WO2013013165A2 - Use of lna compounds for therapeutic inhibition of mir-21 in systemic lupus erythematosus - Google Patents

Abstract

The present invention encompasses compositions of LNA antimiRs as well as methods of their use for the regulation of miR-21 expression. The present invention encompasses methods of treating diseases where miR-21 is up-regulated. An example of such a disease is Systemic Lupus Erythematosus (SLE).

Description

TITLE OF THE INVENTION

USE OF LNA COMPOUNDS FOR THERAPEUTIC INHIBITION OF MIR-21 IN

SYSTEMIC LUPUS ERYTHEMATOSUS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application Serial No. 61/510,283, filed July 21, 2011 and U.S. Patent Application Serial No. 61/511,854, filed July 26, 2011, the contents of all are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Systemic lupus erythematosus (SLE, lupus) is an autoimmune disease, in which a combination of genetic predisposition and possible environmental influences triggers an exaggerated immune reaction against self-antigens and loss of immune tolerance. Antibody production by B cells and aberrant antibody-independent B and T cell functions have placed B and T cells at the center of development of new therapeutic strategies for treatment of SLE (reviewed in (Crispin et al, 2010, Nat Rev Rheumatol 6:317-325; Sanz et al, 2010, Nat Rev Rheumatol 6:326-337)). The tri- congenic B6. Slel.Sle2.Sle3 (B6.Slel23) mouse model bears three lupus susceptibility loci from the NZM2410 lupus-prone strain, backcrossed onto a C57BL/6 (B6) background (Morel et al, 2000, Proc Natl Acad Sci USA 97:6670-6675). B6.Slel23 mice spontaneously develop an autoimmune syndrome that strongly resembles human lupus, characterized by autoantibodies against H2A/H2B/DNA nucleosomes, splenomegaly, lymphadenopathy and immune complex-mediated glomerulonephritis. Anti-histone autoantibodies are detected early in the life of B6.Slel23 mice; however, splenomegaly and kidney disease are not typically present until the age of four to six months (Morel et al, 2000, Proc Natl Acad Sci U S A 97:6670-6675). As in human SLE, autoimmune manifestations in B6.Slel23 mice are associated with lymphocyte signaling defects (reviewed in (La Cava et al, et al, 2009, Lupus 18: 196-201; Liu et al, 2009, Autoimmun Rev 8:214-218)) and perturbation of cell proliferation and apoptosis (Mohan et al, 1997, J Immunol 159:454-465; Mohan et al, 1999, J Immunol 162:6492-6502). MicroRNAs (miRNAs) are approximately 22 nt non-coding RNAs that regulate gene expression post-transcriptionally by mediating translational repression or promoting degradation of their target mRNAs (Filipowicz et al, 2008, Nat Rev Genet 9: 102-114; Nelson et al, 2003, Trends Biochem Sci 28:534-540). Animal miRNAs have emerged as key players in diverse immunological processes, such as B cell lineage commitment, regulation of T cell differentiation, TCR signaling, and regulation of IFN signaling (Lu et al., 2009, Immunology 127:291-298; Tang et al, 2009, Arthritis Rheum 60: 1065-1075; Xiao et al. 2009, Cell 136:26-36), however, their function in autoimmunity and specifically in SLE, remains poorly understood. Reports of induction of lymphoproliferative syndromes in mice by failed interaction of miR-101 with ICOS (Yu et al, 2007, Nature 450:299-303) or by transgenic expression of the miR-17-92 cluster (Xiao et al., 2008, Nat Immunol 9:405-414) highlight an important role for miRNAs in autoimmunity. Furthermore, aberrant miRNA expression in lymphocytes and peripheral blood mononuclear cells (PBMCs) from patients with SLE (Dai et al, 2007, Lupus 16:939-946; Te et al, 2010, PLoS One 5:el0344) and miRNA regulation of signaling pathways involved in the induction or maintenance of lupus (Tang et al, 2009, Arthritis Rheum 60: 1065-1075; Divekar et al., 2011, J Immunol 15:924-930; Pan et al, 2010, J Immunol 184:6773- 6781 ; Zhao et al., 2010, Arthritis Rheum Dec 16. [Epub ahead of print]) add another layer of complexity to the molecular pathways disordered in lupus.

miRNAs are implicated in numerous immunological processes including B cell lineage commitment, TCR signaling, regulation of T cell differentiation and IFN signaling. However, their function in autoimmunity and specifically in systemic lupus erythematosus (SLE) remains poorly understood. There is a long-felt need in the art for a therapeutic method of treating autoimmunity and specifically SLE. The present invention fills this need.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention provides a method of reducing the amount of miR-21 in a peripheral lymphocyte. In one embodiment, the method comprises contacting a peripheral lymphocyte with an oligomer, wherein the oligomer comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21. In one embodiment, the oligomer comprises the sequence of SEQ ID

NO: 1.

In one embodiment, the sequence of miR-21 is SEQ ID NO: 2.

In one embodiment, the oligomer is at least 7-10 contiguous nucleotides in length.

In one embodiment, the oligomer is at least 11-18 contiguous nucleotides in length.

In one embodiment, the oligomer is stabilized against nucleolytic degradation.

In one embodiment, the oligomer comprises a nucleotide analog selected from the group consisting of 2'-0-alkyl-RNA monomers, 2'-amino-DNA, 2'- fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, HNA, ΓΝΑ, 2'-MOE-RNA (2'-0-methoxyethyl-RNA), 2'Fluoro-DNA, and locked nucleic acid (LNA).

In one embodiment, the nucleotide analog is a locked nucleic acid (LNA).

In one embodiment, the antisense oligonucleotide is essentially incapable of recruiting RNaseH.

In one embodiment, least 70% of the nucleotide units of the oligomer are selected from the group consisting of LNA units and 2' substituted nucleotide analogues, and wherein at least 50% of the nucleotide units of the oligomer are LNA units.

In one embodiment, the length of the oligomer is 7, 8 or 9 contiguous nucleotides, wherein the contiguous nucleotide units are independently selected from the group consisting of LNA units and 2' substituted nucleotide analogues.

In one embodiment, at least 30%, such as at least 40%, such as at least

50%, such as at least 60%, such as at least 70% of the nucleotide units of the oligomer are nucleotide analogue units.

In one embodiment, all the nucleotide units of the oligomer are nucleotide analogue units.

In one embodiment, the nucleotide units of the oligomer are independently selected from LNA units and DNA units, and wherein the

oligonucleotide does not comprise a region of more than 5 consecutive DNA units. In one embodiment, at least one of the internucleoside linkages present between the nucleoside units of the contiguous nucleotide sequence is a

phosphorothioate internucleoside linkage.

In one embodiment, all the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

The invention also provides a method of treating a mammal having a systemic autoimmune disease. In one embodiment, the method comprises administering to a mammal in need thereof an effective amount of a nucleic acid molecule wherein the nucleic acid molecule binds to miR-21 in a peripheral lymphocyte in the mammal; and wherein the binding of the nucleic acid to miR-21 in the lymphocyte diminishes the level of expression of miR-21 in the lymphocyte.

The invention provides an in vitro method of reducing the amount of miR-21 in a peripheral lymphocyte, the method comprising contacting a peripheral lymphocyte with an oligomer which comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21.

The invention provides an oligomer which comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21 for use in the treatment of a mammal suffering from a systemic autoimmune disease.

The invention provides a method of using an oligomer which comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21 in the manufacture of a medicament for the treatment of a mammal suffering from a systemic autoimmune disease.

The invention provides a method of reducing the amount of miR-21 in a peripheral lymphocyte, the method comprising contacting a peripheral lymphocyte with a nucleic acid molecule, wherein the nucleic acid molecule comprises a sequence which is substantially complementary to 6 to 15 contiguous nucleotides of miR-21.

In one embodiment, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.

In one embodiment, the sequence of miR-21 is SEQ ID NO: 2.

In one embodiment, the nucleic acid molecule is at least six nucleotides in length.

In one embodiment, the nucleic acid molecule is stabilized against nucleolytic degradation. In one embodiment, the nucleic acid molecule comprises a nucleotide analog.

In one embodiment, the nucleotide analog is a locked nucleic acid

(LNA).

The invention provides a method of treating a mammal suffering from a systemic autoimmune disease, the method comprising administering an effect amount of a nucleic acid molecule to the mammal in need thereof, wherein the nucleic acid molecule comprises a sequence which is substantially complementary from 6 to 22 contiguous nucleotides of miR-21.

In one embodiment, the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.

In one embodiment, the sequence of miR-21 is SEQ ID NO: 2.

In one embodiment, the nucleic acid molecule is at least six nucleotides in length.

In one embodiment, the nucleic acid molecule is stabilized against nucleolytic degradation.

In one embodiment, the nucleic acid molecule comprises a nucleotide analog.

In one embodiment, the nucleotide analogue is a locked nucleic acid (LNA).

In one embodiment, the mammal is a human.

In one embodiment, the systemic autoimmune disease is associated with unregulated expression of miR-21.

In one embodiment, the systemic autoimmune disease is SLE.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1, comprising Figures 1A through ID, is a series of images demonstrating that miR-21 expression in B6.Slel23 is regulated transcriptionally and correlates with age and disease severity. Figure 1A depicts pre-miR-21 and mature miR-21 expression in B6.Slel23 splenic B cells. 20 μg of total splenic B cell RNA from individual age- matched 2, 6 and 12 months old B6.Slel23 and B6 mice were analyzed under denaturing conditions, transferred and hybridized with LNA probe complementary to miR-21. For mass-normalization, the same membrane was stripped and re-probed with mouse snoRNA-429 radiolabeled DNA probe. The data were normalized to snoRNA-429 expression and relative fold-expression values calculated by taking the B6.Slel23/B6 ratio. A shorter exposure of the same membrane was used for the detection of the miR-21. Figure IB depicts that B6.Slel23 disease severity correlates with age. Blood Urea Nitrogen (BUN) of 2, 6 and 12 months old female B6 and B6.Slel23 mice was used as a measure of disease severity. Figure 1C depicts a comparison of spleens extracted from 6 months old B6 and B6.Slel23 mice showing correlation of BUN and splenomegaly. Figure ID depicts qRT-PCR amplification of primary miR-21 (pri-miR-21) using splenic B cell RNA from individual, age-matched 9 months old B6.Slel23 and B6 mice. The average of three individual experiments is shown. Error bars represent SEM.

Figure 2, comprising Figures 2A through 2B, is a series of images demonstrating that silencing of miR-21 in vivo by tiny LNA-antimiR derepresses PDCD4 in B6.Slel23 T cells. Figure 2A depicts three groups of two B6.Slel23 mice that were treated in vivo with tiny LNA antimiR-21 or LNA scramble control compound daily for three days. miR-21 -specific qRT-PCR quantification was performed using total RNA from CD4⁺ CD62L^High CD44^Low T cells FACS purified from LNA antimiR-21 -and LNA scramble compound-treated mice. Mean RQs of the three independent experiments. *p= 8.1 x 10^"6. Figure 2B depicts western blot analysis of PDCD4 expression in CD4⁺CD62L^High CD44^Low T cells derived from in vivo LNA antimiR-21 treated mice and controls. Results are representative of three independent experiments.

Figure 3, comprising Figures 3A through 3C, is a series of images demonstrating that silencing of miR-21 in vivo by tiny LNA-antimiR ameliorates splenomegaly in B6.Slel23 Figure3A depicts in vivo sequestration of miR-21 by tiny LNA antimiR-21 in a slower migrating heteroduplex. Four groups of mice were treated with either saline (n=4), or LNA-scramble control compound (n=4) or tiny LNA antimiR-21 (n=4) over a period of 12 weeks. Native Northern blot analysis of total RNA extracted from liver tissue (top panels) or from purified splenic B220⁺ B cells (lower panels) was performed using a radiolabeled DNA probe complementary to miR-21. The first and second sample in each panel corresponds to in vitro annealed miR-21 : LNA antimiR-21 duplex and synthetic miR-21 , respectively, was used as controls. Figure 3B depicts the effect of in vivo miR-21 silencing in autoimmune splenomegaly. Individual spleens harvested from the four groups of mice treated in the study are shown. Figure 3B depicts mean spleen masses plotted as spleen-mass (mg)/mouse body mass (g) ratios. Error bars represent the SEM of the four independent experiments, ^-values were calculated using an un-paired, two-tailed t test. * =0.0149, ** =0.000127.

