[go: up one dir, main page]

WO2012080461A1 - Miarn dans une maladie articulaire - Google Patents

Miarn dans une maladie articulaire Download PDF

Info

Publication number
WO2012080461A1
WO2012080461A1 PCT/EP2011/073036 EP2011073036W WO2012080461A1 WO 2012080461 A1 WO2012080461 A1 WO 2012080461A1 EP 2011073036 W EP2011073036 W EP 2011073036W WO 2012080461 A1 WO2012080461 A1 WO 2012080461A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
disease
sample
level
individual
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2011/073036
Other languages
English (en)
Inventor
Kathrin Heermeier
Johannes Christof Schneider
Thomas Leeuw
Christiane Metz-Weidmann
Joachim SAAS
Jochen Beninga
Uwe Dietz
Tilo Weiss
Eckart Bartnik
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi SA
Original Assignee
Sanofi SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanofi SA filed Critical Sanofi SA
Publication of WO2012080461A1 publication Critical patent/WO2012080461A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • miRNAs in Joint Disease Field of the Invention concerns microRNAs (miRNA), in particular mir-199a, as an indicator of a tissue status or a disease such as osteoarthritis (OA) and as a target molecule for the discovery of a substance for the treatment of a joint disease. Further provided are methods and compounds for investigation, analysis and/or treatment of a joint disease, especially OA.
  • RA Rheumatoid arthritis
  • AS ankylosing spondylitis
  • Juvenile idiopathic arthritis is characterised in an inflammation of the synovium of the joint and effects children of an age below 16 years. Gout is characterised by recurring attacks of acute inflammatory arthritis which are caused by elevated levels of uric acid in the blood. Septic arthritis is caused by an infection of the joint and psoriatic arthritis may develop in patients suffering from psoriasis.
  • Osteoarthritis is characterised by mechanical abnormalities of the joint such as the degradation of the joints articular cartilage and subchondral bone.
  • OA is one of the most common diseases of the western world with more than 21 million diagnosed symptomatic cases in the USA, Japan, and Germany in 2000. Due to the aging of the population, an ever-increasing population of OA patients has to be faced, whose quality of life is severely affected. In addition, OA carries a tremendous socio-economic burden with direct and indirect costs.
  • the current treatment modalities concentrate on the management of the pain associated with OA, since pharmacological treatment modalities able to slow, stop or even reverse the course of OA and its effects, are long since sought for but still missing.
  • OA can be viewed as the clinical and pathological outcome of a range of disorders that result in structural and functional failure of synovial joints. It occurs when the dynamic equilibrium between the breakdown and the repair of joint tissues is overwhelmed. Structural failure of articular cartilage can result from abnormal mechanical strains injuring healthy cartilage, as well as from failure of pathologically impaired cartilage degenerating under the influence of physiological and mechanical strains. OA is characterised by a progressive degeneration of the articular cartilage, ultimately leading to a functional inability of the affected joint. During the chronical progression of OA the articular cartilage is destroyed setting free the underlying bone tissue, ultimately necessitating the surgical replacement of the affected joint.
  • Morphological changes observed in OA include cartilage erosion as well as a variable degree of synovial inflammation. These changes are attributed to a complex network of biochemical factors, including proteolytic enzymes that lead to a breakdown of the cartilage macromolecules, in particular collagen II, X and aggrecan. Cytokines such as IL-1 and TNF-a which are produced by activated synoviocytes, mononuclear cells or by articular cartilage itself, significantly up regulate matrix-metalloproteinases (MMPs) and cytokine gene expression, and blunt compensatory synthesis pathways.
  • MMPs matrix-metalloproteinases
  • Diagnosis of OA is commonly based on clinical examination of the joints in patients showing a gradual onset of symptoms and signs, with x-rays confirming the diagnosis. Typical changes observable via x-ray include a narrowing of the joint space, marginal osteophytes, subchondral sclerosis, subchondral cyst formation, bony remodelling, and joint effusions. Laboratory tests for the diagnosis of OA are not available as the markers routinely used remain normal in OA. These tests are only useful in order to differentiate OA from others forms of arthritis, especially inflammatory types of arthritis. The diagnosis of OA is thus, only possible at a stage where the degradation of the joint already progressed far enough to be visible on an x-ray. Early onsets of the disease are not detectable as of yet, preventing a timely treatment that could avoid the destruction of the joint in the first place. Improved tools allowing for an identification of OA at an early stage are thus, still desired.
  • DMOAD non-steroidal anti-inflammatory drugs
  • the object of the present invention to provide improved methods for the identification of the disease as well as of substances and compositions suitable for an improved treatment.
  • This is solved by the different aspects of present invention in providing methods and substances on the basis of miRNAs that are differentially expressed during the differentiation process of chondrocytes and cultured bone marrow stem cells (BMCSs).
  • BMCSs bone marrow stem cells
  • the present invention is based on the surprising finding of the inventors that specific inhibition of mir-199a in chondrocytes increased chondrogenic markers whilst the overexpression of mir-199a in chondrocytes lead to an decrease of chondrogenic markers.
  • the invention provides in a first aspect a mir-199a for use as an indicator of a tissue status or a disease and in a second aspect, the use of mir-199a as an indicator of a tissue status or a disease.
  • the invention provides a method of identifying an alteration of a tissue status or a risk for developing and/or identifying the presence and/or monitoring progression of a disease, preferably a joint disease, in an individual, comprising detecting the level of mir-199a in a sample.
  • the present invention provides a method for determining the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease, preferably a joint disease, in an individual, comprising the steps of (a) determining the level of mir-199a in a sample of the individual, and (b) determining the dosage of a pharmaceutical depending on the level of mir-199a in the tested sample.
  • the present invention provides a method for adapting the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a joint disease, comprising the steps of (a) determining the level of mir-199a in a sample,
  • the present invention provides a method of determining the beneficial and/or adverse effects of a substance on a tissue status or the development of a joint disease, comprising the steps of (a) determining the level of mir-199a in a tested sample, (b) determining the level of mir-199a in one or more reference samples, and
  • the present invention provides the use of mir-199a in a method according to any of the aspects three to six.
  • the present invention provides a kit for use in a method according to any of the aspects three to six., comprising one or more means of detecting mir- 199a.
  • the present invention provides for the use of the kit of the eighth aspect in a method according to any of the aspects three to six.
  • the present invention provides one or more nucleic acids for detecting a mir-199a gene, gene product or functionally active variant thereof in a method according to any of the aspects three to six.
  • the present invention provides a peptide, polypeptide or protein for detecting a mir-199a gene, gene product or functionally active variant thereof in a method according to any of the aspects three to six.
  • the present invention provides for the use of a nucleic acid according to aspect ten, or of a peptide, polypeptide or protein according to aspect eleven in a method according to any of the aspects three to six.
  • the present invention provides a method of screening for a mir- 199a agonist, wherein the method comprises the steps of (a) providing mir-199a or the mir-199a gene, (b) providing a test compound, and (c) measuring or detecting the influence of the test compound on mir-199a or the mir-199a gene.
  • the present invention provides a mir-199a agonist for the alteration of a tissue status or the prevention or treatment of a joint disease.
  • the present invention provides a pharmaceutical comprising the mir-199a agonist according to the fourteenth aspect.
  • the present invention provides a method of alteration of a tissue status or prevention or treatment of a joint disease, wherein a therapeutically effective amount of the pharmaceutical according to aspect fifteen is administered to an individual at risk of developing or suffering from a joint disease.
  • Fig.1 a mir-199a expression in chondrocyte pellets during the cultivation period
  • Fig.1 b expression of mir-22, mir-140, mir-146a and mir-199a in chondrocyte pellets during the cultivation period
  • Fig.2 mir-199a expression in mesenchymal stem cell pellets during the cultivation period
  • Fig.3 Effects of mir-199a overexpression on the expression levels of two marker genes and two marker proteins in three different experiments: collagen II (A); aggrecan (B); ALP (C); MMP-1 (D); Fig.4: Effects of mir-199a knock down on the expression levels of two marker genes and 1 marker protein in three different experiments: collagen II (A); aggrecan (B); ALP (C)
  • Fig.5 Relative changes in the expression of the two marker genes collagen II and aggrecan in response to mir-199a overexpression or knock-down
  • the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (lUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • nucleic acid molecules are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a
  • nucleobase a five-carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups.
  • a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers.
  • nucleic acid molecules include but are not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • polynucleotide and “nucleic acid” are used
  • nucleic acids can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. &
  • Nucleic acids may be degraded by endonucleases or exonucleases, in particular by DNases and RNases which can be found in the cell. It is, therefore, advantageous to modify the nucleic acids in order to stabilize them against degradation, thereby ensuring that a high concentration of the nucleic acid is maintained in the cell over a long period of time (Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94;
  • Such stabilization can be obtained by introducing one or more internucleotide phosphorus groups or by introducing one or more non-phosphorus internucieotides.
  • Modified internucleotide phosphate radicals and/or non- phosphorus bridges in a nucleic acid which can be employed in one of the uses according to the invention contain, for example, methyl phosphonate,
  • non-phosphorus internucleotide analogues contain, for example, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamidate bridges and/or thioether bridges. It is also the intention that this modification should improve the durability of a pharmaceutical composition which can be employed in one of the uses according to the invention.
  • Nucleic acids may be selected from the group consisting of a polynucleotide probe, a primer(s) (e.g. a primer pair), preferably a primer(s) for polymerase chain reaction (PCR), reverse transcription (RT) reaction, or DNA sequencing, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), a microRNA (miRNA), and a small interfering RNA (siRNA).
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • miRNA microRNA
  • siRNA small interfering RNA
  • a probe hybridizes to single-stranded nucleic acid (DNA or RNA) whose nucleotide sequence allows for nucleotide pairing due to complementarity between the probe and the target sequence.
  • the length of a probe depends on the intended use as well as the required specificity of the probe.
  • a probe is 20-500 (i.e. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500) nucleotides long, preferably 20- 100 nucleotides, more preferably 20-50.
  • For detection of microRNA probes are between 12 and 30 nucleotides. Probes are used in various experimental set ups such as but not limited to Southern and Northern Blots, for real-time PCR and In situ
  • a probe may be unlabeled, directly labelled, or indirectly labelled, such as with biotin to which a streptavidin complex may later bind.
  • Said label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • suitable labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which are or can be made detectable.
  • a label may be incorporated into nucleic acids at any position, e.g. at the 3' end, at the 5' end or internally.
  • probe also encompasses nucleic acids differing in the composition of their backbone such as but not limited to peptide nucleic acids (PNAs), locked nucleic acids (LNAs), glycol nucleic acids (GNAs) and threose nucleic acids (TNAs).
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • GNAs glycol nucleic acids
  • TAAs threose nucleic acids
  • primer refers to a single-strand oligonucleotide which typically serves as a starting point for DNA-replicating enzymes.
  • a primer binds to or hybridises with a DNA template and typically comprises a sequence being
  • a primer may also comprise additional sequences e.g. sequences serving as nuclease cleavage sites (e.g. Bam H1 , Hind III, etc.).
  • the length of a primer is chosen depending on the intended use. For instance, primers used for the amplification of DNA in Polymerase- Chain Reactions (PCR) typically have a length of at least 10 nucleotides, preferably between 10 to 50 (i.e. 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, 45, 50) nucleotides, more preferably between 15 and 30 nucleotides.
  • primers Shorter primers of at least 5 nucleotides are used for sequencing of DNA templates. Also encompassed in the term “primer” are “degenerate primers” which are a mixture of similar, but not identical primers. A primer may be tagged or labelled with a marker molecule detectable by spectroscopic, photochemical,
  • miRNA and variations such as “miRNA” and “miR” include human miRNAs, mature single stranded miRNAs, precursor miRNAs (pre-miR), and variants thereof, which may be naturally occurring.
  • miRNA also includes primary miRNA transcripts and duplex miRNAs.
  • the name of a specific miRNA refers to the mature miRNA.
  • mir-199a refers to a mature miRNA sequence derived from pre- mir-199a. Sequences for miRNAs, including human mature and precursor sequences (also called stem-loop sequences), are reported in the following database:
  • This miRNA sequence is predicted based on homology to a verified miRNA from mouse ["Identi fication of novel genes coding for small expressed RNAs" Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T Science. 294:853-858(2001 ), "Reduced accumulation of specific microRNAs in colorectal neoplasia” Michael MZ, O' Connor SM, van Hoist Pellekaan NG, Young GP, James RJ Mol Cancer Res. 1 :882-891 (2003)]. Its
  • Landgraf P Rusu M, Sheridan R, Sewer A, lovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foa R, Schliwka J, Fuchs U, Novosel A, Muller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M Cell. 129: 1401 -1414(2007).].
  • MicroRNAs are endogenous, small RNA molecules that are implied in the regulation of gene expression at the translation level. Moreover, they are part of the cell's RNA interference (RNAi) mechanism. MiRNAs are encoded by genes transcribed from DNA but are not translated into protein (non-protein-coding RNA). They have been first discovered in caenorhabditis elegans and exist in both plants and animals. The sequences of many miRNAs are highly conserved among different species, suggesting that the miRNA pathway is part of a relatively old and important regulatory mechanism (Grosshans et al. J Cell Biol 156: 17-21 (2002)).
  • MiRNAs have been found to regulate about 30% of mammalian genes (Czech, NEJM 354: 1 194-1 195 (2006); Mack, Nature Biotech. 25:631 -638 (2007); Eulalio, et al., Cell 132:9-14 (2008)).
  • miRNAs repress protein production by blocking translation or causing transcript degradation, thereby regulating gene expression.
  • a single miRNA may target 250-500 different mRNAs, proving this class of RNA to be an extremely important mediator of a wide range of cellular functions.
  • Many of the genes regulated by miRNAs are disease-causing genes. Thus, any miRNA-modulating functionality has great therapeutic potential.
  • miRNAs are first expressed from the genome as RNA transcripts called primary miRNAs (pri-miRNAs). They are transcribed by RNA Polymerase II, and form hairpin structures. In the nucleus, the dsRNA-specific ribonuclease Drosha processes the pri-miRNAs into shorter, about 70- to 100-nucleotide long stem-loop structures known as pre-miRNAs, which are then exported out into the cytoplasm, likely by
  • RNA-induced silencing complex RISC
  • siRNAs small or short interfering RNAs
  • the mature miRNA is integrated into the RISC complex, and binds to a messenger RNA (mRNA) molecule that has a significantly, though often not perfectly, complementary sequence.
  • mRNA messenger RNA
  • the passenger strand, miRNA * may be degraded.
  • Translation of the mRNA bound by the miRNA-RISC complex is then repressed, resulting in reduced expression of the corresponding gene.
  • the bound mRNA is cleaved or deadenylated and degraded.
  • a single precursor contains more than one mature miRNA sequence. In other instances, multiple precursor miRNAs contain the same mature sequence.
  • Proteins usable in the present invention can be further modified by chemical modification.
  • chemical modification comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids.
  • Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility.
  • Chemical modifications applicable to the variants usable in the present invention include without limitation: PEGylation, glycosylation of non-glycosylated parent polypeptides, or the modification of the glycosylation pattern present in the parent polypeptide.
  • variant is to be understood as a polynucleotide or protein which differs in comparison to the polynucleotide or protein from which it is derived by one or more changes in its length or sequence.
  • polypeptide or polynucleotide from which a protein or nucleic acid variant is derived is also known as the parent polypeptide or polynucleotide.
  • variant comprises "fragments" or
  • “derivatives” of the parent molecule typically, “fragments” are smaller in length or size than the parent molecule, whilst “derivatives” exhibit one or more differences in their sequence in comparison to the parent molecule.
  • modified molecules such as but not limited to post-translationally modified proteins (e.g.
  • glycosylated biotinylated, phosphorylated, ubiquitinated, palmitoylated, or
  • variants are constructed artificially, preferably by gene-technological means whilst the parent polypeptide or
  • polynucleotide is a wild-type protein or polynucleotide.
  • variants also naturally occurring variants are to be understood to be encompassed by the term "variant" as used herein.
  • the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent molecule, i.e. is functionally active.
  • a "variant" as used herein can be characterised by a certain degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 80% sequence identity to its parent polypeptide.
