Methods for diagnosing and / or treating acute or chronic liver, kidney or lung disease - Patents.com

Abstract

The present invention relates to a CNNM4 inhibitor for use in treating acute or chronic liver disease, renal disease and / or lung disease in a subject, and to a pharmaceutical composition comprising a therapeutically effective amount of a CNNM4 inhibitor and a pharmaceutically acceptable excipient or carrier. Further, the present invention relates to a method for diagnosing liver disease, renal disease or lung disease in a subject, and a method for identifying a compound potentially useful for alleviating CNNM4-mediated disease or pathology induced in a cell.

Description

[Technical field]

[0001] The present invention relates to the field of diagnosis and treatment of acute or chronic diseases. More specifically, the present invention relates to the use of CNNM4 as a marker of said diseases and to inhibitors of CNNM4 for treating liver disease, kidney disease and lung disease. [Background technology]

[0002] 2. Background of the Invention Chronic liver disease encompasses a group of liver pathologies from different etiologies. Nonalcoholic fatty liver disease (NAFLD), with a prevalence of 20-30% of the world's population, includes a spectrum of liver disorders ranging from simple lipid accumulation steatosis to the development of steatohepatitis (NASH), characterized by steatosis accompanied by inflammation and sometimes fibrosis. NAFLD is a rather benign and reversible condition, while about 20% of patients with NASH develop cirrhosis. Importantly, cirrhosis is an irreversible condition characterized by the deposition of extracellular matrix that leads to liver failure. Finally, approximately one-quarter of patients with fibrosis develop hepatocellular carcinoma (HCC), the most common form of liver cancer and the fifth leading cause of morbidity and mortality worldwide. Very few patients are eligible for therapeutic intervention at the time of diagnosis, and survival rates are indeed poor, ranging from only 6 to 20 months after diagnosis.

[0003] On the other hand, the liver can also be acutely damaged. This organ plays a central role in drug metabolism and clearance and can develop drug-induced liver injury (DILI) in case of drug overdose. This condition is the leading cause of acute liver failure and transplantation in the United States and most of Europe. Approximately 30,000 patients develop acetaminophen (APAP)-induced liver injury annually, 29% of whom undergo liver transplantation. To date, the only standard therapy is based on treatment with N-acetylcysteine, which is unlikely to salvage the liver, and therefore other therapies are needed.

[0004] Considering the wide range of liver pathologies and their prevalence among the population, both in terms of health and in terms of economy, the development of new effective treatments is required. It should also be mentioned that, since liver pathologies are relatively asymptomatic in their early stages, in addition to treatment, diagnostic tools are also needed to detect the disease as early as possible in order to stop its progression.

[0005] Renal fibrosis results in the excessive accumulation of extracellular matrix (ECM) seen in almost all types of chronic liver disease. As in the liver, the pathogenesis is a progressive process leading to end-stage renal failure, a destructive disease requiring dialysis or kidney transplantation. The process of ECM development and accumulation can be caused by two alterations. On the one hand, several cellular pathways have been identified as the main avenues by which ECM-producing cells arise in pathological conditions, and on the other hand, defective matrix degradation makes the exact action and mechanism of ECM-degrading enzymes increasingly complex. Although many therapeutic interventions seem to be effective in animal models, the translation of these promising results to humans remains elusive.

[0006] Pulmonary fibrosis is a disease that results in damage and scarring of lung tissue. Thickening and stiffness lead to obstruction and shallow breathing as fibrosis worsens. In most cases, the cause of the condition remains unknown and therefore it is usually known as idiopathic pulmonary fibrosis (IPF). In recent years, anti-fibrotic therapies have been developed, but there have been some attempts to identify key biomarkers that could direct more specific treatments. Summary of the Invention

[0007] The inventors unexpectedly found that CNNM4 is overexpressed in a group of different pathologies in both human samples and animal models.These pathologies include chronic liver pathologies such as NASH, cirrhosis and HCC, or acute pathologies such as DILI.CNNM4 overexpression has also been found in renal fibrosis mouse models.The inventors also found that in animal models of the disease, inhibition of CNNM4 reduces the level of biomarker indicators.

[0008] Thus, in a first aspect, the present invention relates to a CNNM4 inhibitor for use in medicine.

[0009] In a further aspect, the present invention relates to a CNNM4 inhibitor for use in the treatment of an acute or chronic disease in a subject, wherein the disease is selected from the group consisting of liver disease, renal disease and pulmonary disease.

[0010] In another aspect, the present invention provides an in vitro method for diagnosing liver disease, kidney disease, or lung disease in a subject, comprising: (a) determining the expression level of CNNM4 in a sample from the subject; and (b) comparing said level to a reference value. comprising An increase in the expression level of CNNM4 in said sample relative to a reference value indicates that said patient is suffering from a liver disease, a kidney disease or a lung disease.

[0011] In yet another aspect, the present invention relates to the use of specific reagents for determining the expression level of CNNM4 for in vitro diagnosis of liver disease, kidney disease or lung disease in a subject.

[0012] In a further aspect, the present invention relates to an in vitro method of alleviating an induced disease or pathology in a cell, comprising contacting said cell with a specific CNNM4 inhibitor in an amount effective to reduce the activity, level or function of CNNM4 in said cell.

[0013] In a final aspect, the present invention relates to an in vitro method for identifying a compound potentially useful for alleviating a CNNM4 mediated disease or pathology induced in a cell, comprising contacting said cell with a candidate compound in an amount effective for reducing the activity, level or function of CNNM4 in the cell, or in an amount effective for reducing lipid accumulation relative to a reference value or reducing relative mitochondrial ROS, wherein a candidate compound that inhibits CNNM4 activity, reduces lipid accumulation relative to a reference value or reduces mitochondrial ROS relative to a reference value in a CNNM4 mediated disease or pathology induced in the cell is identified as a compound potentially useful for treating and / or preventing a CNNM4 mediated liver, kidney and / or lung disease. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows hepatic CNNM4 expression determined by IHC in human samples from different pathologies of DILI and chronic liver disease and in mouse models. *p<0,05 vs. healthy; **p<0,01 vs. healthy; ***p<0,001 vs. healthy. [Diagram 2] Figure 2A shows CNNM4 expression as determined by qPCR of CNNM4 mRNA levels in human liver samples from healthy subjects compared to samples from patients with fatty liver and NASH. Figure 2B shows CNNM4 expression as determined by qPCR of CNNM4 mRNA levels in an in vivo mouse model. Figure 2C shows CNNM4 expression as determined by qPCR of CNNM4 mRNA levels in an in vitro mouse cell model. *p<0.05 vs. healthy. [Diagram 3]Figure 3A shows that lipid content in NASH-induced primary hepatocytes is restored to healthy levels upon treatment with siRNA CNNM4, Figure 3B shows that ROS-induced inflammation is also reversed in treated mice, and Figure 3C shows that DILI-induced cell death is ameliorated when hepatocytes are treated with siRNA therapy. *p<0.05 vs. healthy; **p<0.01 vs. healthy; ***p<0.001 vs. healthy; #p<0.05 vs. NASH; ##p<0.01 vs. NASH; ###p<0.001 vs. DILI. [Figure 4] Figure 4A shows that lipid content in NASH-induced human cells is restored to healthy levels when treated with siRNA CNNM4. Figure 4B shows that lipid content in NASH-induced human cells is restored to healthy levels when treated with shRNA CNNM4. *p<0.05 vs. healthy; **p<0.01 vs. healthy; #p<0.05 vs. NASH control; ##p<0.01 vs. NASH control. [Diagram 5] Figure 5A shows that lipid content in NASH-induced primary hepatocytes does not return to healthy levels when treated with siRNA CNNM1, siRNA CNNM2 or siRNA CNNM3. Figure 5B shows that magnesium supplementation does not reduce lipid content in primary hepatocytes in the case of CNNM4 overexpression. Figure 5C shows that siRNA CNNM4 treatment in primary hepatocytes reduces lipid accumulation caused by magnesium deficiency. ***p<0.001 vs. healthy; ##p<0.01 vs. NASH model / Mg2+-free + siRNA φ (empty population). [Figure 6] Figure 6 shows the parameters analyzed to monitor NAFLD progression after siRNA CNNM4 therapy. Figure 6A shows Sudan Red as an indicator of lipid content reduction, Figure 6B shows GPT in serum as an indicator of liver damage, Figure 6C shows DHE as an indicator of ROS-induced inflammation, and Figure 6D shows αSMA as an indicator of fibrosis. *p<0.05 vs. siRNA φ (null set); **p<0.01 vs. siRNA φ (null set). [Figure 7]Figure 7 shows that pharmacological inhibition of CNNM4 by 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one has the same effect as siRNA CNNM4 therapy in NASH-induced hepatocytes. Figure 7A shows that treated hepatocytes have decreased lipid levels, and Figure 7B shows that treated hepatocytes have increased intracellular magnesium levels. *p<0.05 vs. untreated; ***p<0.001 vs. untreated. [Figure 8] Figure 8 shows CNNM4 expression determined by IHC in animal samples with renal fibrosis. ***p<0.001 vs. healthy. [Figure 9] Figure 9A shows CNNM4 expression determined in TCGA (The Cancer Genome Atlas) primary tumor samples of hepatocellular carcinoma (LIHC) compared to normal tissue. Figure 9B shows CNNM4 expression determined in TCGA (The Cancer Genome Atlas) primary tumor samples of lung adenocarcinoma (LUAD) compared to normal tissue. Description of the Invention

[0015] As mentioned before, the present inventors unexpectedly found that CNNM4 is overexpressed in a group of different pathologies in both human samples and animal models.These pathologies include chronic liver pathologies such as NASH, liver cirrhosis and HCC, or acute pathologies such as DILI.CNNM4 overexpression has also been found in renal fibrosis mouse models.The present inventors also found that CNNM4 inhibition reduces the level of biomarker indicators in animal models of said diseases.

[0016] Medical Uses of CNNM4 Inhibitors Thus, in a first aspect, the present invention relates to a CNNM4 inhibitor for use in medicine (ie for use as a medicament).

[0017] In a second aspect, the present invention relates to a CNNM4 inhibitor for use in the treatment of an acute or chronic disease in a subject, said disease being selected from the group consisting of liver disease, renal disease and pulmonary disease.

[0018] As used herein, the term "CNNM4" refers to "Cyclin and CBS domain divalent metal cation transport mediator" (Ancient Conserved Domain Protein, ACDP, also known as Cyclin M or CNNM). CNNM is a membrane protein encoded by four genes CNNM1, CNNM2, CNNM3 and CNNM4 that are evolutionarily expressed in all adult tissues during development, except for CNNM1, which is expressed primarily in the brain. CNNM plays an important role in the transport of magnesium ions (Mg2+) across cell membranes in various organs (Funato et al., 2014. J Clin Invest.124(12):5398-5410). CNNM4 corresponds to the human gene identified in the Ensembl database by ID number ENSG00000158158 (from release 93 of July 2018). According to Ensembl database, CNNM4 encodes at least four transcripts or splice variants.Therefore, the present disclosure relates to variant CNNM4-201 (ENST00000377075.2) and other three variants CNNM4-204 (ENST00000496186.5), CNNM4-203 (ENST00000493384.1) and CNNM4-202 (ENST00000482716.5).CNNM4 gene encodes protein "metal transporter CNNM4" identified by Q6P4Q7 (according to version 130 of October 10, 2018) in Uniprot database.

[0019] As used herein, the term " Inhibitors" (inhibitor) is understood as any substance or compound that can specifically silence, reduce, suppress and / or block the expression of the gene by suppressing the transcription of the gene CNNM4, thus avoiding the formation of any of the transcripts of the gene, or by promoting the degradation of any of the transcripts of the gene CNNM4, or by specifically reducing, suppressing and / or blocking the expression of the encoded protein, as well as any compound that inhibits the activity of the CNNM4 protein. In one embodiment, a CNNM4 inhibitor is any substance or compound that specifically silences, reduces, suppresses and / or blocks the expression of the gene by suppressing the transcription of the gene CNNM4, thus avoiding the formation of any of the transcripts of the gene, or by promoting the degradation of any of the transcripts of the gene CNNM4, or by specifically reducing, suppressing and / or blocking the expression of the encoded protein.

[0020] The terms "transcript" or "transcript" as used herein refer to RNA derived from transcription of a gene.

[0021] The expression of a protein or nucleic acid is considered to be reduced when its level is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., not present) relative to the reference value. The reference value refers to the mRNA or protein level in a control subject, which may be a subject that does not suffer from a particular disease and a subject that is generally considered healthy. Alternatively, the reference value may refer to the mRNA or protein level in a subject before administration of an inhibitor. In the context of the present invention, the reference value refers to the protein or mRNA level of CNNM4 in a control subject or a subject before administration of an inhibitor. In one embodiment, the control subject is a subject that does not suffer from liver disease, kidney disease or lung disease.

[0022] The activity of CNNM4 protein is considered to be inhibited when said activity is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., not present) with respect to the reference value. In one embodiment, the reference value refers to the activity of CNNM4 protein in a sample from a control subject, which may be a subject that does not suffer from a particular disease and a subject that is generally considered healthy. Alternatively, the reference value refers to the activity of CNNM4 protein in a sample from a subject before administration of a CNNM4 inhibitor. In the context of the present invention, the reference value refers to the activity of CNNM4 protein in a control subject or a subject before administration of a CNNM4 inhibitor. In one embodiment, the control subject is a subject that does not suffer from liver disease, kidney disease or lung disease.

[0023] "Reference value" as used herein refers to a test value that is used as a reference for the value / data obtained from a sample. Reference value (or reference level) can be an absolute value, a relative value, a value with upper and / or lower limits, a series of values, an average value, a median value, an average value, or a value expressed by referring to a control value or a reference value. Reference value can be based on the value obtained from an individual sample, such as, for example, a value obtained from a test sample but at a previous time point. Reference value can be based on a number of samples, such as a value obtained in a sample population or a value based on a pool of samples including or excluding test samples.

[0024] Suitable methods for determining the expression level of CNNM4 are known in the art and include any suitable method for determining the expression and / or protein level of CNNM4 gene.In certain embodiments, the reference value is the level of mRNA transcribed from CNNM4 gene in the absence of the inhibitor of the present invention.

[0025] Suitable methods for determining the expression level of the CNNM4 gene include, but are not limited to, standard assays for determining the expression level of mRNA, such as qPCR, RT-PCR, RNA protection analysis, Northern blot, RNA dot blot, in situ hybridization, microarray technology, tag-based methods such as serial analysis of gene expression (SAGE) methods, including variations such as LongSAGE and SuperSAGE, microarrays, fluorescent in situ hybridization (FISH) (including variations such as Flow-FISH, qFiSH and double fusion FISH (D-FISH)), and the like.

