Patent Application
20130231383
The present invention relates to a novel usage of SMS2. More specifically, the present invention relates to a pharmaceutical composition for treating a metabolic syndrome, which uses SMS2.
The induction of obesity has been known to be involved in an action of accelerating eating via proteins that are expressed in the hypothalamus such as neuropeptide Y5 receptor, or melanocortin receptor, an action of accelerating the synthesis of fatty acids via acyl-CoA carboxylase (ACC) or the like. Currently, as an anti-obesity drug, a neuropeptide Y5 receptor antagonist, a melanocortin receptor antagonist and an ACC inhibitor or the like have been developed. However, to date, an effective anti-obesity drug has not been found.
Fatty liver is a disease, which a neutral fat accumulates in the liver, and obesity are deemed as a major cause of developing it. However, similarly to an anti-obesity drug, an effective drug for treating fatty liver has not been found to date. In addition, regarding diabetes which is deemed as a representative example of a metabolic syndrome and which is deemed as a fundamental cause of lifestyle related diseases such as obesity or circulatory system diseases, sulfonylurea drugs, biguanides, alphaglucosidase and azolidine derivatives and the like have been developed. However, problems for side effect and efficacy thereof exist. Therefore, a more effective drug for treatment is expected to be developed.
A sphingomyelin synthetase (SMS) is an enzyme which synthesizes sphingomyelin(SM), a sphingolipid most abundant in a cell membrane, and plays an important role in cell death and survival (see, NPLs 1 and 2). SMS was identified in 2004, and are known to be present as two kinds, SMS1 and SMS2. SMS1 is known to be expressed in the Golgi body and be involved in de novo synthesis of SM. On the other hand, SMS2 is expressed in the Golgi body and cell membrane and unknown for its physiological function in detail (see, NPL 3) and merely suggested in NPLs 4-6 for the possibility of involving in arteriosclerosis.
To date, methods for ameliorating a metabolic syndrome by using neutral fat metabolism/absorbance inhibitors or inhibiting agents, agents for accelerating neutral fat metabolism or the like have been attempted. Specifically, these are drugs for ameliorating hyperlipidemia and type II diabetes by synthetic ligands of intranuclear receptor PPARs, drugs for ameliorating hyperlipidemia via inhibiting cholesterol synthesis by statin agents, or the like.
To date, an action of inducing obesity via controlling the synthesis of sphingolipids have not been known. A SMS inhibitor developed as anti-diabetes drug, anti-obesity drug or a drug for treating fatty liver does not exist, either.
On the other hand, RNAi is a phenomenon found by Fire et al in 1998 (NPL 7), in which a double stranded RNA strongly suppresses the expression of a target gene homologous thereto. It is recently taken notice since it is easier than conventional transgenic methods using a vector or the like, has a high specificity to the target and can be applied to a gene therapy. Among molecules that mediate RNAi, a short chain interfering RNA (siRNA) is advanced for its application (NPL 8). However, it has not been developed as an SMS inhibitor. It has not been known to have an effect of ameliorating a metabolic syndrome as an SMS inhibitor, either.
The present invention relates to decreasing body weight, decreasing visceral fat, decreasing triglycerides in the liver and ameliorating obesity and fatty liver by suppressing the synthesis of sphingolipids, as well as a drug for treating a metabolic syndrome such as diabetes.
Regarding the above problem, it is found that the above disease can be treated or prevented by suppressing and controlling the expression of sphingomyelin synthetases, in particular, SMS2 and the present invention has been completed. Therefore, the present invention provides the followings:
A pharmaceutical composition for treating or preventing a metabolic syndrome, comprising a nucleic acid that suppresses the expression of SMS2.
The pharmaceutical composition according to Item 1, wherein said nucleic acid is an siRNA.
The pharmaceutical composition according to Item 2, wherein said siRNA consists of any one or more selected from the group consisting of siRNAs of the following (a)-(o):
(a) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 1 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 2 <SMS2-i6>;
(b) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 3 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 4 <SMS2-i7>;
(c) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 5 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 6 <SMS2-i8>;
(d) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 7 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 8 <SMS2-i104>;
(e) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 9 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 10 <SMS2-i105>;
(f) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 11 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 12 <SMS2-i106>;
(g) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 13 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 14 <SMS2-i107>;
(h) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 15 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 16 <SMS2-i108>;
(i) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 17 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 18 <SMS2-i109>;
(j) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 21 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 22 <SMS2-i3>;
(k) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 23 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 24 <SMS2-i11>;
(l) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 39 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 40 <SMS2-i1>;
(m) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 41 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 42 <SMS2-i2>;
(n) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 45 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 46 <SMS2-i5>; and
(o) an siRNA of any of (a)-(n), wherein one to several nucleotides are added, inserted, deleted or substituted in one or both of the base sequences, and having an activity of suppressing the expression of SMS2.
The pharmaceutical composition according to Item 1, wherein said nucleic acid is an antisense nucleic acid.
The pharmaceutical composition according to Item 1 or 4, wherein said nucleic acid is an antisense nucleic acid comprising a locked nucleic acid (LNA).
The pharmaceutical composition according to any of Item 1, 4 or 5, wherein said nucleic acid is an antisense nucleic acid comprising a locked nucleic acid (LNA) at 5′ end and an LNA at 3′ end.
The pharmaceutical composition according to Item 4, 5 or 5A, wherein said antisense nucleic acid consists of any one or more of SEQ ID NOs: 81-92.
The pharmaceutical composition according to any one of Items 1-5, 5A or 6, wherein said metabolic syndrome is one or two or more selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver.
The pharmaceutical composition according to any one of Items 1-5, 5A, 6, 7 or 7A, wherein said nucleic acid is comprised in an amount effective to suppress the expression of SMS2.
The pharmaceutical composition according to any one of Items 1-5, 5A, 6 or 7, further comprising a pharmaceutically acceptable carrier.
An siRNA set forth in any one selected from the group consisting of the following (a)-(o):
(a) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 1 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 2 <SMS2-i6>;
(b) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 3 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 4 <SMS2-i7>;
(c) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 5 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 6 <SMS2-i8>;
(d) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 7 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 8 <SMS2-i104>;
(e) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 9 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 10 <SMS2-i105>;
(f) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 11 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 12 <SMS2-i106>;
(g) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 13 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 14 <SMS2-i107>;
(h) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 15 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 16 <SMS2-i108>;
(i) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 17 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 18 <SMS2-i109>;
(j) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 21 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 22 <SMS2-i3>;
(k) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 23 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 24 <SMS2-i11>;
(l) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 39 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 40 <SMS2-i1>;
(m) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 41 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 42 <SMS2-i2>;
(n) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 45 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 46 <SMS2-i5>; and
(o) an siRNA of any of (a)-(n), wherein one to several nucleotides are added, inserted, deleted or substituted in one or both of the base sequences, and having an activity of suppressing the expression of SMS2.
The siRNA according to Item 8, for treating or preventing a metabolic syndrome.
The siRNA according to Item 9, wherein said metabolic syndrome is one or two or more selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver.
Use of siRNA according to any one of Items 8-10, for the manufacture of a medicine for treating or preventing a metabolic syndrome.
Use according to Item 10A, wherein said metabolic syndrome is one or two or more selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver.
A locked nucleic acid (LNA)-containing nucleic acid consisting of any one or more of SEQ ID NOs: 81-92.
The locked nucleic acid (LNA)-containing nucleic acid according to Item 11, for treating or preventing a metabolic syndrome.
The locked nucleic acid (LNA)-containing nucleic acid according to Item 12, wherein said metabolic syndrome is one or two or more selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver.
A method for treating or preventing a metabolic syndrome, said method comprising: administering the pharmaceutical composition according to any one of Items 1-5, 5A, 6, 7, 7A or 7B to a subject in need of said treatment or prevention.
The method according to Item 14, wherein said metabolic syndrome is one or two or more selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver.
A method for treating or preventing a metabolic syndrome, said method comprising: administering the siRNA according to any one of Items 8-10, 10A or 10B to a subject in need of said treatment or prevention.
The method according to Item 16, wherein said metabolic syndrome is one or two or more selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver.
A method for treating or preventing a metabolic syndrome, said method comprising: administering the locked nucleic acid (LNA)-containing nucleic acid according to any one of Items 11-13 to a subject in need of said treatment or prevention.
The method according to Item 18, wherein said metabolic syndrome is one or two or more selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver.
Relating to the present invention, it has been known that a metabolic syndrome result from complex factors. Therefore, it is likely that an occurrence of a medicament having a new mechanism of action can supplement the weakness of conventional drugs.
The present inventors has developed a method for ameliorating a metabolic syndrome by controlling sphingolipid metabolism, thereby indirectly affecting neutral lipid synthesis/metabolism, which has been not known to date.
The present inventors propose a method for ameliorating a metabolic syndrome by controlling sphingomyelin synthetases such as SMS2, a synthetase of sphingomyelin that is one of sphingolipids, to affect the synthesized amount of triacylglycerol. SMS produces equimolar amount of diacylglycerol in synthesizing sphingomyelin. From the experiments exemplified herein, it is demonstrated that the diacylglycerol is likely to be a material for the synthesis of triglycerol, a representative neutral lipid. Specifically, as an illustrative example, increase in body weight of SMS2 gene-deficient mice fed by a high fat diet was significantly decreased compared to a control group and closer to weight of mice fed by normal diet. That is, it can be expected that by controlling the activity of SMS, the synthesized amount of triacylglycerol is controlled and consequently a metabolic syndrome are ameliorated.
Then, it has been considered that a therapeutic effect on a metabolic syndrome can be expected by inhibiting SMS2 function and, for example, application of an RNAi (RNA interference) method that is a novel molecular-specific knockdown method, to a therapy has been investigated.
An RNAi method is a technique that quickly suppresses the expression of a specific gene at a gene level by using a short interfering dsRNA (siRNA (small interfering RNA)) (Non-patent Document 7 and 8). The present inventors have selected multiple target sequences from SMS2 mRNA sequence, synthesized their siRNA and found that knockdown of SMS2 can be linked to healing diseases.
Conventionally, there has not been drugs for ameliorating a metabolic syndrome that are via controlling the synthesis of sphingolipids. Therefore, the present invention enables it possible to make a medicament having a new mechanism of action.
Preferred embodiments of the present invention are explained below. Throughout the present specification, unless specifically referred to, an expression in a singular form is to be understood to encompass the concept of its plurality form. Therefore, unless specifically referred to, singular form articles (for example, “a”, “an”, “the” or the like in English, and corresponding articles and adjectives or the like in other languages) are to be understood to encompass the concept of their plurality form. Furthermore, terms used herein, unless specifically referred to, are to be understood to be used in the meaning usually used in the art. Therefore, unless defined otherwise, all technical terms and scientific terms herein have the same meaning as generally recognized by those skilled in the art to which the present invention belongs. If they contradict, the present specification (including the definition) governs.
The definition of terms specifically used herein are listed.
As used herein, “sphingomyelin synthetase” (also referred herein as “SMS”) refers to an enzyme that synthesizes sphingomyelin (also referred herein as “SM”) wherein it converts ceramide into sphingomyelin in the presence of phosphatidylcholine (also referred herein as “PC”), and which plays an important role in cell death and survival (see, Non-patent Documents 1 and 2). Here, phosphatidylcholine after conversion is converted into diacylglycerol. As sphingomyelin synthetases, typically, SMS 1 and SMS2 are known, as well as homologs (see, Non-patent Documents 1 and 2). SMS1 is known to be expressed in the Golgi body and be involved in de novo synthesis of SM. On the other hand, SMS2 is expressed in the Golgi body and cell membrane, and its physiological function is not known in detail (see, Non-patent Document 3). In addition, GenBank Accession numbers of SMS1 are NM—147156 (human) and NM—144792 (mouse), and those of SMS2 are BC028705.1 (human), BC041369.2 (human) and NM—028943 (mouse).