  • polynucleotide variant in the context of the present invention exhibits at least 80% sequence identity to its parent polynucleotide.
  • sequence identity is used throughout the specification with regard to polypeptide and
  • This expression preferably refers to a
  • sequence identity can be determined via sequence alignments.
  • sequence alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g.
  • sequence matching may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX).
  • BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410.
  • Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.
  • Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 : 154-162) or Markov random fields.
  • Shuffle-LAGAN Brudno M., Bioinformatics 2003b, 19 Suppl 1 : 154-162
  • Markov random fields Markov random fields.
  • Hybridization can also be used as a measure of sequence identity or homology between two nucleic acid sequences.
  • a nucleic acid sequence encoding miRNAs or a portion thereof, can be used as a "hybridization probe” according to standard
  • hybridization techniques The hybridization of a mir-199a probe to DNA or RNA from a test source is an indication of the presence of the mir-199a DNA or RNA in the test source.
  • Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1 -6.3.6, 1991 .
  • “Moderate hybridization conditions” are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in 1X SSC, 0.1 % SDS at 50°C.
  • Highly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
  • tissue refers to an ensemble of cells of the same origin which fulfil a specific function concertedly.
  • a tissue include but are not limited to bone, cartilage, connective tissue, muscle tissue, nervous tissue, and epithelial tissue. Multiple tissues together form an "organ” to carry out a specific function.
  • organ include but are not limited to joint, skeleton, muscle, blood, brain, heart, liver, kidney, stomach, and skin.
  • a joint is formed of many different tissues such as but not limited to bone, cartilage, synovium, muscle, ligament, and tendon.
  • joint refers to a location where two or more bones make contact.
  • the term may refer to a movable joint or an unmovable joint.
  • a joint may be a fibrous joint wherein the bones are attached by dense connective tissue rich in collagen or a cartilaginous joint wherein the bones are connected by cartilage.
  • a joint may also be a synovial joint wherein the bones are not directly connected but have a synovial cavity and are attached by a capsule of articular cartilage which is associated with accessory ligaments and tendons.
  • Synovial joints include but are not limited to ball-and-socket-joints, such as the hip or shoulder joints; ellipsoid joints and flat joints, such as the joints of the wrist; hinge joints, such as elbows, knees, ankles or the joints of fingers and toes; saddle joints such as the joint of the thumb; and pivot joint, such as the joint of atlas and axis.
  • tissue status means whether tissue fails to fulfil its function due to an illness or injury.
  • status of a tissue may be characterised by a specific morphology of such tissue or may be characterised by the expression of one or more specific molecules such as but not limited to peptides, proteins, and nucleic acids, or combinations thereof.
  • the status of a tissue may be regarded as "healthy” or “normal” in case it resembles the condition of such tissue when being free from illness or injury and efficiently fulfilling its specific function.
  • the status of a tissue may be regarded as "degenerative”, “diseased” or “abnormal” in case such tissue fails to fulfil its function due to an illness or injury. Additionally or
  • the status of a tissue may be regarded as "degenerative”, “diseased” or “abnormal” in case the morphology of the tissue or its molecule expression pattern is "altered” or “changed” in comparison to normal tissue.
  • the morphology of a tissue or the expression pattern of specific molecules in a tissue may be an indicator for the state of a tissue.
  • a tissues status include but are not limited to tissue degradation such as cartilage degradation, bone degradation, and degradation of the synovium, tissue inflammation such as cartilage inflammation, or inflammation of the synovium, tissue remodelling such as bone remodelling or cartilage remodelling, sclerosis, liquid accumulation, or proliferative tissue such as proliferation in wound healing processes, cyst formations, or in cancer.
  • disease and “disorder” are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a tissue, an organ or an individual is not able to efficiently fulfil its function anymore.
  • a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a tissue, an organ or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease.
  • a progression of a disease is typically characterised by an increase or decrease of such symptoms or signs which may indicate a "worsening" or “bettering” of the disease.
  • tissue, an organ or an individual being at "risk of developing" a disease is in a healthy state but shows potential of a disease emerging.
  • risk of developing is typically in a healthy state but shows potential of a disease emerging.
  • a disease is associated with early or weak signs or symptoms of such disease. In such case, the onset of the disease may still be prevented by treatment.
  • a disease include but are not limited to traumatic diseases, inflammatory diseases, infectious diseases, cutaneous conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, and various types of cancer.
  • joint disease refers to the abnormal condition of the joints, in particular those due to injurious, traumatic, degenerative, inflammatory, infectious or autoimmune causes.
  • Joint diseases include but are not limited to arthritis, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), juvenile idiopathic arthritis (JIA), gout, septic arthritis, psoriatic arthritis, and osteoarthritis (OA), cancer, such as chondrosarcoma, osteosarcoma, fibrosarcoma, and multiple myeloma, tendinitis, bursitis, fractures, and damage to cartilage or bone.
  • RA rheumatoid arthritis
  • AS ankylosing spondylitis
  • JIA juvenile idiopathic arthritis
  • OA osteoarthritis
  • cancer such as chondrosarcoma, osteosarcoma, fibrosarcoma, and multiple myeloma, tendinitis, bursitis
  • Symptoms of a disease are implication of the disease noticeable by the tissue, organ or organism having such disease and include but are not limited to pain, weakness, tenderness, strain, stiffness, and spasm of the tissue, an organ or an individual.
  • Signals or “signals” of a disease include but are not limited to the change or alteration such as the presence, absence, increase or elevation, decrease or decline, of specific indicators such as biomarkers or molecular markers, or the development, presence, or worsening of symptoms.
  • indicator refers to a sign or signal for a condition or is used to monitor a condition.
  • a condition refers to the biological status of a cell, tissue or organ or to the health and/or disease status of an individual.
  • An indicator may be the presence or absence of a molecule, including but not limited to peptide, protein, and nucleic acid, or may be a change in the expression level or pattern of such molecule in a cell, or tissue, organ or individual.
  • An indicator may be a sign for the onset,
  • indicators relating to arthritic diseases such as OA include but are not limited to transcription factors such as Sox-5, Sox-6, Sox-9, Nfatl , pitxl , FoxO, HIF2A, SAF-1 , RUNX-2, cytokines such as IL-1 ⁇ , IL-2, IL-7, IL-12, IL-18, GM-CFS, TNF-a, NF-KB, and INF- ⁇ , phosphatases such as Alkaline Phosphatase (ALP), proteases such as metalloproteases (e.g.
  • ALP Alkaline Phosphatase
  • proteases such as metalloproteases (e.g.
  • one or more of these indicators are expressed or activated to a higher or lower level than in a tissue, organ, or individual not suffering from arthritis. For instance, the levels of the extracellular matrix components collagen II and X, as well as
  • aggrecan and CD-RAP are lowered in a tissue, organ, or individual suffering or at risk of developing OA in comparison a tissue, organ, or individual not suffering or at risk of developing OA. Further, the level of the transcription factor Sox9 as well as the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-4 are decreased in a tissue, organ, or individual suffering or at risk of developing OA in comparison a tissue, organ, or individual not suffering or at risk of developing OA.
  • the levels of the cytokine IL-1 , IL-6 and TNF-a as well as of the metalloproteases MMP-1 , MMP-2, MMP-3 and MMP-9, and phosphatase ALP are enhanced in a tissue, organ, or individual suffering or at risk of developing OA in comparison a tissue, organ, or individual not suffering or at risk of developing OA.
  • preferred indicators of a tissue status or a disease are miRNAs, including but not limited to mir-21 , mir-22, mir-140, mir-146a, and mir-199a.
  • Mir-199a is particularly preferred as an indicator of diseases such as arthritis, in particular OA, in an individual.
  • an "individual” means any mammal, reptile or bird that may benefit from the present invention.
  • an individual is selected from the group consisting of laboratory animals (e.g. mouse, rat or rabbit), domestic animals (including e.g. guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, duck, camel, cat, dog, turtle, tortoise, snake, or lizard), or primates including chimpanzees, bonobos, gorillas and human beings. It is particularly preferred that the "individual” is a human being.
  • sample or “sample of interest” are used interchangeably herein, referring to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual.
  • samples include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, perichondrium, capsule, and connective tissue.
  • samples are cell cultures or tissue cultures such as but not limited to cultures of cartilage, bone, synovium, bone marrow cells, chondrocytes, chondroblasts, chondroclasts, synovial cells, osteocytes, osteoclasts, osteoblasts stem cells and/or mesenchymal stem cells.
  • Analysis of a sample may be accomplished on a visual or chemical basis. Visual analysis includes but is not limited to microscopic imaging or radiographic scanning of a tissue, organ or individual allowing for morphological evaluation of a sample.
  • Chemical analysis includes but is not limited to the detection of the presence or absence of specific indicators or alterations in their amount or level.
  • reference sample refers to a sample which is analysed in a substantially identical manner as the sample of interest and whose information is compared to that of the sample of interest. A reference sample thereby provides a standard allowing for the evaluation of the information obtained from the sample of interest.
  • a reference sample may be derived from a healthy or normal tissue, organ or individual, thereby providing a standard of a healthy status of a tissue, organ or individual. Differences between the status of the normal reference sample and the status of the sample of interest may be indicative of the risk of disease development or the presence or further progression of such disease or disorder.
  • a reference sample may be derived from an abnormal or diseased tissue, organ or individual thereby providing a standard of a diseased status of a tissue, organ or individual. Differences between the status of the abnormal reference sample and the status of the sample of interest may be indicative of a lowered risk of disease development or the absence or bettering of such disease or disorder.
  • a reference sample may also be derived from the same tissue, organ, or individual as the sample of interest but has been taken at an earlier time point. Differences between the status of the earlier taken reference sample and the status of the sample of interest may be indicative of the progression of the disease, i.e. a bettering or worsening of the disease over time.
  • a reference sample was taken at an earlier or later time point in case a period of time has lapsed between taking of the reference sample and taking of the sample of interest. Such period of time may represent years (e.g. 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 years), months (1 , 2, 3, 4, 5, 6, 7, 8,
  • lowered or “decreased” level of an indicator refer to the level of such indicator in the sample being reduced in comparison to the reference or reference sample.
  • elevated or “increased” level of an indicator refer to the level of such indicator in the sample being higher in comparison to the reference or reference sample.
  • a miRNA that is detectable in higher amounts in synovial fluid of one individual suffering from OA than in synovial fluid of individuals not suffering from OA has an elevated level.
  • an elevated level in a sample indicates the presence of OA or increased susceptibility or increased probability to develop OA.
  • a reference sample may be "treated differently” or “exposed differently” than a sample of interest in case both samples are treated in a substantially identical way except from a single factor.
  • single factors include but are not limited to the time of exposure, the concentration of exposure, or the temperature of exposure to a certain substance.
  • a sample of interest may be exposed to a different dosage of a certain substance than the reference sample or may be exposed for a different time interval than the reference sample or may be exposed at a different temperature than the reference sample.
  • Different dosages to which a sample of interest may be exposed to include but are not limited to the 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold and/or 1000-fold increased or decreased dosage of the dosage the reference sample is exposed to.
  • Different exposure times to which a sample of interest may be exposed to include but are not limited to the 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 100-fold and/or 1000-fold longer or shorter time period than the exposure of the reference.
  • Different temperatures of exposure to which a sample of interest may be exposed to include but are not limited to the 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold and/or 1000-fold increased or decreased temperature than the exposure of the reference.
  • a sample of interest may be exposed to a 10-fold increased concentration of a substance than the reference sample. The analysis of both samples is then conducted in a substantially identical manner allowing determining the effects, i.e.
  • agonist refers to a substance that causes an action in a tissue, organ or individual such as receptor-signalling, gene expression, protein synthesis, and protein degradation. Typically, agonists act by binding to the active site or to allosteric sites of a receptor molecule thereby, triggering a specific reaction.
  • agonists include but are not limited to nucleic acid molecules, such as mRNA or miRNA, or proteins, such as hormones, cytokines, growth factors,
  • antagonist refers to a substance blocking the action of an agonist.
  • antagonists act by binding to the active site or to allosteric sites of a receptor molecule, or interact with unique binding sites not normally involved in the regulation of the activity of the receptor.
  • an antagonist competes with the agonist at structurally-defined binding sites or alters the binding site of the agonist in a manner that the agonist is not able to cause the action it would normally cause due to its binding.
  • the antagonist activity may be reversible or irreversible depending on the longevity of the interaction of the antagonist-receptor complex. Examples for antagonists include but are not limited to nucleic acid molecules, such as siRNAs or miRNAs, or proteins such as hormones, cytokines, growth factors or neurotransmitter, antibodies, or transcription factors.
  • a mimic refers to a substance that imitates the action of a target molecule (e.g. a target molecule such as an agonist or an antagonist in a tissue, organ or individual) such as receptor-signalling, gene expression, protein synthesis, and protein degradation. Accordingly, a mimic may be structurally different from the target molecule (e.g. agonist or antagonist) it imitates but may still effect the same specific reaction, e.g. by binding to the active site or to allosteric sites of a receptor molecule (i.e. by exerting at least one of the activities of the agonist or antagonist). Typically, a mimic fulfils the same function as the respective agonist or antagonist, i.e. is functionally equivalent. For example, a mimic of mir-199a is a substance that exerts at least one of the activities of mir-199a, has the function of mir-199a and/or imitates the action of mir-199a.
  • a target molecule e.g. a target molecule such as an agonist
  • receptor refers to a molecule such as a protein or polynucleotide, to which one or more specific signalling molecules bind.
  • Signalling molecules may act as agonist or antagonist including without limitation nucleic acid molecules, such as siRNAs or miRNAs, or proteins such as hormones, cytokines, growth factors or neurotransmitter, antibodies or transcription factors.
  • Receptors may be localised at the plasma membrane of a cell, within the cytoplasm and/or in intracellular compartments.
  • treat means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in an individual that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in individuals that were previously symptomatic for the disorder(s).
  • prevent means preventing that such disease or disorder occurs in patient.
  • “Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • active ingredient refers to the substance in a pharmaceutical composition or formulation that is biologically active, i.e. that provides pharmaceutical value.
  • a pharmaceutical composition may comprise one or more active ingredients which may act in conjunction with or independently of each other.
  • the active ingredient 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 but not limited to those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • composition intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
  • carrier refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, or vehicle with which the therapeutically active ingredient is administered.
  • Such pharmaceutical carrier can be liquid or solid.
  • Liquid carrier include but are not limited to sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • a saline solution is a preferred carrier when the pharmaceutical composition is administered intracavitary.
  • Solid form preparations are suitable for oral administration and include powders, tablets, pills, capsules, lozenges, cachets, suppositories, and dispersible granules.
  • a solid excipient can be one or more substances, which may also act as diluents, flavouring agents, binders, preservatives, tablet disintegrating agents, or an
  • the excipient is preferably a finely divided solid, which is in a mixture with the finely divided active ingredient.
  • the active ingredient of the present invention is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from 5% to 80%, more preferably from 20% to 70% of the active compound.
  • Suitable pharmaceutical "excipients” include starch, glucose, lactose, sucrose, gelatine, pectin, dextrin, malt, rice, flour, chalk, silica gel, magnesium carbonate, sodium stearate, magnesium stearate, glycerol monostearate, talc, sodium chloride,
  • tragacanth methylcellulose, sodium carboxymethylcellulose, dried skim milk, glycerol, propylene, glycol, water, ethanol a low melting wax, cocoa butter, and the like.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