[0026] Suitable methods for determining the expression level of CNNM4 protein include, but are not limited to, conventional quantification using, for example, an antibody that can specifically bind to CNNM4 protein and then quantifying the resulting antibody-antigen complex.There is a wide range of well-known assays that can be used in the present invention using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies), including, among others, Western blot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry and immunohistochemistry techniques, techniques based on the use of biochips or protein microarrays that contain specific antibodies, or assays based on colloidal precipitation in the form of dipsticks, etc.Other methods for detecting and quantifying the level of the protein of interest include affinity chromatography techniques, ligand binding assays, etc.

[0027] In a preferred embodiment, the inhibitor of the expression of CNNM4 is selected from the group consisting of protein, nucleic acid, small molecule or combination thereof.Alternatively, the inhibitor of the expression of CNNM4 is selected from the group consisting of protein, nucleic acid or combination thereof.Preferably, the inhibitor of the expression of CNNM4 is selected from the group consisting of neutralizing antibody or its functional fragment, antagonist, soluble binding protein, soluble receptor mutant, non-functional derivative, antisense polynucleotide, RNA interference oligonucleotide, phosphorodiamidate morpholino oligomer (PMO), miRNA, siRNA, methylated siRNA, processed siRNA, shRNA, antisense RNA, dicer substrate 27-mer duplex, aptamer, DNAzyme, ribozyme, triplex forming oligonucleotide (TFO), small molecule and combination thereof.

[0028] In some embodiments, the inhibitor of CNNM4 is a neutralizing antibody or a functional fragment thereof. As used herein, the term "antibody" refers to a protein that contains at least one immunoglobulin variable region, such as an amino acid sequence that provides an immunoglobulin variable domain or a sequence of an immunoglobulin variable domain. A "functional fragment" is any fragment of an antibody that can retain its ability to inhibit CNNM4. An antibody can, for example, contain a heavy chain variable (H) region (abbreviated herein as VH) and a light chain variable (L) region (abbreviated herein as VL). Generally, an antibody contains two heavy chain variable regions and two light chain variable regions. The term "antibody" encompasses antigen-binding antibody fragments (e.g., single chain antibodies, nanobodies (VHH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments and dAb fragments) of IgA, IgG types (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM (and subtypes thereof) as well as complete antibodies, e.g., intact and / or full-length immunoglobulins. The heavy and light chain variable regions can be further subdivided into hypervariable regions called "complementarity determining regions" ("CDRs") interspersed with more conserved regions called "framework regions" (FRs). The extensions of FRs and CDRs have been precisely defined (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., The United States Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The Kabat definitions are used herein. Each heavy and light chain variable region is generally formed of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The VH or VL chain of an antibody may further comprise all or part of a heavy or light chain constant region, thereby forming a heavy chain (HC) or light chain (LC) immunoglobulin, respectively. The immunoglobulin light and heavy chains may be linked by disulfide bridges. The heavy chain constant region generally comprises three constant domains, CH1, CH2 and CH3. The light chain constant region generally comprises a CL domain. The heavy and light chain variable regions comprise a binding domain that interacts with an antigen. The constant region of an antibody generally mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term antibody encompasses both antibodies formed by heavy and light chains and single chain antibodies.

[0029] As used herein, the term "heavy chain" or "HC" encompasses both full-length heavy chains and fragments thereof. A full-length heavy chain comprises a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH3 domain is at the carboxyl-terminus.

[0030] As used herein, the term "light chain" encompasses a full-length light chain and fragments thereof. A full-length light chain contains a variable region domain, VL, and a constant region domain, CL. Like the heavy chain, the light chain variable region domain is at the amino-terminus of the polypeptide.

[0031] As used herein, the term "single chain antibody" refers to a genetically engineered molecule containing a light chain variable region and a heavy chain variable region joined by a suitable peptide linker formed as a genetically fused single chain molecule.

[0032] As used herein, the term "nanobody" refers to a single domain antibody (sdAb), which is an antibody fragment consisting of a single monomeric variable antibody domain. Like complete antibodies, nanobodies can selectively bind to a specific antigen.

[0033] As used herein, the term "antibody mimetic" refers to any compound that can specifically bind to an antigen in a manner similar to an antibody, but is not necessarily structurally related to an antibody. A "mimetic" of a compound includes compounds in which the chemical structure of the compound required for functional activity is replaced with other chemical structures that mimic the conformation of the compound. Examples of mimetics include peptide compounds in which the peptide backbone is replaced with one or more benzodiazepine molecules (see, e.g., James, GL et al. (1993) Science 260: 1937-1942) or oligomers that mimic peptide secondary structures by using amide bond equivalents and / or modifying the natural peptide backbone, including chain extension or incorporation of heteroatoms, examples of which include azapeptides, oligocarbamates, oligoureas, beta-peptides, gamma-peptides, oligo(phenyleneethynylene), vinylogous sulfonopeptides, poly-N-substituted glycines (peptoids), and the like. Methods for preparing peptidomimetic compounds are well known in the art and are set forth, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[0034] As used herein, the term antibody also refers to "non-immunoglobulin agents" as binding agents other than immunoglobulins, based on differences in molecular properties, topology, or scaffolds. The term scaffold is meant to represent a protein framework that may have altered amino acids or sequence insertions that confer different functions to the protein variants, usually related to binding to specific targets. Examples of such non-immunoglobulin agents are well known in the art and include, but are not limited to, peptide aptamers, nucleic acid aptamers, affibody molecules, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptide monobodies, and other protein scaffolds are reviewed in Binz et al., 2005 (Nat. Biotech. 23:1257-68), which is incorporated herein by reference.

[0035] According to the present invention, antibodies can be "humanized" to reduce immunogenicity in human individuals. Humanized antibodies improve the safety and efficacy of monoclonal antibody therapy. One common method of humanization is to produce a monoclonal antibody in any suitable animal (e.g., mouse, rat, hamster) and replace its constant region with a human constant region, and antibodies engineered in this way are called "chimeras." Another common method is "CDR grafting," which replaces non-human V-FR with human V-FR. In this CDR grafting method, all residues except the CDR region are of human origin.

[0036] In another embodiment, the inhibitor of the expression of CNNM4 is an antagonist. As disclosed herein, an "antagonist" is a type of receptor ligand or compound that blocks or attenuates a biological response by binding to and blocking the receptor, rather than activating it as an agonist does. Alternatively, another proteinaceous agent that can downregulate the activity of CNNM4 can be its non-functional derivative (i.e., dominant negative). Peptides and others that mimic these non-functional derivatives can be synthesized using solid-phase peptide synthesis methods that are well known in the art and are described in more detail by John Morrow Stewart and Janis Dillaha Young, [Solid Phase Peptide Syntheses (2nd Edition, Pierce Chemical Company, 1984)]. Synthetic peptides can be purified by preparative high performance liquid chromatography, and their composition can be confirmed by amino acid sequencing.

[0037] Another class of CNNM4 inhibitors according to the present invention comprises specific soluble binding proteins. These binding proteins do not bind to CNNM4 by themselves. However, they form a complex with the relevant binding site of CNNM4, thereby preventing CNNM4 from interacting with effector proteins. In another embodiment, said CNNM4 binding inhibitor is a soluble receptor mutant that is generated by proteolytic cleavage of membrane-bound receptor or by translation of alternatively spliced ​​receptor RNA. Many of these soluble receptors lack the transmembrane and cytoplasmic domains that are usually found in membrane-bound receptors.

[0038] In another particular embodiment, the CNNM4 inhibitor is a nucleic acid that specifically binds to the CNNM4 gene or a transcription product of said gene and blocks the expression of said gene.

[0039] "Nucleic acid" as used herein means a biological polymer (polynucleotide, polynucleic acid) of nucleotides linked together by phosphodiester bonds. The nucleotides of a nucleic acid may additionally or instead be linked by phosphorothioate or phosphorodithioate bonds, if chemically synthesized. Depending on the type of sugar of the nucleotide (ribose or deoxyribose), two types are distinguished: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). As used herein, it relates to either natural or non-natural nucleic acids, and "oligonucleotide" and "polynucleotide" are used interchangeably in the context of the present invention. In the context of the present invention, "natural nucleotide" means a nucleotide that can be purified from a natural source. "Non-natural nucleotide" is defined as one that is produced using a recombinant expression system and optionally purified, one that is chemically synthesized, etc. Where applicable, for example in the case of chemically synthesized molecules, these nucleic acids may comprise nucleoside analogues, e.g. analogues with chemically modified bases or sugars, backbone modifications, etc. Nucleic acid sequences are represented in the 5'-3' direction unless otherwise indicated.

[0040] In some embodiments, the inhibitor of CNNM4 is antisense polynucleotide.As used herein, "antisense polynucleotide" refers to antisense polynucleotide or sense polynucleotide that comprises single-stranded nucleic acid sequence (RNA or DNA) that can bind to target mRNA sequence (sense) or DNA sequence (antisense).In a preferred embodiment, antisense polynucleotide is antisense RNA.In another embodiment, the inhibitor of CNNM4 is RNA interference oligonucleotide.

[0041] In another embodiment, the inhibitor of CNNM4 is a miRNA. The term "microRNA" or "miRNA" refers to a short, single-stranded RNA molecule, generally around 21-23 nucleotides in length, that can regulate gene expression. miRNAs can be synthetic (i.e., recombinant) or natural. Natural miRNAs are encoded by genes that are transcribed from DNA and processed from the primary transcript ("pri-miRNA") into a short stem-loop structure ("pre-miRNA"), and finally into the mature miRNA. Mature miRNA molecules are partially complementary to one or more mRNA molecules and downregulate gene expression through a process similar to RNA interference or by inhibiting translation of the mRNA.

[0042] In another embodiment, the inhibitor of CNNM4 is an siRNA. The term "small interfering RNA" ("siRNA") refers to a small inhibitory RNA duplex that induces the RNA interference pathway. Small interfering RNA (siRNA) is generated in cells by enzymatic cleavage of long dsRNA by the RNase-III class endoribonuclease Dicer. siRNA associates with the RNA-induced silencing complex (RISC) in a process facilitated by Dicer. The Dicer substrate RNAi method exploits the binding between Dicer and RISC loading seen when RNA is processed by Dicer. Conventional 21-mer siRNAs are chemically synthesized RNA duplexes that mimic the Dicer product and bypass the need for Dicer processing. Dicer substrate RNAs are chemically synthesized RNA duplexes optimized for Dicer processing. These molecules can be of variable length (generally 18-30 base pairs) and contain various complementarities to their target mRNA in the antisense strand. In certain embodiments, the inhibitor of CNNM4 is a Dicer substrate 27-mer duplex.

[0043] Some, but not all, siRNAs have unpaired overhanging bases at the 5' or 3' end of sense strand and / or antisense strand.The term "siRNA" includes the duplex of two individual strands.As used herein, siRNA molecule is not limited to RNA molecule, but further includes nucleic acid having one or more chemically modified nucleotides, such as morpholino.

[0044] In a preferred embodiment, the CNNM4 inhibitor is an siRNA. An siRNA is a nucleic acid that mediates RNA interference (iRNA). In a particular embodiment, the siRNA comprises sequences having SEQ ID NO: 13 and SEQ ID NO: 14. In another particular embodiment, the siRNA comprises any of the sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 50, or SEQ ID NO: 51. In another particular embodiment, the siRNA comprises a double-stranded siRNA against human CNNM4, preferably targeting the sequence 5'-GCGAGAGCAUGAAGCUGUAUGCACU-3' (SEQ ID NO: 25) (Yamazaki D, et al. (2013), PLoS Genet 9(12): e1003983). In a further particular embodiment, the siRNA comprises at least one of the sequences having the SEQ ID NOs shown in Table 1. These sequences target either mouse CNNM4 or human CNNM4 as shown, or in some cases both mouse CNNM4 and human CNNM4.

[0045] [Table 1] TIFF0007672337000002.tif179156

[0046] In another embodiment, the inhibitor of CNNM4 is shRNA. As used herein, the term "shRNA" or "short hairpin RNA" refers to double-stranded RNA (dsRNA) in which two strands are linked by a non-interfering strand to the nucleotide between the 3' end of one strand and the 5' end of the corresponding other strand to form a duplex structure. shRNA can be used to silence target gene expression by RNA interference, because once processed, shRNA becomes located in RNA-induced silencing complex (RISC) and targets RISC to mRNA with complementary sequence. RISC can cleave this mRNA or suppress its translation. In a particular embodiment, shRNA comprises a sequence having SEQ ID NO:52.

[0047] In another embodiment, the inhibitor of CNNM4 is sgRNA. As disclosed herein, the term single guide RNA ("sgRNA") refers to a chimeric non-coding RNA that contains a targeting sequence (crRNA sequence) that specifically recognizes a gene region and a CRISPR enzyme. The crRNA region is a 20-nucleotide sequence that is homologous to the gene region of interest and directs CRISPR enzyme activity. As disclosed herein, the CRISPR enzyme can be any known in the art, such as Cas9 and Cpf1.

[0048] As disclosed herein, CRISPR is a genome editing method. The use of this technology in genome editing has been well described in the art, for example, in U.S. Pat. No. 8,697,359 and references cited herein. Briefly, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes and non-coding RNA elements (sgRNAs) that can program the specificity of CRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPR systems have been identified in a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of repeat sequence arrays (direct repeats) inserted with short stretches of non-repetitive sequences (spacers). The non-coding CRISPR arrays are transcribed and cleaved within the direct repeats into short crRNAs containing individual spacer sequences, which guide the Cas nuclease to the target site (protospacer). Type II CRISPR is one of the better-characterized systems and performs targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of pre-crRNA and mediates processing of pre-crRNA into mature crRNA, which contains individual spacer sequences. Third, the mature crRNA:tracrRNA complex guides Cas9 to the target DNA by Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA adjacent to the protospacer adjacent motif (PAM) that is further required for target recognition. Finally, Cas9 mediates cleavage of the target DNA to create a double-strand break within the protospacer.

[0049] One major advantage of the CRISPR-Cas9 system compared to traditional gene targeting and other programmable endonucleases is the ease of multiplexing, allowing multiple genes to be mutated simultaneously using multiple sgRNAs, each targeting a different gene. Furthermore, two sgRNAs that flank a genomic region can be used to delete or invert the segment between them.