“Metabolic syndrome” or “metabolic disease” herein are used in the broadest meaning used in the art, and refer to states wherein hyperglycemia, dyslipidemia or hypertension are caused with a common factor of visceral fat obesity and are previously called as “lifestyle related disease”, “adult diseases” or the like. All these terms are exchangably used herein. Metabolic syndrome or metabolic disease herein specifically comprise at least one of diseases such as diabetes, obesity, dyslipidemia (excess of visceral fat), fatty liver or the like, or states such as decreased expression of adiponectin. Furthermore, since arteriosclerosis is often developed after some years as a result of metabolic syndrome, it is understood that “metabolic syndrome” does not comprise arteriosclerosis in general but comprises the arteriosclerosis caused by metabolic syndrome (for example, diabetes, obesity or the like).
“Anti-diabetes” herein refer to a drug capable of treating (for example, suppressing, preventing or the like) diabetes. Diabetes is used in the meaning usually used in the art and refers to a state wherein a blood glucose level (glucose concentration in blood) is pathologically high, and diabetes is largely divided into type 1 diabetes which is caused by not secreting insulin from the pancreas and type 2 diabetes which is caused by decreased function of insulin. In the present specification, diabetes encompasses type I and type II and is not constricted to its type. In the present specification, “type I anti-diabetes” is referred when specifically referring to type I and “type II antidiabetes” is referred when specifically referring to type II.
Here, insulin resistance syndrome is a major cause of diabetes (in particular, type 2 diabetes) and is a fundamental cause for lifestyle related diseases such as obesity and circulatory system disease. Insulin resistance means a state which a response to insulin of peripheral tissues is dull. Since, as a result of a response to insulin being dull, since peripheral tissues cannot respond to insulin, they cannot uptake blood glucose and state in which blood glucose level is high is maintained. Since pancreas releases a large amount of insulin so as to decrease blood glucose, the concentration of insulin in blood becomes high.
Therefore, by ameliorating insulin resistance, blood glucose level decreases and thus it is useful for treating or preventing the above lifestyle related diseases. An index of amelioration of insulin resistance is that the concentration of insulin in blood is decreased when measured. In addition, increased concentration of adiponectin in blood is another index. In view of above, a drug for increasing adiponectin or a drug which decreases the concentration of insulin in blood while maintaining blood glucose level at a normal level becomes a drug for ameliorating insulin resistance and thus is also effective for treating diabetes or obesity, circulatory system diseases (arteriosclerosis, hypertension or the like) and metabolic syndrome resulting from insulin resistance.
“Anti-obesity drug” herein refers to a drug capable of treating (for example, suppressing, preventing or the like) obesity. “obesity” is one of metabolic syndromes and can be recognized as one of diseases. Obesity state can include, for example, excess of body weight, excess of fat or the like. If BMI measured as a body mass index is 25 or more, it is deemed as obesity. When body fat percentage is used, 18% or more is a criteria. An effect of treating obesity can be judged by measuring body weight, measuring fat, measuring triglyceride accumulated in fat, or the like.
“Drug for decreasing body weight” herein refers to a drug which decreases body weight or suppresses feeding. “decrease in body weight” or “decreasing body weight” herein must be interpreted in the broadest manner to encompass a case of decreasing the current body weight as well as a case of suppressing increase in body weight and maintaining the current body weight. Decrease in body weight can be judged by measuring body weight, measuring fat, measuring triglyceride accumulated in fat, or measuring the amount of feeding.
“Drug for decreasing visceral fat” herein refers to a drug which decreases visceral fat or prevent increase in visceral fat. Visceral fat is used in the meaning usually used in the art and refers to fat accumulated around viscera among body fat. Decrease in visceral fat can be judged by measuring white fat cell mass, measuring with abdominal CT image, or the like. An object for decreasing visceral fat can include diseases or symptoms such as excess of visceral fat or dyslipidemia. “Dyslipidemia” refers to a state in which fat included in blood is excess or short, and was previously also called as hyperlipidemia. Dyslipidemia is one of metabolic syndromes and can be recognized as one of diseases.
“Drug for treating fatty liver” herein refers to a drug capable of treating (for example, suppressing, preventing) fatty liver. “Fatty liver” is used in the meaning usually used in the art and refers to a status in which a large amount of fat accumulates in the liver. As a cause for it, alcoholic, nutritive, diabetic, drug-induced causes or the like are known. Fatty liver is said to be partly developed to cirrhosis. In the sense, the present invention can be recognized to be a drug for preventing cirrhosis. An effect of treating fatty liver can be judged by measuring triglyceride (TG) in fat, measuring liver weight, grossly inspecting liver, or the like.
“Drug for increasing adiponectin expression” herein refers to a drug which increases the expression of adiponectin. Adiponectin is used in the meaning usually used in the art, is a secretory protein that is secreted from fat cell and is said to be negatively correlated with visceral fat amount, and said to be an index for so-called metabolic diseases. Increase in adiponectin expression can be judged, for example, by obtaining fat, extracting mRNA thereof and measuring the expression amount of adiponectin mRNA, by immunological measurement using an anti-adiponectin antibody, or the like.
“Expression” of a gene, polynucleotide, polypeptide or the like herein refers to that such gene or the like undergo in vivo a certain action to become another form. Preferably, it refers to that gene, polynucleotide or the like are transcribed and translated into a polypeptide form. It can be also one embodiment of the expression that they are transcribed so as to produce mRNA. More preferably, such forms of polypeptide can be one after post-translation processing.
Therefore, “decrease” in “expression” of gene, polynucleotide, polypeptide or the like herein refers to that the amount of expression is significantly decreased when the agent of the present invention is acted, compared to a case it is not acted. Preferably, decrease in expression encompasses decrease in the expression amount of a polypeptide. “Increase” in “expression” of gene, polynucleotide, polypeptide or the like herein refers to that the amount of expression is significantly increased when the agent of the present invention is acted, compared to a case it is not acted. Preferably, increase in expression encompasses increase in the expression amount of a polypeptide. “Induction” of “expression” of a gene herein refers to that a cell is acted by an agent so as to increase the expression amount of the gene. Therefore, induction of expression encompasses allowing the gene to be expressed when the expression of the gene is not found at all, and increasing the expression of the gene when the expression of the gene has been already found.
“Detection” or “quantification” of a gene expression (for example, mRNA expression, and polypeptide expression) herein can be achieved by using a suitable method, for example, including measurement of mRNA and immunological measuring methods. As molecular biological measuring methods, for example, Northern blot method, dot blot method or PCR method or the like are exemplified. As immunological measuring methods, for example, ELISA method using a microtiter plate, RIA method, fluorescent antibody method, Western blot method, immunohistological staining method or the like are exemplified. In addition, as a quantifying method, ELISA method or RIA method or the like are exemplified. It can be also carried out by a gene analyzing method using an array (for example, DNA array, and protein array). A DNA array is broadly in reviewed in (Shujunsha Co., Ltd., ed., Saibo Kogaku Bessatsu [Cell Engineering, Separate zvolume], “DNA Maikuroarei to Saishin PCR Ho [DNA Microarray and the Latest PCR Method]”). A protein array is described in detail Nat. Genet. 2002 December; 32 Suppl: 526-32. In addition to the above, a method for analyzing gene expression include, but not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation, two-hybrid system, in vitro translation or the like. Additional such analyzing method is described, for example, in Genomu Kaiseki Jikken Ho [A Experimental Method for Genome Analysis], Nakamura Yusuke Rabo Manyuaru [Laboratory Manual by Yusuke Nakamura], Yusuke Nakamura ed., Yodosha Co., Ltd. (2002), the description of which are all incorporated herein as reference.
“Expression amount” herein refers to an amount in which a polypeptide or mRNA is expressed in a cell of interest or the like. Such expression amount includes expression amount of the polypeptide of the present invention at a protein level evaluated by any suitable methods using the antibody of the present invention including immunological measuring methods such as ELISA method, RIA method, fluorescent antibody method, Western blot method, immunohistological staining method or the like, or expression amount of the polypeptide of the present invention at an mRNA level evaluated by any suitable method including molecular biological measuring methods such as Northern blot method, dot blot method, PCR method or the like. “Change of expression amount” means increase or decrease in expression amount of a polypeptide of present invention at a protein level or mRNA level evaluated by any suitable method including the above immunological measuring methods or molecular biological measuring methods.
“A material (for example, nucleic acid) that suppresses the expression (of a gene such as SMS2)” herein is not specifically limited as long as it is a material that suppresses the transcription of mRNA of a target gene, a material (for example, a nucleic acid) that degrades a transcribed mRNA, or a material that suppresses the translation of a protein from mRNA. As such materials, nucleic acids such as siRNA, an antisense oligonucleotide, a ribozyme or expression vector thereof or the like are exemplified. Among them, siRNA and expression vector thereof are preferred, and siRNA is specifically preferred. A “material that suppresses gene expression” also includes proteins, peptides, and other small molecules, besides the above.
Here, the target gene in the present invention is an SMS2 gene.
“siRNA” herein is an RNA molecule having a double stranded RNA portion consisting of 15-40 bases, and has a function of cleaving the mRNA of a target gene having a sequence complementary to an antisense strand of the siRNA, thereby suppressing the expression of the target gene. Specifically, the siRNA in the present invention is an RNA comprising a double stranded RNA portion which consists of a sense RNA strand consisting of a sequence homologous to a contiguous RNA sequence in the mRNA of a SMS2 gene, and an antisense RNA strand consisting of a sequence complementary to the sense RNA sequence. Designing and producing such siRNA and variant siRNA described below fall within the scope of technique of those skilled in the art.
The length of the double stranded RNA portion in base is 15-40 bases, preferably 15-30 bases, more preferably 15-25 bases, further more preferably 18-23 bases and most preferably 19-21 bases. It is understood that the upper and lower limits are not such specific ones and can be any combination of the listed ones. The terminal structure of sense strand or antisense strand of siRNA can be suitably selected, without specific limitation, depending on its purpose, and can be, for example, one with blunt end, or one with protruding end (overhung), and one with 3′ protruding end is preferred. siRNA which has overhangs consisting of several bases, preferably 1-3 bases, more preferably 2 bases at 3′ ends of the sense RNA strand and antisense RNA strand often has a excellent effect of suppressing the expression of its target gene and is thus preferred. The kind of the base constituting the overhung is not specifically limited and can be any of bases that constitute an RNA or bases that constitute a DNA. Preferred overhung sequence can include dTdT (2 bp of deoxyTs) at 3′ end or the like. For example, preferred siRNA includes, but not limited to, all siRNA whose sense and antisense strands have dTdT (2 bp of deoxyTs) at their 3′ ends.
Furthermore, siRNA wherein one to several nucleotides are deleted, substituted, inserted, and/or added in one or both of the sense strand or antisense strand of the above siRNA can be also used. Here, one to several bases are, but not specifically limited to, preferably 1-4 bases, more preferably 1-3 bases, most preferably 1-2 bases. Specific examples of such variation include, but not limited to, one wherein the number of 3′ overhung portion is 0-3 bases, one wherein the base nucleotide of 3′ overhung portion is changed into other base sequence, or one wherein the lengths of the above sense RNA strand and antisense RNA strand differ in 1-3 bases due to insertion, addition or deletion of base(s), one wherein the base(s) in the sense strand and/or antisense strand is replaced with other base(s), or the like. However, in the variant siRNAs it is necessary that the sense strand and antisense strand hybridize and that such variant siRNAs have an ability of suppressing a gene expression similarly to siRNA without any variation.
