Treatment methods for diseases related to ciliopathy

Abstract

To provide a method for treating a ciliopathy-associated disease.SOLUTION: The method for treating a ciliopathy-related disease comprises administering to a subject in need thereof an effective amount of a compound that targets at least one G protein-coupled receptor (GPCR). Provided is a method for identifying a therapeutic agent for the treatment of a disease having ciliopathy, the method comprising: providing an animal model system for a ciliopathy to investigate a putative therapeutic agent; administering a destructive agent to the animal; treating the administered animal with the putative therapeutic agent; comparing the measurable phenotype of the treated animal with that of an untreated animal; and identifying the therapeutic target as a therapeutic agent for ciliopathy when a measurable phenotype of the treated animal is reduced compared to that of the untreated animal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This specification is an international application under the Patent Cooperation Treaty, claiming the interests of U.S. Provisional Application No. 62 / 572,051, filed on 13 October 2017. The contents of the aforementioned application are incorporated herein by reference in their entirety. [Background technology]

[0002] Cilia are cell surface projections based on microtubules arising from basal bodies, i.e., membrane-bound centrioles. Primary cilia are non-motile sensory organelles present as a single copy on the surface of most stunted or differentiated mammalian cells. Cilia sense changes in flow and mediate essential signaling pathways in development and tissue homeostasis, such as Hedgehog, Wnt / PCP, and cAMP / PKA signaling. Intraciliary transport (IFT) selects cargo at the base of the cilia and transports axonemal components necessary for cilia construction, as well as proteins involved in ciliary signaling. Once cilia are formed, the control of ciliary membrane composition relies on individual molecular machines, including a barrier against membrane proteins entering the cilia in a specialized region at the base of the cilia called the transition zone, and a transport adapter called BBSome (a complex of the Valde-Vidl syndrome (BBS) protein and other proteins that are components of the basal body and involved in the transport of cargo to the primary cilia) that controls the localization of G protein-coupled receptors (GPCRs) to the cilia. Ciliation requires the coordination of many processes. For cilia to be generated, a complex harmony of cell cycle regulation, vesicular transport, and ciliary elongation must occur at precise timings. The importance of producing and maintaining properly differentiated cilia during embryonic development and in adult physiological function is best highlighted by many human diseases associated with cilia.

[0003] Ciliopathy is a group of human disorders directly caused by defects in the formation or function of cilia. Primary ciliopathy results in multifunctional and highly diverse abnormalities, consistent with the widespread tissue distribution of primary cilia and their broad range of functions. Patients with primary ciliopathy present with a combination of various skeletal defects, including kidney and retinal abnormalities, central nervous system defects that can lead to intellectual disability, liver defects (including cysts), obesity, and abnormalities in limb length, number of fingers (polydactyly), left / right axis organization (situs inversus), and craniofacial patterning. Specific abnormalities in the cilia that connect photoreceptors can also lead to retinal degeneration and blindness. Examples of primary ciliopathy include nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), Valde-Vidl syndrome (BBS), Meckel-Gruber syndrome (MKS), orofacial-finger syndrome (OFD), and Jeune syndrome (JATD).

[0004] Nephronoplasia (NPHP) is an autosomal recessive nephropathy characterized by massive interstitial fibrosis, tubular basement membrane thickening, and cystic formation, leading to end-stage renal disease (ESRD) in childhood. NPHP can occur alone or, in a syndrome hereafter referred to as nephronoplasia-associated ciliopathy (NPHP-RC), it may be associated with different extrarenal conditions (e.g., retinal dystrophy, hepatic fibrosis, skeletal dysplasia, etc.).

[0005] NPHP is caused by 21 NPHP genes that are known to be the primary cause in 60% of cases. Given the high genetic heterogeneity of NPHP and the numerous mechanistic pathways that have been discussed, it is clear that there is no single pathology leading to NPHP. Renal histology of NPHP points to common endpoints of tubular damage and fibrosis, which may have multiple triggers. While each paper describing a new gene discovery seems to provide a clearer molecular diagnosis, it appears to be more confusing regarding the underlying signaling pathways of the disease.

[0006] There remains a great need to characterize the molecular basis of ciliary diseases, including NPHP, which are largely unknown, and to improve the diagnosis and treatment of these diseases. [Overview of the project]

[0007] In one embodiment, the disclosure relates to a method for treating at least one ciliopathy-related disorder in a subject, the method comprising administering to the subject a therapeutically effective dose of at least one agent targeting at least one G protein-coupled receptor (GPCR). In the embodiment, the ciliopathy-related disorder is caused by a homozygous deletion at the NPHP1 locus. In the embodiment, the ciliopathy-related disorder is caused by a heterozygous deletion at the NPHP1 locus and a heterozygous or homozygous loss of function (LOF) at a second locus. In the embodiment, the ciliopathy-related disorder is caused by a heterozygous deletion in one allele of NPHP1 and an LOF mutation in the other allele. In the embodiment, the ciliopathy-related disorder 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 nephronopharyngeal plaque (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), all variant forms of JBTS having further features, e.g., polydactyly, ocular defects, retinal dystrophy, renal cysts, oral frenulum, and hepatic fibrosis; Valde-Vidl syndrome (BBS), Meckel-Gruber syndrome (MKS), orofacial finger syndrome (OFD), end-stage renal disease caused by large homozygous deletion of NPHP1, and renal and retinal ciliopathy associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations, and any ciliopathy caused by the NPHP gene. In certain embodiments, the at least one agent is CP-544326, and the at least one GPCR is EP2. In certain embodiments, the effective dose is 100 pM to 5 μM. In a particular embodiment, the at least one disease is nephronitis.