[0050] Thus, Cas9 is the hallmark protein of type II CRISPR-Cas systems and is a large monomeric DNA nuclease that is guided to DNA target sequences flanked by a PAM (protospacer adjacent motif) sequence motif by a complex of two non-coding RNAs: CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains that are homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, whereas the RuvC-like domain cleaves the non-complementary strand, resulting in the introduction of a blunt break in the target DNA. Heterologous expression of Cas9 together with sgRNA allows the introduction of site-specific double-strand breaks (DSBs) in genomic DNA of living cells from various organisms. For application in eukaryotes, the Cas9 protein has been successfully used in Streptococcus pyogenes ( Streptococcus pyogenes ), but instead uses a codon-optimized variant of Cas9 originating from Staphylococcus aureus ( Staphylococcus aureus )Cas9 (SaCas9), Campylobacter jejuni ( Campylobacter jejuni Other suitable Cas9 orthologues, such as Cas9 (CjCas9), can also be used.

[0051] Single guide RNA (sgRNA) is the second component of the CRISPR / Cas system that forms a complex with Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA and tracrRNA. The sgRNA guide sequence located at its 5' end confers target specificity to DNA. Thus, sgRNAs with different target specificities can be created by modifying the guide sequence. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs are expressed using plant RNA polymerase III promoters such as U6 and U3.

[0052] Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.

[0053] "crRNA" or CRISPR RNA means an RNA sequence that includes a protospacer element and additional nucleotides complementary to the tracrRNA.

[0054] "tracrRNA" (trans-activating RNA) refers to an RNA sequence that hybridizes to crRNA and binds to a CRISPR enzyme, such as Cas9, thereby activating a nuclease complex to introduce a double-stranded break at at least one specific site in alpha-, gamma- and / or omega gliadin in the genomic sequence.

[0055] "Protospacer element" refers to the portion of the crRNA (or sgRNA) that is complementary to the genomic DNA target sequence (usually around 20 nucleotides in length). This may also be known as the spacer or target sequence.

[0056] "sgRNA" (single guide RNA) refers to the combination of tracrRNA and crRNA as a single RNA molecule, preferably including a linker loop (connecting tracrRNA and crRNA into a single molecule). "sgRNA" may also be referred to as "gRNA", and these terms are used interchangeably herein. The sgRNA or gRNA provides both target specificity and anchorage / binding ability for Cas nucleases. gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule. The sgRNA can be provided as an RNA molecule or as a DNA molecule that is transcribed into a functional sgRNA.

[0057] "CRISPR enzyme" refers to an RNA-guided DNA endonuclease that can be associated with the CRISPR system. In particular, such an enzyme binds to the tracrRNA sequence. In one embodiment, the CRISPR enzyme is a Cas protein ("CRISPR-associated protein"), preferably Cas9 or Cpf1.

[0058] In another embodiment, the inhibitor of CNNM4 is an aptamer. As used herein, the term "aptamer" refers to an oligonucleotide that selectively binds to a target ligand but does not catalyze a subsequent chemical reaction. The term "peptide aptamer" refers to a short variable peptide domain that is attached at both ends to a protein scaffold and binds to a specific target molecule. The variable loop length is generally composed of 10-20 amino acids, and the scaffold can be any protein with good solubility and compaction properties. The term "nucleic acid aptamer" or "DNA aptamer" as used herein refers to a short DNA strand that has been engineered by iterative selection to bind to a specific molecular target.

[0059] In another embodiment, the inhibitor of CNNM4 is a DNAzyme. As used herein, the term "DNAzyme" refers to a DNA oligonucleotide, usually catalytic, that can carry out a specific chemical reaction.

[0060] In another embodiment, the inhibitor of CNNM4 is a ribozyme.As used herein, the term "ribozyme" or "RNA enzyme" or "catalytic RNA" refers to an RNA molecule that catalyzes chemical reactions.Ribozymes can be used to hydrolyze phosphodiester bonds in other RNA.Nucleic acids that have the ability to inhibit the expression of CNNM4 can contain one or more modifications in the nucleobase, sugar, and / or internucleotide bond.

[0061] In another embodiment, the inhibitor of CNNM4 is a triplex-forming oligonucleotide. As used herein, the term "triplex-forming oligonucleotide (TFO)" refers to an oligonucleotide capable of forming a triplex, which binds in a sequence-specific manner into the major groove of double-stranded DNA through the formation of hydrogen bonds. All of the inhibitors described above may be subject to one or more modifications. Modifications to one or more residues of the nucleic acid backbone may comprise one or more of the following: sugar modifications at 2', such as 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-methoxyethoxy, 2'-fluoro (2'-F), 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 2'-O-(N-methylcarbamate); sugar modifications at 4', including 4'-thio, 4'-CH2-O-2' bridge, 4-(CH2)2-O-2' bridge; intercalating nucleic acid (INA); twisted intercalating nucleic acid (TINA); hexitol nucleic acid (HNA); arabino nucleic acid (ANA); cyclohexene nucleic acid (CNA); cyclohexenyl nucleic acid (CeNA); threose nucleic acid (TNA); morpholine oligonucleotides; gapmers; mixmers; incorporation of arginine-rich peptides; addition of 5'-phosphates to synthetic RNA; RNA aptamers ((Que-Gewirth NS, Gene Ther. 2007 Feb;14(4):283-91); RNA aptamers controlled by antidotes in the context of a particular RNA aptamer (see Oney S, Oligonucleotides. 2007 Fall;17(3):265-74)), or any combination thereof.

[0062] Modifications of one or more nucleoside linkages of a nucleic acid may comprise one or more of the following: phosphorothioate, phosphorodithioate, phosphoramidate, phosphorodiamidate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, and phosphoroanilidate, or any combination thereof.

[0063] Locked Nucleic Acids (LNA), also commonly called inaccessible RNA, are modified RNA nucleotides. The ribose group of LNA nucleotides is modified with an extra bridge (O2', C4'-methylene bridge) connecting carbons 2' and 4'. This bridge "locks" the ribose in the 3'-endo conformation, which is typically found in A-form DNA or RNA. LNA nucleotides can also be mixed with DNA or RNA bases in a nucleic acid as desired. Such oligomers are commercially available.

[0064] Peptide nucleic acid (PNA) is an artificially synthesized polymer whose backbone consists of repeating units of N-(2-aminoethyl)-glycine linked by peptide bonds. Various purine and pyrimidine bases are linked to the backbone by methylene-carbonyl bonds.

[0065] Intercalating nucleic acids (INA) are modified nucleic acid analogs that comprise a normal deoxyribonucleotide covalently linked to a hydrophobic intercalation.

[0066] Hexitol nucleic acid (HNA) is a nucleotide formed by a natural nucleobase and a phosphorylated 1,5-anhydrohexitol backbone. The molecular association between HNA and RNA is more stable than that between HNA and DNA and between natural nucleic acids (dsDNA, dsRNA, DNA / RNA). Other synthetically modified oligonucleotides include ANA (arabinonucleic acid), CNA (cyclohexene nucleic acid), CeNA (cyclohenexyl nucleic acid), and TNA (threose nucleic acid).

[0067] Morpholinos are synthetic molecules that are the product of redesigning natural nucleic acid structures. Structurally, morpholinos differ from DNA or RNA in that they have standard nucleobases, but the bases are linked to a six-membered morpholine ring rather than a deoxyribose / ribose ring, and the nonionic phosphorodiamidate bonds between the subunits are replaced by anionic phosphodiester bonds. Morpholinos are sometimes referred to as PMOs (phosphorodiamidate morpholino oligonucleotides). The six-membered morpholine ring has the chemical formula O(CH2CH2)2NH.

[0068] Gapmers, or "gapped oligomeric compounds", are RNA-DNA-RNA chimeric oligonucleotide probes in which DNA windows or gaps, known as "wings", have been inserted into normal or modified RNA oligonucleotides. This modification increases the stability of the oligonucleotide in vivo and makes the probe more amenable to interacting with the target, allowing shorter probes to be used effectively. Preferably, these wings are modified 2'-O-methyl (OMe) or 2'-O-methoxyethyl (MOE) oligonucleotides that protect the internal blocks from nuclease degradation. Additionally, the nucleotides forming the gaps or wings can be linked by phosphodiester or phosphorothioate bonds, thereby making them resistant to RNase degradation. Additionally, the nucleotides forming the wings can also be modified by the incorporation of bases linked by 3'-methylphosphonate bonds.

[0069] In another embodiment, the inhibitor of CNNM4 is a small molecule. As used herein, the term "small molecule" refers to an organic compound with a low molecular weight (<900 Daltons) that can regulate biological processes. Small molecules can inhibit a specific function of a protein or disrupt protein-protein interactions. In a preferred embodiment, the small molecule is a rhodanine derivative known as 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (PubChem CID 91383855) or 2-[5-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-furan-2-yl]-benzoic acid as described in Park, H., et al. 2008. Bioorg Med Chem Lett. 18(7):2250-5 (Chemspider ID 1014170).

[0070] In another embodiment, the inhibitor of CNNM4 is any combination of the above mentioned inhibitors.

[0071] As used herein, the term " treatment " includes any type of therapy aimed at terminating, preventing, ameliorating and / or reducing susceptibility to a disease state as described herein. Thus, "treatment" as used herein includes any treatment of a disease state or disorder in a mammal, including a human, to obtain a desired pharmacological and / or physiological effect. The effect may be prophylactic in that the disorder or its symptoms are completely or partially prevented, and / or may be therapeutic in that the disorder and / or adverse effects that may result from the disorder are partially or completely cured. It includes any treatment of disease in a mammal, particularly a human, including (a) extending survival; (b) reducing the risk of death from the disease; (c) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as suffering from it; (d) inhibiting the disease, i.e., arresting its onset (e.g., slowing down the rate of progression of the disease); and (e) palliating the disease, i.e., causing the disease to regress.

[0072] As used herein, " disease " means a condition of a living animal or one of its parts that impairs normal function and is generally evidenced by distinct signs and symptoms. The animal of the preferred embodiment is a mammal, preferably a human.

[0073] As used herein, " Acute illness " means a condition that is severe and of sudden onset.

[0074] As used herein, " chronic disease " refers to a condition that develops over a long period of time. Acute illnesses can lead to chronic illnesses.

[0075] term" subject ", "patient" or "individual" are used interchangeably herein to refer to any member of the animal kingdom, and may be a vertebrate such as a mammal, fish, bird, reptile, or amphibian, including a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. Preferably, the subject is a mammal, more preferably a human.

[0076] According to the present invention, the disease is selected from the group consisting of liver disease, kidney disease and lung disease. In one embodiment, the disease is a non-proliferative disease (i.e., the disease is a disease other than cancer).

[0077] As used herein, " Liver disease" is acute or chronic damage to the liver, usually caused by infection, injury, exposure to drugs or toxic compounds, alcohol, impurities in food, and an abnormal increase in normal substances in the blood, autoimmune processes, or genetic defects (such as hemochromatosis). The exact cause of the injury may not be known. Liver disease can be classified as acute or chronic liver disease based on the duration of the disease. In acute liver diseases, such as acute hepatitis and acute liver failure (ALF), the history of the disease does not exceed six months. Liver disease of longer duration is classified as chronic liver disease. Non-limiting examples of common liver diseases include cirrhosis, liver fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic ischemia-reperfusion injury, primary biliary cirrhosis (PBC), and hepatitis, including viral hepatitis and alcoholic hepatitis. The most common forms of viral hepatitis are hepatitis B and hepatitis C (HBV and HCV, respectively). Chronic hepatitis can lead to cirrhosis of the liver. Death of liver cells by a process known as apoptosis is common in all forms of liver disease. Apoptosis of liver cells leads to liver fibrosis and other liver diseases.

[0078] Liver disease, particularly drug-induced liver disease (Drug-Induced Liver Injury or DILI), manifests itself clinically with a variety of symptoms, including no specific information. Non-limiting examples of symptoms include loss of appetite, fatigue, dizziness, weight loss, nausea, vomiting, fever, pain in the right upper abdominal region, joint pain, muscle pain, itching, rash, discoloration of stool, including jaundice of the eyes and skin.

[0079] As the experts in this field know, the presence of active liver disease is often detected by the presence of increased enzyme levels in blood.In particular, blood levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) that exceed clinically acceptable normal range are known to be indicators of ongoing liver damage.In order to evaluate the progression of liver disease during medical treatment, it is clinically used to follow up liver disease patients periodically with respect to ALT and AST blood levels.The reduction of increased ALT and AST to acceptable normal range is considered as clinical evidence reflecting the reduction of the severity of ongoing liver damage in patients.In certain embodiments, liver disease is caused by any kind of liver damage.

[0080] The term "liver injury" as used herein refers to any type of liver injury (injury), including chronic and acute injury and pathological changes present in liver cells or liver tissue. Pathologies of liver injury may include, but are not limited to, degeneration of liver cells, vasculitis of the liver, simple or focal necrosis present in the liver, inflammatory cell infiltration or fibroblast proliferation in the liver and portal tract, or hepatomegaly, and cirrhosis, hepatoma resulting from severe liver injury, etc. Liver disease results from injury to the liver. Liver injury may be caused by toxins, including alcohol, some drugs, impurities in food, abnormal increase in normal substances in the blood, by infection, or by autoimmune disorders. In some cases, liver injury resulting from injury to the liver includes, but is not limited to, fatty liver, cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and alpha-1 antitrypsin deficiency. Liver injuries include, but are not limited to, cirrhosis, liver fibrosis, nonalcoholic fatty disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic ischemia-reperfusion injury, hepatitis including viral hepatitis and alcoholic hepatitis, and primary biliary cirrhosis (PBC).

[0081] In a preferred embodiment, the liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), Budd-Chiari syndrome and hepatitis, or any combination thereof. In a more preferred embodiment, the liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), Budd-Chiari syndrome and hepatitis, or any combination thereof. In a more preferred embodiment, the liver disease is selected from drug-induced liver injury (DILI), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), or any combination thereof. In a more preferred embodiment, the liver disease is selected from drug-induced liver injury (DILI), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), or any combination thereof.

[0082] As used herein, "fibrosis" is the formation of excess fibrous connective tissue in an organ or tissue in a repair or reactive process. It can be reactive, benign, or pathological. When in response to injury, it is called scarring, and when fibrosis arises from a single cell lineage, it is called fibroma. Physiologically, fibrosis serves to deposit connective tissue, which can interfere with or completely inhibit the structure and function of the underlying organ or tissue. Fibrosis can be used to describe the pathological condition of excess deposition of fibrous tissue, as well as the process of connective tissue deposition in healing. When defined by the pathological accumulation of extracellular matrix (ECM) proteins, fibrosis results in scarring and thickening of affected tissue, which is essentially an exaggerated wound healing response that interferes with normal organ function. Fibrosis can affect any organ. In a preferred embodiment, the fibrosis is liver fibrosis.