  • adjuvant refers to agents that augment, stimulate, activate, potentiate, or modulate the immune response to the active ingredient of the composition at either the cellular or humoral level, e.g. immunologic adjuvants stimulate the response of the immune system to the actual antigen, but have no immunological effect themselves.
  • adjuvants include but are not limited to inorganic adjuvants (e.g. inorganic metal salts such as aluminium phosphate or aluminium hydroxide), organic adjuvants (e.g. saponins or squalene), oil-based adjuvants (e.g. Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g. IL-1 ⁇ , IL-2, IL-7, IL-12, IL-
  • inorganic adjuvants e.g. inorganic metal salts such as aluminium phosphate or aluminium hydroxide
  • organic adjuvants e.g. saponins or squalene
  • oil-based adjuvants e.g. Freund's complete adjuvant and Freund's incomplete adjuvant
  • cytokines e.g. IL-1 ⁇ , IL-2, IL-7, IL-12, IL-
  • particulate adjuvants e.g. immuno-stimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g. monophosphoryl lipid A, or muramyl peptides), synthetic adjuvants (e.g. non-ionic block copolymers, muramyl peptide analogues, or synthetic lipid A), or synthetic polynucleotides adjuvants (e.g polyarginine or polylysine).
  • administering includes in vivo administration, as well as
  • tissue ex vivo such as vein grafts.
  • oral, topical, transdermal and/or intracavitary (e.g. intravenous) administration is preferred.
  • a “therapeutically effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose.
  • the effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration.
  • the effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.
  • the present invention provides mir-199a for use as an indicator of a tissue status or a disease.
  • mir-199a for use as an indicator of a tissue status or a disease.
  • mir-199a is indicative of a tissue status or a disease.
  • the presence, absence or altered amounts of mir-199a are a sign for the presence, absence or progression of a tissue status or disease.
  • an altered tissue status is preferably characterised by the tissue exhibiting different mir-199a level than normal tissue or the same tissue at an earlier time point.
  • a disease status is preferably characterised by an organ or individual exhibiting different mir-199a level in comparison to a normal organ or individual or in comparison to the same organ or individual at an earlier time point.
  • the present invention relates to the use of mir-199a as an indicator of a tissue status or a disease. Due to the different expression levels of mir-199a during disease development and/or progression, mir-199a is used as an indicator of a tissue status or a disease.
  • the level of mir-199a is indicative of (a) a tissue status or a disease and/or (b) the risk of developing an altered tissue status or a disease and/or (c) the suffering of an individual from an altered tissue status or a disease and/or (d) the progression or a stage of a tissue status or a disease, in an individual.
  • the level of mir-199a is the expression level of mir-199a or the amount of mir-199a present.
  • the expression level of mir-199a and/or the amount of mir-199a present are indicative of (a) a tissue status or a disease and/or (b) the risk of developing an altered tissue status or a disease and/or (c) the suffering of an individual from an altered tissue status or a disease and/or (d) the progression or a stage of a tissue status or a disease, in an individual.
  • the level of mir-199a is the level of mir-199a in an individual or in a sample of an individual.
  • an alteration in the mir-199a level indicates a change in tissue status or disease such as the worsening or bettering of a tissue status or disease.
  • a lowered level of mir-199a is indicative of a worsening of a tissue status or disease.
  • An elevated level of mir-199a is indicative of a bettering of a tissue status or disease.
  • An alteration in the mir-199a level is also indicative of the risk of developing an altered tissue status or a disease, more preferably a lowered level of mir-199a is indicative of the risk of developing a degenerative tissue status or disease.
  • An altered mir-199a level preferably a lowered mir-199a level, is also indicative of an individual suffering from an altered tissue status or a disease.
  • an altered mir-199a level e.g. an elevated or lowered level of mir-199a, indicates the progression or a stage of a tissue status or a disease, in an individual.
  • a lowered mir-199a level is indicative of a worsening of a tissue status or disease.
  • the level of mir-199a is indicative of a tissue status wherein the tissue is selected from the group consisting of bone, cartilage, connective tissue, muscle tissue, nervous tissue, and epithelial tissue.
  • tissue status is tissue degradation such as but not limited to cartilage degradation, bone degradation, and degradation of the synovium, tissue inflammation such as cartilage inflammation, or inflammation of the synovium, tissue remodelling such as bone remodelling or cartilage remodelling, sclerosis, liquid accumulation, or proliferative tissue such as proliferation in wound healing processes, cyst formations, or in cancer.
  • tissue status relates to tissue degradation or tissue inflammation, more preferably to cartilage degradation, degradation of the synovium, cartilage inflammation and/or inflammation of the synovium.
  • the level of mir-199a is indicative of a disease wherein the disease is a joint disease such as but not limited to arthritis, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), juvenile idiopathic arthritis (JIA), gout, septic arthritis, psoriatic arthritis, and osteoarthritis (OA), cancer, such as chondrosarcoma, osteosarcoma, fibrosarcoma, and multiple myeloma, tendinitis, bursitis, fractures, and/or damage to cartilage or bone.
  • the disease is arthritis, more preferably osteoarthritis.
  • the present invention provides for a method of identifying (a) an alteration of a tissue status or the presence of a disease and/or (b) the risk of
  • mir-199a is detected on a RNA or DNA level, more preferably via known means such as but not limited to antibodies, primers or nucleic acid probes able to specifically bind and/or hybridise to mir-199a RNA or cDNA.
  • the level of mir-199a is the level of mir-199a in an individual or in a sample of an individual.
  • the level of mir-199a is the expression level of mir-199a or the amount of mir-199a present in an individual or in a sample of an individual.
  • the individual is a mammal, reptile or bird. More preferably, the individual is selected from the group consisting of laboratory animals (e.g. mouse, rat or rabbit), domestic animals (including e.g. guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, duck, camel, cat, dog, turtle, tortoise, snake, or lizard), or primates including macacae, chimpanzees, bonobos, gorillas and human beings. It is particularly preferred that the individual is a human being.
  • laboratory animals e.g. mouse, rat or rabbit
  • domestic animals including e.g. guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, duck, camel, cat, dog, turtle, tortoise, snake, or lizard
  • primates including macacae, chimpanzees, bonobos, gorillas and human beings. It is particularly preferred that the individual is a human being.
  • the sample of an individual is one or more solid samples such as but not limited to tissue extracts, cartilage, bone, synovium, perichondrium, capsule, skin, and
  • connective tissue or one or more fluid samples such as but not limited to blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid.
  • the sample is selected from the group consisting of cartilage, synovium, capsule, blood, plasma, serum, urine, and synovial fluid.
  • the sample is cartilage, synovium, serum, and/or synovial fluid.
  • the level of mir-199a is detected via any known method using known means for detection of nucleic acids such as specific nucleic acids or polypeptides able to binding the miRNA in question. Suitable methods include but are not limited to Polaymerase- Chain-Reaction (PCR) techniques such as reverse transcriptase PCR or quantitative real time PCR, hybridisation techniques such as Northern Blot, in situ hybridization, or Chip hybridisation, immunohistological or immunohistochemical or
  • PCR Polaymerase- Chain-Reaction
  • HPLC Chromatography
  • the means of detecting the level of mir-199a present in an individual or in a sample of an individual include but are not limited to those typically used in above mentioned methods. These include but are not limited to one or more sets of primers capable of specifically detecting mir-199a RNA or cDNA, (e.g. for use in a quantitative RT PCR), one or more nucleic acid probes able to specifically hybridise to mir-199a RNA or cDNA under standard conditions (e.g. for use in Northern Blot or Chip hybridisation techniques), one or more antibodies able to specifically detect mir-199a (e.g. for use in immunohistological or immunohistochemical or immunochemical techniques, e.g. detection of mir-199a in histological tissue sections or mir-199a immobilized on suitable carriers like membranes, chips, ELISA plates etc.).
  • primers capable of specifically detecting mir-199a RNA or cDNA
  • nucleic acid probes able to specifically hybridise to mir-199a RNA or cDNA under standard
  • the level of mir-199a in said individual or sample of an individual is compared to the level of mir-199a in one or more references and/or reference samples.
  • the reference is selected from the group consisting of a healthy individual, a diseased individual or the same individual as the tested individual at an earlier or later time point.
  • the reference is a value representative for the level of mir-199a in absence of an altered tissue status or disease, in presence of an altered tissue status or disease or for an increased or decreased risk of developing an altered tissue status or disease.
  • the reference sample is preferably derived from a healthy individual, a diseased individual, or from the same individual as the sample of interest.
  • the reference sample was taken from the same individual as the sample of interest
  • the reference sample was preferably taken at an earlier or later time point then the sample of interest.
  • the time period which has lapsed between taking of the reference sample and taking of the sample of interest preferably represents years (e.g. 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 years), months (1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 months), weeks (e.g. 1 , 2, 3, 4, 5, 6, 7, 8 weeks), days (e.g.
  • the reference sample is a reference sample with a level of mir-199a representative for a healthy individual or representative for the presence or absence of an altered tissue status or disease or representative for an increased or decreased risk of developing an altered tissue status or disease.
  • a lowered level of mir-199a in the sample of interest in comparison to said reference indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
  • a similar level of mir-199a in the sample of interest in comparison to said reference indicates (a) an alteration of the tissue status or an improvement or absence of a disease and/or (b) a decreased risk to develop a deteriorated tissue status or a disease and/or (c) a declined progression of the deteriorated tissue status or the disease.
  • an elevated level of mir-199a in the sample of interest in comparison to said reference indicates (a) an alteration of the tissue status or an improvement or absence of a disease and/or (b) a decreased risk to develop a deteriorated tissue status or a disease and/or (c) a declined progression of the deteriorated tissue status or the disease.
  • a similar level of mir-199a-a indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
  • a lowered level of mir-199a indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
  • an elevated level of mir-199a indicates (a) an alteration of the tissue status or an improvement or absence of a disease and/or (b) a decreased risk to develop a deteriorated tissue status or a disease and/or (c) a declined progression of the deteriorated tissue status or the disease.
  • a lowered level of mir- 199a-a in the sample of interest indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
  • an elevated level of mir- 199a-a in the sample of interest indicates (a) an alteration of the tissue status or an improvement or absence of a disease and/or (b) a decreased risk to develop a deteriorated tissue status or a disease and/or (c) a declined progression of the deteriorated tissue status or the disease.
  • a similar level of mir- 199a-a in the sample of interest indicates (a) a similar risk to develop a deteriorated tissue status or a disease and/or (b) a stagnation in the progression of a deteriorated tissue status or a disease, and/or (c) a persistence of the deteriorated tissue status or the disease in the individual.
  • the present invention provides a method for determining the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease in an individual, comprising the steps of (a) determining the level of mir-199a in a sample of the individual, and optionally determining the level of mir-199a in a reference or reference sample for comparison with the level of mir-199a in the sample of interest, and (b) determining the dosage of a pharmaceutical depending on the level of mir-199a in the sample of interest, optionally depending on the comparison of the levels of mir-199a in the sample of interest and the reference or reference sample.
  • levels of mir-199a are preferably detected on a RNA or DNA level, more preferably via known means such as but not limited to antibodies, primers or nucleic acid probes able to specifically bind and/or hybridise to the mir-199a gene, gene product(s) and/or functionally active variant(s) thereof.
  • Samples can be fluid or solid, preferably fluid sample are body fluid sample such as but not limited to blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, and solid sample are a tissue sample, such as but not limited to cartilage, bone, synovium, perichondrium, capsule, skin, and connective tissue.
  • the reference sample is preferably derived from a healthy individual, a diseased individual, or from the same individual as the sample of interest. Where the reference sample was taken from the same individual as the sample of interest, the reference sample was preferably taken at an earlier or later time point then the sample of interest.
  • the reference sample is a reference sample with a level of mir-199a representative for a healthy individual or representative for the presence or absence of an altered tissue status or disease or representative for an increased or decreased risk of developing an altered tissue status or disease.
  • the necessity for maintenance or alteration, i.e. an increase or decrease, of the dosage of the pharmaceutical is determined upon determination of the mir-199a levels in the sample, preferably in comparison to a reference or reference sample.
  • the reference or reference sample is derived from a healthy individual or an individual with a decreased risk of developing an altered tissue status or a disease or with a level of mir-199a representative of the absence of an altered tissue status or disease, and a lowered level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, the necessity of a maintained dosage or of an increased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is determined.
  • the reference or reference sample is derived from a healthy individual or an individual with a decreased risk of developing an altered tissue status or a disease or with a level of mir-199a representative of the absence of an altered tissue status or disease, and an elevated level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, the necessity of a maintained dosage or of an decreased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is determined.
  • the reference or reference sample is derived from a healthy individual or an individual with a decreased risk of developing an altered tissue status or a disease or with a level of mir-199a representative of the absence of an altered tissue status or disease, and a similar level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, the necessity of a maintained dosage or of an decreased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is determined.
  • the reference or reference sample is derived from a diseased individual or an individual with an increased risk of developing an altered tissue status or a disease or a value representative of the presence of an altered tissue status or disease, and a similar level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, the necessity of a maintained dosage or of an increased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is determined.
  • the reference or reference sample is derived from a diseased individual or an individual with an increased risk of developing an altered tissue status or a disease or a value representative of the presence of an altered tissue status or disease, and an elevated level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, the necessity of a maintained dosage or of an decreased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is determined.
  • the reference or reference sample is derived from a diseased individual or an individual with an increased risk of developing an altered tissue status or a disease or a value representative of the presence of an altered tissue status or disease, and a lower level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, the necessity of a maintained dosage or of an increased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is determined.
  • the necessity of a maintained dosage or of an increased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is determined.
  • the necessity of a maintained dosage or of an decreased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is determined.
  • the necessity of a maintained dosage or of an increased or of a decreased dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is determined.
  • the present invention provides a method for adapting the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease, comprising the steps of (a) determining the level of mir-199a in a sample, (b) determining the level of mir-199a in one or more references or reference samples, (c) examining the tested sample as to whether the level of mir-199a present in said sample of interest is different from the level in the one or more references or reference samples, and (d) adapting the dosage of a pharmaceutical depending on whether the level of mir-199a in the sample of interest is different from the level in the one or more references or reference samples.
  • RNA or DNA levels are preferably detected on a RNA or DNA level, more preferably via known means such as but not limited to antibodies, primers or nucleic acid probes able to specifically bind and/or hybridise to the mir-199a gene, gene product(s) and/or functionally active variant(s) thereof.
  • Samples can be fluid or solid, preferably fluid sample are body fluid sample such as but not limited to blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, and solid sample are a tissue sample, such as but not limited to cartilage, bone, synovium, perichondrium, capsule, skin, and connective tissue.
  • the dosage of the pharmaceutical is maintained or altered, i.e. increase or decrease, upon determination of the mir-199a levels in the sample in comparison to the one or more reference or reference sample.
