WO2014058920A1 - Methods of targeting mir-223 expression to ablate leukemic cells - Google Patents

Description

TITLE

METHODS OF TARGETING MIR-223 EXPRESSION TO ABLATE LEUKEMIC CELLS

Inventors: Clay B. Marsh, Melissa G. Piper, and Yijie Wang

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States Provisional Application

No. 61/710,899, filed October 8, 2012, the entire disclosure of which is expressly incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

[0002] The invention relates to the treatment of cancers, in particular to the treatment of leukemia. The invention additionally provides novel methods and compositions for the improvement of anti-cancer therapies, such as chemotherapy and other conventional cancer therapies.

STATEMENT REGARDING SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted via

EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy, created on October 7, 2013, is named 54848_PCT_SeqList_OSIF_2012_317.txt, and is 796 bytes in size.

BACKGROUND

[0004] MicroRNAs (miRNAs) are non-coding RNAs of 19-25 nucleotides in length that regulate gene expression by inducing translational inhibition or cleavage of their target mRNA through base pairing to partially or fully complementary sites. The miRNAs are involved in critical biological processes, including development, cell differentiation, apoptosis and proliferation.

[0005] Identification of miRNAs that are differentially-expressed in leukemic cells would aid in treating, diagnosing, and prognosticating leukemia. Furthermore, the identification of miRNAs useful as targets for antagomirs, or anti-miRNAs, may help to treat leukemia.

SUMMARY OF THE INVENTION

[0006] In a first broad aspect, there is provided herein a method of treating leukemia in a subject having leukemia by administering an agent that inhibits miR-223 expression in a cell of the subject.

[0007] In certain embodiments, the agent is an antagomir of miR-223, an anti-miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR- 223, or a double stranded RNA that complementary base-pairs with miR-223.

[0008] In another aspect, there is provided herein a method of increasing the efficacy of an anticancer treatment in a subject having leukemia by administering at least one anti-cancer treatment and an agent that inhibits miR-223 expression in a cell of the subject. [0009] In another aspect, there is provided herein a method of treating leukemia in a subject having leukemia, the method comprising administering an effective amount of an agent that inhibits miR-223 expression in a cell of the subject, wherein the agent that inhibits miR-223 expression is a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1, or its complement.

[00010] In certain embodiments, the cell is a leukemic cell.

[00011] In certain embodiments, the cell is myeloid leukemic cell.

[00012] In certain embodiments, the cell is a lymphocytic leukemic cell.

[00013] In certain embodiments, the cell is present in a subject.

[00014] In certain embodiments, the subject is a human.

[00015] In certain embodiments, the subject has acute myelogenous leukemia.

[00016] In certain embodiments, the subject has chronic lymphocytic leukemia.

[00017] In another aspect, there is provided herein a method of increasing the efficacy of anticancer treatment in a subject having leukemia, comprising: (i) administering at least one anti-cancer treatment to the subject having leukemia; and (ii) administering an effective amount of an agent that inhibits miR-223 expression in a cell of the subject; wherein the efficacy of the anti-cancer treatment is increased, and wherein the agent that inhibits miR-223 expression is a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1, or its complement.

[00018] In certain embodiments, an increase in the efficacy of an anti-cancer treatment is evidenced by increased remission of the cancer in the subject, relative to a suitable control.

[00019] In another aspect, there is provided herein a method of increasing the sensitivity of a leukemic cell to the cytotoxic effects of an anti-cancer agent, comprising administering to the leukemic cell an effective amount of an agent that inhibits miR-223 in a cell of the subject, wherein the sensitivity of the cell to the anti-cancer agent is increased, and wherein the agent that inhibits miR- 223 is a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1, or its complement.

[00020] In certain embodiments, the agent that inhibits miR-223 and the anti-cancer agent are coadministered.

[00021] In certain embodiments, an increase in the sensitivity of the leukemic cell to the anticancer agent is evidenced by the death of the leukemic cell.

[00022] In another aspect, there is provided herein a pharmaceutical composition for treating leukemia comprising an inhibitor of miR-223, wherein the inhibitor of miR-223 comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, or its complement.

[00023] In certain embodiments, the inhibitor of miR-223 is an antagomir of miR-223, an anti- miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR-223, or a double stranded RNA that complementary base-pairs with miR-223.

[00024] In another aspect, there is provided herein a pharmaceutical composition for treating leukemia comprising at least one anti-cancer agent and an inhibitor of miR-223, wherein the inhibitor of miR-223 comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, or its complement.

[00025] In another aspect, there is provided herein a method for determining the efficacy of a cancer therapy in a subject, comprising administering at least one therapeutic agent to the subject and subsequently measuring the expression of miR-223 in a sample from the subject.

[00026] In certain embodiments, the sample is a blood sample.

[00027] In certain embodiments, the sample is a plasma sample.

[00028] In certain embodiments, the sample is a bone marrow sample.

[00029] In certain embodiments, a decrease in the expression of miR-223 relative to a suitable control is indicative of successful treatment.

[00030] In yet another aspect, there is provided herein a method comprising: determining the level of monocytes in a subject; and administering a therapeutically effective amount of an inhibitory nucleic acid that is complementary to miR-223 to a subject determined to have an increased production of monocytes; whereby differentiation of the monoctyes is reduced; hematopoietic cell production in the subject's marrow is decreased; and /or, release of microvesicles produced from macrophages is decreased.

[00031] In another aspect, there is provided herein a method of treating leukemia in a subject in need thereof, the method comprising: obtaining a sample of hematopoietic progenitor cells from the subject; contacting the hematopoietic stem progenitor cells with a vector comprising a nucleic acid sequence that is an antagomir of miR-223; and, introducing the cell into the same subject.

[00032] In certain embodiments, the agent that decreases miR-223 expression in a cell comprises a vector comprising a nucleic acid sequence that is at least 90% identical to the antagomir of miR- 223.

[00033] In certain embodiments, the nucleic acid is at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and all the intermediate percentages between 90% and 100%, identical to the antagomir of miR-223.

[00034] In certain embodiments, the agent decreases the level of miR-233 in hematopoietic progenitor cells.

[00035] In another aspect, there is provided herein a method of treating leukemia in a subject in need thereof comprising: administering to the subject an agent comprising a sequence complementary to miR-223, wherein expression or activity of miR-223 in target cells of the subject is inhibited following administration of the agent, thereby treating the leukemia.

[00036] In certain embodiments, the agent comprises a nucleic acid sequence similar to one or more of: an antagomir of miR-223, an anti-miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR-223, or a double stranded RNA, all of which complementary base-pair with miR-223. [00037] In certain embodiments, the antisense oligonucleotide comprises at least one chemical modification.

[00038] In certain embodiments, the antisense oligonucleotide comprises a 2'-0-methyl oligoribonucleotide.

[00039] In certain embodiments, the antisense oligonucleotide comprises a sequence that is complementary to a mature miR-223 sequence.

[00040] In certain embodiments, the antisense oligonucleotide comprises a sequence that is complementary to miR-223.

[00041] In certain embodiments, the antisense oligonucleotide is encoded by an expression vector, and wherein the antisense oligonucleotide is under the transcriptional control of a promoter.

[00042] In certain embodiments, the promoter is a macrophage-specific promoter.

[00043] In certain embodiments, the agent includes a virus or a non-virus vector.

[00044] In certain embodiments, administering comprises oral, transdermal, sustained release, controlled release, delayed release, suppository, sublingual, intravenous or direct injection administration of the antisense oligonucleotide.

[00045] In certain embodiments, the method of treating leukemia in a subject in thereof further comprises administering to the subject a second therapy.

[00046] In certain embodiments, the method of treating leukemia in a subject in thereof is carried out in conjunction with at least one additional therapy modality.

[00047] In certain embodiments, the additional therapy modality is selected from the group consisting of bone marrow transplant, cord blood cell transplant, surgery, chemotherapy, radiation therapy, immunotherapy and a combination thereof.

[00048] In certain embodiments, the additional therapy modality is administered at the same time as the antisense oligonucleotide.

[00049] In certain embodiments, the additional therapy modality is administered either before or after the antisense oligonucleotide.

[00050] In certain embodiments, treating comprises improving one or more symptoms of the leukemia.

[00051] In certain embodiments, progression of leukemia is inhibited in the subject following administration of the antisense oligonucleotide.

[00052] In certain embodiments, a nucleic acid construct is used for the preparation of a medicament for treating cancer in a human subject.

[00053] In certain embodiments, a nucleic acid construct is used for the preparation of a medicament for inhibiting leukemic progression in a human subject.

[00054] In certain embodiments, a nucleic acid construct is used for the preparation of a medicament for inhibiting metastasis in a human subject. [00055] In certain embodiments, a nucleic acid construct is used for the preparation of a medicament for reducing or alleviating a symptom associated with a neoplastic disorder.

[00056] In certain embodiments, treating comprises delaying the transition from a preleukemic condition to leukemia.

[00057] In another aspect, there is provided herein a pharmaceutical composition comprising an antagonist of miR-223 and a pharmaceutically acceptable carrier, excipient or diluent.

[00058] In another aspect, there is provided herein a pharmaceutical composition wherein the antagonist of miRNA-223 comprises a sequence that is complementary to the mature sequence of miRNA-223.

[00059] In another aspect, there is provided herein a pharmaceutical composition comprising a miR-223 molecule or an antagomir thereof or a variant thereof for use in the positive or negative modulation of macrophage-derived micro vesicles, whereby the use in the modulation relates to the treatment of a leukemic disease.

[00060] In another aspect, there is provided herein a kit comprising i) one or more dosage units of the pharmaceutical compositions described herein; and ii) instructions for administering the nucleic acid construct to a subject in need thereof.

[00061] In another aspect, there is provided herein a kit containing i) one or more dosage units of a nucleic acid construct sufficient for one or more courses of treatment for a cell or microvesicle expressing miR-223; and ii) instructions for administering the nucleic acid construct to a subject in need thereof.

[00062] Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

[00063] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[00064] FIGS. 1A - ID: Microvesicle production during macrophage differentiation:

[00065] FIG. 1A: THP-lcells were treated with vehicle (inset) or alternatively with PMA to induce differentiation and observed by electron microscopy, n=3.

[00066] FIG. IB: Freshly isolated peripheral blood monocytes were untreated for 30 minutes (inset) or GM-CSF for 4 hours then cultured overnight, n=6. Images were captured with an AMT camera (Advanced Microscopy Techniques, Danvers, MA). Representative images are shown in (FIG. 1A and FIG. IB) and microvesicles are indicated by the white arrows in both panels.

[00067] FIG. 1C: THP-1 cells and peripheral blood monocytes were treated with PMA and GM- CSF, respectively. Microvesicle concentration containing both Annexin V positive and negative events from the MV gated region was quantified by flow cytometry. Shown is the average concentration +S.E.M, (n=6)

[00068] FIG. ID: The vitrified microvesicle samples were transferred to a Gatan Cryo holder (Pleasanton, CA) and visualized with Tecnai G2 F20 ST TEM at 200kV. Images were captured using a 4k x 4k Gatan Ultrascan CCD camera at a magnification of 38,000x. Representative cryo-TEM images of microvescicles collected using a 16,000x g centrifugation from the culture supernatant of PMA-treated THP-1 cells are shown. Large (>200 nm diameter) and small micro vesicles (30-100 nm diameter) (inset) are present. Scale bar = 100 nm in the figure and inset.

[00069] FIG. 2: Microvesicle uptake by peripheral blood monocytes. Shown are confocal images from GM-CSF-treated donor monocytes stained with D3 84, a phospholipid membrane dye (red stain) (upper left panel) and SytoRNA Select, an RNA-select stain (green stain) (lower left panel). After 48 hours, microvesicles from GM-CSF-stimulated monocytes were collected. 100,000 microvesicles/ml were incubated with unstained naive recipient cells. Cells were imaged using the Zeiss LSM 510 multiphoton confocal microscope (Thomwood, NY). Background fluorescence was subtracted using unstained cells. Transfer of fluorescent microvescicles from the GM-CSF-stimulated cells was apparent in the recipient cell membranes and cytoplasma as indicated by the appearance of the red dye as well as transferred RNA as indicated by the green stain (right panel). The differential interference contrast (DIC) was used to visualize the morphology of the cells without fluorescence. Data is representative of three independent monocyte donors and three monocyte recipients.

[00070] FIGS. 3A - 3F: Cellular Differentiation induced by microvesicles:

[00071] FIGS. 3A and 3B: THP-1 cells (FIG. 3 A) and monocytes (FIG. 3B) were treated with microvesicles from PMA- or GM-CSF-stimulated cells, respectively. After 48 hours, adherent cells possessing macrophage -like phenotype were stained with crystal violet and dye uptake was measured at 550 nm. As controls, THP-1 cells were treated with vehicle (DMSO), PMA or microvesicles from untreated cells while monocytes were left untreated (media only) or treated with GM-CSF. The absorbance was measured and shown as the average Optical Density +S.E.M (n=3).

[00072] FIGS. 3C and 3D: CD206, CD16 and CCR5 surface expression by flow cytometry. Quantification of surface marker expression is presented as average fluorescence +S.E.M (n=3). Significance was determined comparing to vehicle or freshly isolated monocytes as indicated.

[00073] FIG. 3E: Monocytes cells were treated with GM-CSF or microvesicles from GM- CSF- stimulated cells for 48 hours. Real-time RT-PCR was performed using primers for CD 16, CD206, or CCR5. Data are normalized over the average CT value of housekeeping genes GAPDH and CAP-1 and expressed as relative fold increase of in gene expression compared to freshly isolated monocytes. Data represents the average fold change in expression +S.E.M for three independent experiments.

[00074] FIG. 3F: Freshly isolated monocytes were treated with GM-CSF or microvesicles from GM-CSF-stimulated cells for 48 hours. Phagocytic SRBC were counted from 100 cells under fluorescent microscope. Total number of phagocytes and total number of SRBC within 100 phagocytes is presented as phagocytic index. Data shown is the average fold change +S.E.M from independent recipient blood donors treated with microvesicles generated from three different donors.

[00075] FIGS. 4A - 4D: Gene expression profiling after microvesicle treatment. Microarray was performed using Human Genome U133 Chips from RNA isolated from THP-1 cells (n=3) and peripheral blood monocytes from three individual monocyte donors to three different monocyte recipients. Each treatment was performed in three independent experiments:

[00076] FIG. 4A: Principle component analysis (PC A) mapping between THP-1 cells treated with vehicle (DMSO), PMA, or microvesicles from PMA-stimulated cells.