[0083] As used herein, "liver fibrosis" is the scarring process that corresponds to the liver's response to injury.Similar to how skin and other organs heal wounds by depositing collagen and other matrix components, the liver also repairs injury by depositing new collagen.Liver fibrosis is the first stage of liver scarring.Subsequently, if more parts of the liver are scarred, it can lead to liver cirrhosis.

[0084] As used herein, "veno-occlusive liver disease" or "hepatic sinusoidal obstruction syndrome (SOS)" is characterized by hepatomegaly, right upper quadrant pain, jaundice, and ascites, and is seen most frequently in patients undergoing hematopoietic cell transplantation (HCT), and less commonly in the non-transplant setting after use of certain chemotherapeutic agents, oral ingestion of alkaloid toxins, after high-dose radiation therapy, or with liver transplantation. The hepatic venous outflow obstruction in SOS is due to obstruction of the terminal hepatic venules and hepatic sinusoids.

[0085] As used herein, "drug-induced liver injury (DILI)" means damage to the liver that occurs following the use of one or more drugs.

[0086] As used herein, "steatosis", also called fatty change, is a process that represents the abnormal retention of lipids in cells. It reflects the disturbance of the normal process of synthesis and excretion of triglyceride fats. Excess lipids accumulate in vesicles and displace the cytoplasm. If the vesicles are large enough to distort the nucleus, the condition is known as macrodroplet steatosis, otherwise the condition is known as microdroplet steatosis. While minor injuries are not particularly harmful to the cell, large accumulations destroy cellular components, and in severe cases, cells may even burst. Risk factors associated with steatosis are varied and include diabetes, protein malnutrition, hypertension, cytotoxins, obesity, anoxia, and sleep apnea. The liver is the major organ of lipid metabolism, so it is most often associated with steatosis, but it can occur in any organ, commonly the kidney, heart, and muscle. In a preferred embodiment, steatosis is caused in the liver.

[0087] As used herein, "nonalcoholic steatohepatitis (NASH)" is a syndrome that develops in non-alcoholic patients that causes liver damage that is histologically indistinguishable from alcoholic hepatitis. It most often develops in patients with at least one of the following risk factors: obesity, dyslipidemia, and glucose intolerance. The etiology is not fully understood but appears to be related to insulin resistance (e.g., obesity or metabolic syndrome). Most patients are asymptomatic. Laboratory findings include increased aminotransferase levels. A biopsy is required to confirm the diagnosis.

[0088] As used herein, "cirrhosis" refers to late, progressive liver fibrosis characterized by distortion of liver architecture and formation of regenerative nodules.Cirrhosis is generally considered irreversible in its advanced stages, at which point the only therapeutic option may be liver transplantation.Reversal of cirrhosis (in its early stages) is possible in some forms of liver disease after treatment of the underlying cause.Cirrhotic patients are susceptible to various complications, and their life expectancy is usually significantly reduced.

[0089] As used herein, "hepatocellular carcinoma (HCC)" is an invasive tumor that is often seen in the setting of chronic liver disease and cirrhosis. HCC is generally diagnosed later in its course, with a median survival after diagnosis of approximately 6-20 months. The mainstay of treatment is surgical resection, but the majority of patients are ineligible due to tumor extent or underlying liver failure.

[0090] As used herein, "Budd-Chiari syndrome" (BCS) is defined as hepatic venous outflow obstruction regardless of level or mechanism of obstruction, provided that the obstruction is not due to cardiac disease, pericardial disease, or sinusoidal obstruction syndrome (veno-occlusive disease). Primary Budd-Chiari syndrome exists when there is obstruction primarily due to venous processes (thrombosis or phlebitis), whereas secondary Budd-Chiari exists when there is compression or invasion of the hepatic veins and / or inferior vena cava by a lesion of extravenous origin (e.g., malignancy).

[0091] As used herein, "hepatitis" refers to inflammation of the liver, regardless of cause. Hepatitis can be caused by several conditions, including drug toxicity, immune disorders, and viruses. Hepatitis is characterized by jaundice, liver enlargement, and fever.

[0092] As used herein, " kidney disease " is acute or chronic damage to the kidney. It refers to diseases that occur in the kidney due to various reasons including external factors, internal factors, genetic factors, etc. Non-limiting examples of renal diseases include nephritis, nephrosis, renal fibrosis, thin glomerular basement membrane (TGBM), minimal change disease (MCD), membranous glomerulonephritis (MGN), focal segmental glomerulosclerosis (FSGS), DM nephropathy, IgA nephropathy (IgAN), tubulointerstitial nephritis (TIN), Henoch-Schönlein purpura (HSP) nephritis, acute tubular injury, BK virus nephropathy, acute cellular rejection, chronic antibody-mediated rejection, chronic active antibody-mediated rejection, chronic calcineurin inhibitor toxicity, acute kidney injury, chronic kidney disease, ischemic kidney disease, glomerulonephritis, lupus nephritis, polycystic kidney disease, pyelonephritis, kidney stones, renal tuberculosis, renal tumors, chronic renal failure, end stage renal failure, sepsis, kidney injury caused by liver injury, and the like.

[0093] In preferred embodiments, the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis and renal fibrosis.

[0094] As used herein, "acute kidney injury (AKI)" or "acute renal failure (ARF)" is a sudden loss of kidney function that develops within 7 days. Non-limiting causes include damage to kidney tissue caused by reduced renal blood flow (renal ischemia) from any cause (e.g., low blood pressure), exposure to substances harmful to the kidney, inflammatory processes within the kidney, or obstruction of the urinary tract that prevents urine flow. AKI is diagnosed based on characteristic laboratory findings such as increased blood urea nitrogen and creatinine, or the kidneys' inability to produce sufficient amounts of urine. AKI can result in several complications, including metabolic acidosis, high potassium levels, uremia, changes in fluid balance, and effects on other organ systems, including death. Patients who have suffered AKI may have a high risk of chronic kidney disease in the future.

[0095] As used herein, "chronic kidney disease (CDK)" refers to a type of kidney disease that shows a gradual loss of kidney function over a period of months or years. It is generally asymptomatic in the early stages. Later, swelling of the legs, fatigue, vomiting, loss of appetite, or confusion may occur. Comorbidities may include heart disease, hypertension, bone disease, or anemia. Non-limiting causes of chronic kidney disease include diabetes, hypertension, glomerulonephritis, and polycystic kidney disease. Risk factors include a family history of the condition. Diagnosis is generally by blood tests to measure glomerular filtration rate and urine tests to measure albumin. Further tests such as ultrasound or kidney biopsy may be performed to determine the underlying cause.

[0096] As used herein, "nephritis" refers to inflammation of the kidney, and may include the glomerulus, tubule, or interstitial tissue surrounding the glomerulus and tubule. Non-limiting causes include infection, toxins, and autoimmune disorders. Nephritis includes glomerulonephritis (inflammation of the glomerulus) and interstitial nephritis or tubulointerstitial nephritis (inflammation of the spaces between renal tubules).

[0097] As used herein, "nephrosis" refers to any degenerative disease of the kidney tubules. Nephrosis may be caused by kidney disease or may be a complication of another disorder, particularly diabetes. Diagnosis is made by urine testing for the presence of protein, blood testing for lower than normal levels of protein, and examination of the swelling.

[0098] In a preferred embodiment, the fibrosis is renal fibrosis. As used herein, "renal fibrosis" refers to fibrosis affecting the kidney.

[0099] As used herein, " respiratory disease " means any pathology affecting the organs and tissues that enable gas exchange in higher organisms, and includes pathologies of the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, pleura and thoracic cavity, and the nerves and muscles of respiratory function.

[0100] As used herein, " Pulmonary disease " is any respiratory disease that affects the lungs. Pulmonary diseases can be acute or chronic damage to the lungs. Non-limiting examples of pulmonary diseases include asthma, chronic obstructive pulmonary disease, chronic or acute bronchitis, cystic fibrosis emphysema, acute respiratory distress syndrome, bacterial pneumonia, tuberculous pulmonary embolism, and lung cancer.

[0101] In a preferred embodiment, the pulmonary disease is pulmonary fibrosis. Pulmonary fibrosisPulmonary fibrosis" refers to a respiratory disease in which scarring occurs in lung tissue, leading to respiratory problems. Scarring, which is the accumulation of excess fibrous connective tissue (a process called fibrosis), leads to thickening of the walls and causes a decrease in oxygen delivery to the blood. As a result, patients suffer from shortness of breath. Symptoms of pulmonary fibrosis include shortness of breath, chronic thirst, dry cough, fatigue and weakness, chest discomfort including chest pain, loss of appetite and rapid weight loss. As disclosed herein, pulmonary fibrosis may also be a secondary effect of other diseases and / or certain treatments that include non-invasive administration for systemic and local delivery of therapeutic agents to the lungs, such as intranasal and oral inhalation administration. Non-limiting examples of diseases and conditions that can cause pulmonary fibrosis as a secondary effect include inhalation of environmental and occupational pollutants such as exposure to metals in asbestosis, silicosis and certain gases; hypersensitivity pneumonitis (most often resulting from inhalation of dust contaminated with bacteria, fungi, or animal products); smoking; some connective tissue diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma, sarcoidosis and granulomatosis with polyangiitis; infections; certain medications, e.g., amiodarone, bleomycin (pinjangmycin), busulfan, methotrexate, apomorphine and nitrofurantoin; and radiation therapy to the chest.

[0102] In certain embodiments, the CNNM4 inhibitor for use in the present invention can be administered as part of a pharmaceutical composition, where the CNNM4 inhibitor is administered together with a pharma- ceutical acceptable excipient.Thus, the present invention relates to a pharmaceutical composition comprising a CNNM4 inhibitor as described herein and a pharma- ceutical acceptable excipient for use in medicine (i.e., as a treatment).The present invention further relates to a pharmaceutical composition comprising a CNNM4 inhibitor as described herein and a pharma- ceutical acceptable excipient for use in treating acute or chronic disease in a subject, where the disease is preferably selected from the group consisting of liver disease, renal disease and pulmonary disease.

[0103] "Pharmaceutical composition", as used herein, refers to compositions and molecular entities that are physiologically tolerated and do not generally produce allergic reactions or similar adverse reactions such as gastric upset, dizziness, etc., when administered to humans or animals. Preferably, the term "pharmaceutical acceptable" means that it is approved by a state or federal regulatory agency or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans.

[0104] The term "excipient" refers to a vehicle, diluent or adjuvant administered with an active ingredient. Such pharmaceutical excipients can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or saline and aqueous dextrose and glycerol solutions are preferably used as vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" or "Handbook of Pharmaceutical Excipients" by EW Martin, 21st Edition, 2005, Rowe CR; Paul JS; Marian EQ, 6th Edition.

[0105] A suitable amount of an inhibitor as defined above can be formulated together with pharma- ceutically acceptable excipients and / or carriers to obtain a pharmaceutical composition for use in medicine, in particular for use in the treatment of liver, kidney and / or lung diseases.

[0106] Suitable pharma- ceutically acceptable vehicles include, for example, water, salt solutions, alcohol, vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, aromatic oils, mono- and diglycerides of fatty acids, fatty acid esters, petroleum, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0107] The pharmaceutical composition comprising the inhibitor as defined above may be in any dosage form deemed appropriate for the selected route of administration, for example, systemic administration (e.g., intravenous, subcutaneous, intramuscular injection), oral administration, parenteral administration or local administration (for which the pharmaceutical composition includes the necessary pharma- ceutical acceptable excipients for formulation for the desired method of administration). In addition, the composition comprising the inhibitor as defined above may be administered intranasally or sublingually, which allows for systemic administration by a non-invasive mode of administration. Intraventricular administration may also be appropriate. In a preferred embodiment, the route of administration is the intravenous route. In another preferred embodiment, the route of administration is the subcutaneous route.

[0108] If necessary, the inhibitor for use in the present invention is included in a composition that also includes a solubilizing agent and a local anesthetic to improve pain at the injection site.Generally, the ingredients are supplied individually or mixed together in unit dosage form, for example as lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active drug.If the composition is administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0109] In the case of other than intravenous administration, the composition may contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may be a solution, suspension, emulsion, gel, polymer, or sustained release formulation. The composition may be formulated with conventional binders and carriers as known in the art. The formulation may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc., which are inert carriers with well-established functionality in pharmaceutical manufacturing. Various delivery systems are known and can be used to administer the therapeutic agent of the present invention, including encapsulation in liposomes, microparticles, microcapsules, etc.

[0110] The solid dosage form for oral administration may include conventional capsules, sustained release capsules, conventional tablets, sustained release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels.In these solid dosage forms, the active compound may be mixed with at least one inert excipient such as sucrose, lactose or starch.Such dosage forms may also comprise additional substances other than inert diluents, such as magnesium stearate, as is common practice.In the case of capsules, tablets, effervescent tablets and pills, the dosage form may also comprise a buffering agent.Tablets and pills may be provided with an enteric coating.

[0111] Liquid dosage forms for oral administration may include pharma- ceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents, such as water, commonly used in the art. These compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

[0112] Injectable preparations, for example, aqueous or oily suspensions, sterile injections can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Acceptable vehicles and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. Sterile oils are also commonly used as solvents or suspending media.

[0113] For topical administration, the compounds of the present invention can be formulated as creams, gels, lotions, liquids, pomades, spray solutions, dispersions, solid bars, emulsions, microemulsions and the like, which can be formulated according to conventional methods using suitable excipients such as, for example, emulsifiers, surfactants, thickeners, colorants, and combinations of two or more thereof.

[0114] In addition, the inhibitors as defined above may be administered in the form of a transdermal patch or iontophoresis device. Suitable transdermal patches are known in the art.

[0115] Several drug delivery systems are known and can be used to administer the inhibitors as defined above, including, for example, encapsulation in liposomes, microbubbles, emulsions, microparticles, microcapsules, cationic lipids, and the like. Cationic lipids are also called "bolaamphiphiles" or "bolas" and consist of hydrophobic chains with one or more head groups with a positive charge at each end. The required amount can be administered as a single unit or in sustained release form. Non-limiting examples of non-invasive specific treatments include the use of viral vectors for airway gene delivery to the lungs, such as lentiviruses and adenoviruses, as well as polymer-based nanotechnology, which can also be administered locally to the lungs by inhalation. The method of administration can be any technique known in the art, such as using any of the most commonly used devices for respiratory delivery, including nebulizers, metered dose inhalers, and dry powder inhalers.

