JP2014521600A - MicroRNA-31 compositions and methods for use in autoimmune diseases - Google Patents

Abstract

  The present disclosure provides a method of treating systemic lupus erythematosus and other autoimmune conditions.

Description

  Autoimmune conditions are associated with high morbidity and, in some cases, high mortality. The symptoms of an autoimmune condition affect the ability to work and may otherwise affect the patient's quality of life.

  Systemic lupus erythematosus (SLE) is a multifactorial autoimmune condition characterized by chronic activation of the immune system and multiple immunological phenotypes (Fairhurst et al., 2006; Anders et al. , 2009). Many of the molecular abnormalities identified explain specific established cell and cytokine abnormalities in lupus. T cells from lupus patients display some signaling abnormalities (Nambiar et al., 2004; Fujii et al., 2006; Kong et al., 2003; Multon et al., 2011).

  Interleukin-2 (IL-2) is a multifunctional cytokine produced primarily by T cells and is essential for T cell activation, proliferation and contraction (Lieberman et al., 2010). It has been reported that IL-2 production is reduced in SLE T cells and that transcriptional regulators involved in transcription or repression of IL-2 production are imbalanced in SLE T cells (Lieberman et al. , 2010; Tenblock et al., 2004; Katsari et al., 2005; Jung et al., 2005; Herndon et al., 2002; Solomou et al., 2001).

  There is a great need for improved methods and compositions used for diagnosis, management and treatment of autoimmune conditions. The present disclosure provides methods and compositions applicable to the treatment, evaluation, diagnosis and prognosis of subjects with autoimmune conditions such as systemic lupus erythematosus. In certain embodiments, the methods provided herein are based on a correlation between microRNA-31 expression and IL-2 expression in a subject.

  In a first aspect, the present disclosure provides a method of increasing IL-2 expression in a subject having an autoimmune condition and requiring increased expression of interleukin-2 (IL-2). Such methods include administering to the subject an effective amount of a composition that increases microRNA-31 expression or decreases RhoA expression. In certain embodiments, the subject in need thereof is a subject with systemic lupus erythematosus. In other embodiments, the subject in need thereof is a mechanism of action, etiology, correlation with IL-2 dysregulation, or a correlation with T cell dysregulation, particularly regulatory T cell dysregulation. A subject with another autoimmune condition that shares one or more characteristics with SLE, such as sharing.

  In a second aspect, the present disclosure provides a method of increasing the expression of interleukin-2 (IL-2) in a T cell of a subject having an autoimmune condition. Such methods include contacting the T cells with an effective amount of a composition that increases microRNA-31 expression or decreases RhoA expression. Contacting the T cells may be in vivo, eg, by administering the composition to a subject. Alternatively, contacting the T cells may be in vitro, for example by adding the composition to a cell culture medium. In certain embodiments, the subject having an autoimmune condition has systemic lupus erythematosus. In other embodiments, the subject in need thereof is a mechanism of action, etiology, correlation with IL-2 dysregulation, or a correlation with T cell dysregulation, particularly regulatory T cell dysregulation. A subject with another autoimmune condition that shares one or more characteristics with SLE, such as sharing.

  In a third aspect, the disclosure provides a method of treating an autoimmune condition, wherein the effective amount of increasing microRNA-31 expression or reducing RhoA expression to treat the autoimmune condition. There is provided a method comprising administering the composition to a subject in need of treatment of an autoimmune condition. In certain embodiments, the autoimmune condition is systemic lupus erythematosus. In other embodiments, the condition is a mechanism of action, etiology, correlation with IL-2 dysregulation, or sharing of correlation with T cell dysregulation, particularly dysregulation of regulatory T cells, etc. Another autoimmune condition that shares one or more characteristics with SLE.

  In a fourth aspect, the present disclosure provides a method of reducing the expression of RhoA in a subject having an autoimmune condition. Such methods include administering to the subject an effective amount of a composition that increases the expression of microRNA-31. In certain embodiments, the autoimmune condition is systemic lupus erythematosus. In other embodiments, the condition is a SLE, such as a mechanism of action, etiology, correlation with IL-2 dysregulation, or sharing of correlation with T cell dysregulation, particularly dysregulation of regulatory T cells. Another autoimmune condition that shares one or more characteristics with.

  In a fifth aspect, the present disclosure provides a method of reducing RhoA expression in T cells of a subject having an autoimmune condition. Such a method involves contacting the T cell with an effective amount of a composition that increases expression of microRNA-31. Contacting the T cells may be in vivo, eg, by administering the composition to a subject. Alternatively, contacting the T cells may be in vitro, for example by adding the composition to a cell culture medium. In certain embodiments, the subject having an autoimmune condition has systemic lupus erythematosus. In other embodiments, the subject in need thereof is a mechanism of action, etiology, correlation with IL-2 dysregulation, or a correlation with T cell dysregulation, particularly regulatory T cell dysregulation. A subject with another autoimmune condition that shares one or more characteristics with SLE, such as sharing.

  The present disclosure contemplates that the following embodiments may be applied to any of the foregoing aspects of the present disclosure and / or to any of the following embodiments of the present disclosure. In certain embodiments, the subject is a human subject. In other embodiments, the subject is a non-human subject. In certain embodiments, the subject has systemic lupus erythematosus and has symptoms of disease flare, eg, symptoms of disease flare prior to administration of the composition. In other embodiments, the subject has systemic lupus erythematosus and does not exhibit symptoms of disease flare prior to administration of the composition. In certain embodiments, the symptoms of SLE include lupus nephritis. In certain embodiments, the subject has another autoimmune condition, such as a mechanism of action, etiology, correlation with IL-2 dysregulation, or T cell dysregulation, particularly dysregulation of regulatory T cells. Sharing one or more features with the SLE, such as In certain embodiments, the other autoimmune condition is rheumatoid arthritis (RA) or type I diabetes.

  In certain embodiments, the T cell contacted or affected by the composition is an activated T cell, eg, an activated T cell in vitro or in vivo.

  In certain embodiments of any of the foregoing, the composition comprises a nucleic acid comprising a nucleotide sequence that increases the expression of microRNA-31. In certain embodiments, the composition comprises a nucleic acid comprising a nucleotide sequence that reduces RhoA expression. In other embodiments, the composition comprises a polypeptide or small organic molecule that increases the expression of microRNA-31 and / or decreases the expression of RhoA. This disclosure contemplates embodiments in which expression of a transcript and / or protein is increased or decreased by increasing or decreasing expression.

  In certain embodiments of any of the foregoing, the composition comprises microRNA-31 added externally. Such microRNA-31 may correspond to natural microRNA-31 or may include synthetic nucleic acids. In certain embodiments, the composition comprises a short interfering nucleic acid (siNA), such as a microRNA, siRNA or antisense oligonucleotide. In certain embodiments, the composition comprises siNA that can hybridize to RhoA.

  In certain embodiments of any of the foregoing, decreasing RhoA expression comprises decreasing RhoA transcript expression. In other embodiments, reducing RhoA expression comprises reducing RhoA protein expression. In certain embodiments, increasing IL-2 expression comprises increasing IL-2 transcript expression. In other embodiments, increasing IL-2 expression comprises increasing IL-2 protein expression.

  In certain embodiments of any of the foregoing, the method further comprises one or more assay steps. For example, in certain embodiments, the method comprises assaying IL-2 expression in a sample taken from the subject at some time after administration of the composition. In other embodiments, the method further comprises assaying RhoA expression in a sample taken from the subject at some time after administration of the composition. Such samples may be collected by or at the same facility that administers the therapeutic composition, or such samples may be collected by different providers and / or at different facilities.

  In certain embodiments, the sample used for the assay step comprises a blood sample. For example, the assay may be performed using a whole blood sample, using lymphocytes separated from a blood sample, or using serum or some other component of blood. The sample is prepared based on the particular assay used. In other embodiments, the sample used in the assay step comprises a bone marrow sample. Regardless of the sample used for the assay step, in certain embodiments, assaying for expression of IL-2 comprises assaying expression of IL-2 transcript, such as in T cells. In other embodiments, assaying for expression of IL-2 comprises assaying for expression of IL-2 protein in the sample. Similarly, regardless of the sample used for the assay step, in certain embodiments, assaying for RhoA expression comprises assaying RhoA transcript expression, such as in T cells. In other embodiments, assaying RhoA expression comprises assaying RhoA protein expression in a sample.

  In a sixth aspect, the present disclosure provides a method. The methods assay for the presence, absence or amount of a biomarker selected from microRNA-31, RhoA or IL-2 in a subject having an autoimmune condition, such as systemic lupus erythematosus, to which a compound has been administered. And determining whether to adjust the dosing regimen or dosing regimen of the compound subsequently administered to the subject based on the presence, absence or amount of the biomarker assayed. In certain embodiments, the autoimmune condition is systemic lupus erythematosus. In other embodiments, the autoimmune condition is a mechanism of action, etiology, a correlation with IL-2 dysregulation, or a shared correlation with T cell dysregulation, particularly regulatory T cell dysregulation, etc. Share one or more features with the SLE.

  In certain embodiments, the subject is a human subject. In other embodiments, the subject is a non-human subject. In certain embodiments, the subject has systemic lupus erythematosus and exhibits symptoms of disease flare. In other embodiments, the subject has systemic lupus erythematosus and does not exhibit symptoms of disease flare. In certain embodiments, the symptoms of SLE include lupus nephritis. In certain embodiments, the subject has another autoimmune condition, such as a mechanism of action, etiology, correlation with IL-2 dysregulation, or T cell dysregulation, particularly dysregulation of regulatory T cells. Sharing one or more features with the SLE, such as In certain embodiments, the other autoimmune condition is rheumatoid arthritis (RA) or type I diabetes.

  In certain embodiments, the sample used for the assay step comprises a blood sample. For example, the assay may be performed using a whole blood sample, using lymphocytes separated from a blood sample, or using serum or some other component of blood. The sample is prepared based on the particular assay used. In other embodiments, the sample used comprises a bone marrow sample. Regardless of the sample used for the assay step, in certain embodiments, assaying for expression of IL-2 comprises assaying expression of IL-2 transcript, such as in T cells. In other embodiments, assaying for expression of IL-2 comprises assaying for expression of IL-2 protein in the sample. Similarly, regardless of the sample used for the assay step, in certain embodiments, assaying for RhoA expression comprises assaying RhoA transcript expression, such as in T cells. In other embodiments, assaying RhoA expression comprises assaying RhoA protein expression in a sample.

  In certain embodiments, the compound comprises a steroid or an immunosuppressive drug. In other embodiments, the compound comprises a composition of the present disclosure. In certain embodiments, the compound comprises a composition that increases expression of microRNA-31 or decreases expression of RhoA. In certain embodiments, the compound comprises IL-2.

  In certain embodiments, the biomarker examined is microRNA-31. In other embodiments, the biomarker examined is RhoA. In other embodiments, the biomarker examined is IL-2. In still other embodiments, one or more combinations of the aforementioned biomarkers are examined. Biomarkers may be examined at the transcript level or at the protein level.

  In a seventh aspect, the present disclosure provides a method of diagnosing an autoimmune condition such as systemic lupus erythematosus. The method includes taking a sample from a subject suspected of being an autoimmune condition, such as systemic lupus erythematosus; and assaying for expression of microRNA-31 or RhoA in the sample.

  In certain embodiments, the subject is a human subject. In other embodiments, the subject is a non-human subject. In certain embodiments, the subject has systemic lupus erythematosus and exhibits symptoms of disease flare. In other embodiments, the subject has systemic lupus erythematosus and does not exhibit symptoms of disease flare. In certain embodiments, the symptoms of SLE include lupus nephritis. In certain embodiments, the subject has another autoimmune condition, such as a mechanism of action, etiology, correlation with IL-2 dysregulation, or T cell dysregulation, particularly dysregulation of regulatory T cells. Sharing one or more features with the SLE, such as In certain embodiments, the other autoimmune condition is rheumatoid arthritis (RA) or type I diabetes.

  In certain embodiments, the sample used for the assay step comprises a blood sample. For example, the assay may be performed using a whole blood sample, using lymphocytes separated from a blood sample, or using serum or some other component of blood. The sample is prepared based on the particular assay used. In other embodiments, the sample used comprises a bone marrow sample. Regardless of the sample used for the assay step, in certain embodiments, assaying for expression of IL-2 comprises assaying expression of IL-2 transcript, such as in T cells. In other embodiments, assaying for expression of IL-2 comprises assaying for expression of IL-2 protein in the sample. Similarly, regardless of the sample used for the assay step, in certain embodiments, assaying for RhoA expression comprises assaying RhoA transcript expression, such as in T cells. In other embodiments, assaying RhoA expression comprises assaying RhoA protein expression in a sample.

  In an eighth aspect, the present disclosure provides a method of monitoring treatment of an autoimmune condition such as systemic lupus erythematosus. The method detects microRNA-31 or RhoA expression in a sample from a subject undergoing treatment for an autoimmune condition, such as systemic lupus erythematosus; and microRNA-31 or RhoA expression, Comparing to expression in a sample from the same subject taken prior to treatment or at an early time point during the treatment period. In such methods, treatment from the increase in microRNA-31 or decrease in RhoA in a sample taken at a later time point during the treatment period relative to a sample taken before treatment or at an earlier time point during the treatment period. The effectiveness of the treatment is demonstrated, thereby monitoring the treatment.

  In certain embodiments, the subject is a human subject. In other embodiments, the subject is a non-human subject. In certain embodiments, the subject has systemic lupus erythematosus and exhibits symptoms of disease flare. In other embodiments, the subject has systemic lupus erythematosus and does not exhibit symptoms of disease flare. In certain embodiments, the symptoms of SLE include lupus nephritis. In certain embodiments, the subject has another autoimmune condition, such as a mechanism of action, etiology, correlation with IL-2 dysregulation, or T cell dysregulation, particularly dysregulation of regulatory T cells. Sharing one or more features with the SLE, such as In certain embodiments, the other autoimmune condition is rheumatoid arthritis (RA) or type I diabetes.

  In certain embodiments, the sample used for the assay step comprises a blood sample. For example, the assay may be performed using a whole blood sample, using lymphocytes separated from a blood sample, or using serum or some other component of blood. The sample is prepared based on the particular assay used. In other embodiments, the sample used comprises a bone marrow sample. Regardless of the sample used for the assay step, in certain embodiments, assaying for expression of IL-2 comprises assaying expression of IL-2 transcript, such as in T cells. In other embodiments, assaying for expression of IL-2 comprises assaying for expression of IL-2 protein in the sample. Similarly, regardless of the sample used for the assay step, in certain embodiments, assaying for RhoA expression comprises assaying RhoA transcript expression, such as in T cells. In other embodiments, assaying RhoA expression comprises assaying RhoA protein expression in a sample.

  In a ninth aspect, the present disclosure provides a method of treating a subject having an autoimmune condition such as systemic lupus erythematosus. The method comprises comparing the expression of microRNA-31 or RhoA from a sample taken from a subject prior to the start of a specific treatment of an autoimmune condition to a standard range reflecting expression in a sample from a healthy subject. In addition, the expression of microRNA-31 is below the standard range or the expression of RhoA is above the standard range, indicating, for example, that it is sensitive to the treatment of systemic lupus erythematosus. The method may include siNA hybridizing to microRNA-31, RhoA or IL-2 protein, or another composition of the present disclosure, if the subject is confirmed to be susceptible to treatment of systemic lupus erythematosus. Treating a subject with an effective amount of the composition; detecting expression of microRNA-31 or RhoA in the sample after treatment from the subject; and expression of microRNA-31 or RhoA in the sample after treatment Is further compared to expression in a sample taken prior to the start of a particular treatment.

  In certain embodiments, the subject is a human subject. In other embodiments, the subject is a non-human subject. In certain embodiments, the subject has systemic lupus erythematosus and exhibits symptoms of disease flare. In other embodiments, the subject has systemic lupus erythematosus and does not exhibit symptoms of disease flare. In certain embodiments, the symptoms of SLE include lupus nephritis. In certain embodiments, the subject has another autoimmune condition, such as a mechanism of action, etiology, correlation with IL-2 dysregulation, or T cell dysregulation, particularly dysregulation of regulatory T cells. Sharing one or more features with the SLE, such as In certain embodiments, the other autoimmune condition is rheumatoid arthritis (RA) or type I diabetes.

  In certain embodiments, the sample used for the assay step comprises a blood sample. For example, the assay may be performed using a whole blood sample, using lymphocytes separated from a blood sample, or using serum or some other component of blood. The sample is prepared based on the particular assay used. In other embodiments, the sample used comprises a bone marrow sample. Regardless of the sample used for the assay step, in certain embodiments, assaying for expression of IL-2 comprises assaying expression of IL-2 transcript, such as in T cells. In other embodiments, assaying for expression of IL-2 comprises assaying for expression of IL-2 protein in the sample. Similarly, regardless of the sample used for the assay step, in certain embodiments, assaying for RhoA expression comprises assaying RhoA transcript expression, such as in T cells. In other embodiments, assaying RhoA expression comprises assaying RhoA protein expression in a sample.

  The disclosure may be combined with any one or more of the foregoing aspects and embodiments and / or combined with any of the embodiments described in the detailed description and examples. I think I can do it.