[00077] FIG. 4B: Freshly isolated monocytes treated with GM-CSF or microvesicles from GM- CSF-stimulated monocytes were compared to freshly isolated monocytes by PCA. THP-1 cells treated with PMA or microvesicles from PMA-stimulated THP-1 cells expressed similar genes and were different from vehicle-treated cells. Gene expression was similar in monocytes incubated with GM-CSF or microvesicles from GM-CSF-stimulated cells but different compared to freshly isolated monocytes.

[00078] FIG. 4C: Venn diagrams generated using pair-wise comparisons for significantly expressed genes in cells treated as indicated showed 126 significantly co-expressed genes between THP-1 cells treated with PMA or microvesicles from PMA-stimulated THP-1 cells.

[00079] FIG. 4D: 407 significantly co-expressed genes were found between the monocytes incubated with either GM-CSF or microvesicles from GM-CSF-stimulated monocytes.

[00080] FIGS. 5A - 5D: Cellular uptake of microvesicles from GM-CSF-stimulated monocytes:

[00081] FIGS. 5A - 5C: Donor monocytes cells were stained with D384 and SytoRNA Select then treated with GM-CSF. Microvesicles from GM-CSF-stimulated monocytes were collected and added to different cells lines as indicated. Representative confocal images using the Zeiss LSM 510 multi photon confocal microscope are shown. Microvesicles obtained from three individual monocyte donors revealed the uptake of the microvesicles by (FIG. 5A) A549 lung epithelial cells (FIG. 5B) CCL-204 lung fibroblasts and (FIG. 5C) human umbilical vein endothelial cells (HUVECs).

[00082] FIG. 5D: Quantification of microvesicle transfer from three independent experiments is shown (average +S.E.M). Cellular morphology was visualized without fluorescence using differential interference contrast (DIC).

[00083] FIGS. 6A - 6C: Cellular uptake of functional miR-223 contained in microvesicles from GM-CSF-stimulated monocytes does not require recipient cell transcriptional activity for expression:

[00084] FIG. 6A: A549, CCL-204 or HUVEC cells were incubated with microvesicles from GM-CSF-stimulated monocytes for two days. RNA was then isolated and expression for miR-223, miR-29b and miR-34a was measured by qRT-PCR. Shown is the average fold increase +S.E.M (n=4) in miR-223 levels compared to untreated cells. A significant increase after treatment with GM-CSF- stimulated microvesicles is observed for miR-223 (* p < 0.05) while miR-29b and miR-34a levels remained unchanged.

[00085] FIG. 6B: A549 cells (5 x 10⁶ cells/condition) were treated with vehicle (DMSO) or actinomycin D at the indicated concentrations for 6 hours. The cells were washed and culture media was replaced in the absence or presence of microvesicles. After 24 hours, RNA was isolated and miR-223 expression measured by qRT-PCR. Fold increase was determined by comparing the samples to the cells treated only with DMSO. Shown is the average +S.E.M for cells treated with microvesicles from four independent donors.

[00086] FIG. 6C: A549 cells were transfected with the luciferase reporter vector containing miR- 223 recognition sequence, a control luciferase vector lacking the sequence or co-transfected with miR-223 precursor. After 18-20 hours incubation, the cells were treated with or without

microvesicles from GM-CSF-stimulated monocytes. The cells were cultured for another 24 hours then lysed and the luciferase activity was measured. The data are expressed as fold-decrease of luciferase activity of cells transfected with the luciferase reporter containing miR-223 recognition sequence over the vector lacking the recognition sequence for each culture condition. Shown is the average data +S.E.M from A549 cell treated with microvesicles generated from four independent monocyte -derived microvesicle donors.

[00087] FIGS. 7A - 7F: Antagomir to miR-223 reduces macrophage differentiation and cell survival. In 6 well plates, THP-1 cells (0.5 x 10⁶) (FIG. 7A) and monocytes (1.0 x 10⁶ ) (FIG. 7B) were plated in 2.4 ml of media then transfected with miR-223 antagomir and incubated with PMA or GM-CSF, respectively in X-VIVO 15 medium. Both non-adherent and adherent cells were collected and analyzed for cellular differentiation and survival. Cellular adherence was measured by crystal violet assay in the antagomir-transfected THP-1 cells and monocytes. Shown is the average absorbance for crystal violet +S.E.M (n=3 in duplicate). Antagomir-transfected THP-1 cells (FIG. 7C) and monocytes (FIG. 7D) were analyzed for gene expression as indicated. The average relative copy number is shown +S.E.M from three independent experiments. Caspase-3 assay was performed for THP-1 cells (n=3) (FIG. 7E) and monocytes (n=5) (FIG. 7F) transfected with the miR-223 antagomir then treated with PMA or GM-CSF, respectively. Shown is the average fold change +S.E.M compared to untransfected cells.

[00088] FIGS. 8A - 8C: Characterization of GM-CSF-stimulated microvesicles:

[00089] FIG. 8A: Using the LSRII flow cytometry, FITC conjugated polystyrene beads (0.2- 0.8 μιη) were visualized by FSC vs SSC (left panel) and FITC vs SSC (middle panel) and indicated by arrows to each designated gated region. A gate encompassing all the boxes was drawn and designated as microvesicles (MVs). All subsequent analyses used events present in this gate. In the right panel, a representative plot of microvesicles from GM-CSF-derived macrophages (n=6) and collected using a 16,000x g centrifugation is shown. [00090] FIG. 8B: Microvesicles were collected from GM-CSF treated cells that were either were left unstained or stained with SytoRNA Select FITC. Events in the MV gated region (FIG. 8A) were further analyzed by flow cytometry, as indicated. Shown is a representative plot from three individual donors.

[00091] FIG. 8C: The isolated microvesicles were unstained or stained with Annexin V FITC and Hoechst to quantifying the presence of phosphatidylserine and DNA, respectively. Events in the MV gated region as shown in FIG. 8A were further analyzed as indicated. Shown is a representative plot from three different donors. The GM-CSF stimulated microvesicles were 71.58 ±2.70% negative for Annexin V (average ±S.E.M, n=3) and 6.87 ±1.52% positive for DNA (average ±S.E.M, n=3).

[00092] FIGS. 9A and 9B: Differential centrifugations results in similar micro vesicle size distribution:

[00093] FIG. 9A: Representative cryo-TEM images of microvesicles secreted from PMA-treated THP-1 cells spun at centrifuged at 160,000x g. Both small (30-100 nm) and large (>200 nm) microvesicles are apparent. Scale bar= 50 nm.

[00094] FIG. 9B: Dynamic light scattering (DLS) number- weighted distributions of the hydrodynamic diameter of THP-1 cell-secreted microvesicles obtained from samples centrifuged at 16,000x g (open bars) and 160,000x g (filled bars). The relative % of microvesicle diameters> 200 nm is less than 1.44% (16,000x g samples) and 0.19% (160,000x g samples).

[00095] FIGS. 10A and 10B: PMA-Stimulated Microvesicles Bind THP-1 Cells and Stimulate Monocyte Adherence:

[00096] FIG. 10A: Donor THP-1 cells were stained with D384 and SytoRNA Select then treated with PMA. The PMA-stimulated microvesicles were collected and added to either THP-1 cells. Representative confocal image of THP-1 cells from three independent experiments reveal the uptake of the microvesicles.

[00097] FIG. 10B: Monocytes were treated with PMA-stimulated microvesicles. After 48 hours, adherent cells possessing macrophage-like phenotype were stained with crystal violet. Dye uptake was measured at 550 nm.

[00098] FIGS. 11A - 11D: Decreased Expression of miR-223 and Survival Genes in Cells Transfected with miR Antagomir. THP-1 cells or freshly isolated monocytes were transfected with an antagomir to miR-223 and cultured in the presence of PMA or GMCSF, respectively. After 18 hours, the cells were analyzed for miRNA and gene expression. Expression of miR-223 but not miR-191 was decreased in the transfected (FIG. 11 A) THP1 cells and (FIG. 11B) monocytes. Antagomir- transfected (FIG. 11C) THP-1 cells and (FIG. 11D) monocytes stimulated with PMA or GM-CSF, respectively, were analyzed for gene expression as indicated. The average relative copy number is shown ±S.E.M from three independent experiments.

[00099] FIG. 12: Table showing associated Network Functions of Co-Expressed Genes. [000100] FIG. 13: Table showing molecular and Cellular Functions of Co-Expressed Genes.

[000101] FIG. 14: Table showing associated Systems and Functions of Co-Expressed Genes.

[000102] FIG. 15: Table showing normalized miRNA Expression in Micro vesicles from GM- CSF-Treated Monocytes (n=3).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

[000103] General Description

[000104] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[000105] Definitions and General Discussion

[000106] It is to be understood that the descriptions herein are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise.

[000107] Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[000108] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g. " is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. " is synonymous with the term "for example."

[000109] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[000110] Also, the use of "comprise", "contain", and "include", or modifications of those root words, for example but not limited to, "comprises", "contained", and "including", are not intended to be limiting. The term "and/or" means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, "X and/or Y" can mean "X" or "Y" or "X and Y".

[000111] The term "combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.

[000112] It is understood that a miRNA is derived from genomic sequences or a gene. In this respect, the term "gene" is used for simplicity to refer to the genomic sequence encoding the precursor miRNA for a given miRNA. However, embodiments of the invention may involve genomic sequences of a miRNA that are involved in its expression, such as a promoter or other regulatory sequences.

[000113] The terms "miR," "mir" and "miRNA" generally refer to microRNA, a class of small RNA molecules that are capable of modulating RNA translation (see, Zeng and Cullen, RNA, 9(1):112-123, 2003; Kidner and Martienssen Trends Genet, 19(l): 13-6, 2003; Dennis C, Nature, 420(6917):732, 2002; Couzin J, Science 298(5602):2296-7, 2002, each of which is incorporated by reference herein). Generally, microRNAs (miRNAs) are single-stranded RNA molecules that regulate gene expression. MicroRNAs are generally 21-23 nucleotides in length. MicroRNAs are processed from primary transcripts known as pri-miRNA to short stem-loop structures called precursor (pre)-miRNA and finally to functional, mature microRNA. Mature microRNA molecules are partially-complementary to one or more messenger RNA molecules, and their primary function is to down-regulate gene expression. MicroRNAs regulate gene expression through the RNAi pathway. "miRNA" generally refers to a single-stranded molecule, but in specific embodiments, molecules implemented in the invention will also encompass a region or an additional strand that is partially (between 10 and 50% complementary across length of strand), substantially (greater than 50% but less than 100% complementary across length of strand) or fully complementary to another region of the same single-stranded molecule or to another nucleic acid. Thus, nucleic acids may encompass a molecule that comprises one or more complementary or self -complementary strand(s) or

"complement(s)" of a particular sequence comprising a molecule. For example, precursor miRNA may have a self -complementary region, which is up to 100% complementary miRNA probes of the invention can be or be at least 60, 65, 70, 75, 80, 85, 90, 95, or 100% complementary to their target.

[000114] As used herein interchangeably, a "miR gene product," "microRNA," "miR," or

"miRNA" refers to the unprocessed or processed RNA transcript from a miR gene. As the miR gene products are not translated into protein, the term "miR gene products" does not include proteins. The unprocessed miR gene transcript is also called a "miR precursor," and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miR precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, RNAse III (e.g., E. coli RNAse III)) into an active 19- 25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the

"processed" miR gene transcript or "mature" miRNA.

[000115] The active 19-25 nucleotide RNA molecule can be obtained from the miR precursor through natural processing routes (e.g. , using intact cells or cell lysates) or by synthetic processing routes (e.g., using isolated processing enzymes, such as isolated Dicer, Argonaut, or RNAse III). It is to be understood that the active 19-25 nucleotide RNA molecule can also be produced directly by biological or chemical synthesis, without having to be processed from the miR precursor.

[000116] "MicroRNA seed sequence," "miRNA seed sequence," "seed region" and "seed portion" generally refer to nucleotides 2-7 or 2-8 of the mature miRNA sequence. The miRNA seed sequence is typically located at the 5' end of the miRNA.

[000117] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[000118] Adjunctive therapy: A treatment used in combination with a primary treatment to improve the effects of the primary treatment.

[000119] Agent and/or drug: Generally refer to any therapeutic agents (e.g., chemotherapeutic compounds and/or molecular therapeutic compounds), antisense therapies, radiation therapies, or surgical interventions, used in the treatment of a particular disease or disorder.

[000120] Candidate agent: A compound selected for screening to determine if it can function as a therapeutic agent. The candidate agents can be any type of agent, such as a protein, peptide, small molecule, antibody or nucleic acid. In some embodiments, the candidate agent is a cytokine. In some embodiments, the candidate agent is a small molecule. Screening includes both high-throughput screening and screening individual or small groups of candidate agents. "Incubating" includes a sufficient amount of time for an agent to interact with a cell or tissue. "Contacting" includes incubating an agent in solid or in liquid form with a cell or tissue. "Treating" a cell or tissue with an agent includes contacting or incubating the agent with the cell or tissue.

[000121] Clinical outcome: Refers to the health status of a patient following treatment for a disease or disorder or in the absence of treatment. Clinical outcomes include, but are not limited to, an increase in the length of time until death, a decrease in the length of time until death, an increase in the chance of survival, an increase in the risk of death, survival, disease-free survival, chronic disease, metastasis, advanced or aggressive disease, disease recurrence, death, and favorable or poor response to therapy.

[000122] Control: Generally refers to a sample or standard used for comparison with an experimental sample, such as a sample obtained from a subject. In some embodiments, the control is a sample obtained from a healthy subject. In some embodiments, the control is cell/tissue sample obtained from the same subject. In some embodiments, the control is a historical control or standard value (i.e., a previously tested control sample or group of samples that represent baseline or normal values, such as the level in a control sample). In other embodiments, the control is a sample obtained from a healthy subject, such as a donor. Test samples and control samples can be obtained according to any method known in the art. In some embodiments, the control may be a non-cancerous cell/tissue sample obtained from the same patient, or a cell/tissue sample obtained from a healthy subject, such as a healthy tissue donor. Tumor samples and non-cancerous cell/tissue samples can be obtained according to any method. For example, tumor and non-cancerous samples can be obtained from cancer patients that have undergone resection, or they can be obtained by extraction using a hypodermic needle, by microdissection, or by laser capture.

[000123] Cytokines: Proteins produced by a wide variety of hematopoietic and non-hematopoietic cells that affect the behavior of other cells. Cytokines are important for both the innate and adaptive immune responses.

[000124] Decrease in survival: As used herein, "decrease in survival" refers to a decrease in the length of time before death of a patient, or an increase in the risk of death for the patient.