Furthermore, the siRNA can be a molecule with a structure of one closed end, for example, siRNA with a hairpin structure (Short Hairpin RNA; shRNA). shRNA is an 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 sequence, and a liker sequence that links the both strands, and wherein the sense strand portion and the antisense strand portion hybridize to form a double stranded RNA portion.
It is desirable that siRNA does not exhibit so-called off-target effect in clinical use. Off-target effect refers to an effect of suppressing the expression of a gene different from a target gene which partly has a homology to the siRNA used. In order to avoid the off-target effect, it is possible to pre-confirm by using a DNA microarray or the like that a candidate siRNA does not have a cross reactivity. In addition, it is possible to avoid off-target effect by confirming with known databases provided by NCBI (National Center for Biotechnology Information) or the like whether there is any gene other than a target gene which comprises a portion highly homologous to the sequence of a candidate siRNA.
In order to make the siRNA of the present invention, known methods such as methods using chemical synthesis and methods using genetic recombination technique can be suitably used. In methods using chemical synthesis, a double stranded RNA can be synthesized by using conventional methods based on sequence information. In methods using genetic recombination technique, it can be made by constructing an expression vector encoding a sense strand sequence or antisense strand sequence, introducing the vector into a host cell, then obtaining the sense strand RNA or antisense strand RNA which are produced by transcription, respectively. In addition, a desired double stranded RNA can be made by expressing shRNA comprising a sense strand of a specific sequence of a target gene, an antisense strand consisting of a sequence complementary to the sense strand sequence, and a linker sequence liking the both strands, and forming a hairpin structure.
Regarding siRNA, as long as it has an activity of suppressing the expression of a target gene, all or part of the nucleic acid that constitutes the siRNA can be a natural nucleic acid or modified nucleic acid.
In the nucleic acid of the present invention that suppresses the expression of a gene such as SMS2 or the like, a modified nucleic acid can be used. A modified nucleic acid means one wherein a nucleoside (base portion, sugar portion) and/or internucleoside bond portion are modified and which has a different structure from that of a native nucleic acid. A “modified nucleoside” that constitutes a modified nucleic acid includes abasic nucleoside; arabinonucleoside, 2′-deoxyuridine, alpha-deoxyribonucleoside, beta-L-deoxyribonucleoside, other nucleoside having sugar modification; peptide nucleic acid (PNA), peptide nucleic acid bearing phosphate group (PHONA), locked nucleic acid (LNA), morpholino nucleic acid or the like. The nucleoside having sugar modification includes substituted pentoses such as 2′-O-methylribose, 2′-deoxy-2′-fluororibose, 3′-O-methylribose or the like; 1′,2′-deoxyribose; arabinose; substituted arabinose; nucleosides whose sugar is modified in hexose and alpha-anomer. Such nucleosides can be a modified base wherein the base portion is modified. Such modified bases include, for example, pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil or the like; purines such as 6-methyladenine, 6-thioguanosine or the like; and other heterocyclic bases, or the like.
A “modified internucleoside bond” that constitutes a modified nucleic acid includes, for example, non-native intranucleoside bonds such as alkyl linker, glyceryl linker, amino linker, poly(ethylene glycol) bond, methylphosphonate internucleoside bond; methylphosphothioate, phosphotriester, phosphothiotriester, phosphorothioate, phosphodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidate or the like.
As a nucleic acid sequence included in the double stranded siRNA of the present invention, the sequences set forth in the Sequence Listing can be preferably used. The nucleotide sequences of these siRNA are shown in Table 1. In Table 1, shown in capital letters are sense RNA sequence and antisense RNA sequence, and shown in lower-case letters or d+capital letters (meaning deoxy forms) are 3′ end overhung sequences.
“Transgenic” herein refers to integration of a specific gene into an organism (or cell or the like), or an organism (for example, including animals (such as a mouse)) (or cell or the like) wherein such gene is integrated or deleted or suppressed. Among transgenic organisms (or cell or the like), one wherein a gene is deleted or suppressed is referred to as a knockout organism (or cell or the like). Therefore, “knockout”, when referring to animal or cell or the like, means states wherein a native gene which is targeted does not function or is not expressed.
“Transgenesis” herein refers to introducing a gene of interest into a cell, tissue or animal, conceptually encompasses “transformation”, “transduction” and “transfection” or the like, and can be realized by any technique known in the art. In addition, “transgenesis” also encompasses any of one wherein a site to be introduced is not limited and one via homologous recombination wherein a site to be introduced is limited. A method for transgenesis include, but not limited to, for example, methods using retrovirus, plasmid, vector or the like, or electroporation method, methods using particle gun (gene gun), calcium phosphate method. A cell used for transgenesis can be any cell, and use of an undifferentiated cell (for example, fibroblast or the like) is preferred.
“Preventing” herein refers to, by any means, not allowing the occurrence of or at least delaying a disease, disorder or symptom which the present invention targets before occurrence of the disease, disorder or symptom, or not allowing the occurrence of a disorder even if any cause itself of the disease, disorder or symptom occurs.
“Treating” herein refers to stopping the progression of a disease, disorder or symptom which the present invention targets, or completely or partly arresting or ameliorating a disease, disorder or symptom which the present invention targets.
“Treatment” herein refers to somewhat affecting a disease, disorder or symptom or preventing a subject from being in such a disease, disorder or symptom, and can encompass therapy and prevention. In narrower sense, “treatment” refers to the above action after occurrence thereof compared to “prevention”.
“Cell death inducing drug” refers to any drug capable of inducing cell death. In the present invention, any material can be used as long as it is a drug capable of inducing cell death by biding to cholesterol in cell membrane and destroying the cell membrane. In an SMS deficient cell, cyclodextrins can be used so as to induce cell death, but it is understood that the drug is not limited to them. “Cyclodextrin” as referred to herein is used in broader sense, refers to any cyclic oligosaccharide and derivatives thereof which several molecules of D-glucose are bound via alpha(1->4) glycoside bond, and refers to any one of substituted (for example, substituent includes, but not limited to, alkyl group, hydroxylalkyl group or the like) or unsubstituted cyclodextrins. Cyclodextrin which can be used as a cell death inducing drug includes, for example, methyl-alpha-cyclodextrin, methyl-beta-cyclodextrin, 2-hydroxylpropyl-beta-cyclodextrin, alpha-cyclodextrin, beta-cyclodextrin or the like (for reference, see, J. Biol. Chem. 270, 29, 17250-17256, 1995; J. Biol. Chem. 275, 44, 34028-34034; Japanese Laid-Open Publication No. 2007-332128 or the like).
“Reconstructed cell” herein refers to a cell produced by introducing a gene of interest into it, optionally after deleting a specific gene by knocking out it. Reconstructed cell can include cells wherein at least one sphingomyelin synthetase (for example, SMS 1, SMS2 or both of SMS1 and SMS2) are transgenically introduced into a cell wherein the sphingomyelin synthetase SMS 1 and SMS2 of the present invention are knocked out, or the like.
It is understood that “medicament” herein is interpreted in the broadest sense in the art, comprise any drug and encompass pharmaceuticals under the Parmaceutical Affaris Law, quasi-drugs or the like as well as any usage of drug and composition or the like which is intended for treatment or prevention by osteogenesis. As such examples, applications in medical art, dental art or the like are listed, for example, a gene therapeutic agent or the like is listed. Usually, a medicament comprises a solid or liquid excipient and can optionally comprise additives such as a disintegrating agent, a flavoring agent, a delayed releasing agent, a lubricant, a binding agent, a coloring agent or the like. The form of a medicament includes, but not limited to, tablets, injections, capsules, granules, powders, fine granules, controlled release formulations or the like.
It should be understood that while description of preferred embodiments is described below, the embodiments are illustrative of the present invention and the scope of the present invention is not limited to such preferred embodiments. It should be also understood that those skilled in the art can readily carry out modification, alternation or the like within the scope of the present invention with reference to the preferred embodiments below.
(A Nucleic Acid which Suppresses the Expression of SMS2)
The present invention provides a nucleic acid which suppresses the expression of SMS2. The nucleic acid of the present invention has a function that suppresses the translation or transcription or the like of a nucleic acid. Such a nucleic acid can include an antisense nucleic acid, a nucleic acid having RNAi effect (for example, siRNA), a nucleic acid having a ribozyme activity, or the like. The nucleic acid which suppresses the expression of SMS2 of the present invention can comprise a modified nucleic acid.
Such a nucleic acid (for example, siRNA, antisense nucleic acid) is used for ameliorating, treating or preventing a metabolic syndrome.
A preferred embodiment of the nucleic acid which suppresses the expression of SMS2 of the present invention such as siRNA of SMS2 can include, for example, nucleic acids selected from the group consisting of the following (a)-(c): (a) an antisense nucleic acid toward a transcript of a gene encoding an SMS2 protein or part thereof, (b) a nucleic acid having a ribozyme activity which specifically cleaves a transcript encoding an SMS2 protein; and (c) a nucleic acid having an effect of inhibiting the expression of a gene encoding SMS2 protein via RNAi effect (for example, siRNA).
SMS2 can include, typically, SEQ ID NO: 79 (human)(Locus: NM—152621, 6246 bp), SEQ ID NO: 80 (mouse)(NM—028943, 5791 bp)(full length sequence of SMS2) or the like. However, it is understood that besides above, any sequence known as SMS2 can be used as a target. As such sequences, sequences referred to by a plurality of Accession numbers on genome databases (for example, on nucleotide databases, besides above, NM—001136257, NM—001136258, BC041369, BC028705 (human) or the like are searched, and on protein databases, NP—001129730, NP—689834, NP—001129729, Q8NHU3, AAH41369, AAH28705, Q86VZ5 (the precedings are human), NP—083219 (mouse) or the like) are searched on the public gene database NCBI.
Even if it is not above proteins, for example, proteins which have a high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) to the sequences described in these Accession numbers and have functions which the above proteins have (for example, a function of synthesizing sphingomyelin in a cell, or the like), are encompassed in proteins which the present invention targets. It is understood that proteins consisting of an amino acid sequence wherein one or more of amino acids are added, deleted, substituted, or inserted in the amino acid sequences set forth by the Accession numbers related to the above proteins and wherein the number of amino acids that are changed is usually within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids and most preferably within 3 amino acids are also encompassed. Alternatively, ones having a high homology to the DNA sequences set forth by the Accession numbers related to the above nucleotide sequences are also encompassed. High homology means 50% or more, preferably 70% or more, more preferably 80% or more, further more preferably 90% or more (for example, 95% or more, further 96%, 97%, 98% or 99% or more) of homology. Such homology can be determined by mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7). Alternatively, the sequence which the present invention targets can be one which hybridizes under stringent condition to the DNA sequences set forth by Accession numbers related to the above nucleotide sequences. Here, “stringent condition” can include, for example, “2×SSC, 0.1% SDS, 50“C”, “2×SSC, 0.1% SDS, 42 degrees Celsius”, “1×SSC, 0.1% SDS, 37 degrees Celsius”, more stringent condition can include conditions of “2×SSC, 0.1% SDS, 65 degrees Celsius”, “0.5×SSC, 0.1% SDS, 42 degrees Celsius” and “0.2×SSC, 0.1% SDS, 65 degrees Celsius”.