[0116] Slow-release forms and suitable materials and methods for their preparation are described, for example, in "Modified-Release Drug Delivery Technology", Rathbone, MJ Hadgraft, J. and Roberts, MS (eds.), Marcel Dekker, Inc., New York (2002), and "Handbook of Pharmaceutical Controlled Release Technology", Wise, DL (ed.), Marcel Dekker, Inc. New York, (2000). In one embodiment of the present invention, the orally administrable form of the inhibitor according to the present invention is a slow-release form further comprising at least one coating or matrix. Coatings or slow-release matrices include, but are not limited to, natural polymers, semi-synthetic or synthetic water-insoluble modified waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic or synthetic plasticizers, or combinations of two or more thereof. Enteric coatings can be applied using conventional methods known to those skilled in the art, for example as described in Johnson, JL, "Pharmaceutical tablet coating", Coatings Technology Handbook (2nd ed.), Satas, D. and Tracton, AA (eds.), Marcel Dekker, Inc. New York, (2001), Carstensen, T., "Coating Tablets in Advanced Pharmaceutical Solids", Swarbrick, J. (ed.), Marcel Dekker, Inc. New York (2001), 455-468.

[0117] In one embodiment, the CNNM4 inhibitor is an siRNA. Methods for in vivo delivery of siRNA are known in the art (Davis, ME et al., Nature 2010 April 15; 464(7291): 1067-1070).

[0118] In some embodiments, siRNA is conjugated to at least one ligand.Ligand is preferably specific to the receptor expressed on the surface of target cell.For siRNA targeted to hepatocyte, at least one ligand is preferably GalNAc derivative or GalNAc derivative.

[0119] In certain embodiments, the siRNA is used at a concentration of 0.1 μg / μl to 2.5 μg / ml. In preferred embodiments, the siRNA is at least 0.1 μg / μl, at least 0.2 μg / μl, at least 0.3 μg / μl, at least 0.4 μg / μl, at least 0.5 μg / μl, at least 0.6 μg / μl, at least 0.7 μg / μl, at least 0.8 μg / μl, at least 0.9 μg / μl, at least 1 μg / μl, at least 1.1 μg / μl, at least 1.2 μg / μl, at least 1. In another preferred embodiment, the siRNA is used at a concentration of 0.3 μg / μl, at least 1.4 μg / μl, at least 1.5 μg / μl, at least 1.6 μg / μl, at least 1.7 μg / μl, at least 1.8 μg / μl, at least 1.9 μg / μl, at least 2 μg / μl, at least 2.1 μg / μl, at least 2.2 μg / μl, at least 2.3 μg / μl, at least 2.4 μg / μl, at least 2.5 μg / μl, or more. In another preferred embodiment, the siRNA is used at a concentration of 0.75 μg / μl.

[0120] In certain embodiments, the siRNA is used at a dose of 0.1-10 mg / kilogram of patient. In preferred embodiments, the siRNA is used at a dose of at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1 mg / kg, at least 5 mg / kg, or at least 10 mg / kg.

[0121] In a preferred embodiment, the CNNM4 inhibitor is administered once a day, twice a day, three times a day or more.In another preferred embodiment, the CNNM4 inhibitor is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week or more.In a more preferred embodiment, the CNNM4 inhibitor is administered twice a week.

[0122] Alternatively, the present invention relates to a method for treating liver disease, kidney disease and / or lung disease, comprising administering the CNNM4 inhibitor or pharmaceutical composition according to the present invention to a subject in need thereof.All terms and embodiments described above are equally applicable to this aspect of the present invention.

[0123] Alternatively, the present invention relates to a CNNM4 inhibitor or pharmaceutical composition according to the present invention for the manufacture of a medicament for treating liver disease, kidney disease and / or lung disease. All terms and embodiments described above are equally applicable to this aspect of the present invention.

[0124] Method for diagnosing liver disease, kidney disease or lung disease In another aspect, the present invention provides an in vitro method for diagnosing liver disease, kidney disease and / or lung disease in a subject, comprising: (a) determining the expression level of CNNM4 in a sample from the subject; and (b) comparing said level to a reference value. comprising An increase in the expression level of CNNM4 in said sample relative to a reference value indicates that said patient is suffering from a liver disease, a kidney disease or a lung disease.

[0125] The terms "liver disease", "kidney disease", "lung disease", "subject", "CNNM4 gene" and "reference" are as previously defined in relation to other aspects of the invention. All specific and preferred embodiments of the other aspects of the invention relating to these terms apply fully to this aspect.

[0126] The term "diagnosis" as used herein refers to both the process of attempting to determine and / or identify a possible disease in a subject, i.e., diagnostic methods, and the diagnosis reached by this process, i.e., diagnostic diagnosis. It can therefore also be considered as an attempt to classify an individual's condition into separate and distinct categories that allow medical decisions regarding treatment and prognosis to be made. As the skilled artisan will appreciate, such a diagnosis is preferably, but need not be, accurate in 100% of the subjects diagnosed. However, the term requires that a statistically significant portion of subjects can be identified as suffering from or predisposed to a disease, in this case, specifically liver disease, kidney disease or lung disease. Those skilled in the art can use various well-known statistical evaluation tools to determine whether a population is statistically significant, for example, by determining confidence intervals, determining p-values, Student's test, Mann-Whitney, etc. (see Dowdy and Wearden, 1983). Preferred confidence intervals are at least 50%, at least 60%, at least 70%>, at least 80%>, at least 90%) or at least 95%. The p-value is preferably 0.05, 0.025, 0.001 or lower. The term "diagnosing" as disclosed herein refers to the process of determining which disease or condition explains the symptoms and signs of a person.

[0127] In a particular embodiment, the method for diagnosing liver disease, kidney disease or lung disease of the present invention comprises determining the expression level of CNNM4. The method for determining the expression level of CNNM4 is as disclosed elsewhere herein. In a preferred embodiment, the method for diagnosing liver disease, kidney disease or lung disease of the present invention comprises determining the expression level of CNNM4 protein. In a more preferred embodiment, the method for diagnosing liver disease, kidney disease or lung disease of the present invention comprises determining the expression level of CNNM4 protein by immunohistochemistry.

[0128] In some embodiments, if the expression level of CNNM4 is increased relative to the reference value, the patient is diagnosed as suffering from liver disease, kidney disease and / or lung disease.The method for determining the expression level of CNNM4 has been disclosed previously.In some embodiments, if the expression level of CNNM4 is increased relative to the reference value by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% or more, the patient is diagnosed as suffering from liver disease, kidney disease and / or lung disease.

[0129] As used herein, "sample" or "biological sample" refers to a biological material isolated from a subject. A sample may contain any material suitable for detecting the expression level of a gene and may be a material comprising the genetic material of a subject. A biological sample may comprise cellular and / or non-cellular material of a subject, preferably cellular material. In certain embodiments, a sample comprises the genetic material of a subject under study, such as DNA, genomic DNA (gDNA), complementary DNA (cDNA), RNA, mRNA, etc. In certain embodiments, the genetic material is RNA. A sample may be isolated from a biological tissue or fluid, such as blood, saliva, plasma, serum, urine, cerebrospinal fluid (CSF), feces, nasal, oral or buccal swabs, specimens, specimens obtained from biopsies, and paraffin-embedded tissue samples, liver tissue, lung tissue, or kidney tissue. Procedures for isolating samples are well known to those skilled in the art. A biological sample may contain any biological material suitable for detecting the expression level of CNNM4 and may comprise cellular and / or non-cellular material from a subject. Preferably, the sample used for determining the expression level of CNNM4 is the sample that can be obtained using minimally invasive procedures.In a preferred embodiment, the sample is liver, kidney and / or lung sample or biopsy.

[0130] Before analyzing a sample, it is often desirable to carry out one or more operations to prepare said sample in order to separate the molecules to be determined from other molecules present in the sample. In certain embodiments, these molecules are nucleic acids, DNA and / or RNA. These sample preparation operations include operations such as concentration, suspension, extraction of intracellular material (e.g., nucleic acids from tissue samples / whole cells, etc.), nucleic acid amplification, fragmentation, transcription, labeling and / or extension reactions. These methods are well known to those skilled in the art. Commercial kits for mRNA purification are also available, including, but not limited to, the miRNeasy Mini Kit from Qiagen, the miRNA Life Technologies isolation kit from Sigma-Aldrich, the mirPremier the microRNA isolation kit, and the High Pure miRNA isolation kit from Roche. In certain embodiments, RNA integrity is analyzed using the RNA 6000 Nano Chips (Agilent Technologies, Palo Alto, CA, USA) of the Agilent 2100 bionalizer.

[0131] In one embodiment, a therapeutically effective amount of a CNNM4 inhibitor is administered to a patient when the patient is diagnosed with liver disease, renal disease and / or lung disease.

[0132] As used herein, the term "therapeutically effective amount" is understood to mean any amount of inhibitor according to the present invention that can inhibit the expression of CNNM4.In a preferred embodiment, if it is found that CNNM4 expression is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% with respect to the reference value, a therapeutically effective amount of CNNM4 inhibitor is administered to said patient.

[0133] In a preferred embodiment, the inhibitor is selected from the group consisting of neutralizing antibodies or functional fragments thereof, antagonists, soluble binding proteins, soluble receptor mutants, non-functional derivatives, antisense polynucleotides, RNA interference oligonucleotides, phosphorodiamidate morpholino oligomers (PMOs), miRNAs, siRNAs, shRNAs, sgRNAs, dicer substrate 27-mer duplexes, aptamers, DNAzymes, ribozymes, triplex-forming oligonucleotides (TFOs), small molecules, and combinations thereof. In a more preferred embodiment, the CNNM4 inhibitor is an siRNA. In an even more preferred embodiment, the siRNA comprises at least one of the nucleic acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 50, or SEQ ID NO: 51, or any of the sequences having the SEQ ID NOs shown in Table 1. In an even more preferred embodiment, the siRNA comprises the nucleic acid sequences shown herein as SEQ ID NO: 7 and SEQ ID NO: 8.

[0134] In a preferred embodiment, the liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), Budd-Chiari syndrome and hepatitis, or any combination thereof. In another preferred embodiment, the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis and renal fibrosis, or any combination thereof. In another preferred embodiment, the pulmonary disease is pulmonary fibrosis.

[0135] All terms and embodiments previously described are equally applicable to this aspect of the invention.

[0136] Use of the Reagents for In Vitro Diagnosis of Disease In a further aspect, the present invention relates to the use of specific reagents for determining the expression level of CNNM4 for the in vitro diagnosis of liver, kidney or lung diseases in a subject.

[0137] The specific reagents for determining the expression level of CNNM4 can be provided in kit format. In the context of the present invention, a "kit" or an "assay device" is understood as a product or device that contains various reagents necessary for carrying out the method for determining the expression level of CNNM4, packed to allow transportation and storage. Suitable materials for packing the components of the kit include quartz, plastic (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, envelopes, etc. In addition, these kits can include instructions for simultaneous, sequential or separate use of the various components contained in the kit. The instructions can be in the form of printed matter or electronic support that can store the instructions, such as electronic storage media (magnetic disks, tapes, etc.), optical media (CD-ROM, DVD), etc. that are readable by the subject. Additionally or alternatively, the medium can include an internet address that provides the instructions.

[0138] The expression "specific reagent for determining the expression level of CNNM4" refers to a compound or a set of compounds that allows the determination of the expression level of the gene, both by determining the mRNA level or by determining the protein level. Thus, the first type of reagent includes a probe that can specifically hybridize with the mRNA encoded by said gene. The second type of reagent includes a compound that specifically binds to the protein encoded by the marker gene, preferably includes an antibody, but they may also be specific aptamers.

[0139] Thus, in certain embodiments, the reagent specific for determining the expression level of CNNM4 is selected from the group consisting of a set of probes capable of specifically hybridizing to CNNM4 mRNA and a set of primer pairs capable of specifically amplifying CNNM4 mRNA, or the reagent specific for determining the expression level of CNNM4 is an antibody that specifically binds to CNNM4 polypeptide.

[0140] In preferred embodiments, the reagents sufficient for determining the expression level of CNNM4 comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents sufficient for determining the expression levels of the genes forming the kit.

[0141] In a preferred embodiment, the first component of the kit of the invention comprises a probe capable of specifically hybridizing to the above-mentioned gene.

[0142] The term "capable of specifically hybridizing" as used herein refers to conditions that allow two polynucleotides to hybridize under highly stringent or moderately stringent conditions.

[0143] The "stringency" of a hybridization reaction is easily determined by those skilled in the art and is generally an empirical calculation that depends on the probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures tend to make them less so. For further details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0144] "Stringent conditions" or "high stringency conditions", as defined herein, generally refer to (1) low ionic strength and high temperature, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate, at 50° C. for washing; (2) denaturing agents, e.g., formamide, e.g., 50% (v / v) formamide (containing 0.1% bovine serum albumin) / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5 (containing 750 mM sodium chloride, 75 mM sodium citrate), at 42° C. during hybridization; or (3) 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, followed by washes in 0.2x SSC (sodium chloride / sodium citrate) and 50% formamide at 42°C, followed by a high stringency wash consisting of 0.1x SSC containing EDTA at 55°C.

[0145] "Moderately stringent conditions" can be specified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of less stringent washing solutions and hybridization conditions (e.g., temperature, ionic strength, and SDS%) than those described above. An example of moderately stringent conditions is washing of the filter in 1×SSC at about 37-50° C., after overnight incubation in a solution comprising 20% ​​formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA. Those skilled in the art will know how to adjust temperature, ionic strength, etc., as necessary to accommodate factors such as probe length.

[0146] When the expression level of CNNM4 is determined by measuring the level of the polypeptide encoded by said gene, the kit according to this use comprises a reagent that can specifically bind to said polypeptide.For this purpose, antibody arrays such as those described by De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge et al. (2000) Nucleic Acids Res. 28, e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; WO01 / 40803 and WO99 / 51773A1 can be useful. The antibodies of the array include immunological agents capable of binding to ligands with high affinity, including IgG, IgM, IgA, IgD and IgE, as well as antibody-like molecules having antigen-binding sites, such as Fab', Fab, F(ab')2, single domain antibodies or DABS, Fv, scFv, etc. Techniques for preparing said antibodies are very well known to those skilled in the art and include the methods described by Ausubel et al. (Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons (1992)).

[0147] The antibodies of the array can be applied at high speed, for example, using commercially available robotic systems (such as those produced by Genetic Microsystems or Biorobotics). The substrate of the array can be nitrocellulose, plastic, quartz, or a porous material, such as acrylamide, agarose, or another polymer. In another embodiment, cells producing specific antibodies to detect the proteins of the invention can be used by means of culturing them on the array filter. After inducing expression of the antibody, it is immobilized on the filter at the array location where the producing cells were localized. This array of antibodies can be contacted with a labeled target, and the binding level of the target to the immobilized antibody can be determined. If the target is not labeled, a sandwich-type assay can be used in which a second labeled antibody specific for a polypeptide that binds to the polypeptide immobilized on the support is used. Quantitation of the amount of polypeptide present in the sample at each point of the array can be stored in a database as an expression profile. The array of antibodies can be produced in duplicate and used to compare the binding profiles of two different samples.

