JP2006500073A - Antisense compounds, methods and compositions for treating MMP-12 related inflammatory disorders - Google Patents

Abstract

The present invention relates to antisense oligonucleotide compounds for use in modulating the function of a nucleic acid molecule encoding mammalian MMP-12. More particularly, the present invention relates to nucleobases of 8 to 50 lengths that can specifically hybridize to a nucleic acid molecule encoding MMP-12, thereby inhibiting expression of the MMP-12 protein product. As well as their pharmaceutical compositions and methods of use thereof. The antisense oligonucleotide compounds are used to treat diseases such as inflammatory bowel disease, rheumatoid arthritis, psoriasis, emphysema and asthma.

Description

The present invention relates to oligonucleotides as antisense compounds. More specifically, the present invention relates to antisense oligonucleotides that can act as specific inhibitors of metalloproteinase 12 (in the present invention "MMP-12"), as well as their pharmaceutical compositions and uses thereof Provide a method.

  Metalloproteinases represent a superfamily of proteinases (enzymes) whose number has increased dramatically in recent years. These enzymes are classified into families and subfamilies based on structural and functional considerations (Hooper, 1994). Examples of metalloproteinases include matrix metalloproteinases (MMP) such as collagenases (MMP1, MMP8, MMP13), gelatinases (MMP2, MMP9), stromelysin (MMP3, MMP10, MMP11), matrilysin (MMP7), metalloelastase ( MMP12), enamelin (MMP19), MT-MMP (MMP14, MMP15, MMP16, MMP17); reprolysin (adaplysin), or the MDC family includes secretase and siddase. ), For example, TNF converting enzymes (ADAM10 and TACE); Enzymes such as Rose single proteinase (PCP); and other metalloproteinases, such as aggrecanase, the endothelin converting enzyme family and include the angiotensin converting enzyme family.

  Metalloproteinases (referred to in this way because of the presence of metal ions in their active centers) are mainly composed of extracellular matrix components, which are processes necessary for the growth and remodeling of tissues under normal physiological conditions. Involved in degradation.

  MMP-12 (also referred to as macrophage elastase or metalloelastase) was first cloned from mice by Shapiro et al. (1992) and also from humans by the same group in 1993 (Shapiro et al., 1993).

  Perhaps not surprisingly, metalloproteinases are implicated in the pathogenesis and progression of many disease types and conditions because of the widespread involvement of metalloproteinases in all kinds of processes. For example: various inflammatory and allergic diseases such as joint inflammation (especially rheumatoid arthritis, osteoarthritis and gout), gastrointestinal inflammation (especially inflammatory bowel disease, ulcerative colitis, gastritis, And Crohn's disease), skin inflammation (especially psoriasis, eczema, dermatitis); tumor metastasis or invasion; diseases associated with uncontrolled degradation of the extracellular matrix, such as osteoarthritis; bone resorption disease ( Diseases related to ectopic angiogenesis; diabetes, periodontal disease (eg gingivitis), corneal ulceration, skin ulceration, post-operative conditions (eg colon adhesions), and Enhanced collagen remodeling associated with skin wound healing; demyelinating diseases of the central and peripheral nervous system (eg, multiple sclerosis); Alzheimer's disease; observed in cardiovascular disease Extracellular matrix remodeling, for example, restenosis which, and atherosclerosis; asthma; rhinitis; and involved in chronic obstructive pulmonary disease (COPD).

Considerable scientific evidence has shown that uncontrollable binding matrix metalloproteinase (MMP) activity is involved in the progression of many diseases observed with MMP, and as a result Inevitably, inhibiting these enzymes has become an attractive target for therapeutic intervention (Matritian, 1992; Emonard et al., 1990; Docherty, 1990).

  Realizing the transition of MMP inhibitors to clinical conditions always comes with associated problems. Extensive inhibition of MMP in clinical conditions results in musculoskeletal stiffness and pain (Rasmussen and McCann, 1997). These side effects and effects associated with widespread inhibition can be enhanced with prolonged administration. Accordingly, it can be extremely advantageous to provide selective MMP inhibitors that can only prevent the activity of a particular subject MMP.

There is a study that MMP-12 is required for the development of cigarette smoke-induced emphysema in prior art mice (Hautamaki et al., 1997). This is further supported by a publication that showed that transgenic mice without functional integrin αvβ6 develop MMP-12-dependent emphysema (Morris et al., 2003).

  Rapid breakdown of the extracellular matrix of articular cartilage is an important feature in both rheumatoid arthritis and osteoarthritis pathologies, and current evidence indicates that inappropriate MMP synthesis is a key event It suggests. In addition, various MMPs can hydrolyze the membrane-bound precursor of the proinflammatory cytokine tumor necrosis factor α (TNF-α) (Gearing et al., 1994). This cleavage results in mature soluble TNF-α and MMP inhibitors can block the production of TNF-α both in vitro and in vivo (Mohler et al., 1994 and McGeehan et al., 1994).