Those skilled in the art can suitably obtain a protein functionally equivalent to the above proteins among the above proteins having a high homology by using a method of measuring an activity of synthesizing sphingomyelin. Specific methods for measuring the activity are illustratively described in the Examples. In addition, those skilled in the art can suitably obtain an endogenous gene in other organism which corresponds to the gene based on the base sequence of the gene. Here, in the present specification, protein and gene in organism other than a human which corresponds to the above protein and gene, or protein and gene functionally equivalent to the above protein and gene are, in some times, merely described with the above names.
As a method of inhibiting the expression of a specific endogenous gene such as SMS2, methods using an antisense technique are well known to those skilled in the art. As an action that an antisense nucleic acid inhibits the expression of a target gene, a plurality of factors as follows exist. That is, inhibition of starting transcription by triplex formation, inhibition of transcription by forming a hybrid with a portion wherein opened loop structure is locally made by an RNA polymerase, inhibition of transcription by forming a hybrid with an RNA which is being synthesized, inhibition of splicing by hybrid formation at junction of an intron and an exon, inhibition of splicing by forming hybrid with a spliceosome forming site, inhibition of translocation from nucleus to cytoplasm by hybrid formation with mRNA, inhibition of splicing by forming hybrid with a capping site or poly(A) addition site, inhibition of starting translation by forming hybrid with translation initiator-binding site, inhibition of translation by forming hybrid with ribosome binding site near start codon, inhibition of elongation of peptide chain by forming hybrid with translated region or polysome binding site of mRNA, and inhibition of gene expression by forming hybrid with nucleic acid-protein interacting site, or the like. As explained above, an antisense nucleic acid inhibits the expression of a target gene by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shin Seikagaku Jikken Koza [New Lectures on Biochemical Experiment] 2, Kakusan [Nucleic acid] IV, Idenshi no Fukusei to Hatsugen [Gene Replication and Expression], The Japanese Biochemical Society, ed., Tokyo Kagaku Dojin, 1993, 319-347.).
The antisense nucleic acid used in the present invention can inhibit the expression and/or function of the gene encoding SMS2 described above via any actions above. As one embodiment, if an antisense sequence is designed which is complementary to untranslated region near 5′ end of mRNA of the gene encoding SMS2 described above, it is considered to be effective in inhibiting the translation of the gene. In addition, a sequence complementary to the coding region or 3′ untranslated region can be also used. As such, a nucleic acid comprising an antisense sequence of not only the translated regions of the gene encoding the above SMS2 but also the untranlated regions is also encompassed by antisense nucleic acid used in the present invention. An antisense nucleic acid used is liked downstream of a suitable promoter and is preferably liked, 3′ thereto, to a sequence comprising a transcription termination signal. A nucleic acid thus prepared can be transformed into a desired animal (cell) by using known methods. The sequence of an antisense nucleic acid is desirably a sequence complementary to a gene encoding an endogenous SMS2 which the animal (cell) to be transformed has or part thereof, but are not necessarily completely complement as long as it can effectively suppress the expression of the gene. The transcribed RNA is preferably 90% or more, most preferably 95% or more complementary to the transcript of a target gene. In order to effectively inhibit the expression of a target gene by using an antisense nucleic acid, it is preferably that the length of the antisense nucleic acid is at least 12 bases or more and less than 25 bases, but the antisense nucleic acid of the present invention is not necessarily limited to such length and can be, for example, 11 bases or less, 100 bases or more, or 500 bases or more. The antisense nucleic acid can be composed of DNA only, but can comprise nucleic acids other than DNA, for example, a locked nucleic acid (LNA). As an embodiment, the antisense nucleic acid used in the present invention can be an antisense nucleic acid comprising a locked nucleic acid (LNA) at 5′ end and an LNA at 3′ end. Such an LNA-containing antisense nucleic acid can include, but not limited to, SEQ ID NOs: 81-92 or the like. In addition, in an embodiment of the present invention which uses an antisense nucleic acid, using, for example, the method described in Hirashima and Inoue, Shin Seikagaku Jikken Koza [New Lectures on Biochemical Experiment] 2, Kakusan [Nucleic acid] IV, Idenshi no Fukusei to Hatsugen [Gene Replication and Expression], The Japanese Biochemical Society, ed., Tokyo Kagaku Dojin, 1993, 319-347, based on the nucleic acid sequences of SMS2 set forth in SEQ ID NO: 79 or 80, antisense sequences can be designed. As sequences that can be referred to, SEQ ID NO: 1-18, 21-24, 39-42, 45, 46 or the like can be used, but not limited thereto. For example, those such as SEQ ID NOs: 81-92 described in Example 10 are preferably used, but not limited thereto. These antisense nucleic acids can be confirmed for the effect of the antisense of the present invention by the methods exemplified in Examples 7 and 8 which use mice and HepG2 cell.
The inhibition of the expression of SMS2 can be carried out by utilizing a ribozyme or a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having a catalytic activity. Various ribozymes have various activities. Among them, a study focusing a ribozyme as an enzyme that cleaves an RNA enabled it possible to design a ribozyme that site-specifically cleaves an RNA. Some ribozymes are 400 nucleotides or more such as Group I intron type or M1 RNA included in RNase P, others called as hammer head type and hairpin type have about 40 nucleotides of activity domain (Mokoto Koizumi and Eiko Otsuka, Tanpakushitsu Kakusan Koso [Protein, Nucleic Acid and Enzyme], 1990, 35, 2191).
For example, autocleaving domain of a hammerhead type ribozyme cleaves 3′ of C 15 of a sequence G13U14C15, and base-paring of U14 and A9 is important for the activity and it was demonstrated that A15 or U15 instead of C15 can be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). If a ribozyme whose substrate-binding site is complementary to an RNA sequence near its target site is designed, an RNA-cleaving ribozyme which recognizes sequences UC, UU or UA in the target RNA like a restriction enzyme can be made (Koizumi, M. et al., FEBS Lett, 1988, 239, 285., Mokoto Koizumi and Eiko Otsuka, Tanpakushitsu Kakusan Koso [Protein, Nucleic Acid and Enzyme], 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059).
Furthermore, a hairpin type ribozyme is also useful for the purpose of the present invention. The ribozyme is found in, for example, a minus strand of satellite RNA of tobacco ringspot virus (Buzayan, J M., Nature, 1986, 323, 349). It was demonstrated that a target specific RNA cleaving ribozyme can be also made from a hairpin type ribozyme (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751, Yo Kikuchi, Chemistry and Biology, 1992, 30, 112.). As described above, by specifically cleaving the transcript of a gene encoding SMS2 with a ribozyme, the expression of the gene can be inhibited.
Furthermore, the suppression of the expression of an endogenous gene such as SMS2 can be also carried out by RNA interference (abbreviated as “RNAi”, hereinafter) with a double stranded RNA having an identical or similar sequence to its target gene sequence. RNAi is a recently noted technique wherein when a double stranded RNA (dsRNA) is directly taken into a cell, the expression of a gene having a sequence homologous to the dsRNA is suppressed. In a mammalian cell, it is possible to induce RNAi by using a short chain dsRNA (siRNA). RNAi has many advantages over a knockout mouse that the effect is stable, experiment for it is easy, the cost is reasonable or the like. For siRNA, it is also described in detail in other postions herein.
As described above, those skilled in the art can suitably produce the “siRNA” of the present invention based on the base sequences of the genes encoding SMS2 described above which the double stranded RNA targets. As an example, a sense strand of the duplex RNA portion includes, but not limited to, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 21 and 23 or the like. It can be suitably carried out by those skilled in the art within the scope of usual trial to select any contiguous RNA region of the mRNA that is a transcript of SMS2 sequence and then make a double stranded RNA corresponding to the region. It can be also suitably carried out by those skilled in the art with known methods to select an siRNA sequence having a stronger RNAi effect among mRNA sequences that are the transcript of the sequence. In addition, if one strand is known, those skilled can also readily know the base sequence of the other strand (a complementary chain). Those skilled in the art can suitably make an siRNA by using a commercially available nucleic acid synthesizer. Alternatively, for the synthesis of a desired RNA, a usual contract service for synthesis can be used.
Therefore, in one embodiment, the present invention is an siRNA of SMS2 (for example, SEQ ID NO: 79 or 80 which are the full length sequences of SMS2). Such siRNA specifically includes, but not limited to, siRNA of any one selected from the group consisting of the following (a)-(o), which are siRNAs based on the sequences which the prevent inventors have originally designed:
(a) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 1 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 2 <SMS2-i6>;
(b) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 3 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 4 <SMS2-i7>;
(c) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 5 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 6 <SMS2-i8>;
(d) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 7 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 8 <SMS2-i104>;
(e) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 9 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 10 <SMS2-i105>;
(f) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 11 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 12 <SMS2-i106>;
(g) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 13 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 14 <SMS2-i107>;
(h) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 15 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 16 <SMS2-i108>;
(i) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 17 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 18 <SMS2-i109>;
(j) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 21 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 22 <SMS2-i3>;
(k) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 23 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 24 <SMS2-i11>;
(l) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 39 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 40 <SMS2-i1>;
(m) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 41 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 42 <SMS2-i2>;
(n) an siRNA wherein one strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 45 and the other strand of the duplex RNA portion is a base sequence set forth by SEQ ID NO: 46 <SMS2-i5>; and
(o) an siRNA of any of (a)-(n), wherein one to several nucleotides are added, inserted, deleted or substituted in one or both of the base sequences, and having an activity of suppressing the expression of SMS2.
siRNA in the present invention is not necessarily a pair of double stranded RNA to its target sequence, but can be a mixture of multiple pairs of double stranded RNA toward regions comprising the target sequence. Here, siRNA as a nucleic acid mixture corresponding to a target sequence can be suitably made by those skilled in the art with a commercially available nucleic acid synthesizer and DICER enzyme. Alternatively, for the synthesis of a desired RNA, a usual contract service for synthesis can be used. Here, siRNA of the present invention encompasses so-called “cocktail siRNA”. Furthermore, not all nucleotides in the siRNA in the present invention are necessarily ribonucleotide (RNA). That is, in the present invention, one or plurality of ribonucleotide(s) that constitutes siRNA can be a corresponding deoxyribonucleotide. The “corresponding” refers to being same kind of base (adenine, guanine, cytosine, thymine (uracil)) with different sugar structures. For example, a corresponding deoxyribonucleotide to a ribonucleotide having adenine refers to a deoxyribonucleotide having adenine. In addition, the above “plurality” refers to, but not specifically limited to, preferably as small number as about 2-5.
Furthermore, a DNA (a vector) capable of expressing the RNA of the present invention can also be encompassed by preferred embodiment of the nucleic acid of the present invention that is capable of suppressing the expression of SMS2. For example, a DNA (a vector) capable of expressing the double stranded RNA of the present invention is a DNA having a structure wherein a DNA encoding one strand of the double stranded RNA and a DNA encoding the other strand of the double stranded RNA are liked to promoters so that each of the DNA can be expressed.
Those skilled in the art can suitably make the DNA of the present invention by general genetic engineering techniques. More specifically, the expression vector of the present invention can be made by suitably inserting a DNA encoding the RNA of the present invention into various known expression vectors.
(A Medicament for a Metabolic Syndrome and a Method for Treating or Preventing a Metabolic Syndrome)
In another aspect, the present invention provides a pharmaceutical composition for treating or preventing a metabolic syndrome, comprising a nucleic acid that suppresses the expression of SMS2. Here, it is understood that as a nucleic acid that suppresses the expression of SMS2 used, any nucleic acid described in (A nucleic acid which suppresses the expression of SMS2) herein can be used.
