Antisense oligonucleotide for regulating CUBN expression

Abstract

The present invention provides a method for improving renal function, and a compound for regulating the expression and function of CUBN and can be used in the method for improving renal function.　Renal function was successfully improved using a CUBN inhibitor.

Description

Antisense oligonucleotides for regulating the expression of CUBN

[0001] The present invention relates to antisense oligonucleotides for regulating the expression of CUBN.

[0002] Cubilin (CUBN) has been reported to be involved in the reabsorption of albumin and vitamin D-binding protein in the kidney, and in the absorption of vitamin B12 (VB12) in the small intestine (Non-Patent Document 1). Mutations in the CUBN gene have been reported to be associated with autosomal recessive Imerslund-Grasbeck syndrome (IGS); chronic benign proteinuria (PROCHOB), etc. IGS is also called selective vitamin B12 malabsorption syndrome, which results in vitamin B12 deficiency and presents with megaloblastic anemia and neurological symptoms such as peripheral neuropathy, cognitive impairment, and dementia. On the other hand, PROCHOB is caused by abnormalities in the CUBN gene, which is responsible for the reabsorption of albumin and low-molecular-weight proteins in the proximal renal tubules and small intestine. Albuminuria (proteinuria) is generally recognized as a risk factor for kidney damage and is observed in all chronic kidney diseases associated with glomerular disorders.

[0003] Kidney Int 2016;89:58-67

[0004] The object of the present invention is to provide a method for improving kidney function; and a compound for regulating the expression and function of CUBN that can be used in the method for improving kidney function.

[0005] As a result of intensive studies, the inventors have newly found that, contrary to the above-mentioned common technical knowledge, kidney function can be improved by deliberately reducing the expression and function of CUBN, the mutation of which causes albuminuria, a risk factor for kidney damage. The present invention includes the following aspects.

[0006] [1] An antisense oligonucleotide having a length of 10 to 30 nucleotides, wherein the antisense oligonucleotide targets the CUBN gene and is at least 90% complementary to SEQ ID NO: 1 or SEQ ID NO: 2. [2] The antisense oligonucleotide according to [1] having a sequence that is at least 90% complementary to Exon 37 or 15 of CUBN. [3] The antisense oligonucleotide according to [1] comprising a sequence of at least 10 consecutive nucleotides having at least 90% homology to any of the sequences described in SEQ ID NOs: 138 to 366 or 399 to 408. [4] The antisense oligonucleotide according to [1], wherein some or all of the constituent nucleosides contain modified sugars. [5] A pharmaceutical composition containing the antisense oligonucleotide according to any one of [1] to [4].

[0007] [1A] A pharmaceutical composition for improving renal function in a subject, comprising a CUBN inhibitor. [2A] The pharmaceutical composition according to [1A], wherein the CUBN inhibitor is siRNA, shRNA, miRNA, antisense oligonucleotide or decoy oligonucleotide against the CUBN gene, or an antibody, antibody fragment or aptamer against the CUBN gene product. [3A] The pharmaceutical composition according to [2A], wherein the antisense oligonucleotide against the CUBN gene is the antisense oligonucleotide according to any one of [1] to [4]. [4A] The pharmaceutical composition according to any one of [1A] to [3A], wherein the subject exhibits albuminuria. [5A] A pharmaceutical composition for treating or preventing focal segmental glomerulosclerosis (FSGS) in a subject, comprising a CUBN inhibitor.

[0008] [1B] Use of a CUBN inhibitor in the manufacture of a pharmaceutical product for improving renal function in a subject. [2B] The use according to [1B], wherein the CUBN inhibitor is an siRNA, shRNA, miRNA, antisense oligonucleotide or decoy oligonucleotide against the CUBN gene, or an antibody, antibody fragment or aptamer against the CUBN gene product. [3B] The use according to [2B], wherein the antisense oligonucleotide against the CUBN gene is an antisense oligonucleotide according to any one of [1] to [5]. [4B] The use according to any one of [1B] to [3B], wherein the subject exhibits albuminuria. [5B] Use of a CUBN inhibitor in the manufacture of a pharmaceutical product for the treatment or prevention of focal segmental glomerulosclerosis (FSGS) in a subject.

[0009] [1C] A CUBN inhibitor for use in improving the renal function of a subject. [2C] The CUBN inhibitor according to [1C], wherein the CUBN inhibitor is an siRNA, shRNA, miRNA, antisense oligonucleotide or decoy oligonucleotide against the CUBN gene, or an antibody, antibody fragment or aptamer against the CUBN gene product. [3C] The CUBN inhibitor according to [2C], wherein the antisense oligonucleotide against the CUBN gene is an antisense oligonucleotide according to any one of [1] to [5]. [4C] The CUBN inhibitor according to any one of [1C] to [3C], wherein the subject exhibits albuminuria. [5C] A CUBN inhibitor for use in the treatment or prevention of focal segmental glomerulosclerosis (FSGS) in a subject.

[0010] [1D] A method for improving the renal function of a subject, comprising administering a CUBN inhibitor to the patient; preferably, a method that reduces the burden on the kidneys and consequently improves renal function by suppressing or inhibiting the reabsorption of albumin or low molecular weight proteins by CUBN. [2D] The method according to [1D], wherein the CUBN inhibitor is siRNA, shRNA, miRNA, antisense oligonucleotide or decoy oligonucleotide against the CUBN gene, or an antibody, antibody fragment or aptamer against the CUBN gene product. [3D] The method according to [2D], wherein the antisense oligonucleotide against the CUBN gene is the antisense oligonucleotide described in any one of [1] to [5]. [4D] The method according to any one of [1D] to [3D], wherein the subject exhibits albuminuria. [5D] A method for treating or preventing focal segmental glomerulosclerosis (FSGS) in a subject, comprising administering a CUBN inhibitor to the patient; preferably, a method that reduces the burden on the kidneys by inhibiting the reabsorption of albumin and low molecular weight proteins by CUBN, thereby reducing or preventing the symptoms of focal segmental glomerulosclerosis (FSGS).

[0011] [1E] A method for producing a drug that improves the renal function of a target, comprising: 1) a step of preparing a CUBN inhibitor; 2) a step of administering the CUBN inhibitor in vitro to cells expressing CUBN; 3) a step of detecting the amount of CUBN expression in the cells; and 4) a step of identifying a CUBN inhibitor with a high inhibitory effect on expression from the amount of CUBN expression detected in step 3), and producing a drug that improves the renal function of a target using the identified CUBN inhibitor. [2E] The method according to [1E], wherein in step 4), the magnitude of expression suppression is determined by comparing the amount of CUBN expression in the cells expressing CUBN before administration of the CUBN inhibitor with the amount of CUBN expression detected in step 3). [3E] A method for producing a drug that improves the renal function of a target, comprising: 1) a step of preparing a CUBN inhibitor; 2) a step of administering the CUBN inhibitor in vivo to a non-human subject exhibiting renal impairment; 3) a step of detecting a renal function marker in the non-human subject; and 4) a step of producing a drug that improves the renal function of a target using the administered CUBN inhibitor if improvement in renal function is observed from the renal impairment marker detected in step 3). [4E] The method according to [3E], wherein the renal function marker comprises at least one selected from the group consisting of KIM-1, NGAL, L-FABP, MCP-1, and Uromodulin. [5E] The method according to [3E] or [4E], wherein in step 4) the presence or absence of improvement in renal function is determined by comparing the renal function marker in the non-human subject before administration of the CUBN inhibitor with the renal function marker detected in step 3). [6E] The method for producing a CUBN gene according to any one of [1E] to [5E], wherein the CUBN inhibitor is a siRNA, shRNA, miRNA, antisense oligonucleotide or decoy oligonucleotide against the CUBN gene, or an antibody, antibody fragment or aptamer against the CUBN gene product. [7E] The method for producing a CUBN gene according to [6E], wherein the antisense oligonucleotide against the CUBN gene is an antisense oligonucleotide according to any one of [1] to [5].