[000125] Detecting level of expression: For example, "detecting the level of miR or miRNA expression" refers to quantifying the amount of miR or miRNA present in a sample. Detecting expression of the specific miR, or any microRNA, can be achieved using any method known in the art or described herein, such as by qRT-PCR. Detecting expression of miR includes detecting expression of either a mature form of miRNA or a precursor form that is correlated with miRNA expression. Typically, miRNA detection methods involve sequence specific detection, such as by RT-PCR. miR- specific primers and probes can be designed using the precursor and mature miR nucleic acid sequences.

[000126] Expression vector: Generally refers to a nucleic acid construct that can be generated recombinantly or synthetically. An expression vector generally includes a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Generally, the gene expression is placed under the control of certain regulatory elements, such as constitutive or inducible promoters.

[000127] miR expression: As used herein, "low miR expression" and "high miR expression" are relative terms that refer to the level of miRNAs found in a sample. In some embodiments, low and high miR expression is determined by comparison of miRNA levels in a group of control samples and test samples. Low and high expression can then be assigned to each sample based on whether the expression of miRNA in a sample is above (high) or below (low) the average or median miR expression level. For individual samples, high or low miR expression can be determined by comparison of the sample to a control or reference sample known to have high or low expression, or by comparison to a standard value. Low and high miR expression can include expression of either the precursor or mature forms of miRNA, or both. [000128] In some embodiments, miR expression is measured relative to certain small non-coding RNAs (ncRNAs) that are expressed both abundantly and stably, making them good endogenous control candidates.

[000129] Operably linked: Generally describes the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue- specific regulatory elements, and enhancers. A gene or coding region is the to be "operably linked to" or "operatively linked to" or "operably associated with" the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.

[000130] Subject: As used herein, the term "subject" includes human and non-human animals. The preferred patient for treatment is a human. "Patient" and "subject" are used interchangeably herein.

[000131] Pharmaceutically acceptable vehicles: The pharmaceutically acceptable carriers

(vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents.

[000132] Preventing, treating or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. The terms "prevent," "preventing" and "prevention" generally refer to a decrease in the occurrence of disease or disorder in a subject. The prevention may be complete, e.g., the total absence of the disease or disorder in the subject. The prevention may also be partial, such that the occurrence of the disease or disorder in the subject is less than that which would have occurred without the present invention. "Preventing" a disease generally refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease.

[000133] Screening: Generally refers to the process used to evaluate and identify candidate agents that affect such disease. Expression of a microRNA can be quantified using any one of a number of techniques known in the art and described herein, such as by microarray analysis or by qRT-PCR. In some embodiments, screening comprises contacting the candidate agents with cells. The cells can be primary cells obtained from a patient, or the cells can be immortalized or transformed cells.

[000134] Small molecule: A molecule, typically with a molecular weight less than about 1000 Daltons, or in some embodiments, less than about 500 Daltons, wherein the molecule is capable of modulating, to some measurable extent, an activity of a target molecule.

[000135] Therapeutic: A generic term that includes both diagnosis and treatment.

[000136] Therapeutic agent: A chemical compound, small molecule, or other composition, such as an antisense compound, antibody, protease inhibitor, hormone, chemokine or cytokine, capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

[000137] Therapeutically-effective amount: A quantity of a specified pharmaceutical or therapeutic agent sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. The effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition.

[000138] Pharmaceutically acceptable vehicles: Generally refers to such pharmaceutically acceptable carriers (vehicles) as would be generally used. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 20 Edition, describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[000139] Pharmaceutically acceptable salt: Generally refers to any salt (e.g., obtained by reaction with an acid or a base) of a compound of the present invention that is physiologically tolerated in the target animal (e.g., a mammal). Salts of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene -2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and the like.

Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate, methanesulionate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NW4+ (wherein W is a CI -4 alkyl group), and the like. For therapeutic use, salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound

[000140] MicroRNA detection and uses

[000141] In some methods herein, it is desirable to identify miRNAs present in a sample.

[000142] The sequences of precursor microRNAs (pre-miRNAs) and mature miRNAs are publicly available, such as through the miRBase database, available online by the Sanger Institute (see Griffiths-Jones et al., Nucleic Acids Res. 36:D154-D158, 2008; Griffiths-Jones et al., Nucleic Acids Res. 34:D140-D144, 2006; and Griffiths-Jones, Nucleic Acids Res. 32:D109-D111, 2004). The sequences of the precursor and mature forms of the presently disclosed preferred family members are provided herein.

[000143] Detection and quantification of RNA expression can be achieved by any one of a number of methods well known in the art (see, for example, U.S. Patent Application Publication No.

2006/0211000 and US Pat. No. 7,955,848, herein incorporated by reference). Using the known sequences for RNA family members, specific probes and primers can be designed for use in the detection methods described below as appropriate.

[000144] In some cases, the RNA detection method requires isolation of nucleic acid from a sample, such as a cell or tissue sample. Nucleic acids, including RNA and specifically miRNA, can be isolated using any suitable technique. For example, phenol-based extraction is a common method for isolation of RNA. Phenol-based reagents contain a combination of denaturants and RNase inhibitors for cell and tissue disruption and subsequent separation of RNA from contaminants.

Phenol-based isolation procedures can recover RNA species in the 10-200-nucleotide range (e.g., precursor and mature miRNAs, 5S and 5.8S ribosomal RNA (rRNA), and Ul small nuclear RNA (snRNA)). In addition, extraction procedures such as those using TRIZOL™ or TRI REAGENT™, will purify all RNAs, large and small, and are efficient methods for isolating total RNA from biological samples that contain miRNAs and small interfering RNAs (siRNAs).

[000145] In some embodiments, use of a microarray is desirable. A microarray is a microscopic, ordered array of nucleic acids, proteins, small molecules, cells or other substances that enables parallel analysis of complex biochemical samples. A DNA microarray consists of different nucleic acid probes, known as capture probes that are chemically attached to a solid substrate, which can be a microchip, a glass slide or a microsphere-sized bead. Microarrays can be used, for example, to measure the expression levels of large numbers of messenger RNAs (mRNAs) and/or miRNAs simultaneously.

[000146] Microarrays can be fabricated using a variety of technologies, including printing with fine -pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays.

[000147] Microarray analysis of miRNAs, for example (although these procedures can be used in modified form for any RNA analysis) can be accomplished according to any method known in the art (see, for example, PCT Publication No. WO 2008/054828; Ye et al., Nat. Med. 9(4):416-423, 2003; Calin et al., N. Engl. J. Med. 353(17): 1793-1801, 2005, each of which is herein incorporated by reference). In one example, RNA is extracted from a cell or tissue sample, the small RNAs (18-26- nucleotide RNAs) are size-selected from total RNA using denaturing polyacrylamide gel electrophoresis. Oligonucleotide linkers are attached to the 5' and 3' ends of the small RNAs and the resulting ligation products are used as templates for an RT-PCR reaction with 10 cycles of amplification. The sense strand PCR primer has a fluorophore attached to its 5' end, thereby fluorescently labeling the sense strand of the PCR product. The PCR product is denatured and then hybridized to the microarray. A PCR product, referred to as the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed. The spot will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA.

[000148] In an alternative method, total RNA containing the small RNA fraction (including the miRNA) extracted from a cell or tissue sample is used directly without size-selection of small RNAs, and 3' end labeled using T4 RNA ligase and either a fluorescently-labeled short RNA linker. The RNA samples are labeled by incubation at 30°C for 2 hours followed by heat inactivation of the T4 RNA ligase at 80°C for 5 minutes. The fluorophore-labeled miRNAs complementary to the corresponding miRNA capture probe sequences on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed. The microarray scanning and data processing is then carried out.

[000149] There are several types of microarrays that can be employed, including spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays and spotted long oligonucleotide arrays. In spotted oligonucleotide microarrays, the capture probes are

oligonucleotides complementary to miRNA sequences. This type of array can be hybridized with amplified PCR products of size-selected small RNAs from two samples to be compared (such as noncancerous tissue and cancerous or sample tissue) that are labeled with two different fluorophores. Alternatively, total RNA containing the small RNA fraction (including the miRNAs) can be extracted from the two samples and used directly without size-selection of small RNAs, and 3' end labeled using T4 RNA ligase and short RNA linkers labeled with two different fluorophores. The samples can be mixed and hybridized to one single microarray that is then scanned, allowing the visualization of up-regulated and down-regulated miRNA genes in one assay.

[000150] In pre-fabricated oligonucleotide microarrays or single-channel microarrays, the probes are designed to match the sequences of known or predicted miRNAs. There are commercially available designs that cover complete genomes (for example, from Affymetrix or Agilent). These microarrays give estimations of the absolute value of gene expression and therefore the comparison of two conditions requires the use of two separate microarrays.

[000151] In some embodiments, use of quantitative RT-PCR is desirable. Quantitative RT-PCR (qRT-PCR) is a modification of polymerase chain reaction used to rapidly measure the quantity of a product of polymerase chain reaction. qRT-PCR is commonly used for the purpose of determining whether a genetic sequence, such as a miR, is present in a sample, and if it is present, the number of copies in the sample. Any method of PCR that can determine the expression of a nucleic acid molecule, including a miRNA, falls within the scope of the present disclosure. There are several variations of the qRT-PCR method known in the art, three of which are described below.

[000152] Methods for quantitative polymerase chain reaction include, but are not limited to, via agarose gel electrophoresis, the use of SYBR Green (a double stranded DNA dye), and the use of a fluorescent reporter probe. The latter two can be analyzed in real-time.

[000153] Various methods of screening candidate agents can be used to identify therapeutic agents for the treatment of disease. Methods of detecting expression levels of RNA and proteins are, but not limited to, microarray analysis, RT-PCR (including qRT-PCR), in situ hybridization, in situ PCR, and Northern blot analysis. In one embodiment, screening comprises a high-throughput screen. In another embodiment, candidate agents are screened individually.

[000154] The candidate agents can be any type of molecule, such as, but not limited to nucleic acid molecules, proteins, peptides, antibodies, lipids, small molecules, chemicals, cytokines, chemokines, hormones, or any other type of molecule that may alter cancer disease state(s) either directly or indirectly.

[000155] It will be understood in methods described herein that a cell or other biological matter such as an organism (including patients) can be provided a miRNA or miRNA molecule

corresponding to a particular miRNA by administering to the cell or organism a nucleic acid molecule that functions as the corresponding miRNA once inside the cell. The form of the molecule provided to the cell may not be the form that acts a miRNA once inside the cell. Thus, it is contemplated that in some embodiments, biological matter is provided a synthetic miRNA or a nonsynthetic miRNA, such as one that becomes processed into a mature and active miRNA once it has access to the cell's miRNA processing machinery. In certain embodiments, it is specifically contemplated that the miRNA molecule provided to the biological matter is not a mature miRNA molecule but a nucleic acid molecule that can be processed into the mature miRNA once it is accessible to miRNA processing machinery. The term "nonsynthetic" in the context of miRNA means that the miRNA is not "synthetic," as defined herein. Furthermore, it is contemplated that in embodiments that concern the use of synthetic miRNAs, the use of corresponding nonsynthetic miRNAs is also considered, and vice versa. It will be understand that the term "providing" an agent is used to include "administering" the agent to a patient.

[000156] In certain embodiments, methods also include targeting a miRNA to modulate in a cell or organism. The term "targeting a miRNA to modulate" means a nucleic acid will be employed so as to modulate the selected miRNA. In some embodiments, the modulation is achieved with a synthetic or non-synthetic miRNA that corresponds to the targeted miRNA, which effectively provides the targeted miRNA to the cell or organism (positive modulation). In other embodiments, the modulation is achieved with a miRNA inhibitor, which effectively inhibits the targeted miRNA in the cell or organism (negative modulation).

[000157] In some embodiments, the miRNA targeted to be modulated is a miRNA that affects a disease, condition, or pathway. In certain embodiments, the miRNA is targeted because a treatment can be provided by negative modulation of the targeted miRNA. In other embodiments, the miRNA is targeted because a treatment can be provided by positive modulation of the targeted miRNA.

[000158] In certain methods, there is a further step of administering the selected miRNA modulator to a cell, tissue, organ, or organism (collectively "biological matter") in need of treatment related to modulation of the targeted miRNA or in need of the physiological or biological results discussed herein (such as with respect to a particular cellular pathway or result like decrease in cell viability). Consequently, in some methods there is a step of identifying a patient in need of treatment that can be provided by the miRNA modulator(s). It is contemplated that an effective amount of a miRNA modulator can be administered in some embodiments. In particular embodiments, there is a therapeutic benefit conferred on the biological matter, where a "therapeutic benefit" refers to an improvement in the one or more conditions or symptoms associated with a disease or condition or an improvement in the prognosis, duration, or status with respect to the disease. It is contemplated that a therapeutic benefit includes, but is not limited to, a decrease in pain, a decrease in morbidity, a decrease in a symptom. For example, with respect to cancer, it is contemplated that a therapeutic benefit can be inhibition of tumor growth, prevention of metastasis, reduction in number of metastases, inhibition of cancer cell proliferation, inhibition of cancer cell proliferation, induction of cell death in cancer cells, inhibition of angiogenesis near cancer cells, induction of apoptosis of cancer cells, reduction in pain, reduction in risk of recurrence, induction of chemo- or radiosensitivity in cancer cells, prolongation of life, and/or delay of death directly or indirectly related to cancer.

[000159] Furthermore, it is contemplated that the miRNA compositions may be provided as part of a therapy to a patient, in conjunction with traditional therapies or preventative agents. Moreover, it is contemplated that any method discussed in the context of therapy may be applied as preventatively, particularly in a patient identified to be potentially in need of the therapy or at risk of the condition or disease for which a therapy is needed.

[000160] In addition, certain methods concern employing one or more nucleic acids corresponding to a miRNA and a therapeutic drug. The nucleic acid can enhance the effect or efficacy of the drug, reduce any side effects or toxicity, modify its bioavailability, and/or decrease the dosage or frequency needed. In certain embodiments, the therapeutic drug is a cancer therapeutic. Consequently, in some embodiments, there is a method of treating cancer in a patient comprising administering to the patient the cancer therapeutic and an effective amount of at least one miRNA molecule that improves the efficacy of the cancer therapeutic or protects non-cancer cells. Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments.