  The observation that the majority of MMP-12 is macrophage specific supports the discovery that macrophages require MMP-12 to allow macrophage migration to the inflammatory site (Shipley et al., (1996). Moreover, macrophages themselves provide a means to maintain inflammation, in which they are involved in the production of many proinflammatory inflammatory cytokines. Thus, targeting MMP-12 and thereby reducing macrophage migration may provide a new therapeutic potential for treating inflammatory conditions.

  In this regard, few selective MMP-12 inhibitors appear to be reported, and non-selective or non-selective MMP-12 inhibitors may treat any disease in any mammal. Approved for or on the market. Accordingly, there is a need to find new compounds that are effective selective MMP inhibitors and have an acceptable therapeutic index of toxicity / efficacy that allows clinical use in the prevention and treatment of related disease states. is there.

  The object of the present invention is to provide such selective compounds in the form of antisense oligonucleotides.

The present invention provides antisense oligonucleotide compounds for use by modulating the function of a mammalian MMP-12-encoding nucleic acid molecule and ultimately by regulating the production of MMP-12. More particularly, the invention relates to nucleic acids 8-50 in length that can specifically hybridize to a nucleic acid molecule encoding MMP-12, thereby blocking the production of the MMP-12 protein product. A compound comprising a base is provided. Further provided are methods and compositions for modulating the expression of MMP-12 in a cell or tissue, the method contacting said cell or tissue with one or more antisense compounds or compositions of the invention. Including that. This is accomplished by providing an antisense compound that specifically hybridizes to the nucleic acid encoding the MMP-12 protein product.

  The invention is further defined in the appended claims, which are incorporated into this invention by reference.

DESCRIPTION OF THE DRAWINGS The present invention is explained in more detail in the following description, examples and accompanying drawings:
FIG. 1 shows RT-PCR analysis of MMP-12 expression in biopsy samples from patients with either ulcerative colitis (A) or Crohn's disease (B). The experimental protocol is outlined in Example 2. (Symbol: M is a base pair marker; H indicates a biopsy from a completely normal healthy individual; C indicates a biopsy sample taken from a non-inflamed area; and T is (A biopsy taken from an inflamed area of the same patient shows the number in parentheses indicates the patient number and the horizontal bar displays C and T biopsy samples from the same patient) . α-actin is used as a loading control and represents the expression state of a housekeeping gene that is commonly used to demonstrate equal mRNA input in all RT-PCR reactions.

FIG. 2 shows a histogram showing the four different criteria used to evaluate the improvement in the degree of inflammation in the gastrointestinal tract after administration of the antisense compound. In this example, the antisense compound was obtained according to SEQ ID NO: 3 and the experimental protocol was that used in Example 2. (Symbols: black bars indicate healthy animals that received only standard drinking water (healthy controls); hatched bars indicate 2.5% DSS that induces inflammation of the colon in the drinking water. 2 shows an animal (disease control) in which colitis was administered.
Plaid bars represent animals that received the antisense compound outlined in Example 7 (SEQ ID NO: 3) in addition to DSS in their drinking water). Thick black bars indicate negative controls versus sick animal controls. A thin black bar shows a comparison between the sick animal control and the MMP-12 antisense treated group. Histologically, it is graded from 0 to 4 according to the scale shown in Table 1. Significance is shown as * P <0.05, ** P <0.001 and *** P <0.0005. Error bar: SEM.

  FIG. 3 shows a histological section of mouse colon tissue obtained in Example 2. A) Normal marginal epithelium, healthy colon with normal Lieberkun glands, few inflammatory cells, B) Loss of marginal epithelium, impaired crypt structure, and large inflammatory cells Inflamed colon showing extensive invasion, C) Injured but maintained marginal epithelium, normalized crypts, little infiltration of inflammatory cells, recovery of inflammation Shown colon treated with MMP-12 antisense obtained in SEQ ID NO: 3. A layer of mucus can be observed on top of the marginal epithelium. Bar = 50 μm.

DETAILED DESCRIPTION OF THE INVENTION Prior to the disclosure and description of the method of the present invention, the present invention is not limited to the specific configurations, process steps and materials disclosed in the present invention, but such configurations, process steps. It should be understood that the materials can be somewhat modified. Also, since the scope of the present invention is limited only by the appended claims and their equivalents, the terminology used in the present invention is used only for the purpose of describing particular embodiments and is not limited. It should also be understood that there is no intention to do so.

  In the context of the present invention, “antisense” as used in “antisense molecule” and “antisense sequence” means a single-stranded RNA or DNA molecule that is complementary to the mRNA portion of the target gene. Antisense molecules base pair with the mRNA, thereby preventing translation of the mRNA into protein. Consequently, the term “antisense therapy” refers to an anti-sense that specifically hybridizes to a target nucleic acid and modulates its function or translation, eg, by suppressing or reducing the expression of the gene product encoded by said sequence. It means a method using a sense compound.

  In the context of the present invention, “complementary” means the ability for precise pairing between two nucleotides.

  In the context of the present invention, “hybridization” means hydrogen bonding, such as Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding between complementary nucleoside or nucleotide bases. Thus, complementarity and hybridization are terms used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an oligonucleotide and a DNA or RNA target. .