Alternatively, the present invention provides a nucleic acid (for example, siRNA or an antisense nucleic acid) which suppresses the expression of SMS2 for treating or preventing a metabolic syndrome. It is understood that as a nucleic acid which suppresses the expression of SMS2 used for treating or preventing a metabolic syndrome, any nucleic acid described in (A nucleic acid which suppresses the expression of SMS2) herein can be used.
Preferably, the nucleic acid of the present invention that suppresses the expression of SMS2 is siRNA or an antisense nucleic acid, and the above specific siRNAs or antisense nucleic acids can be exemplified.
In various embodiments, it is understood that a metabolic syndrome which the present invention targets is selected from the group consisting of obesity, diabetes, dyslipidemia and fatty liver. Therefore, when the nucleic acid or medicament of the present invention is used, site to be applied or kind of a disease is not limited as long as the disease is a metabolic disease and it is applied to for example, obesity, diabetes, dyslipidemia and fatty liver or the like as an object. The above disease can be one with which other disease concurrently occurs.
In an embodiment, the medicament of the present invention further comprises a pharmaceutically acceptable excipient. When the composition of the present invention is used as a medicament or pharmaceuticals, the dosage form includes, for example, tablets, powders, fine granules, granules, coated tablets, controlled release formulations, capsules, injections or the like. The pharmaceuticals can comprise an excipient, optionally, additives such as a binding agent, a disintegrating agent, a lubricant, a flavoring agent, a coloring agent, a delayed releasing agent or the like. In a case of an oral formulation, as an excipient, for example, lactose, corn starch, sucrose, glucose, mannitol, sorbite, crystalline cellulose or the like; as a binding agent, for example, polyvinyl alcohol, polyvinylether, methylcellulose, hydroxylpropylcellulose, gum arabic, tragacanth, gelatin, shellac, polyvinylpyrrolidone, block copolymers or the like; as a disintegrating agent, for example, starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin or the like; as a lubricant, for example, magnesium stearate, talc, polyehylene glycol, silica, hydrogenated vegetable oils or the like; as a flavoring agent, for example, cocoa powder, peppermint oil, cinnamic powder or the like can be used, but not limited thereto. Optionally, it can be coated so as to be a controlled release formulation or enteric coated formulation. In a case of a formulation for injection, a pH modulating agent, a solubilizing agent, an isotonic agent, a buffering agent or the like can be used, but not limited thereto.
The medicament of the present invention can be mixed with a physiologically acceptable carrier, excipient or diluent or the like as above, and orally or parenterally administered as a pharmaceutical composition. As an oral formulation, it can be in dosage forms above such as granules, powders, tablets, capsules, solvents, emulsions, or suspensions. As a parenteral formulation, dosage forms such as injections, drops, medicines for external use, inhalant (nebulizer) or suppository or the like can be selected. As injections, hypodermic injections, intramuscular injections, intraperitoneal injections, intracranial injections, or intranasal injections or the like can be shown. As a medicine for external use, transnasal drugs or ointments or the like can be shown. Technique for formulating the above dosage form so as to comprise the medicament of the present invention as a major ingredient is known.
For example, tablets for oral administration can be made by adding to the nucleic acid or medicament of the present invention an excipient, a disintegrating agent, a binding agent, a lubricant or the like, mixing them and compression molding them. As an excipient, lactose, starch or mannitol or the like are generally used. As a disintegrating agent, calcium carbonate or carboxymethyl cellulose calcium or the like are generally used. As a binding agent, gum arabic, carboxymethyl cellulose or polyvinylpyrrolidone is used. As a lubricant, talc or magnesium stearate or the like are known.
Tablets comprising the nucleic acid or medicament of the present invention can be made with known coating so as to make them to be masked or a enteric coated formulation. As a coating agent, ethylcellulose or polyoxyethylene glycol or the like can be used.
Injections can be also obtained by dissolving the nucleic acid or medicament of the present invention as a major ingredient with a suitable dispersing agent, or dissolving or dispersing them in a disperse medium. Depending on selecting the disperse medium, any dosage form of an aqueous solvent or an oily solvent can be taken. For making an aqueous solvent, a distilled water, a saline, or Ringer's solution or the like are used as a disperse medium. For an oily solvent, various vegetable oils or propylene glycol or the like is used as a disperse medium. In such a case, preservative agents such as paraben or the like can be optionally added. Into injections, known isotonic agents such as sodium chloride or glucose or the like can be also added. Furthermore, analgesics such as benzalkonium chloride or procaine hydrochloride can be added.
The nucleic acid or medicament of the present invention can be also made to be a medicament for external use by making it to be a solid, liquid or semi-solid composition. A solid or liquid composition can be made as a medicament for external use by making them to be similar to the composition as described above. A semi-solid composition can be prepared by optionally adding a thickening agent to a suitable solvent. For the solvent, water, ethyl alcohol or polyethylene glycol or the like can be used. For a thickening agent, bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone or the like are generally used. To the composition, a preservative agent such as benzalknoum hydrochloride or the like can be added. It can also be made to be a suppository by combining with, as a carrier, a oily substrate such as cacao butter or an aqueous gel substrate such as cellulose derivatives.
When the nucleic acid or medicament of the present invention is used as a gene therapic agent, a method of directly administering the nucleic acid or medicament of the present invention by an injection, as well as a method of administering a vector wherein the nucleic acid is incorporated are listed. As the vector, an adenoviral vector, an adeno-associated viral vector, a herpesviral vector, a vaccinia viral vector, a retroviral vector, a lentiviral vector or the like are listed, and it can be effectively administerd by using these viral vectors.
It is also possible to introduce the nucleic acid or medicament of the present invention into a phospholipid vesicle such as a liposome and administer it. A vesicle retaining an siRNA or shRNA can be introduced into a given cell by lipofection method. Then, the cell thus obtained is administered systemically, for example, intravenously, intraarterially or the like. It can be also administered locally to site or the like of obesity, diabetes, dyslipidemia and fatty liver. Since siRNA exerts a highly excellent effect of specific and post-transcriptional suppression in vitro but is quickly degraded by a nuclease activity in a serum and has limited duration time in vivo, a more optimal and effective delivery system has been sought to be developed. As one example, by Ochiya, T et al; Nature Med., 5: 707-710, 1999, Curr. Gene Ther., 1:31-52, 2001, it was reported that when atelocollagen that is a biocompatible material is mixed with a nucleic acid to form a complex, it has a effect of protecting the nucleic acid from degrading enzymes in vivo and thus is a carrier which is very suitable as a carrier for an siRNA. Therefore, such a form can be used, but a method for introducing the nucleic acid or medicament of the present invention is not limited thereto. Since it is not degraded in vivo quickly by nucleic acid degrading enzymes in a serum, lasting a long term effect can be achieved in such a way. For example, in Takeshita F. PNAS.(2003) 102(34) 12177-82, Minakuchi Y Nucleic Acids Research(2004) 32(13) e109, it was reported that atelocollagen derived from a bovine skin forms a complex with a nucleic acid and has an effect of protecting the nucleic acid from degrading enzymes in vivo, which is thus very suitable as a carrier for an siRNA. Such techniques can be used.
The nucleic acid or medicament of the present invention is administered to a mammal including a human in a necessary amount (an effective amount) which is within the scope of dosage amount that is deemed as safe. The dosage amount for the nucleic acid or medicament of the present invention can be suitably determined, ultimately by the judgment of a doctor or vetenarian in view of the kind of dosage form, administration method, age or body weight of a patient, symptom of a patient or the like. As an example, although it varies depending on age, sex, symptom, route for administration, times of administration, dosage form, for example, the dosage amount in a case of an adenovirus is about 106-1013 per one administration per day and is administered at interval of a week-8 weeks.
It is also possible to use a commercially available kit for transgenesis (for example, Adeno Express: Clontech) in order to introduce an siRNA or shRNA into a tissue or organ of interest.
The medicament or pharmaceutical composition of the present invention can further comprise an effective ingredient. Such additional medicaments that can be comprised are variously considered depending on its purpose.
In another aspect, the present invention provides use of a nucleic acid (for example, siRNA, antisense nucleic acid) that suppresses the expression of SMS2 of the present invention for the manufacture of a medicament for treating or preventing a metabolic syndrome. Here, it is understood that as a nucleic acid that suppresses the expression of SMS2 of the present invention, any nucleic acid described in the above section of (A nucleic acid which suppresses the expression of SMS2) or the present section of (A medicament for a metabolic syndrome and a method for treating or preventing a metabolic syndrome) can be used.
In another aspect, the method of the present invention is a method for manufacturing a pharmaceutical composition or medicament for treating or preventing a metabolic syndrome comprising a nucleic acid that suppresses the expression of SMS2, comprising mixing the nucleic acid that suppresses the expression of SMS2 with a pharmaceutically acceptable excipient. Besides a medicament form, food, health food, functional food or the like which are approved by the authorities can be smilarly manufactured. In such cases, in place of a pharmaceutically excepient, a secondary ingredient can be used depending on its purpose.
An effective amount of the medicament or pharmaceutical composition of the present invention refers to an amount in which the medicament or pharmaceutical composition of the present invention can exert an intended pharmaceutical efficacy, and the smallest concentration among such effective amounts is sometime referred herein as a minimal effective amount, which can be suitably determined by those skilled in the art based on the description of the present specification. For determining such effective amounts, besides actual administration, animal model or the like can be used. The present invention is also useful in determining such effective amounts.
In a further aspect, the present invention provides a method for treating or preventing a metabolic syndrome, said method comprising: administering the nucleic acid (for example, siRNA, antisense nucleic acid) that suppresses the expression of SMS2 of the present invention to a subject in need of said treatment or prevention. Here, it is understood that as a nucleic acid that suppresses the expression of SMS2 which can be used, any nucleic acid described in the above section of (A nucleic acid which suppresses the expression of SMS2) or the present section of (A medicament for a metabolic syndrome and a method for treating or preventing a metabolic syndrome) can be used.
Administration to an individual is as explained in the present section of (A medicament for a metabolic syndrome and a method for treating or preventing a metabolic syndrome), any method can be used for it, and it can be generally carried out by methods known to those skilled in the art, for example, intraarterial injection, intravenous injection, hypodermic injection or the like. Dosage amounts vary depending on body weight or age of a patient, administration method or the like, but those skilled in the art (doctors, veterinarians, pharmacists or the like) can suitably select a reasonable dosage amount.
An individual that is an object for the method for treatment or prevention of the present invention is not specifically limited as long as it is an organism that can develop a metabolic syndrome, but preferably a human.
The amount of an effective ingredient used in the method for treatment or prevention of the present invention can be readily determined by those skilled in the art by taking into consideration purpose of use, objective diseases (kind, severity or the like), age, body weight, sex, anamnesis of a patient, form or kind of cell, or the like. Frequency at which the method of treatment of the present invention is carried out on a subject (or a patient) can be also readily determined by those skilled in the art in view of purpose of use, objective diseases (kind, severity or the like), age, body weight, sex, anamnesis of a patient, course of treatment or the like. Such frequency includes, for example, administration once per day to once per several months (for example, once per week to once per month). It is preferable to carry out administration once per day to once per month with observing the course.
Type and amount of ingredients used in the method of treatment of the present invention can be readily determined by those skilled in the art in view of purpose of use, objective diseases (kind, severity or the like), age, bodyweight, sex, anamnesis of a patient, form or kind of site of a patient to be administered or the like, based on information obtained by the method of the present invention (for example, information regarding a disease). Frequency at which the method of monitoring of the present invention is administered on a subject (or a patient) can be also readily determined by those skilled in the art in view of purpose of use, objective diseases (kind, severity or the like), age, body weight, sex, anamnesis of a patient, course of treatment or the like. Frequency for monitoring a status of a disease includes, for example, monitoring once per day to once per several months (for example, once per week to once per month). It is preferable to carry out monitoring once per day to once per month with observing the course.