FIG. 5 shows that miR-31 expression is decreased in lupus patients compared to normal control (NC) subjects. (A) Expression of miR-31 in T cells, B cells and monocytes was determined by Taqman quantitative PCR. Results are shown as mean ± standard error of SEM mean, corrected for expression of B cells from 3 healthy donors. (B) Independent validation by Taqman quantitative PCR on the expression of miR-31 in T cells from 32 lupus patients and 11 normal controls is shown (P <0.0001). The above results are shown as mean ± SEM. P values were determined by Mann-Whitney U test. The expression of miR-31 is shown. (A) Taqman quantitative PCR analysis of expression for miR-31 in activated primary T cells 24 hours after transfection of miR-31 mimic or control (Ctrl). (B) Taqman quantitative PCR analysis of miR-31 expression in activated primary T cells 24 hours after transfection of antagomir-31 or control. It is an experimental result which shows that miR-31 controls the expression of IL-2 and the activity of IL-2 promoter. (A) Primary T cells were stimulated with PMA and ionomycin, and miR-31 levels were measured at various times by Taqman quantitative PCR. RNU48 levels were used for expression correction. Each bar is the average of 3 independent experiments. (B) IL-2 in activated primary T cells 24 hours after transfection of miR-31 mimic or control mimic (Ctrl) with antagomir-31 or antagomir control (Ctrl) It is the quantitative PCR analysis regarding expression. The histogram shows the fold change in mRNA expression relative to the control corrected with the housekeeping gene RPL13A. (C) Detection of IL-2 levels in culture supernatants of activated primary T cells transfected with miR-31 mimics or control mimics and Antagomir-31 or Antagomir controls did. B and C values are the mean ± SEM of 4 representative healthy donors. (D) Linear correlation analysis of IL-2 and miR-31 expression in activated T cells of lupus patients (n = 15). (E) Jurkat cells were co-transfected with miR-31 mimic or control mimic and IL-2 promoter-luc reporter plasmid. PGL3-basic-luc was used for transfection correction. Cells were unstimulated or stimulated with PMA and ionomycin for 24 hours and relative promoter activity was measured by a dual luciferase assay. Each bar is the average of 3 independent experiments. (F) miR-31 enhanced the luciferase activity of the IL-2 promoter in a dose-dependent manner. The results are the results of three representative experiments. *** P <0.01; *** P <0.001. The experimental results reveal that miR-31 is a negative regulator of RhoA expression. (A) The expression level of RhoA mRNA in activated T lymphocytes transfected with miR-31 mimic or control mimic was analyzed by RT-PCR. Data are from three independent experiments. Results are shown as mean ± SEM. (B) Immunoblot analysis of RhoA protein expression in T cells (n = 3) 48 hours after transfection of miR-31 mimic or control mimic. (C) RhoA expression in T cells from SLE patients (n = 32) and normal controls (n = 11) was measured by RT-PCR. The above results are shown as mean ± SEM. P values were determined by Mann-Whitney U test. (D) Linear correlation analysis of RhoA and miR-31 expression in primary T cells of lupus patients (n = 32). The effect | action of siRNA with respect to RhoA is shown. (A) Quantitative PCR analysis of RhoA expression in primary T cells after transfection of siRNA-1, siRNA-2 or control. (B) Immunoblot analysis of RhoA expression in primary T cells after transfection of two siRNAs against RhoA (siR-1 and siR-2) or control. The above results are shown as mean ± SEM. P values were determined by Mann-Whitney U test. It is an experimental result which shows that knockdown of RhoA by siRNA controls IL-2 production and the activity of IL-2 promoter. (A) Quantitative PCR analysis for expression of IL-2 in activated primary T cells after 24 hours transfection of miR-31 mimic or control mimic and two siRNAs. (B) IL-2 levels in culture supernatants of activated primary T cells transfected with miR-31 mimic, control mimic or two siRNAs were detected by ELISA. A and B values are the mean ± SEM of 3 representative healthy donors. (C) Dual Jurkat cells co-transfected with a pGL3-basic-luc reporter vector containing IL-2 promoter and RhoA siRNA, miR-31 mimic or control mimic Luciferase assay. Data are shown as relative luciferase activity of cells transfected with miR-31 and siRNA relative to a control of 3 independent experiments. It is an experimental result which clarifies the role of miR-31, RhoA, and IL-2 in the activated T cell derived from a SLE patient. (A) Expression of miR-31 in activated T cells of 15 SLE patients and 10 normal controls (NC) was detected by Taqman quantitative PCR. (B) RhoA expression was quantified by RT-PCR of the above samples. (C) IL-2 protein levels in the supernatants of activated T cells from 12 SLE patients and 10 normal controls were measured by ELISA. (D) ELISA analysis of IL-2 expression in culture supernatants of activated T cells from SLE patients (n = 3) after transfection of miR-31 mimic or control mimic. The above results are shown as mean ± SEM. P values were determined by Mann-Whitney U test.

  Table 1 summarizes the clinical characteristics of patients used in the study shown in FIG. 1B.

  Table 2 summarizes the clinical characteristics of patients used in the study shown in FIG.

(I) Overview Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disorder characterized by chronic activation of the immune system and multiple immunological phenotypes. Part of this disclosure is based on the identification of autoimmune disease mechanisms involving signaling through microRNA-31, RhoA and IL-2.

  MicroRNAs (miRNAs) are a class of single-stranded non-coding RNAs that serve as important post-transcriptional regulators of gene expression (Denli et al., 2004; Gregory et al., 2004). In animals, miRNA usually forms incomplete base pairs with the 3 'untranslated region (UTR) of the target gene and regulates target gene expression by translational inhibition or mRNA degradation (Ambros. 2004).

  Without being bound by theory, a portion of this disclosure is based on the examples described below. The examples reflect studies in which low expression of endogenous miR-31 levels was identified in SLE T cells. It was also found that miR-31 is a positive regulator of IL-2 production in activated T cells, and that RhoA expression is inversely correlated with miR-31 expression. Such control appears to occur through control of IL-2 promoter activity. Furthermore, RhoA expression was very high in lupus T cells compared to healthy volunteers.

(Ii) Definitions Before continuing with the more detailed description of the present disclosure, it is to be understood that the present disclosure is not limited to a particular composition or process step and may therefore vary. As used in this specification and the appended claims, the singular forms “a” and “the”, unless the context clearly indicates otherwise, It should be noted that it includes multiple meanings.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, the Consistency Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed. , 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed. , 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press is a common dictionary of terms used in this disclosure for those skilled in the art.

  As used herein, “and / or” indicates that each of the two specified features or elements may be considered to be individually disclosed features or elements, including or not including the other. It is convenient to keep it. For example, “A and / or B” individually disclosed (i) A, (ii) B, and (iii) A and B, respectively, as if each was individually described herein. Can be considered.

(Iii) Compositions This section of the specification describes compositions for use in any of the claimed methods. This disclosure is described based on any combination of functional and / or structural features, and any such composition of this disclosure is intended to be used in any of the methods described herein. Yes.

  Part of this disclosure is based on positive and negative correlations and mechanistic associations between microRNA-31, RhoA, and IL-2. In light of this, the present disclosure provides compositions that are useful in vivo and in vitro in various methods, such as therapeutic and diagnostic methods. Compositions of the present disclosure include compositions that contain nucleic acids, polypeptides or small organic molecules as active ingredients. Further, in certain embodiments, the composition of the present disclosure is a cellular composition, such as a composition that incorporates a cell to be expressed and contains a nucleic acid or polypeptide.

  The term “composition of the present disclosure” is used to refer to any such composition useful in the present method, such as useful in a diagnostic or therapeutic method. Any composition of the present disclosure may be used in any of the methods described herein. Furthermore, any of the compositions of the present disclosure can be described using any of the structural and / or functional features described herein.

Nucleic acid “Nucleic acid” as used herein generally refers to molecules (single stranded, two or more strands) of these derivatives or analogs containing DNA, RNA or nucleobases. Nucleobases are naturally found, for example, in DNA (eg, adenine “A”, guanine “G”, thymine “T” or cytosine “C”) or RNA (eg, A, G, uracil “U” or C). Of purine or pyrimidine bases. The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide”, each as a subgenus of “nucleic acid”. The term nucleic acid described herein includes, but is not limited to, microRNA (whether natural or synthetic), siNA, and antisense RNA. The nucleic acid is at least at most or approximately 3 nucleotides long, 4 nucleotides long, 5 nucleotides long, 6 nucleotides long, 7 nucleotides long, 8 nucleotides long, 9 nucleotides long, 10 nucleotides long, 11 nucleotides long, 12 nucleotides long 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 Nucleotide length, 26 nucleotide length, 27 nucleotide length, 28 nucleotide length, 29 nucleotide length, 30 nucleotide length, 31 nucleotide length, 32 nucleotide length, 33 nucleotide length, 34 nucleotide length 35 nucleotide length, 36 nucleotide length, 37 nucleotide length, 38 nucleotide length, 39 nucleotide length, 40 nucleotide length, 41 nucleotide length, 42 nucleotide length, 43 nucleotide length, 44 nucleotide length, 45 nucleotide length, 46 nucleotide length, 47 nucleotide Length, 48 nucleotide length, 49 nucleotide length, 50 nucleotide length, 51 nucleotide length, 52 nucleotide length, 53 nucleotide length, 54 nucleotide length, 55 nucleotide length, 56 nucleotide length, 57 nucleotide length, 58 nucleotide length, 59 nucleotide length, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides 140 nucleotide length, 150 nucleotide length, 160 nucleotide length, 170 nucleotide length, 180 nucleotide length, 190 nucleotide length, 200 nucleotide length, 250 nucleotide length, 300 nucleotide length, 350 nucleotide length, 400 nucleotide length, 450 nucleotide length, 500 Nucleotide length, 550 nucleotide length, 600 nucleotide length, 650 nucleotide length, 700 nucleotide length, 750 nucleotide length, 800 nucleotide length, 850 nucleotide length, 900 nucleotide length, 950 nucleotide length, or 1000 nucleotide length, or any derivable therefrom The length of the range may be sufficient. In certain embodiments, these lengths correspond only to the length of the functional portion of the nucleic acid portion and do not include the length of any vector sequence or any conjugate portion. Such lengths cover the length of processed miRNA or siNA, miRNA molecule or siNA molecule, precursor miRNA or siNA, vectors containing miRNA or siNA, control nucleic acids, and other molecules, probes and primers. It will be appreciated that in certain embodiments, this molecule does not comprise a natural molecule, regardless of the length of the nucleic acid. For example, an externally provided molecule differs in, for example, sequence, length, modification, strand number, or some other property compared to an endogenously occurring molecule. In many embodiments, human miRNAs are 19-24 nucleotides in length, whereas miRNA precursors are generally 62-110 nucleotides. A nucleic acid described herein may have a region that is the same or complementary to another nucleic acid. It is intended that a complementary or identical region may be at least 5 consecutive residues, but a complementary or identical region specifically includes at least at most that region, Or approximately 5 contiguous nucleotides, 6 contiguous nucleotides, 7 contiguous nucleotides, 8 contiguous nucleotides, 9 contiguous nucleotides, 10 contiguous nucleotides, 11 contiguous nucleotides, 12 contiguous nucleotides, 13 contiguous nucleotides, 14 contiguous nucleotides, 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides Contiguous nucleotides, 22 contiguous nucleotides, 2 Contiguous nucleotides, 24 contiguous nucleotides, 25 contiguous nucleotides, 26 contiguous nucleotides, 27 contiguous nucleotides, 28 contiguous nucleotides, 29 contiguous nucleotides, 30 contiguous nucleotides, 31 contiguous Nucleotides, 32 contiguous nucleotides, 33 contiguous nucleotides, 34 contiguous nucleotides, 35 contiguous nucleotides, 36 contiguous nucleotides, 37 contiguous nucleotides, 38 contiguous nucleotides, 39 contiguous nucleotides 40 contiguous nucleotides, 41 contiguous nucleotides, 42 contiguous nucleotides, 43 contiguous nucleotides, 44 contiguous nucleotides, 45 contiguous nucleotides, 46 contiguous nucleotides 47 contiguous nucleotides, 48 contiguous nucleotides, 49 contiguous nucleotides, 50 contiguous nucleotides, 51 contiguous nucleotides, 52 contiguous nucleotides, 53 contiguous nucleotides, 54 contiguous nucleotides, 55 Contiguous nucleotides, 56 contiguous nucleotides, 57 contiguous nucleotides, 58 contiguous nucleotides, 59 contiguous nucleotides, 60 contiguous nucleotides, 70 contiguous nucleotides, 80 contiguous nucleotides, 90 contiguous nucleotides Nucleotides, 100 contiguous nucleotides, 110 contiguous nucleotides, 120 contiguous nucleotides, 130 contiguous nucleotides, 140 contiguous nucleotides, 150 contiguous nucleotides, 160 contiguous nucleotides Contiguous nucleotides, 170 contiguous nucleotides, 180 contiguous nucleotides, 190 contiguous nucleotides, 200 contiguous nucleotides, 250 contiguous nucleotides, 300 contiguous nucleotides, 350 contiguous nucleotides, 400 contiguous nucleotides Nucleotides, 450 contiguous nucleotides, 500 contiguous nucleotides, 550 contiguous nucleotides, 600 contiguous nucleotides, 650 contiguous nucleotides, 700 contiguous nucleotides, 750 contiguous nucleotides, 800 contiguous nucleotides, Contemplates 850 contiguous nucleotides, 900 contiguous nucleotides, 950 contiguous nucleotides, or 1000 contiguous nucleotides. It will be appreciated that in certain embodiments, regardless of the length of the nucleic acid molecule used, the nucleic acid molecule is a fragment and corresponds to a shorter than the full-length sequence of the natural molecule.

  The nucleic acid may further include a vector such as, but not limited to, a plasmid or a virus. The vector may encode a nucleic acid molecule before processing or a mature molecule after processing (eg, pre-processing or post-processing miRNA or siRNA).

  As described herein, “synthetic nucleic acid” means a nucleic acid that does not have the chemical structure or sequence of a natural nucleic acid. Thus, the term “synthetic miRNA” refers to a “synthetic nucleic acid” that is not isolated from a cell but artificially produced, but can function in a cell or under physiological conditions in some cases. It will be understood.

  As used herein, “stringent conditions” or “high stringency” allows for hybridization between or within one or more nucleic acid strands, including complementary sequences, of random sequences. This is a condition where hybridization does not occur. Under stringent conditions, there is very little mismatch between the nucleic acid and the target strand. Such conditions are well known and are preferred for applications requiring high selectivity. Non-limiting applications include isolation of nucleic acids such as genes or nucleic acid segments thereof, or detection of at least one specific mRNA transcript or nucleic acid segment thereof, and the like.

  Stringent conditions may include low salt and / or high temperature conditions obtained, for example, with about 0.02 M to about 0.5 M NaCl at a temperature of about 42 ° C. to about 70 ° C. The temperature and ionic strength of the desired stringency depends on the length of the specific nucleic acid, the length of the target sequence and the nucleobase content, the charge composition of the nucleic acid, and the formamide, tetramethylammonium chloride or hybridization mixture It will be understood that it is determined in part by the presence or concentration of other solvents.

  These ranges, compositions and conditions of hybridization are described merely as non-limiting examples, and the desired stringency of a particular hybridization reaction is often one or more positive controls or It will be understood that this is determined empirically compared to the negative control. Depending on the envisaged use, various hybridization conditions can be employed to achieve varying degrees of nucleic acid selectivity relative to the target sequence. In one non-limiting example, identification or isolation of related target nucleic acids that do not hybridize to nucleic acids under stringent conditions can be achieved by hybridization at low temperature and / or high ionic strength. Such conditions are referred to as “roast stringency” or “roast stringency conditions”, and non-limiting examples of roast stringency include a temperature range of about 20 ° C. to about 50 ° C., about 0.15 M to about Examples include hybridization performed with 0.9 M NaCl. Low stringency conditions or high stringency conditions may be further modified to suit individual applications.

siNA
siNA is a class of nucleic acid molecules that can mediate sequence-specific RNAi, such as short interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), short interference. Refers to oligonucleotides, short interfering nucleic acids, short interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. Furthermore, as used herein, the term RNAi is intended to be equivalent to other terms used to describe sequence-specific RNA interference, eg post-transcriptional gene silencing, ie epigenetics. doing. For example, when siNA molecules are used, the gene can be epigenetically suppressed at one or both of the post-transcriptional level and the pre-transcriptional level. In one non-limiting example, epigenetic control of gene expression by siNA molecules of this technique can be triggered by siNA mediating modification of chromatin structure to alter gene expression. For this reason, siNA can be used to therapeutically modulate polypeptide or protein levels. Such control may be direct or indirect, for example, by inhibiting the expression of a protein that is itself a repressor of the protein of interest.

  The siNA may be a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target nucleic acid molecule or part thereof. The sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. siNA can be constructed from two separate oligonucleotides, where one strand is the sense strand, the other is the antisense strand, and the antisense and sense strands are self-complementary. In some embodiments, each strand comprises a nucleotide sequence that is complementary to the nucleotide sequence of the other strand, eg, the antisense strand and the sense strand form a double-stranded or double-stranded structure, eg, double-stranded The regions are about 1 base pair, about 2 base pairs, about 3 base pairs, about 4 base pairs, about 5 base pairs, about 6 base pairs, about 7 base pairs, about 8 base pairs, about 9 base pairs, about 10 Base pair, about 11 base pair, about 12 base pair, about 13 base pair, about 14 base pair, about 15 base pair, about 16 base pair, about 17 base pair, about 18 base pair, about 19 base pair, about 20 Base pairs, about 21 base pairs, about 22 base pairs, about 23 base pairs, about 24 base pairs, or about 25 or more base pairs.

  The antisense strand may include a nucleotide sequence that is complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof, and the sense strand may include a nucleotide sequence that corresponds to the target nucleic acid sequence or a portion thereof. In some embodiments, the siNA can be constructed from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA use a nucleic acid linker or non-nucleic acid. Linked by a linker. The siNA may be a polynucleotide comprising a hairpin secondary structure having a self-complementary sense region and an antisense region, in which case the antisense region comprises a nucleotide sequence of another target nucleic acid molecule or part thereof. Complementary nucleotide sequences are included, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA may be a circular single-stranded polynucleotide having a stem comprising two or more loop structures and self-complementary sense and antisense regions, in which case the antisense region comprises the target nucleic acid molecule or its Comprising a nucleotide sequence complementary to a portion of the nucleotide sequence, the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the circular polynucleotide is processed in vivo or in vitro to yield RNAi Can be active siNA molecules capable of mediating.