[0009] 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 the 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 that of an untreated animal or cell model; and (c) identifying the test agent as a therapeutic agent for treating the ciliopathy-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 a Danio rerio (zebrafish) or an nphpl knockout (KO) mouse model (nphpl- / -). In certain embodiments, the animal model is prepared by administering one or more disruptors. In certain embodiments, the one or more disruptors include morpholino. In certain embodiments, the morpholino inhibits the expression of at least one nephrocystin (NPHP), for example, NPHP4. In certain embodiments, the measurable phenotype is selected from the group consisting of body surface curvature, prerenal cysts, lateral cardiac defects, and excretory cavity 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), Valde-Vidl syndrome (BBS), Meckel-Gruber syndrome (MKS), orofacial-finger syndrome (OFD), end-stage renal disease caused by large homozygous deletion of NPHP1, and renal and retinal ciliopathy associated with NPHP1, NPHP4, and NPHP6 / CEP290 mutations.

[0010] In one embodiment, the present disclosure relates to a GPCR agonist for use in the treatment of at least one ciliopathy-related disease. In a particular 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-21 0669, 18a, AGN-21 0961, ED-1 17, CP-533536, and combinations thereof. In a particular embodiment, 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 nephronopharyngeal plaque (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), Valde-Vidl syndrome (BBS), Meckel-Gruber syndrome (MKS), orofacial-finger syndrome (OFD), end-stage renal disease caused by large homozygous deletion of NPHP1, and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations.

[0011] In certain embodiments, the animal model is prepared by administering one or more disruptors. In certain embodiments, the one or more disruptors include a CRISPR / Cas9 system that mediates sgRNA-directed gene deletions. In certain embodiments, the CRISPR / Cas9 system inhibits the expression of at least one nephrocystin (NPHP), for example, 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 bodies. In certain embodiments, the at least one disease is selected from the group consisting of nephronopharyngeal plaque (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS) and related disorders (JSRD), Valde-Vidl syndrome (BBS), Meckel-Gruber syndrome (MKS), orofacial-finger syndrome (OFD), end-stage renal disease caused by large homozygous deletion of NPHP1, and renal and retinal ciliopathies associated with NPHP1, NPHP4, NPHP6 / CEP290 mutations.

[0012] For a further understanding of the nature, purpose, and merits of this disclosure, please refer to the following detailed description, which should be read in conjunction with the following drawings. In the following drawings, similar reference numerals indicate similar elements. [Brief explanation of the drawing]

[0013] [Figure 1] Figures 1A-1D show renal epithelial cells derived from urine. 1A: Normal control, 1B: NPHP patient with NPHP1 deletion (Pt1), 1C: RT-PCR comparison, 1D: Immunoblot comparison. [Figure 2] This document describes an automated in vitro assay for quantifying ciliation in target cells. [Figure 3] This shows that the proportion of ciliated cells derived from NPHP patients (PT1) is significantly lower than that of control cells (CTRL). [Figure 4] This is a schematic diagram showing the steps of a novel ciliary system assay. [Figure 5](A) Fluticasone, (B) Pheniramine, (C) Verapamil, (D) ML-141, (E) Mitoxantrone, (F) Tropisetron, (G) Ethopropazine, (H) Cyproheptadine, (I) Paclitaxel, and (J) Simvastatin on ciliogenesis are shown compared with DMSO. [Figure 6] The effect of alprostadil on ciliogenesis is shown compared with DMSO. [Figure 7A] The dose - response of alprostadil on ciliogenesis is shown compared with DMSO. [Figure 7B] The corresponding semi - logarithmic representation for IC50 determination is 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] A meta - analysis of the results obtained from multiple ciliogenesis experiments with alprostadil treatment, showing the data for each experiment separately (A - D). [Figure 10] The stability of PGE1 under experimental conditions is shown. [Figure 11A] The effects of alprostadil (PGE1), dinoprost (PGE2), and 16,16 - dimethyl - PGE2 (dmPGE2) on ciliogenesis are shown. [Figure 11B] The effect of alprostadil (PGE1) on cell lines derived from patients with NPHP1 deletion is shown. [Figure 11C] A meta - analysis of cilia is shown. [Figure 12] The effect of PGE2 on ciliogenesis is shown. [Figure 13] The expression profiles of EP1 - 4 in human kidney tissues by Western blot and in human retina by immunohistochemistry are shown. [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 mRNA expression of genes encoding EP1-4 receptors in multiple control cell lines and renal epithelial cell lines derived from multiple NPHP patients. [Figure 15] The prostaglandin (PG) modulators (agonists and antagonists) whose effects on ciliary formation were investigated are shown. [Figure 16A] A meta-analysis of cilia is shown. [Figure 16B] This shows cells derived from NPHP patients treated with CP-544326. [Figure 16C] The corresponding semi-logarithmic representation is shown. [Figure 17A] This shows the effect of L-902.688 on ciliary formation. [Figure 17B] This study demonstrates the effects of CP-544326 and alprostadil on ciliation. [Figure 17C] This shows the effects of CP-544326 on patient-derived cells. [Figure 17D] A meta-analysis of cilia is shown. [Figure 18] This shows RNA extracted by RLT or Qiazol for microarray analysis. [Figure 19] This shows microarray data of samples analyzed using hierarchical clustering. [Figure 20] This shows microarray data of samples analyzed using hierarchical clustering. [Figure 21] This shows microarray data of samples analyzed using hierarchical clustering. [Figure 22] This section summarizes the microarray data obtained from RLT extraction samples. [Figure 23] This section summarizes the microarray data obtained from Qiazol-extracted samples. [Figure 24A]This shows that there are no significant differences between microarray data obtained from various doses. [Figure 24B] This shows that there are no significant differences between microarray data obtained from various doses. [Figure 25] This document describes the process of multi-omics analysis of the drug's effect on ciliary formation. [Figure 26] The phenotypic analysis of the effects of alprostadil on ciliation is shown (A-E). [Figure 27] This shows differential expression of drugged and druggable genes via imRNA. [Figure 28A] This paper presents pathway analysis from multi-omics data regarding the downstream interactions of prostaglandin E1 (alprostadil), and related target opportunities. [Figure 28B] This paper presents a path analysis of upstream interactions of NPHP1 from multi-omics data, along with related target opportunities. [Figure 28C] This paper presents pathway analysis from multi-omics data regarding direct interactions related to NPHP1-20 genes, and shows related target opportunities. [Figure 29] The NPHP4 MO model for zebrafish is shown. [Figure 30] This document describes the drug treatment protocol for the NPHP4 MO model of zebrafish. [Figure 31] Summarize the effects of morpholino injection on zebrafish (A-C). [Figure 32] (A) Typical axial curvature of zebrafish, and (B, C) the effect of alprostadil on axial curvature of zebrafish are shown. [Figure 33] (A) A typical prerenal cyst in zebrafish, and (B, C) the effects of alprostadil on prerenal cysts in zebrafish. [Figure 34] (A, B) shows the effect of dinoprostone on the curvature of the body axis in zebrafish, and (C) shows the effect of dinoprostone on the prerenal cysts in zebrafish. [Figure 35]This shows the effects of CP-544326 on the pronenary cysts of zebrafish. [Figure 36] The 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] This shows periodate-Schiff staining of the retina in wt and Nphp1- / - mice. [Figure 38B] This shows a semi-automated method for quantifying the thickness of the retinal layer. [Figure 38C] The quantification of retinal layer thickness in HphpV' mice is shown in comparison to that of wt mice. [Figure 39A] Immunohistochemical staining of Cep290 as a ciliary marker, and rhodopsin and PNA (peanut agglutinin lectin) as photoreceptor markers for the outer segment (OS) and inner / outer segments, respectively, is shown in wt and Nphp1- / - mouse retina. [Figure 39B] Immunohistochemical staining of Cep290 as a ciliary marker, and rhodopsin and PNA (peanut agglutinin lectin) as photoreceptor markers for the outer segment (OS) and inner / outer segments, respectively, is shown in wt and Nphp1- / - mouse retina. [Figure 40] The electroretinogram of Nphp1- / - mice is shown in comparison to that of wt mice. [Figure 41] This shows the expression of the EP2 receptor in wt and Nphp1- / - mice. [Figure 42] A test design according to one embodiment of this disclosure is shown. [Figure 43] This study demonstrates the effect of CP-544326 on the ratio of ONL / OPL retinal layer thickness in Nphp1- / - mice. [Figure 44]This study demonstrates the effect of CP-544326 on the mislocalization of green-labeled rhodopsin in ONL in NphpV' mice. [Figure 45] This study demonstrates the effects of CP-544326 on the electroretinogram of Nphp1- / - mice. [Modes for carrying out the invention]