The present invention can be used as a kit or the like, in such a case, it can be accompanied with written instructions. “Written instructions” herein refers to ones wherein the method of treatment of the present invention is described to a human who carries out the administration such as a doctor, a patient or the like. The written instructions describe, for example, a statement that instructs to suitably administer the medicament or the like of the present invention. The written instructions are made according to the form which is defined by the authorities of a country in which the present invention is carried out (for example, Ministry of Health, Labour and Welfare in Japan, Food and Drug Administration in the United State of America (FDA) or the like), and explicitly describes that it is approved by the authorities. The written instructions is so-called appendant document (package insert), which is usually provided in paper media but not limited thereto, and can be provided, for example, in forms such as electronic media (for example, homepages provided by internet, electronic mail).
(General Techniques)
Techniques of manufacturing a medical devise, techniques of formulation, techniques of microfabrication, molecular biological methods, biochemical methods, microbiological methods, saccharide chain related methods used in herein are well known and routine in the art, and described in for example, Maniatis, T. et al.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and 3rd Ed. (2001); Ausubel, F. M., et al. eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc., NY, 10158 (2000); Innis, M. A. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press; Innis, M. A. et al. (1995) PCR Strategies, Academic Press; Sninsky, J. J. et al. (1999) PCR Applications: Protocols for Functional Genomics, Academic Press; Gait, M. J. (1985) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991) Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R. L. et al. (1992) The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994) Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996) Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (1996) Bioconjugate Techniques, Academic Press; Method in Enzymology 230, 242, 247, Academic Press, 1994; Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume], Idenshi Donyu Oyobi Hatsugen Kaiseki Jikken Ho [Experimental Methods for Transgenesis & Expression Analysis], Yodosha, 1997 or the like, relevant portions (that can be all) of which are incorporated herein by reference.
Methods of culture used in the present invention are described and supported in for example, Dobutsu Baiyo Saibo Manyuaru [Animal Cultured Cell Manual], Seno et al. ed., Kyoritsu Syuppan, 1993 or the like, the entire description of which are incorporated herein.
Hereinabove, the present invention has been described with showing preferred embodiments for easiness of understanding. While the present invention is described hereinbelow based on examples, the above description and the examples below are provided for illustrative purpose only, but not provided for a purpose for limiting the present invention. Therefore, the scope of the present invention is not limited by embodiments or examples that are specifically described herein, but limited only by the Claims.
Handling animals used in the examples below were in accordance with the criteria defined in Hokkaido University.
Abbreviations used are as follows:
SMS: sphingomyelin synthetase
SMS1, SMS2: names of genes
KO: knockout
wKO: double knockout
MEF: mouse embryonic fibroblast
FBS: fetal bovine serum
DMEM: Dulbecco's modified Eagle medium
TG: triglyceride
SM: sphingomyelin
Me-beta-CD: methyl-beta-cyclodextrin
HFD: high fat diet
ND: normal diet
WAT: white fat cell mass
WT: wild type
As a mouse SMS, three isoforms have been found to date, SMS 1 which was isolated by an expression cloning method, and SMS2 and SMSr which were identified as its homologs. SMS1 knockout (SMS1-KO) mice and SMS2 knockout (SMS2-KO) mice were both made by using the targeting vectors shown in
4 weeks old male wild type mice (WT; C57BL6) and SMS2-KO mice were administered a high fat diet (HFD; 58Y1, testDiet) and a control normal diet (ND; AIN76A, testDiet), respectively, and change in body weight were measured for 9 weeks. As a result, as shown in
WT and SMS2 KO mice to which HFD were administered similarly to 2) (12 weeks later) were subjected to abdominal section, and fat attached to epididymis was obtained and then the weights were measured. As a result, as shown in
WT and SMS2 KO mice to which HFD were administered similarly to 2) (12 weeks later) were subjected to abdominal section, and the livers thereof were obtained. Triglyceride in the liver homogenate were quantified by Triglyceride Quantification Kit (Biovision). The methods were in accordance with the writen instructions of the kit. The protein amount in the homogenate was quantified with BCA protein assay (Pierce) and used for correction of triglyceride amounts. As a result, as shown in
Furthermore, the result of comparing triglyceride amount in liver homogenate (mg/mg protein) is shown in
WT and SMS2 KO mice to which HFD were administered similarly to 2) (12 weeks later) were subjected to abdominal section, and fat attached to epididymis was obtained, mRNA thereof was extracted and then the mRNA expression amount of adiponectin was measured by QPCR (Thermal Cycler Dice TP800, TAKARA BIO INC.). At that time, correction was made with the mRNA expression amount of GAPDH in the same samples. As reagents, PrimeScript RT-reagent Kit (TAKARA BIO INC.) was used for the first strand cDNA synthesis and SYBR premix EX Taq II (TAKARA BIO INC.) was used for QPCR. As PCR primers,
were used.
As a result, as shown in
From the above results, it was found that there was possibility that the accumulation of neutral fat in the liver is suppressed by suppressing SMS2.
Next, mRNA expression amount of an insulin receptor in adipose tissues in WT and SMS2KO mice was analyzed by a quantitative PCR. The relative expression amount of insulin receptor was investigated when wild type (WT) and SMS2-KO mice were administered a high fat diet (HFD; 60% fat diet) or a control normal diet (ND). The primer sequence and control primer sequence used are shown. The quantitative PCR was carried out in the same way as described in 5).
Insulin Receptor (IR)
The result is shown in
From above, in the SMS2-KO mice, decrease in expression of an insulin receptor by HFD as observed in the wild type did not occur and the expression amount was almost similar to that with ND. From the above results, it was suggested that in adipose tissues of the SMS2-KO mice, decrease in the expression of insulin receptor by HFD as observed in the WT did not occur, and thus the SMS2-KO mice did not become insulin resistance (
Next, the effect of 60% fat diet administration on Glut4 in adipose tissues was investigated. The relative expression amount of Glut4 was compared between a wild type (WT) and SMS2-KO mice to which a high fat diet (HFD; 60% fat diet) or a control normal diet (ND) were administered. The primer sequences and control primer sequences used are shown.
GLUT4:
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) that is an endogenous control gene:
The result is shown in
The control normal diet (ND) or high fat diet (HFD; 60% fat diet) described in “2) Investigation of the effect of 60% fat diet administration on body weight” above were administered to wild type (WT) or SMS2KO mice. The mice which were further grown for 9 weeks were used for carrying out glucose tolerance test. The mice were fasted for 24 hours and intraperitoneally administered 2 g/kg of glucose. Blood glucose levels were measured in the blood obtained from tail vein immediately before administration, 15 minutes after administration, at 30 minutes after administration, at 60 minutes after administration, and at 120 minutes after administration. For the measurement, a glucometer was used. As a result, it was found between the wild type and SMS2-KO mice to which the high fat diet was administered, that at 120 minutes after administration, the blood glucose level was significantly decreased in the SMS2 KO/HFD group (P<0.05;
From the results, it could be confirmed that they become significantly sensitive to glucose load.
Next, plasma insulin amount was measured. Wild type (WT) and SMS2-KO mice were administered a normal diet from the time of 8 weeks old for 4 weeks, and then fasted for 24 hours, and then administered a normal diet. Blood insulin concentration was measured at 24 hours fasting and 1 hour after the normal diet administration. The results are shown in
Next, the effect of a high fat diet on blood glucose level was investigated. 4 weeks old wild type (WT) and SMS2-KO mice were administered a high fat diet (HFD; 60% fat diet) for 2 weeks. Blood glucose level was measured at the time of 4 hours fasting before the high fat diet administration and at the time of 4 hours fasting after 2 weeks of high fat diet administration. As shown in
Next, the effect of a high fat diet on blood insulin amount was investigated. Wild type (WT) and SMS2-KO mice were administered a high fat diet (HFD; 60% fat diet) for 2 weeks, then fasted for 4 hours, and then blood insulin amount was measured. As a result, as shown in
Next, the blood glucose concentration in response to insulin was investigated. 4 weeks old wild type (WT) and SMS2-KO mice were administered a high fat diet (HFD; 60% fat diet) for 6 weeks, and insulin were intraperitoneally administered in 0.5 U/kg. The results of blood glucose concentrations that were measured before administration and at 15, 30, 60, 90 and 120 minutes after administration are shown in
As also apparent from
In the SMS2-KO mice, plasma insulin amount was decreased compared to the WT. It was thus demonstrated that the SMS2KO mice did not become insulin resistance. Therefore, the SMS2-KO mice was sensitive to insulin, and insulin resistance was not raised therein as in the wild type mice (see, in particular,
From the above results, it could be found that there is possibility that diabetes can be treated, in particular, insulin resistance can be ameliorated, by inhibiting SMS2.
As described in Shin Baiyo Saibo Jikken Ho [New Cultured Cell Experimental Method], Yodosha Co., Ltd., Chapter 4, Shyodai Baiyo Seniga Saibo [A Primary Cultured Fibroblast], p. 66-70, the SMS1-KO mouse and SMS2-KO mouse made in Example 1 were mated and from the thus obtained fetus of double knockout that were deficient in both SMS1 and SMS2 (SMS1, 2-wKO), an MEF (mouse embryonic fibroblast) was isolated. The MEF was infected with a retrovirus that was released into the culture supernatant of 293T cell stably retaining pMFG-SV40Tst, thereby immortalizing MEF.
a) Preparation of a Viral Solution
As a virus producing cell, NIH3T3 ecotroic producer cell psiCRE-MFGtsT (RCB1119; SV40 T antigen-expressing virus producer cell; see, http://www2.brc.riken.jp/lab/cell/detail.cgi?cell_no=RCB1119&type=1) was used. The psiCRE-MFGtsT cell releases a retrovirus expressing SV40tsT into a medium and it infects a mouse host cell. A subconfluent psiCRE-MFGtsT cell was cultured in a DMEM including 10% FBS overnight under conditions of 37 degrees Celsius, 5% CO2. At the next day, the supernatant was recovered as a viral solution and filtered with a 0.20 mm syringe filter (available from CORNING).
b) Viral Infection and Cloning of Cell Strains
In the culture of MEF, the medium was replaced with 5 mL of DMEM including 10% FBS, 1 mL of the viral solution, it was cultured for 24 hours under conditions of 34 degrees Celsius, 5% CO2 and infected with the virus, then added 5 mL of DMEM including 10% FBS and further cultured overnight. At the next day, the medium was replaced with 10 mL of DMEM including 10% FBS. The medium was exchanged every 2-3 days, and immortalized MEF cell strains were established. Furthermore, some cell strains were cloned from the immortalized MEF and one of them was designated as ZS2.
A retroviral vector was made by incorporating SMS1 and SMS2 with a V5 tag at C terminus added into pQCXIP (Clontech). Using this plasmid and GP2-293 cell (Clontech), retroviruses expressing SMS 1 or SMS2, respectively were made and, in accordance with the instructions of Clontech, infected into the ZS2 cell that was one of the cell strains cloned in 1). Uninfected cells were removed with 4 microgram/ml puromycin, cell ZS2/SMS1 that overexpreesed SMS1 and cell strain ZS2/SMS2 that overexpressed SMS2 were obtained. On three these cell strains, a Western blot was carried out with an anti-V5 mab (invitrogen) antibody. In the SMS1 expressing cell and the SMS2 expressing cell, as shown in
The function, in particular, SMS activity and TG synthesis, of the ZS2, ZS2/SMS 1 and ZS2/SMS2 cells made in Example 2.