  In some embodiments, the siNA comprises two strands of RNA. In certain embodiments, the siNA comprises two strands of DNA. The siNA may optionally be a hybrid comprising one strand of RNA and one strand of DNA. One strand or both strands may also contain a mixture of RNA and DNA. In some embodiments, a strand of siNA (eg, a strand of siRNA) is about 5 to about 60 nucleotides in length (eg, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides About 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides. Leotide, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides 45 nucleotides 46 nucleotides 47 nucleotides 48 nucleotides, about 49 Nucleotides, about 50 nucleotides, about 51 nucleotides, about 52 nucleotides, about 53 nucleotides, about 54 nucleotides, about 55 nucleotides, about 56 nucleotides, about 57 nucleotides, about 58 nucleotides or about 59 nucleotides). The siNA strand may optionally exceed 60 nucleotides.

  The siNA may also comprise a single stranded polynucleotide having a nucleotide sequence that is complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof (eg, in this case, the siNA molecule is a target nucleic acid sequence or a portion thereof). In this case, the single-stranded polynucleotide may further comprise a terminal phosphate group, such as 5′-phosphate or 5 ′, 3′-diphosphate. Good.

  In certain embodiments, the siNA molecule may further comprise another sense and antisense sequence or sense and antisense regions, where the sense and antisense regions are nucleotides known in the art. Or covalently linked by non-nucleotide linker molecules, or alternately non-covalently linked by ionic, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and / or stacking interactions. In certain embodiments, the siNA molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In some embodiments, the siNA molecule interacts with the nucleotide sequence of the target gene to cause inhibition of expression of the target gene.

  In some embodiments, one or more nucleotides of the siNA is a modified base that is substituted with another nucleotide compared to an unmodified standard siNA (eg, native siNA), a deletion And / or inserted (eg, siNA 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 Nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides are substituted, modified bases, deleted and / or inserted) . The in vivo or in vitro function of these modified siNAs may be the same as, or may change (eg, increase or decrease) as with standard siNAs. The altered function is typically detectable and in some cases increases within 100-fold of the function induced by standard siNA (eg, 2 fold increase, 4 fold increase, 6 fold increase, 8 fold increase) 10 times increase, 15 times increase, 20 times increase, 25 times increase, 30 times increase, 35 times increase, 40 times increase, 45 times increase, 50 times increase, 55 times increase, 60 times increase, 65 times increase, 70 Fold increase, 75 fold increase, 80 fold increase, 85 fold increase, 90 fold increase, 95 fold increase) or fall within 100 fold (eg, 1/2, 1/4, 1/6, 8 1/10, 1/15, 1/15, 1/25, 1/25, 30/35, 1/40, 1/45, 45/50, 55 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 1/90 It decreased to 1 of 95 minutes). In certain embodiments, the methods of the present disclosure include administering a composition comprising siNA. In other words, siNA molecules are externally added to cells or administered to patients. These externally added siNA molecules are expressed after their introduction. Without being bound by theory, in certain embodiments, externally added siNA molecules contribute to upregulation of endogenous expression of the transcript or protein. In other embodiments, the expression of a particular molecule is increased by externally adding siNA molecules, whereby overexpression of a particular molecule is a cell.

MicroRNA
MicroRNA (synonymous herein and also referred to as “miRNA”) is a class of non-coding regulatory RNA. Natural miRNAs are generally about 15-30 nucleotides in length, and precursor microRNAs are up to 80-120 nucleotides in length. The term “miRNA” generally refers to a single-stranded molecule, but in certain embodiments, is partially complementary to another region of the same single-stranded molecule or to another nucleic acid (10-50% of the total length of the strand). It further includes regions or additional strands that are complementary), substantially complementary (more than 50% complementary to the full length of the strand but less than 100% complementary), or fully complementary. For this reason, miRNAs can include single-stranded molecules, double-stranded molecules or partially single-stranded molecules. For example, a precursor miRNA may have a self-complementary region that is up to 100% complementary.

  Many microRNAs are highly conserved in many species, but in some are species specific. MicroRNAs regulate gene expression after transcription primarily by binding to the 3 'untranslated region (UTR) of their control target mRNA. MicroRNAs are involved in cell proliferation, differentiation and apoptosis, as well as other cellular, molecular and developmental pathways. It will be appreciated that some miRNAs are derived from genomic sequences or genes. In this context, for the sake of brevity, the term “gene” is used to refer to the genomic sequence encoding the precursor miRNA of a certain miRNA. However, some embodiments may include genomic sequences of miRNAs involved in miRNA expression, such as promoters or other regulatory sequences. The term “recombinant” may also be used, which generally refers to a molecule that has been manipulated in vitro, or a replication or expression product of such a molecule.

  Native miRNA is a regulatory RNA that acts as a recognition component of the complex RNA-induced silencing complex (RISC) riboprotein complex. The gene encoding miRNA is longer than the processed mature miRNA molecule. Genomic microRNAs exist as diverse forms such as individual genes, gene clusters of multiple microRNAs, or within introns of genes that encode proteins. The miRNA is initially transcribed as a primary transcript, an average of about 1.2 Kb pri-miRNA consisting of an RNA transcript, or transcribed into an intron of a long transcript encoding the protein.

  pri-miR is processed in the cell nucleus by the Drosha enzyme into a short, approximately 70-120 nucleotide stem-loop structure called pre-miRNA or precursor miRNA. These pre-miRNAs are then processed in the cytoplasm by interaction with the endonuclease Argonaut, Dicer and others to become mature functional miRNAs and RISC complexes are produced.

  MicroRNA generally inhibits translation by forming a base pair with a partially complementary sequence within the 3 'untranslated region (UTR) of the control target mRNA or promotes degradation of the mRNA. Individual messenger RNA (mRNA) may be the target of several miRNAs, or a single miRNA may control multiple target mRNAs. MicroRNAs can coordinately control a series of genes that encode proteins with related functions, bringing complexity and potential to gene regulation.

  The microRNA may be labeled, used for array analysis, or applied for diagnosis, therapy or prognosis. This RNA may have been produced endogenously by the cell, or may have been synthesized or produced chemically or by recombinant techniques. MicroRNA can be isolated and / or purified. In this specification, human miRNA molecules are often referred to using the prefix “hsa-miR-”. Unless otherwise stated, miRNAs referred to in this application are human sequences, and non-human miRNA sequences can be determined and prepared from these sequences (eg, for use in non-human subjects).

  In some embodiments, miRNAs that do not correspond to known human miRNAs may be used.

  In some embodiments, one or more nucleotides of the miRNA are deleted, which is a modified base that is replaced with another nucleotide compared to an unmodified standard miRNA (eg, a native miRNA). And / or inserted (eg, 1 nucleotide, 2 nucleotide, 3 nucleotide, 4 nucleotide, 5 nucleotide, 6 nucleotide, 7 nucleotide, 8 nucleotide, 9 nucleotide, 10 nucleotide, 11 nucleotide, 12 nucleotide of miRNA) 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides are substituted, modified bases, deleted, and / or inserted And it is). The in vivo or in vitro function of such a modified miRNA may be the same as that of a standard miRNA or may be altered (eg, increased or decreased). The altered function is typically detectable and in some cases increases within 100 fold of the function induced by the standard miRNA (eg, 2 fold increase, 4 fold increase, 6 fold increase, 8 fold increase) 10 times increase, 15 times increase, 20 times increase, 25 times increase, 30 times increase, 35 times increase, 40 times increase, 45 times increase, 50 times increase, 55 times increase, 60 times increase, 65 times increase, 70 Fold increase, 75 fold increase, 80 fold increase, 85 fold increase, 90 fold increase, 95 fold increase) or fall within 100 fold (eg, 1/2, 1/4, 1/6, 8 1/10, 1/15, 1/15, 1/25, 1/25, 30/35, 1/40, 1/45, 45/50, 55 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 90 minutes , Reduced to 1 of 95 minutes).

  In some embodiments, the present disclosure is based on an assay of the expression level of microRNA-31 in a cell or sample. When assaying expression, this disclosure contemplates not only determining endogenous expression levels, but also assaying expression after therapeutic intervention, in certain embodiments, in certain embodiments, A combination of endogenous expression and expression of nucleic acid introduced from outside may be evaluated.

Nucleic Acid Modifications Any of the modifications described below can be applied to nucleic acids such as miRNAs and siRNAs as needed. It should be understood that a nucleic acid comprising a specific nucleic acid, such as miRNA-31, may further comprise a nucleic acid modification. Examples of modifications include RNA backbone, sugar or base changes, and various combinations thereof. Any suitable number of backbone linkages, sugars and / or bases may be modified in miRNAs or other nucleic acids (eg, independently about 5%, 10%, 15%, 20%, 25 %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to 100% skeletal linkage , Modify sugars and / or bases; or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Or modify 20 backbone bonds, sugars and / or bases). Unmodified miRNA nucleosides are those in which any one of the bases adenine, cytosine, guanine, thymine or uracil is bound to the 1 ′ carbon of β-D-ribo-furanose.

  The modified base is a nucleotide base other than adenine, guanine, cytosine and uracil at position 1 '. Non-limiting examples of modified bases include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, Aminophenyl, 5-alkylcytidine (eg, 5-methylcytidine), 5-alkyluridine (eg, ribothymidine), 5-halouridine (eg, 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (eg, 6 -Methyluridine), propyne and the like. Other non-limiting examples of modified bases include nitropyrrolyl (eg, 3-nitropyrrolyl), nitroindolyl (eg, 4-nitroindolyl, 5-nitroindolyl, 6-nitroindolyl), hypoxanthinyl, isoinosinyl , 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methyl Benzimidazole, 3-methylisocarbolistyryl, 5-methylisocarbostyrylyl, 3-methyl-7-propynylisocarbolistyryl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9 -Mechi -Imidazopyridinyl, pyrrolopyridinyl, isocarboxtyryl, 7-propynylisocarbostyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl , Phenyl, naptalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl and the like.

  In some embodiments, for example, the nucleic acid may comprise a modified nucleic acid molecule with a modified phosphate backbone. Non-limiting examples of backbone modifications include phosphorothioate modification, phosphorodithioate modification, methylphosphonate modification, phosphotriester modification, morpholino modification, amidate carbamate modification, carboxymethyl modification, acetamidate modification, polyamide modification, sulfonate modification , Sulfonamide modifications, sulfamate modifications, formacetal modifications, thioformacetal modifications, and / or alkylsilyl modifications. In some cases, the naturally occurring ribose sugar moiety of a nucleoside is substituted with a hexose sugar, a polycyclic heteroalkyl ring or a cyclohexenyl group. In some cases, the hexose sugar is allose, altrose, glucose, mannose, gulose, idose, galactose, talose, or derivatives thereof. The hexose may be D-hexose, glucose or mannose. In some cases, the polycyclic heteroalkyl group may be a bicyclic ring containing one oxygen atom in the ring. In some cases, the polycyclic heteroalkyl group is bicyclo [2.2.1] heptane, bicyclo [3.2.1] octane or bicyclo [3.3.1] nonane.

  Nitropyrrolyl nucleobases and nitroindolyl nucleobases are members of a class of compounds known as universal bases. A universal base is a compound that can replace any of the four natural bases without substantially affecting the melting behavior or activity of the oligonucleotide duplex. Oligonucleotide duplexes containing 3-nitropyrrolyl nucleobases can be stabilized by stacking interactions alone, unlike hydrogen-bonding interactions found in natural nucleobases and useful for stabilization. Nitropyrrolyl nucleobases lack specificity for specific complementary bases because there are no large hydrogen bonding interactions. Furthermore, 4-nitroindolyl, 5-nitroindolyl and 6-nitroindolyl also show little specificity for the four natural bases. The procedure for the preparation of 1- (2'-0-methicbeta.-D-ribofuranosyl) -5-nitroindole is described in Gaubert, G. et al. Wengel, J .; Tetrahedron Letters 2004, 45, 5629. Other universal bases include hypoxanthinyl, isoinosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzoimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl and their structural derivatives .

  Difluorotolyl is a non-natural nucleobase that acts as a universal base. Difluorotolyl is an isostere of the natural nucleobase thymine. However, difluorotolyl, unlike thymine, does not show significant selectivity for any of the natural bases. Other aromatic compounds that serve as universal bases include 4-fluoro-6-methylbenzimidazole and 4-methylbenzimidazole. In addition, the relatively hydrophobic isocarbostyryl derivatives 3-methylisocarbolistyryl, 5-methylisocarbostyril, and 3-methyl-7-propynylisocarbostyril also contain only natural bases. It is a universal base that destabilizes oligonucleotide duplexes only slightly compared to. Other non-natural nucleobases include 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-propynylisocarbolistyryl , Propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naptalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and These structural derivatives are mentioned. For a more detailed discussion, including synthetic procedures for difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole and other non-natural bases described above, see Schweitzer et al. , J .; Org Chem. 59: 7238-7242 (1994).

  A composition comprising a nucleic acid also includes a nucleic acid that has been modified, ie, contains a non-natural internucleoside linkage. Such non-natural internucleoside linkages have desirable properties such as, for example, increased cellular uptake, improved affinity for other oligonucleotides or nucleic acid targets, and enhanced stability in the presence of nucleases, so that the natural form Is often selected. The oligomeric compounds of the present invention may have one or more modified internucleoside linkages. As defined herein, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom.

  Suitable modified internucleoside linkages that include phosphorus include phosphorothioate internucleoside linkages. Examples of other modified oligonucleotide skeletons (internucleoside linkages) containing a phosphorus atom therein include, for example, chiral phosphorothioates having ordinary 3′-5 ′ linkages, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, Methyl and other alkyl phosphonates such as 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3′-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphos Luamidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates, their 2'-5 'linked analogs, and one or more nucleotides During coupling 3'-3 ', 5'-5' include those having inverted polarity is or 2'-2 'linkage. Oligonucleotides with reverse polarity are single 3'-3 'linkages, ie abasic (the nucleobase is missing or instead has a hydroxyl group) at the most 3' internucleotide linkage Contains the resulting single inverted nucleoside residue. Various salts, mixed salts and free acid forms are also included.

  The nucleotide analog may further comprise a “locked” nucleic acid. With certain compositions, endogenous nucleic acids can be essentially “anchor” or “locked” to a particular structure. Anchoring the sequence helps to prevent the dissociation of the nucleic acid siNA complex and thus not only prevents copying of the endogenous sequence, but also allows for the labeling, modification and / or cloning of the endogenous sequence. You can also. Locked structures are used as stable structures that can control gene expression (ie inhibit or enhance transcription or replication), or can label or otherwise modify endogenous nucleic acid sequences. Or it can be used to isolate, ie clone, endogenous sequences.

  Nucleic acid molecules need not be limited to molecules containing only RNA or DNA, but further include chemically modified nucleotides and non-nucleotides. The percentage of non-nucleotides or modified nucleotides is 1% to 100% (eg, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40% of nucleic acids About 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% non-nucleotides or modified nucleotides Or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20% are non-nucleotides or modified nucleotides). In certain embodiments, the nucleic acid is missing a nucleotide comprising 2'-hydroxyl (2'-OH). In certain embodiments, the nucleic acid does not require the presence of a nucleotide having a 2′-hydroxy group to mediate a function, so the nucleic acid comprises a ribonucleotide (eg, a nucleotide having a 2′-OH group). It does not have to be included. However, such nucleic acid molecules that do not require the presence of ribonucleotides to support certain functions are linked linkers or other linked or linked groups that contain one or more nucleotides with 2′-OH groups. May have moieties or chains. In some cases, the nucleic acid molecule may comprise ribonucleotides at about 5%, about 10%, about 20%, about 30%, about 40% or about 50% of the nucleotide positions.

Biomarkers Provided herein are microRNA (also referred to herein as “miRNA”) biomarkers and other biomarkers. Specific biomarkers used in the method include miRNA-31, RhoA and IL-2. The use of biomarkers will be discussed in more detail in a later section of this application. The present disclosure contemplates detecting protein levels and / or transcript levels. As an example, biomarkers can be detected using probes and primers that detect transcript levels and antibodies that detect protein levels.

Conjugates In certain embodiments, the present disclosure includes administering a composition comprising a nucleic acid comprising a nucleotide sequence as an active ingredient. In other words, in certain embodiments, the methods of the present disclosure include administration of an exogenous nucleic acid molecule that is expressed after introduction into the cell. In certain embodiments, such nucleic acids may be added to one or more moieties or conjugates that enhance the activity, cell distribution or cellular uptake of the resulting oligomeric compound. In one embodiment, such modified nucleic acid compositions are prepared by covalently attaching a conjugate group to a functional group, such as a hydroxyl group or an amino group. Conjugate groups of the present disclosure include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacological properties of the oligomer, and groups that improve the pharmacokinetic properties of the oligomer. Typical conjugate groups include cholesterol, carbohydrates, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. Groups that enhance pharmacological properties include groups that promote nucleic acid uptake, groups that increase resistance to degradation, and / or groups that enhance hybridization with RNA. Groups that improve pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion. Exemplary conjugate groups are disclosed in International Application No. PCT / US92 / 09196, filed Oct. 23, 1992, the entire disclosure of which is incorporated herein. Conjugate moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-trityl thiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al. Bioorg.Med.Chem.Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 33-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero -3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 3651-3651, 3651) , Palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine moiety or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. , 1996, 277, 923-937), but is not limited thereto.

  Furthermore, the nucleic acid compounds of the present disclosure may be linked or conjugated with one or more moieties that facilitate active or passive transport, localization, or compartmentalization of the oligomeric compounds. Cell localization includes, but is not limited to, localization in the nucleus, nucleolus or cytoplasm. Compartmentation includes, but is not limited to, any induction into cell compartments such as the nucleus, nucleolus, mitochondria, or implantation into the cell membrane.