  • the reference or reference sample is derived from a healthy individual or an individual with a decreased risk of developing an altered tissue status or a disease or with a level of mir-199a representative of the absence of an altered tissue status or disease, and a lowered level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is maintained or increased.
  • the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is maintained or decreased.
  • the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is maintained, decreased or minimized.
  • the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is maintained or increased.
  • the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is maintained or decreased.
  • the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is maintained or increased.
  • a lowered level of mir- 199a in the sample of interest in comparison to said reference or reference sample is determined, the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual, is maintained or increased.
  • the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is maintained or decreased.
  • the dosage of a pharmaceutical beneficial for the alteration of a tissue status or the prevention or treatment of a disease in an individual is maintained, increased or decreased.
  • the present invention provides a method of determining the beneficial and/or adverse effects of a substance on a tissue status or the development of a disease, comprising the steps of (a) determining the level of mir-199a in a sample of interest, (b) determining the level of mir-199a in one or more references or reference samples, and (c) examining the sample of interest as to whether the level of mir-199a present in said sample of interest is different from the level in the one or more references or reference samples, wherein the sample of interest was exposed differently to said substance than the one or more references or reference samples.
  • RNA or DNA level levels of mir-199a are detected on a RNA or DNA level, more preferably via known means such as but not limited to antibodies, primers or nucleic acid probes able to specifically bind and/or hybridise to the mir-199a gene, gene product(s) and/or functionally active variant(s) thereof.
  • Samples can be fluid or solid, preferably fluid sample are body fluid sample such as but not limited to blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, and solid sample are a tissue sample, such as but not limited to cartilage, bone, synovium, perichondrium, capsule, skin, and connective tissue.
  • the beneficial and/or adverse effects of a substance on a tissue status or the development of a disease is determined upon determination of the mir-199a levels in the sample, preferably in comparison to a reference or reference sample.
  • the reference or reference sample is derived from a healthy individual or an individual with a decreased risk of developing an altered tissue status or a disease or with a level of mir-199a representative of the absence of an altered tissue status or disease, and a lowered level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, an adverse effect of said substance on a tissue status or the development of a disease is determined.
  • the reference or reference sample is derived from a healthy individual or an individual with a decreased risk of developing an altered tissue status or a disease or with a level of mir-199a representative of the absence of an altered tissue status or disease, and an elevated level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, a beneficial effect of said substance on a tissue status or the development of a disease is determined.
  • the reference or reference sample is derived from a healthy individual or an individual with a decreased risk of developing an altered tissue status or a disease or with a level of mir-199a representative of the absence of an altered tissue status or disease, and a similar level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, it is determined that said substance has no effect or only very limited effect on a tissue status or the development of a disease.
  • the reference or reference sample is derived from a diseased individual or an individual with an increased risk of developing an altered tissue status or a disease or a value representative of the presence of an altered tissue status or disease, and a similar level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, it is determined that said substance has no effect or only very limited effect on a tissue status or the development of a disease.
  • the reference or reference sample is derived from a diseased individual or an individual with an increased risk of developing an altered tissue status or a disease or a value representative of the presence of an altered tissue status or disease, and an elevated level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, a beneficial effect of said substance on a tissue status or the development of a disease is determined.
  • the reference or reference sample is derived from a diseased individual or an individual with an increased risk of developing an altered tissue status or a disease or a value representative of the presence of an altered tissue status or disease, and a lowered level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, an adverse effect of said substance on a tissue status or the development of a disease is determined.
  • a lowered level of mir- 199a in the sample of interest in comparison to said reference or reference sample is determined, an adverse effect of said substance on a tissue status or the development of a disease is determined.
  • the reference or reference sample is derived from the same individual as the individual of interest at an earlier time point, and an elevated level of mir-199a in the sample of interest in comparison to said reference or reference sample is determined, a beneficial effect of said substance on a tissue status or the
  • the present invention provides the use of mir-199a in a method according to any of the previous aspects of the present invention.
  • mir-199a is provided in a method of identifying (a) an alteration of a tissue status or the presence of a disease and/or (b) the risk of developing an altered tissue status or a disease and/or (c) monitoring the progression or the stage of a tissue status or a disease in an individual, comprising detecting the level of mir-199a, as described in detail above.
  • the use of mir-199a is also provided in a method for determining the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease in an individual, comprising the steps of (a) determining the level of mir-199a in a sample of the individual, and optionally
  • determining the level of mir-199a in a reference or reference sample for comparison with the level of mir-199a in the sample of interest and (b) determining the dosage of a pharmaceutical depending on the level of mir-199a in the sample of interest, optionally depending on the comparison of the levels of mir-199a in the sample of interest and the reference or reference sample, as described in detail above.
  • mir-199a is envisaged in a method for adapting the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease, comprising the steps of (a) determining the level of mir-199a in a sample, (b) determining the level of mir-199a in one or more references or reference samples, (c) examining the tested sample as to whether the level of mir-199a present in said sample of interest is different from the level in the one or more references or reference samples, and (d) adapting the dosage of a pharmaceutical depending on whether the level of mir-199a in the sample of interest is different from the level in the one or more references or reference samples, as described in detail above.
  • mir-199a in a method of determining the beneficial and/or adverse effects of a substance on a tissue status or the development of a disease, comprising the steps of (a) determining the level of mir-199a in a sample of interest, (b) determining the level of mir-199a in one or more references or reference samples, and (c) examining the sample of interest as to whether the level of mir-199a present in said sample of interest is different from the level in the one or more references or reference samples, wherein the sample of interest was exposed differently to said substance than the one or more references or reference samples, as described in detail above, is provided for.
  • the use of mir-199a in either of the above indicated methods comprises the use of the mir-199a gene, mir- 199a gene product(s) and/or functionally active variant(s) thereof.
  • the sample of interest is a solid and/or fluid sample.
  • the solid sample is preferably a tissue sample and the fluid sample a body fluid sample.
  • the tissue sample is selected from the group consisting of cartilage, bone, synovium, perichondrium, capsule, skin, and connective tissue
  • the fluid sample is selected from the group consisting of blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid.
  • the sample is selected from the group consisting of cartilage, synovium, capsule, blood, plasma, serum, urine, and synovial fluid.
  • the sample cartilage, synovium, serum, and/or synovial fluid.
  • the sample is a cell culture or tissue culture sample, preferably selected from the group consisting of cartilage, bone, synovium, bone marrow cells, chondrocytes, chondroblasts, chondroclasts, synovial cells, osteocytes, osteoclasts, osteoblasts, stem cells and/or mesenchymal stem cells.
  • the present invention provides a kit for use in any of the aspects of the present invention, comprising one or more means of detecting mir-199a.
  • the one or more means for detecting mir-199a are means for determining the expression levels of mir-199a, more preferably on a gene and/or RNA level. It is particularly preferred that the one or more means for detecting mir-199a are selected from the group consisting of nucleic acid, preferably DNA or RNA, peptide, and protein, preferably monoclonal or polyclonal antibodies.
  • means for detecting mir-199a are either naturally occurring nucleic acid such as DNA or RNA or chemically modified nucleic acids with complementarity to the sequence of mir-199a and/or its precursor.
  • the kit further comprises (a) a container, and/or (b) a data carrier.
  • the data carrier comprises information such as but not limited to (i) instructions concerning methods for identifying the risk for developing and/or identifying the presence and/or monitoring progression of a joint disease, (ii) instructions for use of the means for detecting mir-199a, preferably in a sample, more preferably in a sample from an individual and/or of the kit, (iii) quality information such as information about the lot/batch number of the means for detecting mir-199a and/or of the kit, the manufacturing or assembly site or the expiry or sell-by date, information concerning the correct storage or handling of the kit, (iv) information concerning the composition of the buffer(s), diluent(s), reagent(s) for detecting mir- 199a and/or of the means for detecting mir-199a, (v) information concerning the interpretation of information obtained when performing the above-mentioned methods identifying and/or monitoring progression of a joint disease.
  • the present invention provides the use of the kit of the eighth aspect in a method of any of the methods of the present invention is provided for.
  • kits in a method of identifying (a) an alteration of a tissue status or the presence of a disease and/or (b) the risk of developing an altered tissue status or a disease and/or (c) monitoring the progression or the stage of a tissue status or a disease in an individual, comprising detecting the level of mir-199a, as described in detail above.
  • kit for determining the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease in an individual, comprising the steps of (a) determining the level of mir-199a in a sample of the individual, and optionally determining the level of mir-199a in a reference or reference sample for comparison with the level of mir-199a in the sample of interest, and (b) determining the dosage of a pharmaceutical depending on the level of mir-199a in the sample of interest, optionally depending on the comparison of the levels of mir-199a in the sample of interest and the reference or reference sample as described in detail above.
  • kit for adapting the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease, comprising the steps of (a) determining the level of mir-199a in a sample, (b) determining the level of mir-199a in one or more references or reference samples, (c) examining the tested sample as to whether the level of mir-199a present in said sample of interest is different from the level in the one or more references or reference samples, and (d) adapting the dosage of a pharmaceutical depending on whether the level of mir-199a in the sample of interest is different from the level in the one or more references or reference samples as described in detail above.
  • kits in a method of determining the beneficial and/or adverse effects of a substance on a tissue status or the development of a disease, comprising the steps of (a) determining the level of mir- 199a in a sample of interest, (b) determining the level of mir-199a in one or more references or reference samples, and (c) examining the sample of interest as to whether the level of mir-199a present in said sample of interest is different from the level in the one or more references or reference samples, wherein the sample of interest was exposed differently to said substance than the one or more references or reference samples, as described in detail above, is provided for.
  • the present invention provides for one or more nucleic acids for detecting a mir-199a gene, gene product(s) or functionally active variant(s) thereof in a method of any of the aspects of the present invention as described in detail above.
  • the nucleic acid is a nucleic acid either occurring naturally such as DNA or RNA, or chemically modified.
  • the nucleic acid is selected from the group consisting of a nucleic acid probe, a polyamide or peptide nucleic acid (PNA), microRNA (miRNA), small interfering RNA (siRNA), locked nucleic acid (LNA), primers for polymerase chain reaction (PCR), primers for reverse transcription (RT) reaction, and primers for DNA sequencing. It is particularly preferred that such nucleic acid exhibits a strong binding to mir-199a.
  • exemplary LNAs of about 8
  • nucleotides are suitable for detecting mir-199a, as long as the sequence is unique within the entirety of microRNA sequences.
  • the present invention provides a peptide, polypeptide or protein for detecting a mir-199a gene, gene product(s), functionally active variant(s) thereof and/or a label on a microRNA in a method of any of the aspects of the present invention as described in detail above.
  • the peptide, polypeptide or protein is a protein ligand, preferably an antibody, a fragment or derivate thereof, a darpin or an anticalin, or the polypeptide or peptide is a probe, preferably a mass spectrometry probe.
  • the protein ligand is an antibody that has been prepared recombinantly and, where appropriate, modified, such as chimaeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-stranded antibodies and F(ab) or F(ab) 2 fragments (see, for example,
  • protein scaffolds against mir-199a are used as protein ligands, e.g. anticalins which are based on lipocalin (Beste et al. (1999) Proc. Natl. Acad. Sci. USA, 96, 1898-1903).
  • the natural ligand-binding sites of the lipocalins can be altered, for example by means of a "combinatorial protein design" approach, in such a way that they bind to selected haptens, here to mir-199a (Skerra, 2000, Biochim. Biophys. Acta, 1982, 337-50).
  • Other known protein scaffolds are known as being alternatives to antibodies for molecular recognition (Skerra (2000) J. Mol. Recognit., 13, 167-187).
  • the present invention provides for the use of the nucleic acid of the tenth aspect of the present invention and/or of a peptide, polypeptide or protein of the eleventh aspect of the present invention in a method of any of the aspects of the present invention.
  • the nucleic acids is used in naked form, in the form of gene transfer vectors or complexed with liposomes or gold particles.
  • gene transfer vectors are viral vectors, for example adenoviral vectors or retroviral vectors (Lindemann et al. (1997), Mol. Med., 3, 466-76; Springer et al. (1988) Mol. Cell., 2, 549-58).
  • Complexes with liposomes usually achieve a very high efficiency of transfection, in particular of skin cells (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 146-a79-85).
  • small, unilamellar vesicles composed of cationic lipids are prepared by ultrasonicating the liposome suspension.
  • the DNA is bound ionically on the surface of the liposomes in a ratio which is such that a positive net charge remains and all the plasmid DNA is complexed by the liposomes.
  • DOTMA 1,2-dioleyloxypropyl-3-trimethylammonium bromide
  • DOPE 1,2-dioleyloxypropyl-3-trimethylammonium bromide
  • lipid formulations employed by Feigner, P. L. et al. (1987), Proc. Natl. Acad. Sci USA, 84, 7413 - 7414, a large number of lipid formulations have by now been synthesized and tested for their efficiency in transfecting a variety of cell lines (Behr et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 6982-6986; Zhao and Huang (1991 ), Biochim. Biophys. Acta, 1 189, 195-203; Feigner et al. (1994) J. Biol. Chem., 269, 2550-2561 ).
  • lipid formulations are DOTAP N-[1 -(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate or DOGS
  • Auxiliary substances which increase the transfer of nucleic acids into the cell can, for example, be proteins or peptides which are bound to the DNA or synthetic peptide-DNA molecules which enable the nucleic acid to be transported into the nucleus of the cell (Schwartz et al. (1999) Gene Therapy 6:282; Branden et al. (1999) Nature Biotech., 17, 784).
  • Auxiliary substances also include molecules which enable nucleic acids to be released into the cytoplasm of the cell (Planck et al. (1994) J. Biol. Chem., 269, 12918; Kichler et al. (1997) Bioconj.
  • Mir-199a or the mir-199a gene or a functionally active variant thereof may be used as a target molecule for the discovery of a mir-199a agonist or mimic thereof.
  • mir-199a or the mir-199a gene or a functionally active variant thereof are brought into direct or indirect contact with a test compound and the influence of the test compound on mir-199a or the mir-199a gene is measured or detected.
  • test compound is a chemical molecule such as but not limited to non-polymeric organic compounds, lipids, carbohydrates, peptides, preferably peptides with about 10 to about 80 amino acids, in particular with 10 to 25 amino acids and oligonucleotides, preferably with about 10 to about 90 nucleotides, in particular with 15 to 25 nucleotides.
  • chemical molecule such as but not limited to non-polymeric organic compounds, lipids, carbohydrates, peptides, preferably peptides with about 10 to about 80 amino acids, in particular with 10 to 25 amino acids and oligonucleotides, preferably with about 10 to about 90 nucleotides, in particular with 15 to 25 nucleotides.
  • small chemical molecules in particular non-polymeric organic compounds, either synthesized in a laboratory or found in nature, with a preferred molecular weight of about 200 g/mole to about 1500 g/mole, in particular 400 g/mole to 1000 g/mole.
  • the test compound alters the level of mir-199a such as increases or decreases the expression level or the amount of mir-199a present.
  • test compounds increasing the level of mir-199a are particularly preferred.
  • a method of screening for a mir-199a agonist or mimic thereof comprises the steps of (a) providing mir-199a or the mir-199a gene, (b) providing a test compound, and (c) measuring or detecting the influence of the test compound on mir-199a or the mir-199a gene.