1) An SMS Activity
The cells made in Example 2 were spread in 6-well plates each (2×105 cell/well), was cultured overnight in DMEM including 10% FBS, then the mediums were replaced with DMEM without serum, and BODIPY-CS-ceramide (molecular probe) adjusted to a final concentration of 1 micromolar was added and they were allowed to react at 37 degrees Celsius for 30 minutes. After the cells were detached by pipetting, they were solubilized with 100 microliter of a solubilizing buffer (20 mM Tris-HCl, pH7.5, 0.2% Triton X-100, 1×complete (Roche), 1 mM PMSF) and the protein amounts were measured with BCA protein assay (Pierce). Lipids were extracted from the solubilized solution with Bligh & Dyer method, and using HPTLC (Merck), BODIPY-ceramide and the produced BODIPY-C5-sphingomyelin were quantified with Fla7000 (Fuji film) and the SMS activity was calculated. The SMS activity was corrected for protein amount. As shown in
2) TG Synthesis
The cells made in Example 2 were spread into 6-well plates (2×105 cell/plate), cultured overnight in DMEM with 10% FBS, then the mediums was DMEM without serum, and [14C] oleic acid (American Radiolabeled Chemicals) at a final concentration of 1 microcurie was added and allowed to react at 37 degrees Celsius for 30 minutes. After the cells were detached by pipetting, they were solubilized with 100 microliter of a solubilizing buffer (20 mM Tris-HCl, pH 7.5, 0.2% Triton X-100, 1×complete (Roche), 1 mM PMSF) and the protein amounts were measured with BCA protein assay (Pierce). Lipids were extracted from the solubized solution with Bligh & Dyer method, and using HPTLC (Merck), [14C] oleic acid and the produced [14C] oleic acid-triglyceride were quantified with Fla7000 (Fuji film) and the TG amounts were calculated. As shown in
In the Example, screening siRNAs toward SMS2 was carried out. Specific procedures were as follows.
siRNAs shown below whose sequences were orinally designed were synthesized and transformed into Hepa1c1c7 cells or HeLa cells. By measuring SMS2 mRNA amounts 48 hours after, the rates of suppressing SMS2 expression by siRNAs (knockdown rates) were compared. Both cells were cultured in DMEM including 10% FCS (fetal calf serum) under conditions of 5% CO2 and 37 degrees Celsius. At the day before transfection, the cells were spread into 12-well plates and cultured for 24 hours. With RNAiMAX (Invitrogen), each siRNA was transfected at a final concentration of 100 nM, and 48 hours after, the mRNA amounts of SMS2 were measured. After treating with DNase I using RNeasy Mini Kit (QIAGEN) according to the attached manual, total RNA was recovered and cDNA was synthesized by using SuperScriptIII First Strand Synthesis System (Invitrogen) according to the attached manual. By carrying out a realtime quantitative PCR on the synthesized cDNA as templates, the mRNA amounts of SMS2 were quantified. In the realtime quantitative PCR, SYBR Green PCR Master Mix (ABI) was used as a reaction reagent and the measurement was carried out with Applied Biosystems 7500 Realtime PCR Symtem (ABI). cDNA was normalized among samples with the expression amount of G3PDH.
As realtime quantitative PCR primer sequences used for measuring the expression amounts of human and mouse SMS2,
were used, as realtime quantitative PCR primer sequences used for measuring the expression amounts of human G3PDH.
were used, as realtime quantitative PCR primer sequences used for measuring the expression amounts of mouse G3PDH,
were used.
The sequences of mouse specific siRNAs used are shown below.
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 40)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 42)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 22)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 44).
The sequences of siRNAs used that are homologous between a human and a mouse are shown below.
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 46)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 2)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 4)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 6).
The sequences of human specific siRNAs used are shown.
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 8)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 10)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 12)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 14)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 16)
Its antisense strand that is a complementary sequence thereto (SEQ ID NO: 18)
As a result, as shown in
As shown in
Next, it was investigated to confirm whether SMS2-i8 suppressed SMS2 gene expression in the mouse ZS2 cell and decreased SM on its cell membrane. As shown in
Into 6-well dishes, 2×105 of ZS2/SMS2 cells suspended in 2 ml of 10% FCS/DMEM medium were spread and various siRNAs were mixed with a commercially aviable transformation reagent RNAiMAX (Invitrogen) and added at a final concentration of 100 nM. Following the manufacturer's protocol, transformation was carried out. After 24 hours, the mediums was replaced with 0.3% FCS OPTIPRO SFM (GIBCO), and cultured for additional 24 hours, then Me-beta-CD was added at a final concentration of 5 mM and they were further cultured at 5% CO2 and 37 degrees Celsius for 16 hours. Seisaibo Su Sokutei Shiyaku [Reagent for measuring living cell counts]including WST8 was added at 10 microliter/well, and further cultured for 6 hours at 5% CO2 and 37 degrees Celsius. Living cell counts were measured following the protocol.
When the ZS2/SMS2 cell was knocked out with siRNA (SMS2-18) toward a human SMS2, the cell became Me-beta-CD-sensitive, and the cell growth was significantly decrease at final Me-beta-CD concentration of 5 mM (test for a significant difference test P<0.01).
Therefore, it is recognized that due to that SMS2 of the ZS2/SMS2 cell is knocked down with the siRNA, SM on the cell membrane is decreased and the ZS2/SMS2 cell became Me-beta-CD-sensitive. Therefore, it is recognized that the siRNA has an activity of decreasing SM on a cell membrane.
The sequences of the siRNA used as a control (CTR-i) are shown.
Its antisense strand (SEQ ID NO: 20).
In the Example, by using two kinds of siRNAs toward a mouse SMS2 (SMS2-13, SMS2-i11), SMS2 in Hepa1c1c7 cell (ATCC) that is a mouse liver parenchymal cell strain was knocked down and the effect was confirmed.
Into 12-well dishes, 1×104 of the cells were spread. After 24 hours, transformation was carried out using a commercially aviable transformation reagent RNAiMAX (Invitrogen) following the manufacturer's protocol. The final concentration of siRNA was 100 nM. After 48 hours, the cells were solubilized with 0.5 ml of Trizol (Invitrogen), and RNA was extracted following the protocol. Reverse transcription into cDNA was carried out following usual methods by using a commercially available kit Superscript III (Invitrogen). A quantitative PCR was carried out by using the above primers and a commercially available kit (ABI).
In the cases using any of the siRNA sequences, 80% or more knockdown was confirmed (
The sequences of mouse SMS2 specific siRNAs used are shown.
Its antisense strand that is complementary sequence thereto (SEQ ID NO: 22)
Its antisense strand that is complementary sequence thereto (SEQ ID NO: 24).
In order to administer these siRNAs (i3 and i11) which were confirmed for their knocking down activity at cell level to mice, so-called siRNA loaded liposomes were made. The siRNA loaded liposomes were made with reference to the following paper (Nature Biotechnology, Vol. 28, No. 2, p172-176, 2010). They were made by mixing lipids and siRNAs with ethanol injection method. Compared to the paper, lipid composition was slightly modified to be C18PEG:Dlin-KC2-DMA:DPPC:Chol.=2:56.6:3.5:34.3(%). When the particle diameters of the liposomes made were measured, they were about 100 nm in the cases that any of the siRNAs were used, and their zeta potential were almost neutral.
Next, leptin deficient obesity mice were prepared by feeding a normal diet to C57BL/6JHamSlc-ob/ob (Japan SLC, hereinafter, ob/ob mice). The siRNA loaded liposomes made as described above were administered to the ob/ob mice (5 weeks old, female) in 1.25 mg/kg as siRNA amount by injecting into their tail vein (n=3). On the day 3 after administration, the livers were recovered and knockdown rate was measured by the same method as the method of quantifying RNA from cell shown in
siRNA loaded liposomes were administered into 7 weeks old ob/ob mice (male) in 1.25 mg/kg as siRNA amount by injecting into their tail vein (n=3). After administration, the body weight and amount of feeding were measured for 10 days. At 10 days after administration, the liver tissues of the ob/ob mice were recovered after 4 hours fasting. The results of measuring the change of body weight at the time when 10 days has elapsed after administration and average amount of feeding for the 10 days are shown in
Extraction of lipid components from the liver was in accordance with Folch method. 0.5 g of livers were homogenized and placed into microtubes, added 0.1M KCl and filled up to 1.4 mL, stirred well and then transferred into 15 mL Falcon tubes. Then, 6 mL of a mixture solution of CHCl3 and MeOH (2:1) was added and stirred at room temperature for 2 hours. 2 mL of distilled water was added, stirred well and left for 5 minutes. They were centrifuged at 3000 rpm for 10 minutes, and the lower layer (organic layer) was recovered to 720 microliter. After completely removing organic solvents by evaporation, yellow transparent lipid component was obtained. The extracted triglyceride was measured with Triglyceride E-Test Wako (GPO-DAOS method: Wako Pure Chemical Industries, Ltd.) following the attached manual. The results of measuring triglyceride amount in the liver are shown in
From the results, since decrease in liver triglyceride by two different kinds of siRNA sequences toward a mouse SMS2 could be confirmed, it could be confirmed that the effect was not so-called off-target effect but raised by suppression of SMS2 expression. From the above results, suppression of visceral fat accumulation and suppression of fatty liver were confirmed.
In addition, since the ob/ob model is also a model for diabetes, it is understood from the experimental results of Example 8 or the like that the siRNAs of the present invention can be also expected for effect on diabetes.
HepG2 cell that is a human liver parenchymal cell was transformed with a control siRNA sequence CTR-i, SMS2-i104 or SMS2-i109. Knockdown rates of SMS2 after 48 hours were measured by the same method as for
After confirming the knockdown, HepG2 cells were transformed with 3 kinds of siRNAs: CTR-i, SMS2-i104 or SMS2-i109, and after 24 hours, FCS of the medium was changed to 0.5%. After additional 24 hours, oleic acid dissolved in ethanol was mixed with BSA, sonicated with a bath-type sonicator, and the dispersed oleic acid-BSA mixture was added to the medium. The final concentration of oleic acid added was 0.4 mM and that of BSA added was 1%. After additional 24 hours, the medium was removed, the cells was washed with PBS twice, then 10% formalin-PBS solution was added and the cells were fixed for 10 minutes. Then, the formalin solution was removed from the cells, the cells were washed with PBS three times to completely remove PBS. AdipoRed (NileRed) (Lonza) solution that is a fluorescent reagent capable of specifically staining triglyceride was added to a PBS solution at a final concentration of 4% and the PBS solution was added to the cells. After 5 minutes, the cells were observed with a fluorescent microscopy (
Next, the TG amount in these lipid droplets was calculated as luminance by quantifying the amount of fluorescence in the images. BZII Analysis Software of KEYENCE was used as a measuring software. As a result, when the cell was transformed with SMS2-i104 or SMS2-i109, the amount of fluorescence was significantly decreased compared to when transformed with CTR-i. It was thus fond that uptake or conversion into TG of oleic acid or both were inhibited in the human liver parenchymal cells. As such, the effect of suppressing TG accumulation is also caused in the inhibition of caveolin above or the like. In addition, generally speaking, it is known that an effect of suppressing TG accumulation within cells raises an anti-obesity effect. Therefore, also in the analysis at cell level that uses a human siRNA by inhibiting SMS2, an effect of suppressing liver TG accumulation by knockdown it in a mouse liver as well as an effect that suggests suppression of visceral fat accumulation and suppression of fatty liver could be confirmed.