  An antisense compound is non-sense-sensitive when the compound interferes with the normal function of the target DNA or RNA by binding to the target DNA or RNA molecule, causing loss of potency, and in situations where specific binding is desired. Specific hybridization is possible if there is a sufficient degree of complementarity to prevent non-specific binding to a specific target sequence.

  Moreover, in the context of the present invention, “hybridization under stringent conditions” refers to standards well known to those skilled in the art for temperature and buffer (Ausubel et al., 1991).

  As is well known in the art, “functionally homologous” is a sequence that probably has structurally low homology to the disclosed sequence, but in vivo in either a healthy or diseased organism. Means that it encodes a highly similar protein that exhibits a homologous function, for example, the same protein or a similar cellular function.

  As is well known in the art, “functionally inserted” or “operably inserted” means that the insertion of a sequence into the host genome is in a direction and position such that correct expression of the sequence occurs. , If applicable, indicates that the expression is in conjunction with a promoter and / or enhancer such that correct expression of the sequence occurs.

  In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) of gene expression. In the context of the present invention, the preferred form of regulation of gene expression is inhibition and the preferred target is mRNA.

The present invention provides oligonucleotide compounds for use in modulating the function of a nucleic acid molecule encoding mammalian MMP-12 and controlling the amount of MMP-12 ultimately produced. More specifically, the compound is an antisense oligonucleotide complementary to MMP-12 mRNA. This modulation is achieved by providing an antisense compound that specifically hybridizes to the nucleic acid encoding the MMP-12 protein product, thereby inhibiting translation of MMP-12. In one embodiment, the target sequence is a human, the antisense compound is preferably SEQ ID NO: 1 ^{(GenBank (R)} Accession No. NM-002,426), or functionally equivalent to those homologues Hybridize.

In another embodiment, the target sequence is SEQ ID NO: 2 ^{(GenBank (R)} Accession No. M82831), or a sequence of mouse functionally equivalent homologue thereof.

  Antisense compounds directed to one or other of the above target sequences or their equivalents according to the present invention preferably comprise from about 8 to about 50 nucleobases in length. Antisense oligonucleotides containing about 8 to about 30 nucleobases (ie, about 8 to about 30 linked nucleosides in length) are particularly preferred, with oligonucleotides containing about 16 to about 24 nucleobases being most preferred preferable.

The antisense oligonucleotide according to the present invention is either a DNA molecule or an RNA molecule. The present invention relates to MMP-12, as defined above and in particular as set forth in SEQ ID NOs: 3-14 (see Table 2 and the attached sequence listing made with PatentIn 3.1). Nucleic acid molecules in the form of antisense oligonucleotide molecules that can specifically hybridize to the encoding nucleic acid molecule and thereby block production of the MMP-12 protein product are made available.

  In a further aspect of the invention, the term “oligonucleotide” means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or a mimetic thereof. The term also encompasses oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides with non-naturally occurring modifications. As is known in the art, the phosphate group in an oligonucleotide structure is generally said to form the internucleoside backbone of the oligonucleotide. The natural bond or backbone of RNA and DNA is a 3 'to 5' phosphodiester bond.

  Particular examples of preferred antisense compounds useful in the present invention include modified backbones or oligonucleotides containing non-natural internucleoside linkages. These modifications introduce certain desirable properties (such as reduced toxicity properties, increased stability against nuclease degradation, and enhanced cellular uptake) to oligonucleotides that are not obtainable by naturally occurring oligonucleotides. Making it possible.

  According to a further embodiment, the antisense oligonucleotide comprises at least one modified nucleobase, which contains an uncrosslinked oxygen atom of the antisense nucleic acid backbone, methane phosphate, methyl phosphate, and Phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-amino phosphoramidates Chemical modification by substitution with a component selected from the group consisting of dating and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, and thionoalkylphosphotriesters Possible it is.

  According to one embodiment, the substitution can occur at one or more nucleotides independently selected from the last three nucleotides at the 3 'end and / or the 5' end of the oligonucleotide. Substitutions may also occur at any position along the length of the oligonucleotide, or in fact all intranucleoside linkages may be modified. Preferably, the oligonucleotide comprises at least one modified sugar component nucleobase, and the modified sugar component can be a 2'-O-methoxyethyl sugar component.

  The antisense agent can also be an antisense agent composed of DNA or RNA, or an analog or mimic of DNA or RNA, which can include the following: methylphosphonate, N3 ′ → P5′-phosphoramidate, morpholino, peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinosyl nucleic acid (ANA), fluoro-arabinosyl nucleic acid (FANA) methoxy-ethyl nucleic acid (MOE), but not limited thereto . Preferably, the antisense agent is a homopolymer or heteropolymer comprising the DNA or RNA, or analogs or mimic combinations of DNA or RNA.

  In further embodiments, the antisense compounds of the invention can be used for therapy and as prophylaxis. With respect to treatment, animals, preferably humans, who may be suffering from a disease or disorder associated with inappropriate MMP-12 expression that can be treated by modulating the expression of MMP-12 are therapeutically or prophylactically effective. Treated by administering an amount of an antisense compound of the invention. The compounds of the present invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention can be useful prophylactically (ie, delay the onset of a disease or condition suspected of involving MMP-12).