Figure 4, comprising Figures 4A through 4B, is a series of images demonstrating that silencing of miR-21 in vivo results in altered CD4/CD8 T cell ratios and reduced populations of Fas receptor-expressing B cells. Four groups of B6.Slel23 mice were treated in vivo with antimiR-21 inhibitor or controls for 12 weeks, as described elsewhere herein. Splenocytes from B6.Slel23 mice injected with saline (left panels), LNA scramble control (middle panels) or LNA antimiR-21 (right panels) were stained with appropriate fluorophore-conjugated antibodies and subjected to flow cytometry. Figures 4A depicts top panels: CD4⁺ and CD8⁺ populations from the four treatment groups plotted as the percentage of viable lymphocytes. Horizontal bar indicates the mean value. Top Right: Chart of the mean CD4/CD8 ratios in the four treatment groups. */?=0.0135, ** =0.0025. Bottom panels: representative flow cytometry plots from one treatment group. Figure 4B depicts top panels: FasR⁺ IgD^" populations from the four treatment groups plotted as the percentage of CD19⁺ B220⁺ lymphocytes. Horizontal bar indicates the mean value. Bottom panels: representative flow cytometry plots from one treatment group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is partly based on the discovery that miR-21 is up-regulated in an SLE animal model. In some instances the up-regulation of miR-21 is related to enhancement of miR-21 gene transcription. Accordingly, the present invention encompasses compositions comprising an inhibitor of miR-21 as well as methods of their use for treating, attenuating, alleviating, or preventing a systemic autoimmune disease such as Systemic Lupus erythematosus (SLE or lupus).

The present invention provides compositions and methods that are useful in reducing miRNA and pre-miRNA levels, in e.g., a mammal, such as a human. In particular, the present invention provides specific compositions and methods that are useful for reducing levels of miRNAs miR-21 for the purposes of treating a systemic autoimmune disease such as SLE.

The present invention relates to the discovery that administration of an inhibitor of miR-21 in an SLE animal model reverses splenomegaly, which is one of the cardinal manifestations of autoimmunity. In some instances, treatment with an inhibitor of miR-21 in the SLE animal model alters CD4/CD8 T cell ratios, reduces Fas receptor-expressing lymphocyte populations and de-represses the expression of programmed cell death protein 4 (PDCD4) in T cells.

In one embodiment, the inhibitor of the invention that can be used to treat SLE is an antisense oligomer that targets miR-21. Preferably, the inhibitor is a nucleic acid molecule that comprises a Locked Nucleic Acid (LNA) that is capable of inhibiting miR-21. To the extent that it may aid in the understanding of the invention with respect to LNA, U.S. Patent Application Publication Nos. 201 1/0077288 and 2010/0286234 are incorporated herein by reference.

In one embodiment, the invention provides a method for systemic delivery of an unconjugated, saline- formulated Locked Nucleic Acid-modified oligonucleotide (LNA-antimiR) that effectively antagonizes a desired microRNA such as microRNA-21 (e.g, miR-21). Accordingly, the invention also includes a pharmaceutical composition comprising an anti-microRNA oligonucleotide. Methods of treating diseases sensitive to treatment with an anti-microRNA oligonucleotide, using the compositions according to the invention are also provided.

Definitions

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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

"Antisense," as used herein, refers to a nucleic acid sequence which is complementary to a target sequence, such as, by way of example, complementary to a miR-21 sequence, including, but not limited to, a mature hsa-miR-21 sequence, or a sub-sequence thereof. Typically, an antisense sequence is fully complementary to the target sequence across the full length of the antisense nucleic acid sequence.

The phrase "at risk" as used herein refers to a subject with a greater than average likelihood of developing a disease or disorder associated with clinical features due to up-regulation of miR-21.

The term "autoimmune disease" as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, cutaneous lupus, multiple sclerosis, myasthenia gravis, myositis, dermatomyositis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, systemic sclerosis, limited scleroderma, vasculitis, psoriatic arthritis, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others.

"Complementary" as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are substantially complementary to each other when at least about 50%, preferably at least about 60% and more preferably at least about 80% of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

The term "dysregulation" as used herein describes an over- or under- expression of miR-21 present and detected in a body sample obtained from an individual as compared to miR-21 present in a sample obtained from one or more normal, not-at-risk individuals, or from the same individual at a different time point. In some instances, the level of miR-21 expression is compared with an average value obtained from more than one not-at-risk individuals. In other instances, the level of miR-21 expression is compared with a miR-21 level assessed in a sample obtained from one normal, not-at-risk sample. In yet another instance, the level of miR-21 expression in the putative at-risk individual is compared with the level of miR-21 expression in a sample obtained from the same individual at a different time.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system. The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

"Fragment" as the term is used herein, is a nucleic acid sequence that differs in length (i.e., in the number of nucleotides) from the length of a reference nucleic acid sequence, but retains essential properties of the reference molecule. Preferably, the fragment is at least about 50% of the length of the reference nucleic acid sequence. More preferably, the fragment is at least about 75% of the length of the reference nucleic acid sequence. Even more preferably, the fragment is at least about 95% of the length of the reference nucleic acid sequence.

As used herein, the term "gene" refers to an element or combination of elements that are capable of being expressed in a cell, either alone or in combination with other elements. In general, a gene comprises (from the 5' to the 3' end): (1) a promoter region, which includes a 5' nontranslated leader sequence capable of functioning in any cell such as a prokaryotic cell, a virus, or a eukaryotic cell (including transgenic animals); (2) a structural gene or polynucleotide sequence, which codes for the desired protein; and (3) a 3' nontranslated region, which typically causes the termination of transcription and the polyadenylation of the 3' region of the RNA sequence. Each of these elements is operably linked by sequential attachment to the adjacent element.

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 5'- ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

As used herein, "homology" is used synonymously with "identity." As used herein, "hybridization," "hybridize(s)" or "capable of hybridizing" is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. Complementary sequences in the nucleic acids pair with each other to form a double helix. The resulting double-stranded nucleic acid is a "hybrid." Hybridization may be between, for example two complementary or partially complementary sequences. The hybrid may have double-stranded regions and single stranded regions. The hybrid may be, for example, DNA:DNA, RNA:DNA or DNA:RNA. Hybrids may also be formed between modified nucleic acids (LNA compounds). One or both of the nucleic acids may be immobilized on a solid support. Hybridization techniques may be used to detect and isolate specific sequences, measure homology, or define other

characteristics of one or both strands. The stability of a hybrid depends on a variety of factors including the length of complementarity, the presence of mismatches within the complementary region, the temperature and the concentration of salt in the reaction or nucleotide modifications in one of the two strands of the hybrid.

Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) or 100 mM MES, 1 M Na, 20 mM EDTA, 0.01% Tween-20 and a temperature of 25- 50°C are suitable for allele-specific probe hybridizations. In a particularly preferred embodiment, hybridizations are performed at 40-50°C. Acetylated BSA and herring sperm DNA may be added to hybridization reactions. Hybridization conditions suitable for microarrays are described in the Gene Expression Technical Manual and the GeneChip Mapping Assay Manual available from Affymetrix (Santa Clara, CA).

The term "inhibit," as used herein, means to suppress or block an activity or function by at least about ten percent relative to a control value. Preferably, the activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system.

Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

As used herein, "isolated" means altered or removed from the natural state through the actions of a human being. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

As used herein, "microRNA" or "miRNA" describes small non-coding RNA molecules, generally about 15 to about 50 nucleotides in length, preferably 17- 23 nucleotides, which can play a role in regulating gene expression through a process termed RNA interference (RNAi). RNAi describes a phenomenon whereby the presence of an RNA sequence that is complementary or antisense to a sequence in a target gene messenger RNA (mRNA) results in inhibition of expression of the target gene. miRNAs are processed from hairpin precursors of about 70 or more nucleotides (pre-miRNA) which are derived from primary transcripts (pri-miRNA) through sequential cleavage by RNAse III enzymes.

By "modification" is meant any alteration of any nucleic acid of the invention. Modifications can be made, by way of non-limiting examples, to increase stability in vivo, to avoid or diminish immune stimulation or to enhance affinity to a target. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the removal of terminal sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine. Other

modifications include, but are not limited to, other modifications as described elsewhere herein. Other modifications known in the art will be readily understood by the skilled artisan to be included herein. A "mutation," as used herein, refers to a change in nucleic acid or polypeptide sequence relative to a reference sequence (which is preferably a naturally-occurring normal or "wild-type" sequence), and includes translocations, deletions, insertions, and substitutions/point mutations. A "mutant," as used herein, refers to either a nucleic acid or protein comprising a mutation.

"Naturally occurring" as used herein describes a composition that can be found in nature as distinct from being artificially produced. For example, a nucleotide sequence present in an organism, which can be isolated from a source in nature and which has not been intentionally modified by a person in the laboratory, is naturally occurring.

By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate,

methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'- end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.

The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences."

As used herein, "polynucleotide" includes cDNA, RNA, DNA/RNA hybrid, anti-sense RNA, ribozyme, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatized, synthetic, or semi-synthetic nucleotide bases. Also, included within the scope of the invention are alterations of a wild type or synthetic gene, including but not limited to deletion, insertion, substitution of one or more nucleotides, or fusion to other polynucleotide sequences.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in an inducible manner.

"Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.

A "sample," as used herein, refers to a biological sample from a subject, including but is not limited to tissue, blood, saliva, feces, and urine. A sample can also be any other source of material obtained from a subject.

The terms "subject," "patient," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

"Synthetic mutant" includes any purposefully generated mutant or variant protein or nucleic acid. Such mutants can be generated by, for example, chemical mutagenesis, polymerase chain reaction (PCR) based approaches, or primer- based mutagenesis strategies well known to those skilled in the art.

The term "target" as used herein refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed by the invention include, but are not restricted to, oligonucleotides, nucleic acids, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes referred to in the art as anti-probes.

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptosm of pathology, for the purpose of diminishing or eliminating those signs or symptoms.

As used herein, "treating a disease or disorder" means reducing the frequency or severity with which a sign or symptom of a disease or disorder is experienced by a patient.

The phrase "therapeutically effective amount," as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease, disorder or condition, including alleviating signs or symptoms of a disease, disorder or condition.

"Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

The term "LNA" refers to a bicyclic nucleoside analogue, known as

"Locked Nucleic Acid." LNA may refer to an LNA monomer, or, when used in the context of an "LNA oligonucleotide," LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues. LNA nucleotides are characterised by the presence of a linker group (such as a bridge) between C2' and C4' of the ribose sugar ring - for example as shown as the biradical R^4* - R^2* as described below.

The LNA used in the oligonucleotide compounds of the invention preferably has the structure of the eneral formula I

wherein for all chiral centers, asymmetric groups may be found either R or S orientation; wherein X is selected from -0-, -S-, -N(R^N*)-, -C(R⁶R^6*)-, such as, in some embodiments -0-;

B is selected from hydrogen, optionally substituted Ci-4-alkoxy, optionally substituted Ci-4-alkyl, optionally substituted Ci-4-acyloxy, nucleobases including naturally occurring and nucleobase analogues, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; preferably, B is a nucleobase or nucleobase analogue;

P designates an internucleotide linkage to an adjacent monomer, or a 5'-terminal group, such internucleotide linkage or 5'-terminal group optionally including the substituent R⁵ or equally applicable the substituent R^5*;

P* designates an internucleotide linkage to an adjacent monomer, or a 3'-terminal group;

R^4* and R^2* together designate a bivalent linker group consisting of 1 - 4 groups/atoms selected from -C(R^aR^b)-, -C(R^a)=C(R^b)-, -C(R^a)=N-, -0-, -Si(R^a)₂-, -S-, -SO2-, -N(R^a)-, and >C=Z, wherein Z is selected from -0-, -S-, and -N(R^a)-, and R^a and R^b each is independently selected from hydrogen, optionally substituted C_1-12- alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, optionally substituted Ci-12-alkoxy, C2-i2-alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, Ci-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,

heteroarylcarbonyl, amino, mono- and di(Ci-6-alkyl)amino, carbamoyl, mono- and di(Ci-6-alkyl)-amino-carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci_6- alkyl)amino-Ci_6-alkyl-aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci_6- alkanoyloxy, sulphono, Ci_6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂), wherein for all chiral centers, asymmetric groups may be found in either R or S orientation, and;

each of the substituents R^1*, R², R³, R⁵, R^5*, R⁶ and R^6*, which are present is independently selected from hydrogen, optionally substituted Ci-12-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci_ 12-alkoxy, C2-i2-alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, C_1-12- alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6- alkyl)amino, carbamoyl, mono- and di(Ci_6-alkyl)-amino-carbonyl, amino-Ci-6-alkyl- aminocarbonyl, mono- and di(Ci_6-alkyl)amino-Ci_6-alkyl-aminocarbonyl, Ci-6-alkyl- carbonylamino, carbamido, Ci_6-alkanoyloxy, sulphono, Ci-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene; wherein R^N is selected from hydrogen and Ci-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and R^N*, when present and not involved in a biradical, is selected from hydrogen and Ci-4-alkyl; and basic salts and acid addition salts thereof. For all chiral centers, asymmetric groups may be found in either R or S orientation.