[000161] Inhibitors of miRNAs can be given to achieve the opposite effect as compared to when nucleic acid molecules corresponding to the mature miRNA are given. Similarly, nucleic acid molecules corresponding to the mature miRNA can be given to achieve the opposite effect as compared to when inhibitors of the miRNA are given. For example, miRNA molecules that increase cell proliferation can be provided to cells to increase proliferation or inhibitors of such molecules can be provided to cells to decrease cell proliferation. For example, these embodiments can be contemplated in the context of the different physiological effects observed with the different miRNA molecules and miRNA inhibitors disclosed herein. These include, but are not limited to, the following physiological effects: increase and decreasing cell proliferation, increasing or decreasing apoptosis, increasing transformation, increasing or decreasing cell viability, reduce or increase viable cell number, and increase or decrease number of cells at a particular phase of the cell cycle. Methods are also contemplated to include providing or introducing one or more different nucleic acid molecules corresponding to one or more different miRNA molecules. It is contemplated that the following, at least the following, or at most the following number of different nucleic acid molecules may be provided or introduced: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range derivable therein. This also applies to the number of different miRNA molecules that can be provided or introduced into a cell.

[000162] miRNA nucleic acid" generally refers to RNA or DNA that encodes a miR as defined above, or is complementary to a nucleic acid sequence encoding a miR, or hybridizes to such RNA or DNA and remains stably bound to it under appropriate stringency conditions. Particularly included are genomic DNA, cDNA, mRNA, miRNA and antisense molecules, pri-miRNA, pre-miRNA, mature miRNA and miRNA seed sequences. Also included are nucleic acids based on alternative backbones or including alternative bases. MiRNA nucleic acids can be derived from natural sources or synthesized.

[000163] It is to be understood that a miRNAs or pre-miRNAs can be 18-100 nucleotides in length, and more preferably from 18-80 nucleotides in length. For example, mature miRNAs can have a length of 19-30 nucleotides, preferably 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MicroRNA precursors typically have a length of about 70-100 nucleotides and have a hairpin conformation. Thus, once a sequence of a miRNA or a pre-miRNA is known, a miRNA antagonist that is sufficiently complementary to a portion of the miRNA or the pre-miRNA can be designed according to the rules of Watson and Crick base pairing. As used herein, the term

"sufficiently complementary" generally means that two sequences are sufficiently complementary such that a duplex can be formed between them under physiologic conditions. A miRNA antagonist sequence that is sufficiently complementary to a miRNA or pre-miRNA target sequence can be 70%, 80%, 90%, or more identical to the miRNA or pre-miRNA sequence.

[000164] In one embodiment, the miRNA antagonist contains no more than 1, 2 or 3 nucleotides that are not complementary to the miRNA or pre-miRNA target sequence. In another embodiment, the miRNA antagonist is 100% complementary to a miRNA or pre-miRNA target sequence.

Sequences for miRNAs are available publicly through the miRBase registry (Griffiths-Jones, et al., Nucleic Acids Res., 36(Database Issue):D154-D158 (2008); Griffiths-Jones, et al., Nucleic Acids Res., 36(Database Issue):D140-D144 (2008); Griffiths-Jones, et al., Nucleic Acids Res., 36(Database Issue):D109-Dl l l (2008)).

[000165] A "miR-specific inhibitor" may be an anti-miRNA (antagomir or anti-miR)

oligonucleotide. Anti-miRNAs may be single stranded molecules. Anti-miRs may comprise RNA or DNA or have non-nucleotide components. Anti-miRs anneal with and block mature microRNAs through extensive sequence complementarity. In some embodiments, an anti-miR may comprise a nucleotide sequence that is a perfect complement of the entire miRNA. In some embodiments, an anti-miR comprises a nucleotide sequence of at least 6 consecutive nucleotides that are

complementary to the seed region of a microRNA molecule at positions 2-8 and has at least 50%, 60%, 70%, 80%, or 90% complementarity to the rest of the miRNA. In other embodiments, the anti- miR may comprise additional flanking sequence, complimentary to adjacent primary (pri-miRNA) sequences. Chemical modifications, such as 2'-0-methyl; locked nucleic acids (LNA); and 2'-0- methyl, phosphorothioate, cholesterol (antagomir); 2'-0-methoxyethyl can be used. Chemically modified anti-miRs are commercially available from a variety of sources, including but not limited to Sigma-Proligo, Ambion, Exiqon, and Dharmacon.

[000166] The miRNA antagonists can be oligomers or polymers of RNA or DNA, and can contain modifications to their nucleobases, sugar groups, phosphate groups, or covalent internucleoside linkages. In certain embodiment, modifications include those that increase the stability of the miRNA antagonists or enhance cellular uptake of the miRNA antagonists. In one embodiment, the miRNA antagonists are antagomirs, which have 2'-0-methylation of the sugars, a phosphorothioate backbone and a terminal cholesterol moiety.

[000167] In some embodiments, miR-specific inhibitors possess at least one microRNA binding site, mimicking the microRNA target (target mimics). These target mimics may possess at least one nucleotide sequence comprising 6 consecutive nucleotides complementary to positions 2-8 of the miRNA seed region. Alternatively, these target mimics may comprise a nucleotide sequence with complementarity to the entire miRNA. These target mimics may be vector encoded. Vector encoded target mimics may have one or more microRNA binding sites in the 5' or 3' UTR of a reporter gene. The target mimics may possess microRNA binding sites for more than one microRNA family. The microRNA binding site of the target mimic may be designed to mismatch positions 9-12 of the microRNA to prevent miRNA-guided cleavage of the target mimic.

[000168] In an alternative embodiment, a miR-specific inhibitor may interact with the miRNA binding site in a target transcript, preventing its interaction with a miRNA.

[000169] The terms "miRNA specific inhibitor" and "miRNA antagonist," generally refer to an agent that reduces or inhibits the expression, stability, or activity of a miRNA. A miRNA antagonist may function, for example, by blocking the activity of a miRNA (e.g., blocking the ability of a miRNA to function as a translational repressor and/or activator of one or more miRNA targets), or by mediating miRNA degradation. Exemplary miRNA antagonists include nucleic acids, for example, antisense locked nucleic acid molecules (LNAs), antagomirs, or 2'O-methyl antisense RNAs targeting a miRNA.

[000170] The phrase "inhibiting expression of a target gene" generally refers to the ability of an RNAi agent, such as a siRNA, to silence, reduce, or inhibit expression of a target gene. The another way, to "inhibit," "down-regulate," or "reduce," it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the RNAi agent.

[000171] For example, in one embodiment, inhibition, down-regulation, or reduction contemplates inhibition of the target mRNA below the level observed in the presence of, for example, a siRNA molecule with scrambled sequence or with mismatches.

[000172] To examine the extent of gene silencing, a test sample (e.g., a biological sample from organism of interest expressing the target gene(s) or a sample of cells in culture expressing the target gene(s)) is contacted with a siRNA that silences, reduces, or inhibits expression of the target gene(s). Expression of the target gene in the test sample is compared to expression of the target gene in a control sample (e.g., a biological sample from organism of interest expressing the target gene or a sample of cells in culture expressing the target gene) that is not contacted with the siRNA. Control samples (i.e., samples expressing the target gene) are assigned a value of 100%. Silencing, inhibition, or reduction of expression of a target gene is achieved when the value of the test sample relative to the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10% or 0%. Suitable assays include, e.g., examination of protein or mRNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ

hybridization, ELISA, microarray hybridization, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[000173] An "effective amount" or "therapeutically effective amount" of a miR-specific inhibitor is an amount sufficient to produce the desired effect, e.g., inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the miR-specific inhibitor. Inhibition of expression of a target gene or target sequence by a miR-specific inhibitor is achieved when the expression level of the target gene mRNA or protein is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the expression level of the target gene mRNA or protein of a control sample. The desired effect of a miR-specific inhibitor may also be measured by detecting an increase in the expression of genes down-regulated by the miRNA targeted by the miR-specific inhibitor.

[000174] By "modulate" is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up-regulated or down-regulated, such that expression, level or activity is greater than or less than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," but the use of the word "modulate" is not limited to this definition.

[000175] Non-limiting examples of suitable sequence variants of miRNA can include:

substitutional, insertional or deletional variants. Insertions include 5' and/or 3' terminal fusions as well as intrasequence insertions of single or multiple residues. Insertions can also be introduced within the mature sequence. These, however, can be smaller insertions than those at the 5' or 3' terminus, on the order of 1 to 4 residues, 2 residues, and/or 1 residue.

[000176] Insertional sequence variants of miRNA are those in which one or more residues are introduced into a predetermined site in the target miRNA. Most commonly insertional variants are fusions of nucleic acids at the 5' or 3' terminus of the miRNA.

[000177] Deletion variants are characterized by the removal of one or more residues from the miRNA sequence. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding miRNA, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. However, variant miRNA fragments may be conveniently prepared by in vitro synthesis. The variants typically exhibit the same qualitative biological activity as the naturally-occurring analogue, although variants also are selected in order to modify the characteristics of miRNA.

[000178] Substitutional variants are those in which at least one residue sequence has been removed and a different residue inserted in its place. While the site for introducing a sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target region and the expressed miRNA variants screened for the optimal combination of desired activity. Various suitable techniques for making substitution mutations at predetermined sites in DNA having a known sequence can be used.

[000179] Nucleotide substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs; i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions, deletion, insertions or any combination thereof may be combined to arrive at a final construct.

[000180] Changes may be made to decrease the activity of the miRNA, and all such modifications to the nucleotide sequences encoding such miRNA are encompassed.

[000181] An "isolated nucleic acid or DNA" is generally understood to mean chemically synthesized DNA, cDNA or genomic DNA with or without the 3' and/or 5' flanking regions. DNA encoding miRNA can be obtained from other sources by, for example: a) obtaining a cDNA library from cells containing mRNA; b) conducting hybridization analysis with labeled DNA encoding miRNA or fragments thereof in order to detect clones in the cDNA library containing homologous sequences; and, c) analyzing the clones by restriction enzyme analysis and nucleic acid sequencing to identify full-length clones.

[000182] As used herein nucleic acids and/or nucleic acid sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTN using default parameters) are generally available. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[000183] The term "detecting the level of miR expression" generally refers to quantifying the amount of such miR present in a sample. Detecting expression of a miR, or any microRNA, can be achieved using any method, such as by qRT-PCR. Detecting expression of a miR includes detecting expression of either a mature form of the miR or a precursor form that is correlated with the miR expression. For example, miRNA detection methods involve sequence specific detection, such as by RT-PCR. miR-specific primers and probes can be designed using the precursor and mature miR nucleic acid sequences, and may include modifications which do not change the function of the sequences.

[000184] The terms "low miR- expression" and "high miR- expression" are relative terms that refer to the level of miR/s found in a sample. In some embodiments, low miR- and high miR- expression are determined by comparison of miR/s levels in a group of test samples and control samples. Low and high expression can then be assigned to each sample based on whether the expression of a miR in a sample is above (high) or below (low) the average or median miR expression level. For individual samples, high or low miR expression can be determined by comparison of the sample to a control or reference sample known to have high or low expression, or by comparison to a standard value. Low and high miR expression can include expression of either the precursor or mature forms of miR, or both.

[000185] In some instances, a disease reference standard or sample may be used. A reference standard may comprise miR levels indicative of a known cancer. A reference standard may be a composite of samples derived from cancer tissues. Comparison of test results with a disease reference and/or control can be used in diagnostic methods. In some embodiments, a test sample is processed at the same time as one or more disease reference samples and one or more normal, non-diseased, control samples.

[000186] As used herein, the term "effective amount" refers to an amount of an agent that is sufficient to effect a therapeutically significant ablation of leukemic cells in a subject diagnosed with leukemia. The therapeutically effective amount amounts to be administered will depend on the severity of the condition and individual subject parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art; however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reason.

[000187] The actual dosage amount of a composition administered to the subject, such as a human subject, can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[000188] As used herein, the term "agent" refers to a nucleic acid sequence or a vector. The nucleic acid sequence can have modifications such as 2' O-methylation and 3' end cholesterol found in antagomirs and locked nucleic acid oligonucleotides.

[000189] As used herein, the term "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand nucleic acid of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the template nucleic acid is DNA. In another aspect, the template is RNA. Suitable nucleic acid molecules are DNA, including genomic DNA, ribosomal DNA and cDNA. Other suitable nucleic acid molecules are RNA, including mRNA, rRNA and tRNA. The nucleic acid molecule can be naturally occurring, as in genomic DNA, or it may be synthetic, i.e., prepared based up human action, or may be a combination of the two. The nucleic acid molecule can also have certain modification such as 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), cholesterol addition, and phosphorothioate backbone; and certain ribonucleoside that is linked between the 2'- oxygen and the 4'-carbon atoms with a methylene unit.

[000190] As used herein, "identity," in the context of two or more nucleic acids sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions, such as when using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 nucleotides in length, or more preferably over a region that is 50-100 nucleotides in length. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[000191] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[000192] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described by Altschul et al. (J. Mol. Biol. 215:403-410 (1990), which is incorporated by reference herein). (See also Zhang et al., Nucleic Acid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res. 25:3389-402 (1997), which are incorporated by reference herein). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information internet web site. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-9 (1992), which is incorporated by reference herein) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[000193] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), which is incorporated by reference herein). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference amino acid sequence if the smallest sum probability in a comparison of the test amino acid to the reference amino acid is less than about 0.1, more typically less than about 0.01, and most typically less than about 0.001.

[000194] Antagomirs are RNA-like oligonucleotides that harbor various modifications for RNAse protection and pharmacologic properties, such as enhanced tissue and cellular uptake. They differ from normal RNA by, for example, complete 2'-0-methylation of sugar, phosphorothioate backbone and, for example, a cholesterol-moiety at 3'-end. Antagomirs may be used to efficiently silence endogenous miRNAs by forming duplexes comprising the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing. Antagomir RNAs may be synthesized using standard solid phase oligonucleotide synthesis protocols.

[000195] Locked nucleic acid (LNA)-modified oligonucleotides are distinctive 2'-0-modified RNA in which the 2'-0-oxygen is bridged to the 4'-position via a methylene linker to form a rigid bicycle, locked into a C3'-endo (RNA) sugar conformation. The LNA modification leads to the

thermodynamically strongest duplex formation with complementary RNA known. Consequently, a biological activity is often attained with very short LNA oligonucleotides. For example, an 8 nt fully- modified LNA oligomer complementary to a structural loop inhibited 50% of self-splicing of group I introns from rRNA genes in pathogenic organisms whereas DNA and RNA oligonucleotides were ineffective. Short fully-modified LNA oligonucleotides designed against telomerase were active in cellular assays, compared to mismatched negative controls. Furthermore, LNAs display excellent mismatch discrimination. The synthesis and incorporation of LNA bases can be achieved by using standard DNA synthesis chemistry.