Next, using, for example, the method described in Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751, Yo KIKUCHI, Kagaku to Seibutsu [Chemistry and Biology], 1992, 30, 112, based on the nucleic acid sequences of SMS2 set forth in SEQ ID NO: 79 or 80, ribozyme sequences are designed.
These can be confirmed for the effect of the ribozyme of the present invention by the methods described in Examples 7 and 8 which use mice and HepG2 cell.
In the Example, the effectiveness of antisense nucleic acids designed based on a homologous region (a continuous homologous region of 13 mer or more) between human and mouse SMS2 nucleotide sequences was demonstrated.
The antisense oligonucleotides were designed and made, and knockdown experiments in human HEK 293 cell were carried out with them. “Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents”, Nucleic Acid Research 2010, Vol 38, No. 1 was referred to for designing their sequence structure.
Sequences and forms of 13 mer antisense oligonucleotides used in the Example are shown below. The nucleic acids used were an LNA-containing nucleic acid that is also called as an LNA type Gapmer antisense oligonucleotide. Capital letters mean an LNA (Locked nucleic acid) and lower-case letters mean DNA. LNA is a kind of BNA (Bridged Nucleic Acid). 10 kinds or more of BNAs are known. LNA is an artificial nucleic acid wherein positions 2′ and 4′ of the sugar are crosslinked with —O—CH2—, thereby its conformation is fixed in N-form and which is available from Funakoshi Corporation or the like. In the oligonucleotides, all nucleic acids are linked via phosphorothioate modified backbone. All Cs in LNA portions are methylcytosines. In the examples below, 3 locked nucleic acids (LNAs) are included at 5′ end and 2 locked nucleic acids are included at 3′ end.
Using the 12 kinds of LNA type Gapmer antisense oligonucleotides made above, knockdown experiments in human HEK 293 cell were carried out. The LNA type Gapmer antisense oligonucleotide was added in a naked form at a final concentration of 5 micromolar to the cell culture solution. Quantitative PCR carried out 72 hours after transformation were carried out. G3PDH was used as an endogenous control.
As primer sequences used for measuring the expression amount of human SMS2,
were used, and as primer sequences used for measuring the expression amount of human G3PDH,
were used.
As a result, the suppression of the gene expression of SMS2 was confirmed in any embodiment with the above oligonucleotides compared to cells to which saline was administered (cells to which antisense was not added) and 50% or more suppression in the gene expression of SMS2 was confirmed with SMS2-13-006, SMS2-13-007, SMS2-13-008, SMS2-13-012, SMS2-13-014, SMS2-13-017 and SMS2-13-019 <SEQ ID NOs: 82, 83, 84, 88, 89, 90 and 91, respectively>compared to cells to which saline was administered (cells to which antisense was not added).
While, as described above, the present invention has been illustrated with preferred embodiments of the present invention, it is understood that the scope of the present invention should be interpreted only by the Claims. It is understood that the content of the patents, patent applications and literatures referred herein should be incorporated herein for reference same as if the content thereof itself is specifically described herein. The present application claims priority to U.S. Provisional Application Ser. No. 61/385,377 that was filed on Sep. 22, 2010. It is understood that the entire content of it is to be incorporated as constituting a part of the present specification in a similar manner to as if it is specifically described herein.
The present invention provides a drug for treating and preventing a metabolic disease.
SEQ ID NO: 1: a sequence of the sense strand portion of the duplex portion of SMS2-i6
SEQ ID NO: 2: a sequence of the antisense strand of the duplex portion of SMS2-i6
SEQ ID NO: 3: a sequence of the sense strand portion of the duplex portion of SMS2-i7
SEQ ID NO: 4: a sequence of the antisense strand of the duplex portion of SMS2-i7
SEQ ID NO: 5: a sequence of the sense strand portion of the duplex portion of SMS2-i8
SEQ ID NO: 6: a sequence of the antisense strand of the duplex portion of SMS2-i8
SEQ ID NO: 7: a sequence of the sense strand portion of the duplex portion of SMS2-i104
SEQ ID NO: 8: a sequence of the antisense strand of the duplex portion of SMS2-i104
SEQ ID NO: 9: a sequence of the sense strand of the duplex portion of SMS2-i105
SEQ ID NO: 10: a sequence of the antisense strand of the duplex portion of SMS2-i105
SEQ ID NO: 11: a sequence of the sense strand of the duplex portion of SMS2-i106
SEQ ID NO: 12: a sequence of the antisense portion of the duplex portion of SMS2-i106
SEQ ID NO: 13: a sequence of the sense strand of the duplex portion of SMS2-i107
SEQ ID NO: 14: a sequence of the antisense strand of the duplex portion of SMS2-i107
SEQ ID NO: 15: a sequence of the sense strand of the duplex portion of SMS2-i108
SEQ ID NO: 16: a sequence of the antisense strand of the duplex portion of SMS2-i108
SEQ ID NO: 17: a sequence of the sense strand of the duplex portion of SMS2-i109
SEQ ID NO: 18: a sequence of the antisense strand of the duplex portion of SMS2-i109
SEQ ID NO: 19: a sequence of the sense strand of the duplex portion of CTR-i
SEQ ID NO: 20: a sequence of the antisense strand of the duplex portion of CTR-i
SEQ ID NO: 21: a sequence of the sense strand of the duplex portion of SMS2-i3
SEQ ID NO: 22: a sequence of the antisense strand of the duplex portion of SMS2-i3
SEQ ID NO: 23: a sequence of the sense strand of the duplex portion of SMS2-i11
SEQ ID NO: 24: a sequence of the antisense strand of the duplex portion of SMS2-i11
SEQ ID NO: 25: a Fw primer for adiponectin used in Example 1
SEQ ID NO: 26: a Rv primer for adiponectin used in Example 1
SEQ ID NO: 27: a Fw primer for insulin receptor (IR)
SEQ ID NO: 28: a Rv primer for insulin receptor (IR)
SEQ ID NO: 29: a Fw primer for GLUT4
SEQ ID NO: 30: a Rv primer for GLUT4
SEQ ID NO: 31: a Fw primer for hypoxanthine-guanine phosphoribosyltransferase (HPRT)
SEQ ID NO: 32:a Rv primer for hypoxanthine-guanine phosphoribosyltransferase (HPRT)
SEQ ID NO: 33: a Fw primer for realtime quantitative PCR used for measuring the expression amount of human and mouse SMS2
SEQ ID NO: 34: a Rv primer for realtime quantitative PCR used for measuring the expression amount of human and mouse SMS2
SEQ ID NO: 35: a Fw primer for human G3PDH
SEQ ID NO: 36: a Rv primer for human G3PDH
SEQ ID NO: 37: a Fw primer for mouse G3PDH
SEQ ID NO: 38: a Rv primer for mouse G3PDH
SEQ ID NO: 39: a sequence of the sense strand portion of the duplex portion of SMS2-i1
SEQ ID NO: 40: a sequence of the antisense strand of the duplex portion of SMS2-i1
SEQ ID NO: 41: a sequence of the sense strand portion of the duplex portion of SMS2-i2
SEQ ID NO: 42: a sequence of the antisense strand of the duplex portion of SMS2-i2
SEQ ID NO: 43: a sequence of the sense strand of the duplex portion of SMS2-i4
SEQ ID NO: 44: a sequence of the antisense strand of the duplex portion of SMS2-i4
SEQ ID NO: 45: a sequence of the sense strand of the duplex portion of SMS2-i5
SEQ ID NO: 46: a sequence of the antisense strand of the duplex portion of SMS2-i5
SEQ ID NO: 47: a sequence of the sense strand of SMS2-i6
SEQ ID NO: 48: a sequence of the antisense strand of SMS2-i6
SEQ ID NO: 49: a sequence of the sense strand of SMS2-i7
SEQ ID NO: 50: a sequence of the antisense strand of SMS2-i7
SEQ ID NO: 51: a sequence of the sense strand of SMS2-i8
SEQ ID NO: 52: a sequence of the antisense strand of SMS2-i8
SEQ ID NO: 53: a sequence of the sense strand of SMS2-i104
SEQ ID NO: 54: a sequence of the antisense strand of SMS2-i104
SEQ ID NO: 55: a sequence of the sense strand of SMS2-i105
SEQ ID NO: 56: a sequence of the antisense strand of SMS2-i105
SEQ ID NO: 57: a sequence of the sense strand of SMS2-i106
SEQ ID NO: 58: a sequence of the antisense strand of SMS2-i106
SEQ ID NO: 59: a sequence of the sense strand of SMS2-i107
SEQ ID NO: 60: a sequence of the antisense strand of SMS2-i107
SEQ ID NO: 61: a sequence of the sense strand of SMS2-i108
SEQ ID NO: 62: a sequence of the antisense strand of SMS2-i108
SEQ ID NO: 63: a sequence of the sense strand of SMS2-i109
SEQ ID NO: 64: a sequence of the antisense strand of SMS2-i109
SEQ ID NO: 65: a sequence of the sense strand of CTR-i
SEQ ID NO: 66: a sequence of the antisense strand of CTR-i
SEQ ID NO: 67: a sequence of the sense strand of SMS2-i3
SEQ ID NO: 68: a sequence of the antisense strand of SMS2-i3
SEQ ID NO: 69: a sequence of the sense strand of SMS2-i11
SEQ ID NO: 70: a sequence of the antisense strand of SMS2-i11
SEQ ID NO: 71: a sequence of the sense strand of SMS2-i1
SEQ ID NO: 72: a sequence of the antisense strand of SMS2-i1
SEQ ID NO: 73: a sequence of the sense strand of SMS2-i2
SEQ ID NO: 74: a sequence of the antisense strand of SMS2-i2
SEQ ID NO: 75: a sequence of the sense strand of SMS2-i4
SEQ ID NO: 76: a sequence of the antisense strand of SMS2-i4
SEQ ID NO: 77: a sequence of the sense strand of SMS2-i5
SEQ ID NO: 78: a sequence of the antisense strand of SMS2-i5
SEQ ID NO: 79: a nucleic acid sequence of human SMS2
SEQ ID NO: 80: a nucleic acid sequence of mouse SMS2
SEQ ID NO: 81: a sequence of an SMS2-13-003 antisense nucleic acid used in Example 10
SEQ ID NO: 82: a sequence of an SMS2-13-006 antisense nucleic acid used in Example 10
SEQ ID NO: 83: a sequence of an SMS2-13-007 antisense nucleic acid used in Example 10
SEQ ID NO: 84: a sequence of an SMS2-13-008 antisense nucleic acid used in Example 10
SEQ ID NO: 85: a sequence of an SMS2-13-009 antisense nucleic acid used in Example 10
SEQ ID NO: 86: a sequence of an SMS2-13-010 antisense nucleic acid used in Example 10
SEQ ID NO: 87: a sequence of an SMS2-13-011 antisense nucleic acid used in Example 10
SEQ ID NO: 88: a sequence of an SMS2-13-012 antisense nucleic acid used in Example 10
SEQ ID NO: 89: a sequence of an SMS2-13-014 antisense nucleic acid used in Example 10
SEQ ID NO: 90: a sequence of an SMS2-13-017 antisense nucleic acid used in Example 10
SEQ ID NO: 91: a sequence of an SMS2-13-019 antisense nucleic acid used in Example 10
SEQ ID NO: 92: a sequence of an SMS2-13-020 antisense nucleic acid used in Example 10
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/005325 | 9/21/2011 | WO | 00 | 5/20/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61385377 | Sep 2010 | US |