In some embodiments, R^4* and R^2* together designate a biradical consisting of a groups selected from the group consisting of C(R^aR^b)-C(R^aR^b)-,

C(R^aR^b)-0-, C(R^aR^b)-NR\ C(R^aR^b)-S-, and C(R^aR^b)-C(R^aR^b)-0-, wherein each R^a and R^b may optionally be independently selected. In some embodiments, R^a and R^b may be, optionally independently selected from the group consisting of hydrogen and ci-₆alkyl, such as methyl, such as hydrogen.

In some embodiments, R^4* and R^2* together designate the biradical -O-

CH(CH₂OCH₃)- (2'0-methoxyethyl bicyclic nucleic acid - Seth at al, 2010, J. Org. Chem) - in either the R- or S- configuration.

In some embodiments, R^4* and R^2* together designate the biradical -O- CH(CH₂CH₃)- (2'O-ethyl bicyclic nucleic acid - 2010, Seth et al, J. Org. Chem 75: 1569-1581). - in either the R- or S- configuration.

In some embodiments, R^4* and R^2* together designate the biradical -O- CH(CH₃)-. - in either the R- or S- configuration. In some embodiments, R^4* and R^2* together designate the biradical -0-CH₂-0-CH₂- - (2010, Seth et al, J. Org. Chem 75: 1569-1581).

In some embodiments, R^4* and R^2* together designate the biradical -O-

NR-CH₃- (2010, Seth et al., J. Org. Chem 75: 1569-1581).

In some embodiments, the LNA units have a structure selected from the following group:

1 * 2 3 5 5 *

In some embodiments, R , R , R , R , R are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci_6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation.

1 * 2 3 5 5 *

In some embodiments, R , R , R , R , R are hydrogen. In some embodiments, R^1*, R², R³ are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci_6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation.

In some embodiments, R^1*, R², R³ are hydrogen.

In some embodiments, R⁵ and R^5* are each independently selected from the group consisting of H, -CH₃, -CH₂-CH₃,- CH₂-0-CH₃, and -CH=CH₂. Suitably in some embodiments, either R⁵ or R^5* are hydrogen, whereas the other group (R⁵ or R^5* respectively) is selected from the group consisting of C1-5 alkyl, C2-6 alkenyl, C2-6 alkynyl, substituted Ci_6 alkyl, substituted C2-6 alkenyl, substituted C2-6 alkynyl or substituted acyl (-C(=0)-); wherein each substituted group is mono or poly substituted with substituent groups independently selected from halogen, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl, substituted C₂-6 alkynyl, OJi, SJ_b NJ1J2, N₃, COOJi, CN, 0-C(=0)NJiJ₂, N(H)C(=NH)NJ,J₂ or N(H)C(=X)N(H)J₂ wherein X is O or S; and each Ji and J2 is, independently, H, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, Ci-6 aminoalkyl, substituted Ci-6 aminoalkyl or a protecting group. In some embodiments either R⁵ or R^5* is substituted Ci_6 alkyl. In some embodiments either R⁵ or R^5* is substituted methylene wherein preferred substituent groups include one or more groups independently selected from F, NJ1J2, N₃, CN, OJi, SJi, 0-C(=0)NJiJ₂, N(H)C(=NH)NJ, J₂ or N(H)C(0)N(H)J₂. In some embodiments each Ji and J₂ is, independently H or Ci_6 alkyl. In some embodiments either R⁵ or R^5* is methyl, ethyl or methoxymethyl. In some embodiments either R⁵ or R^5* is methyl. In a further embodiment either R⁵ or R^5* is ethylenyl. In some embodiments either R⁵ or R^5* is substituted acyl. In some embodiments either R⁵ or R is C(=0)NJiJ₂. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such 5' modified bicyclic nucleotides are disclosed in WO

2007/134181, which is hereby incorporated by reference in its entirety.

In some embodiments B is a nucleobase, including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine, such as a nucleobase referred to herein, such as a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil, and/or a modified or substituted nucleobase, such as 5- thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2'thio-thymine, 5-methyl cytosine, 5- thiozolo-cytosine, 5-propynyl-cytosine, and 2,6-diaminopurine.

In some embodiments, R^4* and R^2* together designate a biradical selected from -C(R^aR^b)-0-, -C(R^aR^b)-C(R^cR^d)-0-, -C(R^aR^b)-C(R^cR^d)-C(R^eR^f)-0-, - C(R^aR^b)-0-C(R^cR^d)-, -C(R^aR^b)-0-C(R^cR^d)-0-, -C(R^aR^b)-C(R^cR^d)-, -C(R^aR^b)-

C(R^cR^d)-C(R^eR^f)-, -C(R^a)=C(R^b)-C(R^cR^d)-, -C(R^aR^b)-N(R^c)-, -C(R^aR^b)-C(R^cR^d)- N(R^e)-, -C(R^aR^b)-N(R^c)-0-, and -C(R^aR^b)-S-, -C(R^aR^b)-C(R^cR^d)-S-, wherein R^a, R^b, R^c, R^d, R^e, and R^f each is independently selected from hydrogen, optionally substituted Ci_i₂-alkyl, optionally substituted C₂_i₂-alkenyl, optionally substituted C₂_ i₂-alkynyl, hydroxy, Ci_i₂-alkoxy, C₂_i₂-alkoxyalkyl, C₂_i₂-alkenyloxy, carboxy, Ci_i₂- alkoxycarbonyl, Ci_i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6-alkyl)amino, carbamoyl, mono- and di(Ci_6-alkyl)-amino- carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci_6-alkyl- aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci_6-alkanoyloxy, sulphono, Ci-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂). For all chiral centers, asymmetric groups may be found in either R or S orientation.

In a further embodiment R^4* and R^2* together designate a biradical (bivalent group) selected from -CH₂-0-, -CH₂-S-, -CH₂-NH-, -CH₂-N(CH₃)-, -CH₂- CH₂-0-, -CH₂-CH(CH₃)-, -CH₂-CH₂-S-, -CH₂-CH₂-NH-, -CH₂-CH₂-CH₂-, -CH₂-CH₂- CH₂-0-, -CH₂-CH₂-CH(CH₃)-, -CH=CH-CH₂-, -CH₂-0-CH₂-0-, -CH₂-NH-0-, -CH₂- N(CH₃)-0-, -CH₂-0-CH₂-, -CH(CH₃)-0-, and -CH(CH₂-0-CH₃)-0-, and/or, -CH₂- CH₂-, and -CH=CH- For all chiral centers, asymmetric groups may be found in either R or S orientation.

In some embodiments, R^4* and R^2* together designate the biradical

C(R^aR^b)-N(R^c)-0-, wherein R^a and R^b are independently selected from the group consisting of hydrogen, halogen, Ci-₆ alkyl, substituted Ci-₆ alkyl, C₂-₆ alkenyl, substituted C₂_6 alkenyl, C₂_6 alkynyl or substituted C₂_6 alkynyl, Ci_6 alkoxyl, substituted Ci_6 alkoxyl, acyl, substituted acyl, Ci_6 aminoalkyl or substituted Ci_6 aminoalkyl, such as hydrogen, and; wherein R^c is selected from the group consisting of hydrogen, halogen, Ci_₆ alkyl, substituted C_h alky!, C₂_6 alkenyl, substituted C₂_6 alkenyl, C₂_6 alkynyl or substituted C₂_6 alkynyl, Ci_6 alkoxyl, substituted Ci_6 alkoxyl, acyl, substituted acyl, Ci_6 aminoalkyl or substituted Ci_6 aminoalkyl, such as hydrogen.

In some embodiments, R^4* and R^2* together designate the biradical

C(R^aR^b)-0-C(R^cR^d) -0-, wherein R^a, R^b, R^c, and R^d are independently selected from the group consisting of hydrogen, halogen, Ci-₆ alkyl, substituted Ci-₆ alkyl, C₂_6 alkenyl, substituted C₂_6 alkenyl, C₂_6 alkynyl or substituted C₂_6 alkynyl, Ci_6 alkoxyl, substituted Ci_6 alkoxyl, acyl, substituted acyl, Ci_6 aminoalkyl or substituted Ci_6 aminoalkyl, such as hydrogen.

In some embodiments, R^4* and R^2* form the biradical -CH(Z)-0-, wherein Z is selected from the group consisting of C_h alky!, C₂_6 alkenyl, C₂_6 alkynyl, substituted Ci_6 alkyl, substituted C₂_6 alkenyl, substituted C₂_6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio; and wherein each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJi, NJiJ₂, SJi, N₃, OC(=X)Ji, OC(=X)NJiJ₂, NJ³C(=X)NJiJ₂ and CN, wherein each J J₂ and J₃ is, independently, H or Ci_6 alkyl, and X is O, S or NJi. In some embodiments Z is Ci-6 alkyl or substituted Ci-6 alkyl. In some embodiments Z is methyl. In some embodiments Z is substituted Ci_6 alkyl. In some embodiments said substituent group is Ci-6 alkoxy. In some embodiments Z is CH₃OCH2-. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in U.S. Pat. No. 7,399,845 which is hereby incorporated by reference in its entirety. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. In

1 * 2 3 * 5 5* some embodiments, R , R , R are hydrogen, and one or both of R^J, R^J may be other than hydrogen as referred to above and in WO/2007/134181.

In some embodiments, R^4* and R^2* together designate a biradical which comprise a substituted amino group in the bridge such as consist or comprise of the biradical -CI¾-N( R^c)-, wherein R^c is Ci _ u alkyloxy. In some embodiments R^4* and R^2* together designate a biradical -Cq₃q₄-NOR -, wherein q₃ and q₄ are independently selected from the group consisting of hydrogen, halogen, Ci-6 alkyl, substituted C e alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, C e alkoxyl, substituted C e alkoxyl, acyl, substituted acyl, C e aminoalkyl or substituted Ci_6 aminoalkyl; wherein each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, OJi, SJi, NJ1J2, COOJi, CN, 0-C(=0)NJiJ₂, N(H)C(=NH)N J1J2 or N(H)C(=X=N(H)J₂ wherein X is O or S; and each of Ji and J2 is,

independently, H, Ci_6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C e aminoalkyl or a protecting group. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in Int. Pat. App. Pub. No.

WO/2008/150729 which is hereby incorporated by reference in its entirety. In some

1 * 2 3 5 5 *

embodiments, R , R , R , R , R are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyl, substituted C e alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, C e alkoxyl, substituted Ci_6 alkoxyl, acyl, substituted acyl, C e aminoalkyl or substituted C e aminoalkyl. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. In some

1* 2 3 5 5 *

embodiments, R , R , R are hydrogen and one or both of R^J, R^J may be other than hydrogen as referred to above and in Int. Pat. App. Pub. No. WO 2007/134181. In some embodiments R^4* and R^2* together designate a biradical (bivalent group) C(R^aR^b)-0-, wherein R^a and R^b are each independently halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted C1-C12 alkoxy, OJi SJi, SOJi, SO2J1, NJ1J2, N₃, CN, C(=0)OJi, C(=0)NJiJ₂, C(=0)Ji, 0-C(=0)NJiJ₂, N(H)C(=NH)NJiJ₂, N(H)C(=0)NJiJ₂ or N(H)C(=S)NJiJ₂; or R^a and R^b together are =C(q3)(q4); q₃ and q₄ are each, independently, H, halogen,

or substituted C1-C12 alkyl; each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, Ci-Ce alkyl, substituted Ci- Ce alkyl, C₂- Ce alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, OJ_b SJ_b NJ1J2, N₃, CN, C(=0)OJi, C(=0)NJiJ₂, C(=0)Ji, 0-C(=0)NJiJ₂, N(H)C(=0)NJiJ₂ or N(H)C(=S)NJiJ₂. and; each Ji and J₂ is, independently, H, Cl-C₆ alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C1-C6 aminoalkyl, substituted C1-C6 aminoalkyl or a protecting group. Such compounds are disclosed in Int. Pat. App. Pub. No.