[0012] The present invention provides a method for improving renal function, and a compound that modulates the expression and function of CUBN, which can be used in the method for improving renal function.

[0013] The following shows the expression of CUBN in the kidney in Example 2-1. The following shows the expression levels of CUBN in the renal cortex (A), the ileum (B), and albuminuria (albumin / creatinine ratio: U-ACR) (C) in Example 2-2. The following shows the expression levels of CUBN in the renal cortex (A), the ileum (B), and albuminuria (albumin / creatinine ratio: U-ACR) (C) in Example 2-3. The following shows the expression levels of CUBN in the renal cortex (A), the ileum (B), and albuminuria (albumin / creatinine ratio: U-ACR) (C) in Example 2-4. The following shows the expression levels of CUBN in the renal cortex (A), CUBN in the ileum (B), and albuminuria (albumin / creatinine ratio: U-ACR) (C) in Example 2-6. The following shows the expression levels of CUBN in the renal cortex (A), Kim-1 (Havcr1) in the renal cortex (B), MCP-1 (Ccl2) in the renal cortex (C), and collagen 1a1 (Col1a1) in the renal cortex (D) in Example 3-1. The following shows the expression levels of CUBN in the renal cortex (A), Kim-1 (Havcr1) in the renal cortex (B), MCP-1 (Ccl2) in the renal cortex (C), and collagen 1a1 (Col1a1) in the renal cortex (D) in Example 3-2. The following shows the expression levels of CUBN in the renal cortex (A), Kim-1 (Havcr1) in the renal cortex (B), blood urea nitrogen levels (C), and the changes in urinary KIM-1 levels after administration of Adriamycin (D) in Example 3-3. The following shows the immunohistochemical staining images of the kidneys of individuals administered Adriamycin and PBS with KIM-1 antibody (A and B) in Example 3-3, and the immunohistochemical staining images of the kidneys of individuals administered Adriamycin and TOA206V with KIM-1 antibody (C and D). The following shows the number of animals by severity determined from the regenerated tubular image in pathological examination in Example 3-3 (A), the number of animals by severity determined from the KIM-1 antibody immunopositive image (B), and the number of animals by severity determined from the glomerulosclerosis image (C).The severity criteria were as follows: for regenerated tubules, a regenerated tubule percentage of approximately 75% or more in the renal cortex was classified as Severe, approximately 40-75% as Moderate, approximately 20-40% as Slight, and approximately 20% or less as Minimal. For KIM-1 antibody immunopositivity, a KIM-1 positive tubule percentage of approximately 40% or more in the renal cortex was classified as Severe, approximately 20-40% as Moderate, approximately 10-20% as Slight, and approximately 10% or less as Minimal. For glomerular sclerosis, severe sclerosis or collapse was observed as Severe, mild adhesion or partial sclerosis in many glomeruli was classified as Moderate, mild adhesion or mild sclerosis in some glomeruli was classified as Slight, and only mild changes such as glomerular enlargement or mesangial expansion were observed as Minimal.

[0014] Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but are not necessarily limited thereto. The object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and those skilled in the art will be able to easily reproduce the present invention from the description herein. The embodiments and specific examples of the invention described below are examples of preferred embodiments of the present invention and are provided for illustrative or explanatory purposes only; the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein, within the intent and scope of the present invention as disclosed herein.

[0015] In this specification, “comprising” may include “substantially comprising,” “essentially comprising,” “consisting essentially of,” and “consisting of.”

[0016] Antisense oligonucleotides (sometimes called antisense nucleic acids or ASOs) are oligonucleotides that target target RNA (mRNA, pre-RNA, miRNA, etc.) present in cells. Antisense oligonucleotides include RNA-degrading antisense nucleic acids, which specifically cleave the target by adding nucleic acids that form a hybrid with the target RNA (mRNA, miRNA, etc.) using RNase H activity, which specifically hydrolyzes the phosphodiester bond of RNA hybridized to DNA, and splicing-regulating antisense nucleic acids, which act by strongly binding to splice sites and inhibiting the binding of proteins involved in splicing. Such oligonucleotides have a base portion and a main chain portion in terms of structure. In one embodiment of the present invention, the main chain portion includes a "sugar" portion and a "nucleoside bond". A "nucleoside bond" refers to a group or bond that forms a covalent bond between the sugars of adjacent nucleosides in an oligonucleotide.While not particularly limited, the main chain portion may include: 1) Naturally occurring main chain portions (for example, the sugar and phosphate group (ester) called deoxyribose that constitute DNA; the sugar and phosphate group (ester) called ribose that constitute RNA); 2) Naturally occurring main chain portions in which the phosphodiester (PO) portion, which is the nucleoside bond, is replaced with alkylphosphonates (methylphosphonate, methoxypropylphosphonate, etc.); phosphate triesters; phosphorothioates (PS), etc.; 3) Naturally occurring main chain portions in which the sugar (ribose; β-D-ribosyl and β-D-2'-deoxyribosyl) is replaced with sugar substitutes or other furanosyl sugars, etc. (hereinafter also referred to as modified sugars) [For example, while not particularly limited, 3-1) Naturally occurring main chain portions in which the 2' position of the sugar (ribose) is replaced with 2'-OMe; 2'-MOE; 2'-F, etc., resulting in a modified non-bicyclic sugar; 3-2) The naturally occurring main chain sugar (ribose) has been replaced with a bicyclic sugar (examples include 2',4'-BNA (LNA), ENA, (S)-cEt, AmNA, GuNA, scpBNA, etc., where the 2' and 4' positions are cross-linked); and 3-3) The naturally occurring main chain sugar (ribose) has been replaced with a methylenemorpholine ring skeleton, which is a sugar substitute; 4) The main chain may have a structure different from the naturally occurring main chain (for example, peptide nucleic acids (such as those with N-(2-aminoethyl)glycine linked by an amide bond); phosphorodiamidate morpholino oligonucleotides (PMOs) (such as those with a methylenemorpholine ring skeleton linked via a phosphorodiamidate group)); and combinations thereof (for example, 2) + 3) = the nucleoside bond and sugar portion are both substituted).Furthermore, although not particularly limited, the base portion may be selected from a) ordinary bases (A, G, T, C, U); b) modified bases (hypoxanthine (inosine (I) as a nucleotide); xanthine (xanthosine (X)); 7-methylguanine (7-methylguanosine (m7G)); 5,6-dihydrouracil (dihydrouridine (D)); 5-methylcytosine (5-methylcytidine (mC)); 5-hydroxymethylcytosine (5-hydroxymethylcytidine) etc.); and c) nucleic acid analogs (aminoallyl nucleotides; isoguanine and isocytosine; 2-amino-6-(2-thienyl)purine and pyrrole-2-carbaldide) etc.).

