WO2014024820A1 - Pharmaceutical composition for treating new chronic kidney disease and method for screening medicine for new chronic kidney disease - Google Patents

Abstract

The present invention relates to use of the GALNT2, COL8A2, FRYL or TIMP1 genes as a target for treating chronic kidney diseases or fibrotic diseases, a therapeutic agent for chronic kidney diseases or fibrotic diseases that targets the GALNT2, COL8A2, FRYL or TIMP1 genes, and a method for screening the therapeutic agent for chronic kidney diseases or fibrotic diseases that targets the GALNT2, COL8A2, FRYL or TIMP1 genes.

Description

Novel pharmaceutical composition for treating chronic kidney disease and screening method for novel therapeutic agent for chronic kidney disease

The present invention relates to a pharmaceutical composition for treating chronic kidney disease or fibrotic disease, which contains a substance that suppresses the expression of a specific gene. Specifically, the present invention relates to a pharmaceutical composition for the treatment of chronic kidney disease or fibrosis including a substance that suppresses expression of GALNT2, COL8A2, FRYL or TIMP1 gene, and expression of COL8A2, GALNT2, FRYL or TIMP1 gene The present invention relates to a method for screening a therapeutic substance for chronic kidney disease or fibrotic disease based on fluctuation.

The number of patients with renal dysfunction continues to increase worldwide, and the increase in the number of end-stage renal failure patients receiving dialysis treatment is a medical economic problem. In recent years, the concept of chronic kidney disease (CKD) has been advocated, and awareness of the importance of prevention and treatment is increasing. Chronic kidney disease refers to a condition in which "finding of kidney disease such as urine protein positivity" or "decreased renal function (glomerular filtration rate is less than 60 mL / min / 1.73 m2)" continues for 3 months or more.

Hypertension, diabetes, abnormal lipid metabolism, smoking, etc. are risk factors for the onset and progression of chronic kidney disease, and the spread of metabolic syndrome due to lifestyle changes is causing a concern that the number of patients will continue to grow. Chronic kidney disease is not only a risk of progression of end-stage renal failure, but is also a risk factor for cardiovascular events, causing myocardial infarction, heart failure, cerebral infarction and death.

In addition to lifestyle guidance and dietary guidance, anti-hypertensive therapies such as angiotensin receptor antagonists and calcium antagonists are being used as preventive and therapeutic methods for chronic kidney disease. However, the effects obtained by existing therapies cannot be said to be sufficient, and there is a demand for better preventive / therapeutic agents for renal dysfunction.

The pathological features of chronic kidney disease include fibrosis of glomeruli and tubulointerstitium. The pathological features of end stage renal failure are markedly parenchymal cell loss and fibrosis. It is known that patients who show tubulointerstitial fibrosis in patients with chronic kidney disease progress more rapidly in renal function deterioration than patients who do not show fibrosis.

Fibrosis can occur in any tissue, but can progress by a common mechanism, regardless of the type of trigger for its onset.
On the other hand, structures and structures of animal tissues and organs are maintained by fibers such as collagen, but when the tissues are injured in some way, they are restored to the original tissues by a wound healing process accompanied by collagen production. However, if the tissue is subjected to multiple immunological, chemical, mechanical, metabolic, or other injuries or the extent of the injuries is large, excessive fibrous connective tissue accumulation may occur. Such accumulation of connective tissue is irreversible, and if fibers increase abnormally, a fibrotic disease in which tissues and organs do not function normally is caused.

In recent years, RNA oligonucleotide strands of about 21 bases complementary to a desired gene (mRNA) and double-stranded RNA (small RNA interfering RNA) (siRNA) consisting of the antisense RNA oligonucleotide strand have been developed, and can be arbitrarily used in mammalian cells. It became possible to suppress the expression of the gene (Non-patent Document 1). Currently, siRNA and antisense oligo technologies are actively used in life science research, but there are no disease treatment siRNA or antisense oligos sold as pharmaceuticals.

The object of the present invention is to suppress the expression of type 1 Collagen (COL1A1) gene by suppressing the expression of a novel target gene for chronic kidney disease or fibrosis, ie, its expression, thereby causing chronic kidney disease or fibrosis To provide a pharmaceutical composition for the treatment of chronic kidney disease or fibrotic disease.

The present inventors have introduced GALNT2, COL8A2 which are genes that significantly reduce the expression of COL1A1 gene by suppressing the gene expression by introducing a human gene siRNA library into human tubular epithelial cell line (HK2). The present invention has been completed by identifying the FRYL and TIMP1 genes.