[000196] An anti-sense oligonucleotide of miR-223 has a sequence that is complementary to the mature miR-223. Complementary pairing between an anti-sense oligonucleotide of miR-223 and miR-223 produces a duplex RNA that is highly susceptible to RNase degradation.

[000197] Methods for increasing efficacy of Anti-Cancer Treatments

[000198] Described herein is a method for increasing the efficacy of an anti-cancer treatment in a subject having a cancer, comprising administering to the subject an effective amount of an agent that inhibits miR-223 expression in a cell of a subject, and additionally administering at least one additional anti-cancer treatment to the subject. The additional anti-cancer treatment can be any anticancer treatment known to those of skill in the art. Such suitable anti-cancer treatments include, but are not limited to, chemotherapy, radiation therapy and combinations thereof {e.g., chemoradiation). Also contemplated are methods of increasing the efficacy of non-conventional cancer therapies as well.

[000199] As used herein, chemotherapy refers to the administration of one or more chemical substances or drugs for the treatment of cancer. A suitable chemotherapeutic agent for the methods of the invention can be any chemical substance known to be useful for treating cancer, for example, DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors,

metalloproteinase inhibitors, antisense nucleic acids, triple -helix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial or exotoxic agents. Examples of suitable agents include, but are not limited to, cytidine arabinoside, methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin), cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, CPT-11 , taxol and derivatives thereof, tamoxifen, dacarbazine, rituximab, daunorubicin, Ι-β-D-arabinofuranosylcytosine, imatinib, fludarabine, docetaxel and FOLFOX4.

[000200] As used herein, radiation therapy refers to the use of high energy radiation for the treatment of cancer. High energy radiation for use in radiation therapy may be provided by X-rays, gamma ray and neutrons, among others. Different methods of radiation therapy are well known in the art and are suitable. These methods include, but are not limited to, external beam radiation, brachytherapy, intensity-modulated radiotherapy (IMRT), implant radiation, systemic radiation and stereotactic radiotherapy.

[000201] The terms "subject," "individual" and "patient" are defined herein to include animals such as mammals, including but not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species. In one embodiment, the subject is a mammal (e.g. , a human) afflicted with one or more forms of leukemia. As described herein, suitable form of leukemia for treatment by the methods of the invention include acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia

[000202] According to the methods described herein, the efficacy of a cancer treatment is increased by administering an effective amount of an agent that inhibits miR-223 in a cell of the subject. The agent may be co-administered (i.e., simultaneously) with a chemotherapeutic agent, or is may be administered separately. In one embodiment, the agent is a nucleic acid sequence that is at least 90% identical to miR-223. In another embodiment, the agent is an antagomir of miR-223. In further embodiments, the agent is an anti-miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR-223, or a double stranded RNA that complementary base- pairs with miR-223.

[000203] An increase in the efficacy of an anti-cancer treatment can be evidenced by increased remission of the cancer in the subject relative to a suitable control. Cancer remission can be determined according to accepted clinical standards, such as a decrease in the number of cancer cells in the subject and/or a decrease in tumor mass and/or size. The number of leukemic cancer cells in a subject can be determined by direct measurement.

[000204] In one embodiment, the sensitivity of a cancer cell to the cytotoxic effects of an anticancer agent is increased by providing an effective amount of an agent that inhibits miR-223 in a cell of a subject. By providing the agent to the subject, the sensitivity of the cell to an anti-cancer agent is increased. [000205] In one embodiment, the described herein is a method of increasing the sensitivity of a cancer cell to the cytotoxic effects of an anti-cancer agent, comprising providing at least one anticancer agent to a cancer cell and additionally providing an effective amount of an agent that inhibits miR-223 in a cell of a subject, wherein the agent that inhibits miR-223 is a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1, or its complement. In another embodiment, the agent is at least 90% identical to the antagomir of miR-223. In another embodiment, the agent is an antagomir of miR-223. In further embodiments, the agent is an anti-miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR-223, or a double stranded RNA that complementary base-pairs with miR-223.

[000206] As used herein, the phrase "cytotoxic effects of an anti-cancer agent" refers to any cell damage and/or death that results from the administration of the agent. An increase in the sensitivity of a cancer cell to the cytotoxic effects of an anti-cancer agent can be evidenced by an increase in the severity of cell damage and/or an increase in the quantity or rate of cell death that results from administration of at least one anti-cancer agent and at least one miR gene product, relative to a control cell in which only an anti-cancer agent is provided. Techniques for assessing cytotoxicity (e.g. , cell damage and/or death) are well known in the art and include, but are not limited to, assays that monitor cell proliferation, cell growth and cell death (e.g., apoptosis, necrosis).

[000207] In a particular embodiment, the cancer cells are leukemic cells. In one embodiment, the cancer cell is an in vivo cell (e.g. , a myeloid cancer cell in a mammal (e.g. , a human)). In other embodiments, the cancer cell is obtained from a patient sample (e.g. , biopsy, blood) or it may be obtained from an established cancer cell line or a cell lined derived from a cancer cell of a patient sample. In certain embodiments, the cancer cell is from a patient sample that was obtained from a subject who has a form of leukemia, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.

[000208] In a related embodiment, the compositions of the invention further comprise at least one chemotherapeutic agent. Many chemotherapeutic agents that are useful for the treatment of cancer are well known in the art. Suitable chemotherapeutic agents for use in the compositions of the invention are described herein. In certain embodiments, the amount of chemotherapeutic agent administered per dose is from about 0.0001 to 1000 mg/kg, about 0.5 to 70 mg/kg or about 1 to 50 mg/kg.

[000209] Pharmaceutical compositions are characterized as being sterile and pyrogen-free. As used herein, "pharmaceutical compositions" include compositions for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example, as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.

[000210] In one embodiment, the present pharmaceutical compositions comprise an agent that inhibits miR-223, wherein the agent is a nucleic acid sequence that is at least 90% identical to miR- 223 having SEQ ID NO: 1, or its complement (e.g. , 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically acceptable carrier. In another embodiment, the agent is at least 90% identical to the antagomir of miR-223. The pharmaceutical compositions of the invention can also comprise an antagomir of miR-223, an anti-miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR-223, or a double stranded RNA, which are encapsulated by liposomes and a pharmaceutically-acceptable carrier.

[000211] In one embodiment, a nucleic acid or vector administered to the subject cells comprise a non-cationic lipid for cytoplasmic and/or nuclear delivery, wherein the nucleic acid or vector is stable and is used in biological extracellular fluids typically found in animals, particularly blood serum.

[000212] Liposomes, spherical, self-enclosed vesicles composed of amphipathic lipids, can be employed as vectors for in vivo administration of therapeutic agents. In particular, the so-called long circulating liposomes formulations which avoid uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen, have found commercial applicability. Such long-circulating liposomes include a surface coat of flexible water soluble polymer chains, which act to prevent interaction between the liposome and the plasma components which play a role in liposome uptake.

[000213] In one embodiment, the liposome encapsulates the nucleic acid sequences, vectors or even the viral particles. In one embodiment, the nucleic acid sequences or vectors are condensed with a cationic polymer, e.g. , PEI, polyamine spermidine, and spermine, or a cationic peptide, e.g. , protamine and poly-lysine, and encapsulated in the lipid particle. The liposomes can comprise multiple layers assembled in a step-wise fashion.

[000214] Lipids may include relatively rigid varieties, such as sphingomyelin, or fluid types, such as phospholipids having unsaturated acyl chains. "Phospholipid" refers to any one phospholipid or combination of phospholipids capable of forming liposomes. Phosphatidylcholines (PC), including those obtained from egg, soybeans or other plant sources or those that are partially or wholly synthetic, or of variable lipid chain length and unsaturation are suitable for use. Synthetic, semisynthetic and natural product phosphatidylcholines including, but not limited to,

distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg

phosphatidylcholine (HEPC), dipalmitoylphosphatidylcholine (DPPC) and

dimyristoylphosphatidylcholine (DMPC) are suitable phosphatidylcholines for use. Further, phosphatidylglycerols (PG) and phosphatic acid (PA) are also suitable phospholipids for use and include, but are not limited to, dimyristoylphosphatidylglycerol (DMPG),

dilaurylphosphatidylglycerol (DLPG), dipalmitoylphosphatidylglycerol (DPPG),

distearoylphosphatidylglycerol (DSPG) dimyristoylphosphatidic acid (DMPA),

distearoylphosphatidic acid (DSPA), dilaurylphosphatidic acid (DLPA), and dipalmitoylphosphatidic acid (DPP A). Distearoylphosphatidylglycerol (DSPG) may be a preferred negatively charged lipid when used in formulations. Other suitable phospholipids include phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, and phosphatidic acids containing lauric, myristic, stearoyl, and palmitic acid chains. For the purpose of stabilizing the lipid membrane, in certain embodiments, an additional lipid component, such as cholesterol, can be added; non-limiting examples of lipids for producing liposomes include phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in further combination with cholesterol (CH). According to one embodiment, a combination of lipids and cholesterol for producing the liposomes comprise a PE:PC:Chol molar ratio of 3: 1: 1. Further, incorporation of polyethylene glycol (PEG) containing phospholipids is also contemplated.

[000215] In addition, in order to prevent the uptake of the liposomes into the cellular endothelial systems and enhance the uptake of the liposomes into the target of interest, the outer surface of the liposomes may be modified with a long-circulating agent. The modification of the liposomes with a hydrophilic polymer as the long-circulating agent can prolong the half-life of the liposomes in the blood.

[000216] Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

[000217] Pharmaceutical compositions can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g. , tromethamine hydrochloride), chelants (such as, e.g. , DTPA or DTPA- bisamide) or calcium chelate complexes (e.g. , calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (e.g. , calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions can be packaged for use in liquid form, or can be lyophilized.

[000218] For solid pharmaceutical compositions, conventional nontoxic solid pharmaceutically- acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For example, a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, preferably 25% -75%, of the miR-223 inhibiting agent. A pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01- 20% by weight, preferably 1%-10% by weight, of the miR-223 inhibiting agent encapsulated in a liposome, as described above, and a propellant. A carrier can also be included as desired, e.g. , lecithin for intranasal delivery.

[000219] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from at least about 1 microgram/kg/body weight, including about 5, about 10, about 50, about 100, about 200, about 350 weight, about 500 microgram/kg/body weight; or at least about 1 milligram (mg)/kg/body weight, such as about 5, about 10, about 50, about 100, about 200, about 350, about 500, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.

[000220] One skilled in the art can also readily determine an appropriate dosage regimen for administering a compound that inhibits miRNA expression to a given subject, as described herein. Suitable compounds for inhibiting miRNA gene expression include double-stranded RNA (such as short- or small-interfering RNA or "siRNA"), antisense nucleic acids, and enzymatic RNA molecules, such as ribozymes. Each of these compounds can be targeted to a given miRNA gene product and interfere with the expression (e.g., by inhibiting translation, by inducing cleavage and/or degradation) of the target miRNA gene product. For example, expression of a given miRNA gene can be inhibited by inducing RNA interference of the miRNA gene with an isolated double-stranded RNA ("dsRNA") molecule which has at least 90%, at least 95%, at least 98%, at least 99%, 100%, or any intermediate percentage between 90% and 100% sequence homology with at least a portion of the miRNA gene product.

[000221] Also provided herein is a pharmaceutical pack or kit comprising one or more dosage units of a nucleic acid construct sufficient for one or more courses of treatment for a cell or microvesicle expressing miR-223. Associated with such pharmaceutical pack or kit are instructions for administering the nucleic acid construct. Optionally associated with such pharmaceutical pack or kit is a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human consumption.

[000222] Examples

[000223] Certain embodiments of the present invention are defined in the Examples herein. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

[000224] As used herein, the term "leukemia" and "leukemic cancer" refer to all cancers of the hematopoietic and immune systems (blood and lymphatic system). These terms refer to a progressive, malignant disease of the blood-forming organs, marked by distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Myelomas refer to other types of tumors of the blood, bone marrow cells. Lymphomas refer to tumors of the lymph tissue. [000225] Reagents

[000226] Reagents for antagomir transfection, fluorescently labeling microvesicles, cell culture, RNA extraction, and miRNA expression were obtained from Life Technologies (Grand Island, NY) unless otherwise indicated. Housekeeping PCR primers except CAPl were obtained from Real Time Primers, LLC (Elkins Park, PA). Primers to measure gene expression were purchased from Qiagen (Valencia, CA). Flow cytometry reagents including antibodies, Annexin V and instrumentation were from BD Biosciences (San Jose, CA). Transmission electron microscopes were from FEI (Hillsboro, OR). Chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[000227] Human Sample Processing and Cell Culture

[000228] Peripheral human blood monocytes were isolated from American Red Cross buffy coats or healthy volunteers following informed consent (IRB# 2011H0007). Briefly, diluted blood was layered over Lymphocytes Separation Medium (d=1.077) (Cellgro, Manassas, VA). CD14+ monocytes were isolated using the Monocyte Isolation Kit from Miltenyi Biotech (Auburn, CA). Monocytes ( 1-10 >< 10⁶ cells/ml) in X-VIVO 15 serum-free media (Lonza, Walkers ville, MD) were supplemented with 10 μg/ml polymyxin B in the absence or presence of 50 ng/ml of rhGM-CSF (Berlex Laboratories Inc., Montville, NJ). The monocytes were washed after 4 hours and cultured in fresh media without added rhGM-CSF. Primary cells were subjected to morphological assessment to confirm response to GM-CSF prior to subsequent use and collection of microvesicles.

[000229] Human monocytic THP-1 cells, lung fibroblasts CCL-204 and lung epithelial A549 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). THP-1 cells were grown in RPMI 1640 supplemented with 10% FBS (Hyclone Laboratories, Logan, UT) and 1% PSA. To induce differentiation, THP-1 cells (1 x 10⁶ cells/ml) were grown in X-VIVO 15 media and treated with 65 nM phorbol 12-myristate 13-acetate (PMA). Cells treated with 0.001% dimethyl sulfoxide (DMSO) served as a vehicle control. Lung fibroblasts and epithelial cells were cultured in DMEM medium containing 10% FBS and 1% PSA. Human umbilical vein endothelial cells (HUVEC) and endothelial cell medium were purchased from ScienCell Research Laboratories (Carlsbad, CA). The cells were maintained in cell culture flask coated with 15 pg/ml ibronectin.

[000230] Antagomir Transfection

[000231] THP-1 cells or monocytes were transfected with the miR-223 antagomir (100 nM) (Life Technologies and SwitchGear Genomics, Menlo Park, CA) using the siPORT NeoFX transfection reagent according to manufacturer's instructions. The anti-miR miRNA Inhibitor Negative Control #1 (100 nM) served as a negative control. Cells were then cultured for 6-24 hours prior to analysis.