[0017] One embodiment of the present invention is an antisense oligonucleotide targeting the CUBN gene. Cubilin (CUBN; sometimes referred to as IFCR, Gp280, etc.) is a gene specifically expressed in the epithelium of the kidney and small intestine (HGNC: 2548 NCBI Gene: 8029 Ensembl: ENSG00000107611 OMIM(R): 602997 UniProtKB / Swiss-Prot: O60494), and functions as an endocytosis receptor that plays a role in the metabolism of lipoproteins, vitamins, and iron by promoting their uptake. Although Cubilin does not have a transmembrane domain, it binds to a single-pass transmembrane amino acid-less (AMN) and localizes on the cell membrane surface. In humans, the CUBN gene product is located on chromosome 10 (10p13) and is a gene of approximately 300,000 base pairs (https: / / www.ncbi.nlm.nih.gov / gene / 8029) (Sequence ID 1). It is transcribed from DNA into mRNA precursor, and then spliced ​​to form mRNA with 67 exons (Sequence ID 2). Exons 1-67 of human CUBN correspond to Sequence IDs 4-70, respectively. In mice, it is located on chromosome 2 (2A1; 29.86 cM) and contains 67 exons (https: / / www.ncbi.nlm.nih.gov / gene / 65969), and the sequence of a full-length mRNA of 11262 bp has been reported (Sequence ID 3). Exons 1-67 of mouse CUBN correspond to Sequence IDs 71-137, respectively.

[0018] In this specification, "complementary" with respect to an oligonucleotide or a part thereof (hereinafter referred to as "oligonucleotide, etc.") means that when the oligonucleotide, etc. and the nucleic acid base sequence of a nucleic acid or a part thereof are aligned in opposite directions, they form so-called Watson-Crick type base pairs (for example, adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methylcytosine (mC) and guanine (G), etc.). In this specification, "n%" (0 ≤ n ≤ 100) in "n% complementary" with respect to an oligonucleotide means the percentage of nucleic acid bases that the oligonucleotide, etc. forms Watson-Crick type base pairs with the nucleic acid or a part thereof when the nucleic acid base sequence of the oligonucleotide, etc. and the nucleic acid base sequence of a part thereof are aligned in opposite directions. "n%" is calculated by dividing the number of nucleic acid bases of the oligonucleotide, etc. that are complementary to the nucleic acid base at the corresponding position in the nucleic acid or a part thereof by the total number of nucleic acid bases of the oligonucleotide, etc. In this specification, "having homology" means that a nucleic acid or a region thereof, or an oligonucleotide or a sequence thereof (hereinafter abbreviated as "nucleic acid, etc.") has the same or similar sequence as another nucleic acid, etc. In this specification, "having n% homology" means that "n%" (0 ≤ n ≤ 100) is the percentage of nucleic acid bases that are identical in nucleic acid, etc. α to nucleic acid, etc. β when the sequences of one nucleic acid, etc. (referred to as nucleic acid, etc. α) and another nucleic acid, etc. (referred to as nucleic acid, etc. β) are aligned in the same direction. "n%" is calculated by dividing the number of nucleic acid bases in nucleic acid, etc. α that are identical to the nucleic acid bases at corresponding positions in nucleic acid, etc. β by the total number of nucleic acid bases in nucleic acid, etc. α. Here, cytosine and 5-methylcytosine, and uracil and thymine are considered identical for the calculation.

[0019] The antisense oligonucleotide in one embodiment of the present invention is not particularly limited in length as long as it functions as an antisense oligonucleotide, but may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. The nucleotide length is preferably 12 to 28, more preferably 14 to 26, and even more preferably 16 to 24.

[0020] It has also been reported that various SNPs exist in CUBN. Therefore, sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology to these sequences can be considered equivalent to the human CUBN gene genomic sequence, human mRNA sequence, and mouse mRNA sequence, respectively. Accordingly, an antisense oligonucleotide targeting the CUBN gene in one embodiment of the present invention may be 100% complementary to any of the sequences described in Sequence ID No. 1 to 3, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology thereto.

[0021] In one embodiment of the present invention, the antisense oligonucleotide targeting the CUBN gene may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% complementary to the sequence described in any of SEQ ID NOs: 1 to 3.

[0022] In one embodiment of the present invention, an antisense oligonucleotide has a sequence consisting of at least 10, 11, 12, 13, 14, 15, 16, 17, 18 consecutive nucleotides in its sequence, which corresponds to Sequence ID No. 2, specifically 671-688, 783-800, 1359-1378, 1877-1896, 1878-1897, 1879-1898, 1880-1899, 1881-1900, 1882-1901, 1991-2008, 1993-2010, 2288-2305, 2563-2582, 2564-2583, 2565-2584, 2566-2583, 2 567-2584, 3581-3600, 4038-4055, 4039-4056, 4061-4078, 4062-4079, 4286-4303, 4333-4352, 4609-4628, 5084-5101, 5424-5443, 5469-5488, 5470- 5489, 5471-5490, 5472-5491, 5599-5618, 5600-5619, 5601-5620, 5604-5623, 5605-5624, 5608-5627, 5610-5629, 5611-5630, 5612-5631, 5613-5632, 5614-5631, 5908-5927, 5913-5932, 6674-6693, 7431-7448, 8285-8302, 8529-8546, 8530-8547, 9961-9978, 10924-10943, 10935-10954, 10946-1096 5, 10957-10976, 10968-10987, 10979-10998, 11002-11021, 11013-11032, 11024-11043, 11035-11054, 11046-11065, 11058-11077, 11062-11081, 111 79-11198, 11197-11216, 11303-11322, 11369-11388, 11380-11399, 11391-11410, 11402-11421, 11413-11432, 11424-11443, 11435-11454, 11446-1 1465, 11457-11476, 11468-11487, 11479-11498, 11490-11509, 11501-11520, 11513-11532, 11528-11547, 11548-11567, 11561-11580, 11578-11597,11590-11609, 11639-11658, 11686-11705, 11699-11718, 11712-11731, 11745-11764, 11754-11773, 11765-11784, 11776-11795, 11821-11840, 11832-11851, 11843-118 Sequence 62, 11854-11873, 11865-11884, 11876-11895, 11887-11906, and 11898-11917 may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% complementary to any of these sequences.