  • a mir-199a agonist or mimic thereof may be used for the alteration of a tissue status or the prevention or treatment of a joint disease .
  • An agonist for mir-199a or mimic thereof can be any molecule or composition that elevates or increases the function or elevates the expression level or amount of mir- 199a present. Examples comprise, but are not limited to: 1 .
  • a nucleic acid such as an oligonucleotide, oligoribonucleotides, especially dsRNA, ssRNA, siRNA, miRNA, shRNA etc, as known in the art, 2.
  • An agonistic peptide or protein such as a protein stabilising or promoting the function of the miRNA in question, an agonistic antibody or fragment thereof or a transcriptional agonist of the miRNA in question, and/or 3. a small molecule compound with agonistic action.
  • the agonist of the present invention can be in the form of a natural product extract, either in crude or in purified form. The extract can be produced according to standard
  • Such agonist or mimic thereof may be used for the treatment of a joint disease, in particular a degenerative joint disease such as osteoarthritis.
  • the mir-199a agonist or mimic thereof may be used to treat joint pain, in particular by reducing the joint pain in degenerative joint diseases.
  • the mir-199a agonist or mimic thereof may be used for the production of a medicament for the treatment of a joint disease and/or for the treatment of the joint pain as specified above.
  • a pharmaceutical may comprising a mir-199a agonist or mimic thereof for use in the prevention or treatment of a joint disease,.
  • the pharmaceutical may further comprises a pharmaceutically acceptable carrier and/or excipient and optionally one or more additional active substances.
  • the additional active substances may be effective against the same or against a different disease or disorder or condition. Accordingly, additional active substances comprise those having a beneficial effect in the treatment of a joint disease and/or for the treatment of the joint pain as specified above.
  • additional active substances directed at the treatment of different diseases or disorder or conditions suitable to be administered in combination with the active ingredient of the present invention are comprised.
  • additional active substances affecting further miRNAs and/or their gene product(s), such as agonist or antagonists to individual miRNAs are encompassed.
  • Additional active substances also comprise food supplements such as but not limited to vitamins, minerals, fiber, fatty acids, or amino acids, considered beneficial in a persons diet and/or in the treatment of a joint disease and/or for the treatment of the joint pain as specified above and/or in any other disease, disorder or condition.
  • the mir-199a agonists or mimic thereof are usually formulated with one or more pharmaceutically acceptable additives or auxiliary substances, such as physiological buffer solution, e.g. sodium chloride solution, demineralized water, stabilizers, such as protease or nuclease antagonists, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or sequestering agents such as EDTA, gel formulations, such as white vaseline, low-viscosity paraffin and/or yellow wax, etc. depending on the kind of administration.
  • physiological buffer solution e.g. sodium chloride solution
  • demineralized water demineralized water
  • stabilizers such as protease or nuclease antagonists, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or sequestering agents such as EDTA
  • gel formulations such as white vaseline, low-viscosity paraffin and/or yellow wax, etc. depending on the kind of administration.
  • Suitable further additives are, for example, detergents, such as, for example, Triton X-100 or sodium desoxycholate, but also polyols, such as, for example, polyethylene glycol or glycerol, sugars, such as, for example, sucrose or glucose, zwitterionic compounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or a protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or zwitterionic compounds are preferred.
  • the physiological buffer solution preferably has a pH of approx. 6.0-8.0, expecially a pH of approx. 6.8-7.8, in particular a pH of approx. 7.4, and/or an osmolarity of approx. 200-400 milliosmol/liter, preferably of approx. 290-310 milliosmol/liter.
  • the pH of the medicament is in general adjusted using a suitable organic or inorganic buffer, such as, for example, preferably using a phosphate buffer, tris buffer
  • MOPS buffer 3-morpholino- 1 -propanesulphonic acid
  • Phosphate buffer is suitable, for example, for injection and infusion solutions.
  • the mir-199a agonists or mimic thereof may also be formulated with a nanoparticle, a liposome or other lipid complexation, or may be conjugated to a protein, peptide, nucleic acid (e.g. an aptamer) or gel, e.g. based on hyaluronic acid, or similar slow release formulations.
  • a protein e.g. an aptamer
  • gel e.g. based on hyaluronic acid, or similar slow release formulations.
  • the pharmaceutical is preferably in unit dosage form.
  • the preparation may be subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, an injection vial, a tablet, a cachet, or a lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • a method of alteration of a tissue status or prevention or treatment of a joint disease comprises the administration of a therapeutically effective amount of the
  • Intracavitary administration includes but is not limited to intravenous and intra-articular administration.
  • the pharmaceutical can be administered in a conventional manner, e.g. by means of oral dosage forms, such as, for example, tablets or capsules, by means of the mucous membranes, for example the nose or the oral cavity, in the form of dispositories implanted under the skin, by means of injections, infusions or gels which contain the medicaments according to the invention. It is further possible to administer the medicament topically and locally in order to treat the particular joint disease as described above, if appropriate, in the form of liposome complexes. Furthermore, the treatment can be carried out by means of a transdermal therapeutic system (TTS), which makes possible a temporally controlled release of the medicaments.
  • TTS transdermal therapeutic system
  • TTS are known for example, from EP 0 944 398 A1 , EP 0 916 336 A1 , EP 0 889 723 A1 or EP 0 852 493 A1.
  • Injection solutions are in general used if only relatively small amounts of a solution or suspension, for example about 1 to about 20 ml, are to be administered to the body. Intracavitary administration includes but is not limited to infusions and injections. Infusion solutions are in general used if a larger amount of a solution or suspension, for example one or more litres, are to be administered.
  • the formulation according to the invention should be diluted briefly before administration to such an extent that an at least approximately isotonic solution is obtained.
  • An example of an isotonic solution is a 0.9% strength sodium chloride solution.
  • tissue status is tissue damage, preferably selected from the group consisting of damage to bone, cartilage, synovium, perichondrium, capsule, and connective tissue and/or is characterised by altered level of regulatory molecules, signalling molecules, and/or degenerative molecules present in the tissue.
  • the disease is a joint disease, preferably selected from the group consisting of osteoarthritis, rheumatoid arthritis, psoriatic arthritis, septic arthritis, Ankylosing spondylitis, Bursitis (inflammation), Dermatomyositis, Fibromyalgia, Gouty arthritis, Juvenile chronic arthritis (Still's disease), Mixed Connective Tissue Disease, Polymyalgia rheumatica, Polymyositis, Reactive arthritis (Reiter's Syndrome), Scleroderma, Shoulder tendinitis, Sjorgen's Sydrome, Systemic Lupus Erythemetosis and/or is characterised by a physical or metabolic injury of the joint indicated by an inflammation of the joint or a degradation of the joint, more preferably wherein the disease is osteoarthritis.
  • compositions described in the invention include antagonists of mir-199a., that function as miRNA antagonists that can be introduced to a patient to treat and reduce one or more of the conditions associated with joint diseases, especially with osteoarthritis.
  • compositions described in the invention include agonists of mir-199a / antagonists of mir-199a, that function as miRNAs / miRNA agonist/antagonists that can be introduced to a patient to treat and reduce one or more of the conditions associated with inflammatory diseases, especially with osteoarthritis.
  • the detection of the miRNA can be performed by any known method using known means for detection of nucleic acids such as specific nucleic acids able to binding the miRNA in question.
  • One aspect of present invention concerns the use of a means for detecting the miRNA in question amount in a biological sample for the manufacture of a diagnostic means for diagnosing a malfunction of the carbohydrate or lipid metabolism.
  • the means to detect the amount (i.e. the steady state level) of the miRNA in question present in a biological sample can be any means to detect the miRNA in question. This can be for example a set of primers capable of specifically detecting the miRNA in question or the cDNA, e.g.
  • Another means can e.g. be a nucleic acid probe able to specifically hybridise to miRNA in question mRNA or cDNA under standard conditions, e.g. for use in Northern Blot or Chip hybridisation techniques.
  • Another means can e.g. be an antibody able to specifically detect the miRNA in question e.g. for use in immunohistological or immunohistochemical or immunochemical techniques (e.g. detection of miRNA in question in histological tissue sections or miRNA in question immobilized on suitable carriers like membranes, chips, ELISA plates etc.).
  • the invention concerns a method for identifying a joint disease such as OA or an increased risk for developing a joint disease, such as OA in an individual, which comprises examining a sample taken from the individual, as to whether the level of mir-199a and/or the activity of mir-199a present in said sample is different from that of one or more reference samples. The presence of an elevated level indicating an increased risk of said individual to suffer from or to develop a joint disease such as OA.
  • lowered level refers to the amount of the miRNA in a sample of tissue or tissue fluid known to have, under certain circumstances, detectable levels If the miRNA in question (such as cellular or tissue extract, synovial fluid, serum or plasma), especially from an individual to be tested, compared to the amount of the miRNA in comparable sample from a cohort or cohorts that do not have the joint disease, especially OA.
  • a miRNA that is detectable in higher amounts in synovial fluid of one individual suffering from OA than in synovial fluid of individuals not suffering from OA has an elevated level.
  • an elevated level in a patient sample indicates the presence of OA or increased susceptibility or increased probability to develop OA.
  • the level of the miRNA marker will be compared to a control to determine whether the level is reduced or elevated.
  • the control may be an external control, such as the miRNA in a sample from a patient known to be free of the joint disease especially OA.
  • the external control may be the miRNA in a sample from a tissue/tissue fluid known not to have detectable amounts (or always the same amount, irrespective of the disease-state of the individual) of the miRNA in question or a known amount of a synthetic RNA.
  • An internal control may be a miRNA from the tissue/tissue fluid sample being tested.
  • the identity of a miRNA control may be the same as or different from the patient serum or plasma miRNA being measured.
  • an antagonist for a miRNA can be any molecule or composition that lowers or inhibits the function or amount of the miRNA in question. Examples comprise, but are not limited to:
  • a nucleic acid such as an oligonucleotide, oligoribonucleotides, especially dsRNA, ssRNA, siRNA, miRNA, shRNA, ssDNA, ssDNA/RNA hybrids or chemically modified nucleic acids etc., as known in the art.
  • An antagonistic peptide or protein such as a protein destabilising or inhibiting the function of the miRNA in question, an antagonistic antibody or fragment thereof or a transcriptional antagonist of the miRNA in question,
  • composition comprising one or more of 1 -4 and possibly an excipient.
  • One subject matter of the present invention is, therefore, the use of a antagonist for the treatment of a joint disease, in particular a degenerative joint disease such as osteoarthritis.
  • the miRNA antagonist can also be used to treat the joint pain, in particular by reducing the joint pain in degenerative joint diseases.
  • the miRNA antagonist can be used for the production of a medicament for the treatment of a joint disease and/or for the treatment of the joint pain as specified above.
  • the term "chemical molecule” encompasses non- polymeric organic compounds, lipids, carbohydrates, peptides, preferably peptides with about 10 to about 80 amino acids, in particular with 10 to 25 amino acids and oligonucleotides, preferably with about 10 to about 90 nucleotides, in particular with 15 to 25 nucleotides.
  • small chemical molecules in particular non- polymeric organic compounds, either synthesized in a laboratory or found in nature, with a preferred molecular weight of about 200 g/mole to about 1500 g/mole, in particular 400 g/mole to 1000 g/mole.
  • the antagonist of the present invention can be in the form of a natural product extract, either in crude or in purified form.
  • the extract can be produced according to standard procedures, such as water and/or alcohol and/or organic solvent extraction and/or column chromatography and/or precipitation from an animal, plant or microbial source, like snake poison, leaves or microbial fermentation broths.
  • binding protein or "binding peptide” refers to a class of proteins or peptides which bind and inhibit mir-199a including, without limitation, polyclonal or monoclonal antibodies, antibody fragments and protein scaffolds directed against mir-199a, e.g. anticalins which are directed against mir-199a.
  • antibody or antibody fragment is also understood as meaning antibodies or antigen-binding parts thereof, which have been prepared recombinantly and, where appropriate, modified, such as chimaeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-stranded antibodies and F(ab) or F(ab) 2 fragments (see, for example, EP-B1 -0 368 684, US 4,816,567, US 4,816,397, WO 88/01649,
  • protein scaffolds against mir-199a e.g. anticalins which are based on lipocalin
  • the natural ligand-binding sites of the lipocalins for example the retinol-binding protein or the bilin-binding protein, can be altered, for example by means of a "combinatorial protein design" approach, in such a way that they bind to selected haptens, here to mir-199a (Skerra, 2000, Biochim. Biophys. Acta, 1982, 337-50).
  • Other known protein scaffolds are known as being alternatives to antibodies for molecular recognition (Skerra (2000) J. Mol. Recognit., 13, 167-187).
  • nucleic acids against the mir-199a gene or mir-199a itself refers to double- stranded or single stranded DNA or RNA which, for example, inhibit or activate the expression of the mir-199a gene or the activity of mir-199a and includes, without limitation, antisense nucleic acids, aptamers, siRNAs (small interfering RNAs), miRNAs, shRNAs (short hairpin RNAs) and ribozymes.
  • the nucleic acids can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990)
  • Aptamers are nucleic acids which bind with high affinity to a polypeptide, here mir-199a.
  • Aptamers can be isolated by selection methods such as SELEX (see e.g. Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981 ) from a large pool of different single-stranded RNA molecules.
  • Aptamers can also be synthesized and selected in their mirror-image form, for example as the L-ribonucleotide (Nolte et al. (1996) Nat. Biotechnol., 14, 1 1 16-9; Klussmann et al. (1996) Nat. Biotechnol., 14,
  • Nucleic acids may be degraded by endonucleases or exonucleases, in particular by DNases and RNases which can be found in the cell. It is, therefore, advantageous to modify the nucleic acids in order to stabilize them against degradation, thereby ensuring that a high concentration of the nucleic acid is maintained in the cell over a long period of time (Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94;
  • WO 95/1 1910; WO 98/37240; WO 97/291 16 can be obtained by introducing one or more internucleotide phosphorus groups or by introducing one or more non-phosphorus internucleotides.
  • Suitable modified internucleotides are compiled in Uhlmann and Peyman (1990), supra (see also Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94; WO 95/1 1910;
  • Modified internucleotide phosphate radicals and/or non- phosphorus bridges in a nucleic acid which can be employed in one of the uses according to the invention contain, for example, methyl phosphonate,
  • non-phosphorus internucleotide analogues contain, for example, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamidate bridges and/or thioether bridges. It is also the intention that this modification should improve the durability of a pharmaceutical composition which can be employed in one of the uses according to the invention.
  • siRNAs as tools for RNA interference in the process to down regulate or to switch off gene expression, here mir-199a gene expression, is e.g. described in Elbashir, S. M. et al. (2001 ) Genes Dev., 15, 188 or Elbashir, S. M. et al. (2001 ) Nature, 41 1 , 494.
  • Ribozymes are also suitable tools to inhibit the translation of nucleic acids, here the
  • RAK gene because they are able to specifically bind and cut the mRNAs. They are e.g. described in Amarzguioui et al. (1998) Cell. Mol. Life Sci., 54, 1 175-202; Vaish et al. (1998) Nucleic Acids Res., 26, 5237-42; Persidis (1467) Nat. Biotechnol., 15, 921 -2 or Couture and Stinchcomb (1996) Trends Genet., 12, 510-5.
  • nucleic acids described are preferably meant to be used or act as antagonists of mir-199a.
  • the mir-199a antagonists of the present invention are usually formulated with one or more pharmaceutically acceptable additives or auxiliary substances, such as physiological buffer solution, e.g. sodium chloride solution, demineralized water, stabilizers, such as protease or nuclease antagonists, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or sequestering agents such as EDTA, gel formulations, such as white vaseline, low-viscosity paraffin and/or yellow wax, etc. depending on the kind of administration.
  • physiological buffer solution e.g. sodium chloride solution
  • demineralized water demineralized water
  • stabilizers such as protease or nuclease antagonists, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or sequestering agents such as EDTA
  • gel formulations such as white vaseline, low-viscosity paraffin and/or yellow wax, etc. depending on the kind of administration.
  • Suitable further additives are, for example, detergents, such as, for example, Triton X-100 or sodium desoxycholate, but also polyols, such as, for example, polyethylene glycol or glycerol, sugars, such as, for example, sucrose or glucose, zwitterionic compounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or a protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or zwitterionic compounds are preferred.
  • the physiological buffer solution preferably has a pH of approx. 6.0-8.0, expecially a pH of approx. 6.8-7.8, in particular a pH of approx. 7.4, and/or an osmolarity of approx.