WO/2009/006478, hereby incorporated in its entirety by reference.

In some embodiments, R^4* and R^2* form the biradical - Q -, wherein Q is C(_qi)(q2)C(q₃)(q₄), C(_qi)=C(q₃), C[=C(_qi)(q2)]-C(q₃)(q₄) or C(qi)(q₂)- C[=C(q₃)(q₄)]; qi, q₂, q₃, q₄ are each independently. H, halogen, Ci-12 alkyl, substituted Ci-12 alkyl, C2-12 alkenyl, substituted Ci-12 alkoxy, OJi, SJi, SOJi, SO2J1, NJ1J2, N₃, CN, C(=0)OJi, C(=0)-NJiJ₂, C(=0) J_b -C(=0)NJiJ₂, N(H)C(=NH)NJiJ₂, N(H)C(=0)NJiJ₂ or N(H)C(=S)NJiJ₂; each Ji and J₂ is, independently, H, Ci_₆ alkyl, C2-6 alkenyl, C2-6 alkynyl, Ci_6 aminoalkyl or a protecting group; and, optionally wherein when Q is C(qi)(q2)(q₃)(q₄) and one of q₃ or q₄ is CH₃ then at least one of the other of q₃ or q₄ or one of qi and q2 is other than H. In some embodiments, R^1*, R², R³, R⁵, R^5* are hydrogen. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in Int. Pat. App. Pub. No. WO/2008/154401, which is hereby incorporated by reference in its entirity. In

1 * 2 3 5 5 *

some embodiments, R , R , R , R , R are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci_6 alkoxyl, substituted Ci_6 alkoxyl, acyl, substituted acyl, Ci_6 aminoalkyl or substituted Ci_6

1 * 2 3 5 5 *

aminoalkyl. In some embodiments, R , R , R , R , R are hydrogen. In some

1* 2 3 5 5 *

embodiments, R , R , R are hydrogen and one or both of R^J, R^J may be other than hydrogen as referred to above and in WO 2007/134181 or WO2009/067647 (alpha-L- bicyclic nucleic acids analogs).

In some embodiments the LNA used in the oligonucleotide compounds of the invention preferably has the structure of the general formula II:

wherein Y is selected from the group consisting of -0-, -CH₂0-, -S-, -

NH-, N(R^e) and/or -CH₂-; Z and Z* are independently selected among an

internucleotide linkage, R^H, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety (nucleobase), and R^H is selected from hydrogen and Ci-4-alkyl; R^a, R^b R^c, R^d and R^e are, optionally independently, selected from the group consisting of hydrogen, optionally substituted Ci-12-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci-12-alkoxy, C2-i2-alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, C_1-12- alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6- alkyl)amino, carbamoyl, mono- and di(Ci_6-alkyl)-amino-carbonyl, amino-Ci-6-alkyl- aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci-6-alkyl-aminocarbonyl, Ci-6-alkyl- carbonylamino, carbamido, Ci_6-alkanoyloxy, sulphono, Ci_6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂); and R^H is selected from hydrogen and Ci-4-alkyl. In some embodiments R^a, R^b R^c, R^d and R^e are, optionally independently, selected from the group consisting of hydrogen and Ci_6 alkyl, such as methyl. For all chiral centers, asymmetric groups may be found in either R or S orientation, for example, two exemplary stereochemical isomers include the beta-D and alpha-L isoforms, which may be illustrat as follows:

Specific exemplary LNA units are shown below:

-D-oxy-LNA

β-D-amino-LNA

The term "thio-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both beta- D and alpha-L-configuration.

The term "amino-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from -N(H)-, N(R)-, CH₂-N(H)-, and -CH₂- N(R)- where R is selected from hydrogen and Ci-4-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.

The term "oxy-LNA" comprises a locked nucleotide in which Y in the general formula above represents -0-. Oxy-LNA can be in both beta-D and alpha-L- configuration.

The term "ENA" comprises a locked nucleotide in which Y in the general formula above is -CH₂-0- (where the oxygen atom of -CH₂-0- is attached to the 2'-position relative to the base B). R^e is hydrogen or methyl. In some exemplary embodiments LNA is selected from beta-D-oxy- LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA. Description

The present invention is based in part on the discovery that expression of endogenous microRNAs (miRNAs) or pre-microRNAs (pre-miRNAs) can be inhibited using an antisense nucleic acid molecule that targets a desired miRNA or pre-miRNA (e.g., antimiRs). The present invention provides specific compositions and methods that are useful in reducing miRNA and pre-miRNA levels, in e.g., a mammal, such as a human. In particular, the present invention provides compositions and methods that are useful for reducing levels of the miRNA miR-21 for the treatment of a systemic autoimmune disease, such as SLE, in a mammal.

The present invention is related to the discovery that expression of miR-21 is up-regulated in B and T lymphocytes in a SLE animal model compared to the expression of miR-21 in an otherwise identical healthy animal model.

Accordingly, the invention provides compositions and methods for treating a systemic autoimmune disease such as SLE by silencing miR-21 in vivo using an antisense oligomer that targets miR-21.

In one embodiment, the invention comprise administering a composition comprising an antisense oligomer that targets miR-21 to a mammal exhibiting up-regulated levels of miR-21 or determined to be at risk for developing up-regulated levels of miR-21. The methods of the present invention further comprise administering a composition comprising an antisense oligomer that targets miR-21 to a mammal that has been diagnosed with SLE, or who has symptoms or signs of SLE.

The invention may be practiced in any subject diagnosed with, or at risk of developing SLE. Preferably, the subject is a mammal and more preferably, a human.

Composition

The present invention includes antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS)

oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compositions of the invention may elicit the action of one or more enzymes or structural proteins or may induce allosteric conformational changes to RNA binding proteins to modify the function of the target nucleic acid(s).

While the preferred form of an antisense composition is a single- stranded antisense oligonucleotide, in many species the introduction of double- stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products.

In the context of this invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are a preferred form of the compounds of the invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

The present invention relates to compositions comprising an inhibitor of a microRNA (miRNA) preferably the inhibitor is a nucleic acid molecule. In one embodiment, nucleic acid molecule of the invention is a single-stranded, double stranded, partially double stranded or hairpin structured chemically modified oligonucleotide that comprises at least 6 or more contiguous nucleotides substantially complementary to an endogenous miRNA and more particularly agents that include 6 or more contiguous nucleotides substantially complementary to the 5' end sequence of an miRNA or pre-miRNA. As used herein partially double stranded refers to double stranded structures that contain less nucleotides than the complementary strand. In general, such partial double stranded agents will have less than 75% double stranded structure, preferably less than 50%, and more preferably less than 25%, 20% or 15% double stranded structure.

In one embodiment, the nucleic acid molecule of the invention is an antisense oligomer that targets a RNA target in a cell or a mammal, preferably a human. In one embodiment, the RNA target is selected from the group consisting of a microRNA (miRNA), a pre-miRNA, a mRNA, a non-coding RNA, a viral RNA, and the like. Preferably, the microRNA is miR-21.

In one embodiment, the antisense oligomer comprises a nucleic acid that targets miR-21 (e.g., antimir-21 oligomer). In another embodiment, the antisense oligomer is a LNA antimiR-21. Preferably, the LNA antimiR-21 is an 8-mer have the sequence of GATAAGCT (SEQ ID NO: 1).

The oligomer may, in some embodiments, be either i) fully complementary to a sub-sequence of contiguous nucleotides present in the RNA target, or ii) comprises no more than a single mismatch with the complement of a subsequence of contiguous nucleotides present in said RNA target. As such the oligonucleotide is an antisense oligonucleotide in that it is either fully complementary to the (corresponding) target sequence, or comprises no more than a single mismatch with the target sequence. The RNA target is typically associated with a medical condition or disease, and may in some embodiments, be a microRNA (miRNA) or a mRNA, for example. The oligomer may therefore be, for example, an antimiR, a microRNA mimic, a microRNA blockmir, or an antisense oligomer.

In one embodiment, the oligonucleotide is designed to be essentially incapable of recruiting RNAseH. Oligonucleotides that are essentially incapable of recruiting RNAseH are well known in the literature, in example see WO2007/112754, WO2007/1 12753, or PCT/DK2008/000344. Oligonucleotides of the invention may be designed to comprise a mixture of affinity enhancing nucleotide analogs, such as in non-limiting example 2'-0-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'- fluoro-DNA monomers, LNA monomers, arabino nucleic acid (ANA) monomers, 2'- fluoro-ANA monomers, HNA monomers, ΓΝΑ monomers, 2'-MOE-RNA (2'-0- methoxyethyl-RNA), 2'Fluoro-DNA, and LNA.

In a further embodiment, the oligonucleotide does not include any DNA or RNA nucleotides, but is solely composed of affinity enhancing nucleotide analogs. In some embodiments, the oligonucleotide only comprises one type of affinity enhancing nucleotide analogs together with DNA and/or RNA. In some embodiments, the oligonucleotide is composed solely of one or more types of nucleotide analogs, such as in non-limiting example 2'-0-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, arabino nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, ΓΝΑ monomers, 2'-MOE- RNA (2'-0-methoxyethyl-RNA), 2'Fluoro-DNA, and LNA.

In one embodiment, the oligomer of the invention includes at least 6 or more contiguous nucleotides substantially complementary to a target sequence of an miRNA or pre-miRNA nucleotide sequence. Preferably, the oligomer of the invention includes a nucleotide sequence sufficiently complementary to hybridize to a miRNA target sequence of about 6 to 25 nucleotides, preferably about 15 to 23 nucleotides, more preferably 8-15. More preferably, the target sequence differs by no more than 1, 2, or 3 nucleotides from the sequence of miR-21. The sequence of miR-21 is

UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO: 2)

In another aspect, the length of the oligomer of the invention can contribute to the biochemical function of the inhibitor of miRNA with respect to the ability to decrease expression levels of a desired miRNA, such as miR-21. An antimiR-21 oligomer of the invention can be, for example, from about 6 to 30 nucleotides in length, preferably about 8 to 15 nucleotides in length (e.g., 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length). In some instances, antimiR-21 oligomer may require at least 6 nucleotides in length for optimal function.

In one embodiment, the oligomer of the invention comprises the sequence of GATAAGCT (SEQ ID NO: 1).

The oligomer of the invention can be further stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. In one embodiment, the oligomer includes a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0- MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0- dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-

DMAEOE), or 2'-0~N-methylacetamido (2'-0-NMA). In a particularly preferred embodiment, the oligomer includes at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the antagomir include a 2'-0-methyl modification.

In one embodiment, the contiguous nucleotide sequence of the oligomer comprises of at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as 95%, such as 100% LNA units. The remaining units may be selected from the non-LNA nucleotide analogs referred to herein in, such those selected from the group consisting of 2'-0-alkyl-RNA unit, 2'-OMe-RNA unit, 2'-amino-DNA unit, 2'-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, ΓΝΑ unit, and a 2'MOE RNA unit, or the group 2'- OMe RNA unit and 2'-fluoro DNA unit.

In one embodiment, the oligomer is modified in positions 3 to 8. The design of this sequence may be defined by the number of non-LNA units present or by the number of LNA units present. In some embodiments of the former, at least one, such as one, of the nucleotides in positions three to eight, counting from the 3' end, is a non-LNA unit. In one embodiment, at least two, such as two, of the nucleotides in positions three to eight, counting from the 3' end, are non-LNA units. In one embodiment, at least three, such as three, of the nucleotides in positions three to eight, counting from the 3' end, are non-LNA units. In one embodiment, at least four, such as four, of the nucleotides in positions three to eight, counting from the 3' end, are non-LNA units. In one embodiment, at least five, such as five, of the nucleotides in positions three to eight, counting from the 3' end, are non-LNA units. In one embodiment, all six nucleotides in positions three to eight, counting from the 3' end, are non-LNA units.

In one embodiment, the oligomer comprises of a contiguous nucleotide sequence which consists only of LNA units.

In one embodiment, the oligomer comprises a contiguous nucleotide sequence of repeating pattern of nucleotide analog and naturally occurring

nucleotides, or one type of nucleotide analog and a second type of nucleotide analogs. The repeating pattern, may, for instance be every second or every third nucleotide is a nucleotide analog, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2' substituted nucleotide analog such as 2'MOE of 2' fluoro analogs as referred to herein, or, in some embodiments selected form the groups of nucleotide analogs referred to herein. It is recognized that the repeating pattern of nucleotide analogs, such as LNA units, may be combined with nucleotide analogs at fixed positions (e.g. at the 5' or 3' termini of the molecule).