[0023] In one embodiment of the present invention, the antisense oligonucleotides are 671-688, 783-800, 1359-1378, 1877-1896, 1878-1897, 1879-1898, 1880-1899, 1881-1900, 1882-1901, 1991-2008, 1993-2010, 2288-2305, 2563-2582, 2564-2583, 2565-2584, 2566-2583, 2567-2584, 3581-3600, 4038-4055, 4039-4056, 4061-4078, 4062- 4079, 4286-4303, 4333-4352, 4609-4628, 5084-5101, 5424-5443, 5469-5488, 5470-5489, 5471-5490, 5472-5491, 5599-5618, 5600-5619, 5601-562 0, 5604-5623, 5605-5624, 5608-5627, 5610-5629, 5611-5630, 5612-5631, 5613-5632, 5614-5631, 5908-5927, 5913-5932, 6674-6693, 7431-7448, 82 85-8302, 8529-8546, 8530-8547, 9961-9978, 10924-10943, 10935-10954, 10946-10965, 10957-10976, 10968-10987, 10979-10998, 11002-11021, 1 1013-11032, 11024-11043, 11035-11054, 11046-11065, 11058-11077, 11062-11081, 11179-11198, 11197-11216, 11303-11322, 11369-11388, 11380 ~11399, 11391~11410, 11402~11421, 11413~11432, 11424~11443, 11435~11454, 11446~11465, 11457~11476, 11468~11487, 11479~11498, 11490~11 509, 11501-11520, 11513-11532, 11528-11547, 11548-11567, 11561-11580, 11578-11597, 11590-11609, 11639-11658, 11686-11705, 11699-11718,The sequences 11712–11731, 11745–11764, 11754–11773, 11765–11784, 11776–11795, 11821–11840, 11832–11851, 11843–11862, 11854–11873, 11865–11884, 11876–11895, 11887–11906, and 11898–11917 may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% complementary to any of these sequences.

[0024] In one embodiment of the present invention, an antisense oligonucleotide has a sequence consisting of at least 10, 11, 12, 13, 14, 15, 16, 17, 18 consecutive nucleotides in its sequence, which corresponds to 1877-1896, 1878-1897, 1879-1898, 1880-1899, 1881-1900, 1882-1901, 5599-5618, and 5600-561. 9, may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% complementary to any of the sequences at positions 9, 5601–5620, 5604–5623, 5605–5624, 5608–5627, 5610–5629, 5611–5630, 5612–5631, 5613–5632, or 5614–5631.

[0025] In one embodiment of the present invention, the antisense oligonucleotide may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% complementary to any of the sequences 1877-1896, 1878-1897, 1879-1898, 1880-1899, 1881-1900, 1882-1901, 5599-5618, 5600-5619, 5601-5620, 5604-5623, 5605-5624, 5608-5627, 5610-5629, 5611-5630, 5612-5631, 5613-5632, or 5614-5631 of Sequence ID No. 2.

[0026] In one embodiment of the present invention, the antisense oligonucleotide is preferably complementary to the exon region of the CUBN gene. It is not particularly limited, but it may be complementary to the sequences of human exons 1, 7, 8, 12, 15, 16, 17, 19, 25, 27, 28, 29, 31, 34, 37, 38, 39, 43-44, 48, 49-50, 52-53, 54, 62, or 67, and among these, it is preferably complementary to the sequence of human exon 15 (SEQ ID NO: 18) or 37 (SEQ ID NO: 40).

[0027] The antisense oligonucleotide in one embodiment of the present invention may include part or all of the sequences described in any of SEQ ID NOs: 138-366 or 399-408. The sequence of the antisense oligonucleotide in one embodiment of the present invention may be the sequence described in any of SEQ ID NOs: 138-366 or 399-408.

[0028] An antisense oligonucleotide in one embodiment of the present invention may include a sequence consisting of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% homology with any of the sequences represented by SEQ ID NOs: 138-366 and 399-408.

[0029] The antisense oligonucleotide in one embodiment of the present invention may contain a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% homology with the antisense oligonucleotide represented by any of SEQ ID NOs: 138-366 or 399-408. The sequence of the antisense oligonucleotide in one embodiment of the present invention may contain a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% homology with the antisense oligonucleotide represented by any of SEQ ID NOs: 138-366 or 399-408.

[0030] The antisense oligonucleotide in one embodiment of the present invention is not particularly limited as long as it functions as an antisense oligonucleotide, but may be composed of any or a combination thereof of the main chain portions 1) to 4) described above. The nucleoside bonds of the antisense oligonucleotide in one embodiment of the present invention may be partially or entirely composed of nucleoside bonds defined in the main chain portion 2). The nucleoside bonds may include phosphorothioates, or all of the nucleoside bonds may be phosphorothioates. The sugar portion of the antisense oligonucleotide in one embodiment of the present invention may include modified sugars defined in the main chain portion 3).

[0031] The base portion of the antisense oligonucleotide in one embodiment of the present invention may be any of a) to c) above or a combination thereof.

[0032] In one embodiment of the present invention, the antisense oligonucleotide is a so-called gapmer. The gapmer includes two external regions (called "wings") and a central or internal region (called the "gap"). Of the two wings, the 5' end is called the "5'-wing" and the 3' end is called the "3'-wing". The 5'-wing, gap, and 3'-wing are linked by nucleoside bonds. In this embodiment, the two wings have RNase enzyme resistance, while the gap contributes to recognition by RNaseH, thereby allowing the oligonucleotide to function as an RNA-degrading antisense nucleic acid. Furthermore, when the entire antisense oligonucleotide is formed from a mixture of the above-mentioned main chain portion 1) and the above-mentioned main chain portions 2) to 4) (a so-called mixedmer), it can function as a splicing-controlled antisense nucleic acid.

[0033] Each wing of the gapmer in one embodiment of the present invention may contain 1 to 8, 2 to 8, 3 to 8, or 3 to 5 nucleosides. In other words, the nucleotide length of each wing may be 1 to 8, 2 to 8, 3 to 8, or 3 to 5. The wing of the gapmer in one embodiment of the present invention has nucleosides whose sugar portion is a modified sugar, and at least the sugar portion of the nucleosides adjacent to the gap is a modified sugar. Each nucleoside of the wing of the gapmer in one embodiment of the present invention has modified sugars with one or two exceptions. All nucleosides of the wing of the gapmer in one embodiment of the present invention may have 3) modified sugars and / or 4) a backbone portion having a structure different from the naturally occurring backbone portion.

[0034] In one embodiment of the present invention, the gap in the gapmer may contain 6 to 16, 6 to 14, 8 to 14, or 8 to 12 nucleosides. In other words, the nucleotide length of the gap may be 6 to 16, 6 to 14, 8 to 14, or 8 to 12.

[0035] In one embodiment of the present invention, the gap of the gapmer has a β-D-2'-deoxyribosyl nucleoside as its sugar portion, and the sugar portion of at least the nucleoside adjacent to the wing may be β-D-2'-deoxyribosyl. In one embodiment of the present invention, the sugar portion of each nucleoside in the gapmer of the gapmer may be β-D-2'-deoxyribosyl, with one exception. In one embodiment of the present invention, the sugar portion of all nucleosides in the gapmer of the gapmer may be β-D-2'-deoxyribosyl.

[0036] An antisense oligonucleotide in one embodiment of the present invention includes a conjugate group. In this specification, “conjugate group” means an atomic group attached to the oligonucleotide. The conjugate group may comprise, but is not limited to, a “conjugate portion” and a “conjugate linker” that attaches the conjugate portion to the oligonucleotide. The “conjugate linker” means an atomic group comprising at least one bond that links the conjugate portion to the oligonucleotide. The “conjugate portion” means an atomic group attached to the oligonucleotide via the conjugate linker. The conjugate group may be attached to the 5' and / or 3' end of the antisense oligonucleotide. In one embodiment of the present invention, the conjugate group may comprise at least one N-acetylgalactosamine (GalNAc), at least two N-acetylgalactosamines (GalNAc), or at least three N-acetylgalactosamines (GalNAc) as the conjugate portion.