Ribozymes The composition of the present disclosure may comprise a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that can cleave single-stranded nucleic acids such as mRNAs that have complementary regions. To this end, the ribozyme (eg, hammerhead ribozyme; described in Haselhoff and Gerlach, 1988, Nature 334: 585-591) is used to catalytically cleave the mRNA transcript, thereby Can inhibit translation. For example, a ribozyme having specificity for a nucleic acid molecule encoding RhoA can be designed based on the nucleotide sequence of RhoA.

  Regardless of the particular nucleic acid comprising the composition used, this disclosure contemplates a composition comprising a nucleic acid that increases miRNA-31 expression and a composition comprising a nucleic acid that reduces RhoA expression. Particularly preferred compositions can be used to increase IL-2 expression. It will be appreciated that some nucleic acid compositions can serve, for example, to increase endogenous expression of microRNA-31. On the other hand, in certain embodiments, increased expression of microRNA-31 may be caused by externally transferring microRNA-31 to the cell in a form that allows expression upon introduction into the cell, eg, as described in the Examples. Do by giving. Administration of an externally provided molecule (single stranded, double stranded, partially double stranded, etc.) containing a sequence that mimics the sequence of endogenous microRNA-31 upon expression is referred to as a microRNA-31 mimic This is also an example of a siNA molecule. In certain embodiments, the microRNA-31 mimetic is an oligonucleotide (single stranded, double stranded, partially double stranded) comprising a sequence corresponding to that of natural human microRNA-31 or pre-microRNA-31. Including a main chain).

  In certain embodiments, a composition of the present disclosure comprises siNA that inhibits expression of RhoA, eg, siRNA that hybridizes to RhoA. In another embodiment, a composition of the present disclosure comprises siNA that increases endogenous expression of microRNA-31. In other embodiments, the compositions of the present disclosure may include sequences of microRNA-31 (natural sequences or modified variants thereof), such that the expression of microRNA-31 is increased by the expression of an externally supplied nucleic acid. A nucleic acid comprising a nucleotide sequence corresponding to

  Nucleic acid compositions can be isolated, generated and / or delivered using methods well known in the art. In one embodiment, a composition of the present disclosure is a nucleic acid that increases microRNA-31 expression or decreases RhoA expression and is part of an expression vector that expresses the nucleic acid in a suitable host. Contains nucleic acids. In particular, such nucleic acids may have a promoter, such as a heterologous promoter, which promoter is an inducible or constitutive promoter, optionally a tissue-specific promoter.

  Delivery of the nucleic acid to the subject may be direct delivery, in which case the subject is contacted directly with the nucleic acid or a vector carrying the nucleic acid, or may be indirect delivery, in which case cells are first transformed with the nucleic acid in vitro. And then transplanted into the subject. These two approaches are known as in vivo gene therapy or ex vivo gene therapy, respectively. Thus, in certain embodiments, the disclosure includes the use of cell compositions.

  Isolated and / or recombinant nucleic acids, such as miRNA gene products, can be obtained using a number of standard techniques. For example, miRNA gene products can be chemically synthesized using one of several methods. In one method, the miRNA gene product is chemically synthesized using a suitably protected ribonucleoside phosphoramidite and a conventional DNA / RNA synthesizer. Suppliers of synthetic RNA molecules or reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Rockford, Ill.). Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruchem (Glasgow, UK), but are not limited thereto.

  Alternatively, nucleic acids such as miRNA gene products can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from plasmids include, but are not limited to, U6 or H1 RNA pol III promoter sequences, or cytomegalovirus promoters. The recombinant plasmid may further comprise an inducible promoter or a regulatable promoter to allow the cancer cell to express the miRNA gene product.

  Nucleic acid gene products such as miRNA gene products expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques.

  For example, selection of suitable plasmids for expression of miRNA gene products, methods for inserting nucleic acid sequences into plasmids to express gene products, and methods for delivering recombinant plasmids to target cells are reviewed from a number of publications. be able to. See, for example, Zeng et al., The entire disclosure of which is incorporated herein by reference. , Molecular Cell 9: 1327-1333 (2002); Tuschl, Nat. Biotechnol 20: 446-448 (2002); Brummelkamp et al. , Science 296: 550-553 (2002); Miyagishi et al. Nat. Biotechnol. 20: 497-500 ((2002); Paddison et al., Genes Dev. 16: 948-958 (2002); Lee et al., Nat. Biotechnol. 20: 500-505 (2002); and Paul et al. , Nat.Biotechnol.20: 505-508 (2002).

  miRNA gene products can also be expressed from recombinant viral vectors. It is contemplated that the miRNA gene product may be expressed from two separate recombinant viral vectors or from the same viral vector. RNA expressed from recombinant viral vectors can be isolated from cultured cell expression systems by standard techniques or used directly to infect cells in vitro or in vivo.

  The recombinant viral vector may comprise a sequence encoding the miRNA gene product and any promoter suitable for expression of the RNA sequence. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequence, or the cytomegalovirus promoter.

  Any viral vector can be used, as long as it can incorporate the coding sequence of the miRNA gene product (or its complementary sequence), eg, adenovirus (AV); adeno-associated virus (AAV); retrovirus (For example, but not limited to, vectors derived from lentivirus (LV), rhabdovirus, murine leukemia virus); herpes virus and the like. The tropism of a viral vector can be altered as needed by pseudotyping the vector with an envelope protein or other surface antigen from another virus, or by using another viral capsid protein instead. it can.

  Methods for transfection of eukaryotic cells include direct injection of nucleic acids into the nucleus or pronucleus of cells; electroporation; introduction by liposomes or via lipophilic materials; nucleic acid delivery via receptors, gene guns or There are, but are not limited to, particle accelerating or particle accelerating; calcium phosphate precipitation and viral vector mediated transfection.

For example, the liposome-introduced compound (ie, for example, DOTAP (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethyl-ammonium methyl sulfate, Boehringer-Mannheim) or Equivalents such as lipofectin may be transfected, the amount of nucleic acid used is not critical to the practice of the present invention, acceptable results use 0.1-100 micrograms of nucleic acid / 10 ⁵ cells For example, a ratio of about 0.5 micrograms of plasmid vector in 3 micrograms of DOTAP per 10 ⁵ .

  The foregoing provides a detailed description of the compositions of the present disclosure, particularly compositions using nucleic acids. In certain embodiments, the composition used in the claimed method comprises a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and optionally Provided as part of the vector. In certain embodiments, the nucleic acid comprises a nucleotide sequence that is an oligonucleotide, such as a siNA molecule, that corresponds to a functional portion of a natural nucleic acid sequence. In addition, the compositions of the present disclosure include compositions in which the active agent is a polypeptide such as an antibody or a small organic molecule. Any of the classes of compounds may be formulated and administered as a composition or pharmaceutical composition.

  Polypeptides and peptide fragments: In certain embodiments, the compound is a polypeptide or peptide fragment. Exemplary polypeptides or peptide fragments include wild type sequences as well as variant sequences. A variant polypeptide comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a particular wild-type polypeptide.

  In addition to polypeptides and peptide fragments, the present invention further contemplates isolated nucleic acids comprising nucleotide sequences that encode the polypeptides and fragments. The term nucleic acid, as used herein, is intended to include fragments as equivalents, such fragments having substantially the same function as the full-length nucleic acid sequence from which it is derived. Equivalent nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, eg, allelic variants, and thus include, for example, sequences that differ from the nucleotide sequence of the native nucleotide sequence. Equivalent sequences include sequences that differ from known wild type or variant sequences due to the degeneracy of the genetic code. The equivalent sequence hybridizes to the native nucleotide sequence under stringent conditions (ie, corresponding to a temperature about 20-27 ° C. below the melting temperature (Tm) of the DNA duplex formed in about 1M salt). It may further comprise a nucleotide sequence that soybeans. A further example of stringent hybridization conditions includes a 0.2 × SSC wash step at 65 ° C. It will be understood that an equivalent nucleotide sequence encodes a polypeptide that retains the activity of the polypeptide encoded by the native nucleotide sequence.

  Equivalent nucleotide sequences used in the methods described herein further include sequences that are at least 60% identical to a nucleotide sequence. In another embodiment, the nucleotide sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleotide sequence of the native sequence.

  Nucleic acids having sequences that differ from the nucleotide sequence encoding a particular polypeptide due to the degeneracy of the genetic code are also within the scope of the invention. Such nucleic acids functionally encode equivalent peptides, but differ from wild-type sequences known in the art in terms of sequence due to the degeneracy of the genetic code. For example, there are several amino acids that are specified in two or more triplets. Codons that define the same amino acid, ie, synonymous codons (eg, CAU and CAC each encode histidine) may result in “silent” mutations that do not affect the amino acid sequence. However, it is expected that there will be DNA sequence polymorphisms that reliably lead to amino acid sequence changes. One skilled in the art will recognize that these changes in one or more nucleotides of a nucleic acid encoding a polypeptide (up to about 3-5% of the nucleotides) exist among individuals of a particular species due to natural allelic changes. You will understand that there are cases.

  Antibodies: exemplary compounds further include antibodies. An antibody may have very high affinity and specificity for a particular epitope. Without being bound by theory, an antibody can exert or induce a specific effect on a cell, tissue or organism by inhibiting or enhancing the activity of intracellular proteins and signaling pathways.

  Monoclonal or polyclonal antibodies can be made using standard protocols (see, eg, Antibodies: A laboratory manual ed. By Harlow and Lane (Cold Spring Harbor Press: 1988)). Mammals such as mice, hamsters or rabbits can be immunized with immunogenic peptides. Techniques for conferring immunogenicity on proteins or peptides include conjugation with carriers or other techniques well known in the art. The immunogenic portion of the protein may be administered in the presence of an adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. A standard ELISA or other immunoassay may be used with the immunogen as the antigen to assess antibody levels. An antibody may be immunospecific for a specific protein, may be immunospecific for a specific family of proteins, or may have a low immunospecificity Note that it may cross-react with multiple proteins. Antibodies that are immunospecific do not substantially cross-react with heterologous proteins. Substantially non-reactive means that the binding affinity of the antibody to the heterologous protein is at least one order of magnitude, more preferably at least two orders of magnitude, even more than the binding affinity of the antibody to the protein for which the antibody is immunospecific. Preferably it means at least 3 orders of magnitude lower.

  It should also be pointed out that antibodies such as antibodies that detect IL-2 or RhoA expression are particularly useful in diagnostic methods.

  Peptide mimetics: In other embodiments, the present invention contemplates that the agent is a peptidomimetic. Peptidomimetics are compounds based on or derived from peptides and proteins. Peptidomimetics can be obtained by structural modification of the amino acid sequence of a known protein using unnatural amino acids, conformational restraint, isosteric replacement and the like . This peptide mimetic constitutes a continuum of structural space between the peptide and the non-peptide synthetic structure.

  Exemplary peptide mimetics appear to increase specificity and / or efficacy, such as being non-hydrolyzable (eg, increasing stability to proteases or other physiological conditions that degrade the corresponding peptide). Furthermore, it may have such characteristics as increasing cell permeability due to subcellular localization. For illustrative purposes, peptide analogs of the invention are described, for example, in benzodiazepines (see, for example, Freidinger et al. In Peptides: Chemistry and Biology, GR Marshall ed., ESCOM Publisher: Leiden, Netherlands). , Substituted gamaractam rings (Garvey et al. In Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p123), C-7 mimics (et. and Biology, GRM rshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptide (Ewenson et al. (1986) J Med Chem 29: 295; and Ewenson et al. in Peptides: Sept. Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide core (Nagai et al. (1985) Tetrahedron Lett. Et al. 1986) J Chem Soc erkin Trans 1: 11231), β-aminoalcohol (Gordon et al. (1985) Biochem Biophys Res Commun 126: 419; and Dann et al. (1986) Biochem Biophys Res Commun 134: 71, diaminoketone (Natalajan et al.). (1984) Biochem Biophys Res Commun 124: 141), and methyleneamino modification (Roark et al. In Peptides: Chemistry and Biology, G.). R. Marshall ed. , ESCOM Publisher: Leiden, Netherlands, 1988, p134). In addition, generally see Session III: Analytical and synthetic methods, in Peptides: Chemistry and Biology, G. et al. R. Marshall ed. , ESCOM Publisher: Leiden, Netherlands, 1988).

  In addition to the various side chain substitutions that may be made to create the present peptide mimetics, the present invention specifically contemplates the use of mimetics that have fixed peptide secondary structure conformations. Many surrogates have been developed to replace peptide amide bonds. Frequently used substitutes for amide linkages include the following groups (i) trans-olefins, (ii) fluoroalkenes, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides. .

  In addition, peptide peptidomimetics based on more significant modifications of the peptide backbone may be used. Peptide mimetics that fall into this category include (i) retroinverso analogs and (ii) N-alkylglycine analogs (so-called peptoids).

  Furthermore, combinatorial chemistry methods (for example, WO 99/48897 pamphlet) have also influenced the development of new peptide mimetics. For example, one embodiment of a so-called “peptide morphing” strategy focuses on the creation of a random library of peptide analogs containing various peptide bond surrogates.

  In an exemplary embodiment, the peptidomimetic can be derived as a retro-inverso analog of the peptide. Retro-inverso analogs can be made according to methods known in the art, such as those described by Sisto et al., US Pat. No. 4,522,752. As a general guide, the site most prone to proteolysis is typically changed, but it is optional to use an amide bond that is less prone to degradation when switching to a mimetic. The final product or its intermediate can be purified by HPLC.

  In another illustrative embodiment, the peptidomimetic can be obtained as a retro-enatio analog of the peptide. Such retroenantiomeric analogs can be synthesized using commercially available D-amino acids (or analogs thereof) and standard solid phase or liquid phase peptide synthesis techniques. For example, in a preferred solid phase synthesis method, a suitably amino-protected (t-butyloxycarbonyl, Boc) residue (or analog thereof) is covalently bound to a solid support, such as a chloromethyl resin. The resin is washed with dichloromethane (DCM) and the BOC protecting group is removed by treatment with DCM containing TFA. The resin is washed and neutralized and then the Boc protected D-amino acid is introduced by conjugation with diisopropylcarbodiimide. The resin is washed again and the cycle is repeated in turn for each remaining amino acid. When the synthesis of the protected retroenantiomeric peptide is complete, the protecting group is removed and the peptide is cleaved from the solid support by treatment with hydrofluoric acid / anisole / dimethylsulfide / thioanisole. The final product is purified by HPLC to give a pure retroenantiomeric analog.

  In yet another illustrative embodiment, trans-olefin derivatives may be made for any of the polypeptides. Transolefin analogs are described in Y.W. K. Shue et al. (1987) Tetrahedron Letters 28: 3225, and also by other methods known in the art. It will be appreciated that depending on the nature of the reagents used, it may be necessary to modify the cited procedures or other available procedures.

  Furthermore, it is also possible to link a pseudo dipeptide synthesized by the above method to another pseudo dipeptide to produce a peptide analog having several olefin functional groups instead of amide functional groups.

  Yet another class of peptidomimetic derivatives are phosphonate derivatives. The method for synthesizing such phosphonate derivatives may be modified from a known synthesis scheme. For example, see Boots et al. in Peptides: Chemistry and Biology, (Essci Science Publishers, Leiden, 1988, p. 118); Petrillo et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co. Rockland, IL, 1985).

  Many other peptidomimetic structures are also known in the art and can be readily modified for the design of peptidomimetics. For illustration, the peptidomimetics are 1-azabicyclo [4.3.0] nonane substitutes (see Kim et al. (1997) J. Org. Chem. 62: 2847), or N-acyl piperazic acid (Xi et al. (1998) J. Am. Chem. Soc. 120: 80), or 2-substituted piperazine moieties as a regulated amino acid analog (Williams et al. (1996) J. Am. Med. Chem. 39: 1345-1348). In still other embodiments, certain amino acid residues are substituted with aryl and biaryl moieties, such as monocyclic or bicyclic aromatic nuclei or monocyclic or bicyclic heteroaromatic nuclei, or diaromatic nuclei. , Aromatic nuclei, heteroaromatic nuclei or diheteroaromatic nuclei may be substituted.

  Small organic or inorganic molecules: In certain embodiments, the compound is a small organic or inorganic molecule. Small organic or inorganic molecules promote or inhibit the function of a particular protein or class of proteins. By small organic or inorganic molecule is meant a molecule comprising carbon having a molecular weight of less than 5000 amu, preferably less than 2500 amu, more preferably less than 1500 amu, even more preferably less than 750 amu, less than 500 amu, or less than 250 amu.

  Small organic or inorganic molecules can be readily identified by screening a library of organic molecules and / or chemical compounds to identify compounds with the desired function. Alternatively, a single compound or a small number of candidate compounds may be screened individually or in combination. In certain embodiments, small molecules (eg, inorganic or organic molecules) are non-peptidyl compounds that contain or do not contain 2 or less, 1 or less peptide bonds and / or sugar bonds.

  RhoA small molecule inhibitors are good examples of small molecules that can be used in the compositions of the present disclosure. Several small molecule inhibitors are commercially available. They can be used as active agents per se or as a starting point for medicinal chemistry to produce other small molecule RhoA inhibitors.

(Iv) Autoimmune conditions The present disclosure provides methods for diagnosing, monitoring and treating autoimmune conditions using any of the compositions of the present disclosure. Any of the disclosed compositions described herein can be used in a subject having or suspected of having any of the autoimmune conditions described herein. In certain embodiments, the methods provided herein are based on a correlation between microRNA-31 expression and IL-2 expression in a subject with a particular autoimmune condition. In certain embodiments, the autoimmune condition is SLE or is a mechanism of action, pathogenesis, correlation with IL-2 dysregulation, or T cell dysregulation, particularly regulatory T cell dysregulation Another autoimmune condition that shares one or more features with SLE, such as sharing a correlation with. In certain embodiments, the autoimmune condition is systemic lupus erythematosus, a flare or remission SLE. In other embodiments, the autoimmune condition is correlated with decreased expression of IL-2 and / or decreased expression of microRNA-31. In certain embodiments, the symptoms or signs of SLE include lupus nephritis.