[0014] This disclosure is not limited to the specific embodiments described below, and variations of such embodiments may be made and may be included in the scope of the appended claims. Furthermore, the terminology used is intended to describe specific embodiments and is not intended to limit them.

[0015] In this specification and the appended claims, "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure belongs.

[0016] NPHP patient Nephronoplasia (NPHP) is a recessive tubulointerstitial ciliopathy characterized by progressive kidney destruction, leading to end-stage renal disease (ESRD). ESRD caused by NPHP can develop between a few months of age (childhood NPHP) and over 60 years of age (adult NPHP), with over 17% of cases occurring 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. Complete deletion of the NPHP1 locus (NPHP1(del)) accounts for over 20% of NPHP cases. Traditionally, the rare disease portal Orphanet has reported a global frequency of approximately 1 in 100,000 (Canada 1 / 50,000, the United States 1 / 900,000, Finland 1 / 100,000, France 1 / 50,000). Currently, there is no treatment for NPHP.

[0017] Ciliopathy is often caused by mutations in the gene encoding the transition zone (TZ) protein or in the intraciliary transport (IFT) component (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 cilia at the proximal end of the axoneme, which controls the movement 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 several 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 ciliation and / or epithelialization, leading to the development of fibrosis and cysts in NPHP patients. The IFT apparatus selects cargo at the base of the cilia and transports axonemal components necessary for ciliary construction, as well as proteins involved in ciliary signaling. The IFT-B complex, consisting of 16 different 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 the IFT-A subunit have been identified in NPHP-associated ciliopathy, with only three IFT-B subunits associated with nephronophthenia (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, accessory proteins and GPCRs are also essential factors for ciliary function and maintenance.

[0019] Regarding the NPHP module, Nphp4 mutant mice developed retinal degeneration but did not develop renal cysts or severe ciliary deficiency. Males were infertile and exhibited reduced sperm motility (Won, J. et al., Hum. Mol. Genet., 20:482-96, 2011). Similarly, targeted disruption of Nphpl in these mice (deletion of the last C-terminal exon 20) did not result in nephronophthria, but showed rapid retinal degeneration starting at P14-P21 (Jiang, S. et al., Hum. Mol. Genet., 17:3368-79, 2008) and caused male infertility (Jiang, S. et al., Hum. Mol. Genet., 18:1566-77, 2009). Cep290 knockout mice lack photoreceptor-binding cilia and are unable to mature motile ependymal cilia, which is 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 adult-onset ESRD patients (random sampling for etiology), NPHP due to homozygous whole gene deletion of NPHP1 (NPHP(del)) has a prevalence of 1 in 200 patients (0.5%) in all adult-onset ESRD cases (Snoek, R. et al., J.Am.Soc.Nephrol., 29:772-9, 2018). The incidence was significantly higher in patients with ESRD onset between 18 and 50 years of age (prevalence 0.9%), but NPHP can develop up to age 61. They conclude that because their method underestimates the total number of causative mutations, NPHP is a relatively frequent single-gene factor in adult-onset ESRD and may be underdiagnosed in current routine clinical practice.