  In still other embodiments, the antisense compounds of the invention are useful in investigating and diagnosing human subjects because they hybridize to nucleic acids encoding MMP-12 and take advantage of this fact. This is to make it easier to design sandwiches and other analyzes as possible. Hybridization of the antisense oligonucleotide of the present invention with a nucleic acid encoding MMP-12, and the resulting suppression / inhibition of MMP-12 expression can be detected by means well known in the art. For example, radiolabeling of antisense compounds, RNase protection analysis can demonstrate specific hybridization of antisense compounds to target mRNA of MMP-12. Various means known in the art to detect a decrease in MMP-12 levels can be used, such as antibody detection of MMP-12 or enzyme-based activity analysis. In other embodiments, the antisense compound is used in a method of inhibiting the expression of MMP-12 in a cell or tissue, wherein the cell or tissue is treated with a therapeutically effective dose of the invention in vivo or in vitro. Contacting a compound or composition thereby inhibiting expression of MMP-12. Preferably, said inhibition suppresses MMP-12 dependent processes in human subjects. The MMP-12 dependent process is most preferably one of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, rheumatoid arthritis, psoriasis, emphysema and asthma.

  Another embodiment of the invention relates to a method of diagnosing inflammatory bowel disease in a human subject, the method comprising screening for the presence or absence of expression of MMP-12, wherein the expression of MMP-12 is , An indicator of inflammatory bowel disease.

  Because the compounds of the present application are selective, the compounds of the present application are expected to be useful for long-term therapy with reduced complications associated with various known inhibitors. Thus, the compounds of the present application are useful for the treatment of various diseases and conditions mediated by MMP-12, while these selective inhibitors are particularly useful for the treatment of disorders having significant inflammatory components. is there.

As an alternative to targeted antisense delivery, targeted ribozymes may be used. The term “ribozyme” refers to an RNA-based enzyme that can target and cleave specific base sequences of both DNA and RNA. Ribozymes can either target cells directly in the form of RNA oligonucleotides incorporating ribozyme sequences, or can be introduced into cells as expression vectors encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same way as described for antisense polynucleotides. Ribozyme sequences may also be modified in much the same way as described for antisense polynucleotides. For example, the ribozyme sequence may incorporate non-Watson-Crick bases, form a mixed RNA / DNA oligonucleotide, or modify the phosphodiester backbone.

  Another alternative to antisense is the use of so-called “RNA interference” (RNAi). Double stranded RNA (dsRNA) can cause gene silencing in a number of in vivo environments, including Drosophila, Caenorhabditis elegans, planaria, hydra, trypanosoma, fungi , Plants and mammals. RNAi's natural function and, together with repression, remove genomes from invasion by mobile genetic elements such as retrotransposons and viruses that produce abnormal RNA or dsRNA in host cells when activated. It appears to protect (Jensen et al., 1999; Ketting et al., 1999; Ratcliff et al., 1999; Tabara et al., 1999). Specific mRNA degradation prevents transposons and viral replication, but several viruses can overcome or prevent this process by expressing proteins that suppress PTGS (Lucy et al., 2000). Double-stranded RNA molecules can be produced by a method comprising the following steps: (a) synthesizing two RNA strands each having a length of 19-25, for example 19-23 nucleotides (see above) The RNA strand can form a double stranded RNA molecule, preferably at least one strand has a 3′-overhang of 1-5 nucleotides), (b) a target specific nucleic acid Binding of the synthesized RNA strand under conditions that form double stranded RNA molecules capable of mediating modification of RNA, particularly RNA interference and / or DNA methylation. In one embodiment, the antisense RNAi comprises at least 8 nucleotide portions comprised in one of the sequences of SEQ ID NOs: 3-14 and has a total length of 25 or fewer nucleotides.

  dsRNA is usually administered as a pharmaceutical composition. Such administration can be performed by known methods in which the nucleic acid is introduced into the desired target cell in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation, microinjection, and viral methods. Such a method is taught in Current Protocols in Molecular Biology, Ausubel et al. (1993).

  The invention also makes available a pharmaceutical composition, said composition comprising a compound or antisense agent as described above and a pharmaceutically acceptable formulation and composition, carrier or diluent. Preferably, the pharmaceutical composition further comprises a colloidal dispersion system. The pharmaceutical compositions of the invention can be administered in a number of ways, depending largely on whether the local, surface or systemic mode of administration is optimal for the condition to be treated. These various modes of administration can be, for example, surface (eg on the skin), topically (eg ocular and various mucous membranes such as vaginal, nasal and rectal delivery), orally or parenterally, and to the lungs The mode of administration.

  The manufacture of such compositions and formulations is generally known to those skilled in the pharmaceutical and pharmaceutical arts and is applicable to the formulation of the compositions of the present invention.