[0037] One embodiment of the present invention is a pharmaceutical composition comprising the above-mentioned antisense oligonucleotide. Such a pharmaceutical composition may also contain other active ingredients, a pharmacologically acceptable carrier, and the like, in addition to the antisense oligonucleotide.

[0038] One embodiment of the present invention is a pharmaceutical composition or method for improving renal function in a subject. The subject is mainly mammals, and includes, but is not limited to, humans and non-human animals (including companion animals such as dogs and cats, industrial livestock such as cattle and pigs, and laboratory animals such as mice and rats). In this specification, "improving renal function" may include "preventing or treating renal damage," "preventing or treating renal inflammation," and "preventing or treating renal fibrosis." That is, "improving renal function" may include "preventing or treating renal damage," "preventing or treating renal inflammation," and "preventing or treating renal fibrosis." The kidneys primarily play a role in maintaining fluid homeostasis by filtering and excreting waste products and excess water from the blood to produce urine; however, this function can decline due to various factors. The present invention can be used to restore or maintain such function. Recovery or maintenance involves monitoring for markers of renal dysfunction such as Kim-1 (Kidney Injury Molecule 1), NGAL (neutrophil gelatinase-associated lipocalin), L-FABP (liver-type fatty acid-binding protein), NAG (N-acetyl-β-D-glucosaminidase), clasterin, cystatin C, β2-microglobulin, and the inflammatory marker MCP-1 (monocyte chemoattractant protein). 1) RANTES (regulated on activation, normal T cell expressed and secret / CCR5), a tissue fibrosis marker such as Col1a1 (collagen 1A1), can be evaluated as a decrease or maintenance. Such markers can also be detected from isolated samples derived from the subject (urine, blood (including plasma and serum), kidney tissue, etc.).Without particular limitation, administration of the pharmaceutical composition according to the present invention may reduce the detectable markers of renal dysfunction, inflammation, or fibrosis by at least 5%, 10%, 20%, or 30% compared to before administration of the pharmaceutical composition or in a patient without administration of the pharmaceutical composition. Alternatively, recovery or maintenance can be evaluated by pathological methods. In one embodiment of the present invention, such a pharmaceutical composition or method may use a CUBN inhibitor. A CUBN inhibitor means a compound that can reduce the function of CUBN in the kidney and is not limited to the above-mentioned oligonucleotides. Without particular limitation, this also includes siRNA, shRNA, miRNA, or decoy oligonucleotides against the CUBN gene, or antibodies, antibody fragments, or aptamers against the CUBN gene product. Without particularly limiting the mechanism of action, such inhibitors can reduce the burden on the kidney and thereby improve renal function by suppressing or inhibiting the reabsorption of albumin and low molecular weight proteins by CUBN.

[0039] In one embodiment of the present invention, the decline in renal function may be caused by the administration of a drug to the subject. Various drugs are metabolized or excreted from the body after administration. In this process, damage to the kidneys is often caused.

[0040] In one embodiment of the present invention, the decline in renal function may be due to nephrotic syndrome, focal segmental glomerulosclerosis (FSGS), etc. Accordingly, the present invention includes pharmaceutical compositions or methods for the treatment or prevention of nephrotic syndrome, focal segmental glomerulosclerosis (FSGS), etc. Focal segmental glomerulosclerosis is one of the kidney diseases that cause nephrotic syndrome, and is a disease in which a part of the glomerulus, which is a spherical cluster of capillaries in the kidney, hardens (hardens), resulting in high concentrations of proteinuria, hematuria, hypertension, edema, etc.

[0041] "Treatment of nephrotic syndrome, focal segmental glomerulosclerosis (FSGS), etc." includes the alleviation and remission of various symptoms (such as edema, hematuria, and hypertension) associated with nephrotic syndrome, focal segmental glomerulosclerosis, etc. as a result of administration of the pharmaceutical composition according to the present invention. Although not particularly limited, "treatment of focal segmental glomerulosclerosis (FSGS)" may include an improvement in the prognosis value. Such a prognosis value can be obtained from the results of examining pathological tissues collected by renal biopsy by ordinary methods.

[0042] "Prevention of nephrotic syndrome, focal segmental glomerulosclerosis (FSGS), etc." includes suppressing the occurrence of various symptoms (such as edema, hematuria, and hypertension) associated with nephrotic syndrome, focal segmental glomerulosclerosis, etc. as a result of administration of the pharmaceutical composition according to the present invention. Although not particularly limited, "prevention of focal segmental glomerulosclerosis" may include no change in the prognosis value. Such a prognosis value can be obtained from the results of examining pathological tissues collected by renal biopsy by ordinary methods.

[0043] One embodiment of the present invention is a method for screening a pharmaceutical composition in vivo or in vitro for improving renal function in a subject or for treating or preventing focal segmental glomerulosclerosis (FSGS). By manufacturing or obtaining an inhibitor of CUBN and detecting the inhibitory activity of the expression function of CUBN or renal function markers and inflammatory markers by the method described herein, it becomes possible to obtain a highly effective pharmaceutical composition.

[0044] An inhibitor of CUBN can be manufactured or obtained by methods known in the art. For example, an antisense oligonucleotide can be synthesized using a commercially available automatic nucleic acid synthesizer and then purified using a reverse phase column or the like. Alternatively, an antisense oligonucleotide can also be ordered from a manufacturer (such as Nippon Shokubai Co., Ltd.) by specifying the nucleobase sequence, modification site, and type and obtained.

[0045] The inhibitory activity of cubilin expression can be measured in an in vitro experimental system using cells that express cubilin. Without particular limitation, NMuli cells (derived from mouse hepatocytes), mouse cubilin-expressing HEK293 cells (HEK / mCubilin), HEK293 cells (derived from human fetal kidney cells), MIA PaCa-2 cells (derived from human pancreatic cancer), etc. are preferred. A cubilin inhibitor is administered to cells that express cubilin, and its expression inhibitory activity is detected by RT-PCR, ELISA, etc. Without particular limitation, for the production of a highly effective pharmaceutical, in the cell culture medium, at an added concentration of 1000 nM, 300 nM, 100 nM, 60 nM, 50 nM, 30 nM, 20 nM, 17 nM, 10 nM, or 3 nM, compared to the expression level before administration, at least 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, a cubilin inhibitor that causes expression inhibition can be used.

[0046] The non-human subject used for screening is preferably a subject with renal dysfunction. Without particular limitation, those known as model experimental animals for renal dysfunction are preferred. As an example, puromycin aminonucleoside (PAN), which is a drug that directly damages glomerular epithelial cells; adriamycin; daunomycin, etc. are administered to produce model experimental animals, etc. The renal function disorder marker in the non-human subject may be detected from an isolated sample derived from the subject, or may be detected from the excised kidney tissue after euthanasia of the non-human subject. Without particular limitation, in a single administration or repeated administration at a dose of 5 to 60 mg / kg, if any one of the renal function disorder marker or the inflammatory marker is at least 5% or more, 10% or more, 20% or more, or 30% or more better than the sample before administration or the sample of the vehicle-administered subject, it can be used for the production of a highly effective pharmaceutical composition.