  In certain embodiments, the subject has another autoimmune condition, such as a mechanism of action, etiology, correlation with IL-2 dysregulation, or T cell dysregulation, particularly dysregulation of regulatory T cells. Sharing one or more features with the SLE, such as In certain embodiments, the other autoimmune condition is rheumatoid arthritis (RA), type I diabetes, or autoimmune thyroid disease. In certain embodiments, the other autoimmune condition is scleroderma. In certain embodiments, the autoimmune condition is correlated with decreased expression of IL-2 compared to healthy subjects.

  Exemplary autoimmune conditions including symptoms of the above conditions are described below. In addition, a few examples of animal models used for the evaluation of diagnostic and therapeutic agents for autoimmune conditions are also provided. These animal models, or other animal models known in the art, can be used to develop the compositions of the present disclosure for use in methods of diagnosis or treatment. “Treating” means alleviating or eliminating one or more symptoms of a condition by administration of the composition over one or more doses. As used herein, the term “symptoms” is used in a broad sense and refers to signs and symptoms of a condition, whether observable directly by a patient or by medical examination, Also referred to as downstream complication of disease that can be alleviated by treatment.

Systemic lupus erythematosus (SLE)
Systemic lupus erythematosus, often abbreviated as SLE, is a systemic autoimmune condition that can affect any part of the body. SLE is a type III hypersensitivity reaction caused at least in part by the formation of antibody-immune complexes.

  SLE most often has deleterious effects on the heart, joints, skin, lungs, blood vessels, liver, kidneys and nervous system. The course of the disease is unpredictable and the duration of the disease (called flare) and remission occur alternately This disease occurs 9 times more often in women than in men, especially in women of fertile age.

  There is currently no cure for SLE, and symptom management has generally been limited to the use of general immunosuppression with cyclophosphamide, corticosteroids and other immunosuppressive agents.

  Common symptoms of SLE include arthritis; fatigue; general discomfort, anxiety or poor physical condition (fatigue); joint pain and swelling; muscle pain; nausea and vomiting; and skin rash. Symptoms can also include abdominal pain; hematuria; discoloration of fingers during pressure or cold; numbness and tingling; and erythema of the skin. In some patients, SLE has lung or kidney damage. In some cases, patients with SLE develop a specific kidney condition called lupus nephritis. The present disclosure is based on the fact that a subject in need of treatment may have any one or more of these symptoms and any one or more improvements in these symptoms. I am thinking of being able to measure

Skin Symptoms Skin symptoms are very common, with 30% to 50% of subjects suffering from a classic cheek rash, also called a butterfly rash. Some patients may also have thick red scaly spots (called discoid lupus) on the skin. Other skin symptoms include hair loss; mouth, vagina, nasal and / or urinary ulcers; skin lesions; and small tears of delicate tissue around the eyes.

Musculoskeletal Symptoms Joint pain is a common symptom of SLE and it is estimated that 90% of patients report joint pain and / or muscle pain at some point in the course of the disease. A possible link between rheumatoid arthritis and SLE has been suggested.

Hematologic symptoms Up to 50% of SLE patients are anemic. In addition, SLE patients appear to have an increased incidence of thrombotic disorders, antiphospholipid syndrome, characterized by the presence of autoantibodies to phospholipids in serum. Characteristics of antiphospholipid antibody syndrome include prolonged partial thromboplastin time and positivity of antiphospholipid antibody test. If these findings are observed, it is called “Loops anticoagulant positivity”.

Cardiac Symptoms Cardiac symptoms of SLE include inflammation of various parts of the heart such as pericardium (pericarditis), myocardium (myocarditis) and endocardium (endocarditis). In SLE patients, atherosclerosis is likely to occur at a high frequency and, if present, progresses faster than observed in the general population.

Pulmonary Symptoms Patients with SLE often have various symptoms of lung and pleural inflammation. Such inflammation can cause pleurisy, pleural effusion, lupus pneumonitis, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary embolism, pulmonary hemorrhage and atrophic pulmonary syndrome.

Kidney Symptoms Lupus nephritis is an inflammation of the kidney and is a severe complication of systemic lupus erythematosus (SLE). SLE is characterized by natural self-reactive and immune multi-organ injury of B and T cells. In the kidney, lupus nephritis can reduce function. Patients with lupus nephritis eventually develop renal failure and may require dialysis or kidney transplantation.

  WHO classified lupus nephritis into five classes based on biopsy results. This classification was defined in 1982 and revised in 1995. Class I is micromesangial glomerulonephritis that is histologically normal under an optical microscope but has mesangial deposits observed under an electron microscope. Class II is based on the presence of mesangial proliferative lupus nephritis. Class III is focal proliferative nephritis. Class IV is diffuse proliferative nephritis. Class V is membranous nephritis and is characterized by extreme edema and protein loss. Class VI is glomerulosclerosis. The present disclosure contemplates treating patients with lupus nephritis classified as either class I, II, III, IV, V or VI. In certain embodiments, the patient has lupus nephritis classified as class III or higher, class IV or higher, or class V or higher. In certain embodiments, the patient has lupus nephritis classified as class VI.

  Symptoms of lupus nephritis include hematuria, foam urine, hypertension, proteinuria, fluid retention and edema. Other symptoms include signs and symptoms of renal fibrosis and / or renal failure. Lupus nephritis can lead to renal failure and even end-stage renal disease if left untreated.

  Lupus nephritis can be diagnosed and / or monitored by renal biopsy as well as by using blood or urine tests. Such tests may be used to monitor symptom improvement during or after treatment. In certain embodiments, the treatment includes any one or more improvements in these indicators. As an example, lupus nephritis can be diagnosed and / or determined by assessing the amount of blood and / or protein in the urine. Treatment may include reducing the amount of blood and / or protein in the urine, for example to normal or near normal levels. Lupus nephritis may also be diagnosed and / or determined by evaluating creatinine and / or urea levels in the blood and / or by an estimate of glomerular filtration rate based on the creatinine score.

Psychiatric Symptoms The American College of Rheumatology defines 19 neuropsychiatric syndromes of systemic lupus erythematosus, which can occur through effects on the central and peripheral nervous systems. The most common neuropsychiatric disorder in SLE patients is headache. Other common symptoms include cognitive dysfunction, mood disorders, cerebrovascular, convulsions, polyneuropathy, anxiety disorders and psychosis. Other less commonly observed symptoms include acute confusion, Guillain-Barre syndrome, aseptic meningitis, autonomic neuropathy, demyelinating syndrome, mononeuropathy, chorea, myasthenia gravis, myelopathy, brain Neuropathy and plexus disorders are included.

Neurological symptoms Neurological symptoms increase the morbidity and mortality of SLE. Lupus neurological symptoms are known as neuropsychiatric systemic lupus erythematosus (NPSLE). One aspect of this is severe damage to epithelial cells at the blood brain barrier.

Systemic Symptoms The most common systemic symptom of SLE is fatigue. Fatigue may be due to a variety of factors such as anemia, hypothyroidism, pain, depression, poor quality sleep and disease activity.

  The American College of Rheumatology has set eleven criteria to classify and operate the definition of SLE in clinical trials. This classification scheme was not for use as a separate diagnostic scheme, but for use in clinical trials. To identify patients in a clinical trial, if any 4 of the following 11 symptoms are present simultaneously or over time at two different time points: SLE: buccal rash; discoid Rash; serositis or pericarditis; oral ulcers (including oral or nasopharynx); arthritis (especially non-erosive arthritis of two or more peripheral joints); photosensitivity; hematological disorders, especially hemolytic anemia, Leukopenia, lymphocyte count reduction or thrombocytopenia; kidney disorders, especially protein or cell casts in urine; positive antinuclear antibody test; immunological disorders, especially anti-Sm, anti-double-stranded DNA or anti-nuclear Positive phospholipid test; and neuropathy, especially convulsions or psychosis.

  A more comprehensive approach is used when diagnosing individual patients. For example, some people with antiphospholipid syndrome may have SLE but not the above four criteria. Furthermore, SLE may exhibit symptoms and characteristics other than those listed in the above criteria.

  The quality of life of SLE patients can be improved by flare prevention. Warning signs of imminent flare include increased fatigue, pain, rash, fever, abdominal discomfort, headache and dizziness. Early recognition of warning signs and good communication with a physician may help patients remain active, reduce pain, and reduce visits.

  Currently, treatment options for SLE include flare prevention and its severity and duration, as well as the management of individual symptoms. Treatment includes corticosteroids, antimalarials, and nonsteroidal anti-inflammatory drugs. Certain types of lupus nephritis require several outs of cytotoxic drugs, such as cyclophosphamide and mycophenolate.

  Disease-modifying anti-rheumatic agents (DMARDs) can also be used to suppress flare and disease processes and reduce the need for steroid use. Commonly used DMARDs include antimalarial agents such as plaquinyl, and immunosuppressive agents such as methotexate and azathioprine.

  Corticosteroids and immunosuppressants are also used for disease control and flare prevention. However, the use of steroids can cause Cushing's syndrome and other side effects.

  Because most people with SLE suffer from varying amounts of chronic pain, different types of drugs are often used for pain management. Pain management typically begins with over-the-counter drugs, such as non-steroidal anti-inflammatory drugs. However, mild or intense opiates may be required for management of moderate or severe pain.

  In certain embodiments, the present disclosure provides a method of treating SLE in a subject with SLE and / or increasing IL-2 levels by administering an effective amount of a composition of the present disclosure. Administration of the compositions of the present disclosure can be used to reduce one or more symptoms of SLE and / or reduce the frequency or severity of flare. In certain embodiments, administration of a composition of the present disclosure is used to treat SLE in patients with lupus nephritis. In such cases, treatment of SLE may include treating lupus nephritis, for example, by alleviating the symptoms of lupus nephritis. In certain embodiments, the treatment comprises treating a skin symptom of SLE. In certain embodiments, the treatment alleviates one or more symptoms of lupus nephritis. In certain embodiments, treatment includes reducing, delaying or eliminating the need for dialysis. In certain embodiments, treatment includes reducing, delaying or eliminating the need for kidney transplantation. In certain embodiments, the treatment comprises delaying or preventing the progression of lupus nephritis to renal failure or end-stage renal disease.

  The present invention relates to a method of treating SLE, including the treatment of lupus nephritis, alone or in combination with one or more other drugs, biologics, or other therapeutic regimens. In part, a method comprising administering a composition of the present disclosure is contemplated. Other regimens selected as part of the treatment regimen can be selected depending on the severity of the patient's disease and primarily the particular condition being targeted. By way of example, in certain embodiments, a composition of the present disclosure may be used as an analgesic or other pain management drug (eg, to help relieve pain associated with arthritic symptoms or other symptoms), anti-inflammatory drugs , Steroids, immunosuppressants, antimalarials, and cytotoxic drugs may be administered as part of a treatment regimen. Other examples of agents and treatment regimes that can be used in combination include diet, exercise, stress management, acupuncture and physical therapy. As other specific examples, agents and therapies limited to alleviating the symptoms of lupus nephritis, such as antihypertensives, protein, potassium and / or sodium reduced diets, cytotoxic drugs, and / or immunosuppressants Use. Treatment methods include treatment in combination with dialysis, kidney transplantation, or one or more other renal failure therapeutics.

  Symptoms of the disease can be reproduced in animal models, and such models are easily used to confirm the effectiveness of monotherapy or combination therapy. For this reason, many mouse models have been developed (Foster, Relevance of Systemic Lupus Erythematosus Nephritis Animal Models to Human Disease. Seminephrol. 1999, 19 (1): 124). Such models are described in the host-graft interaction (Bruijn et al., Murine Chronic Graft-Versus-Host Disease as a Model for Lupus Nephritis. Am J Pathol., 1988, 130 (3): 639-643. Mice (Takahashi et al., Suppression of Experiential Lupus Nephritis by Aberrant Expression of the C, and Anti-the 3). An ti-DNA Antibodies in the Pathology of Lupus Nephritis-The Emerging Machinery. Autoimmunity Reviews, 2008, 7 (4): 317-321). These and other available animal models may be used in the course of developing diagnostic or therapeutic agents. In addition, in vitro systems, such as blood and tissue samples from subjects who have previously been diagnosed with SLE, may be used. Exemplary tissue samples include blood samples, bone marrow samples, and T cell cultures obtained after isolation of T cells from the blood sample.

Type I Diabetes Type 1 diabetes (type 1 diabetes, IDDM, or previously referred to as juvenile diabetes) is a form of diabetes that results from autoimmune destruction of pancreatic insulin-producing beta cells. Thereafter, insulin is deficient and glucose in blood and urine increases. Classic symptoms include polyuria, heavy drinking, overeating, and weight loss.

  Currently, type 1 diabetes generally results in death if not treated with insulin, which must be continued throughout the patient's lifetime. Even with treatment, complications of hypoglycemia and hyperglycemia can occur, including convulsions, loss of consciousness, and prolonged peripheral nerve and blood vessel damage.

  In certain embodiments, the compositions of the present disclosure may be used in the treatment of type I diabetes and / or to increase the expression of IL-2 in a subject with type I diabetes. Without being bound by theory, the use of such compositions can help to stop autoimmune attacks on pancreatic cells and thus help reduce or eliminate dependence on exogenous insulin. In certain embodiments, the compositions of the present disclosure are used in combination with insulin, but the use of such compositions reduces the frequency of requiring insulin injections and / or increases patient blood glucose levels. / The magnitude of the descent may be small.

  Symptoms of the disease can be reproduced in animal models, and such models are easily used to confirm the effectiveness of monotherapy or combination therapy. There are many animal models of type I diabetes, and many such available models are summarized in a recent review (Rees (2005) Diabetic Medicine 22: 359-370).

Rheumatoid arthritis Rheumatoid arthritis is a persistent disease that can affect joints in any part of the body, but is most commonly seen in the hands, wrists and knees. In rheumatoid arthritis, the immune system mistakenly attacks itself, thickening the intima of the joint. Thereafter, inflammation may spread around the tissue and eventually destroy cartilage and bone. In more severe cases, rheumatoid arthritis can affect other areas of the body such as the skin, eyes and nerves.

  Symptoms of rheumatoid arthritis include fatigue, fever, rash, joint inflammation, pain, tenderness around the affected joint, stiffness, redness and warmth around the affected joint, and reduced range of motion. Absent.

  Rheumatoid arthritis develops regardless of age, but is generally observed from 25 to 55 years old. Rheumatoid arthritis is 2-3 times more common in women than in men. Rheumatoid arthritis is the second most common form of arthritis, affecting 2.1 million people in the United States alone.

  For some people, rheumatoid arthritis can cause muscle weakness, tendon contraction, and destruction of the epiphysis, which can eventually deform the limbs. Current therapies include drugs to control pain, joint swelling and inflammation. These drugs include non-steroidal anti-inflammatory drugs, corticosteroids, mitotic inhibitors (methotrexate and cyclophosphamide), and anti-TNFα drugs to systemically suppress inflammatory responses. Other treatments include diet, exercise therapy and physical therapy aimed at helping to maintain muscle strength and range of motion, thereby delaying the effects of the disease causing disability.

  The methods of the present disclosure can be used in the treatment of rheumatoid arthritis and / or to increase the expression of IL-2 in patients with rheumatoid arthritis. In certain embodiments, the methods of the present disclosure are used to reduce one or more symptoms of rheumatoid arthritis. In other words, “treating” is intended to include reducing or eliminating one or more symptoms of rheumatoid arthritis. Further, the present disclosure provides that the composition of the present disclosure can be administered alone or as part of a treatment regimen with other agents useful for reducing one or more symptoms of rheumatoid arthritis. I'm also thinking about what to do. For example, the compositions of the present disclosure may be administered with analgesics, anti-inflammatory agents, and rheumatoid arthritis biologicals (eg, Humira, Simponi, Remicade, etc.).

  Symptoms of the disease can be reproduced in animal models, and such models are easily used to confirm the effectiveness of monotherapy or combination therapy. Many animal models of rheumatoid arthritis exist in the art. As a non-limiting example, BioMedCode Helas SA has created an animal model of an inflammatory condition. Many of the company's models have been approved by the FDA for testing potential treatments for RA. Such models include Tg197 mice and Tg5433 mice.

  Other animal models for arthritis and rheumatoid arthritis include, but are not limited to, the models described in the following publications: Hammer et al. 1990, Cell 63: 1099-1112; Haqqi et al. 1992, PNAS 89: 1253-1255; Keffer et al. 1991, EMBO Journal 10: 4025-4031; Pelletier et al. , 1997, Arthritis Rheum 40: 1012-1029; Trentham et al. , 1977, Journal of Experimental Medicine 146: 857-868; Wooley et al. , 1981, Journal of Experimental Medicine 154: 688-700. Recently, Bina and Wilder reviewed many available rheumatoid arthritis models. Bina and Wilder, 1999, Molecular Medicine Today 5: 367-369.

Autoimmune thyroid disease Autoimmune thyroid disease, eg, autoimmune thyroiditis, also called Hashimoto thyroiditis or chronic lymphocytic thyroiditis, is a condition in which the immune system attacks the thyroid gland. The thyroid gland, a small gland in front of the neck, is chronically inflamed and reduces the production of the thyroid hormones triiodothyronine and thyroxine. Because these hormones are used almost everywhere in the body, autoimmune thyroid disease can have widespread and severe effects and many symptoms.