That is, the present invention provides the following [1] to [21].
[1] A pharmaceutical composition comprising a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene, or a substance that suppresses the activity of a protein encoded by these genes.
[2] The pharmaceutical composition according to [1], wherein the substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or the substance that suppresses the activity of the protein encoded by these genes is a nucleic acid.
[3] The pharmaceutical composition according to [2], wherein the nucleic acid is siRNA or antisense oligonucleotide or an expression vector thereof.
[4] A COL1A1 gene expression inhibitor comprising a substance that suppresses expression of GALNT2, COL8A2, FRYL, or TIMP1 gene, or a substance that suppresses the activity of a protein encoded by these genes.
[5] The COL1A1 gene expression inhibitor according to [4], wherein the substance that suppresses the expression of the GALNT2, COL8A2, FRYL, or TIMP1 gene or the substance that suppresses the activity of the protein encoded by these genes is a nucleic acid.
[6] The COL1A1 gene expression inhibitor according to [5], wherein the nucleic acid is siRNA, an antisense oligonucleotide, or an expression vector thereof.
[7] A pharmaceutical composition for treating a disease associated with the COL1A1 gene, comprising a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene, or a substance that suppresses the activity of a protein encoded by these genes.
[8] The pharmaceutical composition according to [7], wherein the substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or the substance that suppresses the activity of the protein encoded by these genes is a nucleic acid.
[9] The pharmaceutical composition according to [8], wherein the nucleic acid is siRNA or antisense oligonucleotide or an expression vector thereof.
[10] The pharmaceutical composition according to any one of [7] to [9], wherein the disease associated with the COL1A1 gene is chronic kidney disease or fibrotic disease.
[11] (1) A step of bringing a cell into contact with a test substance,
(2) measuring the expression level of the GALNT2, COL8A2, FRYL, or TIMP1 gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; (3) including a step of selecting the test substance as a COL1A1 gene expression inhibitor when the expression of the gene in the cell contacted with the test substance is lower than the expression of the gene in a control cell;
A screening method for a COL1A1 gene expression inhibitor.
[12] (1) A step of contacting a cell with a test substance,
(2) measuring the expression level of the GALNT2, COL8A2, FRYL, or TIMP1 gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; (3) selecting the test substance as a therapeutic substance for a disease associated with the COL1A1 gene when the expression of the gene in the cell contacted with the test substance is lower than the expression of the gene in a control cell; Including,
A screening method for a therapeutic agent for a disease associated with the COL1A1 gene.
[13] A polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted, and / or added. And a method for screening a therapeutic substance for a disease associated with the COL1A1 gene, which comprises using a polypeptide having N-acetylgalactosamine transfer activity.
[14] (1) A substrate for GALNT2 protein, a test substance and 1: a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or 2: one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 Contacting a polypeptide having an amino acid sequence deleted, substituted, and / or added and having N-acetylgalactosamine transfer activity;
(2) comparing the N-acetylgalactosamine transfer activity of the polypeptide contacted with the test substance with the N-acetylgalactosamine transfer activity of the polypeptide not contacted with the test substance, and
(3) When the N-acetylgalactosamine transfer activity of the polypeptide contacted with the test substance is lower than the N-acetylgalactosamine transfer activity of the polypeptide not contacted with the test substance, the test substance is transferred to the COL1A1 gene. Selecting as a therapeutic agent for a disease associated with
A screening method for a therapeutic agent for a disease associated with the COL1A1 gene.
[15] The method according to any one of [12] to [14], wherein the disease associated with the COL1A1 gene is chronic kidney disease or fibrotic disease.
[16] A method for treating a disease associated with the COL1A1 gene, comprising a step of administering a substance that suppresses expression of the GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes.
[17] A substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene, or the activity of a protein encoded by these genes, used for treatment of a disease associated with the COL1A1 gene.
[18] Use of a substance that suppresses the expression of the GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes for the manufacture of a therapeutic agent for a disease associated with the COL1A1 gene.
[19] A method for treating chronic kidney disease or fibrotic disease, comprising a step of administering a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes.
[20] A substance that suppresses the expression of a GALNT2, COL8A2, FRYL, or TIMP1 gene or a protein encoded by these genes, which is used for treatment of chronic kidney disease or fibrotic disease.
[21] Use of a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes for the manufacture of a therapeutic agent for chronic kidney disease or fibrotic disease.

According to the present invention, it is possible to suppress the expression of COL1A1 gene by suppressing the expression of GALNT2, COL8A2, FRYL or TIMP1 gene, and the substance which suppresses the expression of GALNT2, COL8A2, FRYL or TIMP1 gene is a chronic kidney It can be used as an active ingredient of a pharmaceutical composition for treating diseases or fibrotic diseases. The pharmaceutical composition according to the present invention provides a therapeutic agent having a new mechanism of action that specifically suppresses the expression of a specific gene, and is more toxic than conventional therapeutic agents for chronic kidney disease or fibrotic disease. It is a big feature that there is little. Furthermore, the present invention also provides a method for screening a substance for treating chronic kidney disease or fibrosis using the GALNT2, COL8A2, FRYL or TIMP1 gene as a target. *

Unless otherwise stated, terms used in the present specification are used in the meaning normally used in this field.

In one embodiment, the present invention provides a therapeutic drug for a disease associated with the COL1A1 gene, comprising a substance that suppresses expression of the GALNT2, COL8A2, FRYL, or TIMP1 gene, or a substance that suppresses the activity of the protein encoded by these genes. A composition is provided. Diseases associated with the COL1A1 gene include, for example, chronic kidney disease such as chronic renal failure, chronic nephritis, tubulointerstitial disorder, nephrotic syndrome, polycystic kidney disease, diabetic nephropathy, and nephrosclerosis Disease, and scleroderma, hypertrophic scar, scar contracture, keloid disease, collagen disease, pulmonary fibrosis, fibrosis, silicosis, myelofibrosis, renal systemic fibrosis, Crohn's disease, myocardial fibrosis, and The disease included by fibrosis diseases, such as cirrhosis, can be mentioned.

As shown in Example 1-3 of the present specification, GALNT2, COL8A2, FRYL or TIMP1 was found as a novel target molecule for chronic kidney disease or fibrotic disease.

In the present specification, the “GALNT2 gene” means a gene encoding GALNT2 protein. The base sequence of the human GALNT2 gene and the amino acid sequence of the human GALNT2 protein are known. For example, the base sequence of the human GALNT2 gene and the human GALNT2 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_004481) and published.
In this specification, the human GALNT2 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank. Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included. However, these mutant human GALNT2 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
In this specification, the human GALNT2 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation. Furthermore, the “GALNT2 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank. As mentioned above, the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. However, these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
The GALNT2 protein belongs to the family of GalNAc transferase (GALNT) consisting of at least 20 kinds of GALNT1 to GALNT14, GALNTL1, GALNTL2, GALNTL4, GALNTL5, GALNTL6, and WBSCR17 proteins. GALNT family proteins are enzymes that add N-acetylgalactosamine to serine or threonine residues of a substrate protein and are known to be involved in the first step of substrate protein glycosylation.
There are about 1,200 genes that are elevated in the diabetic nephropathy patient group (DIABETES, 60, 2354-2369, 2011 Supplementary data), but the GALNT2 gene is one of over 1,200 genes. It is mentioned as. However, the relationship between suppression of GALNT2 gene expression and suppression of GALNT2 protein activity and chronic kidney disease or fibrosis is not known.