[0047] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[0048] In vitro study <Inhibition of CUBN expression by antisense oligonucleotides in NMuLi cells (derived from mouse hepatocytes)> Example 1-1 Mouse NMuLi cell lines were purchased from ATCC and incubated in a humidified incubator at 37°C and 5% CO2, in accordance with the supplier's recommendations. ２ Cells were cultured in [specimen name]. Antisense oligonucleotide treatment was performed using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific Co., Ltd.) according to the supplier's instructions, by reverse transfection, seeding 20,000 cells / well in a 96-well plate to the final oligonucleotide concentration shown in Table 2. 48 hours after antisense oligonucleotide treatment, RNA was extracted using RNeasy 96 Kit (Qiagen Co., Ltd.) according to the supplier's instructions. Next, cDNA was synthesized using PrimeScript FAST RT Reagent Kit with gDNA Eraser (Takara Bio Inc.). Gene expression analysis was performed by qPCR using TB Green Premix Ex Taq II FAST qPCR (Takara Bio Inc.). For qPCR, one of the primer sets in Table 4 below was used for mouse CUBN. The housekeeping gene ATP5F1 was used as the control. The knockdown activity (%) of antisense oligonucleotides against mouse CUBN mRNA is shown in Table 2 as 100 minus the reduction in mRNA expression (%) by the test substance compared to the control (untreated cells). In the following tables, [mC] represents 5-methylcytosine, d represents DNA (β-D-2'-deoxyribose), [MOE] represents RNA with a 2'-O-methoxyethyl group introduced into the sugar portion (ribose), [LNA] represents LNA (Locked Nuclear Acid), and [ENA] represents ENA (2'-O,4'-C-Ethylene-bridged Nuclear Acids). All of the antisense oligonucleotides used had the phosphodiester (PO) portion of the naturally occurring main chain replaced with a phosphorothioate (PS).

[0049] <Inhibition of CUBN expression in mouse CUBN-expressing HEK293 cells (HEK / mCUBN) by antisense oligonucleotides> Example 1-2 Mouse CUBN-expressing HEK293 cells were prepared using Flp-In Core System (Thermo Fisher Scientific Co., Ltd.) and Flp-In-293 Cell Line (Thermo Fisher Scientific Co., Ltd.) according to the supplier's instructions, and incubated in a humidified incubator at 37°C and 5% CO2. ２ Cells were cultured in the following manner. Antisense oligonucleotide treatment was performed using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific Co., Ltd.) according to the supplier's instructions, by reverse transfection, seeding 30,000 cells / well in a 96-well half-area plate to the final oligonucleotide concentration shown in Table 3. 48 hours after antisense oligonucleotide treatment, cDNA was synthesized using SuperPrep II Cell Lysis & RT Kit for qPCR (Toyobo Co., Ltd.) according to the supplier's instructions. Gene expression analysis was performed by qPCR using TB Green Premix Ex Taq II FAST qPCR (Takara Bio Inc.). For qPCR, one of the following mouse CUBN primer sets shown in Table 4 was used. For the control, the knockdown activity (%) of antisense oligonucleotides using the housekeeping gene ATP5F1 against mouse CUBN mRNA was calculated as 100 minus the reduction in mRNA expression (%) by the test substance compared to the control (untreated cells), as shown in Table 3.

[0050] All of the antisense oligonucleotides used had the phosphodiester (PO) portion of the naturally occurring main chain replaced with a phosphorothioate (PS).

[0051]

[0052] <Inhibition of CUBN expression by antisense oligonucleotides in HEK293 cells (derived from human fetal kidney cells)> Example 1-3 Human HEK293 cell line was purchased from ATCC and incubated in a humidified incubator at 37°C and 5% CO2 according to the supplier's recommendations. ２ Cells were cultured in [specimen name]. Treatment with the test substance was performed by forward transfection using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific Co., Ltd.) according to the supplier's instructions, seeding 75,000 cells / well in a 48-well plate so that the final oligonucleotide concentration was as shown in Table 5. 48 hours after treatment with the test substance, RNA was extracted using RNeasy Mini Kit (Qiagen Co., Ltd.) according to the supplier's instructions. Next, cDNA was synthesized using PrimeScript RT Master Mix (Takara Bio Inc.). Gene expression analysis was performed by qPCR using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio Inc.). For qPCR, one of the primer sets listed in Table 7 below was used as the human CUBN primer set. The housekeeping gene ATP5F1 was used as the control. The knockdown activity (%) of the test substance against human CUBN mRNA is shown in Table 5 as 100 minus the reduction in mRNA expression (%) by the test substance compared to the control (untreated cells).

[0053] All of the antisense oligonucleotides used had the phosphodiester (PO) portion of the naturally occurring main chain replaced with a phosphorothioate (PS).

[0054] <Inhibition of CUBN Expression by Antisense Oligonucleotides in MIA PaCa-2 Cells (Derived from Human Pancreatic Cancer)> Example 1-4 Human MIA PaCa-2 cells were purchased from ATCC and cultured in a humidified incubator at 37°C and 5% CO2 according to the supplier's recommendations. Antisense oligonucleotide treatment was performed using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific Co., Ltd.) according to the supplier's instructions, by reverse transfection, seeding 20,000 cells / well in a 96-well half-area plate to the final oligonucleotide concentration shown in Table 6. 48 hours after antisense oligonucleotide treatment, cDNA was synthesized using SuperPrep II Cell Lysis & RT Kit for qPCR (Toyobo Co., Ltd.) according to the supplier's instructions. Gene expression analysis was performed using qPCR with TB Green Premix Ex Taq II FAST qPCR (Takara Bio Inc.). One of the primer sets in Table 7 below was used for qPCR. One of the primer sets in Table 7 below was used as the primer set for human CUBN for qPCR. Human B2m (beta-2-microglobulin), a housekeeping gene, was used as the control. The knockdown activity (%) of antisense oligonucleotides against human CUBN mRNA is shown in Table 6 as 100 - the amount of mRNA expression reduced by the test substance (%) compared to the control (untreated cells).

[0055] All of the antisense oligonucleotides used had the phosphodiester (PO) portion of the naturally occurring main chain replaced with a phosphorothioate (PS).

[0056] Vivo Study <Administration of CUBN Antisense Oligonucleotides to Normal Mice> Example 2-1 To confirm whether CUBN antisense exhibits KD activity in the kidneys, antisense nucleic acids selected from the above were administered to 9-week-old male C57 / BL6 mice (purchased from Jackson Laboratory Japan). Each treatment group consisted of 4 mice. Each mouse received repeated subcutaneous administration of 100 mg / kg of antisense oligonucleotide (TOA007V, TOA039V, or TOA007EV) or Saline for 5 days. Five days after the final administration, the mice were euthanized and their kidneys were collected. The kidneys were then fixed in 10% neutral phosphate-buffered formalin and embedded in paraffin according to standard procedures. The sections were thinly sliced ​​to approximately 3 μm thickness, and the expression status of CUBN was examined immunohistochemically using antibodies with the N-terminus or C-terminus of the CUBN protein as the immunogen.

[0057] As a result, as shown in Figure 1, administration of 100 mg / kg of TOA007V, TOA039V, or TOA007EV significantly reduced the positive reaction to N-terminal and C-terminal CUBN antibodies.