[0148] In one embodiment, the present invention relates to the use of a kit or assay device comprising sufficient reagents for the determination of the average expression level of a polypeptide encoded by the Cnnm4 gene, said reagents being an antibody or set of antibodies that specifically bind to the polypeptide encoded by said gene, said reagents comprising at least 10% of the reagents present in the kit.

[0149] In a particular aspect, the present invention relates to the use of the kit of the present invention for determining the prognosis of a patient suffering from a liver disease, a kidney disease or a lung disease. In a preferred embodiment, the liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), Budd-Chiari syndrome and hepatitis, or any combination thereof. In another preferred embodiment, the kidney disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis and renal fibrosis, or any combination thereof. In another preferred embodiment, the lung disease is pulmonary fibrosis.

[0150] All terms and embodiments previously described are equally applicable to this aspect of the invention.

[0151] Methods for alleviating an induced disease or condition In a final aspect, the present invention relates to an in vitro method of alleviating an induced disease or pathology in a cell, comprising contacting said cell with a specific CNNM4 inhibitor in an amount effective to reduce the activity, level or function of CNNM4 in said cell.

[0152] The term "method of alleviating a disease or pathology induced in a cell" as used herein refers to a method for observing the increase or decrease of a biomarker level characteristic of the disease induced in a cell model of said disease. In a particular embodiment, the disease or pathology induced in a cell is a CNNM4-mediated disease or pathology induced in a cell, said disease being accompanied by an increase in CNNM4 expression relative to a reference value. In an embodiment, the CNNM4-mediated disease or pathology induced is accompanied by an increase in CNNM4 expression relative to a reference value, characterized by an expression level of CNNM4 that is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% or more relative to a reference value. In one embodiment, the reference value corresponds to the expression level of CNNM4 in the same cell line in which a CNNM4-mediated disease or pathology is not induced. The same cell line in which a CNNM4-mediated disease or pathology is not induced can be used as a control.

[0153] In some embodiments, the disease or pathology induced in cells is a disease model for the study of DILI, steatosis, NASH, cirrhosis, HCC, liver fibrosis, renal fibrosis or pulmonary fibrosis.In some embodiments, the disease or pathology induced in cells refers to a culture model cell line.In some embodiments, the cell culture is a primary cell culture.In some embodiments, the disease or pathology induced is induced in an animal model before culturing the cells.In a particular example, the disease or pathology induced in cells is NASH induced in primary hepatocytes.

[0154] In a particular embodiment, the animal model is a NAFLD animal model, in which NAFLD is induced by feeding animals (i.e., C57BL / 6J wild type mice) a methionine (0.1%) and choline (0%) deficient diet for 4 weeks, after which the liver is harvested and relevant cells are cultured.

[0155] In certain embodiments, the animal model is a liver cirrhosis model, in which liver cirrhosis is induced by subjecting animals (i.e., C57BL / 6J wild type mice) to bile duct ligation, sacrificing the animals 1-21 days later, harvesting the liver, and culturing the relevant cells.

[0156] In a specific embodiment, the animal model is a hepatocellular carcinoma (HCC) animal model, and adult GNMT- / - mice as an HCC animal model spontaneously develop HCC. The animals are maintained for 7-9 months, sacrificed, and the livers are harvested and the relevant cells are cultured.

[0157] In a particular embodiment, the animal model is a drug-induced liver injury (DILI) animal model, in which DILI is induced by subjecting animals (i.e., adult C57BL / 6J wild-type mice) to a 500 mg / kg acetaminophen (APAP) treatment to induce acute liver injury. After 48 hours, the animals are sacrificed, the livers are harvested, and relevant cells are cultured.

[0158] As used herein, the term "biomarker" refers to a relevant readout required to follow progression in a particular disease model. In a particular embodiment, the biomarker is the expression level of CNNM4. In a particular embodiment, the biomarker is relative lipid accumulation. In a particular embodiment, the biomarker is relative mitochondrial ROS (reactive oxygen species). In a particular embodiment, the biomarker is the percentage value of TUNEL positive cells. In a particular embodiment, the biomarker is the level of intracellular magnesium accumulation.

[0159] In another embodiment, the present invention relates to a method for identifying a compound that is potentially useful for alleviating CNNM4-mediated disease or pathology induced in a cell, comprising contacting said cell with an effective amount of a candidate compound for reducing the activity, level or function of CNNM4 in the cell, or an effective amount of a candidate compound for reducing relative lipid accumulation or reducing relative mitochondrial ROS to a reference value, and the candidate compound that inhibits CNNM4 activity, reduces relative lipid accumulation or reduces relative mitochondrial ROS to a reference value in the CNNM4-mediated disease or pathology induced in the cell is identified as a compound that is potentially useful for treating and / or preventing CNNM4-mediated liver disease, kidney disease and / or lung disease. In one embodiment, the reference value corresponds to the lipid accumulation level or mitochondrial ROS level in the same cell line that is induced with CNNM4-mediated disease or pathology but is not exposed to the compound. The same cell line that is not induced with CNNM4-mediated disease or pathology can be used as a control.

[0160] Lipid accumulation in a cell model system or mitochondrial ROS levels in a cell system are considered to be reduced if the levels are decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% relative to a reference value.

[0161] In yet another embodiment, the present invention provides a method for identifying a compound potentially useful for treating and / or preventing CNNM4-mediated liver, kidney and / or lung disease, comprising: (a) contacting the candidate compound with a cellular model of a disease; and (b) assaying a marker in the presence of the candidate compound, wherein the marker is selected from the group consisting of: activity of CNNM4 in the presence of the compound, relative lipid accumulation relative to a reference value, or relative mitochondrial ROS relative to a reference value; comprising The present invention relates to methods in which compounds that inhibit CNNM4 activity, reduce relative lipid accumulation relative to a reference value, or reduce relative mitochondrial ROS relative to a reference value are potentially useful compounds for treating and / or preventing liver disease, kidney disease and lung disease.

[0162] In certain embodiments, the candidate compound is selected from the group consisting of a neutralizing antibody or functional fragment thereof, an antagonist, a soluble binding protein, a soluble receptor mutant, a non-functional derivative, an antisense polynucleotide, an RNA interference oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), an miRNA, an siRNA, an shRNA, an sgRNA, an antisense RNA, a Dicer substrate 27-mer duplex, an aptamer, a DNAzyme, a ribozyme, a triplex forming oligonucleoid (TFO), a small molecule, and combinations thereof.

[0163] All terms and embodiments previously described are equally applicable to this aspect of the invention.

[0164] The present invention further discloses the following aspects. 1.CNNM4 inhibitors for medical use.

[0165] 2. A CNNM4 inhibitor for use in treating an acute or chronic disease selected from the group consisting of liver disease, renal disease and pulmonary disease in a subject.

[0166] 3. The liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), and Budd-Chiari syndrome, hepatitis; the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis, and renal fibrosis; and The lung disease is pulmonary fibrosis. CNNM4 inhibitor for the use according to 2 above.

[0167] 4. The CNNM4 inhibitor for use according to any one of 1 to 3 above, wherein said inhibitor is selected from the group consisting of a neutralizing antibody or functional fragment thereof, an antagonist, a soluble binding protein, a soluble receptor mutant, a non-functional derivative, an antisense polynucleotide, an RNA interference oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), an miRNA, an siRNA, an shRNA, an sgRNA, an antisense RNA, a Dicer substrate 27-mer duplex, an aptamer, a DNAzyme, a ribozyme, a triplex forming oligonucleotide (TFO), a small molecule, and a combination thereof.

[0168] 5. The CNNM4 inhibitor for use according to 4 above, wherein the small molecule is 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one or 2-[5-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-furan-2-yl]-benzoic acid.

[0169] 6. An in vitro method for diagnosing a liver disease, a kidney disease, or a lung disease in a subject, comprising: (a) determining the expression level of CNNM4 in a sample from the subject; and (b) comparing said level to a reference value. comprising The method, wherein an increase in the expression level of CNNM4 in the sample relative to a reference value indicates that the patient is suffering from liver disease, kidney disease or lung disease.

[0170] 7. The method according to claim 6, wherein the sample is a liver biopsy, a kidney biopsy or a lung biopsy.

[0171] 8. The liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), and Budd-Chiari syndrome, hepatitis; the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis, and renal fibrosis; and The lung disease is pulmonary fibrosis. 8. The method according to claim 6 or 7.

[0172] 9. Use of a specific reagent for determining the expression level of CNNM4 for in vitro diagnosis of liver disease, kidney disease or lung disease in a subject.

[0173] 10. The liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), and Budd-Chiari syndrome, hepatitis; and the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis, and renal fibrosis; The lung disease is pulmonary fibrosis. 9. The use as defined in claim 9.

[0174] 11. The use described in 9 or 10 above, wherein the reagent specific for determining the expression level of CNNM4 is selected from the group consisting of a set of probes capable of specifically hybridizing to CNNM4 mRNA and a set of primer pairs capable of specifically amplifying CNNM4 mRNA, or the reagent specific for determining the expression level of CNNM4 is an antibody that specifically binds to CNNM4 polypeptide.

[0175] 12. An in vitro method for alleviating a disease or pathology induced in a cell, comprising contacting the cell with a specific CNNM4 inhibitor in an amount effective to reduce the activity, level or function of CNNM4 in the cell.

[0176] 13. An in vitro method for identifying a compound potentially useful for alleviating a CNNM4-mediated disease or pathology induced in a cell, comprising contacting the cell with a candidate compound in an amount effective to reduce the activity, level or function of CNNM4 in the cell, or with an amount effective to reduce lipid accumulation relative to a reference value or reduce relative mitochondrial ROS, wherein a candidate compound that inhibits CNNM4 activity, reduces lipid accumulation relative to a reference value, or reduces mitochondrial ROS relative to a reference value in a CNNM4-mediated disease or pathology induced in the cell is identified as a compound potentially useful for treating and / or preventing CNNM4-mediated liver disease, kidney disease and / or lung disease.

[0177] 14. The method according to claim 12 or 13, wherein the inhibitor or compound is selected from the group consisting of a neutralizing antibody or a functional fragment thereof, an antagonist, a soluble binding protein, a soluble receptor mutant, a non-functional derivative, an antisense polynucleotide, an RNA interference oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), an miRNA, an siRNA, an shRNA, an sgRNA, an antisense RNA, a Dicer substrate 27-mer duplex, an aptamer, a DNAzyme, a ribozyme, a triplex forming oligonucleotide (TFO), a small molecule, and a combination thereof.

[0178] The present invention is as follows. [1] A CNNM4 inhibitor for use in treating an acute or chronic disease in a subject, wherein the disease is selected from the group consisting of liver disease, kidney disease and lung disease. [2] the liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), and Budd-Chiari syndrome, hepatitis; the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis, and renal fibrosis; and The lung disease is pulmonary fibrosis. A CNNM4 inhibitor for use as described in [1] above. [3] The CNNM4 inhibitor for use according to [1] or [2] above, wherein the inhibitor is selected from the group consisting of a neutralizing antibody or a functional fragment thereof, an antagonist, a soluble binding protein, a soluble receptor mutant, a non-functional derivative, an antisense polynucleotide, an RNA interference oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), an miRNA, an siRNA, an shRNA, an sgRNA, an antisense RNA, a Dicer substrate 27-mer duplex, an aptamer, a DNAzyme, a ribozyme, a triplex-forming oligonucleotide (TFO), a small molecule, and a combination thereof. [4] The CNNM4 inhibitor for use according to the above [3], wherein the small molecule is 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one or 2-[5-(4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-furan-2-yl]-benzoic acid. [5] An in vitro method for diagnosing liver disease, kidney disease, or lung disease in a subject, comprising: (a) determining the expression level of CNNM4 in a sample from the subject; and (b) comparing said level to a reference value. comprising The method, wherein an increase in the expression level of CNNM4 in the sample relative to a reference value indicates that the patient is suffering from liver disease, kidney disease or lung disease. [6] The method according to [5] above, wherein the sample is a liver biopsy, kidney biopsy or lung biopsy. [7] the liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), and Budd-Chiari syndrome, hepatitis; the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis, and renal fibrosis; and The lung disease is pulmonary fibrosis. The method according to [5] or [6] above. [8] Use of a reagent specific for determining the expression level of CNNM4 for in vitro diagnosis of liver disease, kidney disease or lung disease in a subject. [9] the liver disease is selected from hepatic fibrosis, veno-occlusive liver disease, drug-induced liver injury (DILI), steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma (HCC), and Budd-Chiari syndrome, hepatitis; the renal disease is selected from acute kidney injury (AKI), chronic kidney disease, nephritis, nephrosis, and renal fibrosis; and The lung disease is pulmonary fibrosis. The use as described in [8] above.

[10] The use described in [8] or [9] above, wherein the reagent specific for determining the expression level of CNNM4 is selected from the group consisting of a set of probes that specifically hybridize to CNNM4 mRNA and a set of primer pairs that can specifically amplify CNNM4 mRNA, or the reagent specific for determining the expression level of CNNM4 is an antibody that specifically binds to CNNM4 polypeptide.

[11] An in vitro method of reducing an induced disease or pathology in a cell, wherein the induced disease or pathology is an investigational disease model of DILI, steatosis, NASH, cirrhosis, HCC, liver fibrosis, renal fibrosis or pulmonary fibrosis, comprising contacting the cell with a specific CNNM4 inhibitor in an amount effective to reduce the activity, level or function of CNNM4 in the cell.

[12] An in vitro method for identifying a compound potentially useful for alleviating a CNNM4-mediated disease or pathology induced in a cell, comprising contacting the cell with a candidate compound in an amount effective to reduce CNNM4 activity, level or function in the cell, or with an amount effective to reduce lipid accumulation relative to a reference value or reduce mitochondrial ROS relative to a reference value, wherein a candidate compound that inhibits CNNM4 activity, reduces lipid accumulation relative to a reference value, or reduces mitochondrial ROS relative to a reference value in a CNNM4-mediated disease or pathology induced in the cell is identified as a compound potentially useful for treating and / or preventing CNNM4-mediated liver disease, kidney disease and / or lung disease.