In the present specification, “COL1A1 gene” means a gene encoding COL1A1 protein. The base sequence of the human COL1A1 gene and the amino acid sequence of the human COL1A1 protein are known. For example, the base sequence of the human COL1A1 gene and the human COL1A1 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_000088) and published.
In this specification, the human COL1A1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank. Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included. However, these mutant human COL1A1 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
In this specification, the human COL1A1 gene includes not only a gene having a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation. Furthermore, the “COL1A1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank. As mentioned above, the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. However, these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
It is known that expression of fibrosis-related genes such as the COL1A1 gene is increased and extracellular matrix overproduction occurs, leading to chronic kidney disease and fibrosis (Nephron Experimental Nephrology, Vol. 102, e71). -e75, see 2006).
Therefore, an expression inhibitor of a fibrosis-related gene such as the COL1A1 gene is a therapeutic agent for chronic kidney disease or fibrosis.

In the present specification, the “αSMA gene” means a gene encoding an αSMA protein. The nucleotide sequence of the human αSMA gene and the amino acid sequence of the human αSMA protein are known. For example, the nucleotide sequence of the human αSMA gene and the amino acid sequence of the human αSMA protein are registered in GenBank (GenBank Accession No. NM_001141945) and published.
In this specification, the human αSMA protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank. Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included. However, these mutant human αSMA proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above-described GenBank.
In the present specification, the human αSMA gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation. Furthermore, the “αSMA gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank. As mentioned above, the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. However, these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
αSMA is known as a marker of fibrosis progression and is known to be upregulated in chronic kidney disease and fibrosis (Histopathology, 47, 276-280, 2005, J Am Soc Nephrol 15) 1-12, 2004, and Virchows Archiv, 450, 41-50, 2007).

In the present specification, “COL8A2 gene” means a gene encoding COL8A2 protein. The base sequence of the human COL8A2 gene and the amino acid sequence of the human COL8A2 protein are known. For example, the base sequence of the human COL8A2 gene and the human COL8A2 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_005202) and published.
In this specification, the human COL8A2 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank. Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included. However, these mutant human COL8A2 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
In this specification, the human COL8A2 gene includes not only a gene having a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation. Furthermore, the “COL8A2 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank. As mentioned above, the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. However, these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.

In the present specification, “FRYL gene” means a gene encoding FRYL protein. The nucleotide sequence of the human FRYL gene and the amino acid sequence of the human FRYL protein are known. For example, the nucleotide sequence of the human FRYL gene and the amino acid sequence of the human FRYL protein are registered in GenBank (GenBank Accession No. NM_015030) and published.
In this specification, the human FRYL protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank. Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included. However, these mutant human FRYL proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above-described GenBank.
In the present specification, the human FRYL gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation. Furthermore, the “FRYL gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank. As mentioned above, the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. However, these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.

In the present specification, the “TIMP1 gene” means a gene encoding a TIMP1 protein. The base sequence of the human TIMP1 gene and the amino acid sequence of the human TIMP1 protein are known. For example, the base sequence of the human TIMP1 gene and the human TIMP1 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_003254) and published.
In this specification, the human TIMP1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank. Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included. However, these mutant human TIMP1 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
In this specification, the human TIMP1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation. Furthermore, the “TIMP1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank. As mentioned above, the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included. The identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA. However, these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.

In the present specification, the “substance that suppresses gene expression” refers to a substance that suppresses the transcription of mRNA of the target gene, a substance that degrades the transcribed mRNA, or a substance that suppresses the translation of the protein from the mRNA. There is no particular limitation. Examples of such substances include siRNA, antisense oligonucleotide, microRNA, ribozyme, and expression vectors thereof. Among them, siRNA or antisense oligonucleotide or an expression vector thereof is preferable, and siRNA or antisense oligonucleotide is particularly preferable. In addition to the above, “substances that suppress gene expression” include proteins, peptides, and other small molecules. In the present invention, the target gene is COL8A2, GALNT2, FRYL, or TIMP1 gene.

In the present specification, the “substance that suppresses the activity of the protein encoded by the gene” is not particularly limited as long as it is a substance that suppresses the function inherent to the target protein. Examples of such substances include antibodies and antagonists.
For example, the substance that suppresses the activity of the GALNT2 protein means a substance that inhibits the activity of the GALNT2 protein imparting N-acetylgalactosamine to the serine or threonine residue of the substrate. As a substrate of GALNT2 protein, ApoC-III, ANGPTL3 and the like are known (see Proceedings of the National Academy of Sciences of the USA, Vol. 109, pages 9893-9898, 2012).

In the present specification, “siRNA” is an RNA molecule having a double-stranded RNA portion consisting of about 15 to about 40 bases, and cleaves the mRNA of the target gene having a sequence complementary to the antisense strand of the siRNA. And has a function of suppressing the expression of the target gene. Specifically, the siRNA in the present invention is an antisense consisting of a sense RNA strand consisting of a sequence homologous to a continuous RNA sequence in mRNA of COL8A2, GALNT2, FRYL or TIMP1 gene, and a sequence complementary to the sense RNA sequence. An RNA comprising a double-stranded RNA portion comprising an RNA strand. The design and production of such siRNAs and mutant siRNAs described below are within the skill of the artisan.