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

[0022] This specification 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, with the aim of identifying novel disease and patient-related insights, thereby enabling scientific discovery and facilitating its transition to patient management. The 100,000 Genomes Project dataset includes patients with rare diseases (and their relatives) and cancer patients. Within this dataset, patients homozygous for NPHP1(del) 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 were recruited as having obvious clinical signs / symptoms of NPHP, e.g., signs / symptoms of renal or ciliopathy, or as patients with congenital renal or urinary tract anomalies (CAKUT). The remaining 3 patients exhibited more complex clinical presentations, possibly involving multiple rare diseases. In addition to homozygotes, 193 NPHP1(del) heterozygous patients were identified across the entire dataset (a frequency of approximately 1 in 200 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 (a combination of NPHP1(del) and LOF mutations at another locus). Furthermore, patients may have additional NPHP1-LOF variants such as splice variants, frameshifts, and nonsense mutations, which may also contribute to clinical NPHP findings.

[0023] The NPHP(del) findings described herein were obtained from research conducted using the Genomics England database. This research was made possible through access to the data and the 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 by NHS clinicians and medical teams who contributed to the data and results applied to this research. Genomics England's Research Environment is managed by Genomics England Limited (a wholly owned subsidiary 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 treatment. In the United States alone, more than 600,000 transplants have been performed in the past 50 years, and demand today is higher than ever. Unfortunately, the availability of donor organs has not kept pace with the demand for transplants. Embodiments of this disclosure include identifying and / or treating patients who are homozygous or heterozygous with respect to NPHP (e.g., NPHP-induced ESRD) and / or patients with NPHP-associated ciliopathy (e.g., NPHP1).

[0025] Cells derived from NPHP patients This specification describes materials and methods for identifying therapeutic agents useful for treating ciliopathy-related diseases or disorders, such as NPHP or NPHP1(del)-related diseases or disorders. Such methods may include the use of patient-derived cell lines. Such developed cell lines can also be used in other related methods, such as monitoring the effectiveness of a given therapeutic agent for ciliopathy-related diseases or disorders or NPHP1(del)-related diseases or disorders.

[0026] To identify compounds for treating ciliopathy-associated diseases, such as NPHP, we obtained cells from NPHP patients and established cell lines. Briefly, exfoliated renal epithelial cells, mostly proximal tubular cells (tbcs) recovered from the urine of NPHP1-deficient patients, were immortalized by retroviral gene transfection with the SV40 T antigen. The cells were fixed and fluorescently labeled with Hoechst (for nuclear staining), anti-Y-tubulin antibody (for basal body staining), and anti-ARL13B antibody (for ciliary staining) for detection using immunofluorescence microscopy. In contrast to most normal urine-derived renal epithelial cells (URECs) which have a single cilia in each cell (Figure 1A), most cells from NPHP patients lack cilia (Figure 1B). The absence of NPHP expression in these NPHP patient-derived cells was further confirmed by RT-PCR (Figure 1C) and immunoblotting (Figure 1D), but the cells from NPHP patients did not show detectable levels of NPHP RNA and NPHP protein expression, respectively.

[0027] Figure 2 shows an automated in vitro assay that can be used to quantify ciliation in target cells. Briefly, cells derived from NPHP patients and control cells were cultured in complete medium at 39C (a temperature unacceptable for SV40 expression), and then ciliation was measured, for example, in units of % cilia, by automated ciliary analysis using immunofluorescence microscopy. The rotating platform may be used for drug screening (Figure 5, A-J) and ciliation analysis of G3 multi-omics datasets (Figure 29, A-E). The Opera Phenix platform may be used for other phenotypic analyses (e.g., ciliation titration of alprostadil and CP-544326, screening of other EP agonists based on ciliation, ciliation using other NPHP1 patient-derived cell lines, and α-tubulin acetylation analysis).

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

[0029] Drug screening The ciliary system assay described above can be used to identify compounds that restore ciliary formation. Figure 4 shows the process of the ciliary system assay, where, for example, cells may be seeded in a cell culture (e.g., a 96-well plate) on day 0, incubated with candidate drugs on day 3, fixed on day 5, and fluorescently labeled with Hoechst, anti-γ-tubulin antibody, and anti-ARL13B antibody. 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 bodies (γ-tubulin, 555 nm), and cilia (ARL13b, 647 nm) may be obtained.

[0030] Using the process shown in Figure 4, cells derived from NPHP patients were treated with several candidate drugs to identify drugs that could restore ciliogenesis. Figure 5, panels A-J, show that fluticasone, pheniramine, verapamil, ML-141, mitoxantrone, tropisetron, etopropazine, cyproheptadine, paclitaxel, and simvastatin, at various test concentrations, did not have a significant effect on ciliogenesis compared to DMSO. Surprisingly, Figure 6 shows that alprostadil significantly restored ciliogenesis in NPHP patient-derived cells compared to DMSO, as indicated by an increase in the percentage of ciliated cells.

[0031] Alprostadil, or prostaglandin E1 (PGE1), has a chemical structure. [ka] It possesses the properties of vasodilation, inhibition of platelet aggregation, and stimulation of intestinal and uterine smooth muscle, which are beneficial for treating heart disease and erectile dysfunction. Alprostadil may act as an agonist by binding to E-type prostaglandin (EP) receptors, which are G protein-coupled receptors (GPCRs), with IC50 values ​​of 36, 10, 1.1, and 2.1 nM for EP1, EP2, EP3, and EP4, respectively. GPCRs stimulate adenylyl cyclase, which subsequently increases intracellular cAMP.