  Within the scope of the present invention, preferred examples of pharmaceutically acceptable salts include: (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine. (B) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid and the like; (c) salts formed with organic acids such as acetic acid , Alginic acid, ascorbic acid, benzoic acid, citric acid, fumaric acid, gluconic acid, maleic acid, methanesulfonic acid, naphthalenedisulfonic acid, naphthalenesulfonic acid, oxalic acid, palmitic acid, polyglutamic acid, p-toluenesulfonic acid, polygalacturon Salts of acids, succinic acid, tartaric acid, tannic acid and the like; and (d) chlorine, bromine and iodine Elemental anions salt formed from such as iodine, but not limited to.

  In still other embodiments, the pharmaceutical compositions and formulations for surface administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Can be mentioned. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

  Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids, or binders may also be desirable.

  Compositions and formulations for parenteral, intrathecal or intraventricular administration include sterile aqueous solutions, which include buffers, diluents and other suitable additives such as penetration enhancers, carrier compounds , And other pharmaceutically acceptable carriers or excipients.

  The pharmaceutical composition of the present invention includes, but is not limited to, solutions, emulsions, and liposome-containing preparations. These compositions can be made from a variety of components, including but not limited to pre-prepared liquids, self-emulsifying solids and self-emulsifying semisolids. In general, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Typically, the manufacture of such compositions involves combining a therapeutic agent with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates, such as Glucose, sucrose or dextrin, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Examples of suitable diluents include neutral buffered saline or saline mixed with non-specific serum albumin.

  The pharmaceutical formulations of the invention can conveniently be present in unit dosage form and can be prepared according to conventional techniques well known in the pharmaceutical industry.

  In yet other embodiments, the compositions of the present invention can be manufactured and formulated as an emulsion, typically an emulsion comprising one liquid in droplets dispersed in another liquid. Heterogeneous systems (Idson, 1988). Examples of naturally occurring emulsifiers used in emulsion formulations include gum arabic, beeswax, lanolin, lecithin and phosphatide. The application of emulsion formulations for the dermal, oral and parenteral routes and methods for their production have been reviewed in the literature (Idson, 1988).

  In one embodiment of the invention, the oligonucleotide and nucleic acid composition can be formulated as a microemulsion. A microemulsion is defined as a system of water, oil and amphiphile and is a single, optically isotropic, thermodynamically stable liquid solution (Rosoff, 1988).

  Another embodiment of the invention is the use of liposomes to transfer and deliver active ingredients to the site of action. Since liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissue, the liposomes begin to fuse to the cell membrane. This phenomenon has facilitated extensive research in the use of liposomes as a potential mode of drug delivery.

  In other embodiments, the use of permeation enhancers can be useful as a mode of drug delivery. Such substances are classified as belonging to one of five broad categories (ie surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants) (Lee et al., 1991).

  In other related embodiments, the composition of the invention comprises one or more antisense compounds that target a first nucleic acid, in particular an oligonucleotide, and one or more that target a second nucleic acid target. Further additional antisense compounds may be included. Two or more combined compounds can be used together or sequentially.

  The formulation of therapeutic compositions and their subsequent administration is considered within the skill of the art. The optimal dosing schedule can be calculated by measuring drug accumulation in the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. After good treatment, it may be desirable to have the patient undergo maintenance therapy to prevent recurrence of the condition, in which case the oligonucleotide is administered in a maintenance dose.

  The invention also relates to a recombinant nucleotide sequence comprising an antisense compound according to the invention. The recombinant nucleotide sequence can be inserted into an expression vector, such as a plasmid or virus, or any other vector known to those skilled in the art. Accordingly, the present invention includes an antisense oligonucleotide sequence operably linked to one or more expression regulators so that in vivo or in vitro expression of the antisense compound can be achieved. The vector that can contain the antisense oligonucleotide can be eukaryotic or prokaryotic.

  One embodiment of the present invention is a method of inhibiting expression of MMP-12 in a cell or tissue, wherein the cell or tissue is a recombinant nucleotide sequence expressed by a recombinant vector in vivo or in vitro. Contact with. The invention also includes host cells transformed with these antisense oligonucleotide sequences operably linked to one or more expression regulators.

  The present invention specifically relates to inappropriate MMPs by administering a therapeutically or prophylactically effective amount of one or more antisense compounds or compositions of the invention designed to modulate MMP-12 expression. Provided are compounds and methods for treating animals, particularly humans, who have or are suspected of having a human disease associated with -12 modulation.

  The invention also provides for transgenic cells themselves as well as transgenic non-human animals. Transgenic animals include animals containing viable transgenic cells or organs, as well as any one of the antisense oligonucleotide sequences of the present invention (SEQ ID NOs: 3-14), or their functional The whole animal is functionally integrated into the genome under the control of an appropriate expression cassette. Such animals are useful as research tools for investigations regarding the etiology of MMP-12-related disorders, progression, diagnosis and treatment of MMP-12-related disorders. As is well known in the art, expression cassettes containing these appropriate promoters and enhancers can be used in vectors so that expression of the desired antisense DNA sequence is achieved, thereby resulting in in vivo or in vitro inhibition of MMP-12 production. It is introduced into the target cells in the form of Thus, in one embodiment, the sequences of the invention may be overexpressed so that intended suppression of the target is achieved in the cell in which the antisense compound is expressed.