In one preferred embodiment, the inhibitor of miR-21 comprises an antisense LNA oligonucleotide. In one specially preferred embodiment, the inhibitor of miR-21 comprises an oligonucleotide which is between 7 and 25 nucleotides long and comprises at least one LNA. In some embodiments, the inhibitor of miR-21 comprises an oligonucleotide which is between 7 and 25 nucleotides long and comprises at least one LNA, and further comprises at least one other affinity increasing nucleotide analog. In some embodiments, the oligonucleotide of the invention comprises phosphorothioate linkages. In one specially preferred

embodiment, the pharmaceutical composition comprise an anti-miR-21 oligomer having the sequence: 5'-gAtaAgCt (SEQ ID NO: 1), wherein Capital letters indicate LNA units. Methods

When the miR-21 inhibitor of the invention is a nucleic acid molecule as well as derivative or variant form of isolated nucleic acid, any number of procedures may be used for the generation of the miR-21 inhibitor of the invention, such as those described, for example in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (2001, Current Protocols in Molecular Biology, Green & Wiley, New York).

As an example, a method for synthesizing nucleic acids de novo involves the organic synthesis of a nucleic acid from nucleoside derivatives. This synthesis may be performed in solution or on a solid support. One type of organic synthesis is the phosphotriester method, which has been used to prepare gene fragments or short genes. In the phosphotriester method, oligonucleotides are prepared which can then be joined together to form longer nucleic acids. For a description of this method, see Narang et al, (1979, Meth. EnzymoL, 68: 90) and U.S. Pat. No. 4,356,270. The phosphotriester method can be used in the present invention to synthesize an isolated antimiR-21 oligomer.

In addition, the compositions of the present invention can be synthesized in whole or in part, or an isolated antimiR-21 oligomer can be conjugated to another nucleic acid using organic synthesis such as the phosphodiester method, which has been used to prepare a tRNA gene. As in the phosphotriester method, the phosphodiester method involves synthesis of oligonucleotides which are subsequently joined together to form the desired nucleic acid.

Another method for synthesizing nucleic acids, described in U.S. Pat. No. 4,293,652, is a hybrid of the above-described organic synthesis and molecular cloning methods. In this process, the appropriate number of oligonucleotides to make up the desired nucleic acid sequence is organically synthesized and inserted sequentially into a vector which is amplified by growth prior to each succeeding insertion.

In addition, molecular biological methods, such as using a nucleic acid as a template for a PCR or LCR reaction, or cloning a nucleic acid into a vector and transforming a cell or tissue with the vector can be used to make large amounts of the nucleic acid of the present invention.

Oligomers of the invention include unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (1994, Nucleic Acids Res. 22: 2183-2196). Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature, preferably different from that which occurs in the human body.

As nucleic acids are polymers of subunits or monomers, many of the modifications described elsewhere herein occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, in a terminal region, e.g., at a position on a terminal nucleotide, or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A component can be attached at the 3' end, the 5' end, or at an internal position, or at a combination of these positions. For example, the component can be at the 3' end and the 5' end; at the 3' end and at one or more internal positions; at the 5' end and at one or more internal positions; or at the 3 ' end, the 5' end, and at one or more internal positions. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, or may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the oligonucleotide. The 5' end can be phosphorylated.

For increased nuclease resistance and/or binding affinity to the target, an oligonucleotide agent, can include, for example, 2'-modified ribose units and/or phosphorothioate linkages. E.g., the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);

polyethyleneglycols (PEG), 0(CH₂CH₂0)_nCH₂CH₂OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; amine, 0-ΑΜΓΝΕ and aminoalkoxy, 0(CH₂)_nAMINE, (e.g., AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

Preferred substitutes include but are not limited to 2'-methoxyethyl, 2'- OCH3, 2'-0-allyl, 2'-C- allyl, and 2'-fluoro.

"Deoxy" modifications include hydrogen (i.e. deoxyribose sugars); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid);

NH(CH₂CH₂NH)_nCH₂CH₂-AMiNE (AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), -NHC(0)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.

One way to increase resistance is to identify cleavage sites and modify such sites to inhibit cleavage. For example, the dinucleotides 5'-UA-3', 5'-UG-3', 5'- CA-3', 5'-UU-3', or 5'-CC-3' can serve as cleavage sites. Enhanced nuclease resistance can therefore be achieved by modifying the 5' nucleotide, resulting, for example, in at least one 5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine is a 2 '-modified nucleotide; at least one 5'-uridine-guanine-3' (5'-UG-3') dinucleotide, wherein the 5 '-uridine is a 2 '-modified nucleotide; at least one 5'- cytidine-adenine-3' (5'-CA-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; at least one 5'-uridine-uridine-3' (5'-UU-3') dinucleotide, wherein the 5'- uridine is a 2'-modified nucleotide; or at least one 5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the 5'-cytidine is a 2 '-modified nucleotide. In certain embodiments, all the pyrimidines of the miRNA inhibitor carry a 2'-modification, and the miRNA inhibitor therefore has enhanced resistance to endonucleases.

In addition, to increase nuclease resistance, the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called "chimeric" oligonucleotides are those that contain two or more different modifications.

With respect to phosphorothioate linkages that serve to increase protection against RNase activity, the miRNA inhibitor can include a

phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence. In one embodiment, the miRNA inhibitor includes a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0- methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0- NMA). In some instances, the miRNA inhibitor includes at least one 2'-0-methyl- modified nucleotide, and in some embodiments, all of the nucleotides of the miRNA inhibitor include a 2'-0-methyl modification.

The 5' -terminus can be blocked with an aminoalkyl group, e.g., a 5'-0- alkylamino substituent. Other 5' conjugates can inhibit 5 '-3' exonucleolytic cleavage. While not being bound by theory, a 5' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5' end of the oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3 '-5'- exonucleases. The oligonucleotide can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, an oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the oligonucleotide and target nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein. Alternatively, the oligonucleotide can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest (e.g., an mRNA, pre-mRNA, or an miRNA).

Any polynucleotide of the invention may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

Therapy

The invention provides a method of reducing the levels of an miRNA or pre-miRNA in a cell of a mammal, preferably a human. Preferably, the miRNA is miR-21. In another aspect, the invention includes reducing the level of an miRNA or pre-miRNA in a lymphocyte. The method includes the step of administering an oligomer of the invention to a mammal in need thereof. Such methods include contacting the cell with an oligomer of the invention for a time sufficient to allow uptake of the oligomer into the cell.

In another aspect the invention features a method of inhibiting miRNA expression (e.g., miR-21) or pre-miRNA expression in a cell, e.g., a cell of a mammal. Preferably, the cell is a lymphocyte. The method includes contacting the cell with an effective amount of an oligomer of the invention, which is substantially

complementary to the nucleotide sequence of the target miRNA (e.g., miR-21) or the target pre-miRNA. Such methods can be performed on a mammalian subject by administering to a subject one of the oligonucleotide agents/pharmaceutical compositions described herein.

In another embodiment, the present invention provides a method of treating a mammal diagnosed with a disease or disorder associated with dysregulated miRNA expression. In another embodiment, the invention further provides a method of treating a mammal diagnosed with a disease or disorder wherein miR-21 expression is a component of the disease or disorder. In still another embodiment, the present invention encompasses methods of treating systemic autoimmune diseases, such as SLE. In yet another embodiment, the invention further comprises a method of treating a systemic autoimmune disease where miR-21 up-regulation is a component of the disease.

In another embodiment, the present invention further comprises a method of decreasing expression of miR-21. In yet another embodiment, the present invention provides a method of inhibiting the expression of miR-21.

The methods of the invention comprise administering a therapeutically effective amount of at least one inhibitor of miR-21 to a mammal wherein the inhibitor attenuates expression of miR-21. In another embodiment, the methods of the invention comprise administering a therapeutically effective amount of at least one inhibitor of miR-21 to a mammal wherein the mammal exhibits a symptom of SLE. In another embodiment, the methods of the invention comprise administering a therapeutically effective amount of at least one inhibitor of miR-21 to a mammal wherein the inhibitor is used to treat a mammal diagnosed with a disease or disorder wherein expression of miR-21 is a component of the disease or disorder. In still another embodiment, the methods of the invention comprise administering a therapeutically effective amount of at least one inhibitor of miR-21 a mammal wherein the inhibitor is used to treat a systemic autoimmune disease such as SLE.

The subject may be diagnosed with a disease or disorder wherein the disease or disorder is characteristic of up-regulation of miR-21 expression as part of the disease's clinical features. In another embodiment, the subject may be at-risk of developing a disease or disorder wherein the disease or disorder is characteristic of up-regulation of miR-21 expression as part of the disease's clinical features.

Examples of a disease or disorder which may be treated using the methods of the present invention include but are not limited to a systemic autoimmune disease such as SLE or lymphoproliferative syndromes other than SLE such as Castleman's disease or other syndromes, or diseases characterized by increased cell proliferation or defective apoptosis. In a preferred embodiment the subject is a mammal. In a more preferred embodiment he subject is a human.

Methods of prophylaxis (i.e., prevention or decreased risk of disease), as well as reduction in the frequency or severity of symptoms associated with up- regulation of miR-21 expression or any related disease or disorder, are encompassed by the present invention.

The method of the invention comprises administering a therapeutically effective amount of at least one inhibitor of miR-21 to a mammal wherein the miR-21 inhibitor is used either alone or in combination with other therapeutic agents to treat a subject. An miR-21 inhibitor may be administered either before, during, after, or throughout the administration of said therapeutic agent. The compositions and methods of the present invention can be used in combination with other treatment regimens, including virostatic and virotoxic agents, antibiotic agents, antifungal agents, anti-inflammatory agents (steroidal and non-steroidal), antidepressants, anxiolytics, pain management agents, (acetaminophen, aspirin, ibuprofen, opiates (including morphine, hydrocodone, codeine, fentanyl, methadone), steroids (including prednisone and dexamethasone), and antidepressants (including gabapentin, amitriptyline, imipramine, doxepin) antihistamines, antitussives, muscle relaxants, bronchodilators, beta-agonists, anticholinergic, corticosteroids, mast cell stabilizers, leukotriene modifiers, methylxanthines, as well as combination therapies, and the like. The invention can also be used in combination with other treatment modalities, such as chemotherapy, cryotherapy, hyperthermia, radiation therapy, and the like.

Various forms of the miR-21 inhibitor of the invention can be administered or delivered to a mammalian cell using known methods in the art (e.g., by virus, direct injection, or liposomes, or by any other suitable methods known in the art or later developed). The methods of delivery can be modified to target certain cells, and in particular, cell surface receptor molecules. As an example, the use of cationic lipids as a carrier for nucleic acid constructs provides an efficient means of delivering the isolated TLR agonist nucleic acid of the present invention.

Various formulations of cationic lipids have been used to deliver nucleic acids to cells (WO 91/17424; WO 91/16024; U.S. Pat. Nos. 4,897,355;

4,946,787; 5,049,386; and 5,208,036). Cationic lipids have also been used to introduce foreign polynucleotides into frog and rat cells in vivo (Holt et al, Neuron 4:203-214 (1990); Hazinski et al, Am. J. Respr. Cell. Mol. Biol. 4:206-209 (1991)). Therefore, cationic lipids may be used, generally, as pharmaceutical carriers to provide biologically active substances (for example, see WO 91/17424; WO

91/16024; and WO 93/03709). Thus, cationic liposomes can provide an efficient carrier for the introduction of polynucleotides into a cell.

Further, liposomes can be used as carriers to deliver a nucleic acid to a cell, tissue or organ. Liposomes comprising neutral or anionic lipids do not generally fuse with the target cell surface, but are taken up phagocytically, and the

polynucleotides are subsequently subjected to the degradative enzymes of the lysosomal compartment (Straubinger et al, 1983, Methods Enzymol. 101 :512-527; Mannino et al, 1988, Biotechniques 6:682-690). However, as demonstrated by the data disclosed herein, an isolated snR A of the present invention is a stable nucleic acid, and thus, may not be susceptible to degradative enzymes. Further, despite the fact that the aqueous space of typical liposomes may be too small to accommodate large macromolecules, the isolated TLR agonist nucleic acid of the present invention is relatively small, and therefore, liposomes are a suitable delivery vehicle for the present invention. Methods of delivering a nucleic acid to a cell, tissue or organism, including liposome-mediated delivery, are known in the art and are described in, for example, Feigner (Gene Transfer and Expression Protocols Vol. 7, Murray, E. J. Ed., Humana Press, New Jersey, (1991)).