  • the pH of the medicament is in general adjusted using a suitable organic or inorganic buffer, such as, for example, preferably using a phosphate buffer, tris buffer
  • Phosphate buffer is suitable, for example, for injection and infusion solutions.
  • the medicament can be administered in a conventional manner, e.g. by means of oral dosage forms, such as, for example, tablets or capsules, by means of the mucous membranes, for example the nose or the oral cavity, in the form of dispositories implanted under the skin, by means of injections, infusions or gels which contain the medicaments according to the invention. It is further possible to administer the medicament topically and locally in order to treat the particular joint disease as described above, if appropriate, in the form of liposome complexes. Furthermore, the treatment can be carried out by means of a transdermal therapeutic system (TTS), which makes possible a temporally controlled release of the medicaments.
  • TTS transdermal therapeutic system
  • TTS are known for example, from EP 0 944 398 A1 , EP 0 916 336 A1 , EP 0 889 723 A1 or EP 0 852 493 A1 .
  • Injection solutions are in general used if only relatively small amounts of a solution or suspension, for example about 1 to about 20 ml, are to be administered to the body.
  • Infusion solutions are in general used if a larger amount of a solution or suspension, for example one or more litres, are to be administered. Since, in contrast to the infusion solution, only a few millilitres are administered in the case of injection solutions, small differences from the pH and from the osmotic pressure of the blood or the tissue fluid in the injection do not make themselves noticeable or only make themselves noticeable to an insignificant extent with respect to pain sensation. Dilution of the formulation according to the invention before use is therefore in general not necessary.
  • the formulation according to the invention should be diluted briefly before administration to such an extent that an at least approximately isotonic solution is obtained.
  • An example of an isotonic solution is a 0.9% strength sodium chloride solution.
  • the dilution can be carried out, for example, using sterile water while the administration can be carried out, for example, via a so-called bypass.
  • nucleic acids can be used in naked form, in the form of gene transfer vectors or complexed with lipids (especially cationic lipids) or gold particles, they may be formulated as liposomes (wherein the nucleic acid is e.g. carried as cargo inside a membraneous lipidlayer such as a lipid bilayer), or may be based on
  • nanoparticles e.g. on dextrane, peptides, or other polymers.
  • gene transfer vectors are viral vectors, for example adenoviral vectors or retroviral vectors (Lindemann et al. (1997), Mol. Med., 3, 466-76; Springer et al. (1988) Mol. Cell., 2, 549-58).
  • Complexes with liposomes usually achieve a very high efficiency of transfection, in particular of skin cells (Alexander and Akhurst, 1465, Hum. Mol.
  • lipid formulations are DOTAP N-[1 -(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate or DOGS
  • Auxiliary substances which increase the transfer of nucleic acids into the cell can, for example, be proteins or peptides which are bound to the DNA or synthetic peptide- DNA molecules which enable the nucleic acid to be transported into the nucleus of the cell (Schwartz et al. (1999) Gene Therapy 6:282; Branden et al. (1999) Nature
  • Auxiliary substances also include molecules which enable nucleic acids to be released into the cytoplasm of the cell (Planck et al. (1994) J. Biol. Chem., 269, 12918; Kichler et al. (1997) Bioconj. Chem, 8, 213) or, for example liposomes (Uhlmann and Peyman (1990), supra).
  • Another, particularly suitable form can be obtained by applying the above-described nucleic acids to gold particles and firing these particles into tissue or cells using what is termed a "gene gun" (Wang et al. (1999) J. Invest. Dermatol. 1 12:775-81 , Tuting et al. (1998) J. Invest. Dermatol. 1 1 1 : 183-8).
  • mir-199a or the mir-199a gene as a target for the discovery of a mir-199a antagonist for the treatment of a joint disease, in particular a degenerative joint disease such as osteoarthritis or and joint disease such as rheumatoid arthritis, and/or for the treatment of joint pain, in particular by reducing the joint pain in degenerative joint diseases.
  • a mir-199a antagonist for the treatment of a joint disease, in particular a degenerative joint disease such as osteoarthritis or and joint disease such as rheumatoid arthritis, and/or for the treatment of joint pain, in particular by reducing the joint pain in degenerative joint diseases.
  • the mir-199a antagonist can be used in form of a medicament as described above.
  • the present invention refers also to a method of screening a mir-199a antagonist, wherein the method comprises the steps of:
  • mir-199a or the mir-199a gene is provided e.g. in an assay system and brought directly or indirectly into contact with a test compound, in particular a biochemical or chemical test compound, e.g. in the form of a chemical compound library. Then, the influence of the test compound on mir-199a or the mir-199a gene is measured or detected. Thereafter, suitable antagonists can be analyzed and/or isolated. For the screening of chemical compound libraries, the use of high-throughput assays are preferred which are known to the skilled person or which are commercially available.
  • chemical compound library refers to a plurality of chemical compounds that have been assembled from any of multiple sources, including chemically synthesized molecules and natural products, or that have been generated by combinatorial chemistry techniques.
  • a heterogeneous assay is an assay which includes one or more washing steps, whereas in a homogeneous assay such washing steps are not necessary. The reagents and compounds are only mixed and measured.
  • Suitable functional assays may be based on the gene expression of mir-199a.
  • the present invention relates to the following aspects:
  • Mir-199a for use as an indicator of a tissue status or a disease.
  • Mir-199a as an indicator of a tissue status or a disease.
  • Mir-199a or use of mir-199a or method according to aspect 5 further comprising comparing the level of mir-199a in said individual or sample to the level of mir-199a in one or more references or reference samples.
  • the reference is selected from the group consisting of a healthy individual, a diseased individual or the same individual as the tested individual at an earlier or later time point or a value representative for the level of mir-199a in absence of the altered tissue status or disease, in presence of an altered tissue status or disease or for an increased or decreased risk of developing an altered tissue status or disease.
  • the reference sample is selected from the group consisting of a reference sample derived from a healthy individual, a reference sample derived from a diseased individual, a reference sample derived from the same individual as the sample of interest taken at an earlier or later time point, and a reference sample with a level of mir-199a representative for a healthy individual or representative for the presence or absence of an altered tissue status or disease or representative for an increased or decreased risk of developing an altered tissue status or disease.
  • an elevated level of mir-199a in the sample of interest indicates (a) an alteration of the tissue status or an improvement or absence of a disease and/or
  • a method for determining the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease in an individual comprising the steps of
  • a method for adapting the dosage of a pharmaceutical for the alteration of a tissue status or the prevention or treatment of a disease comprising the steps of
  • tissue sample is selected from the group consisting of tissue extract, synovial tissue and cartilage
  • body fluid sample is selected from the group consisting of synovial fluid, serum plasma and urine.
  • the one or more means for detecting mir-199a are means for determining the expression levels of mir-199a, preferably on a gene and/or RNA level.
  • the one or more means for detecting mir-199a are selected from the group consisting of nucleic acid, preferably DNA or RNA, a mixture of both or chemically modified nucleic acids, peptide, and protein, preferably monoclonal or polyclonal antibodies.
  • kit according to any of aspects 18 to 20, wherein the kit further comprises (a) a container, and/or
  • quality information such as information about the lot/batch number of the means for detecting mir-199a and/or of the kit, the manufacturing or assembly site or the expiry or sell-by date, information concerning the correct storage or handling of the kit,
  • LNA locked nucleic acid
  • PNA polyamide or peptide nucleic acid
  • miRNA microRNA
  • siRNA small interfering RNA
  • primers for polymerase chain reaction PCR
  • RT reverse transcription
  • nucleic acid according to aspect 24 comprising the nucleotide sequence of at least 8 nucleotides.
  • tissue damage preferably selected from the group consisting of damage to bone, cartilage, synovium, perichondrium, capsule, and connective tissue and/or is characterised by altered level of regulatory molecules, signalling molecules, and/or degenerative molecules present in the tissue.
  • Polymyalgia rheumatica Polymyositis, Reactive arthritis (Reiter's Syndrome), Scleroderma, Shoulder tendinitis, Sjorgen's Sydrome, Systemic Lupus Erythemetosis and/or is characterised by a physical or metabolic injury of the joint indicated by an inflammation of the joint or a degradation of the joint, more preferably wherein the disease is osteoarthritis.
  • a method of identifying an increase in risk for joint disease in a subject comprising:
  • a decrease in the level of mir-199a in the sample is determined as compared to the control, thereby identifying the subject as having an increased risk for joint disease.
  • a method for diagnosing a joint disease in a subject comprising: a) measuring the level of mir-199a in a biological sample from the subject; and b) comparing the level of mir-199a in the sample to the level of mir-199a in a suitable control
  • a decrease in the level of mir-199a in the sample is determined as compared to the control, thereby identifying the subject as having the joint disease.
  • a method for treating a subject having a joint disease comprising: a) measuring the level of mir-199a in a biological sample from the subject; b)comparing the level of mir-199a in the sample to the level of mir-199a in a suitable control; and c) administering to the patient a pharmaceutical composition for treatment of joint disease when a decrease in the level of mir-199a in the sample is determined as compared to the control, thereby treating the subject.
  • the method comprising: a) measuring the level of mir-199a in a biological sample from the subject; b) comparing the level of mir-199a in the sample to the level of mir-199a in a suitable control; and c) decreasing the dosage of a pharmaceutical composition when an increase in the level of mir-199a in the sample is determined as compared to the control or increasing the dosage of the pharmaceutical composition when a decrease in the level of mir- 199a in the sample is determined as compared to the control, thereby determining the dosage of the pharmaceutical in the subject.
  • a method for determining the effect of a substance or test compound on a joint disease comprising: a) measuring the level of mir-199a in a sample treated with the substance or test compound; b) comparing the level of mir-199a in the treat sample to the level of mir-199a in a suitable control; and c) identifying the substance as having an adverse effect if a decrease in the level of mir-199a in the sample is determined as compared to the control or identifying the substance as a having a beneficial effect if an increase in the level of mir-199a in the sample is determined as compared to the control, thereby determining the effect of the substance or test compound on joint disease. 6.
  • any one of aspects 1 -4 wherein the level of mir-199a is detected using one or more nucleic acid probes.
  • the suitable control is a biological sample from a healthy subject. 8. The method of aspect 6, wherein the suitable control is a sample that is not treated with substance.
  • Method of treatment with mir-199a antagonist A method of treating a joint disease in a subject comprising administering a mir-199a agonist to the subject, thereby treating joint disease in the subject.
  • mir-199a agonist is selected from the group consisting of an siRNA, a shRNA, an antisense RNA, and a ribozyme.
  • mir-199a agonist is substantially complementary to at least a portion of the precursor form of mir-199a (SEQ ID NO: 1 ).
  • mir-199a agonist is substantially complementary to at least a portion of the mature form of mir-199a (SEQ ID NO:2) or mir-199a * (SEQ ID NO:3).
  • the miRNA-146a agonist is a nucleic acid molecule of about 6 to about 20 nucleotides in length.
  • miRNA-146a agonist is a LNA-modified or PNA-modified nucleic acid molecule.
  • the joint disease is osteoarthritis.
  • a kit comprising one or more means of detecting mir-199a and a suitable control.
  • Example 1 mir-199a expression profile in chondrocyte and stem cells during their cultivation
  • mir-199a was determined in primary chondrocytes and bone marrow derived mesenchymal stem cells (BMSC) during their cultivation in order to assess the normal fluctuation in the expression level of mir-199a in these cells over the indicated time periods.
  • BMSC bone marrow derived mesenchymal stem cells
  • the chondrocytes were cultivated in pellet culture for 0, 1 , 4, 7 or 14 days in DMEM medium supplemented with 2mM L-glutamine (Sigma-Aldrich), 1 * non essential amino acids (Sigma-Aldrich), 10nM dexamethasone (Sigma-Aldrich), 10pg/ml insulin,
  • BMSC 5.5pg/ml transferrin, 5ng/ml sodium selenite (Sigma-Aldrich), 44pg/ml ascorbic acid (Sigma-Aldrich) and 10ng/ml TGF- ⁇ (R&D Systems).
  • BMSC were cultivated in pellet culture for 0, 1 , 4, 7, 14 or 21 days in the same medium. Chondrocytes and BMSC were harvested and each pellet was processed individually for RNA isolation. For the analysis of secreted proteins supernatants were collected and pooled over the entire culture period. For extraction of the RNA, Pellets were harvested after seven days of culture and separately homogenized with the Mixermill (Retsch). A proteinase K digest was performed onto the homogenized cells.
  • RNA including miRNA was eluted from the Qiagen miRNeasy Plus Micro kit.
  • the RNA was reverse transcribed into cDNA using the Reverse Transcriptase Kit and RNase Inhibitor (Life Technologies) according to the manufacturer's instructions.
  • the mir-199a expression profile in chondrocyte and BMSC was determined via miRNA specific quantitative real time PCR using Taqman probes according to protocols well-known to the skilled person.
  • the ABI Taqman ® microRNA Low density arrays (TLDA, Applied Biosystems, Foster City, CA) were used to profile 668 miRNAs and additional reference genes.
  • the assay includes three steps: a multiplex RT, a preamplification and a singleplex TaqMan PCR. All steps were performed according to the manufacturer's instructions.
  • the Real-time PCR was done on an AB 7900HT Sequence Detection System. For expression analysis the comparative Ct method was applied.
  • Fig. 1 a illustrates the mir-199a expression during the cultivation period in chondrocyte pellets
  • Fig. 2 illustrates the mir-199a expression during the cultivation period in BMSC.
  • the expression level of mir-199a increased in both cell lines with increased term of cultivation.
  • the cultivation conditions were chosen such as to reflect conditions of healthy differentiated cartilage. The increase in mir-199a expression over time is thus, indicative of an increased mir-199a expression in differentiated, healthy cartilage.
  • mir-199a is an indicator of the physiological, differentiated cartilage.
  • This increase in expression level over cultivation time stands in contrast to the relatively stable expression levels of mir-22 and mir-146a in parallel experiments conducted under the same conditions (Fig. 1 b), both micro RNAs identified by the inventors as markers of osteoarthritic cartilage.
  • mir-140 a micro RNA identified by present inventors to be associated with the physiological,
  • mir-199a in comparison to certain controls, e.g. markers of osteoarthritic cartilage such as mir-22 and/or mir- 146a is indicative for a physiological cartilage state and the absence of OA.
  • mir-199a is a indicator of differentiated cartilage.
  • Example 2 Overexpression and Knock down of mir-199a in BMSC
  • the expression of the proteins collagen II, aggrecan, ALP, MMP-1 and MMP-3 is know to be altered in arthritic joints. Their respective expression levels are thus, indicative of the presence or absence of OA.
  • mir-199a the effects of its overexpression or knockdown on these molecules, measured either on mRNA (collagen II, aggrecan) or on protein level (MMP-1 , and MMP-3) or as activity (ALP) are analysed in BMSC after chondrogenic differentiation.
  • BMSC were infected with pseudoviral particles comprising either the DNA encoding for mir-199a (herein further referred to as: mir-199a) or MRP ((herein further referred to as: empty vector)
  • mir-199a had been cloned into pLenti9, a lentiviral vector containing partial sequences of HIV1 LTRs (see map below).
  • Pseudoviral particles were produced as a custom service by Vectalys (Toulouse, France). Infection of BMSCs was carried out with a multiplicity of infection (MOI) of five.
  • MOI multiplicity of infection
  • RNA samples were harvested after 7 days of pellet culture and their RNA isolated following the same protocol as given in Example 1 .
  • cDNA synthesis was performed using the Reverse Transcriptase Kit and RNase Inhibitor (Life Technologies) according to the manufacturer's instructions.
  • the expression levels of the four chondrogenic markers collagen II, aggrecan, Sox9, and collagen X were determined via quantitative real time PCR.
  • TaqMan reactions were performed using TaqMan® Universal PCR Master Mix and the FAMTM-labelled gene specific assays for the target genes ACAN, COL2A1 , COL10A1 , and SOX9 (see table). All gene specific assays consist of FAMTM dye- labeled TaqMan® MGB probes.
  • PCR was performed using the ABI Prism 7900 (Life Technologies). Ribosomal protein L37a (RPL37a) was used as reference gene. For RPL37a the following sequences were selected: reference forward primer:
  • the MGB reference probe was labeled with VIC and had the sequence: CCGCCAGCCACTGTCT.
  • CCGCCAGCCACTGTCT For expression analysis the comparative Ct method was applied.
  • marker proteins i.e. collagen II and aggrecan
  • mir-199a was knocked-down in BMSC (herein further referred to as: mir-199a inh) using miRIDIAN Hairpin Inhibitor hsa-mir- 199a (Dharmacon) and the effects on the expression of the three marker proteins collagen II, aggrecan and ALP were observed in comparison to control cells.
  • BMSCs were treated with miRIDIAN Hairpin Inhibitor hsa-mir-199a at a concentration of 30 nM in monolayer culture.
  • miRIDIAN microRNA Hairpin Inhibitor Negative Control #1 served as negative control.
  • Fig. 5 further illustrates the percentage changes of the expression levels of the marker proteins collagen II and aggrecan due to overexpression or knock-down of mir-199a in comparison to the non-treated controls.
  • SEQ ID NO: 1 Homo sapiens stem-loop sequence of hsa-mir-199a-1 (accession number: MI0000242)
  • SEQ ID NO: 4 Homo sapiens stem-loop sequence of hsa-mir-199a-2 (accession number: MI0000281 )