The length of the double-stranded RNA portion is about 15 to about 40 bases, preferably 15 to 30 bases, more preferably 15 to 25 bases, still more preferably 18 to 23 bases, and most preferably 19 to 21 bases as a base. It is. The end structure of the sense strand or antisense strand of siRNA is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may have a blunt end or a protruding end (overhang) It is preferable that the 3 ′ end protrudes. The siRNA having an overhang consisting of several bases, preferably 1 to 3 bases, more preferably 2 bases, at the 3 ′ end of the sense RNA strand and the antisense RNA strand suppresses the expression of the target gene. In many cases, the effect is large, which is preferable. The type of the overhanging base is not particularly limited, and may be either a base constituting RNA or a base constituting DNA.

Furthermore, siRNA in which 1 to several nucleotides are deleted, substituted, inserted and / or added in one or both of the sense strand or the antisense strand of the siRNA is also used in the chronic kidney disease or fibrosis of the present invention. It can be used in a pharmaceutical composition for treating a disease. Here, the 1 to several bases are not particularly limited, but are preferably 1 to 4 bases, more preferably 1 to 3 bases, and most preferably 1 to 2 bases. Specific examples of such mutations include those in which the number of bases in the overhang portion at the 3 ′ end is 0 to 3, or the base sequence in the overhang portion at the 3 ′ end is changed to another base sequence, or Those in which the length of the sense RNA strand differs from that of the antisense RNA strand by 1 to 3 bases due to insertion, addition or deletion, or in which the base is replaced with another base in the sense strand and / or antisense strand For example, but not limited to. However, it is necessary that the sense strand and the antisense strand can hybridize in these mutant siRNAs, and that these mutant siRNAs have approximately the same level of gene expression suppression ability as siRNA having no mutation.

Furthermore, the siRNA may be a molecule having a closed structure at one end, for example, an siRNA having a hairpin structure (Short hairpin RNA; shRNA). A shRNA is a RNA comprising a sense strand RNA of a specific sequence of a target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand RNA, and a linker sequence connecting both strands. The sense strand portion and the antisense strand portion Hybridize to form a double stranded RNA portion.

It is desirable that siRNA does not show a so-called off-target effect in clinical use. The off-target effect refers to the action of suppressing the expression of another gene that is partially homologous to the siRNA used in addition to the target gene. In order to avoid the off-target effect, it is possible to confirm in advance that there is no cross-reaction for the candidate siRNA using a DNA microarray or the like. In addition, by using a known database provided by NCBI (National Center for Biotechnology Information), etc., by confirming that there is no gene containing a portion having high homology with the candidate siRNA sequence other than the target gene, It is possible to avoid the off-target effect.

In order to produce the siRNA of the present invention, a known method such as a method using chemical synthesis or a method using a gene recombination technique can be appropriately used. In the synthesis method, double-stranded RNA can be synthesized by a conventional method based on sequence information. In the method using gene recombination technology, an expression vector incorporating a sense strand sequence or an antisense strand sequence is constructed, and the sense strand RNA or antisense strand RNA generated by transcription after introducing the vector into a host cell. It can also be produced by acquiring each of the above. In addition, by expressing a sense strand RNA of a specific sequence of the target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand RNA, and a linker sequence connecting both strands, and expressing a shRNA that forms a hairpin structure, Desired double-stranded RNA can also be prepared.

As long as the siRNA has an activity of suppressing the expression of the target gene, a part of the nucleic acid constituting the siRNA may be DNA. The siRNA may be a nucleic acid in which all or part of the nucleic acid constituting the siRNA is modified as long as it has the activity of suppressing the expression of the target gene.
The modified nucleic acid means a nucleic acid having a structure different from that of a natural nucleic acid, in which a nucleoside (base site, sugar site) and / or internucleoside binding site is modified. Examples of the “modified nucleoside” constituting the modified nucleic acid include an abasic nucleoside; an arabino nucleoside, 2′-deoxyuridine, α-deoxyribonucleoside, β-L-deoxyribonucleoside, and other sugars Examples include nucleosides having modifications; peptide nucleic acids (PNA), peptide nucleic acids to which phosphate groups are bound (PHONA), locked nucleic acids (LNA), morpholino nucleic acids and the like. Examples of the nucleoside having a sugar modification include substituted pentasaccharides such as 2′-O-methylribose, 2′-deoxy-2′-fluororibose, and 3′-O-methylribose; 1 ′, 2′-deoxyribose Arabinose; substituted arabinose sugars; nucleosides with hexose and alpha-anomeric sugar modifications. These nucleosides may be modified bases with modified base sites. Examples of such modified bases include pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases.
Examples of the “modified internucleoside linkage” constituting the modified nucleic acid include, for example, alkyl linker, glyceryl linker, amino linker, poly (ethylene glycol) linkage, methylphosphonate internucleoside linkage; methylphosphonothioate, phosphotriester , Phosphothiotriester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidate, etc. Non-natural internucleoside linkages.

As the nucleic acid sequence contained in the double-stranded siRNA of the present invention, the sequences described in SEQ ID NOs: 2 to 25 in the sequence listing can be preferably used. The nucleotide sequences of these siRNAs are shown in Table 1. In Table 1, the uppercase letters indicate the sense RNA sequence and the antisense RNA sequence, and the lowercase letters indicate the 3 ′ end overhang sequence.
For example, the first sequence in Table 1 is a double-stranded siRNA composed of a sense strand shown in SEQ ID NO: 2 and an antisense strand shown in SEQ ID NO: 3, and tt at the 3 ′ end of SEQ ID NO: 2 In addition, ca at the 3 ′ end of SEQ ID NO: 3 is an overhang sequence.
Since these siRNAs remarkably suppress the expression of the COL1A1 gene, the effect of the pharmaceutical composition for treating a disease associated with the COL1A1 gene containing these siRNAs is great.

Oligonucleotides complementary to the mRNA of the target gene are called “antisense oligonucleotides,” and the mRNA functions are suppressed by forming a double strand with the target gene (mRNA). . “Antisense oligonucleotides” are not limited to those that are completely complementary to the target gene (mRNA), but may contain some mismatches as long as they can be stably hybridized with mRNA.