  Mild autoimmune thyroiditis may be asymptomatic. However, in more severe cases, the thyroid gland may become enlarged due to inflammation (thyroid goiter). Gradually, the thyroid gland becomes more damaged and can cause symptoms of hypothyroidism (inactive thyroid gland) such as fatigue, weight gain, body pain, depression and lethargy. Chronic autoimmune thyroid disease can progress to significant hypothyroidism.

  The classification of autoimmune thyroid disease (AITD) includes Hashimoto thyroiditis (HT) or chronic autoimmune thyroiditis and its variants, Graves' disease (GD) and autoimmune atrophic thyroiditis. HT is characterized by the presence of goiter, thyroid autoantibodies against thyroid peroxidase (TPO) and thyroglobulin (Tg) in serum, and varying degrees of thyroid dysfunction. In HT, autoreactive CD4 + T lymphocytes (Th) recruit B cells and CD8 + T cells (CTL) to the thyroid. As the disease progresses, it results in death of thyroid cells and hypothyroidism.

  Both autoantibodies and thyroid-specific cytotoxic T lymphocytes (CTL) have been thought to be involved in the reduction of autoimmune thyroid cells. In GD, TSH-R is the most important autoantigen. Antibodies against this mimic the action of hormones on thyroid cells, stimulating the autonomous production of thyroxine and triiodothyronine to cause hyperthyroidism.

  The methods of the present disclosure may be used in the treatment of autoimmune thyroid disease and / or to increase IL-2 expression in patients with autoimmune thyroid disease. In certain embodiments, the methods of the present disclosure are used to reduce one or more symptoms of autoimmune thyroid disease. In other words, “treating” is intended to include reducing or eliminating one or more symptoms of autoimmune thyroid disease. Furthermore, the present disclosure provides that a composition of the present disclosure can be administered alone or together with other agents useful for reducing one or more symptoms of autoimmune thyroid disease. It is also contemplated that it may be administered as a part.

  Symptoms of the disease can be reproduced in animal models, and such models are easily used to confirm the effectiveness of monotherapy or combination therapy. Two exemplary mouse models are described in Volpe et al. 1993, Horm Metab Res 25: 623-627.

Scleroderma Scleroderma is a chronic autoimmune disease characterized by fibrosis, vascular alteration and autoantibodies. There are two main forms: localized systemic scleroderma and diffuse systemic scleroderma. Skin symptoms of localized systemic scleroderma involve the hands, arms and face. Patients with this form of scleroderma frequently have one or more of the following complications: calcification, Raynaud's phenomenon, esophageal dysfunction, hand sclerosis and telangiectasia.

  Diffuse systemic scleroderma progresses rapidly, affecting a large area of the skin and one or more internal organs, often the kidney, esophagus, heart and / or lungs.

  Scleroderma affects fine blood vessels called arterioles in all organs. First, endothelial cells of arterioles die one after another due to apoptosis together with smooth muscle cells. These cells replace collagen and other fibrous materials. Inflammatory cells, particularly CD4 + helper T cells, infiltrate arterioles and cause further destruction.

  Dermatologic symptoms of scleroderma can be painful, use of the affected area (e.g., use of hands, fingers, toes, feet, etc.) can be impaired and can also deform. Skin ulcers can occur and these ulcers are also susceptible to infections or even gangrene. Ulcered skin can be difficult or slow to heal. In patients with circulatory disorders, such as those with Raynaud's phenomenon, the difficulty of healing skin ulcers may be particularly increased. In certain embodiments, the methods of the present disclosure are used to treat a patient's scleroderma and / or increase IL-2 expression, such as, for example, scleroderma skin symptoms. In certain embodiments, the treatment of scleroderma comprises treating a skin ulcer, such as a finger ulcer. The administration of the composition of the present disclosure can be used to relieve fibrotic and / or inflammatory symptoms of scleroderma in affected tissues and / or organs.

  Scleroderma may affect the heart, kidneys, lungs, joints and gastrointestinal tract in addition to skin symptoms / symptoms. In certain embodiments, treatment of scleroderma comprises treating symptoms of any one or more diseases of these tissues, for example, by alleviating fibrotic and / or inflammatory symptoms.

  In certain embodiments, the methods of the present disclosure are used to treat scleroderma, such as pulmonary fibrosis associated with scleroderma. Administration of the composition of the present disclosure can be used to alleviate fibrotic symptoms of pulmonary scleroderma. For example, the method can be used to improve lung function and / or reduce the risk of death from scleroderma.

  In certain embodiments, the compositions of the present disclosure are used to treat scleroderma, eg, renal fibrosis associated with scleroderma. Administration can be used to relieve fibrotic symptoms of renal scleroderma. For example, the method can be used to improve renal function, reduce proteinuria, relieve high blood pressure, and / or reduce the risk of renal crisis that can lead to fatal renal failure. .

  The methods and compositions of the present disclosure can be used to treat scleroderma. Exemplary symptoms that can be treated include, but are not limited to, pain (including arthralgia), swelling, skin ulcers, skin irritation, rash, decreased range of motion, and reduced ability to perform daily tasks. It is not a thing. Other symptoms that can be treated include lung function (eg, can improve lung function) and kidney function. Improvement of any of these symptoms may include, for example, a decrease in the number of swollen joints, a decrease in the number of painful joints, an increase in range of motion, a progression of healing of ulcerated tissue, a decrease in the number of skin ulcers, Or reduced severity, increased ability to perform daily tasks, decreased skin disorders, decreased dependence on pain medications or other drugs, improved patient self-assessment for pain or quality of life, improved lung function, hypertension It can be measured by a decrease in protein, a decrease in proteinuria, and the like.

  In certain embodiments, a method of treating scleroderma comprises a composition of the present disclosure as part of a therapeutic regimen with one or more other drugs, biologicals, or therapeutic interventions suitable for scleroderma. Administration of the product. In certain embodiments, other drugs, biologics, or therapeutic interventions are suitable for certain symptoms associated with scleroderma. By way of example, the compositions of the present disclosure may be administered as part of a therapeutic regimen with one or more immunosuppressive drugs, such as methotrexate, cyclophosphamide, azathioprine and mycophenolate.

  Further, the treatment methods may include treatment regimens such as diet, exercise therapy, stress management, smoking cessation, acupuncture, massage and / or physical therapy.

  Symptoms of this disease can be reproduced in animal models and such models can be used to easily confirm the effectiveness of monotherapy or combination therapy. As an exemplary model, cyclosporin (Damoiseaux et al., Cyclosporine A-Induced Autoimmunity: An Animal Model for Human Schleroderma. J. Experimental 2000) In addition to rodent models in which scleroderma is induced by transgenic mice, and graft-versus-host models (Yamamoto, Characteristics of Animal Models for Scleriderma. Current Rheumatology Review 1, 200; Clark, A imal Models in Scleroderma.Current Rheumatology Reports, 2005,7 (2): 150-155) there is.

  The present disclosure relates to any of the disclosed compositions associated with treatment of any of the aforementioned conditions (eg, to alleviate one or more symptoms thereof) or with dysregulation of IL-2 expression. It can be used to treat autoimmune conditions.

(V) Methods of use (a) Diagnostic uses In certain embodiments, the disclosure may be used in vivo and / or in vitro to help diagnose, monitor or prognose a subject with an autoimmune condition, or Further provided are diagnostic methods that can be used to help improve the dosage of a drug (eg, including but not limited to a drug comprising a composition of the present disclosure).

  In an exemplary method, one or more (one, two, three) of microRNA-31, RhoA or IL-2 to be treated with a compound (any compound comprising a composition of the present disclosure). Assayed for a biomarker selected from The assay may be an assay for the presence, absence or amount of a biomarker. Suitable assays include quantitative PCR, ELIZA, and the like suitable for detection of transcript or protein expression in a sample. The assay may be performed using cells or using a sample, eg, a blood sample. Based on the assay results, it can be determined whether to adjust subsequent dosing or dosing regimens of the compound. For example, if an increase in IL-2 expression is not observed after one or more administrations of a microRNA-31 stimulant, the dosage or frequency of administration to achieve the desired increase in IL-2 expression It may be advantageous to increase.

  As another example, the present disclosure provides a method of diagnosing an autoimmune condition such as systemic lupus erythematosus. As part of these methods, a sample is taken from a subject suspected of having an autoimmune condition, such as SLE. The sample may be, for example, a bone marrow sample, a blood sample, or a sample of cells separated from the blood sample. Samples may be isolated during periods when the subject suspected of having the condition is experiencing significant symptoms, as well as during periods when symptoms are less acute. Following sample collection, the sample is assayed for expression of microRNA-31 or RhoA, or any one or more of microRNA31, RhoA or IL-2. Assays include protein expression and transcript expression assays.

  As another example, the present disclosure provides a method for monitoring treatment of an autoimmune condition such as systemic lupus erythematosus. To monitor treatment, microRNA-31, IL-2 and / or RhoA expression is detected in a sample of the subject undergoing treatment. This expression is compared to the expression of the biomarker in a sample taken from the same subject before the start of this particular treatment regimen or at an early time point during the treatment period. Comparing the expression of microRNA-31 and / or RhoA and / or IL-2 over one or more time periods provides a way to monitor the progress of treatment. An increase in microRNA-31 or a decrease in RhoA in later samples indicates the effectiveness of the treatment. Similarly, increasing IL-2 in later samples indicates the effectiveness of the treatment.

  As another example, the present disclosure provides a method of treating a subject having an autoimmune condition such as systemic lupus erythematosus. The method includes comparing the expression of microRNA-31 or RhoA from a sample taken from a subject prior to the start of a particular treatment to a standard range that reflects expression in a sample from a healthy subject. A subject is identified as a subject that may be suitable for treatment with a composition of the present disclosure if the expression of microRNA-31 is below the standard range or the expression of RhoA is above the standard range. If the subject is then confirmed to be sensitive to treatment, an effective amount of a composition comprising microRNA-31, siNA hybridizing to RhoA, IL-2 protein, or another composition of the present disclosure is used. Treat the subject.

  In certain embodiments, the method detects the expression of microRNA-31 or RhoA in a sample after treatment from the same subject, and the expression of microRNA-31 or RhoA in the sample after treatment. Further comprising comparing to expression in a sample taken before the start of a particular treatment.

  In certain embodiments, the particular therapeutic agent is a microRNA-31 mimic, such as microRNA-31, siNA that hybridizes to RhoA, or a small molecule inhibitor of RhoA.

  More generally, diagnostic use can be achieved, for example, by contacting the sample to be tested, optionally with a control sample, with a reagent suitable for the detection of nucleic acids or proteins.

  In certain embodiments of any of the foregoing, the diagnostic assay is performed using a human patient or a sample from a human patient. Suitable diagnostic reagents include, but are not limited to, probes and primers suitable for detection of microRNA-31 or RhoA expression and antibodies suitable for detection of RhoA protein expression. Reagents may be labeled to make assay detection and quantification cheap, and detection may depend on the use of secondary reagents.

(B) Therapeutic Uses In certain embodiments, the present disclosure contemplates that the compositions of the present disclosure can be used therapeutically, for example, in the treatment of human or non-human subjects. Any of the compositions described herein can be used to treat any of the autoimmune conditions described herein. Suitable compositions comprise nucleic acids, polypeptides or small molecules as active ingredients. Other suitable compositions include cell compositions, eg, cells that express microRNA-31.

  Exemplary methods include methods that involve contacting or administering a cell to a subject that increases the expression of microRNA-31 or decreases the expression of RhoA. Such compositions can be used, for example, to increase IL-2 expression in a subject having an autoimmune condition. Exemplary compositions include a nucleic acid comprising a nucleotide sequence that increases expression of microRNA-31 or a nucleic acid comprising a nucleotide sequence that decreases expression of RhoA.

  Exemplary conditions that can be treated include, but are not limited to, autoimmune conditions such as SLE or other autoimmune conditions that have the same mechanism of action or etiology. In certain embodiments, the method includes increasing the expression of IL-2 in the subject.

  Suitable compositions include, for example, a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. Other suitable compositions include, for example, nucleic acids comprising siNA, nucleic acids that mimic microRNA-31, or siNA that hybridizes to RhoA. The nucleic acid composition may be single stranded, partially double stranded, or fully double stranded. In certain embodiments, administration of the composition increases endogenous microRNA expression. In other embodiments, administration of the composition comprises the introduction of an exogenous microRNA-31 molecule, eg, an oligonucleotide that expresses microRNA-31 after introduction into the cell.

  Regardless of the particular composition administered, the patient is administered an effective amount. As used herein, the term “effective amount” reduces and / or reduces the severity and / or duration of a disease or disorder; prevents or delays the progression of the disease or disorder; Of a therapeutic agent sufficient to prevent or delay the recurrence, occurrence or onset of one or more symptoms associated with the disease or disorder, or to enhance or ameliorate the effect of another therapeutic agent Say quantity. It will be appreciated that measurable signs of efficacy may not be observable after a single dose.

  “Treating” a condition or disease refers to curing or alleviating at least one symptom of the condition or disease, reducing the frequency of symptoms of the subject's medical condition, or Administering a delayed composition.

  In certain embodiments, the progress and effectiveness of treatment is monitored during and / or after treatment. For example, autoimmune conditions may be determined using methods appropriate to the individual condition, such as blood tests, urine samples, pain and fatigue self-reports, reduced need for pain medications, X-rays, CT scans and the like. Can be monitored. In addition, treatment may improve symptoms, such as reduced pain (eg, reduced pain medications required / used by the patient), reduced dependence on supplemental oxygen, improved appetite, weight gain, reduced fatigue, increased mobility. And monitoring based on the evaluation of the same. Furthermore, the progress and efficacy of the treatment may be monitored using any one or more of the biomarkers microRNA-31, RhoA or IL-2 as described above.

  In certain embodiments, in certain embodiments, the compositions of the present disclosure are administered as part of a therapeutic regimen along with one or more other agents and / or one or more other modes of treatment. To do. Which other agents and / or treatment regimes should be selected may vary depending on the particular disease, patient condition, patient age, symptoms and the like. By way of example, other suitable treatment regimes include, but are not limited to, surgery, dialysis, insulin therapy, diet, physical therapy, smoking cessation, oxygen therapy, ventilatory assistance, acupuncture and the like. . By way of example, other suitable agents include analgesics, narcotics (such as for pain management), anti-inflammatory drugs, immunosuppressants, corticosteroids, antimalarials, and the like. It is not limited. Any one or more of these agents and / or schemes can be used as part of a treatment regimen.

  Any treatment method involving a combination of agents and / or therapeutic agents can achieve such a combination treatment by administering individual components of the treatment simultaneously, sequentially or separately.

  In certain embodiments, the therapeutic method further comprises an assay step of assessing the expression of the subject's microRNA-31, RhoA and / or IL-2 after treatment with the composition of the present disclosure. Such assay steps can assess RNA or protein expression using suitable methods.

(Vii) Dosage and Administration Embodiments of the present disclosure include sterile pharmaceutical formulations that are useful from the perspective of administering the composition to a subject and / or for use in a diagnostic setting. In addition, various administration and delivery routes and methods may be useful. The present disclosure contemplates that any dosage formulation and / or route of administration may be used with any of the compositions of the present disclosure. Furthermore, the present disclosure contemplates that various dosage formulations and routes of administration are applicable to other agents, for example, agents administered as part of a therapeutic regimen.

  Various delivery systems are known and can be used to administer the compositions of the present disclosure. Administration methods include parenteral (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, topical and mucosal (eg, intranasal and oral routes) It is not limited to this. Administration can be systemic or local. It should be noted that when the formulation is administered as part of a combination therapy, the present disclosure contemplates that other agents may be administered by the same route of administration or by different routes of administration. .

  In certain embodiments, the compositions of the present disclosure include a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutically acceptable carrier is water for injection (USP), 5% dextrose solution (D5W) or saline.

  For some formulations, an aqueous formulation is utilized; for other formulations, the formulation may be lipidic. In certain embodiments, the composition comprising an active pharmaceutical agent or a nucleic acid encoding it is an aqueous formulation. In other embodiments, the formulation is lipidic.

  Solutions of active pharmaceutical agents described herein may be prepared as the free base or as pharmacologically acceptable salts. In some embodiments, such agents may also be prepared with water suitably mixed with a surfactant, such as hydroxypropylcellulose. Alternatively, a dispersion may be prepared using glycerol, liquid polyethylene glycol, a mixture thereof, and oil. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for use in injection include sterile aqueous solutions or dispersions and sterile powders prepared as needed with sterile injectable solutions or dispersions. This form is often sterile, easy to operate with a syringe and has some fluidity. This form can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

  The exact dose and dosage regimen used will depend on the route of administration, the particular disease being treated, the severity of the patient's condition, the particular composition used and the like. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  In certain embodiments, particularly for formulations intended for human administration, the formulation is a pyrogen-free formulation that is substantially free of endotoxin and / or related pyrogens. Endotoxins include toxins that are confined within microorganisms and are released only when the microorganisms are destroyed or die. Examples of pyrogens include heat-resistant substances (glycoproteins) that induce heat generation from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock when administered to humans. Even small amounts of endotoxins must be removed from intravenously administered pharmaceutical solutions due to potential adverse effects. The Food and Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram of body weight per hour for intravenous administration of the agent (The United States Pharmaceuticals) Convention, Pharmacopeia Forum 26 (1): 223 (2000)). When therapeutic proteins are administered in quantities of hundreds or thousands of milligrams per kilogram of body weight, harmful and dangerous endotoxins, as well as antibodies, must be removed in trace amounts. In certain embodiments, the level of endotoxin and pyrogen in the composition is less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or 0.01 EU / mg. Less than or less than 0.001 EU / mg.