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des microARN (miARN), en particulier mir-199a, en tant qu'indicateur d'un état pathologique tissulaire ou d'une maladie telle que l'ostéoarthrite (OA) et en tant que molécule cible pour la découverte d'une substance pour le traitement d'une maladie articulaire. L'invention concerne en outre des procédés et des composés pour la recherche, l'analyse et/ou le traitement d'une maladie articulaire, en particulier l'ostéoarthrite (OA).
PCT/EP2011/073036 2010-12-17 2011-12-16 Miarn dans une maladie articulaire Ceased WO2012080461A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP10306446.5 2010-12-17
EP10306446 2010-12-17

Publications (1)

Publication Number Publication Date
WO2012080461A1 true WO2012080461A1 (fr) 2012-06-21

Family

ID=45443095

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2011/073036 Ceased WO2012080461A1 (fr) 2010-12-17 2011-12-16 Miarn dans une maladie articulaire

Country Status (3)

Country Link
AR (1) AR084331A1 (fr)
TW (1) TW201239097A (fr)
WO (1) WO2012080461A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015091963A3 (fr) * 2013-12-19 2015-10-15 Comprehensive Biomarker Center Gmbh Détermination de miarn plaquettaires dans l'infarctus aigu du myocarde
WO2019229489A1 (fr) * 2018-05-31 2019-12-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de mir-146a-5p et de mir-186 en tant que biomarqueurs de l'arthrose
CN112458163A (zh) * 2020-11-19 2021-03-09 杭州市中医院 一种早期痛风肾损害的生物标志物、试剂盒及应用
CN116919977A (zh) * 2023-06-02 2023-10-24 同济大学 mir-199a-3p在制备治疗骨关节炎的药物中的应用

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988001649A1 (fr) 1986-09-02 1988-03-10 Genex Corporation Molecules de liaison de chaines de polypeptide simples
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
WO1993006213A1 (fr) 1991-09-23 1993-04-01 Medical Research Council Production d'anticorps chimeriques - une approche combinatoire
EP0368684B1 (fr) 1988-11-11 1994-03-09 Medical Research Council Clonage de séquences d'immunoglobulines de domaines variables.
WO1995011910A1 (fr) 1993-10-27 1995-05-04 Ribozyme Pharmaceuticals, Inc. 2'-amido et 2'-peptido oligonucleotides modifies
US5582981A (en) 1991-08-14 1996-12-10 Gilead Sciences, Inc. Method for identifying an oligonucleotide aptamer specific for a target
WO1997029116A1 (fr) 1996-02-06 1997-08-14 Cruachem Limited Phosphoramidites de dinucleotides contenant du soufre
WO1998024884A1 (fr) 1996-12-02 1998-06-11 Genpharm International Animaux transgeniques non humains capables de produire des anticorps heterologues
EP0852493A1 (fr) 1995-09-27 1998-07-15 Laboratoires D'hygiene Et De Dietetique Systeme matriciel transdermique
WO1998037240A1 (fr) 1997-02-24 1998-08-27 Tm Technologies, Inc. Agents pharmacologiques a base d'oligonucleotides antisens
EP0889723A1 (fr) 1996-03-25 1999-01-13 LTS Lohmann Therapie-Systeme GmbH & Co. KG Systeme therapeutique transdermique a faible epaisseur de la zone d'application et a grande souplesse, et son procede de fabrication
EP0916336A1 (fr) 1996-03-11 1999-05-19 Fuisz Technologies Ltd. Système de délivrance transdermique
EP0944398A1 (fr) 1996-11-22 1999-09-29 Soltec Research Pty. Ltd. Dispositif d'administration par voie cutanee
WO2009097136A1 (fr) * 2008-02-01 2009-08-06 The Board Of Trustees Of The Leland Stanford Junior University Procédés et compositions se rapportant à des cellules souches de carcinome
WO2009151600A2 (fr) * 2008-06-10 2009-12-17 Tufts University Les protéines smad contrôlent la maturation des arnmi médiée par drosha

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1988001649A1 (fr) 1986-09-02 1988-03-10 Genex Corporation Molecules de liaison de chaines de polypeptide simples
EP0368684B1 (fr) 1988-11-11 1994-03-09 Medical Research Council Clonage de séquences d'immunoglobulines de domaines variables.
US5582981A (en) 1991-08-14 1996-12-10 Gilead Sciences, Inc. Method for identifying an oligonucleotide aptamer specific for a target
WO1993006213A1 (fr) 1991-09-23 1993-04-01 Medical Research Council Production d'anticorps chimeriques - une approche combinatoire
WO1995011910A1 (fr) 1993-10-27 1995-05-04 Ribozyme Pharmaceuticals, Inc. 2'-amido et 2'-peptido oligonucleotides modifies
EP0852493A1 (fr) 1995-09-27 1998-07-15 Laboratoires D'hygiene Et De Dietetique Systeme matriciel transdermique
WO1997029116A1 (fr) 1996-02-06 1997-08-14 Cruachem Limited Phosphoramidites de dinucleotides contenant du soufre
EP0916336A1 (fr) 1996-03-11 1999-05-19 Fuisz Technologies Ltd. Système de délivrance transdermique
EP0889723A1 (fr) 1996-03-25 1999-01-13 LTS Lohmann Therapie-Systeme GmbH & Co. KG Systeme therapeutique transdermique a faible epaisseur de la zone d'application et a grande souplesse, et son procede de fabrication
EP0944398A1 (fr) 1996-11-22 1999-09-29 Soltec Research Pty. Ltd. Dispositif d'administration par voie cutanee
WO1998024884A1 (fr) 1996-12-02 1998-06-11 Genpharm International Animaux transgeniques non humains capables de produire des anticorps heterologues
WO1998037240A1 (fr) 1997-02-24 1998-08-27 Tm Technologies, Inc. Agents pharmacologiques a base d'oligonucleotides antisens
WO2009097136A1 (fr) * 2008-02-01 2009-08-06 The Board Of Trustees Of The Leland Stanford Junior University Procédés et compositions se rapportant à des cellules souches de carcinome
WO2009151600A2 (fr) * 2008-06-10 2009-12-17 Tufts University Les protéines smad contrôlent la maturation des arnmi médiée par drosha