In other related aspects, the invention includes an isolated antimiR-21 nucleic acid operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is preferably capable of delivering the antimiR-21 nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of an isolated antimiR-21 into or to cells.

Such delivery can be accomplished by generating a plasmid, viral, or other type of vector comprising an antimiR-21 nucleic acid operably linked to a promoter/regulatory sequence which serves to introduce the antimiR-21 into cells in which the vector is introduced. Many promoter/regulatory sequences useful for the methods of the present invention are available in the art and include, but are not limited to, for example, the cytomegalovirus immediate early promoter enhancer sequence, the SV40 early promoter, as well as the Rous sarcoma virus promoter, and the like. Moreover, inducible and tissue specific expression of an isolated antimiR-21 nucleic acid may be accomplished by placing an isolated antimiR-21 nucleic acid, with or without a tag, under the control of an inducible or tissue specific

promoter/regulatory sequence. Examples of tissue specific or inducible

promoter/regulatory sequences which are useful for his purpose include, but are not limited to the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter. In addition, promoters which are well known in the art which are induced in response to inducing agents such as metals, glucocorticoids, and the like, are also contemplated in the invention. Thus, it will be appreciated that the invention includes the use of any promoter/regulatory sequence, which is either known or unknown, and which is capable of driving expression of the desired protein operably linked thereto.

Selection of any particular plasmid vector or other vector is not a limiting factor in this invention and a wide plethora of vectors are well-known in the art. Further, it is well within the skill of the artisan to choose particular

promoter/regulatory sequences and operably link those promoter/regulatory sequences to a DNA sequence encoding a desired polypeptide. Such technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (2001, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and elsewhere herein. Pharmaceutical Compositions

For ease of exposition, the formulations, compositions, and methods in this section are discussed largely with regard to unmodified oligonucleotide agents. It should be understood, however, that these formulations, compositions, and methods can be practiced with other oligonucleotide agents, e.g., modified oligonucleotide agents, and such practice is within the invention.

The invention therefore, in some embodiments, provides a method of lowering of the activity of a RNA target in vivo in a primate, wherein said method comprises the administration of an antisense oligonucleotide to a desired RNA target (e.g., miR-21), wherein said antisense oligonucleotide is essentially incapable of recruiting RNAseH.

In the present methods, the miR-21 inhibitor of the invention can be administered to the subject either as a naked oligonucleotide agent, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the oligonucleotide agent. Preferably, the miR-21 inhibitor is administered as a naked oligonucleotide agent.

The miR-21 inhibitor in the invention can be administered to the subject by any means suitable for delivering the agent to the cells of the tissue at or near the area of unwanted target nucleic acid expression (e.g., target miRNA or pre- miRNA expression).

The number of administrations may be more than 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16 or more treatments. As described herein, the actual number of administrations will depend on the nature of disease or disorder, for example. Diseases which may be cured will provide a definite end point to the administration regimen, whereas a disease or disorder may be treated over an extended period of time, effectively controlling symptoms, but may, in some embodiments not provide a cure. In such instances routine/regular administration may be continued for several months or years, until treatment is no longer desirable as determined by the medical practitioner. It will be noted that in some embodiments, administration regimens may be interrupted by a treatment pause, such a period of more than 125 days, or in some embodiments, a period of more than 2, 3, 5, or 6 months.

The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions comprising an inhibitor of miR-21 of the invention to practice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng kg/day and 100 mg/kg/day. In one embodiment, the invention envisions administration of a dose which results in a concentration of the compound of the present invention between 1 μΜ and 10 μΜ in a mammal.

Typically, dosages which may be administered in a method of the invention to an animal, preferably a human, range in amount from 0.5 μg to about 50 mg per kilogram of body weight of the animal. The precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferably, the dosage of the compound will vary from about 1 μg to about 10 mg per kilogram of body weight of the animal. More preferably, the dosage will vary from about 3 μg to about 1 mg per kilogram of body weight of the animal. The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once every three weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, subcutaneous or another route of administration. Other contemplated formulations include saline formulations, projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically -based formulations.

A formulated antimiR-21 composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the antimiR-21 is in an aqueous phase, e.g., in a solution that includes water, this form being the preferred form for administration via inhalation.

The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle as can be appropriate for a crystalline composition), or can be administered directly without the use of a delivery vehicle or particle.

Generally, the antimiR-21 composition is formulated in a manner that is compatible with the intended method of administration.

An antimiR-21 preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an

oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent.

Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as

RNAsin), other RNAs and so forth.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.

As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and

100% (w/w) active ingredient.

In addition to the active ingredient comprising an inhibitor of miR-21 of the invention may further comprise one or more additional pharmaceutically active agents. The compositions and methods of the present invention can be used in combination with other treatment regimens, including virostatic and virotoxic agents, antibiotic agents, antifungal agents, anti-inflammatory agents, as well as combination therapies, and the like. The invention can also be used in combination with other treatment modalities, such as chemotherapy, cryotherapy, hyperthermia, radiation therapy, and the like.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, intratumoral, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low- boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.

Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives;

physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and

pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's

Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : Silencing of mir-21 in vivo ameliorates autoimmune splenomegaly in lupus mice

The present study was designed to investigate whether miRNAs implicated in hematopoiesis, activation of innate immune responses and in apoptosis or cell proliferation are differentially regulated in SLE and whether such miRNAs play a role in the disease course. The results presented herein demonstrate that several differentially regulated miRNAs in B and T lymphocytes derived from B6.Slel23 mice were identified. It was observed that miR-21 expression was up-regulated in these cells regardless of the age of the mice or the severity of their disease. In vivo silencing of miR-21 using a tiny seed-targeting LNA antimiR reversed splenomegaly, which is one of the cardinal manifestations of autoimmunity in B6.Slel23 mice. In addition, administration of antimiR-21 altered CD4/CD8 T cell ratios and reduced Fas receptor-expressing lymphocyte populations.

The results presented herein demonstrate that tiny LNAs can be used to efficiently antagonize endogenous miRNAs in peripheral lymphocytes and can alter the course of a spontaneous genetic disease in mice.

The materials and methods employed in these experiments are now described.

Materials and Methods Mice

Breeder pairs of B6.Slel Sle2Sle3 (B6.Slel23) (Morel et al, 2000, Proc

Natl Acad Sci U S A 97:6670-6675) mice were a kind gift from Dr. Laurence Morel (University of Florida, Gainesville). B6.Slel23 or C57BL/6 (B6) (Jackson

Laboratory, Bar Harbor, ME, USA) mice were bred and maintained in pathogen-free, Institutional Animal Care and Use Committee (IAUCAC)-approved animal facility at the University of Pennsylvania. Care and experimental procedures were approved by the IACUC at the University of Pennsylvania. Blood urea nitrogen (BUN) was measured in freshly drawn cardiac blood (Azostix reagent strips, Siemens Healthcare Diagnostics Inc.). BUN of 5-15 mg/dL was considered consistent with no detectable renal disease; 15-26 mg/dL was consistent with mild disease; 30-40 mg/dL was consistent with moderate disease; and 50-80 mg/dL was consistent with severe disease.

Antibodies

Rat anti-mouse CD19 (1D3), CD44 (IM7), CD44 (30-F 11), CD62L (MEL-14), CD19 (1D3) and hamster anti-mouse CD3e (145-2C1 1), CD69 (H1.2F3), CD1 lc (HL3), were purchased from BD Pharmingen. Rat anti-mouse CD45R (B220; RA3-6B2), CD5 (53-7.3), CD4 (L3T4), CD4 (GK1.5), CD4 (GK1.5), CD 8 (53-6.7), IgD (1 l-26c), hamster anti-mouse CD3s (145-2C1 1), and mouse anti-mouse CD95 (APO-l/FasR; 15A7) were from eBioscience. Splenocyte preparation and lymphocyte purification

All procedures were performed on ice in FACS buffer (IX DPBS (Ca2+/Mg2+ free); 0.5% BSA; 2mM EDTA). Single-cell suspensions were prepared by crushing spleens between frosted glass slides. Cells were blocked with rat anti- mouse CD16/32 (2.4G2) monoclonal antibody (mAb) for 15 min at room temperature (RT) and stained for 30 min on ice with appropriate fluorophor-conjugated antibodies. Dead cells were excluded from FACS by staining with DAPI. FACS sorting was performed at 4°C on a FACSAria cytometer (BD Biosciences) to a purity > 95%. Alternatively, B cells were purified using autoMACS magnetic cell separation columns. Following purification, cells were either pelleted and snap-frozen or processed immediately.

Flow cytometry

All flow cytometry experiments were performed on a Becton

Dickinson FACSCalibur and the acquired data analyzed with the FloJo software package (Tree Star Inc.).

RNA purification and Quantitative real-time PCR

Total RNA was extracted using either Trizol reagent (Invitrogen) or the mzVVana miRNA Isolation Kit (Ambion). For miRNA-specific reverse transcription (RT), 20 ng purified total RNA was reverse-transcribed (TaqMan miRNA-specific RT PCR primers, Applied Biosystems). Quantitative real-time PCR (qRT-PCR) was performed in triplicates using 1 : 10 dilution of the RT product (Applied Biosystems 7500 real-time PCR system, according to the manufacturer's instructions). Small Nucleolar RNA (snoRNA)-429 expression was used as a control. Relative quantification (RQ) of B6.Slel23 miRNA expression was calculated by evaluating 2^"ΔΔα where AACt = ACt Slel23 -ACt wild-type and ACt = Ct miRNA - Ct snoRNA. For qRT-PCR amplification of the primary miR-21 (pri-miR-21), primers (F: 5'-TGA CAT CGC ATG GCT GTA-3'; SEQ ID NO: 3 and R: 5' -GAT GCT GGG TAA TGT TTG AAT G-3'; SEQ ID NO: 4) spanning a 209 nt sequence containing the mouse pre-miR-21, were designed by identifying a mouse genomic sequence, with 91% similarity to the human primary miR-21. Northern blot analysis

Total RNA samples were resolved on 15% urea-polyacrylamide gels (20% polyacrylamide for native Northern blots), transferred to nitrocellulose membranes, hybridized with radiolabeled DNA or LNA probes complementary to miR-21. snoRNA-429 expression was used for normalization. Detection and analysis: Storm 860 (Molecular Dynamics) and ImageQuant (GE).

Protein isolation and Western blot analysis

Purified cell populations were pelleted, washed with PBS then re- suspended in RIPA buffer (Pierce Biotechnology) supplemented with protease and phosphatase inhibitors (Roche Diagnostics). Proteins were resolved on NuPAGE 4- 12% bis-tris polyacrylamide gels (Invitrogen Corp., Carlsbad, CA) and transferred to nitrocellulose membranes (Invitrogen). Primary antibodies: PDCD4 (Cell Signaling) and beta actin (Sigma). Proteins were visualized using the ECL Western Blotting Detection System (GE Healthcare).

In vivo experiments

8-mer antimiR-21 and LNA scramble oligonucleotides were synthesized with a complete PS backbone as described (Patrick et al, 2010, J Clin Invest 120:3912-3916) and formulated in physiological saline (0.9% NaCl). For short- term experiments, six eight-weeks-old female B6.Slel23 mice were randomly assigned to three groups. The mice in each group received three intravenous injections of either LNA antimiR-21 or LNA scramble compounds (25mg LNA/kg mouse body wt.) on consecutive days. The mice were sacrificed within 24 hours after the last dose. For long-term experiments, twelve nine-weeks-old female B6.Slel23 mice were randomly assigned to four groups and mice in each group received intraperitoneal (IP) injections of either LNA antimiR-21, LNA scramble control compound, or saline. The mice were weighed immediately prior to dosing and received a dose of 25 mg LNA/kg mouse body wt. All mice received three initial IP injections on consecutive days and subsequent booster injections of 25 mg/kg every three weeks over a period of three months. The last booster injection was via intravenous delivery. All mice were sacrificed within 24 hours after the last dose.

Statistical analysis Reported B6.Slel23 mean miRNA expression differences represent triplicate measurements from at least 3 independent experiments in individual age-and gender-matched B6 and B6.Slel23 mice. Standard errors and ^-values were calculated with an un-paired, two-tailed Student's t test.

The results of the experiments are now described.