[000232] Transmission Electron Microscopy (TEIVI)

[000233] Suspension THP-I cells or freshly isolated monocytes were centrifuged at 1600x g for 5 minutes then fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.1 M sucrose for 30 minutes. Cells were rinsed, resuspended in 2% agarose, and dehydrated in graded ethanol concentrations for 10 minutes each. Sections were embedded in Spurr resin and polymerized overnight at 60°C. Treated adherent cells were grown in dual chamber slides then fixed and dehydrated in the same manner but embedded in silicon mold and resin filled Beam capsules. TEM sections were cut at 70 nm using EM UC6 ultra-microtome, and collected on 300-mesh grids.

Sections were stained in 2% uranyl acetate and Reynolds lead citrate and imaged with Technai G2 Spirit TEM at 80 kV.

[000234] Cryo-TEM images of microvesicles were obtained from samples centrifuged at 16,000x g and 160,000x g. To prepare vitrified cryo-TEM specimens, 4 pi suspensions of microvesicles were applied to glow discharged lacey carbon coated copper 400-mesh grids (Pacific Grid-Tech, San Francisco, CA) and flash-frozen in liquid ethane at 22°C and 95% relative humidity using an automated Vitrobot Mark IV (FEI). The vitrified samples were visualized with Tecnai G2 F20 ST TEM at 200kV using low dose mode.

[000235] Confocal Microscopy Studies

[000236] THP-I cells or freshly isolated monocytes were suspended in X-VIVO 15 media and treated with either the phospholipid membrane dye, lipophilic carbocyanine DilCi₆(3) (D384, 1.25 μΜ) or 0.625 pM SytoRNA Select that specifically binds RNA molecules. After 10 minutes of incubation at 37°C, the cells were washed, resuspended in fresh media and treated with PMA or GM- CSF for 4 hours. Cells were then washed and cultured for an additional 48 hours. Fluorescent microvesicles were collected and added to recipient cells. Both donor and recipient cells were fixed then mounted on slides using fluorescent mounting solution (Dako, Carpinteria, CA) then imaged using the Zeiss LSM 510 multiphoton confocal microscope (Thornwood, NY). Background fluorescence was subtracted using unstained cells.

[000237] Dynamic Light Scattering

[000238] Micro vesicle size distributions were measured using a Nano Zetasizer Zen3600 (Malvern Instruments Ltd., Worcestershire, UK). Samples were diluted to 50-300 kilocounts per second and equilibrated at 25°C. All measurements were done in triplicates.

[000239] Crystal Violet Assay

[000240] Cellular adherence (6 X 10³ cells/well) was measured using crystal violet uptake in a 12- well plate. After 48 hours of treatment, cells were washed, fixed in 4% paraformaldehyde then stained with 0.5% crystal violet in 2% ethanol for 10 minutes at room temperature. The cells were washed four times with PBS. The incorporated stain was eluted with 0.1 M sodium citrate (pH 4.2) in 50% ethanol. Absorbance was measured at 550 nm using an assay ELISA plate reader (SLT Lab Instruments GmbH, Salzburg, Austria).

[000241] Flow cytometry

[000242] FITC-conjugated beads 0.2- 1 um (Bangs Laboratory, Inc. (Fishers, IN) and

nonfluoroscent bead standards 0.2-2.0 μιη (Spherotech Inc., Libertyville, IL) were used to gate on microvesicles <1 μιη using the LSRII flow cytometer. Microvesicles concentration was calculated. Background from sheath fluid and media only samples was subtracted.

[000243] To determine DNA, RNA and Annexin V expression, microvesicles were incubated with 0.625 μΜ SytoRNA Green Select or 2.5 μΐ Annexin V - FITC and/or 4.86 μΜ Hoechst 33342 for 30 minutes on ice then analyzed on the LSRII flow cytometer.

[000244] Surface expression of macrophage lineage markers was quantified by flow cytometry using the BD Aria flow cytometer. Cells were washed twice with cold buffer (Hank's Balanced Salt Solution containing 0.1% NaN₃ and 1% bovine serum albumin (BSA) then re-suspended in the buffer and blocked with 100 μg/ml human IgG solution (Jackson ImmunoResearch, West Grove, PA). Antibodies recognizing CD16 PE-Cy5, CD206 PE-Cy5 and CCR5-PE-Cy7 were used according to manufacturer' s recommendation. Isotype antibody controls were purchased from Cedarlane

(Burlington, NC).

[000245] GM-CSF ELISA

[000246] Microvesicle -containing supernatants and protein lysates from concentrated microvesicles were analyzed using the Quantikine human GM-CSF ELISA (R&D Systems, Minneapolis, MN). The signal was read at 450 nm on a PerkinElmer Victor X3 Multilabel Plate Reader (PerkinElmer, Waltham, Massachusetts).

[000247] RNA Extraction and Quantitative Real-Time PCR

[000248] Total RNA was isolated using Trizol. An average of 500 ng of RNA was obtained from microvesicles from cells cultured in a 6 well plate (5 x 106 cells/well). Profiling for all known and individual mature miRNAs was performed and analyzed using TaqMan MicroRNA assays.

[000249] RNA (0.5-1 μg) was subjected to cDNA synthesis and quantitative real-time (qRT)-PCR using SYBR Green Master Mix. The average Ct value ± S.E.M (1 5.432 ± 1.72) of GAPDH and CAP- 1 (forward primer 5'-ATTCCCTGGATTGTGAAATAGTC-3' (SEQ ID NO: 2) and reverse primer 5'- ATTAAAGTCACCGCCTTCTGTAG-3 ' (SEQ ID NO: 3)) were used for normalization. Small nuclear (sn) RNA U6 was used to normalize miRNA expression. For microvesicle treated samples, gene and miRNA is expression reported as the relative fold-increase 2('MC') over non-stimulated freshly isolated samples. For antagomir studies, the data was presented as relative copy number (RCN) using 2('Ac')-

[000250] Phagocytosis Assay

[000251] Briefly, the fluorescent antibody coated SRBC were incubated with freshly isolated monocytes or macrophages (1 χ10⁶/μΕ of coated SRBC) at 37°C for 30 minutes then washed with PBS and fixed in 1% paraformaldehyde. An 100 cells were counted per slide using a fluorescent microscope to enumerate phagocytes and calculate phagocytic index.

[000252] Caspase-3 Analysis

[000253] Adherent and non-adherent THP-1 cells and monocytes were collected 18 hours and 6 hours, respectively, post transfection with miR-223 antagomir for caspase-3 activity. Briefly, protein lysates were incubated with the fluorogenic substrate Ac-DEVD-AMC (N-acetyl- (Asp-Glu-Val- Asp)-(7-amino-4-methylcoumarin)) (EMD Millipore, Billerica, MA) and measured with the Cytofluor 4000 fluorometer (Perseptive, Framingham, MA). Caspase-3 activity is presented as the relative fold- increase change in fluorescence over untreated samples per total protein and is expressed as the mean +S.E.M.

[000254] Microarray Analysis

[000255] Changes in gene expression were analyzed in cytokine and microvesicle-treated cells using Human Genome U133 Plus 2.0 Affymetrix GeneChips® (Affymetrix, Santa Clara, CA) (n=4). The mRNA (30 ng) was processed using the Ovation RNA Amplification System and the FL- Ovation" cDNA Biotin Module V2 (both from NuGEN Technologies, Inc., San Carlos, CA). At all steps, the quantity and quality of the preparations were controlled using Nanodrop (NanoDrop Technologies, Wilmington, DE) and Experion Automated Electrophoresis System (BioRad

Laboratories, Hercules, CA). The arrays were scanned by the GeneChip® Scanner 3000 (Affymetrix) using the GeneChip® Operating Software (GCOS) vl.4.0.036. Partek Genomic Suite (Partek Inc., St. Louis, MO) was used for data analysis on .eel files imported using robust multi-chip average with GC content adjustment (GC-RMA) for background correction and normalized by quantile normalization. Genes that were P (present) or M (marginal) in at least two experimental samples of the triplicate per experimental conditions were analyzed and used to perform principal component analysis (PCA) and ANOVA. Genes were statistically corrected by Bonferroni adjustment were further analyzed and used to generate the pair- wise Venn diagrams.

[000256] Pathway Analysis and Prediction

[000257] Genes identified to be commonly expressed between cells treated with either the positive control stimulus or microvesicles were subjected to pathway exploration using the Ingenuity Pathway Analysis (IP A) software (Ingenuity Systems, Redwood City, CA). Top-ranked pathways and their associated biological functions were determined.

[000258] Luciferase Reporter Assay

[000259] A549 cells were transfected with 100 ng of 3'-UTR luciferase reporter vector containing miR-223 target seed sequence or an empty vector (SwitchGear Genomics) using FuGene HD transfection reagent (Roche, Nutley, NJ). As a positive control, as set of cells were co-transfected with miR-223 precursor (150 nM) (Life Technologies). After 18-20 hours, cells were washed and cultured in X-VIVO media alone or microvesicle- containing media generated from GM-CSF- stimulated monocytes for 24 hours prior to measuring luciferase activity with Lights witch Assay Reagent (SwitchGear Genomics) and Victor X3 Microplate Reader.

[000260] Statistical Analysis

[000261] For all comparisons, data are expressed as the mean +S.E.M derived from at least three independent studies. SPSS17 software (SPSS Inc. Chicago, IL) was used to generate an independent sample t-test, and p<0.05 was considered significant. Non-parametric T-test was used for statistical analysis for the analysis of real-time PCR and caspase-3 assay in which greater than 3 independent experiments.

[000262] Example 1: Microvesicle Production during Macrophage Differentiation

[000263] Microvesicle production is induced upon cell activation and growth. Using electron microscopy, increased microvesicle production was apparent in PMA-treated THP-1 cells (FIG. 1A) compared to cells treated with the vehicle (inset). Similarly, GM-CSF-treated monocytes had more microvesicles than untreated cells (FIG. IB). The vesicles ranging between 0.2-1 pm were RNA positive (FIG. 8A and FIG. 8B). The vast majority of the gated microvesicles were Annexin V negative (71.58 ±2.70%,) while only 6.87 ±1.52% were positive for DNA (FIG. 8C). Microvesicle concentration from the 0.2-1 μιη gate increased during macrophage differentiation (FIG. 1C).

[000264] Cryo-TEM images confirmed that secreted microvesicles from PMA-treated THP-1 cells centrifuged at 16,000x g (FIG. ID) and at 160,000x g (FIG. 9A).had similar physical properties. Both samples contained small (<150 nm diameter) and large (>200 nm diameter) spherical microvesicles (FIG. 9B) with non-uniform internal electron densities and similar distributions. Protrusions on the microvesicle surfaces indicated the presence of membrane proteins. The cryo-TEM images also revealed that 3-5% of the >200 nm microvesicles were ruptured in both centrifugation samples, and a greater fraction ruptured in the 160,000x g sample (data not shown).

[000265] Example 2: Microvesicles Uptake and Transfer of RNA Molecules in Monocytes

[000266] Since microvesicles contain both mRNA and miRNA molecules, it was determined whether the microvesicles transferred RNA molecules to recipient cells using similar concentrations of microvesicles reported for human plasma. Fluorescent-labeled microvesicles were incubated with freshly isolated unstained monocytes. Confocal imaging revealed delivery of labeled microvesicles as indicated by the presence of the fluorescent membrane and RNA dyes in unlabeled recipient monocytes (FIG. 2). A similar transfer of membrane and RNA dyes from THP-1 cell-derived microvesicles to naive, unlabeled THP-1 cells (FIG. 10A) was observed.

[000267] Example 3: Microvesicle Treatment Induces Differentiation in Recipient Cells

[000268] It was determined whether macrophage -derived microvesicles induced activation in recipient cells. Both PMA and microvesicles from PMA-stimulated THP-1 cells induced naive THP- 1 cell adherence as measured by crystal violet staining, while DMSO (vehicle) treatment or microvesicles from vehicle-stimulated THP-1 cells did not (FIG. 3A). Similarly, microvesicles from GM-CSF-stimulated cells added to naive monocytes significantly increased their adherence similar to GM-CSF-stimulated cells (FIG. 3B). These observations showed that the cells were differentiating in the presence of microvesicles.

[000269] To confirm that adherence signified cellular differentiation, recipient cells were stained for macrophage -lineage markers: CD16, CD206, and CCR5 that are highly expressed on macrophages compared to monocytes. Treating naive THP-1 cells with PMA or microvesicles from PMA- stimulated THP-1 cells significantly increased the surface expression of macrophage differentiation markers compared to vehicle -treated samples as quantified by flow cytometry (FIG. 3C). For THP1 cells, >97% of the PMA and microvesicle-treated cells were positive for the surface antigens. The mRNA was also increased for these surface antigens. Similarly, monocytes treated with

microvesicles from GM-CSF-stimulated cells also exhibited enhanced surface and mRNA expression for CD 16, CD206, and CCR5 by flow cytometry and qRT-PCR, respectively (FIG. 3D and FIG. 3E, respectively). GM-CSF or microvesicles treated monocytes were 50-55% positive for CCR5 expression while the percent positive cells for CD16 were 11.06 ±3.17% and 24.04 ±17.23% for GM- CSF and microvesicle-treated cells, respectively. However, CD206 percent positive cells were greater in GM-CSF-treated cells (69.42 ±15.37%) compared to microvesicle-treated cells (17.56 ±1.64%). Furthermore, these genes were absent in the microvesicles as confirmed by PCR (data not shown).

[000270] It was also determined whether the macrophages derived from microvesicle-treated cells were functionally active. A significant increase in phagocytic activity in macrophages derived from either GM-CSF or GM-CSF-derived microvesicles was found, compared to freshly isolated monocytes (FIG. 3F).

[000271] As confirmation that this effect was directly attributed to the microvesicles, it confirmed that there was no carryover of either GM-CSF or PMA in the microvesicles inducing recipient cell activity. To address if GM-CSF was retained in the microvesicles preparation, a human GM-CSF ELISA was used. The GM-CSF concentration was <30 pg/ml in both protein lysates from the microvesicles as well as the GM-CSF-stimulated microvesicle preparation used to treat monocytes and less than the minimal doses of GM-CSF required to activate monocytes. Furthermore, the presence of GM-CSF mRNA or other genes that cluster with GM-CSF was not detected, including IL- 4, -5 and -13 in the microvesicles.