[0032] As used herein, “GPCR agonist” includes compositions that activate a GPCR to mimic the action of an endogenous signaling molecule specific to that receptor. “GPCR antagonist” includes compositions that inhibit GPCR activity. GPCR activity can be measured by its ability to bind to effector signaling molecules such as G proteins. “Activated GPCR” is one that is capable of interacting with and activating G proteins. An inhibited receptor may have a reduced ability to bind to an extracellular ligand and / or a reduced ability to productively interact with and activate G proteins.

[0033] For example, GPCR agonist therapy with taprenepagisopropyl can be administered at concentrations of approximately 0.1 mg / kg to 20 mg / kg, 0.5 mg / kg to 20 mg / kg, 1 mg / kg to 20 mg / kg, 2 mg / kg to 20 mg / kg, 3 mg / kg to 20 mg / kg, 4 mg / kg to 20 mg / kg, 5 mg / kg to 20 mg / kg, 6 mg / kg to 20 mg / kg, 7 mg / kg to 20 mg / kg, and 8 mg / kg to 20 mg / kg. The dosage may be 20 mg / kg, approximately 9 mg / kg to approximately 20 mg / kg, approximately 10 mg / kg to approximately 20 mg / kg, approximately 12 mg / kg to approximately 20 mg / kg, approximately 14 mg / kg to approximately 20 mg / kg, approximately 16 mg / kg to approximately 20 mg / kg, or approximately 18 mg / kg to approximately 20 mg / kg, and the frequency may be, for example, daily, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, every ten days, once a week, every two weeks, every three weeks, or once a month.

[0034] To determine the effective concentration of alprostadil for restoring ciliary formation, automated ciliary analysis was performed by alprostadil titration at concentrations of 1 nM to 2 μM (Figure 7A) and 100 pM to 2 μM (Figure 7B). The effective concentrations of the GPCR agonist, e.g., alprostadil, may be approximately 1 pM to 10 μM, 10 pM to 5 μM, 50 pM to 5 μM, 100 pM to 5 μM, 1 nM to 5 μM, 1 nM to 4 μM, 1 nM to 3 μM, 1 nM to 2.5 μM, 1 nM to 2 μM, 10 nM to 2 μM, 100 nM to 2 μM, 500 nM to 2 μM, or approximately 1 μM to 2 μM. Figure 7C shows the corresponding semi-logarithmic representation for IC50 identification, demonstrating that alprostadil significantly increases ciliated cell percentage in a dose-dependent manner in cells derived 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 ciliation in control normal epithelial cells (CTRL) compared to the control (DMSO 0.04%) (Figure 8A). In contrast, alprostadil treatment significantly increases ciliation in cells derived from NPHP patients (PT1) compared to the control (DMSO 0.04%) (Figure 8B). Figure 8C shows that the effect of alprostadil on ciliation in cells derived from NPHP patients is approximately twofold compared to control cells that did not receive alprostadil treatment, i.e., the control (DMSO 0.04%).

[0036] Meta-analysis also shows a nearly linear effect of alprostadil dose on ciliation. Figure 9 (Panels A and B) shows, for example, the effect of alprostadil on ciliation in control normal epithelial cells. 2 The value is 0.9194. Similarly, Figure 9 (Panels C and D) shows the effect of alprostadil on ciliogenesis in cells derived from NPHP patients. 2 The value is 0.8489.

[0037] To determine the stability of alprostadil (PGE1), supernatants were collected 24 and 48 hours after exposure from renal epithelial cells (URECs) derived from urine exposed to different concentrations of alprostadil. The samples were then extracted and divided into equal volumes for analysis on LC / MS / MS and the Polar LC platform. Figure 10 shows that PGE1 is stable under experimental conditions.

[0038] In addition to PGE1, other EP agonists, for example, chemical structure [ka] The chemical structure of prostaglandin E2 (PGE2 or dinoprostone) and its long-acting derivatives is as follows: [ka] 16,16-dimethyl-PGE2 (dmPGE2), which contains [a specific compound], was also investigated for its ability to restore ciliary formation. Figure 11A shows that PGE2 and dmPGE2 had a similar ciliary formation-restoring effect to alprostadil in cells derived from NPHP patients, but no significant effect was observed in control normal cells. In cells derived from NPHP patients, a slight decrease in ciliary formation restoration was observed at the highest concentrations (40 μM dinoprostone and 20 μM dmPGE2), which may be due to cytotoxicity.

[0039] To investigate the effects of alprostadil (PGE1) on NPHP1-deficient cells, cell lines derived from NPHP1(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. Figure 11B shows that alprostadil significantly increased ciliary formation in NPHP1-deficient cells, while alprostadil did not significantly affect ciliary formation in normal control cells, suggesting that alprostadil is effective in restoring ciliary formation in NPHP1-deficient patients.

[0040] The meta-analysis in Figure 11C shows a linear regression analysis of previous data, where the slopes reflect 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 as 1-09-P L4, 1-06-P1, 1-06-P2, and PT-1). Linear regression for control normal epithelial cells shows slope values ​​of 0.7665 to 0.9974, suggesting no effect of alprostadil on ciliogenesis. In contrast, linear regression for cells derived from multiple NPHP patients shows a combined slope value of 1.414 or a slope value range of 1.333 to 1.506, indicating a promoting effect of alprostadil on ciliogenesis.