Differential miRNA expression in B6.Slel23 peripheral lymphocytes

The following experiments were designed to investigate whether miRNAs implicated in hematopoiesis (Xiao et al, 2007, Cell 131 : 146-159); in activation of innate immune responses (Taganov et al, 2006, Proc Natl Acad Sci U S A 103 : 12481-12486); and in apoptosis or cell proliferation (He et al, 2007, Nat Rev Cancer 7:819-822; Rokhlin et al, 2008, Cancer Biol Ther 7: 1288-1296; Yamakuchi et al, 2009, Cell Cycle 8:712-715) are differentially regulated in SLE and whether such miRNAs play a role in SLE pathogenesis or course. To this end, total RNA was isolated from FACS sorted splenic B and T lymphocytes from individual B6.Slel23 mice and their miRNA expression profiles were compared to those of control B6 mice. Quantitative real-time PCR (qRT-PCR) analysis showed that expression of several miRNAs was differentially regulated in B6.Slel23 splenic B cells, naive (CD44^LOCD62L^HI) and memory (CD44^HICD62L^LO) T cells as shown in Table 1, particularly from older mice (12 months of age), which typically have advanced splenomegaly and kidney disease (Morel et al, 2000, Proc Natl Acad Sci U S A 97:6670-6675, Fairhurst et al, 2008, Eur J Immunol 38: 1948-1960). For up-regulated miRNAs, expression differences ranged from 1.8-to 13 -fold. Interestingly, miR-21 expression was up-regulated in lupus B cells as well as in naive and memory T cells compared to B6 controls, regardless of the age of mice as shown in Table 1 and depicted Figure 1A. In contrast, miR-181 and miR-150 expression was down- regulated in most lymphocyte subsets examined in lupus mice compared to B6 controls as shown in Table 1.

Table 1: miRNA expression in B6.Siel23 lymphocyte subsets

miRNA expression in B6.Slel23 B and T lymphocytes a. CD19⁺ splenic B cells from individual B6.Slel23 and age- and gender-matched B6 mice at 2, 6 and 12 months of age were FACS purified. 20 ng total B cell RNA were reverse-transcribed with TaqMan miRNA-specific reverse-transcription primers followed by quantification using Taqman miRNA-specific qRT-PCR assays. B6.Slel23 miRNA relative quantification (RQ) values represent the average of triplicate measurements from at least 3 independent experiments in individual B6.Slel23 mice relative to the expression of the same miRNA in individual B6 B cells. Prior to obtaining RQ values, the Ct values for each miRNA in each sample, were normalized against sno429 5 expression (AC_t). The mean B6.Slel23 miRNA RQ was calculated by evaluating 2^{" AACt} where AAC_t = AC_t sum ave - AC_{t B}6 ave- b. As in a except total splenic CD44^Low CD62L^Hlgh na^'fve T cells were FACS purified from individual mice. c. As in a except total splenic CD44^High CD62L^Low memory T cells were FACS purified from individual mice. d. As in a expect CD5^" B2 and CD5⁺ Bla B cells and CD4⁺ and 10 CD8⁺ T cells were FACS purified from individual 12 months old B6.Slel23 and age- and gender matched B6 mice. Standard error and ^-values were calculated using an un-paired, two-tailed Student's t test.

Differential miRNA expression in B6.Slel23 splenic B Cells

2 mo. (mild disease) 6 mo. (moderate disease) 12 mo. (severe disease) miRNA RO¹ ±SEM v-value RO¹ ± SEM v-value RO¹ ± SEM v-value miR-21 1.7 ±0.15 0.001 2.2 ± 0.42 o.o3ft 5.8 ±0.64 o.ulft miR-34a 0.9 + 0.38 0.670 1.2 · 1.33 0.821 13.2 + 0.65 0.000 mi - 146a 1.0 ±0.51 o.wft o.ft 0.25 0.044 5.3 ±0.75 0.032 miR-221 1.2 ± 0.29 0.504 1.2 ± 0.28 0.398 13.3 ±1.05 0.023 miR-222 1.4 ±0.22 0.040 2.1 0.26 0.015 5.X D.Wi 0.037 miR-223 0.9 · 0.53 0.720 2.3 ±1.61 0.507 11.4 ±0.68 0.006 miR-I42-5p 1.1 ±0.21 0.374 0.4 ± 0.34 0.017 2.7 ± 0.45 0.032 miR- 155 1.3 ± 0.12 0.026 1.2 ± 0.39 0.504 2.8 · 0.53 0.050 miR- 150 o.- 0.45 0.314 0.5 0.2^" II.IWX 0.5 ± 0.18 O.OOft miR-181a 0.5 ±0.20 0.007 0.3 ±0.78 0.069 0.4 ±0.36 0.020

Differential miRNA expression in B6.Slel23 splenic naive T cells

miR-21 2.5 ^■ 0.3- 0.012 3.ft l.oo 0.110 3.0 0.5 0.032 miR-34a 1.5 ±0.91 0.528 4.2 ±1,05 0.094 6.2 · 0.50 0.006 miR- 146a 1.0 ± 1.20 o.';55 4.' 1.5ft 0. 1ft 4.1 ±0.31 0.002 miR-221 0.3 ± 0.47 0.013 0.433 4.0 · 0.39 0.002 miR-222 o.r, 0.4' 0.187 1.2 0.50 o.ft-2 0.5 0.20 0.00ft miR-223 1.2 ±0.55 0.697 0.019 ...........J ,± L, 0.000 miR-142-5p l.o 0.33 o.vor, 1.3 0.5ft 0.550 2.ft o.4ft 0.02- miR- 155 0.5 ±0.12 0.000 0.475 0.000 miR- 150 1.0 ±0.84 o.^>;43 o.- o.ft'; 0.553 o.ft 0.22 (l.(ll)X miR-181a 1.4 ±0.77 0.562 1.0 ±0.97 0.980 0.2 ± 0.66 0.015

Differential miRNA expression in B6.Slel23 memory T cells

miR-21 2.3 o.2ft O.OU3 2.3 0.32 o.oo' 2.'; 0.31 o.oo- miR-34a 1,2 ± 0.46 0.616 0.170 6,1 ± 1.0^a 0.039 miR- 146a 2.0 0.2^" 11.1)1 IX l.y 0.23 (I.OOft 1.8 ±0.31" 0.033 miR-221 0,9±0.29 0.789 2,2±0,33^a 0.024 0.015 miR-222 3.1) o.ftft 0.048 2.5 0.3' 0.025 4.7 ±0.91" 0.03X miR-223 1,1 ±0,44 0.805 1.7 · 0.14 ^' 0.004 0.026 miR-142-5p i.2 ±0.22 o.35ft 1.3 ^■ ο.3' 0.3 x'; .';^■ 0.52 0.724 miR- 155 0.1 ± 0.86^a 0.028 1.3 + 0.30 0 911 o.i + o.: ;i^c 0.0002 mi R- 150 1 3 _* 0.30 0.343 1 1 + 0.51 ) ⁽ 1 805 1.0 + 0. ^■sx ⁽ I.V4 miR-181a 0.8 + 0.33 0.491 0.9 + 0.98 0.934 0.2 + 0.79^a 0.015

The Sle2 locus in B6.Slel23 mice confers expansion of the peritoneal and splenic B l-a B cell compartment (Mohan et al, 1997, J Immunol 159:454-465),

5 even though the B2 subset remains the most abundant. To evaluate the relative

contribution of each cell subset to individual miRNA abundance in the total pool of

splenic CD19⁺ B cells from older SLE mice, the expression of miR-21, miR-146a and miR-155 in Bl-a and B2 subsets have been quantified. The same trend for miR-21

and miR-146a expression in both lupus B cell subgroups has been detected whereas

10 miR-155 was up-regulated only in B2 cells as shown in Table 2. When lupus T cell

subsets were examined, a 10-and 24-fold up-regulation of miR-21 was detected in

CD4+ and CD8+ T cells, respectively, as well as a significant up-regulation of miR- 146a and miR-155 compared to controls as shown in Table 2.

15 Table 2. Differential miRNA expression in B6.Slel23 lymphocyte subsets

CD5^" B2 and CD5⁺ Bla B cells and CD4⁺ and CD8⁺ T cells were FACS purified from individual 12 months old B6.Slel23 and age- and gender matched B6 mice. 20 ng

total B cell RNA were reverse-transcribed with TaqMan miRNA-specific reverse-

20 transcription primers followed by quantification using Taqman miRNA-specific qRT- PCR assays. B6.Slel23 miRNA relative quantification (RQ) values represent the

average of triplicate measurements from at least 3 independent experiments in

individual B6.Slel23 mice relative to the expression of the same miRNA in individual

B6 B cells. Prior to obtaining RQ values, the Ct values for each miRNA in each

25 sample, were normalized against sno429 expression (AC_t). The mean B6.Slel23

miRNA RQ was calculated by evaluating 2^"AACt where AAC_t = AC_tski23 ave - AC_t B6 ave- Standard error and ^-values were calculated using an un-paired, two-tailed Student's t test.

CD5^~ B2 B cells CD5⁺ Bla B cells CD4⁺ T cells CD8⁺ T cells miRNA RQ¹ ± SEM(p-value) RQ¹ ±SEM(p-value) RQ¹ ± SEM(p-value) RQ¹ ± SEM(p-value) miR-21 5.2 U.S5 < < i.< i4\i 2. - 1 .3d ( 0.32V_.) 10.2 ± 0.76 (0.01 1 ) 24.4 ± 0.97 (0.009) miR-146a 1.9 + 1.09 (0.456) 1 .9 · 0.83 (0.3 1 ) 5.4 · 0.48 (0.007 ) 1 9.4 · 0.93 (0.01 0 ) miR- 1 55 2. 1 ⁽ 1.35 i i ).( i x i o.y4 I . O.V54 I 2.y ^{■ (} 1.⁽ 13 i n. 1 1 iiiiiiiiiiiiiiiiiiiiiiiii

30 miR-21 expression is regulated transcriptionally in B6.Slel23 B cells The results presented herein demonstrate that that miR-21 expression was consistently up-regulated in all SLE cell subsets which were investigated.

Notably, the fold change of miR-21 expression in B6.Slel23 B lymphocytes, compared to that from age-and gender-matched B6 mice, positively correlated with the age of mice and thus with severity of their disease as depicted in Figures 1A, IB and 1C. To further dissect the role of miR-21 in SLE, experiments were designed to test whether the miR-21 gene is transcriptionally regulated by comparing the expression of pri-miR-21 in CD19⁺ B cells isolated from B6.Slel23 and B6 mice. qRT-PCR analysis revealed up-regulation of pri-miR-21 transcript in B cells from lupus mice compared to controls (Figure ID). This indicates that at least in part, miR- 21 up-regulation in B6.Slel23 B lymphocytes results from transcriptional activation of miR-21 gene.

In vivo inhibition of miR-21 de-represses PDCD4 expression in B6.Slel23 T cells

Since miR-21 has been shown to regulate apoptosis and cell proliferation pathways (Chan et al, 2005, Cancer Res 65:6029-6033; Cheng et al., 2005, Nucleic Acids Res 33 : 1290-1297; Lu et al, 2008, Oncogene 27:4373-4379; Sayed et al, 2010, J Biol Chem 285:20281-20290; Yang et al, 2004, Mol Cell Biol 24:3894-3906) and was overexpressed in all subsets of lupus lymphocytes, the next set of experiments was designed to test whether in vivo inhibition of miR-21 in

B6.Slel23 mice could affect the course of their disease. The use of LNA-modified antimiR oligonucleotides for silencing of miRNAs in vivo has been previously demonstrated in rodents and non-human primates (Elmen et al. 2008, Nature 452:896- 899; Elmen et al., 2008, Nucleic Acids Res 36: 1 153-1 162; Worm et al, 2009, Nucleic Acids Res 37:5784-5792). Furthermore, recent studies have described an approach that enables efficient antagonism of miR-21 function in vivo using a short LNA- modified seed-targeting antimiR-21 oligonucleotide (Patrick et al, 2010, J Clin Invest 120:3912-3916). Thus, first, a short-term in vivo study was designed where three B6.Slel23 mice were injected intravenously at a dose of 25 mg/kg of saline- formulated antimiR-21 compound on three consecutive days and sacrificed within 24 hours after the last dose. In control experiments, three age and gender-matched B6.Slel23 mice were treated with a LNA scramble compound. Interestingly, this short-term study showed that silencing of miR-21 in vivo results in approximately 20% de-repression of PDCD4 in naive CD4⁺ T cells as depicted in Figure 2. PDCD4 is an inhibitor of translation initiation (Yang et al, 2004, Mol Cell Biol 24:3894-3906, Zakowicz et al, 2005, RNA 1 1 :261-274) and a tumor suppressor (Cmarik et al, 1999, Proc Natl Acad Sci U S A 96: 14037-14042, Jansen et al., 2005, Cancer Res 65:6034- 6041). Expression of PDCD4 is post-transcriptionally regulated by miR-21 (Asangani et al, 2008, Oncogene 27:2128-2136, Frankel et al, 2008, J Biol Chem 283 : 1026- 1033). However, the function of PDCD4 in lymphocytes was not yet understood prior to the present invention.

