the standpoint of the prior art. The foregoing embodiments are presented only by way of example; the scope of the present disclosure is limited only by the attached claims.

Claims

1. A method of treating at least one ciliopathy-related disease in a subject, comprising administering to the subject a therapeutically effective amount of at least one agent that targets at least one G protein-coupled receptor (GPCR).

2. The method of claim 1, wherein the ciliopathy-related disease is caused by a homozygous deletion of the NPHP1 locus.

3. The method of claim 1, wherein the ciliopathy-related disease is caused by a heterozygous deletion of the NPHP1 locus and a heterozygous or homozygous loss of function at a second locus. 4 . The method of claim 1 , wherein the ciliopathy-related disease is caused by a heterozygous deletion in one allele of NPHP1 and a loss-of-function mutation in the second allele. 5 . The method of claim 1 , wherein the ciliopathy-related disease is caused by a loss-of-function mutation in one allele of NPHP1 and a different loss-of-function mutation in the second allele. The method of claim 1 , wherein the at least one agent is an agonist of the at least one GPCR.

7. The method of claim 6, wherein the at least one agent is a prostaglandin.

8. The method of claim 6, wherein 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-210669, 18a, AGN-210961, ED-117, CP-533536, and combinations thereof.

9. The method of claim 6, wherein the at least one GPCR is selected from the group consisting of: EP1, EP2, EP3, and EP4.

10. The method of claim 6, wherein the at least one disease is selected from the group consisting of: renal wasting disease (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 driven by loss of function of NPHP1, and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 alleles and other pathogenic variants or loss-of-function variants.

Images