[000272] To determine whether PMA carry over from THP-1 cell stimulation was responsible for the recipient cell adherence and differentiation, it was found that while microvesicles from PMA- stimulated THP-1 cells induced adherence of naive primary human monocytes, adding PMA directly to these primary monocytes did not induce their differentiation (FIG. 10B). Thus, the phenotypic and differentiation changes in recipient cells treated with microvesicles from GM-CSF-stimulated monocytes or PMA-stimulated THP-1 cells were from the microvesicle content. Since THP-1 cells are growth factor independent, collectively these data further demonstrated that something other than a growth factor was shuttled by the microvesicle to stimulate the differentiation process.

[000273] Example 4: Gene Changes in Microvesicle-Treated Cells Detected by Microarray Analysis

[000274] It was also determined whether microvesicles from GM-CSF-stimulated monocytes or PMA-treated THP-1 cells induced similar gene expression profiles in primary human monocytes or THP-1 cells treated directly with either GM-CSF or PMA, respectively. Principle component analysis (PCA) of the cDNA expression data was performed to determine the percentage of variance among the samples. The gene expression profile of THP-1 cells directly stimulated with PMA compared to cells treated with PMA-stimulated niicrovesicles with THP-1 cells were highly similar and resided in a relative similar dimension (FIG. 4A).

[000275] Similarly, monocytes treated with GM-CSF or GM-CSF-stimulated niicrovesicles were similar using PCA mapping of the microarray data (FIG. 4B). Using pair- wise comparisons, 126 co- expressed genes between cells treated directly with PMA or with niicrovesicles from PMA-stimulated THP-1 cells (FIG. 4C) were observed. Notably, 234 genes were exclusively expressed in the THP-1 cells treated with PMA, while 17 genes were unique in cells treated niicrovesicles from PMA- stimulated THP-1 cells, (FIG. 4C). On the other hand, treating monocytes with GM-CSF or niicrovesicles from GM-CSF-stimulated monocytes induced the co-expression of 407 genes (FIG. 4D). In contrast, 59 genes and 40 genes were exclusively expressed in monocytes treated with GM- CSF or niicrovesicles from GM-CSF-treated cells, respectively (FIG. 4D).

[000276] Using the co-expressed genes in each group from the Venn diagrams in FIG.s 4C and 4D, the predicted top associated network functions, molecular and cellular functions, physiological and systems development functions were analyzed using Ingenuity Pathway Analysis (IP A) software (FIG. 12, FIG. 13, and FIG. 14). The IPA software identified hematological system development as a top ranked pathway in THP-1 cells treated with PMA-stimulated niicrovesicles and in monocytes treated with GM-CSF-stimulated niicrovesicles.

[000277] Example 5: Non-Myeloid Uptake of GM-CSF 'Stimulated Microvesicles

[000278] It was also determined whether GM-CSF-stimulated microvesicles bind to cells other than myeloid lineages and transferred RNA molecules. Therefore, fluorescently labeled GM-CSF- stimulated microvesicles were incubated with epithelial cells (A549), fibroblasts (CCL-204) or endothelial cells (I-IUVECs). Confocal imaging revealed that membrane- and RNA -labeled microvesicles from GM-CSF-stimulated cells were readily taken up by all three cell types (FIG. 5A, FIG. 5B, and FIG. 5C). As observed by flow cytometry, significant increase in florescence of double positive cells for the membrane and RNA dye was apparent after 24 hours of incubation. No further increase in uptake was observed at 48 hours in the micro vesicle -treated cells (FIG. 5D).

[000279] Example 6: GM-CSF -Stimulated Microvesicles Shuttle Functionally Active miR-223 to Target Cells

[000280] To determine whether the microvesicles could transfer miRNAs to target cells, the iniRNA expression in the microvesicles was characterized. As shown in FIG. 15, 186 miRNAs are expressed in the GM-CSF-stimulated microvesicles. GM -CSF-stimulated microvesicles had a high expression of miR-223 (mean normalized expression 53,018.46 ±33,473.39 S.E.M, n=5, FIG. 12) while miR-29b and miR-34a were undetectable.

[000281] Since epithelial cells, fibroblasts, and endothelial cells have low basal expression of miR- 223, miR-29b, and miR-34a, they are an amendable model to measure the shuttling of miR-223. Upon transfer and uptake of GM-CSF-stimulated microvesicles, these target cells expressed significantly higher levels of miR-223 but levels of miR-34a and miR-29b remained unchanged (FIG. 6A). These data are consistent with the direct transfer of miR-223 from microvesicles to target cells. To ensure that the target cells did not upregulate the expression of miR-223 upon microvesicle treatment, A549 cells were treated with actinomycin D prior to microvesicle treatment. As shown in FIG. 6B, similar miR-223 was detected in the micro vesicle-treated cells regardless of the presence of actinomycin D.

[000282] It was also determined whether miR-223 transferred from the GM-CSF-stimulated microvesicles was functional in the recipient cells. To address this, A549 cells were transfected with a 3'-UTR lucif erase reporter vector to which miR-223 binds. As shown in FIG. 6C, transfected cells in media alone displayed robust luciferase production. However, when A549 cells were cultured with GM-CSF-stimulated microvesicles containing miR-223, luciferase activity was decreased significantly in cells transfected with the miR-223 3'-UTR luciferase reporter vector but not the control vector. Since these cells do not normally have miR-223, this showed that miR-223 transferred from the microvesicles downregulated luciferase production. As a positive control, the luciferase reporter vector and the miR-223 precursor were co-transfected, which showed that luciferase production was also decreased in these cells.

[000283] Example 7: Inhibition of miR-223 reduces macrophage differentiation and survival [000284] Since miR-223 is the most abundant miRNA in the microvesicles, the importance of miRNA in macrophage development was examined. Using an antagomir to miR-223, the expression of miR-223 was specifically knocked down in both THP-1 cells and monocytes (FIG. 11 A and FIG.

1 IB). miR-191 which is the second most abundant miRNA the microvesicles was unchanged in the treated cells.

[000285] Phenotypically, miR-223 antagomir-transfected THP-1 cells or monocytes did not become adherent in the presence of PMA or GM-CSF, respectively (FIG. 7 A and FIG. 7B). It was also determined whether the lack of cell adherence was associated with inhibition of cellular differentiation or survival. It was found that the miR-223 antagomir treatment prevented PMA or GM-CSF induction of macrophage markers CCR5 or CD206 (FIG. 7C and FIG. 7D). c-Myc, which does not change during macrophage differentiation, was unaffected by the miR-223 antagomir (FIG. 7C and FIG. 7D). Moreover, it was found that survival was compromised in the miR-233 -transfected cells compared to untreated or scrambled-transfected cells as measured by an increase in caspase-3 activity (FIG. 7E and FIG. 7F) and a concordant significant decrease in BCL-2 and BCL-xL expression (FIG. 11C and FIG. 11D). [000286] Discussion of Examples 1- 7

[000287] Microvesicles are important mediators of cell-cell communication. Released from the endosomal compartment or shed from the cell surface, microvesicles can directly stimulate target cells by receptor-mediated interactions or by transferring bioactive molecules including proteins, mRNAs, miRNAs, and organelles. Circulating microvesicles in the plasma of normal human volunteers contain miRNAs. These microvesicles are most commonly derived from platelets and macrophages.

[000288] Microvesicle production and release are signal and stimuli dependent. Factors such as environmental stress and calcium concentration affect microvesicle release. Additionally, cytokines like IL-1B induce microvesicle shedding from peripheral blood monocytes, demonstrating the impact of cytokines in microvesicle production. GM-CSF and PMA induces the production of microvesicles from monocytes and the myeloid leukemic cell line, THP-1, respectively. Treatment of tumor cells with PMA significantly alters calcium concentration and increases tumor-derived microvesicle release.

[000289] Microvesicle production increased as these stimuli induced cellular differentiation.

Furthermore, these microvesicles drove the differentiation of THP-1 cells and naive monocytes. Myeloid cells generated by tumor microvesicles have an immunosuppressive phenotype and produce cytokines to promote tumor growth. Normal macrophages produce microvesicles to maintain homeostasis and immune cell production. Tumor cells have evolved to take advantage of this phenomenon.

[000290] Dendritic cells use exosomes to communicate with each other. Macrophage-derived microvesicles communicate with a variety of cell types, showing that the impact of these

microvesicles is widespread. Microvesicle treatment of target cells induces genetic and phenotypic changes, not requiring the transcriptional machinery of the target cell.

[000291] Analysis of the gene profile from microvesicle -treated cells reveals a number of cellular processes including cellular metabolism, survival, signaling, membrane integrity, hematopoietic cell development and immune cell homeostasis. There was no carry-over of GM-CSF in microvesicle preparations to contribute to these changes within the target cells. Furthermore, while microvesicles from PMA-stimulated THP-1 cells stimulated primary human monocyte differentiation, adding PMA directly to human monocytes did not induce these cells to differentiate. Since THP-1 cells are growth factor independent and microvesicles mediated their differentiation, this demonstrates that other critical factors were contained in macrophage-derived microvesicles.

[000292] While microvesicles shed from apoptotic bodies can contain the morphogen, Hedgehog, to promote megakaryocyte differentiation, few apoptotic bodies were found in the GM-CSF- or PMA- stimulated microvesicles. The RNA molecules contained in the macrophage-derived microvesicles are shuttled not only to monocytic cells but other cell lineages.

[000293] miR-223 is highly expressed miRNA in macrophage-derived microvesicles. miR-223 expression decreases as a monocytes differentiates to a macrophage. Similarly, miR-223 decreases during osteoclast and erythroid differentiation while increased miR-223 is important in

megakaryocyte production and progenitor cell proliferation. Furthermore, the absence of miR-223 is associated with incomplete granulopoiesis. Therefore, during GM-CSF-induced macrophage differentiation, the monocytic cell selectively releases miR-223 to permit completion of the maturation process. Upon doing so, the miR-223 contained in the macrophage -derived microvesicles can be shuttled to other progenitor lineages to complete their terminal maturation such as granulocytes or megakaryocytes.

[000294] Macrophage-derived microvesicles contain high levels of miR-223 and induced differentiation of recipient monocytes. Several targets of miR-223 include inositol phosphatases that are important in monocyte survival. As such, knocking down miR-223 in monocytes and THP-1 cells impacted cell survival. While cell viability was compromised in miR-223 antagomir-transfected cells, it is shown herein that viable miR-223 antagomir-transfected cells were unable to differentiate into macrophages in the presence of GM-CSF or PMA.

[000295] Microvesicles produced from activated macrophages induce the differentiation of recruited monocytes, activate hematopoietic cell production in the marrow and induce the release of more microvesicles.

[000296] Further Examples

[000297] Therapeutic/Prophylactic Methods and Compositions

[000298] Further described herein are methods of treatment and prophylaxis by administration to a subject an effective amount of a therapeutic compound, i.e., a monoclonal (or polyclonal) antibody, viral vector, mimic and/or antagonist. In a preferred aspect, the therapeutic is substantially purified. The subject is preferably an animal, including but not limited to, animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.

[000299] Various delivery systems are useful to administer a therapeutic compound, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor- mediated endocytosis, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include, but are not limited to, intradermal, intramuscular,

intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The therapeutic compounds are administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[000300] In a specific embodiment, it may be desirable to administer the therapeutic compositions locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration is by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre -neoplastic tissue.

[000301] In a specific embodiment where the therapeutic is a nucleic acid encoding a protein therapeutic the nucleic acid is administered in vivo to modulate expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus.

Alternatively, a nucleic acid therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

[000302] Pharmaceutical compositions.

[000303] Such compositions comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation will suit the mode of administration.

[000304] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

[000305] In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. For example, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition also includes a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it is be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline is provided so that the ingredients are mixed prior to administration.

[000306] The therapeutic formulation can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from

hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[000307] The amount of the therapeutic formulation which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and is determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and is decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems

[000308] Method of treating cancer patients

[000309] This example describes a method of selecting and treating patients that are likely to have a favorable response to treatments with compositions herein.

[000310] A patient diagnosed with cancer ordinarily first undergoes tissue resection with an intent to cure. Tumor samples are obtained from the portion of the tissue removed from the patient. RNA is then isolated from the tissue samples using any appropriate method for extraction of small RNAs that are well known in the art, such as by using TRIZOL™. Purified RNA is then subjected to RT-PCR using primers specific for miR-494 or other differentially expressed miRNAs disclosed, optionally in conjunction with genetic analysis. These assays are run to determine the expression level of the pertinent RNA in the tumor. If differentially expressed miR expression pattern is determined, especially if mutant status is ascertained, the patient is evaluated as to whether the patient is a candidate for treatment with the compositions herein.

[000311] Accordingly, the patient is treated with a therapeutically effective amount of the compositions according to methods known in the art. The dose and dosing regimen of the compositions will vary depending on a variety of factors, such as health status of the patient and the stage of the cancer. Typically, treatment is administered in many doses over time.

[000312] Evaluation of miR levels at different periods of time may be used to determine appropriate dosage, changing therapeutics, ceasing treatment, or initiating a treatment regime.

[000313 ] Methods of Diagnosing Cancer Patients

[000314] In one particular aspect, there is provided herein a method of diagnosing whether a subject has, or is at risk for developing, cancer. The method generally includes measuring the differential miR expression pattern of the miR compared to control. If a differential miR expression pattern is ascertained, the results are indicative of the subject either having, or being at risk for developing, cancer. In certain embodiments, the level of the at least one gene product is measured using Northern blot analysis. Also, in certain embodiments, the level of the at least one gene product in the test sample is less than the level of the corresponding miR gene product in the control sample, and/or the level of the at least one miR gene product in the test sample is greater than the level of the corresponding miR gene product in the control sample.

[000315] In some embodiments, mRNA-containing samples may be obtained from, blood, mucus, sputum, bronchoscopic biopsy, needle biopsy, open biopsy, or video-assisted thoracoscopic surgery.

[000316] Measuring miR Gene Products

[000317] The level of the at least one miR gene product can be measured by reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides; hybridizing the target oligodeoxynucleotides to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the test sample; and, comparing the test sample hybridization profile to a hybridization profile generated from a control sample. An alteration in the signal of at least one miRNA is indicative of the subject either having, or being at risk for developing, lung cancer, particularly EGFR mutant lung cancer.

[000318] Diagnostic and Therapeutic Applications

[000319] In another aspect, provided herein are methods of treating a cancer in a subject, where the signal of at least one miRNA, relative to the signal generated from the control sample, is de -regulated (e.g., down-regulated and/or up-regulated).

[000320] Also provided herein are methods of diagnosing whether a subject has, or is at risk for developing, a cancer associated with one or more adverse prognostic markers in a subject, by reverse transcribing RNA from a test sample obtained from the subject to provide a set of target

oligodeoxynucleotides; hybridizing the target oligodeoxynucleotides to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the test sample; and, comparing the test sample hybridization profile to a hybridization profile generated from a control sample. An alteration in the signal is indicative of the subject either having, or being at risk for developing, the cancer.