Antisense oligonucleotides may be modified. By applying an appropriate modification, the antisense oligonucleotide becomes difficult to be degraded in the living body, and the expression of the target gene can be inhibited more stably. Such modified oligonucleotides include S-oligo type (phosphorothioate type), C-5 thiazole type, D-oligo type (phosphodiester type), M-oligo type (methyl phosphonate type), peptide nucleic acid And modified antisense oligonucleotides such as phosphodiester bond type, C-5 propynyl pyrimidine type, 2-O-propyl ribose, 2′-methoxyethoxy ribose type. Furthermore, the antisense oligonucleotide may be one in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom. Such an antisense oligonucleotide is particularly excellent in nuclease resistance and affinity for RNA. Examples of the antisense oligonucleotide in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom include oligonucleotides such as S-oligo type.
An antisense oligonucleotide (or a derivative thereof) can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer (for example, Applied Biosystems). Examples of the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate.

“Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligoDNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. In the book, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. Specifically, the ribozyme can specifically cleave mRNA or an initial transcription product encoding a target gene within the coding region (including an intron portion in the case of the initial transcription product). The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends adjacent to the portion having the hammerhead structure (about 10 bases in total) are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, it should be a hybrid ribozyme further linked with a tRNA-modified sequence. (Nucleic Acids Res., 29 (13): 2780-2788 (2001)).

Micro RNA (referred to as “miRNA”) is a small RNA that does not encode a protein, and micro RNA is a molecule having a function of controlling gene expression by binding to a complementary site on a target mRNA.

The substance that suppresses the expression of the target gene may be a nucleic acid molecule such as siRNA, antisense oligonucleotide, microRNA or ribozyme and an expression vector encoding the nucleic acid molecule. In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in a mammalian cell to be administered. The promoter to be used is not particularly limited as long as it can function in the mammal to be administered, but for example, polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter), mammalian promoter (eg, CMV promoter, CAG promoter, SV40 promoter) and the like.
The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. In addition, selectable marker genes for selection of transformed cells (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, etc.) You can also
The basic backbone vector used as the expression vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector. Suitable vectors for administration to mammals such as humans include viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, and the like. .

Examples of the expression vector of the present invention include siRNA, antisense oligonucleotide, microRNA, and ribozyme expression vectors. Among them, siRNA or antisense oligonucleotide expression vectors are preferable.
The nucleic acid sequence encoded by the expression vector for siRNA of the present invention is preferably the sequence shown in Table 1. Since the siRNA shown in Table 1 remarkably suppresses the expression of the COL1A1 gene, the effect of the pharmaceutical composition for treating a disease associated with the COL1A1 gene containing these siRNAs is great.

In the present invention, a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of the protein encoded by these genes can be used as an active ingredient of a pharmaceutical composition. The pharmaceutical composition of the present invention can be used as a pharmaceutical composition for treating a disease associated with the COL1A1 gene by administering the pharmaceutical composition in vivo.

In the pharmaceutical composition of the present invention, a single gene expression inhibitor may be used as an active ingredient, or a plurality of gene expression inhibitors may be used as active ingredients. For example, when the expression inhibitory substance of the pharmaceutical composition of the present invention is siRNA, one or more siRNAs may be used as the active ingredient.

The type of disease that is the subject of treatment with the pharmaceutical composition of the present invention is a disease associated with the COL1A1 gene. Examples of diseases associated with the COL1A1 gene include chronic kidney disease such as chronic renal failure, chronic nephritis, tubulointerstitial disorder, nephrotic syndrome, polycystic kidney disease, diabetic nephropathy, and nephrosclerosis , And scleroderma, hypertrophic scar, scar contracture, keloidosis, collagen disease, pulmonary fibrosis, fibrosis, silicosis, myelofibrosis, renal systemic fibrosis, Crohn's disease, myocardial fibrosis, and cirrhosis Diseases included in fibrotic diseases such as
Here, chronic kidney disease is a condition in which “finding of kidney disease such as urine protein positivity” or “reduced renal function (glomerular filtration rate less than 60 mL / min / 1.73 m2)” continues for 3 months or more. Point to.

The pharmaceutical composition of the present invention can employ both oral and parenteral dosage forms.

The pharmaceutical composition of the present invention can be formulated according to a conventional method, and may contain a pharmaceutically acceptable carrier or additive. Such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl. Sodium starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, pharmaceutical Examples of acceptable surfactants include additives.
The additive is selected from the above alone or in appropriate combination depending on the dosage form of the pharmaceutical composition of the present invention. As for the dosage form, in the case of oral administration, it can be administered as a tablet, capsule, fine granule, powder, granule, liquid, syrup or the like, or in an appropriate dosage form. In the case of parenteral administration, pulmonary dosage forms (for example, those using a nephriser etc.), nasal dosage forms, transdermal dosage forms (for example, ointments, creams), injection dosage forms and the like can be mentioned. In the case of an injection dosage form, it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like.

When the substance that suppresses the expression of the target gene is a nucleic acid molecule such as siRNA, antisense oligonucleotide, microRNA or ribozyme, or an expression vector encoding the nucleic acid molecule, the expression is suppressed in phospholipid vesicles such as liposomes. It is also possible to introduce a substance and use the endoplasmic reticulum as the pharmaceutical composition of the present invention.

The dosage of the pharmaceutical composition of the present invention varies depending on age, sex, symptoms, administration route, administration frequency, and dosage form. The administration method is appropriately selected depending on the age and symptoms of the patient. The effective dose is 0.01 μg to 1000 mg, preferably 0.1 μg to 100 μg, per kg body weight. However, the therapeutic agent is not limited to these doses.