Accordingly, one embodiment of the present invention is such a transgenic cell, organ or animal and against such diseases as a model for investigating the nature and / or etiology of MMP-12 related diseases. Their use as models for assessing the effectiveness of pharmaceuticals as well as models for investigating the effects of substances known and suspected to cause such diseases.

  Once the involvement of MMP-12 in an inflammatory disorder is confirmed, the present invention further provides a screening assay to identify substances that modulate the activity of MMP-12 by altering the activation of the MMP-12 molecule. .

  As a substance useful in the screening method of the present invention, a newly synthesized compound, a commercially available compound, a known compound registered in a chemical file but not known for various activities, or obtained by combinatorial chemistry technology. A series of compounds can be used. In addition, culture supernatants of microorganisms, natural components obtained from plants or marine organisms, animal tissue extracts, and the like can be used.

  In this method, a change in the activity level of MMP-12 is easily observed under conditions that allow MMP-12 to interact with a substance that is predicted to be capable of modifying MMP-12 activity. Contacting MMP-12. A preferred mode of change in activity level is inhibition of substances with MMP-12 activity or antagonism.

  In the present invention, analysis for identifying a substance that modifies the specific activity of MMP-12 can be performed, for example, in vitro or cell-based analysis, and monitoring changes in MMP-12 activity. Preferred methods include, for example, measuring MMP-12 proteolytic activity against several known MMP-12 substrates (eg, tropoelastin, osteonectin, vitronectin, and fibronectin) in the form of zymographic analysis. Can be mentioned. Such a method is taught, for example, in Ausubel et al., 1991.

  In other embodiments, screening for compounds having MMP-12 antagonism can be performed using solid phase combinatorial library methods.

  Such a library is constructed, for example, as a one bead-2 compound library such that each bead contains one general quenching fluorogenic substrate and one of a variety of putative inhibitors. be able to. After incubation with MMP-12, beads containing the active inhibitor can be easily recovered and the inhibitor compound structure can be analyzed using, for example, a MALDI-TOF mass spectrometer (Franz et al., 2003). .

  Although the present invention has been specifically described according to certain preferred embodiments, the following examples are provided merely to illustrate the invention and do not limit the invention. The following examples are typical of those that can be used, and other methods known to those skilled in the art may be used instead of these without undue experimentation.

Example 1. Identification of MMP-12 overexpressed in human inflammatory conditions
Appropriate biopsy material collection Biopsies were collected from patients selected based on clinical and pathological evidence of having an inflammatory condition of CD or UC. In each patient, a total of three biopsies were collected from the inflamed area of the colon, and at the same time, three biopsy samples from non-inflamed areas were collected. This was done in a total of 16 different patients (of which 8 were diagnosed with CD (patients 1-8) and the remaining 8 were diagnosed with UC (patients 9-16)). In the UC patient group, 2 are women, 6 are men, and the age range is 29-77 years. Correspondingly, in the CD age group, 3 are women, 5 are men, and the age range is 27-59.

  Biopsy of each anatomical site of one patient is pooled and total RNA isolation is done using Qiagen's Rneasy kit and Pellet Pestel motor homogenizer according to the manufacturer's protocol. It was. Thirty-two total RNA samples were isolated with two samples per patient: an inflamed (target) sample and a non-inflamed (control) sample.

Implementation of RNA cDNA synthesis For the first strand cDNA synthesis, 2 μg of each RNA sample was used and 10 pM oligo-dT-primer dT-joint (5′-TAG TCT ATG ATC GTC GAC GGC TGA TGA AGC GGC CGC TGG Three restriction enzyme cleavage sites (SalI, NotI and BpmI) were introduced into each synthesized cDNA molecule using AGT TTT TTT TTT TTT TTT TTV-3 ′ (SEQ ID NO: 15) Buffer, deoxynucleotide trilin Acid (dATP, dCTP, dGTP and dTTP) and the enzyme reverse transcriptase (Superscript II) were purchased from Gibco BRL and reacted according to the manufacturer's guidelines. The synthesis of The reaction mixture (excluding the enzyme) was preincubated for 5 minutes at 65 ° C. in a PCR machine (PCR splint, Hybaid), cooled on ice and then preheated to 42 ° C. Then, the enzyme Superscript II was added, and the mixture was incubated at 42 ° C. for 1 hour in a PCR machine (PCR Sprint, Hybide).

  For second strand synthesis, a second strand buffer mix (41 μl) is added to the reaction according to the provided protocol (Gibco BRL) and 4 μl E. coli. E. coli polymerase I (New England Biolabs), 1.5 μl of E. coli. coli DNA ligase (New England Biolabs) and 0.7 μl of RNase H (Gibco BRL) were added to a total volume of 160 μl. The reaction was incubated in a PCR machine (PCR splint) at 16 ° C. for 2.5 hours and then purified using the Qiagen PCR purification kit according to the protocol provided. These samples were eluted with elution buffer (32 μl), and each sample (26 μl) was used in the following steps.