[000321] Kits

[000322] Also provided are pharmaceutical packs or kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) is a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[000323] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, reagents for isolating miRNA, labeling miRNA, and/or evaluating an miRNA population using an array are included in a kit. The kit may further include reagents for creating or synthesizing miRNA probes. The kits will thus comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, and components for isolating miRNA. Other kits may include components for making a nucleic acid array comprising

oligonucleotides complementary to miRNAs, and thus, may include, for example, a solid support.

[000324] For any kit embodiment, including an array, there can be nucleic acid molecules that contain a sequence that is identical or complementary to all or part of any of the sequences herein.

[000325] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[000326] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being one preferred solution. Other solutions that may be included in a kit are those solutions involved in isolating and/or enriching miRNA from a mixed sample.

[000327] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also include components that facilitate isolation of the labeled miRNA. It may also include components that preserve or maintain the miRNA or that protect against its degradation. The components may be RNAse-free or protect against RNAses.

[000328] Also, the kits can generally comprise, in suitable means, distinct containers for each individual reagent or solution. The kit can also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented. It is contemplated that such reagents are embodiments of kits of the invention. Also, the kits are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of miRNA.

[000329] It is also contemplated that any embodiment discussed in the context of an miRNA array may be employed more generally in screening or profiling methods or kits of the invention. In other words, any embodiments describing what may be included in a particular array can be practiced in the context of miRNA profiling more generally and need not involve an array per se.

[000330] It is also contemplated that any kit, array or other detection technique or tool, or any method can involve profiling for any of these miRNAs. Also, it is contemplated that any embodiment discussed in the context of an miRNA array can be implemented with or without the array format in methods of the invention; in other words, any miRNA in an miRNA array may be screened or evaluated in any method of the invention according to any techniques known to those of skill in the art. The array format is not required for the screening and diagnostic methods to be implemented.

[000331] The kits for using miRNA arrays for therapeutic, prognostic, or diagnostic applications and such uses are contemplated by the inventors herein. The kits can include an miRNA array, as well as information regarding a standard or normalized miRNA profile for the miRNAs on the array. Also, in certain embodiments, control RNA or DNA can be included in the kit. The control RNA can be miRNA that can be used as a positive control for labeling and/or array analysis.

[000332] The methods and kits of the current teachings have been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the current teachings. This includes the generic description of the current teachings with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[000333] Array Preparation and Screening

[000334] Also provided herein are the preparation and use of miRNA arrays, which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules or precursor miRNA molecules and that are positioned on a support material in a spatially separated organization. Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted. Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.

[000335] Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose -based material of filter arrays. By having an ordered array of miRNA - complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.

[000336] A variety of different array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art. Useful substrates for arrays include nylon, glass and silicon. The arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like. The labeling and screening methods described herein and the arrays are not limited in its utility with respect to any parameter except that the probes detect miRNA; consequently, methods and compositions may be used with a variety of different types of miRNA arrays.

[000337] All publications, including patents and non-patent literature, referred to in this specification are expressly incorporated by reference herein. Citation of the any of the documents recited herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

[000338] While the invention has been described with reference to various and preferred embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof.

[000339] Therefore, it is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.

Claims

CLAIMS What is claimed is:

1. A method of treating leukemia in a subject having leukemia, the method comprising: administering an effective amount of an agent that inhibits miR-223 expression in a cell of the subject;

wherein the agent comprises: a nucleic acid sequence that is at least 90% identical to miR-223 having SEQ ID NO: 1 , or its complement; an antagomir of miR-223; an anti-miR-223

oligonucleotide; an antisense oligonucleotide to miR-223; a locked nucleic acid that anneals to miR- 223, or, a double stranded RNA that complementary base-pairs with miR-223.

2. A method of increasing the efficacy of an anti-cancer treatment in a subject having leukemia, comprising:

(i) administering at least one anti-cancer treatment to the subject having leukemia; wherein the anti-cancer treatment is chemotherapy and/or radiation therapy;

(ii) administering an effective amount of an agent that inhibits miR-223 expression in a cell of the subject; wherein the agent comprises: a nucleic acid sequence that is at least 90% identical to miR-223 having SEQ ID NO: 1 , or its complement; an antagomir of miR-223; an anti-miR-223 oligonucleotide; an antisense oligonucleotide to miR-223; a locked nucleic acid that anneals to miR- 223, or, a double stranded RNA that complementary base-pairs with miR-223; and

(iii) determining that the subject has an increased efficacy of the anti-cancer treatment when the agent inhibits miR-223 expression, as compared to a control.

3. A method of increasing the sensitivity of a leukemic cell to cytotoxic effects of an anti-cancer agent, comprising:

administering to the leukemic cell an effective amount of an agent that inhibits miR- 223 expression in a cell of the subject sufficient to increase the sensitivity of the cell to the anti-cancer agent is increased,

wherein the agent comprises: a nucleic acid sequence that is at least 90% identical to miR-223 having SEQ ID NO: 1 , or its complement; an antagomir of miR-223; an anti-miR-223 oligonucleotide; an antisense oligonucleotide to miR-223; a locked nucleic acid that anneals to miR- 223, or, a double stranded RNA that complementary base-pairs with miR-223.

4. A method comprising: determining the level of monocytes in a subject; and administering a therapeutically effective amount of an inhibitory nucleic acid that is complementary to miR-223 to a subject determined to have an increased production of monocytes; whereby differentiation of the monoctyes is reduced; hematopoietic cell production in the subject's marrow is decreased; and /or, release of microvesicles produced from macrophages is decreased.

5. A method of treating leukemia in a subject in need thereof, the method comprising: obtaining a sample of hematopoietic progenitor cells from the subject; contacting the hematopoietic stem progenitor cells with a vector comprising a nucleic acid sequence that is an antagomir of miR- 223; and, introducing the cell into the same subject.

6. A method of treating leukemia in a subject in need thereof, the method comprising administering an effective amount of an agent that inhibits miR-223 in a cell to a subject, wherein the agent is a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.

7. The method of claim 6, wherein the cell is a progenitor cell.

8. The method of claim 7, wherein the progenitor cell is a hematopoietic progenitor cell.

9. The method of claim 6, wherein the agent is an antagomir of miR-223, an anti- miR-223 oligonucleotide, an antisense oligonucleotide to miR-223 a locked nucleic acid that anneals to miR-223, or a double stranded RNA that complementary base-pairs with miR-223.

10. A method of treating leukemia in a subject in need thereof, the method comprising: ii) obtaining a sample of hematopoietic progenitor cells from the subject; ii) contacting the hematopoietic progenitor cells with a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1; and iii) introducing the cell from step iii) into the same subject.

11. A method of treating a leukemia or pre-leukemic condition in a subject in need thereof comprising:

administering to the myeloid or lymphoid cells of the subject an agonist of miR-222, wherein the agonist of miR-223 is a polynucleotide comprising a pri-miRNA, pre-miRNA, or mature sequence of miR-223, and wherein the leukemia or pre-leukemic condition is reduced in the subject following administration of the agonist.

12. The method of claim 11, wherein the polynucleotide comprises a sequence of SEQ ID NO: 1.

13. The method of claim 11, wherein the polynucleotide is encoded by an expression vector.

14. The method of claim 11, wherein the polynucleotide is comprised within a lipid vehicle.

15. The method of claim 11, further comprising administering a non-miR-223 anti-leukemic therapy to the subject.

16. The method of claim 11, wherein the polynucleotide is operably- linked to a leukemic cell- specific promoter.

17. The method of claim 11, wherein the polynucleotide is double-stranded.

18. The method of claim 11, wherein the polynucleotide is about 18 to about 25 nucleotides in length.

19. The method of claim 11, wherein the polynucleotide is about 70 to about 200 nucleotides in length.

20. The method of claim 11, wherein the polynucleotide is administered by oral, intradermal, intramuscular, intravenous, subcutaneous, inhalational, or intraperitoneal administration.

21. The method of claim 11, wherein the polynucleotide comprises one or more chemical modifications.

22. The method of claim 21, wherein one or more chemical modifications are selected from the group consisting of sugar modifications, backbone modifications, and combinations thereof.

23. The method of claim 22, wherein the sugar modifications are selected from the group consisting of 2'-0-alkyl, 2'-0-methyl, 2'-0-methoxyethyl, 2'-fluoro, and combinations thereof.

24. The method of any one of the preceding claims, wherein the cell is a leukemic cell.

25. The method of any one of the preceding claims, wherein the cell is a myeloid leukemic cell.

26. The method of any one of the preceding claims, wherein the cell is a lymphocytic leukemic cell.

27. The method of any one of the preceding claims, wherein the leukemic cell is present in a subject.

28. The method of any one of the preceding claims, wherein the subject is a human.

29. The method of any one of the preceding claims, wherein the at least one anti-cancer agent is selected from the group consisting of: cytidine arabinoside; methotrexate; vincristine;

etoposide (VP- 16); doxorubicin (adriamycin); cisplatin (CDDP); dexamethasone; arglabin;

cyclophosphamide; sarcolysin; methylnitrosourea; fluorouracil; 5-fluorouracil (5FU); vinblastine; camptothecin; actinomycin-D; mitomycin C; hydrogen peroxide; oxaliplatin; irinotecan; topotecan; leucovorin; carmustine; streptozocin; CPT-11; taxol and derivatives thereof; tamoxifen; dacarbazine; rituximab; daunorubicin; Ι-β-D-arabinofuranosylcytosine; imatinib; fludarabine; docetaxel; and FOLFOX4

30. A method for determining the efficacy of a cancer therapy in a subject, comprising administering at least one therapeutic agent to the subject and subsequently measuring the expression of miR-223 in a sample from the subject.

31. The method of claim 30, wherein the subject has acute myelogenous leukemia.

32. The method of claim 30, wherein the subject has chronic lymphocytic leukemia.

33. The method of claim 30, wherein the sample is a blood sample.

34. The method of claim 30, wherein the sample is a plasma sample.

35. The method of claim 30, wherein the sample is a bone marrow sample.

36. The method of claim 30, wherein a decrease in the expression of miR-223 relative to a suitable control is indicative of successful treatment.

37. A method of any one of the preceding claims, wherein the agent that decreases miR- 223 expression in a cell comprises a vector comprising a nucleic acid sequence that is at least 90% identical to the antagomir of miR-223.

38. A method of any one of the preceding claims, wherein the nucleic acid is at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and all the intermediate percentages between 90% and 100%, identical to the antagomir of miR-223.

39. A method of any one of the preceding claims, wherein the agent decreases the level of miR-233 in hematopoietic progenitor cells.

40. A method of any one of the preceding claims, wherein the antisense oligonucleotide comprises at least one chemical modification.

41. A method of any one of the preceding claims, wherein the antisense oligonucleotide comprises a 2'-0-methyl oligoribonucleotide.

42. A method of any one of the preceding claims, wherein the antisense oligonucleotide comprises a sequence that is complementary to a mature miR-223 sequence.

43. A method of any one of the preceding claims, wherein the antisense oligonucleotide comprises a sequence that is complementary to miR-223.

44. A method of any one of the preceding claims, wherein the antisense oligonucleotide is encoded by an expression vector, and wherein the antisense oligonucleotide is under the transcriptional control of a promoter.

45. A method of the preceding claim, wherein the promoter is a macrophage-specific promoter.

46. A method of any one of the preceding claims, wherein the agent includes a virus or a non-virus vector.

47. A method of any one of the preceding claims, wherein administering comprises oral, transdermal, sustained release, controlled release, delayed release, suppository, sublingual, intravenous or direct injection administration of the antisense oligonucleotide.

48. A method of any one of the preceding claims, carried out in conjunction with at least one additional therapy modality.

49. A method of the preceding claim, wherein the additional therapy modality is selected from the group consisting of bone marrow transplant, cord blood cell transplant, surgery, chemotherapy, radiation therapy, immunotherapy and a combination thereof.

50. A method of any one of the preceding claims, wherein the additional therapy modality is administered at the same time as the antisense oligonucleotide.

51. A method of any one of the preceding claims, wherein the additional therapy modality is administered either before or after the antisense oligonucleotide.

52. A method of any one of the preceding claims, wherein treating comprises improving one or more symptoms of the leukemia.

53. A method of any one of the preceding claims, wherein progression of leukemia is inhibited in the subject following administration of the antisense oligonucleotide.

54. Use of an agent of any one of the preceding claims for the preparation of a medicament for treating cancer in a human subject.

55. Use of an agent of any one of the preceding claims for the preparation of a medicament for inhibiting leukemic progression in a human subject.

56. Use of an agent of any one of the preceding claims for the preparation of a medicament for inhibiting metastasis in a human subject.

57. Use of an agent of any one of the preceding claims for the preparation of a medicament for reducing or alleviating a symptom associated with a neoplastic disorder.

58. A method of any one of the preceding claims, wherein treating comprises delaying the transition from a preleukemic condition to leukemia.

59. A pharmaceutical composition for treating leukemia comprising an inhibitor of miR- 223, wherein the inhibitor of miR-223 comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, or its complement; an antagomir of miR-223, an anti-miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR-223, or a double stranded RNA that complementary base-pairs with miR-223.

60. A pharmaceutical composition of the preceding claim, further comprising at least one anti-cancer agent.

61. The pharmaceutical composition of claim 27, wherein the inhibitor of miR-223 is an antagomir of miR-223, an anti-miR-223 oligonucleotide, an antisense oligonucleotide to miR-223, a locked nucleic acid that anneals to miR-223, or a double stranded RNA that complementary base- pairs with miR-223.

62. A pharmaceutical composition comprising an antagonist of miR-223 and a pharmaceutically acceptable carrier, excipient or diluent.

63. A pharmaceutical composition according to any one of the preceding claims, wherein the antagonist of miRNA-223 comprises a sequence that is complementary to the mature sequence of miRNA-223.

64. A pharmaceutical composition comprising a miR-223 molecule or an antagomir thereof or a variant thereof for use in the positive or negative modulation of macrophage-derived microvesicles, whereby the use in the modulation relates to the treatment of a leukemic disease.

65. A kit comprising i) one or more dosage units of the pharmaceutical compositions of any one of the preceding claims; and ii) instructions for administering the nucleic acid construct to a subject in need thereof.

66. A kit containing i) one or more dosage units of a nucleic acid construct sufficient for one or more courses of treatment for a cell or micro vesicle expressing miR-223; and ii) instructions for administering the nucleic acid construct to a subject in need thereof.