The invention in a further aspect,
(1) A method for treating a disease associated with the COL1A1 gene, comprising a step of administering a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes,
(2) A substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or the activity of a protein encoded by these genes, which is used for treatment of a disease associated with COL1A1 gene,
(3) Use of a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes for the manufacture of a therapeutic agent for a disease associated with the COL1A1 gene,
(4) a method for treating chronic kidney disease or fibrotic disease, comprising a step of administering a substance that suppresses the expression of GALNT2, COL8A2, FRYL or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes;
(5) a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes, and (6) chronic that is used for the treatment of chronic kidney disease or fibrotic disease There is provided use of a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes for the manufacture of a therapeutic agent for kidney disease or fibrotic disease.
The “substance that suppresses gene expression” and “substance that suppresses the activity of the protein encoded by the gene” are as described above.

In another aspect, the present invention relates to a method for screening a therapeutic agent for a disease associated with the COL1A1 gene, which is based on suppression of expression of the GALNT2, COL8A2, FRYL, or TIMP1 gene, or suppression of the activity of a protein encoded by these genes. I will provide a.

The test substance to be subjected to the screening method may be any known compound and novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

In one embodiment, the screening method of the present invention comprises:
(1) a step of bringing cells into contact with a test substance;
(2) measuring the expression level of the GALNT2, COL8A2, FRYL, or TIMP1 gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; (3) selecting the test substance as a therapeutic substance for a disease associated with the COL1A1 gene when the expression of the gene in the cell contacted with the test substance is lower than the expression of the gene in a control cell; Including,
This is a screening method for a therapeutic substance for a disease associated with the COL1A1 gene.

The cell in step (1) of the above method means a cell capable of measuring the expression of GALNT2, COL8A2, FRYL or TIMP1 gene. In this step, cells and a test substance are placed in contact with each other in a culture medium.

Cells capable of measuring the expression of GALNT2, COL8A2, FRYL or TIMP1 gene are directly or indirectly evaluated for the expression level of GALNT2, COL8A2, FRYL or TIMP1 gene products such as transcripts and translation products. A possible cell. A cell capable of directly evaluating the expression level of the gene product can be a cell capable of naturally expressing the gene, while a cell capable of indirectly evaluating the expression level of the gene product includes: Examples include cells that allow reporter assay for the gene transcription regulatory region. As the cells capable of measuring the expression of the gene, animal cells such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys or human mammalian cells can be used. Human-derived cells, particularly human urine Tubular epithelial cell line HK2 cells and human primary tubular epithelial cells are preferred.

A cell enabling a reporter assay for a gene transcription regulatory region is a cell containing a target gene transcription regulatory region and a reporter gene operably linked to the region. The target gene transcription regulatory region and the reporter gene can be inserted into an expression vector. The transcriptional regulatory region of the target gene is not particularly limited as long as it can control the expression of the target gene. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases in the base sequence of the region Examples include a region consisting of a base sequence deleted, substituted or added and having the ability to control the transcription of the target gene. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, the GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

A cell into which a gene transcription regulatory region and a reporter gene operably linked to the region are introduced can be used for quantitative analysis of the expression level of the reporter gene as long as the transcriptional regulatory function of the target gene can be evaluated. As long as it is, it is not particularly limited.

The culture medium in which the cells and the test substance are brought into contact is appropriately selected depending on the type of cells used. For example, a minimal essential medium (MEM) containing about 5 to 20% fetal bovine serum, Dulbecco's modification Minimum essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the type of cells to be used. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 144 hours.

In step (2) of the above method, first, the expression level of the GALNT2, COL8A2, FRYL, or TIMP1 gene in the cells contacted with the test substance is measured. The expression level can be measured by a method known per se in consideration of the type of cells used. For example, when a cell capable of naturally expressing the GALNT2, COL8A2, FRYL or TIMP1 gene is used as a cell capable of measuring the expression of the gene, the expression level is a gene product such as a transcription product or a translation product. Can be measured by a method known per se. For example, the expression level of the transcript can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological technique, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. On the other hand, when a cell capable of measuring a gene transcription regulatory region is used as a cell capable of measuring the expression of GALNT2, COL8A2, FRYL or TIMP1 gene, the expression level can be measured based on the signal intensity of the reporter.

In step (3) of the above method, the expression level of the gene in the cell contacted with the test substance is compared with the expression level of the gene in the control cell.
The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of the gene in the control cell not contacted with the test substance is the expression level measured at the same time, even if it is the expression level measured in advance compared to the measurement of the gene expression level in the cell contacted with the test substance. However, the expression level is preferably measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

In another embodiment, the screening method of the present invention comprises:
A polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted, and / or added; A method for screening a therapeutic substance for a disease associated with the COL1A1 gene, which comprises using a polypeptide having acetylgalactosamine transfer activity.

In another embodiment, the screening method of the present invention comprises:
(1) a substrate for GALNT2 protein, a test substance and 1: a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or 2: deletion of one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1, Contacting a polypeptide having a substituted and / or added amino acid sequence and having N-acetylgalactosamine transfer activity;
(2) a step of comparing the N-acetylgalactosamine transfer activity of the polypeptide contacted with the test substance with the N-acetylgalactosamine transfer activity of the polypeptide not contacted with the test substance, and (3) a poly contacted with the test substance. When the N-acetylgalactosamine transfer activity of a peptide is reduced compared to the N-acetylgalactosamine transfer activity of a polypeptide that does not contact the test substance, the test substance is selected as a therapeutic agent for a disease associated with the COL1A1 gene Process.

The protein that can be used as a substrate for the GALNT2 protein in step (1) of the above method is as described above. The polypeptide having the amino acid sequence represented by SEQ ID NO: 1 includes an isolated polypeptide, a polypeptide in a membrane fraction, and an intracellular polypeptide.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

The following screening was performed with the aim of finding genes that can suppress the expression of fibrosis-related genes by inhibition.
First, as a screening target gene, a group of genes that are highly likely to be drug discovery targets was narrowed down by expression information from all human genes. Specifically, in the expression database of 27 human normal tissues, a group of genes whose relative expression level is below the reference value in all of the heart, brain, spleen, and peripheral leukocytes is extracted as genes with relatively few side effects, and 10,723 genes Got.
Next, in order to reduce the probability of false positives, total RNA is extracted from the human tubular epithelial cell line HK2 used for primary screening, and DNA microarray analysis is performed to remove genes whose levels cannot be detected in the same cells. This narrowed the target to 7,700 genes.
From the above 7,700 genes, 1,994 genes were selected that are likely to be able to regulate activity with low molecular weight compounds and targets that are likely to be high molecular drugs.