  A compound (eg, a composition or other agent of the present disclosure) may be administered to any suitable subject having a biomarker or in need of treatment for a condition described herein. Non-limiting examples of subjects include mammals, humans, apes, monkeys, ungulates (eg, horses, cows, goats, sheep, pigs, buffaloes, camels and the like), dogs, cats, rodents And the like. The subject may be male or female, and the drug may be administered to subjects of a particular age group, such as young, children, adolescents, adults and the like. In certain embodiments, the compositions or other agents of the present disclosure may comprise antibodies, antibody fragments, single chain antibodies, small molecules, nucleic acids, nucleic acid derivatives, miRNA, siNA, peptides, polypeptides and the like as active ingredients. Including things. Various forms of miRNA or siRNA may be delivered, such as miRNA or siRNA after processing, miRNA or siRNA before processing, or a vector encoding miRNA or siRNA before or after processing.

  The methods provided herein include, but are not limited to, delivery of an effective amount of a nucleic acid or expression construct encoding a nucleic acid. An “effective amount” of a pharmaceutical composition is generally defined as an amount sufficient to detectably and repeatedly achieve a specified desired result, for example, to reduce, alleviate, minimize or limit the extent of the disease or its symptoms. Is done. In some embodiments, there may be steps of monitoring biomarkers to assess the effectiveness of the treatment and control toxicity.

  The agent is administered in an amount that is compatible with the dosage formulation and may be therapeutically effective. Nucleic acid injections are delivered by syringe, or any other method used to inject solutions, so long as the nucleic acid and any related components can pass through the needle of the specific gauge required for the injection. be able to. A syringe system that can accurately inject a given amount of solution multiple times at any depth has been described for gene therapy (US Pat. No. 5,846,225).

  In certain embodiments, a composition of the present disclosure comprises a nucleic acid that increases microRNA-31 expression or decreases RhoA expression. In certain embodiments, compositions using such nucleic acids may be administered as gene therapy. Gene therapy is treatment performed by administering an expressed nucleic acid or an expressible nucleic acid to a subject. In this embodiment of the present disclosure, a nucleic acid (such as an antisense, siRNA or microRNA) is produced that mediates a prophylactic or therapeutic effect.

  Any of the gene therapy methods available in the art can be used in accordance with the present disclosure. An exemplary method is described below. For a general review of gene therapy methods, see Goldspiel et al. 1993, Clinical Pharmacy 12: 488; Wu and Wu, 1991, Biotherapy 3:87; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573; Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191; May, 1993, TIBTECH 11: 155. Methods known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manufacture, 19 (N).

  In one embodiment, the composition using the nucleic acid is provided as part of an expression vector that expresses the nucleic acid in a suitable host. In particular, such nucleic acids have a promoter, eg, a heterologous promoter, said promoter being an inducible promoter or a constitutive promoter, optionally a tissue specific promoter.

  Delivery of the nucleic acid to the subject may be direct delivery, in which case the subject is contacted directly with the nucleic acid or a vector carrying the nucleic acid, or may be indirect delivery, in which case cells are first transformed with the nucleic acid in vitro. And then transplanted into the subject. Thus, in certain embodiments, the composition of the present disclosure comprises a cell composition. These two approaches are known as in vivo gene therapy or ex vivo gene therapy, respectively. A suitable vector is a viral vector. In other embodiments, the nucleic acid may be administered as a naked nucleic acid. Furthermore, in certain embodiments, compositions using nucleic acids may be administered by transduction / infection, DEAE-dextran mediated transfection, lipofection, electroporation and iontophoresis.

(Viii) Product The present disclosure provides a medical pack or kit comprising one or more containers containing a composition of the present disclosure. Similarly, the present disclosure provides medical packs or kits suitable for laboratory and / or diagnostic use. The disclosure includes any of the compositions described herein, eg, a composition comprising a nucleic acid comprising a nucleotide sequence that increases expression of microRNA-31, or a nucleic acid comprising a nucleotide sequence that reduces expression of RhoA. It is contemplated that the composition may be packaged and sold as part of the kit. An exemplary such kit is a pharmaceutical kit.

  The present disclosure also provides a formulation as described herein in one or more first containers and an autoimmune condition, eg, SLE, or an autoimmune condition having a mechanism or etiology similar to SLE ( For example, one or more other prophylactic or therapeutic agents useful for the prevention, management or treatment of conditions correlated with decreased expression of IL-2 or conditions resulting from poor T cell control There is also provided a medical pack or kit for inclusion in one or more second containers.

  In an exemplary embodiment, the formulations of the present disclosure are formulated as sterile formulations in single dose vials. These containers may optionally be accompanied by notices in the form prescribed by government agencies that regulate the manufacture, use or sale of medicinal products or biological substances, which may be manufactured, used or administered for human administration. Approval by the institution for sale is indicated.

  In the case of kits sold for laboratory and / or diagnostic purposes, the kit may optionally include notices indicating proper use, safety considerations and any restrictions on use. Further, in the case of a kit sold for laboratory and / or diagnostic purposes, the kit optionally includes one or more other reagents, eg, positive controls useful for a particular diagnostic or laboratory use. Alternatively, a negative control reagent may be included.

  The present disclosure provides kits that can be used in the above methods. In one embodiment, the kit includes a composition as described herein in one or more containers. In another embodiment, the kit comprises a composition as described herein in one or more containers, and an SLE, or autoimmune condition, eg, an autoimmune condition having a mechanism or etiology similar to SLE. One or more other prophylactic or therapeutic agents useful for the prevention, management or treatment of (eg, a condition correlated with decreased expression of IL-2 or a condition resulting from poor T cell control) including. Preferably, the kit is used in addition to instructions for preventing, treating, managing or reducing the disorder (eg, using the described formulation alone or in combination with another prophylactic or therapeutic agent). In addition, information on side effects and dosages is included.

  The present disclosure further encompasses the final pharmaceutical product packaged and labeled. The product includes a unit dosage form suitable for a suitable container or container, such as a glass vial, or other sealed container. In the case of dosage forms suitable for parenteral administration, the active ingredient is sterile and suitable for administration. In certain embodiments, the formulation is suitable for intravenous administration.

  In preferred embodiments, the unit dosage form is suitable for intravenous delivery, intramuscular delivery, intranasal delivery, oral delivery, topical delivery or subcutaneous delivery. Thus, the present disclosure includes solutions that are preferably sterile and suitable for each delivery route.

  As with any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and shipping. In addition, the product of the present disclosure includes instructions for use or other informational material that advises a physician, technician or patient on how to properly prevent or treat the condition in question. In other words, the product includes, but is not limited to, explanatory means to indicate or recommend an administration regimen, including actual doses, monitoring procedures, etc., and other monitoring information.

  Specifically, the present disclosure provides packaging materials such as boxes, bottles, tubes, vials, containers, nebulizers, insufflators, infusion (iv) bags, envelopes and the like, and packaging materials such as A product comprising at least one unit dosage form of a pharmaceutical agent contained therein, wherein the pharmaceutical agent comprises a composition of the present disclosure and the packaging material is associated with SLE using the composition. One or more symptoms, or another autoimmune condition, eg, an autoimmune condition having a mechanism or etiology similar to SLE (eg, a condition correlated with decreased expression of IL-2, or a T cell A condition resulting from poor control of) or one or more symptoms thereof, administering a specific dose and using a specific dosing regimen as described herein More prevention, management, provides a product containing a description means for instructing that may be treated and / or alleviated.

  The present disclosure also includes packaging materials, such as boxes, bottles, tubes, vials, containers, nebulizers, insufflators, infusion (iv) bags, envelopes, and the like, and each included in the packaging material A product comprising at least one unit dosage form of a pharmaceutical agent, wherein one pharmaceutical agent comprises a composition of the present disclosure and the other pharmaceutical agent comprises a prophylactic or therapeutic agent other than the composition of the present disclosure; The packaging material uses the agent to produce one or more symptoms associated with SLE, or another autoimmune condition, such as an autoimmune condition (e.g. IL -2 in relation to decreased expression of -2, or due to poor T cell control) or one or more symptoms thereof, Treated by the use of specific dosing regimens as described Saisho, provides an article of manufacture comprising a description means for indicating that it is possible to prevent and / or reduce.

  In certain embodiments, particularly in the case of a pharmaceutical kit comprising a formulation intended for human administration, the formulation is a pyrogen-free formulation substantially free of endotoxin and / or related pyrogens. Endotoxins include toxins that are confined within microorganisms and are released only when the microorganisms are destroyed or die. Examples of pyrogens include heat-resistant substances (glycoproteins) that induce heat generation from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock when administered to humans. Even small amounts of endotoxins must be removed from intravenously administered pharmaceutical solutions due to potential adverse effects. The Food and Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram of body weight per hour for intravenous administration of the agent (The United States Pharmaceuticals) Convention, Pharmacopeia Forum 26 (1): 223 (2000)). In certain embodiments, the level of endotoxin and pyrogen in the composition is less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or 0.01 EU / mg. Or less than 0.001 EU / mg.

  The present disclosure will now be described with reference to the following examples. These examples are provided for purposes of illustration only and should not be construed as limiting the disclosure in any way to these examples, but rather are apparent from the results of the teachings provided herein. Should be construed to include any such modifications. Generally speaking, the present disclosure utilizes conventional techniques of molecular biology, chemistry, biochemistry, biophysics, recombinant DNA technology and immunology unless otherwise stated. Of course, it will be understood that the specific list and description of the particular equipment and reagents used, size, manufacturer, etc., should not be considered limiting of the disclosure unless specifically limited. . It will be further understood that other devices and reagents that perform similar functions may be used instead at any time.

Example 1-Expression of microRNA-31 We measured the expression level of miR-31 in T cells, B cells and monocytes isolated from PBMC of 3 healthy volunteers. As shown in FIG. 1A, miR-31 was selectively expressed in T cells, suggesting that miR-31 may play a role in T cell function.

  We then investigated the expression of miR-31 in T cells taken from 32 SLE patients and 11 normal controls (NC), as shown in FIG. It was found that miR-31 was significantly down-regulated (P <0.0001). Detailed information about the subjects used in these studies is shown in Table 1.

Example 2-Correlation between microRNA-31 expression and IL-2 expression T cells from lupus patients display many signaling abnormalities that may be involved in the pathogenesis of SLE (Kong et al., 2003). Multon et al. 2011). The following experiment investigated the role that miR-31 plays in the heterogeneous expression of T cell-related cytokines in SLE. As part of these experiments, a microRNA-31 mimetic was designed and used for microRNA-31 expression, and antagomir-31 was designed and used to reduce microRNA-31 expression. Appropriate oligonucleotide controls were also used. As shown in FIGS. 2A and 2B, transfection of these constructs into primary T cells efficiently increased (if mimetics) or decreased microRNA-31 expression (antagomir). in the case of). Successful ectopic expression or silencing of miR-31 in primary T cells was demonstrated using TaqMan quantitative PCR. TaqMan quantitative PCR showed that miR-31 expression increased or decreased 25-fold by miR-31 overexpression or inhibition after transfection, respectively (FIG. 2). In light of the effectiveness of these reagents in modulating miR-31 expression, these reagents were used to further study the signaling of the cells.

  To evaluate whether binding with TCR affects miR-31 expression in a time course study, CD3 + T lymphocytes freshly purified from healthy donors were stimulated with PMA and ionomycin. As shown in FIG. 3A, miR-31 expression was induced after stimulation and peaked after about 12 hours.

  Since activation of T cells via TCR enhances the induction of IL-2, the possibility of the regulation of IL-2 production by miR-31 was evaluated. To address this issue, human primary T cells were transfected with miR-31 mimics or control mimics and then activated with PMA and ionomycin for 24 hours. Intracellular IL-2 mRNA levels and IL-2 protein levels in the supernatant were measured by RT-PCR and ELISA, respectively. As shown in FIGS. 3B and 3C, miR-31 mimics increased IL-2 expression at both the mRNA and protein levels, whereas the inhibitory oligonucleotide Antagomir-31 was transfected. Silencing of endogenous miR-31 by efduction reduced IL-2 production. As shown in FIG. 3D, consistent with these results, a positive correlation was also observed between miR-31 expression levels and IL-2 expression levels in activated lupus T cells (n = 15). It was.

  To ask whether miR-31 controlled IL-2 promoter activity, a luciferase reporter assay was performed. The IL-2 promoter was cloned into a luciferase reporter vector (referred to as IL-2-luc). Jurkat cells co-transfected with IL-2-luc and miR-31 mimics or controls were stimulated with PMA and ionomycin. As shown in FIG. 3E, miR-31 increased IL-2 promoter activity and dose-dependently (FIG. 3F). Taken together, the above data suggests that miR-31 promotes IL-2 production through enhanced activity of the IL-2 promoter. In addition, the relationship between IL-2 and miR-31 was consistently observed in each sample from lupus patients.

Example 3-T cell RhoA
To gain insights into the molecular mechanism of how miR-31 regulates IL-2 production, bioinformatics tools were used to identify potential miR-31 targets. A list of predicted miR-31 targets was obtained from the miRGen database (www.diana.pcbi.upenn.edu/miRGen/v3/miRGen.html) integrating analysis of TargetScan, PicTar and MiRanda. RhoA has been reported to regulate IL-2 gene expression with T cell activation (Helms et al., 2007), and was a candidate target for miR-31 in this system.

  To assess whether miR-31 inhibits Rho cell expression of T cells, primary T cells were transfected with miR-31 mimics or controls, and RhoA expression was measured by RT-PCR and Western blotting. RT-PCR demonstrated that miR-31 inhibits RhoA mRNA expression (FIG. 4A). Western blotting analysis revealed that RhoA protein was reduced by miR-31 transfection (FIG. 4B). In light of the fact that miR-31 expression levels in SLE samples were significantly lower compared to NC-derived samples, we investigated whether RhoA expression was increased in SLE T cells. T cells from 32 SLE patients and 11 NCs showed that RhoA mRNA was significantly upregulated in SLE T cells (FIG. 4C). Furthermore, linear correlation analysis demonstrated that RhoA mRNA expression was negatively correlated with miR-31 expression in primary T cells of lupus patients (FIG. 4D). Taken together, the above data suggest that miR-31 targets RhoA in human primary T cells and RhoA mRNA expression is significantly higher in lupus T cells compared to NC.

Example 4 Inhibition of RhoA Expression To test the hypothesis that miR-31 regulates T cell IL-2 expression via RhoA, the inhibitory effect of RhoA was investigated in this system, and such inhibition was miR It was evaluated whether it had an effect equivalent to -31 overexpression. RNA interference technology was used to knock down the expression of RhoA. As shown in FIGS. 5A and 5B, RhoA siRNA for 48 hours transfected significantly reduced RhoA mRNA and protein levels as observed for overexpression of miR-31 mimics.

  RhoA siRNA or miR-31 mimics were then transfected into primary T cells for 48 hours and stimulated with PMA and ionomycin for 24 hours. Following transfection and stimulation, IL-2 expression was measured. Consistent with the action of miR-31 mimics, knocking down RhoA significantly increased IL-2 mRNA and protein expression (FIGS. 6A and 6B). This additional evaluation supports the conclusion that RhoA knockdown enhanced the activity of the IL-2 promoter (FIG. 6C).

Example 5 Increased Expression Levels of IL-2 in Activated Lupus T Cells Considering that miR-31 is induced in activated T cells and targets RhoA, we control IL-2 production. The expression levels of miR-31, RhoA and IL-2 were investigated in activated T cells isolated from 15 SLE patients and 10 normal controls (NC). Detailed information about the subjects used in these studies is shown in Table 2. These results revealed that miR-31 and IL-2 expression in activated T cells from SLE patients is lower than that observed in NC, while RhoA expression is high (FIGS. 7A, B). And C).

  To further investigate this relationship, miR-31 levels were manipulated to assess any effect on IL-2 production in T cells from SLE patients. Lupus T cells were transfected with miR-31 mimic or control mimic. After 24 hours stimulation with PMA and ionomycin, an increase in IL-2 protein expression was observed in activated lupus T cells transfected with miR-31, as shown in FIG. 7D. This is consistent with the level of repair of IL-2 production observed in lupus T cells from miR-31.

Materials and Methods Below are a summary of some materials and methods utilized in practicing the foregoing Examples 1-5.

  Patients and healthy controls. All SLE patient samples were obtained from the Department of Rheumatology of Renji Hospital (Shanghai, China) and were at least in the 1982 revision of the American College of Rheumatology Standards of the American College of Rheumatology. SLE activity was assessed by SLE disease activity index (SLEDAI). Healthy controls from healthy volunteers had no history of autoimmune disease or immunosuppressive therapy and matched patient, age, gender, and race. All participants were Chinese groups. Peripheral blood samples (10 mL) collected from each subject were collected in tubes containing acid citrate dextroforma A (ACD-A). This study was approved by the Research Ethics Board of Renji Hospital, Shanghai JiaoTong University, School of Medicine.

  Isolation of CD3 + T cells. Peripheral blood mononuclear cells (PBMC) were separated from heparinized whole blood by density gradient centrifugation using Lymphoprep Ficoll-Paque ™ PLUS (GE Healthcare, Charlotte, UK). CD3 + T cells were purified from fresh PBMC by positive selection using magnetic CD3 microbeads (Miltenyi Biotec) according to the manufacturer's protocol. The purity of T lymphocytes was> 95% as analyzed by FACSCalibur (Becton Dickinson).