[0041] Prostaglandins are found in most human tissues and are synthesized from essential fatty acids. Structural differences between various prostaglandins are the main cause of changes in their biological activity. Prostanoids, including prostaglandins, are produced abundantly in the kidney. These prostanoids are produced by the release of arachidonic acid (AA) from membrane phospholipids by phospholipase A2. Arachidonic acid is subjected to the bisoxygenase and peroxidase activity 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, and these classes initiate their action through these subtypes. Cyclooxygenases 1 and 2 (COX1 and COX2) are primary targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but they can be specific, i.e., selective, or non-selective to either isoform. Blocking PGH2 production via COX inhibition can reduce the levels of all downstream plastanoids.

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

[0043] The COX-Lkt / ABCC4-EP4 signaling cascade is required for cilia formation and elongation (in mouse renal collecting duct cells IMCD3 and in a zebrafish model). cAMP-dependent kinase signaling is known to increase anterograde IFTs during ciliation. Lkt / ABCC4-mediated PGE2 signaling affects cAMP levels and promotes ciliation by increasing the anterograde rate of IFTs. PGE2 treatment increases intracellular cAMP during ciliation in IMCD3 cells, but Ca 2+ It does not increase. PGE2 acts autocrinely and / or paracrinely, and cells may respond to PGE2 released from themselves or from their vicinity. In human cancer cells, the interaction between PGE2 and 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 the addition of exogenous PGE2 increased both ciliary 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 ciliation.

[0045] PGE2 is produced by PGE synthase (PGES) and transmits signals by binding to its GPCRs, namely EP1-4. EP1(G qActivation (conjugated with PLC) is transmitted via PLC to intracellular Ca2 + It increases EP3 (conjugated with Gi) activation, which increases intracellular Ca2 via PLC. + It increases and / or inhibits cAMP production via adenylyl cyclase (AC). EP2 or EP4 (both G s Activation (conjugated with AC) 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. Forty-six GPCRs function as drug targets for hypertension, pain, ulcers, allergies, alcoholism, obesity, glaucoma, psychotic disorders, and HIV. One of many major shortcomings is the overall lack of knowledge regarding the association between putative GPCRs and their precise physiological functions or disease states.

[0047] Figure 13 shows that EP1-4 are expressed in the kidney and retina, and that 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 activation of each EP receptor subtype in the kidney. EP receptors regulate vasoconstriction in the afferent arterioles, where EP1 / EP3 acts as a vasoconstrictor and EP2 / EP4 acts as a vasodilator. EP1 / EP4 regulates proximal tubular transport. EP3 and EP4 regulate large ascending limb and distal tubular transport. EP4 promotes renin release from the macula densa. EP2 / EP4 dilates the straight vasodilation. EP1 is Na + EP3 inhibits the reabsorption of H2O, while EP4 promotes the reabsorption of H2O, thereby regulating collector tubule transport.

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

[0049] PGE2 modulator (EP2) Selective agonists and antagonists of the EP2 receptor are shown, for example, by reference in Markovic, T. "Structural features of subtype-selective EP receptor modulators" Drug Discovery Today. 2017, 22(1):57-71. The first class of agonists includes ligands that are structurally similar to the endogenous ligand PGE2 but incorporate key modifications to the ω-lipophilic chain that contribute to improved potency and selectivity. The second class of agonists are 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 holds.

[0050] A third class of agonists includes non-prostanoid N-phenyl-γ-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, possessing an IC50 of 16 nM (Kb=1.8 nM) and exhibiting a >10,000-fold increase in EP2 receptor selectivity compared to other prostanoid receptors.

[0052] Markovic's Figure 5 (incorporated by reference) shows selective agonists of the EP4 receptor, namely (a) derivatives based on a functionalized cyclopentane core, (b) derivatives having a lactam-corresponding moiety of a hydroxycyclopentanone core, and (c) structurally diverse EP4 agonists. To improve bioavailability, tetrazole characteristics were introduced into the a-chain instead of terminal carboxylic acid functional groups, leading to the discovery of L902,688, a sub-nanomolar agonist of the EP4 receptor (EC50 = 0.2 nM). L902,688 has a prostanoid structure. [ka] It holds.

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

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

[0055] Figure 15 shows the PG modulators (agonists and antagonists) whose effects on ciliary formation were investigated.

[0056] Figure 16A shows that CP-544326, a non-prostanoid EP2 agonist, restores ciliation to a level similar to that of alprostadil. Figure 16B shows that CP-544326 restores ciliation in a dose-dependent manner compared to DMSO. Figure 16C is a semi-logarithmic representation of the results in Figure 16B, showing that the titration of CP-544326 yields an EC50 of 11 nM relative to EP2. The restoration of ciliation by the non-prostanoid CP-544326 supports its specificity in its mechanism of action. In contrast, Figure 17A shows that L-902.688, a prostanoid EP4 agonist, does not have a significant effect on ciliation. These results suggest that EP2 plays a more important role than EP4 in ciliation.

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

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

[0059] Figure 19 shows microarray data of samples analyzed by hierarchical clustering. First, the data were clustered by extraction type (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 microarray data of samples analyzed by hierarchical clustering. Data obtained from Qiazol extract samples were clustered by condition, and then by replication, rather than by dose within the treatment or medium / DMSO within the control.

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

[0062] Regarding microarray data obtained from RLT extract samples, there were no significant differences between DMSO and the culture medium, except for four differentially expressed genes without regulated exons / patterns. Figure 22, however, shows nearly the same number of expressed and regulatory genes across the three alprostadil concentrations, comparing the control (DMSO) with alprostadil treatment (0.2 μM, 2 μM, and 10 μM). The top three regulatory genes are also nearly identical, sharing the same signaling pathways, such as cell adhesion and the downregulation of the extracellular matrix.