In vivo inhibition of miR-21 ameliorates splenomegaly in B6.Slel23 mice

Four 9 week-old female SLE mice were treated by injecting with antimiR-21 or LNA scramble compounds at a dose of 25mg/kg or with saline vehicle daily for three consecutive days followed by single injections every three weeks for a duration of nine weeks. All mice were sacrificed within 24 hours after the last dose. Northern blot analysis suggested that miR-21 was sequestered in a slower-migrating heteroduplex with the antimiR-21 in the liver and in B cells as depicted in Figure 3 A. Notably, silencing of miR-21 in vivo resulted in a significant reduction of

splenomegaly in the SLE mice treated with antimiR-21, compared to the LNA scramble and vehicle control treated mice as depicted in Figure 3B and 3C).

Furthermore, a significant decrease in the CD4⁺ to CD8⁺ T cell ratio in the antimiR-21 treated mice and a reduction in the number of Fas receptor-expressing splenic B cells in the antimiR-21 treated mice were observed as depicted in Figure 4 and Table 3. Interestingly, elevated CD4⁺to CD 8⁺ ratios are a characteristic phenotypic alteration in Sle3 bearing T lymphocytes (Mohan et al, 1999, J Immunol 162:6492-6502). Thus, the results presented herein suggest that miR-21 inhibition skews the CD4⁺ to CD8⁺ ratio towards that of the background non-autoimmune strain (Mohan et al, 1999, J

Immunol 162:6492-6502). In the LNA scramble-treated group, an increase in the CD4 to CD8 ratio due to an increase in CD4 cells was observed. Anti-chromatin antibody titers and Blood Urea Nitrogen (BUN) levels were not affected by miR-21 silencing in this study.

Table 3: In vivo miR-21 silencing alters T and B lymphocyte subsets in B6.Slel23.

Mice were treated in vivo with antimiR-21 inhibitors and control compound or saline for 12 weeks, as described in the text. Splenocytes derived from B6.Slel23 mice injected with saline (left column), LNA scramble control (middle) and LNA antimiR- 21 (right) were stained with appropriate fluorophore-conjugated antibodies, subjected to flow cytometry and analyzed with FloJo software.

Treatment Group

Saline LNA Scramble LNA antimiR-21

Total splenocytes (x

10⁶)⁶ 392 ± 74.5 335 ± 82.2 373 ± 106.5

T cells

Events 250 x 10³ 250 x 10³ 250 x 10³

Viable (x 10³)^c 199.3 ± 15.1 201.6 ± 9.5 207.8 d : 3.9

CD4⁺ (x lOY 36.2 ± 7.5 35.6 ± 4.4 39.8 i : 2.5

CD8⁺ (x lOY 7.8 ± 2.1 5.2 ± 0.4 17.0 i : 5.9 *†

CD4⁺:CD8⁺ ratio 4.7 ± 0.6 6.9 ± 1.3†† 2.6 i : 1.1

B cells

Events 1 x 10^b 1 x 10^b 1 x 10^b

Viable (x 10 3') 617.9 ± 77.5 565.2 ± 56.1 607.6 ± 93.0 CD19⁺ B220⁺ (x

265.0 ± 88.2 127.3 ± 88.8 268.8 ± 50.6

10³)^c

FasR⁺ IgD^~ (xlQ³)^e 38.8 ± 19.4 25.4 + 14.7jj 20.9 ± 7.1 |§ aValues represent the mean ± SD (n = 4 mice in each treatment group)

^Spleens were harvested and single-cell suspensions prepared as described in

Materials and Methods. Viable cells were identified on the basis of trypan blue staining.

10 ^cViable lymphocyte populations were identified post-collection. ¾ated on the viable lymphocyte population. "Gated on the CD19⁺ B220⁺ population.

15

* p= 0.007 LNA antimiR-21 compared to LNA scramble

† p= 0.026 LNA antimiR-21 compared to Saline

†† p= 0.053 LNA scramble compared to Saline

20

p= 0.60 LNA antimiR-21 compared to LNA scramble

§ p= 0.13 LNA antimiR-21 compared to Saline

II p= 0.30 LNA scramble compared to Saline

25 Silencing of microRNA-21 in vivo ameliorates autoimmune splenomegaly in lupus mice

The results presented herein demonstrate aberrant miRNA expression in B6.Slel23 lymphocytes at different stages of lupus. Without wishing to be bound by any particular theory, although only a small set of miRNAs was focused on, it is likely that other miRNAs not tested in this study are also differentially regulated. The up-regulation of miR-21 was of particular interest since it is detected in young as well as older mice and thus precedes severe SLE manifestations. Importantly, up- regulation of miR-21 in B and T lymphocytes preceding SLE manifestations was reported recently in the MRL/lpr, another genetic mouse lupus model. Furthermore, miR-21 is also upregulated in human lupus CD4⁺ T and B cells (Te et al, 2010, PLoS One 5:el0344; Pan et al, 2010, J Immunol 184:6773-6781). Combined with the results presented elsewhere herein, it has been demonstrated that miR-21 plays an important role in the disordered immunoregulation in SLE. To investigate the function of miR-21 in lupus, an in vivo miR-21 knockdown study was designed, which demonstrated a significant amelioration in splenomegaly, induced specifically by treatment with the antimiR-21 compound but not the LNA control compound.

The present study is the first to demonstrate that tiny seed-targeting LNAs can be used to efficiently antagonize endogenous miRNAs in peripheral lymphocytes and that pharmacological inhibition of a miRNA using such compounds can alter the course of a spontaneous disease in mice. Furthermore, the present results suggest that miR-21 controls cellular pathways important for cardinal disease manifestations in B6.Slel23 and are consistent with recent reports showing that miR- 21 inhibition reverses lymphosplenomegaly in mice with pro-B cell lymphoma (Medina et al, 2010, Nature 467:86-90). PDCD4 is directly targeted by miR-21 (Asangani et al, 2008, Oncogene 27:2128-2136, Frankel et al, 2008, J Biol Chem 283: 1026-1033; Reis et al, 2010, Mol Cancer 9:238) and regulates pathways involved in apoptosis, cell cycle and differentiation (Reviewed in (Lankat et al, 2009, Biol Cell 101 :309-317)). In pancreatic adenoma, miR-21 silencing induces PDCD4, promotes cell proliferation and inhibits apoptotic cell death (Bhatti et al, 201 1, J Gastrointest Surg 15: 199-208). PDCD4 deficient mice develop B cell lineage lymphomas and their lymphocytes produce increased cytokines with important functions in T cell differentiation, such as IL-10, IL-4 and IFN gamma (Hilliard et al, 2006, J Immunol 177:8095-8102). The function of PDCD4 in SLE is elusive, however it is likely that PDCD4 is involved in pathways contributing to the characteristic immune system phenotype in B6.Slel23. It is also likely that miR-21 participates in the orchestration of the cross talk between hyperactive B and T cells in lupus by regulating cell- signaling pathways. Without wishing to be bound by any particular theory, it is believed that future elucidation of the signaling pathways controlled by miR-21 in SLE lymphocytes can shed light on the molecular complexity of a prototype autoimmune disease and opens new directions for investigations of novel disease biomarkers and therapeutic strategies for treatment of SLE.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A method of reducing the amount of miR-21 in a peripheral lymphocyte, the method comprising contacting a peripheral lymphocyte with an oligomer, wherein the oligomer comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21.

2. The method of claim 1, wherein the oligomer comprises the sequence of SEQ ID NO: 1.

3. The method of claim 1, wherein the sequence of miR-21 is SEQ ID NO: 2.

4. The method of claim 1, wherein the oligomer is at least 7-10 contiguous nucleotides in length.

5. The method of claim 1, wherein the oligomer is at least 11-18 contiguous nucleotides in length.

6. The method of claims 1, wherein the oligomer is stabilized against nucleolytic degradation.

7. The method of claim 1, wherein the oligomer comprises a nucleotide analog selected from the group consisting of 2'-0-alkyl-RNA monomer, 2'- amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, HNA, ΓΝΑ, 2'-MOE-RNA (2'-0-methoxyethyl-RNA), 2'Fluoro-DNA, and locked nucleic acid (LNA).

8. The method of claim 7, wherein the nucleotide analog is a locked nucleic acid (LNA).

9. The method of claim 1, wherein the antisense oligonucleotide is essentially incapable of recruiting RNaseH.

10. The method of claim 1 , wherein at least 70% of the nucleotide units of the oligomer are selected from the group consisting of LNA units and 2' substituted nucleotide analogues, and wherein at least 50% of the nucleotide units of the oligomer are LNA units.

1 1. The method of claim 10, wherein the length of the oligomer is

7, 8 or 9 contiguous nucleotides, wherein the contiguous nucleotide units are independently selected from the group consisting of LNA units and 2' substituted nucleotide analogues.

12. The method of claim 1, wherein at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70% of the nucleotide units of the oligomer are nucleotide analogue units.

13. The method of claim 1, wherein all the nucleotide units of the oligomer are nucleotide analogue units.

14. The method of claim 1, wherein the nucleotide units of the oligomer are independently selected from LNA units and DNA units, and wherein the oligonucleotide does not comprise a region of more than 5 consecutive DNA units.

15. The method of claim 1, wherein at least one of the internucleoside linkages present between the nucleoside units of the contiguous nucleotide sequence is a phosphorothioate internucleoside linkage.

16. The method of claim 1, wherein all the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

17. A method of treating a mammal having a systemic autoimmune disease, the method comprising administering to a mammal in need thereof an effective amount of a nucleic acid molecule wherein the nucleic acid molecule binds to miR-21 in a peripheral lymphocyte in the mammal; and wherein the binding of the nucleic acid to miR-21 in the lymphocyte diminishes the level of expression of miR-

21 in the lymphocyte.

18. An in vitro method of reducing the amount of miR-21 in a peripheral lymphocyte, the method comprising contacting a peripheral lymphocyte with an oligomer which comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21.

19. An oligomer which comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21 for use in the treatment of a mammal suffering from a systemic autoimmune disease.

20. The use of an oligomer which comprises a sequence which is substantially complementary to 6 to 22 contiguous nucleotides of miR-21 in the manufacture of a medicament for the treatment of a mammal suffering from a systemic autoimmune disease.

21. A method of reducing the amount of miR-21 in a peripheral lymphocyte, the method comprising contacting a peripheral lymphocyte with a nucleic acid molecule, wherein the nucleic acid molecule comprises a sequence which is substantially complementary to 6 to 15 contiguous nucleotides of miR-21.

22. The method of claim 21, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.

23. The method of claim 21 , wherein the sequence of miR-21 is SEQ ID NO: 2.

24. The method of claim 21 , wherein the nucleic acid molecule is at least six nucleotides in length.

25. The method of claim 21, wherein the nucleic acid molecule is stabilized against nucleolytic degradation.

26. The method of claim 21, wherein the nucleic acid molecule comprises a nucleotide analog.

27. The method of claim 26, wherein the nucleotide analog is a locked nucleic acid (LNA).

28. A method of treating a mammal suffering from a systemic autoimmune disease, the method comprising administering an effect amount of a nucleic acid molecule to the mammal in need thereof, wherein the nucleic acid molecule comprises a sequence which is substantially complementary from 6 to 22 contiguous nucleotides of miR-21.

29. The method of claim 28, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.

30. The method of claim 28, wherein the sequence of miR-21 is SEQ ID NO: 2.

31. The method of claim 28, wherein the nucleic acid molecule is at least six nucleotides in length.

32. The method of claim 28, wherein the nucleic acid molecule is stabilized against nucleolytic degradation.

33. The method of claim 28, wherein the nucleic acid molecule comprises a nucleotide analog.

34. The method of claim 33, wherein the nucleotide analogue is a locked nucleic acid (LNA).

35. The method of claim 28, wherein the mammal is a human.

36. The method of claim 28, wherein the systemic autoimmune disease is associated with unregulated expression of miR-21.

37. The method of claim 28, wherein the systemic autoimmune disease is SLE.