[13] The method according to

[11] or

[12] above, wherein the inhibitor or compound is selected from the group consisting of a neutralizing antibody or a functional fragment thereof, an antagonist, a soluble binding protein, a soluble receptor mutant, a non-functional derivative, an antisense polynucleotide, an RNA interference oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), an miRNA, an siRNA, an shRNA, an sgRNA, an antisense RNA, a Dicer substrate 27-mer duplex, an aptamer, a DNAzyme, a ribozyme, a triplex-forming oligonucleotide (TFO), a small molecule, and a combination thereof. The present invention will also be described by the following examples, which should be considered as merely illustrative and not limiting the scope of the present invention. EXAMPLES

[0179] material and method Human samples All studies were conducted in accordance with the Declaration of Helsinki and local legislation. The human ethics committees of each hospital approved the study procedures, and all patients gave written informed consent before enrollment in the study.

[0180] Magnesium was quantified in human serum samples from eight healthy subjects, 31 obese patients, and 43 individuals from a clinical trial cohort. Patients were assessed for nonalcoholic fatty liver disease (NAFLD) after excluding alcohol-related disease and viral hepatitis infection.

[0181] Human CNNM4 expression in non-alcoholic fatty liver disease NAFLD was determined in a cohort of 42 patients: 10 healthy patients, 20 patients were diagnosed with steatosis and 12 with NAFLD.

[0182] CNNM4 levels in patients with cirrhosis were determined in a cohort of 12 patients, of which 5 were healthy and 7 were diagnosed with cirrhosis.

[0183] We also determined CNNM4 levels in 47 hepatocellular carcinoma (HCC) patients: 6 patients were healthy and 41 patients were diagnosed with HCC.

[0184] CNNM4 levels were determined in a cohort of 11 drug-induced liver injury (DILI) patients and compared with three healthy patients.

[0185] Finally, 14 human samples with renal fibrosis were analyzed to determine CNNM4 expression: 7 samples were healthy and another 7 were diagnosed with renal fibrosis.

[0186] Animal testing All animal experiments were performed in accordance with the Spanish Guide for Care and use of Laboratory animals and using International Care and Use Committee Standards. All procedures were approved by the CIC bioGUNE Animal Care Committee and the Supervisory Authority (Diputacion de Bizkaia). Mice were kept in a temperature-controlled animal facility (AAALAC-certified) with a 12-h light / dark cycle. G-mice were fed standard chow (Harlan Tekland) and had water available ad libitum.

[0187] NAFLD animal model: 0.1% methionine and choline deficient diet (0.1% MCDD) for CNNM4 determination C57BL / 6J wild-type mice were fed a methionine (0.1%) and choline (0%) deficient diet for 4 weeks. At the end of the treatment, the animals were sacrificed and the livers were divided into several pieces for subsequent analyses, including RNA or protein extraction, formalin fixation for histology and immunohistochemistry, or metabolic analysis. During the treatment, blood was collected once a week for serum analysis.

[0188] Preclinical trials: NAFLD animal models using siRNA therapy C57BL / 6J wild-type mice were fed a methionine (0.1%) and choline (0%) deficient diet for 4 weeks. Two weeks after the start of the diet, mice were divided into two groups, in vivo silencing CNNM4 or untreated siRNA control, and received 200 μl of a 0.75 μg / μl solution of CNNM4-specific in vivo siRNA (Custom Ambion, USA) or control siRNA (Sigma-Aldrich, USA) using Invivofectamine® 3.0 reagent (Invitrogen, USA) according to the manufacturer's instructions. Tail vein injections were performed twice a week until the fourth week. At the end of the treatment, animals were sacrificed and livers were divided into several pieces for subsequent analyses, including RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. During the treatment, blood was collected once a week for serum analysis.

[0189] Liver Cirrhosis Model: Bile Duct Ligament (BDL) Adult C57BL / 6J wild-type mice were subjected to BDL as previously described (Fernandez-Alvarez et al., 2015. Lab Invest. 95(2):223-36). Briefly, mice were anesthetized with 1.5% isofluorane in O2 and laparotomy was performed. The bile duct was isolated from the portal vein and hepatic artery, and the bile duct was sutured and tightened with a surgical knot. Finally, the abdomen was closed and the mice were sacrificed at 24 h, 48 h, 72 h, 3 days, and 21 days. The liver was divided into several pieces for subsequent analyses, including RNA or protein extraction, formalin fixation for histology and immunohistochemistry, or metabolic analysis. During the procedure, blood was collected once a week for serum analysis.

[0190] Hepatocellular carcinoma animal model (HCC): GNMT- / - mice Adult GNMT - / -Mice were allowed to grow for 7-9 months, as described when they spontaneously develop HCC (Wagner et al., 2009. Toxicol Appl Pharmacol. 1;237(2):246; author reply 247). At this point, animals were sacrificed and livers were divided into several pieces for subsequent analyses, including RNA or protein extraction, formalin fixation for histology and immunohistochemistry, or metabolic analysis. During the treatment, blood was collected once a week for serum analysis.

[0191] Drug-induced liver injury (DILI): Acetaminophen (APAP) overdose Adult C57BL / 6J wild-type mice were treated with 500 mg / kg acetaminophen (APAP) to induce acute liver injury. Mice were sacrificed 48 hours after treatment and livers were divided into several pieces for subsequent analyses, including RNA or protein extraction, formalin fixation for histology and immunohistochemistry, or metabolic analysis. During treatment, blood was collected once a week for serum analysis.

[0192] Isolation, culture and processing of primary hepatocytes Primary hepatocytes from 3-month-old wild-type (C57BL / 6J) mice were isolated by perfusion with type I collagenase (Worthington, USA). Briefly, animals were anesthetized with isoflurane (1.5% isoflurane in O2). Then, the abdomen was opened and the inferior vena cava was catheterized. The liver was perfused with buffer A (1x PBS, 5 mM EGTA, 37°C, oxygenated) and the portal vein was cut. Next, the liver was perfused with buffer B (1x PBS, 1 mM CaCl2 37°C and oxygenated) to remove EGTA and finally, with buffer C (1x PBS, 2 mM CaCl2, 0.65 BSA, type I collagenase, 37°C and oxygenated). After buffer C perfusion, the liver was separated from the rest of the body and placed in a Petri dish containing MEM (Gibco, USA). After careful removal of the gallbladder, the liver was mechanically dissociated with forceps. The digested liver was diluted in MEM and filtered through sterile gauze, and the filtered hepatocytes were collected and washed three times (1x4 min at 400 RPM and 2x5 min at 500 RPM) in MEM supplemented with 10% FBS (Gibco) / 1% PSG (Gibco), preserving the supernatant Kupffer cell and hepatic stellate cell isolates. After the final wash, the hepatocytes contained in the pellet were resuspended in 10% FBS 1% PSG MEM for further culture.

[0193] Primary hepatocytes were cultured on collagen-coated culture dishes at 7600 cells / mm in MEM supplemented with 10% FBS / 1% PSG. 2 The cells were seeded at a density of 100 μg / mL and placed in a 5% CO2-95% air incubator at 37°C. After 6 h of attachment, the culture medium and non-adherent hepatocytes were removed with fresh 0% FBS / 1% PSG MEM for the desired treatment (Table 2).

[0194] [Table 2]

[0195] THLE2 cells THLE-2 cells were purchased from ATCC (ATCC® CRL-2706™). They were maintained in Bronchial Epithelial Growth Medium (BEGM™, Lonza) supplemented with BEGM Bullet Kit™ (Lonza) and 10% FBS. They were dissociated with 0.05% trypsin-EDTA and collected in BEGM. After centrifugation at 123 g for 5 min, the supernatant was discarded and the pellet was resuspended.

[0196] Plasmid transfection Plasmids were transfected into primary mouse hepatocytes for overexpression using jetPRIME® (Polyplus, USA) transfection reagent according to the manufacturer's protocol. In a 24-well plate, 0.5 μg of plasmid was added to jePRIME® buffer and vortexed for 10 s before adding 1 μl of jetPRIME® reagent. The mixture was vortexed for 10 s, spun down, and incubated at room temperature for 10 min. Transfections were performed in cell suspension medium, and the transfection mixture was replaced with fresh medium 6 h post-transfection unless indicated.

[0197] Gene silencing by siRNA delivery Cells were transfected with specific siRNAs at a final concentration of 100 nM using DharmaFECT 1 reagent (Dharmacon) according to the manufacturer's protocol. DharmaFECT 1 and siRNAs were individually diluted in 0% FBS / 1% PSG MEM for 5 min at room temperature. The dilutions were then mixed and incubated at room temperature for 20 min. The mixed siRNA transfections were then added to the cell suspension medium and replaced with fresh medium after 6 h. The siRNA transfection volumes (shown for 6-well plates) and sequences are summarized in Table 3.

[0198] [Table 3] TIFF0007672337000005.tif204160

[0199] Gene silencing by shRNA delivery Cells were transfected with specific shRNAs using Lipofectamine® 3000 (ThermoFisher) according to the manufacturer's protocol. 7.5 μl of Lipofectamine and 3 shRNAs were individually diluted in 0.2 ml of culture medium and incubated at room temperature for 5 minutes. After incubation, they were mixed again and incubated at room temperature for 30 minutes before delivery to cells. shRNA transfection volumes are shown for 6-well plates and sequences are SEQ ID NO: 52. 5'-UCUCUGCCUUCAAGGAUGCGGACAAUGAG-3' (SEQ ID NO:52)

[0200] RNA isolation and cDNA expression determination RNA isolation Total RNA from whole liver or cultured cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For cellular mRNA extraction, 5 μg of glycogen (Ambion, USA) was used for the RNA precipitation step to improve visualization of the RNA pellet. RNA concentration was measured spectrophotometrically using a Nanodrop ND-100 spectrophotometer (ThermoFisher Scientific, USA).

[0201] Reverse transcription 1-2 μg of isolated RNA was treated with DNase I (Invitrogen) and used to synthesize cDNA with M-MLV reverse transcriptase (all from Invitrogen) in the presence of random primers and RNase OUT. The resulting cDNA was diluted 1 / 10 (1 / 20 if 2 μg was used) in RNase-free water (Sigma-Aldrich).

[0202] Real-time quantitative PCR (RT-qPCR) qPCR was performed using either a ViiA 7 or QS6 real-time PCR system with SYBR Select Master Mix (Applied Biosystems, USA). 1.5 μl of cDNA containing specific primers was used for a total reaction volume of 6.5 μl in a 384-well plate (Applied Biosystems). All reactions were performed in triplicate. PCR conditions were optimized for the primers with a melting temperature of 60°C and 40 cycles of 30 s each step. Both human (Homo Sapien) and Mus musculus (Mus musculus) primers for each step were designed and synthesized by Sigma Aldrich using primer 3 software from the NCBI-Nucleotide webpage (www.ncbi.nlm.nih.gov / nucleotide). Details of the primer sequences are shown in Tables 4 and 5. After confirming the specificity of the PCR products using melting curves, the data were normalized to the expression of housekeeping genes (GAPDH, ARP).

[0203] [Table 4]

[0204] [Table 5]

[0205] protein Protein extraction and analysis Total protein extraction was performed as indicated. Cells were washed with cold PBS buffer and resuspended in 200 μl of RIPA lysis buffer (1.6 mM NaH2PO4, 8.4 mM Na2HPO4, 0.5% azide, 0.1 M NaCl, 0.1% SDS, 0.1% Triton X-100, 5 mg / ml sodium deoxycholate). Protease and phosphatase inhibitor cocktail (Roche, Switzerland) was added to the lysis buffer. They were centrifuged (13000 rpm, 20 min at 4 °C) and the supernatants (protein extracts) were quantified for total protein content by Bradford protein assay (Bio-Rad) and determined using a Spectramax M3 spectrophotometer (Molecular Devices, USA).

[0206] For frozen liver tissue, approximately 50 μg of tissue was homogenized in 500 μl of buffer using a Precellys 24 tissue homogenizer (Precellys, France). In all cases, the lysates were centrifuged (13000 rpm, 20 min, 4° C.) and the supernatants (protein extracts) were quantified for total protein content by Bradford protein assay or BCA protein assay (Pierce, USA) depending on the type of lysis buffer used, determined using a Spectramax M3 spectrophotometer.

[0207] Western blotting Protein extracts were boiled at 95°C for 5 min in SDS-PAGE sample buffer (250 mM Tris-HCl pH 6.8, 500 mM β-mercaptoethanol, 50% glycerol, 10% SDS and bromophenol blue). Appropriate amounts of protein (5–50 μg), depending on protein abundance and antibody sensitivity, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 3%–15% acrylamide gels (depending on the molecular weight of the protein of interest) using a Mini-PROTEAN electrophoresis system (Bio-Rad). Gels were transferred to nitrocellulose membranes by electroblotting using a Mini Trans-Blot cell (Bio-Rad). Membranes were blocked with 5% nonfat milk in TBS pH 8 (Sigma Aldrich) containing 0.1% Tween-20 (TBST-0.1%) for 1 h at room temperature, washed 3 times for 10 min with TBST-0.1%, and incubated with commercial primary antibodies overnight at 4°C. Primary antibodies and their optimal incubation conditions are detailed in Table 6. Membranes were then washed 3 times for 10 min with TBST-0.1% and incubated for 1 h at room temperature in blocking solution containing secondary antibodies conjugated to horseradish peroxidase (HRP, Table 6). Immunoreactive proteins were detected by using Western Lightning Enhanced Chemiluminescence Reagent (ECL, PerkinElmer, USA) and exposed to Super Rx-N X-ray film (Fuji, Japan) in Curix 60 Developer (AGFA, Belgium).

[0208] [Table 6]

[0209] Staining assay Sudan Red for lipid staining Frozen liver samples included in OCT were sectioned at 10 μm. Sections were washed in 60% isopropanol and then stained with fresh Sudan III (0.5% in isopropanol; Sigma Aldrich) solution for 30 min. Samples were then washed again in 60% isopropanol before counterstaining with hematoxylin and eosin. These sections were then washed in distilled water and mounted in DPX mounting medium. Images were acquired under an upright light microscope (Zeiss).

[0210] Determining ROS with DHE 8 μm sections embedded in OCT were incubated with 150 μM MnTBAP for 1 h at room temperature. Samples were then incubated with 5 μM dihydroethidine (DHE) for 30 min at 37°C, and sections were mounted and nuclear counterstained with Fluoromount-G (Southern Biotech, USA) containing 0.7 mg / l DAPI. Images were acquired using an Axioimager D1 (Zeiss).

[0211] Immunohistochemistry for CNNM4 determination Paraffin-embedded sections (5 μm thick) were retrieved and peroxide blocked depending on the primary antibody used (3% H2O2 in PBS, 10 min, room temperature). For staining with mouse-hosted antibodies in mouse tissues, samples were blocked with goat anti-mouse Fab fragment (Jackson Immunoresearch, USA) (1:10, 1 h, room temperature) and 5% goat serum (30 min, room temperature). Sections were then incubated with CNNM4 primary antibody (Ab191207, Abcam) at 1:100 in DAKO antibody diluent (DAKO) in a humidified chamber, followed by incubation with Envision anti-rabbit (DAKO) HRP-conjugated secondary antibody (30', room temperature). Colorimetric detection was confirmed with Vector VIP chromogen (Vector) and sections were counterstained with hematoxylin. Samples were mounted with DPX mounting medium. Images were acquired with an upright light microscope (Zeiss).