Due to the limited amount of material obtained from amplification biopsy of the 3 ′ end of the cDNA , a pre-amplification step was necessary. For amplification of the 3 ′ end of the cDNA in vitro, cDNA from all samples (26 μl) was digested with the restriction enzyme DpnII (10 U) for 3 hours at 37 ° C. in a volume of 30 μl. The cleaved cDNA was purified again using Qiagen PCR purification kit and the cDNA was eluted with elution buffer (47 μl). The following circular ligation step was performed in a volume of 50 μl containing 44 μl of cDNA cut with DpnII and 2000 U of T4 DNA ligase (New England Biolabs). These reaction mixtures were incubated at 22 ° C. for 1 hour, heat inactivated at 65 ° C. for 10 minutes, and each reaction mixture (25 μl) was used for the amplification step. Prepare a mixture for 5 PCR reactions per sample (5 × 50 μl = 250 μl total) and mix this mixture with 25 μl of cDNA (cut with DpnII and circularly ligated) and 25 μl of 10 × Advantage. 2 PCR buffer (Clontech), 5 μl joint-Not primer (10 pmol / μl; 5′-TGA TGA AGC GGC CGC TGG-3 ′ (SEQ ID NO: 16)), 5 μl joint-Sal primer (10 pmol / μl) 5′-TTC ATC AGC CGT CGA CGA TC-3 ′ (SEQ ID NO: 17), 5 μl of 10 mM dNTP mix, and 5 μl of 50 × Advantage 2 Taq-polymerase (Clontech) PCR mix for each sample Was distributed into five PCR reaction tubes and PCR was performed under the following conditions: 94 ° C for 1 minute, then 16x (94 ° C for 20 seconds, 55 ° C for 20 seconds, 72 ° C for 1 minute) ).

  Four reactions per sample were removed and placed on ice, and the optimal number of cycles was determined using one of the reactions per sample. The optimal number of cycles for all 32 samples was determined to be 18 cycles, so the remaining 4 reactions per sample would contain 2 additional cycles [2 × (94 ° C. for 20 seconds, 55 ° C. for 20 seconds). Second at 72 ° C. for 1 minute)]. Four PCR reactions per sample were pooled (to a total volume of 200 μl), followed by purification using the Qiagen PCR purification kit, eluting with elution buffer (34 μl). The purified reaction solution was used as a starting material for the identification protocol of differentially expressed genes.

Isolation of differentially expressed cDNA from human biopsy (subtraction protocol) Isolation of differentially expressed cDNA was performed using the protocol outlined in von Stein OD, 2001 (with some changes to the protocol). Including).

Screening for differentially expressed genes For construction of a cDNA library, 2000 clones from each subtraction were plated on one 22 cm ² agar plate. From these plates, using a BioPick machine of BioRobotics (Cambridge, UK), 384 colonies were picked and a 384 well plate (100 μl / ampicillin) containing LB medium (70 μl / well). (including Maniatis et al., 1989). Bacterial clones were incubated overnight at 37 ° C. and then used for colony PCR. This PCR was performed in a 384 PCR well plate with a volume of 20 μl per sample. One PCR reaction contains: 2 μl of 10 × PCR buffer, 0.4 μl of Sport-Not primer (10 pmol of 5′-CGT AAG CTT GGA TCC TCT AGA GC-3 ′ (SEQ ID NO: 18)) 0.4 μl of Sport-Sal primer (10 pmol of 5′-TGC AGG TAC CGG TCC GGA ATT CC-3 ′ (SEQ ID NO: 19)), 1.6 μl of dNTP mix (25 mM each nucleotide), 0.4 μl 0.1% bromophenol blue and 0.5 μl of DynAzyme Taq-polymerase (2 U / μl; Finnzyme). A master mix for all reactions was prepared and distributed and then inoculated with 384 plastic replicas. PCR cycle parameters are as follows: 94 ° C. for 2 minutes, 37 × (94 ° C. for 30 seconds; 50 ° C. for 30 seconds, 72 ° C. for 1 minute), and 72 ° C. for 5 minutes.

After amplification, the PCR reaction was performed using a Hybond N ⁺ membrane (Amersham (Am
ersham)) and stopped using a biorobotic Microgrid TAS. All clones were spotted in duplicate and genomic DNA was used as a guide dot. On one filter, 383 genes from all four subtractions were placed. Twenty-four duplicates were made for analysis by hybridization with various radioactive cDNA probes. These filters were then hybridized with radiolabeled subtracted cDNA from all 8 patients. Sixteen filters were used for 16 different hybridization experiments. CDNA (1 μl) was used for labeling with Klenow polymerase. The hybridization protocol used was the Church protocol outlined in Church and Gilbert, 1984.

Phospho-imager Fujifilm BAS1800H
with BAS1800IIIR program, Array vision version 6.0 (Imaging Research, Inc.)
ch Inc)), sequence analysis, and BLAST analysis were used to determine the extent of differential expression and the identity of the isolated differentially expressed genes.