The following experiment was performed as a primary screening. From the human whole gene siRNA library (Silencer Select Human Genome siRNA Library; manufactured by Ambion; 21,585 gene; 64,755 siRNA; 3 siRNA / gene), the focused siRNA library (5,982 siRNAs) of the above 1,994 gene was prepared by cherry picking . Using the focused siRNA library, we searched for a gene whose expression of COL1A1 gene is reduced by suppressing expression in human tubular epithelial cell line HK2. As a screening protocol, first, 1.5 μl (0.375 pmol, final 12.5 nM) of 0.25 μM library siRNA was spotted on a 384 well plate (Corning) coated with Poly-D-Lysine (PDL). In addition, Negative Control # 1 siRNA (NT-1; manufactured by Ambion) was dispensed in the same amount on each plate as a negative control, and used for hit criteria setting.
Meanwhile, Opti-MEM I (Gibco) 3.5 μl and siRNA introduction reagent RNAiMAX (Invitrogen) 0.1 μl were mixed, kept at room temperature for 5 minutes, and then 384-well plate dispensed with the above siRNA Added to. After incubation at room temperature for 20 minutes, add 25 μl of Keratinocyte Serum Free Media (Invitrogen, 0.05 mg / ml bovine pituitary extract and 5 ng / ml human rEGF) containing 5,000 HK2 cells, 37 ° C The cells were cultured for 48 hours in a 5% CO2 environment. Thereafter, 30 μl of Keratinocyte Serum Free Media containing recombinant TGFβ (manufactured by R & D) at a concentration of 20 ng / ml was added and further cultured for 24 hours.
The culture supernatant was removed by decantation, and 25 μl of Buffer FCW (Wash buffer) contained in FastLane Cell SYBR Green Kit (manufactured by Qiagen) was added. After removing Buffer FCW by decantation, 15 μl of Cell Processing Mix (Lysis buffer) also contained in Kit was added and kept at room temperature for 5 minutes to lyse the cells. A cell lysate heated at 75 ° C. for 5 minutes was used as a template for quantitative PCR. For quantitative PCR, FastLane Cell SYBR Green Kit was used, and the expression levels of COL1A1 gene as fibrosis-related gene and GAPDH gene and PPIA gene as internal standards were measured. The PCR primers used to amplify each gene were purchased from Takara Bio Inc. (catalog number; 5060D-40407, primer set ID; COL1A1, CH000497; GAPDH, HA067812; PPIA, HA067810). PCR containing 1.0 μl of the above template and 5.0 μl of 2x QuantiTect SYBR Green RT-PCR Master Mix, Forward primer 5 pmol, Reverse primer 5 pmol, QuantiTect RT Mix 0.1 μl on a 384-well quantitative PCR plate (Applied Biosystems) A reaction solution was prepared, and quantitative PCR was performed using 7900HT Fast Real Time PCR System (Applied Biosystems).
Based on the obtained Cycle threshold value (Ct value), the expression of COL1A1 gene is reduced to 50% or less compared to the negative control siRNA on the same plate by either GAPDH gene correction or PPIA gene correction. SiRNAs were extracted as hit candidate siRNAs. Then, 158 genes were obtained by using, as a primary screening hit candidate, a gene in which two or more of the three sequences of siRNAs for the same gene were hit candidates.

Next, a reproducibility test was performed. A focused siRNA library of the above 158 genes was prepared (474 siRNA, 3 siRNAs / gene), and the degree of expression reduction of the COL1A1 gene when each gene was suppressed by siRNA was evaluated using the same protocol as the primary screening. The reproducibility test was performed at n = 3, and the criteria for hit siRNA was that the expression of the COL1A1 gene was reduced by 40% compared to the negative control. As a result, the reproducibility of four genes, COL8A2, GALNT2, FRYL, and TIMP1, was confirmed, and the gene group was used as a primary screening hit gene.
The results of the primary screening reproducibility test are listed in Table 2.

Of the 4 genes obtained in Example 3, GALNT2, which is considered to be the most promising target gene from the suppression data of COL1A1 gene expression, was evaluated from the second order onward.
Specifically, in the secondary evaluation, the influence of other fibrosis markers on the expression of alpha-smooth muscle actin (αSMA, ACTA2) protein was examined by Western blot. In addition, siRNA used the same sequence as the primary screening (3 siRNA / gene), and Negative Control # 1 siRNA was used as a negative control. 56.75 μl of Opti-MEM I and 1.75 μl of RNAiMAX were mixed and kept at room temperature for 5 minutes, then mixed with 25 μl of 0.1 μM siRNA (final 5 nM) and kept at room temperature for another 20 minutes. This solution was transferred to a 24-well plate, 416.5 μl of Keratinocyte Serum Free Media containing 50,000 HK2 cells was added to each well, and cultured for 48 hours at 37 ° C. in a 5% CO 2 environment. Next, 500 μl of Keratinocyte Serum Free Media containing 2 ng / ml recombinant TGFβ was added by medium exchange, and further cultured for 24 hours. After removing the culture supernatant, the cells were washed with PBS, and the cells were lysed with 70 μl of RIPA buffer. After centrifuging the cell lysate, the protein concentration of the supernatant was measured, and 6 μg of total protein was subjected to SDS polyacrylamide gel electrophoresis. After electrophoresis, it was transferred to a PVDF membrane, blocked using Blocking One (manufactured by Nacalai Tesque), and then reacted with a primary antibody (mouse anti-αSMA antibody, manufactured by Sigma) at 6.5 ° C. overnight. After washing with TBS-T, the mixture was reacted with an HRP-labeled anti-mouse IgG antibody (secondary antibody, manufactured by GE Healthcare) at room temperature for 1 hour. After washing with TBS-T, antigen-antibody reaction was detected using ECL Advance Western Blotting Detection Kit (GE Healthcare) and LAS-3000 (FUJIFILM), and the signal was quantified. The results are shown in Table 3. Among 3 sequences, 2 sequences siRNA showed an increase in αSMA protein level by TGFβ stimulation compared to negative control, indicating that suppression of GALNT2 gene expression also suppressed increase in fibrosis markers at the protein level. .