[0212] Immunofluorescence for αSMA determination For α-SMA staining, 10 μm sections embedded in OCT were incubated with a 1 / 200 dilution of primary antibody conjugated to Cy3 in 2% BSA with 0.01% PBS azide (C6198, Sigma Aldrich) and mounted with Fluoromount-G (Southern Biotech) containing 0.7 mg / l DAPI to counterstain the nuclei. Images were acquired with an Axioimager D1 (Zeiss).

[0213] BODIPY for lipid quantification in primary hepatocytes Primary hepatocytes cultured in lipid-rich medium (OA) or methionine / choline-deficient medium (MDMC) were fixed with 4% paraformaldehyde in PBS (10 min, room temperature) and incubated with 1 mg / ml BODIPY 493 / 503 (Molecular Propbes, Invitrogen) (1 h, room temperature). BODIPY immunocytofluororescence images were acquired using an Axioimager D1 (Zeiss) microscope. Quantification of fat bodies was performed using Frida software and expressed as the average area relative to the total number of cells.

[0214] Data analysis The average sum of intensity or percentage of stained area for each sample was calculated using FRIDA software (http: / / bui3.win.ad.jhu.edu / frida / , John Hopkins University).

[0215] Quantification of liver lipids Thirty milligrams of frozen liver was homogenized in a Potter homogenizer with 10 volumes of ice-cold PBS. Fatty acids were measured in the homogenates using a Wako Chemicals kit (Richmond, VA) and lipids were quantified as described (Folch et al., 1957. J Biol Chem. 226(1):497-509). Briefly, lipids were extracted from 1.5 mg of protein from liver homogenates. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), fatty acids (FAs) and cholesterol (Ch) were separated by thin layer chromatography (TLC) and quantified as described (Ruiz and Ochoa, 1997). Triglycerides (TGs) were measured in lipid extracts using an A. Menarini Diagnostics (Italy) kit.

[0216] Magnesium determination assay Intracellular magnesium levels Primary hepatocytes grown on glass coverslips were supplemented with 2 μM Mag-S-Tz or 1 μM Mag-S-Tz-AM diluted REF (Gruskos et al., 2016. J. Am. Chem. Soc. 138 (44), pp 14639-14649) in 0% FBS / 1% PSG medium and incubated for 30 min or 1 h, respectively, at 37 °C and 5% CO2. After removing the dye-containing medium, a 30 min incubation in 0% FBS / 1% PSG was performed. The coverslips were then washed in 20 mM Tris-HCl, 2.4 mM CaCl2, 10 mM glucose, pH 7.4 buffer and mounted in a Thermostattes perfusion chamber on an Eclipse TE 300-based microspectrofluorometer (Nikon, USA) and visualized by 40x oil immersion fluorescence. Intracellular Mg 2+Levels were determined using the method described by Grynkiewicz (Grynkiewicz et al., 1985. J. Biol. Chem. 260(6):3440-50). The 340 / 380 nm excitation light ratio was determined using a Delta system (Photon Technologies International, Princeton) and calculated from the standard formula: Mg 2+ was converted to concentration.

number

[0217] Extracellular Magnesium Levels Extracellular magnesium was quantified using the QuantiCrom™ Magnesium Assay Kit (BioAssay Systems, USA). Briefly, 5 μl of serum or culture medium was mixed with 200 μl of a 1:1 mixture of Reagent A and Reagent B. After 2 min of incubation at room temperature, the OD was determined at 500 nm wavelength using a Spectramax M3 spectrophotometer (Molecular Devices, USA). Then, 10 μl of EDTA was added and the OD was measured again. 500 Finally, the OD from the standard concentration (2 mg / ml) was determined. 500 The magnesium concentration was calculated by comparing with the above.

[0218] In vitro assay Determination of mitochondrial ROS Mitochondrial ROS was measured using MitoSOXTM Red reagent (Life Technologies) according to the manufacturer's instructions. Briefly, primary hepatocytes and hepatoma cells were incubated with MitoSOX reagent (2.5 μM, 10 min, 37°C, CO2 incubator) in regular culture medium. Cells were then washed twice with PBS and fluorescence was measured using a spectrophotometer at excitation 510 nm and emission 595 nm. Final values ​​were normalized to total protein concentration.

[0219] Determination of cell death by TUNEL Cell death was analyzed by using an in situ cell death detection kit (Roche) according to the manufacturer's instructions, as described above. Cells were exposed to a peroxide block (3% H2O2 in methanol) for 3 min and then incubated with TUNEL dilution buffer containing FITC-conjugated primary antibody (dilution 1 / 50) for 1 h at 37 °C. Sections were mounted in Dako fluorescent mounting medium (Dako). Images were acquired using an Axioimager D1 (Zeiss) microscope, and cell viability was calculated by determining the % of TUNEL-positive cells.

[0220] result Overexpression of CNNM4 in liver pathologies Chronic liver disease includes a group of different pathologies. Methods have been developed to detect CNNM4 expression by immunohistochemistry (IHC) in livers from human liver biopsies and mouse animal models. Here, CNNM4 expression has been shown to be characteristic in DILI and in all stages of chronic liver disease, both in human biopsies and animal models, with overexpression of this protein in all pathologies (Figure 1). These results were confirmed by a method to detect CNNM4 expression by qPCR of CNNM4 mRNA levels in human liver samples from healthy subjects compared to samples from patients with fatty liver and NASH (Figure 2A). CNNM4 expression could also be determined by qPCR of CNNM4 mRNA levels in an in vivo NASH mouse model (Figure 2B), as well as in an in vitro NASH mouse cell model (Figure 2C).

[0221] CNNM4, a new target for treating liver disease The overexpression seen as previously shown by IHC determination of CNNM4 suggests CNNM4 as a potential target for treating liver disease, both for DILI and chronic disease. In vitro studies were performed in which NASH was induced in primary hepatocytes and treated them with siRNA CNNM4 therapy. In the case of NASH model primary hepatocytes, lipid content and reactive oxygen species (ROS)-induced inflammation were measured and both were found to be upregulated in NASH hepatocytes treated with siRNA therapy (Figure 3A and 3B). Another in vitro study was also performed to mimic DILI by acetaminophen overdose and showed the expected cell death in DILI model hepatocytes, which was reversed when cells were treated with targeted siRNA therapy (Figure 3C). Furthermore, we performed in vitro studies in which we induced NASH in human cells and treated them with siRNA CNNM4 or shRNA CNNM4 therapy, and measured lipid content. We found that relative lipid accumulation was restored in both NASH-induced human cells (THLE2 cells) treated with iRNA therapy (Figure 4A) and NASH-induced human cells treated with shRNA therapy (Figure 4B).

[0222] Specificity and requirement of CNNM4 targeting Given the protective effect of siRNA CNNM4 therapy against NASH and DILI, we determined the effect of targeting and silencing other proteins of the CNNM family (CNNM1, CNNM2, and CNNM3). We induced NASH in primary hepatocytes and treated them with siRNA CNNM1, siRNA CNNM2, and siRNA CNNM3. Unlike siRNA CNNM4 therapy, silencing other proteins of the CNNM family was ineffective, indicating the specificity of the treatment based on CNNM4 alone and not on proteins of the CNNM family (Figure 5A). Furthermore, experiments were performed to prove the necessity of the treatment based on CNNM4. In the first case, lipid accumulation was induced in primary hepatocytes by CNNM4 overexpression, similar to that seen in NASH patients and animal models, and then magnesium was added to them. The addition of magnesium was ineffective in reducing lipid accumulation, similar to siRNA CNNM4 treatment (Figure 5B). Secondly, lipid accumulation was induced in primary hepatocytes by magnesium deficiency, a physiological condition similar to that in the case of CNNM4 overexpression. In this case, siRNA CNNM4 therapy reduced lipid accumulation (Figure 5C). Considering these two results, magnesium supplementation is not sufficient to treat NASH, and therefore there is a need for CNNM4-based therapy.

[0223] Preclinical trials of siRNA CNNM4-based therapy To address the efficacy of CNNM4 modulation in cells as well as animals, we developed a preclinical study in NAFLD, the first stage of chronic liver disease. To develop NAFLD, mice were fed a 0.1% methionine and choline deficient diet (0.1% MCDD) for 2 weeks. At this point, after 2 weeks of 0.1% MCDD, one group was treated with siRNA CNNM4 therapy and another group with siCtrl RNA. The animals were sacrificed and various biomarkers were measured to analyze the progression of NAFLD. Sudan red staining assessed lipid accumulation (Figure 6A), blood transaminases indicate liver injury (Figure 6B), DHE staining quantifies inflammation caused by ROS (Figure 6C), and α-smooth muscle actin (αSMA) indicates the progression of fibrosis (Figure 6D). It is observed that NAFLD is alleviated in the group of animals treated with siRNA CNNM4 therapy.

[0224] Pharmacological inhibition of CNNM4 In addition to siRNA therapy, CNNM4 activity can also be modulated pharmacologically. We performed an in vitro assay in which we induced NASH in primary hepatocytes and treated them with a compound known as 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one. We observed that this pharmacological inhibition of CNNM4 had a similar effect as siRNA therapy in NASH-induced hepatocytes, reducing their lipid content (Figure 7A) and causing them to accumulate magnesium (Figure 7B).

[0225] Overexpression of CNNM4 in pathological conditions of various organs As shown in Figure 1, CNNM4 has been found to be overexpressed in various liver pathologies in both animal models and human samples. Notably, fibrosis development is not only seen in the liver but also in other organs such as the kidney, and given some similarities that fibrosis development can be in both tissues, it is possible that CNNM4 is deregulated in this organ. Overexpression of CNNM4 was found in a renal fibrosis mouse model, indicating that CNNM4-based therapy is an effective one for the treatment of this disease (Figure 8). In addition, analysis of TCGA (The Cancer Genome Atlas) data shows that CNNM4 is overexpressed in primary tumor samples of hepatocellular carcinoma (LIHC) and primary tumor samples of lung adenocarcinoma (LUAD) relative to normal tissues (Figure 9A and Figure 9B).

[0226] In summary, the results presented prove that CNNM4 is a suitable target to prevent NAFLD progression and could also improve other liver pathologies (DILI, cirrhosis and HCC), renal fibrosis and lung cancer. In the case of DILI, in vitro studies show a protective effect of siRNA therapy against APAP overdosage, and the determination of CNNM4 in other pathologies shows that it is overexpressed in all of them. Therefore, inhibiting or silencing this protein or its partners by siRNA therapy or pharmacologically represents a suitable method to treat liver diseases, renal fibrosis and lung cancer.

Claims

1. A pharmaceutical composition for the treatment of acute or chronic liver disease in a subject, comprising a CNNM4 inhibitor, the inhibitor is selected from the group consisting of siRNA, shRNA, miRNA, sgRNA, dicer substrate 27-mer duplex, aptamer, DNAzyme, ribozyme, triplex forming oligonucleotide (TFO), antisense polynucleotide, phosphorodiamidate morpholino oligomer (PMO), non-functional CNNM4 derivative and combinations thereof; The liver disease is drug-induced liver injury (DILI) or nonalcoholic steatohepatitis (NASH); Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the inhibitor is selected from siRNA, shRNA, miRNA, sgRNA, dicer substrate 27-mer duplex, aptamer, DNAzyme, ribozyme and triplex forming oligonucleotide (TFO).

3. The pharmaceutical composition of claim 2 , wherein the inhibitor is an siRNA.

4. 1. An in vitro method for aiding in the diagnosis of liver disease, kidney disease or lung disease in a subject, comprising: (a) determining the expression level of CNNM4 in a sample from said subject; and (b) comparing said level to a reference value. comprising an increased expression level of CNNM4 in the sample relative to a reference value indicates that the patient is suffering from a liver disease, a kidney disease or a lung disease; the sample is a liver biopsy, a kidney biopsy or a lung biopsy; the liver disease is selected from drug-induced liver injury (DILI), nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC); the kidney disease is renal fibrosis; and The lung disease is lung adenocarcinoma. method.

5. Use of a reagent specific for determining the expression level of CNNM4 to aid in the in vitro diagnosis of liver disease, kidney disease or lung disease in a subject; The reagent specific for determining the expression level of CNNM4 is selected from the group consisting of a set of probes that specifically hybridize with CNNM4 mRNA and a set of primer pairs that can specifically amplify CNNM4 mRNA, or the reagent specific for determining the expression level of CNNM4 is an antibody that specifically binds to CNNM4 polypeptide; the liver disease is selected from drug-induced liver injury (DILI), nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC); the kidney disease is renal fibrosis; and The lung disease is lung adenocarcinoma. use.

6. 1. An in vitro method of alleviating an induced disease or pathology in a cell, comprising: the induced disease or condition is an investigational disease model of DILI or NASH, comprising contacting the cells with a specific CNNM4 inhibitor in an amount effective to reduce CNNM4 activity, level or function in the cells; The inhibitor is selected from the group consisting of siRNA, shRNA, miRNA, sgRNA, dicer substrate 27-mer duplex, aptamer, DNAzyme, ribozyme, triplex forming oligonucleotide (TFO), antisense polynucleotide, phosphorodiamidate morpholino oligomer (PMO), non-functional CNNM4 derivative and combinations thereof; method.

7. An in vitro method for identifying a compound potentially useful for ameliorating a CNNM4-mediated disease or pathology induced in a cell, comprising: contacting said cell with an amount of a candidate compound effective to reduce CNNM4 activity, level or function in the cell, or an amount of a candidate compound effective to reduce lipid accumulation relative to a reference value or reduce relative mitochondrial ROS, wherein a candidate compound that inhibits CNNM4 activity, reduces lipid accumulation relative to a reference value, or reduces mitochondrial ROS relative to a reference value in an induced CNNM4-mediated disease or condition in the cell is identified as a compound potentially useful for treating and / or preventing CNNM4-mediated liver disease; the inhibitor is selected from the group consisting of siRNA, shRNA, miRNA, sgRNA, dicer substrate 27-mer duplex, aptamer, DNAzyme, ribozyme, triplex forming oligonucleotide (TFO), antisense polynucleotide, phosphorodiamidate morpholino oligomer (PMO), non-functional CNNM4 derivative, and combinations thereof; The liver disease is drug-induced liver injury (DILI) or nonalcoholic steatohepatitis (NASH); method.

Images