Non-Patent Citations (89)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1991, JOHN WILEY & SONS, article "Moderate hybridization conditions", pages: 6.3.1 - 6.3.6
"Current Protocols in Molecular Biology", 1991, JOHN WILEY & SONS, pages: 6.3.1 - 6.3.6
"Helvetica Chimica Acta", 1995, article "A multilingual glossary of biotechnological terms: (IUPAC Recommendations"
A. Z. OSKOWITZ ET AL: "Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 105, no. 47, 1 November 2008 (2008-11-01), pages 18372 - 18377, XP055022187, ISSN: 0027-8424, DOI: 10.1073/pnas.0809807105 *
ALEXANDER; AKHURST, HUM. MOL. GENET., vol. 4, 1995, pages 146 - A79,85
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
AMARZGUIOUI ET AL., CELL. MOL. LIFE SCI., vol. 54, 1998, pages 1175 - 202
ANNALISA PALMIERI ET AL: "Peptide-15 Changes miRNA Expression in Osteoblast-Like Cells", IMPLANT DENTISTRY, vol. 17, no. 1, 1 January 2008 (2008-01-01), pages 100 - 108, XP055022180, ISSN: 1056-6163, DOI: 10.1097/ID.0b013e318166d182 *
BEHR ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 6982 - 6986
BEIGELMAN ET AL., NUCLEIC ACIDS RES., vol. 23, 1995, pages 3989 - 94
BESTE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 1898 - 1903
BRANDÉN ET AL., NATURE BIOTECH., vol. 17, 1969, pages 784
BRANDEN ET AL., NATURE BIOTECH., vol. 17, 1999, pages 784
BRUDNO M., BIOINFORMATICS, vol. 19, no. 1, 2003, pages 154 - 162
BRUDNO, BIOINFORMATICS, vol. 19, no. 1, 2003, pages 154 - 162
CAI ET AL., PROC NATL ACAD SCI U S A., vol. 102, 2005, pages 5570 - 5575
CAI X; LU S; ZHANG Z; GONZALEZ CM; DAMANIA B; CULLEN BR: "Kaposi's sarcoma-associated herpes virus expresses an array of viral microRNAs in latently infected cells", PROC NATL ACAD SCI U S A., vol. 102, 2005, pages 5570 - 5575
COUTURE; STINCHCOMB, TRENDS GENET., vol. 12, 1996, pages 510 - 5
CZECH, NEJM, vol. 354, 2006, pages 1194 - 1195
DIAMOND ET AL., THE NEW ENGLAND JOURNAL OF MEDICINE, 1981, pages 1344 - 1349
DIAMOND, B.A. ET AL., THE NEW ENGLAND JOURNAL OF MEDICINE, 1981, pages 1344 - 1349
E. A. LIN ET AL: "miR-199a*, a Bone Morphogenic Protein 2-responsive MicroRNA, Regulates Chondrogenesis via Direct Targeting to Smad1", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 284, no. 17, 1 January 2008 (2008-01-01), pages 11326 - 11335, XP055022015, ISSN: 0021-9258, DOI: 10.1074/jbc.M807709200 *
ELBASHIR ET AL., GENES DEV., vol. 15, 2001, pages 188
ELBASHIR ET AL., NATURE, vol. 411, 2001, pages 494
ELBASHIR, S. M. ET AL., GENES DEV., vol. 15, 2001, pages 188
ELBASHIR, S. M. ET AL., NATURE, vol. 411, 2001, pages 494
EULALIO ET AL., CELL, vol. 132, 2008, pages 9 - 14
FELGNER ET AL., J. BIOL. CHEM., vol. 269, 1994, pages 2550 - 2561
FELGNER ET AL., PROC. NATL. ACAD. SCI USA, vol. 84, 1987, pages 7413 - 7414
FELGNER, P. L. ET AL., PROC. NATL. ACAD. SCI USA, vol. 84, 1987, pages 7413 - 7414
FU H; TIE Y; XU C; ZHANG Z; ZHU J; SHI Y; JIANG H; SUN Z; ZHENG X: "Identification of human fetal liver miRNAs by a novel method", FEBS LETT., vol. 579, 2005, pages 3849 - 3854, XP004961982, DOI: doi:10.1016/j.febslet.2005.05.064
FURER V ET AL: "The role of microRNA in rheumatoid arthritis and other autoimmune diseases", CLINICAL IMMUNOLOGY, ACADEMIC PRESS, US, vol. 136, no. 1, 1 July 2010 (2010-07-01), pages 1 - 15, XP027074866, ISSN: 1521-6616, [retrieved on 20100603], DOI: 10.1016/J.CLIM.2010.02.005 *
GRIFFITHS-JONES ET AL., NUCLEIC ACIDS RESEARCH, vol. 34, 2006, pages D140 - D144
GRIFFITHS-JONES ET AL., NUCLEIC ACIDS RESEARCH, vol. 36, 2008, pages D154 - D158
GRIFFITHS-JONES ET AL., NUCLEIC ACIDS RESEARCH, vol. 39, 2011, pages D152 - D157
GRIFFITHS-JONES ET AL.: "34", NUCLEIC ACIDS RESEARCH, 2006, pages D140 - D144
GRIFFITHS-JONES, NUCLEIC ACIDS RESEARCH, vol. 32, 2004, pages D109 - D111
GROSSHANS ET AL., J CELL BIOL, vol. 156, 2002, pages 17 - 21
JAYASENA, CLIN. CHEM., vol. 45, 1969, pages 1628 - 50
JAYASENA, CLIN. CHEM., vol. 45, 1999, pages 1628 - 50
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5877
KICHLER ET AL., BIOCONJ. CHEM, vol. 8, 1997, pages 213
KLUG; FAMULOK, M. MOL. BIOL. REP., vol. 20, 1994, pages 97 - 107
KLUG; FAMULOK, MOL. BIOL. REP., vol. 20, 1994, pages 97 - 107
KLUSSMANN ET AL., NAT. BIOTECHNOL., vol. 14, 1996, pages 1112 - 5
LAGOS-QUINTANA ET AL., SCIENCE, vol. 294, 2001, pages 853 - 858
LAGOS-QUINTANA M; RAUHUT R; LENDECKEL W; TUSCHL T: "Identification of novel genes coding for small expressed RNAS", SCIENCE, vol. 294, 2001, pages 853 - 858, XP002971837, DOI: doi:10.1126/science.1064921
LAGOS-QUINTANA; LAGOS-QUINTANA M; RAUHUT R; MEYER J; BORKHARDT A; TUSCHL T ET AL.: "New microRNAs from mouse and human", RNA., vol. 9, 2003, pages 175 - 179, XP002455766, DOI: doi:10.1261/rna.2146903
LANDGRAF ET AL., CELL, vol. 129, 2007, pages 1401 - 1414
LANDGRAF P; RUSU M; SHERIDAN R; SEWER A; LOVINO N; ARAVIN A; PFEFFER S; RICE A; KAMPHORST AO; LANDTHALER M: "A mammalian microRNA expression atlas based on small RNA library sequencing", CELL, vol. 129, 2007, pages 1401 - 1414, XP002490508, DOI: doi:10.1016/j.cell.2007.04.040
LANDGRAF P; RUSU M; SHERIDAN R; SEWER A; LOVINO N; ARAVIN A; PFEFFER S; RICE A; KAMPHORST AO; LANDTHALER M: "A mammalian microRNA expression atlas based on small RNA library sequencing", CELL., vol. 129, 2007, pages 1401 - 1414, XP002490508, DOI: doi:10.1016/j.cell.2007.04.040
LEUENBERGER ET AL., HELVETICA CHIMICA ACTA, 1995
LIM LP; GLASNER ME; YEKTA S; BURGE CB; BARTEL DP: "Vertebrate microRNA enes", SCIENCE, vol. 299, 2003, pages 1540
LIM LP; GLASNER ME; YEKTA S; BURGE CB; BARTEL DP: "Vertebrate microRNA genes", SCIENCE, vol. 299, 2003, pages 1540, XP002409653, DOI: doi:10.1126/science.1080372
LINDEMANN ET AL., MOL. MED., vol. 3, 1997, pages 466 - 76
MACK, NATURE BIOTECH., vol. 25, 2007, pages 631 - 638
MICHAEL ET AL., CANCER RES., vol. 1, 2003, pages 882 - 891
MICHAEL MZ; 0' CONNOR SM; VAN HOLST PELLEKAAN NG; YOUNG GP; JAMES RJ: "Reduced accumulation of specific microRNAs in colorectal neoplasia", MOL CANCER RES., vol. 1, 2003, pages 882 - 891, XP009051992
NELLEN; LICHTENSTEIN, TRENDS BIOCHEM. SCI., vol. 18, 1993, pages 419 - 23
NOLTE ET AL., NAT. BIOTECHNOL., vol. 14, 1996, pages 1116 - 9
NORIHISA FURUKAWA ET AL: "Optimization of a microRNA expression vector for function analysis of microRNA", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 150, no. 1, 2 December 2010 (2010-12-02), pages 94 - 101, XP028148659, ISSN: 0168-3659, [retrieved on 20101210], DOI: 10.1016/J.JCONREL.2010.12.001 *
PERSIDIS, NAT. BIOTECHNOL., vol. 15, 1997, pages 921 - 2
PERSIDIS, NAT. BIOTECHNOL., vol. 15, pages 921 - 2
PLANCK ET AL., J. BIOL. CHEM., vol. 269, 1994, pages 12918
SCHWARTZ ET AL., GENE THERAPY, vol. 6, 1999, pages 282
SKERRA, BIOCHIM. BIOPHYS. ACTA, vol. 1482, 2000, pages 337 - 50
SKERRA, BIOCHIM. BIOPHYS. ACTA, vol. 1982, 2000, pages 337 - 50
SKERRA, J. MOL. RECOGNIT., vol. 13, 2000, pages 167 - 187
SKERRA, MOL. RECOGNIT., vol. 13, 2000, pages 167 - 187
SPRINGER ET AL., MOL. CELL, vol. 2, 1988, pages 549 - 58
SPRINGER ET AL., MOL. CELL., vol. 2, 1988, pages 549 - 58
STEIN, LEUKEMIA, vol. 6, 1992, pages 697 - 74
THOMPSON ET AL., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 80
THOMPSON, J. D.; HIGGINS, D. G.; GIBSON, T. J., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 80
TOKUMITSU WATANABE ET AL: "Dnm3os , a non-coding RNA, is required for normal growth and skeletal development in mice", DEVELOPMENTAL DYNAMICS, vol. 237, no. 12, 1 December 2008 (2008-12-01), pages 3738 - 3748, XP055022186, ISSN: 1058-8388, DOI: 10.1002/dvdy.21787 *
TUTING ET AL., J. INVEST. DERMATOL., vol. 111, 1998, pages 183 - 8
UHLMANN, E.; PEYMAN, A., CHEMICAL REVIEWS, vol. 90, 1990, pages 543 - 584
UHLMANN; PEYMAN, CHEMICAL REVIEWS, vol. 90, 1990, pages 543 - 584
VAISH ET AL., NUCLEIC ACIDS RES., vol. 26, 1998, pages 5237 - 42
WANG ET AL., J. INVEST. DERMATOL., vol. 112, 1999, pages 775 - 81
WINTER, G.; MILSTEIN, C., NATURE, vol. 349, 1991, pages 293 - 299
WINTER; MILSTEIN, NATURE, vol. 349, 1991, pages 293 - 299
YACYSHYN ET AL., GASTROENTEROLOGY, vol. 114, 1998, pages 1142
YACYSHYN, B. R. ET AL., GASTROENTEROLOGY, vol. 114, 1998, pages 1142
YI ET AL., GENES DEV., vol. 17, 2003, pages 3011 - 3016
ZHAO; HUANG, BIOCHIM. BIOPHYS. ACTA, vol. 1189, 1991, pages 195 - 203
ZHAO; HUANG, BIOCHIM. BIOPHYS. ACTA, vol. 1189, 1994, pages 195 - 203
ZHENG; KEMENY, CLIN. EXP. IMMUNOL., vol. 100, 1995, pages 380 - 2

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015091963A3 (fr) * 2013-12-19 2015-10-15 Comprehensive Biomarker Center Gmbh Détermination de miarn plaquettaires dans l'infarctus aigu du myocarde
WO2019229489A1 (fr) * 2018-05-31 2019-12-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de mir-146a-5p et de mir-186 en tant que biomarqueurs de l'arthrose
CN112458163A (zh) * 2020-11-19 2021-03-09 杭州市中医院 一种早期痛风肾损害的生物标志物、试剂盒及应用
CN116919977A (zh) * 2023-06-02 2023-10-24 同济大学 mir-199a-3p在制备治疗骨关节炎的药物中的应用

Also Published As

Publication number Publication date
AR084331A1 (es) 2013-05-08
TW201239097A (en) 2012-10-01

Similar Documents

Publication Publication Date Title
KR101865433B1 (ko) 알파-l 이두로니다아제 (idua)에 대한 자연 안티센스 전사체의 저해에 의한 idua 관련된 질환의 치료
KR101892888B1 (ko) 시르투인 (sirt)에 대한 자연 안티센스 전사체의 저해에 의한 시르투인 (sirt) 관련된 질환의 치료
RU2611187C2 (ru) Лечение заболеваний, связанных с интерферон-регуляторным фактором 8 (irf8), путем ингибирования природного антисмыслового транскрипта к irf8
KR102547432B1 (ko) Tut4/7 발현 조절인자를 포함하는 암 예방 또는 치료용 약학적 조성물
US8980854B2 (en) miRNA compounds for treatment of prostate carcinoma
WO2012080461A1 (fr) Miarn dans une maladie articulaire
KR102584661B1 (ko) 염증 촉진 인자 발현 억제제, 그 유효 성분의 스크리닝 방법, 그 방법에 유용한 발현 카세트, 진단약 및 진단 방법
WO2012080459A1 (fr) Microarn utilisés dans le cadre d'une maladie articulaire
JP5812491B2 (ja) 腫瘍治療剤
WO2011019074A1 (fr) Acide nucléique qui contrôle la fibrose de cellules ou d'organes
WO2012084709A1 (fr) Miarn dans une maladie articulaire
KR102142791B1 (ko) miR-204 억제제의 골관절염 치료 용도
WO2012080460A1 (fr) Microarn utilisés dans le cadre d'une maladie articulaire
KR20240087624A (ko) 마이크로RNA-10a, 30c 및 451a를 이용한 피부 노화 측정용 바이오마커 또는 피부 노화 개선용 조성물
WO2011130464A1 (fr) Régulation traductionnelle de sparc par les micro-arn mir29a, b et c
KR20090103964A (ko) 마이크로ｒｎａ 활성 측정 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11804539

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11804539

Country of ref document: EP

Kind code of ref document: A1