[0058] Example 2-2 To confirm whether CUBN antisense exhibits KD activity in the kidney, antisense nucleic acids selected from the above (TOA007V and TOA007EV) were administered to 9-week-old male C57 / BL6 mice (purchased from Jackson Laboratory Japan). Each treatment group consisted of 4 mice. Each mouse received a single subcutaneous dose of 10 mg / kg or 30 mg / kg of antisense oligonucleotide or saline. One week later, the mice were euthanized after 24 hours of urine collection using a metabolic cage, and the kidneys and ileum were collected. The expression level of CUBN mRNA in the renal cortex or ileum was analyzed by RT-qPCR. Figure 2 shows the ratio of the normalized mean CUBN mRNA expression level in the CUBN antisense treatment group to the normalized mean CUBN mRNA expression level in the saline treatment group (A) and (B), and the urinary albumin / creatinine ratio (C), respectively, in the renal cortex or ileum.

[0059] As shown in Figure 2, the antisense oligonucleotides of the present invention (TOA007V and TOA007EV) suppressed CUBN expression in the renal cortex and increased albuminuria (albumin / creatinine ratio: U-ACR) without significantly suppressing CUBN expression in the ileum.

[0060] Example 2-3 To confirm whether CUBN antisense exhibits KD activity in the kidney, antisense nucleic acids selected from the above were administered to 9-week-old male C57 / BL6 mice (purchased from Jackson Laboratory Japan). Each treatment group consisted of 4 mice. Each mouse received a single intravenous dose of 30 mg / kg of antisense oligonucleotide (TOA039V) or Saline. After 1 week, the mice were euthanized after 24-hour urine collection using a metabolic cage, and the kidneys and ileum were collected. The expression levels of CUBN mRNA in the renal cortex or ileum were analyzed by RT-qPCR. Figure 3 shows the ratio of the normalized CUBN mRNA expression level of the CUBN antisense treatment group to the normalized average CUBN mRNA expression level of the saline treatment group (A) and (B), and the urinary albumin / creatinine ratio (C), respectively.

[0061] As shown in Figure 3, the antisense nucleic acid of the present invention suppressed CUBN expression in the renal cortex and increased albuminuria (albumin / creatinine ratio: U-ACR) without significantly suppressing CUBN expression in the ileum.

[0062] Example 2-4 To confirm whether CUBN antisense exhibits KD activity in the kidney, antisense nucleic acids selected from the above were administered to 9-week-old male C57 / BL6 mice (purchased from Jackson Laboratory Japan). Each treatment group consisted of 4 mice. Each mouse received a single subcutaneous dose of 30 mg / kg of antisense oligonucleotide (TOA045V) or Saline. After 1 week, the mice were euthanized after 24-hour urine collection using a metabolic cage, and the kidneys and ileum were collected. The expression levels of CUBN mRNA in the renal cortex or ileum were analyzed by RT-qPCR. Figure 4 shows the ratio of normalized CUBN mRNA expression levels in the CUBN antisense treatment group to the normalized mean expression levels in the saline treatment group (A) and (B), and the urinary albumin / creatinine ratio (C), respectively.

[0063] As shown in Figure 4, the antisense nucleic acid of the present invention suppressed CUBN expression in the renal cortex and increased albuminuria (albumin / creatinine ratio: U-ACR) without significantly suppressing CUBN expression in the ileum.

[0064] Example 2-5 To confirm whether CUBN antisense exhibits KD activity in the kidney, antisense nucleic acids selected from the above (TOA047V, TOA098V, TOA099V, TOA101V, TOA102V, TOA104V, TOA105V, TOA111V, TOA219V, TOA221V, TOA232V, or TOA410V) were administered to 9-week-old male C57 / BL6 mice (purchased from Jackson Laboratory Japan). Each treatment group consisted of two mice. Each mouse received a single subcutaneous dose of 1 mg / kg, 3 mg / kg, 8 mg / kg, or 10 mg / kg of antisense oligonucleotide or saline. After one week, the mice were euthanized, their kidneys were collected, and the expression level of CUBN mRNA was analyzed by RT-qPCR. Table 8 shows the inhibition rate of normalized CUBN mRNA expression in the CUBN antisense treatment group compared to the normalized average CUBN mRNA expression in the saline treatment group in the renal cortex.

[0065] As shown in Table 8, the antisense oligonucleotides of the present invention (TOA047V, TOA098V, TOA099V, TOA101V, TOA102V, TOA104V, TOA105V, TOA111V, TOA219V, TOA221V, TOA232V, or TOA410V) suppressed the expression of CUBN in the renal cortex.

[0066] Example 2-6 To confirm whether CUBN antisense drugs exhibit KD activity in the kidneys, antisense nucleic acids selected from the above (TOA020V or TOA042V) were administered to 9-week-old male C57 / BL6 mice (purchased from Jackson Laboratory Japan). Each treatment group consisted of 4 mice. Each mouse received two subcutaneous doses of 3 mg / kg, 10 mg / kg, 30 mg / kg, or 100 mg / kg of antisense oligonucleotide or saline. The administration interval was 3 days, and after collecting urine for 24 hours from 2 to 3 days after the second administration, the mice were euthanized, and the kidneys and ileum were collected. The expression level of CUBN mRNA in the renal cortex or ileum was analyzed by RT-qPCR. Figure 5 shows the ratio of normalized CUBN mRNA expression in the CUBN antisense treatment group to the normalized mean CUBN mRNA expression in the saline treatment group (A) and (B), and the urinary albumin / creatinine ratio (C), respectively, in the renal cortex or ileum.

[0067] As shown in Figure 5, the antisense oligonucleotides of the present invention (TOA020V and TOA042V) suppressed CUBN expression in the renal cortex and increased albuminuria (albumin / creatinine ratio: U-ACR) without significantly suppressing CUBN expression in the ileum.

[0068] <Administration of CUBN Antisense to Glomerulosclerosis Model Mice> Example 3-1 The effect of antisense nucleic acids on focal segmental glomerulosclerosis (FSGS) was confirmed using Adriamycin-induced glomerulosclerosis model mice as follows. Eight-week-old male C57BL / 6 mice were used (purchased from Jackson Laboratory Japan). Adriamycin (Adriamycin® for injection: Sandoz Pharma) was administered intravenously at a dose of 10 mg / kg (10 ml / kg of a solution dissolved in physiological saline at a concentration of 1.3 mg / ml) on the first administration day (day 0) and day 9, respectively. The Adriamycin administration group (n=5-8) was repeatedly subcutaneously administered TOA007EV, an antisense nucleic acid, at a dose of 5 or 15 mg / kg on days -7 (7 days prior), -3 (3 days prior), 2, 7, 14, and 21 as the test drug. Saline was administered to the solvent control group. The dosage volume was 10 ml / kg in all cases. The normal group (n=6) did not receive the drug. On the final day of the experiment (day 28), the nephrectomy was performed under isoflurane inhalation anesthesia, and samples were collected from the renal cortex for gene testing and protein evaluation, as well as fixed in neutral buffered formalin for histopathological examination. The mRNA expression levels of CUBN, the tubular damage marker Kim-1 (Havcr1), the inflammation marker MCP-1 (CCl2), and the fibrosis marker collagen 1a1 (Col1a1) were analyzed by RT-qPCR (similar to Example 2-2, etc.). Figure 6 shows the ratios of the normalized mRNA expression levels of CUBN, Kim-1, MCP-1, and collagen 1a1 in the CUBN antisense-treated group to the average normalized mRNA expression levels of CUBN, Kim-1, MCP-1, and collagen 1a1 in the normal group in the renal cortex.