Cell culture and stimulation conditions. Purified T lymphocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and PMA (50 ng / ml, Sigma-Aldrich) at 37 ° C., 5% CO _2. ) And ionomycin (1 μg / ml, Sigma-Aldrich) for various times. The T cell leukemia strain Jurkat was grown in RPMI-1640 medium (Life Technologies, Rockville, MD) containing 10% FBS and 1% penicillin / streptomycin at 37 ° C., 5% CO ₂ . Jurkat cells were also stimulated with PMA (50 ng / ml) and ionomycin (1 μg / ml).

  miRNA mimics, small interfering RNAs and antagomir. Small interfering RNA (siRNA) and miRNA mimics were synthesized by Genepharma (Shanghai, China). The RhoA siRNA sequences referred to in (Ahmed et al., 2005) are as follows: RhoA-siRNA-1: 5′-AAGATTATGACCGTCTGAGGGC-3 ′ (SEQ ID NO: 1); RhoA-siRNA-2: 5 '-AAGGATCTTCGGA ATGATGAG-3' (SEQ ID NO: 2). The sequence of the miRNA-31 mimetic is as follows: Mimic: 5'-AGGCAAGAUUGCUGGCAAUAGCU-3 '(SEQ ID NO: 3). Antagomir-31 and control constructs were ordered from Ribo biology (Guang Dong, China). The sequence of antagomir-31 is as follows: antagomir: 5'-AGCUAUUGCCAGCAUCUUGCCU-3 '(SEQ ID NO: 4). Function acquisition experiments (mimetic-induced down-regulation of target genes normally inhibited by miRNA-31) and loss-of-function experiments (antagomir-induced up-regulation of target genes normally inhibited by miRNA-31) From the combination, the relationship between the miRNA and the target was proved, and the functional analysis of the miRNA became possible. In each of the aforementioned oligonucleotides, the siRNA and miRNA-31 mimics used were double stranded molecules, and antagomir was a single stranded molecule. Each oligonucleotide was expressed exogenously in the cells by transfecting the cells with Lipofectamine 2000 (Invitrogen).

Transfection. Human primary T cells were left in RPMI 1640 for 2 hours and then transfected with miRNA or siRNA oligonucleotides using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Six hours after transfection, T cells were added to fresh complete medium (RPMI-1640 medium supplemented with 10% FBS). After 24 hours, cells were stimulated with PMA and ionomycin for up to 24 hours at 37 ° C., 5% CO ₂ . For inhibition of miR-31, human primary T cells were resuspended in serum-free opti-DMEM medium (GIBCO) and transfected with 500 nM antagomir-31 or scrambled antagomir as a control. Six hours after transfection, RPMI medium supplemented with 500 nM antagomir was added and cells were stimulated 24 hours later as described above.

Quantitative PCR. Total RNA was isolated with TRIzol reagent (Invitrogen). To quantify miRNA, RNA (20 ng) samples were reverse transcribed using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). For real-time PCR, TaqMan MicroRNA assay was used according to the manufacturer's (Applied Biosystems) recommendations. RNU48 (U48) was used as an endogenous control for correction of expression values. For quantitative RT-PCR analysis of mRNA, 500 ng of total RNA was reverse transcribed using PrimeScript RT reagent Kit (Takara Bio Inc., Shiga, Japan). The cDNA was amplified by real-time PCR using SYBR Green (SYBR Premix Ex Taq RT-PCR kit; Takara Bio Inc. (Takara), Shiga, Japan), and cDNA was used using ribosomal protein L13A (RPL13A) as an internal control. The amount was corrected. TaqMan and SYBR Green assays were performed in duplicate using a 7900HT Fast Real-Time PCR instrument (Applied Biosystems). Relative expression levels were calculated using the 2- ^ΔΔct method. The following custom primers were used for real-time PCR using SYBR Green: RPL13A, forward 5′-CCTGGAGGAAGAGAGAGAAAGAGA-3 ′ (SEQ ID NO: 5), reverse 5′-TTGAGGACCCTCTGTGTATTGTCAA-3 ′ (SEQ ID NO: 6). RhoA, forward 5′-TCTTCGGAATGATGAGCAC-3 ′ (SEQ ID NO: 7), reverse 5′-CTTTGGTTCTTTGCTGAACAC-3 ′ (SEQ ID NO: 8). IL-2, forward 5′-TGCCACAATGTACAGGATGC-3 ′ (SEQ ID NO: 9), reverse 5′-GCCTTCTTGGGGCATGTAAAA-3 ′ (SEQ ID NO: 10). Other primers were also commercially available and / or correspond to published sequences. Another primer can also be easily prepared.

  Reporter construct cloning, transient transfection and luciferase assay. PGL3-Basic Luciferase Reporter Vector (Promega) was used for the production of the IL-2 promoter reporter construct. Briefly, an 884 bp promoter region (-59 to +825 bp base) was amplified from genomic DNA by PCR. The primers used were forward 5'-CATTTCATGTGCCCAGGGTG-3 '(SEQ ID NO: 11), reverse 5'-CATTGTGGCAGGAGTTGAG-3' (SEQ ID NO: 12).

The forward and reverse primers were each provided with an Mlu I site and an Xho I site, and the PCR product was ligated to the pGL3-Basic plasmid according to the manufacturer's instructions. Jurkat cells were seeded in 24-well plates at 1 × 10 ⁶ cells / well and transfected 2 hours later. Using Lipofectamine 2000 (Invitrogen) with png3-basic luciferase reporter plasmid (1 ug) containing the IL-2 promoter, miRNA oligonucleotide or siRNA described above, together with 50 ng of pRL-basic-luc vector used to correct transfection efficiency. Were cotransfected into Jurkat cells. After a 24 hour recovery period, transfected cells were left untreated or stimulated with PMA and ionomycin for 24 hours. Luciferase activity was then evaluated by Dual-Luciferase Reporter Assay System (Promega, Madison, WI) performed using a CENTRO XS ³ LB 960 (Berthold Technologies) instrument according to the manufacturer's protocol. The ratio of Renilla luciferase to firefly luciferase was obtained for each well. All experiments were performed in triplicate.

  Enzyme immunoassay (ELISA). The ELISA for IL-2 protein secreted into the cell culture supernatant was quantified using a commercially available kit (Xi Tang Biology, Shanghai, China) according to the manufacturer's protocol.

Western blotting. In a 6-well plate, T cells are seeded at 5 × 10 ⁶ cells / well and miRNA oligonucleotides are transfected into each well to give a final concentration of miR-31 mimic or control mimic of 100 nM, siRNA RhoA or control. The final concentration of was also 100 nM. Six hours after transfection, T cells were added to fresh complete medium. Two days after transfection, cells were lysed and proteins were extracted. The supernatant was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted with the indicated antibody, and detected with Luminol / Enhancer Solution (Pierce, Rockford, IL). Western blotting of RhoA and Tubulin was performed using mouse anti-RhoA antibody (Santa Cruz, CA, 1: 200) and rabbit anti-Tublin antibody (Santa Cruz, CA, 1: 5,000). For quantification of protein bands, the volume tool of the software Quantity One (Bio-Rad) was used according to the manufacturer's manual.

  Data analysis. Data was analyzed using Prism 4 software, version 4.03 (GraphPad Software, San Diego, Calif.). A non-parametric Mann-Whitney test was used to compare gene expression between the two groups, and an unpaired Student t test was used to compare reporter gene activity. A P value (two-sided) was statistically significant when it was less than 0.05.

Sequence Listing SEQ ID NO: 1 (RhoA-siRNA-1) -AAGATTATGACCGTCTGAGGGC
SEQ ID NO: 2 (RhoA-siRNA-2) -AAGGATCTTCGGAATGATGAG
SEQ ID NO: 3 (miRNA-31 mimetic)-AGGCAAGAUGCUGGCAUAGCU
SEQ ID NO: 4 (antagomir of miRNA-31) -AGCUAUUGCCAGCAUCUGUGCU
SEQ ID NO: 5 (forward primer for RPL13A) -CCTGGAGGGAGAAGAGGAAAGAGA
SEQ ID NO: 6 (reverse primer for RPL13A) -TTGAGGGACCTCTGTGATTTTGTCAA
SEQ ID NO: 7 (RhoA forward primer) -TCTTCGGAATGATGACAC
SEQ ID NO: 8 (RhoA reverse primer) -CTTTGGTCTTTGCTGAACAC
SEQ ID NO: 9 (IL-2 forward primer) -TGCCCAAATGTACAGGATGC
SEQ ID NO: 10 (reverse primer for IL-2) -GCCTTCTTTGGGCATGTAAAA
SEQ ID NO: 11 (promoter forward primer) -CATTTCATAGGTCCCAGGTG
Sequence number 12 (reverse primer of a promoter) -CATTGTGGCAGGAGGTTGAG

References Fairhurst et al. (2006) Adv Immunol 92: 1-69.
Anders et al. (2009) Trends Mol Med 15 (12): 553-561.
Nambiar et al. (2004) Methods Mol Med 102: 31-47.
Fujii et al. (2006) Autoimmune Rev 5 (2): 143-144.
Kong et al. (2003) Ann NY Acad Sci 987: 60-67.
Multon et al. (2011) Arthritis Res Ther 13 (2): 207.
Lieberman et al. (2010) J Biomed Biotechnol Article ID 740619 (6 pages) [doi: 10.15155 / 2010/740619]
Tenblock et al. (2004) Int Rev Immunol 23 (3-4): 333-345.
Katsiari et al. (2005) J Clin Invest 115 (11): 3193-3204.
Jung et al. (2005) J Clin Invest 115 (4): 996-1005.
Herndon et al. (2002) Clin Immunol 103 (2): 145-153.
Solomou et al. (2001) J Immunol 166 (6): 4216-4222.
Denli et al. (2004) Nature 432 (7014): 231-235.
Gregory et al. (2004) Nature 432 (7014): 235-240.
Ambros. (2004) Nature 431 (7006): 350-355.
Pauley et al. (2009) J Autoimmun 32 (3-4): 189-194.
Lu et al. (2010) Cell 142 (6): 914-929.
O'Connell et al. (2010) Immunity 33 (4): 607-619.
Pauley et al. (2008) Ann NY Acad Sci 1143: 226-239.
Tang et al. (2009) Arthritis Rheum 60 (4): 1065-1075.
Pan et al. (2010) J Immunol 184 (12): 6773-6781.
Zhao et al. (2011) Arthritis Rheum 63 (5): 1376-1386.
Yu et al. (2010) Cell Mol Immunol 7 (3): 198-203.
Stitrich et al. (2010) Nat Immunol 11 (11): 1057-1062.
Yu et al. (2009) Development 136 (9): 1497-1507.
Curlete et al. (2010) Blood 115 (2): 265-273.
Thai et al. (2007) Science 316 (5824): 604-608.
Rodriguez et al. (2007) Science 316 (5824): 608-611.
Ahmed et al. (2005) Mol Cell Neurosci 28 (3): 509-523.
Helms et al. (2007) J Leukoc Biol 82 (2): 361-369.
Valastyan et al. (2009) Cee 137 (6): 1032-1046.
Katsiari et al. (2006) Autoimmune Rev 5 (2): 118-121.
Setoguchi et al. (2005) J Exp Med 201 (5): 723-735.
Fontenot et al. (2005) Nat Immunol 6 (11): 1142-1151.
Crispin et al. (2009) autoimmune Rev 8 (3): 190-195.
Gomez-Martin et al. (2009) Autoimmune Rev 9 (1): 34-39.
Nambiar et al. (2003) J Immunol 170 (6): 2871-2876.
Nambiar et al. (2003) Arthritis Rheum 48 (7): 1948-1955.
Bodor et al. (2007) J Leukoc Biol 81 (1): 161-167.
Bodor et al. (2000) J Leukoc Biol 67 (6): 774-779.
Bodor et al. (1998) J Biol Chem 273 (16): 9544-9551.
Garcia et al. (2005) J Proteome Res 4 (6): 2032-2042.
Hu et al. (2008) J Rheumatol 35 (5): 804-810.
Creighton et al. (2010) Cancer Res 70 (5): 1906-1915.
Schmidt et al. (2004) Lupus 13 (5): 348-352.
Hagiwara et al. (2000) Cytokine 12 (7): 1035-1041.
Blenman et al. (2006) Lab Invest 86 (11): 1136-1148.
Prud'homme. (2000) J Gene Med 2 (4): 222-232.

Incorporation All publications and patents mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically indicated for incorporation.

  While specific aspects of the disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure, along with such modifications, should be determined with reference to the full scope of equivalents and the specification.

Claims (35)

  A method of increasing interleukin-2 (IL-2) expression in a subject in need of increasing interleukin-2 (IL-2) expression, wherein the expression of microRNA-31 is increased, or Administering to the subject an effective amount of a composition that reduces RhoA expression, wherein the subject in need of increasing interleukin-2 (IL-2) expression is a subject with systemic lupus erythematosus The way that is.   A method of increasing interleukin-2 (IL-2) expression in T cells of a subject with systemic lupus erythematosus, wherein the effective amount increases microRNA-31 expression or reduces RhoA expression Contacting the T cell with the composition of:   A method for treating systemic lupus erythematosus comprising increasing microRNA-31 expression or treating RhoA in a subject in need of treatment for systemic lupus erythematosus to treat said autoimmune condition. Administering an effective amount of the composition that reduces expression.   A method of reducing RhoA expression in a subject in need of reducing RhoA expression, comprising administering to said subject an effective amount of a composition that increases microRNA-31 expression, comprising: The method wherein the subject in need of reduced expression is a subject with systemic lupus erythematosus.   A method of reducing RhoA expression in T cells of a subject having systemic lupus erythematosus, comprising contacting said T cells with an effective amount of a composition that increases microRNA-31 expression.   6. The method of any one of claims 1-5, wherein the subject having systemic lupus erythematosus presents with symptoms of disease flare.   6. The method according to any of claims 1-5, wherein the subject with systemic lupus erythematosus does not present with symptoms of disease flare.   8. The method of claim 6 or claim 7, wherein the subject's condition comprises lupus nephritis.   6. The method of claim 2 or claim 5, wherein the T cell is an in vitro activated T cell.   10. The method according to any one of claims 1 to 9, wherein the composition comprises a nucleic acid comprising a nucleotide sequence that increases the expression of microRNA-31.   11. The method according to any one of claims 1 to 10, wherein the composition comprises a nucleic acid comprising a nucleotide sequence that reduces RhoA expression.   12. A method according to claim 10 or claim 11 wherein the composition comprises microRNA-31.   12. A method according to claim 10 or claim 11 wherein the composition comprises short interfering nucleic acids (siNA).   14. The method of claim 13, wherein the composition comprises siNA capable of hybridizing to RhoA.   15. A method according to any of claims 1 to 14, wherein reducing RhoA expression comprises reducing RhoA transcript expression.   16. The method according to any of claims 1 to 15, wherein reducing RhoA expression comprises reducing RhoA protein expression.   3. The method of claim 1 or claim 2, wherein increasing IL-2 expression comprises increasing IL-2 transcript expression.   18. A method according to any one of claims 1, 2 or 17, wherein increasing IL-2 expression comprises increasing IL-2 protein expression.   19. The method according to any of claims 1-18, further comprising assaying IL-2 expression in a sample taken from the subject at a time after administration of the composition.   20. The method of any of claims 1-19, further comprising assaying RhoA expression in a sample taken from the subject at a time after administration of the composition.   21. The method of claim 19 or claim 20, wherein the sample comprises a blood sample.   22. A method according to any one of claims 19 to 21 wherein the step of assaying IL-2 expression comprises assaying the expression of IL-2 transcripts in T cells.   22. A method according to any of claims 19 to 21 wherein assaying IL-2 expression comprises assaying IL-2 protein expression in the sample.   24. The method of claim 20 or claim 21, wherein assaying RhoA expression comprises assaying RhoA transcript expression in T cells.   23. The method of claim 20 or claim 21, wherein assaying RhoA expression comprises assaying RhoA protein expression in the sample.   Assaying the presence, absence or amount of a biomarker in a subject with systemic lupus erythematosus to which a compound has been administered, wherein the biomarker is selected from microRNA-31, RhoA or IL-2 Determining whether a dosage regimen or dosage regimen of the compound subsequently administered to the subject is adjusted based on the presence, absence or amount of the biomarker assayed.   27. The method of claim 26, wherein the subject with systemic lupus erythematosus presents with symptoms of disease flare.   27. The method of claim 26, wherein the subject with systemic lupus erythematosus does not exhibit symptoms of disease flare.   29. A method according to any of claims 26 to 28, wherein the compound comprises a steroid or an immunosuppressive drug.   29. A method according to any of claims 26 to 28, wherein the compound comprises a composition that increases microRNA-31 or decreases RhoA.   29. A method according to any of claims 26 to 28, wherein the compound comprises IL-2.   A method for diagnosing systemic lupus erythematosus, comprising: taking a sample from a subject suspected of systemic lupus erythematosus; and assaying for expression of microRNA-31 or RhoA in said sample.   35. The method of claim 32, wherein the subject suspected of systemic lupus erythematosus does not exhibit symptoms of disease flare.   A method of monitoring the treatment of systemic lupus erythematosus comprising detecting the expression of microRNA-31 or RhoA in a sample from a subject undergoing treatment of systemic lupus erythematosus; Or comparing said expression of RhoA with expression in a sample from the same subject taken at an earlier time point prior to or during said treatment period; An increase in microRNA-31 or a decrease in RhoA in a sample taken at a later time point during the treatment period relative to a sample taken at the time point indicates the effectiveness of the treatment, thereby monitoring the treatment how to.   A method of treating a subject having systemic lupus erythematosus, wherein the expression of microRNA-31 or RhoA from a sample taken from the subject prior to the start of specific treatment of systemic lupus erythematosus is derived from a healthy subject. Comparing to a standard range reflecting expression in the sample, wherein microRNA-31 expression is below the standard range or RhoA expression is above the standard range, so that systemic lupus erythematosus A step shown to be sensitive to treatment; and if the subject is confirmed to be sensitive to treatment of systemic lupus erythematosus, siRNA or IL-2 protein that hybridizes to microRNA-31, RhoA, Treating the subject with an effective amount of a composition comprising: microRNA-31 in a sample after treatment from the subject; Detecting the expression of RhoA; and comparing the expression of microRNA-31 or RhoA in the sample after said treatment with the expression in said sample taken before the start of said specific treatment .

Images