[0063] Regarding microarray data obtained from Qiazol extract samples, there were no significant differences between DMSO and the culture medium, for example, 33 differentially expressed genes without regulated exons / patterns. However, as shown in Figure 26, when comparing the control (DMSO) with alprostadil treatment (0.2 μM, 2 μM, and 10 μM), nearly the same number of expressed and regulatory genes were present across the three alprostadil concentrations. The top three regulatory genes were also nearly identical, sharing the same signaling pathways, such as downregulation of cell adhesion and the extracellular matrix, and upregulation of interferon signaling.

[0064] Furthermore, Figures 24A and 24B show that two clusters were defined, grouping a total of 310 genes: "Cluster 1" = 120 downregulated genes and "Cluster 2" = 190 upregulated genes. This indicates that no significant differences were detected between the microarray data obtained from various doses.

[0065] Furthermore, pathway analysis by cross-referencing microarray data from patients, regardless of alprostadil treatment, with RNA-seq data from control patients revealed that alprostadil can reverse the gene expression changes observed in cells derived from NPHP patients compared to control cells.

[0066] Multi-omics analysis Figure 25 shows the process of multi-omics analysis of the drug's effect on ciliation. Figures 26A–26E show, for example, the effect of alprostadil on ciliation in five independent experiments, e.g., phenotypic analysis of ciliated cell percentage. These results indicate that, in n=1–5, alprostadil partially restores ciliation at similar magnifications without a dose-dependent response.

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

[0069] InVivo Model Figure 29 shows the results from a zebrafish NPHP4 morpholino (MO) model, in which unicellular wild-type zebrafish embryos were injected with morpholino (e.g., NPHP4 ATG MO) that blocks the NPHP4 mRNA start site from ribosome binding. This morpholino specifically inhibits the translation of NPHP4 mRNA. Zebrafish NPHP4 MO exhibits classic ciliopathy-related phenotypes, including body surface curvature, prerenal cysts, lateral (cardiac looping) defects, and excretory cavity 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 morpholino (e.g., nphp4 ATG MO) at the unicellular stage. At 8 hours post-fertilization (hpf), the injected embryos were treated with the drug or medium in PTU-egg seawater (1 mL in a 12-well plate). At 24 hpf, the drug treatment was refreshed, and pronase was added at 36 hpf to remove the chorionic membrane. At 54 hpf, the phenotype of the zebrafish embryos, particularly surface curvature and glomerular prerenal cysts, was examined using appropriate means, e.g., stereoscopic microscopy and the 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 prerenal cysts in wild-type zebrafish embryos. Furthermore, zebrafish injected with control morpholino, which does not affect NPHP4 expression, also did not exhibit body surface curvature (Figure 31, Panel B) or prerenal cysts (Figure 31, Panel C). In contrast, zebrafish injected with NPHP4 MO showed dose-dependent classic ciliopathy-related phenotypes, including, for example, body surface curvature (Figure 31, Panel B) and prerenal cysts (Figure 31, Panel C).

[0072] Figure 32, Panel A, shows representative axial curvature of zebrafish in four categories: normal, class I, class II, and class III. Figure 35, Panel B, shows that alprostadil treatment (0.5 μM and 5 μM) did not significantly affect the axial curvature of zebrafish NPHP4 MO compared to DMSO treatment (p>0.05, Fisher's exact test). Similarly, using 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 DMSO treatment.

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

[0074] To investigate the effects of dinoprostone (PGE2) on ciliopathy, 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 axial curvature in zebrafish NPHP4 MO compared to DMSO treatment (p=0.0066, Fisher's exact test). Figure 34, Panel B, however, shows that dinoprostone treatment did not significantly affect dorsal curvature in zebrafish NPHP4 MO compared to DMSO treatment (p=0.0577, t-test). Figure 34, Panel C shows that dinoprostone treatment significantly decreased the percentage of severe and mild prerenal cysts and significantly increased the percentage of normal prerenal cysts in zebrafish NPHP4 MO compared to DMSO treatment (p<0.008, Fisher's exact test).

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

[0076] To investigate the in vivo stability of taprenepag isopropyl (PF 04217329, the prodrug of CP-544326) and taprenepag (CP-544326), a pharmacokinetic (PK) study was conducted in wild-type C57BL / 6J mice. Figure 36 shows this PK study design. After intraperitoneal injection of taprenepag isopropyl (1 mg / kg or 8 mg / kg) or taprenepag (8 mg / kg), the concentrations of these compounds in various organs were measured at different time points. The results generally show 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 1. Homozygous or compound heterozygous mutations in NPHP1 are also associated with, for example, Joubert syndrome 4 (brain abnormalities) and Senior-Loken syndrome 1 (retinopathy). NPHP1 KO animals were generated to investigate whether taprenepag can be used for the treatment of these diseases. Nphpl manipulated with CRISPR / Cas9 - / - To establish a mouse model, single-guide RNA was injected into C57BL / 6J embryos to create a 76-bp deletion encompassing the ATG of exon 1 of Nphpl. Nphpl - / - To characterize the natural growth of the Nphpl mouse model, Nphp1 + / + and Nphpl - / - histochemical staining of kidney and retina sections of Nphpl mice was performed. Nphpl - / - The Nphpl mouse model does not show a renal phenotype. In contrast, Nphpl mice at P14 age - / - begin to show a decrease in the thickness of the photoreceptor layer (e.g., inner segment (IS), outer segment (OS), and outer nuclear layer (ONL)) until they are sacrificed at P28, showing rapid retinal degeneration in this model corresponding to ciliopathy-related signs.