[0069] Example 3-2 The effect of antisense nucleic acids on focal segmental glomerulosclerosis (FSGS) was confirmed using an Adriamycin-induced glomerulosclerosis model mouse as follows. Eight-week-old male C57BL / 6 mice were used (purchased from Jackson Laboratory Japan). Adriamycin (Adriamycin® for injection: Sandoz Pharma) was administered intravenously at a dose of 10 mg / kg (10 ml / kg of a solution dissolved in physiological saline at a concentration of 1.3 mg / ml) on the first administration day (day 0) and day 9, respectively. The Adriamycin administration group (n=6-8) received repeated subcutaneous administrations of the antisense nucleic acid TOA219V at a dose of 15 or 30 mg / kg on days -11 (11 days prior), -7 (7 days prior), -3 (3 days prior), 3, 7, 14, and 21 as the test drug. Saline was administered to the solvent control group. The administered solution volume was 10 ml / kg in all cases. The normal group (n=6) did not receive the treatment. On the final day of the experiment (day 28), the nephrectomy was performed under isoflurane inhalation anesthesia, and samples were collected from the renal cortex for gene testing and protein evaluation, as well as fixed in neutral buffered formalin for histopathological examination. The mRNA expression levels of CUBN, the tubular damage marker Kim-1 (Havcr1), the inflammation marker MCP-1 (CCl2), and the fibrosis marker collagen 1a1 (Col1a1) were analyzed by RT-qPCR (similar to Example 2-2, etc.). Figure 7 shows the ratios of the normalized mRNA expression levels of CUBN, Kim-1, MCP-1, and collagen 1a1 in the CUBN antisense-treated group to the average normalized mRNA expression levels of CUBN, Kim-1, MCP-1, and collagen 1a1 in the normal group in the renal cortex.

[0070] As shown in Figures 6 and 7, the antisense oligonucleotides of the present invention suppressed CUBN expression in the renal cortex even under adriamycin treatment conditions, protecting the kidney from adriamycin-induced inflammation (and tubular damage).

[0071] Example 3-3 The effect of antisense nucleic acids on focal segmental glomerulosclerosis (FSGS) was confirmed using an Adriamycin-induced glomerulosclerosis model rat as follows. Male Wistar rats aged 6-7 weeks were used (purchased from Japan SLC). Adriamycin (Adriamycin® for injection: Sandoz Pharma) was administered intravenously at a dose of 6 mg / kg (5 ml / kg of a solution dissolved in physiological saline at a concentration of 1.2 mg / ml). The Adriamycin administration group (n=4-9) received repeated subcutaneous administration of PBS as a solvent and TOA206V, an antisense nucleic acid, as the test drug, 7 days, 6 days, and 7 days before Adriamycin administration. The solvent control group received PBS. The administration volume was 5 ml / kg in all cases, and TOA206V was administered at a dose of 60 mg / kg. In addition, the normal group (n=4) received intravenous administration of physiological saline, and PBS was administered subcutaneously 7, 6, and 7 days before the administration of physiological saline. Urine samples were collected 14, 21, and 28 days after the administration of Adriamycin, and KIM-1 protein, a marker of renal tubular damage, was measured by ELISA. Blood was collected under isoflurane inhalation anesthesia 28 days after the administration of Adriamycin, and blood urea nitrogen (BUN), a marker of renal function, was measured. Furthermore, the left kidney was removed, and the renal cortex was collected for genetic testing. The mRNA expression levels of CUBN and Kim-1 (Havcr1), a marker of renal tubular damage, were analyzed by RT-qPCR (similar to Example 3-1, etc.). The right kidney was also fixed in neutral buffered formalin for histopathological examination.

[0072] The results are shown in Figures 8-10. Figures 8A and 8B show the ratio of normalized CUBN and Kim-1 mRNA expression levels in the CUBN antisense-treated group to the normalized CUBN and Kim-1 mRNA expression levels in the renal cortex of the normal group. Figure 8C shows the average values ​​of blood urea nitrogen for each group. Figure 8D shows the changes in the average values ​​of urinary KIM-1 for each group after administration of Adriamycin. Figures 9A and 9B show immunohistochemical staining images of the kidneys of individuals administered Adriamycin and PBS, and Figures 9C and 9D show immunohistochemical staining images of individuals administered Adriamycin and TOA206V, respectively. Figures 10A-1C show the number of rats classified by severity as determined from pathological examination ((A) Severity determined from regenerated tubular images; (B) Severity determined from KIM-1 antibody immunopositive images; (C) Severity determined from glomerulosclerosis images). The severity criteria were as follows: for regenerated tubules, a regenerated tubule percentage of approximately 75% or more in the renal cortex was classified as Severe, approximately 40-75% as Moderate, approximately 20-40% as Slight, and approximately 20% or less as Minimal. For KIM-1 antibody immunopositivity, a KIM-1 positive tubule percentage of approximately 40% or more in the renal cortex was classified as Severe, approximately 20-40% as Moderate, approximately 10-20% as Slight, and approximately 10% or less as Minimal. For glomerular sclerosis, severe sclerosis or collapse was observed as Severe, mild adhesion or partial sclerosis in many glomeruli was classified as Moderate, mild adhesion or mild sclerosis in some glomeruli was classified as Slight, and only mild changes such as glomerular enlargement or mesangial expansion were observed as Minimal.

[0073] As shown in Figures 8, 9, and 10, the antisense oligonucleotides of the present invention suppressed CUBN expression in the renal cortex even under Adriamycin treatment conditions and showed protective effects against Adriamycin-induced renal dysfunction and tubular damage.

[0074] The present invention provides a method for improving renal function, and compounds that can be used in the method for improving renal function to regulate the expression and function of CUBN.

Claims

1. An antisense oligonucleotide having a length of 10 to 30 nucleotides, wherein the antisense oligonucleotide targets the CUBN gene and is at least 90% complementary to SEQ ID NO: 1 or SEQ ID NO:

2.

2. The antisense oligonucleotide according to claim 1, having a sequence that is at least 90% complementary to Exon 37 or 15 of CUBN.

3. The antisense oligonucleotide according to claim 1, comprising a sequence consisting of at least 10 consecutive nucleotides having at least 90% homology with any of the sequences described in SEQ ID NOs: 138-366 and 399-408.

4. The antisense oligonucleotide according to claim 1, wherein some or all of the constituent nucleosides contain a modified sugar.

5. A pharmaceutical composition containing an antisense oligonucleotide according to any one of claims 1 to 4.

6. A pharmaceutical composition for improving renal function in a subject, comprising a CUBN inhibitor.

7. The pharmaceutical composition according to claim 6, wherein the CUBN inhibitor is a siRNA, shRNA, miRNA, antisense oligonucleotide or decoy oligonucleotide against the CUBN gene, or an antibody, antibody fragment or aptamer against the CUBN gene product.

8. The pharmaceutical composition according to claim 7, wherein the antisense oligonucleotide against the CUBN gene is the antisense oligonucleotide according to any one of claims 1 to 4.

9. The pharmaceutical composition according to claim 6, wherein the subject exhibits albuminuria.

10. A pharmaceutical composition for the treatment or prevention of focal segmental glomerulosclerosis (FSGS) in a subject, comprising a CUBN inhibitor.

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