Furthermore, as a third evaluation, human primary tubule epithelial cells (Renal proximal tubular epithelial cells: RPTEC, manufactured by LONZA) were used, and when each gene was suppressed with siRNA in the same manner as in Example 2, COL1A1 gene stimulated by TGFβ It was investigated whether the increase in the expression of the gene was attenuated. In addition, siRNA used the same sequence as the primary screening (3 siRNA / gene), and Negative Control # 2 siRNA (NT-2; manufactured by Ambion) was used as a negative control. RPTEC was evaluated using two types of lots (Lot0000264325, 0000197809). 69.3 μl of Opti-MEM I and 0.7 μl of RNAiMAX were mixed and kept at room temperature for 5 minutes, then mixed with 30 μl of 0.1 μM siRNA (final 5 nM) and kept at room temperature for another 20 minutes. This solution was transferred to a 24-well plate, 500 μl of REGM Bullet Kit (manufactured by LONZA) containing 75,000 RPTECs was added to each well, and cultured in a 37 ° C., 5% CO 2 environment. After 24 hours, Starvation treatment is performed by adding a REGM Bullet Kit that does not contain additional factors by changing the medium.After 24 hours, a REGM Bullet Kit that contains 10 ng / ml recombinant TGFβ and does not contain additional factors is added by changing the medium. 500 μl was added and cultured for 24 hours. After removing the culture supernatant, Ct values of COL1A1 gene and GAPDH gene are measured using FastLane Cell SYBR Green Kit and 7900HT Fast Real Time PCR System in the same manner as in Example 2, and for each siRNA using GAPDH gene as an internal standard. The expression level of COL1A1 gene was calculated. The results for cells Lot0000197809 are shown in Table 4. In all three siRNAs, the increase in COL1A1 gene expression by TGFβ stimulation was suppressed as compared to the negative control, and this phenotype was also confirmed in the other cell lot (Lot0000264325). This revealed that the GALNT2 gene is a promising target that suppresses the expression of fibrosis markers in human primary tubular epithelial cells.

Furthermore, the effect of GALNT2 expression suppression on gene expression profiles was evaluated. Two types of siRNA (siRNA # 2, # 3) against GALNT2 in which COL1A1 expression-suppressing action was observed were introduced into HK2 cells, and after 48 hours, recombinant TGFβ was added. Further, 24 hours later, total RNA was collected from the cells, and microarray data was obtained. Compared to the negative control siRNA, GALNT2 was associated with multiple fibrosis because COL4A2, P4HA2, SNAI1, etc. were included in addition to COL1A1 as a result of extracting genes that commonly decrease expression in two types of siRNAs against GALNT2. It became clear that the expression of the factor was controlled.

The present invention can be used in the field of pharmaceuticals, particularly in the field of development and production of therapeutic agents for chronic kidney disease or fibrotic diseases.

Claims (13)

1. A pharmaceutical composition comprising a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or a substance that suppresses the activity of a protein encoded by these genes.
2. The pharmaceutical composition according to claim 1, wherein the substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or the substance that suppresses the activity of the protein encoded by these genes is a nucleic acid.
3. The pharmaceutical composition according to claim 2, wherein the nucleic acid is siRNA or antisense oligonucleotide or an expression vector thereof.
4. A COL1A1 gene expression inhibitor comprising a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene, or a substance that suppresses the activity of a protein encoded by these genes.
5. The COL1A1 gene expression inhibitor according to claim 4, wherein the substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or the substance that suppresses the activity of the protein encoded by these genes is a nucleic acid.
6. The COL1A1 gene expression inhibitor according to claim 5, wherein the nucleic acid is siRNA, antisense oligonucleotide, or an expression vector thereof.
7. A pharmaceutical composition for treating a disease associated with the COL1A1 gene, comprising a substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene, or a substance that suppresses the activity of a protein encoded by these genes.
8. The pharmaceutical composition according to claim 7, wherein the substance that suppresses the expression of GALNT2, COL8A2, FRYL, or TIMP1 gene or the substance that suppresses the activity of the protein encoded by these genes is a nucleic acid.
9. The pharmaceutical composition according to claim 8, wherein the nucleic acid is siRNA or antisense oligonucleotide or an expression vector thereof.
10. The pharmaceutical composition according to any one of claims 7 to 9, wherein the disease associated with the COL1A1 gene is chronic kidney disease or fibrosis disease.
11. (1) a step of bringing cells into contact with a test substance;
    (2) measuring the expression level of the GALNT2, COL8A2, FRYL, or TIMP1 gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; (3) selecting the test substance as a therapeutic substance for a disease associated with the COL1A1 gene when the expression of the gene in the cell contacted with the test substance is lower than the expression of the gene in a control cell; Including,
    A screening method for a therapeutic agent for a disease associated with the COL1A1 gene.
12. A polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted, and / or added; A method for screening a therapeutic substance for a disease associated with the COL1A1 gene, which comprises using a polypeptide having acetylgalactosamine transfer activity.
13. The method according to claim 11 or 12, wherein the disease associated with the COL1A1 gene is chronic kidney disease or fibrosis disease.