[0078] To assess retinal degeneration, a semi-automated tool was developed to detect and quantitatively measure the thickness of each retinal layer in five manually marked, separate planes 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 investigate the effect of Nphpl deletion on the structural mechanism of the photoreceptor in this model, immunohistochemical (IH) analysis was performed on Nphp1 + / + and Nphpl - / - The procedure 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 conjugated ciliary staining) or PNA (for OS and IS staining), and detected using immunofluorescence microscopy. Figure 39A shows Nphp1 - / - The mouse model shows that rhodopsin is localized along the OS and exhibits a well-organized photoreceptor structure surrounded by a punctate distribution of Cep290 in the binding cilia. This indicates that the binding cilia are functional in transporting rhodopsin from the IS to the photosensitive OS. In contrast, Nphpl - / - The mice were unable to form binding cilia and showed clear rhodopsin mislocalization in IS and OS, suggesting that rhodopsin transport requires precise formation / maintenance of binding cilia. In agreement, Figure 39B shows Nphp1 + / + In contrast to mice, Nphpl - / - This study demonstrates that mice exhibit similarly pronounced rhodopsin mislocalization in IS / OS and ONL.

[0080] To evaluate the effects of Nphpl deletion on photoreceptor function, electroretinography (ERG) was performed under different light stimuli to assess Nphpl deletion. + / + and Nphp1 - / - The study was performed on mice (Figures 40A-C). Figures 40A and 40B show the a-wave and b-wave of the ERG recorded from the same animal at P21 for a given light intensity. Nphp1 + / + In contrast to mice, Nphp1 - / -Mice exhibit dramatically lower ERG amplitudes at a given light stimulation intensity. Figure 40C is a magnified view of the 'a' wave of the ERG under different light stimulation intensities, where the 'a' wave reflects photoreceptor function.

[0081] Before investigating the effects of CP-544326 on ciliopathy-related phenotypes, the expression of its potential target, EP2, was examined by immunohistochemistry. Fluorescence microscopy revealed that EP2 expression was present in P21-year-old Nphp7 + / + and Nphpl - / - It has been shown that it is well expressed at the protein level in the mouse photoreceptor layers IS and ONL, but the boundary between OS / IS / ONL is Nphpl - / - It was difficult to distinguish between them in mice.

[0082] Figure 42 shows that CP-544326 is Nphp7 - / - This paper describes an experimental design to evaluate the effects on retinal degeneration in a mouse model. Briefly, animals were injected with either the medium or a medium solution of CP-544326 (18 mg / kg) every 3 or 4 days from P6 to P21 (ip). Phenotypic measurements were taken using Nphpl. - / - The aforementioned structural and functional parameters are included for characterizing the mouse model.

[0083] Figure 43 shows the effect of CP-544326 on the thickness of the photoreceptor layer (ONL), expressed as the ONL / OPL ratio calculated from semi-automatic quantification of the retinal layer of IHC sections. Treatment with CP-544326 (18 mg / kg) is Nphp7 - / - The decrease in the ONL / OPL ratio in mice was significantly suppressed compared to the media treatment (p<0.05, Mann-Whitney test). Similarly, CP-544326 treatment (18 mg / kg) was expressed as the parameter "mean green intensity in ONL," which is semi-automatically quantified in IHC sections by fluorescence microscopy, and Nphpl - / - This significantly suppressed rhodopsin mislocalization in mice (p<0.05, independent t-test) (Figure 44).

[0084] To evaluate the effect of CP-544326 on photoreceptor reactivity, electroretinograms (ERGs) were treated with either CP-544326 (18 mg / kg) or the medium under different light stimulation intensities. + / + and Nphp1 - / - The study was conducted in mice. A magnified view of the ERG a-wave (Figure 45) shows that CP-544326 (18 mg / kg) resulted in a slight improvement in the amplitude of the photoreceptor response compared to that of media-treated Nphpl^ mice.

[0085] All references cited herein are incorporated herein by reference in such a way that each reference is specifically and individually indicated to be incorporated by reference. It will be understood that each or more of the above elements may find useful applications in other types of ways different from those described above. Without further analysis, the foregoing is provided to fully illustrate the essence of this disclosure, and others, by applying their current knowledge, will readily adapt it to various applications without omitting features that, from the perspective of the prior art, properly constitute the essential features of the particular aspects of this disclosure as shown in the appended claims or in general. The embodiments described herein are presented only as examples, and the scope of this disclosure is limited only by the following claims.

Claims

1. A pharmaceutical composition comprising an E-type prostaglandin receptor 2 (EP2) agonist for the treatment of nephronophthria, The agonist of EP2 is a pharmaceutical composition comprising prostaglandin E1 (PGE1).

2. The pharmaceutical composition according to claim 1, wherein the nephron aplasia is caused by a homozygous deletion of the NPHP1 gene locus.

3. The pharmaceutical composition according to claim 1, wherein the nephron aplasia is caused by a heterozygous deletion of the NPHP1 gene locus and a heterozygous or homozygous functional deficiency at a second gene locus.

4. The pharmaceutical composition according to claim 1, wherein the nephron aplasia is caused by a heterozygous deletion in one allele of NPHP1 and a loss-of-function mutation in the other allele.

5. The pharmaceutical composition according to claim 1, wherein the nephron aplasia is caused by a loss-of-function mutation in one allele of NPHP1 and a different loss-of-function mutation in the other allele.

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

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

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the dose of the agonist of EP2 is 100 pM to 5 μM.

9. The use of at least one E-type prostaglandin receptor 2 (EP2) agonist for preparing a pharmacopoeia for use in the treatment of nephronophthria, The agonist of at least one EP2 comprises prostaglandin E1 (PGE1).

10. The use according to claim 9, wherein the agonist of EP2 further comprises prostaglandin E2 (PGE2), 16,16-dimethyl-PGE2 (dmPGE2), AGN-210669 (simenepag isopropyl), AGN-210961 (aganepag isopropyl), DE-117 (omidenepag isopropyl), CP-533536 (evatanepag), or a combination thereof.

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