Confirmation of true differential expression In order to confirm the results of expression profiling experiments, RT-PCR analysis was performed using gene-specific primers and primers for α-actin (control). The original cDNA from each of the 8 UC patients and each of the 8 CD patients was used. The cDNA was then diluted 1: 250 with distilled water and 5 μl aliquots were used in one single PCR reaction. 1 × PCR buffer (with Taq-polymerase) (finzyme), 0.5 μl of 25 mM dNTP-mix, 10 pM MMP-12 or α-actin forward and reverse primers, and 1 unit of DynZyme (finzyme Taq- The reaction was performed in a total volume of 50 μl containing polymerase. PCR reactions were performed on a Thermohybaid thermocycler under the following conditions: 94 ° C for 1 minute and N cycle (94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute). ) And 5 minutes at 72 ° C. For MMP-12, 30 cycles were performed (N = 30), and for α-actin, 28 cycles (N = 28) were performed. After completion, 5 μl of each reaction was loaded onto a 1 × TAE agarose gel and then stained with ethidium bromide.

MMP-12 forward: 5′-GAC TTC CTA CTC CAA CGT ATC ACC-3 ′ (SEQ ID NO: 20)
MMP-12 reverse: 5′-CTC AGT CCA AGG ATG TTA GGA AGC-3 ′ (SEQ ID NO: 21)
α-actin forward: 5′-GTG CAG GGT ATT AAC GTG TCA GGG-3 ′ (SEQ ID NO: 22)
α-actin reverse: 5′-CCA ACT CAA AGC AAG TAA CAG CCC ACG G-3 ′ (SEQ ID NO: 23).

  From these analyses, it can be concluded that upregulation of MMP-12 occurs in most cases in both human UC and CD states (see FIG. 1).

Example 2 Analysis of antisense oligonucleotide inhibition of MMP-12 Antisense modulation of MMP-12 expression can be analyzed in various ways well known in the art. For example, MMP-12 mRNA levels can be quantified, for example, by Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). RNA analysis can be performed on total cellular RNA or poly (A) + mRNA. RNA isolation methods are described, for example, in Ausubel et al., 1992. Northern blot analysis is routine in the art and is described, for example, in Ausubel et al., 1992. Real-time quantitative PCR was performed using ABI PRISM.
TM.7700 Sequence Detection System (PE-Applied Biosystems (Applied
Biosystems (available from Foster City, Calif., USA) can be conveniently achieved and used according to the manufacturer's instructions. Other PCR methods are also known in the art.

Similarly, MMP-12 protein levels can be quantified by various methods well known in the art such as immunoprecipitation, Western blot analysis (immunoblot), or ELISA. Antibodies against MMP-12 are commercially available or antibodies can be produced by conventional antibody production methods. A method for producing a polyclonal antiserum is, for example, Au
Subel et al., 1997. The production of monoclonal antibodies is taught, for example, in Ausubel et al., 1997.

  Immunoprecipitation methods well known in the art can be found, for example, in Ausubel et al., 1998. Western blot (immunoblot) analysis is standard in the art and is described, for example, in Ausubel et al., 1997. Enzyme-linked immunosorbent assay (ELISA) is standard in the art and is described, for example, in Ausubel et al., 1991.

Inhibition of inflammation in colitis mouse model using antisense oligonucleotide to MMP-12 An animal model in which inflammation was induced in the large intestine of mice was described in Okayasu et al., 1990. In the model used in the experiments of the present invention, oral dextran sulfate sodium (DSS) is utilized to induce inflammation (Axelsson et al., 1998). DSS can be administered to mice with drinking water, thereby inducing colitis resembling inflammatory bowel disease (IBD) in humans. Those with a MW of about 40-50 kD and containing a high content of sulfur up to about 19% have been shown to be optimal as DSS forms to induce inflammation. In Okayasu, 1990, DSS is administered to animals at a concentration of about 2-5%.

  In this study, a final pH 8.5 (adjusted with NaOH) DSS dissolved in water at a concentration of 2.5% was used. DSS was orally administered to female SPF NMRI mice for 8 consecutive days to induce stable colitis in all individuals. It has been shown that colitis induced in this type of experiment is sufficiently induced even 4-5 days after addition to drinking water (Cooper et al., 1993).

  The antisense substance obtained in SEQ ID NO: 3 was administered rectally to untreated or anesthetized colitis animals. A shortened XRO feeding tube (Vygon, Ecouen, France) was inserted into the rectum to the level of trits ligament, the substance was administered in a volume of 100 μl, and the tube was slowly and carefully withdrawn to prevent leakage of the substance into the rectum. A single dose of antisense (100 μg) in water (100 μl) was administered. Treatment treatment was given once on day 8, and then DSS treatment was continued for 10 days. On day 18, animals were sacrificed for clinical inflammatory parameter analysis and histopathology.

Clinical signs Each mouse was observed once daily during the study period. All signs of health deterioration and any behavioral changes were recorded. Animals that showed signs of severe illness and lost more than 15% of their original weight were killed.

Mortality and mortality during the autopsy experiment were recorded. At the end of the experimental period, the animals were killed by cervical dislocation. The abdomen was opened and the spleen was excised and weighed. The large intestine was excised from the junction of the ileocecal adjoining rectum to the passage near the pubic cartilage. The cecum was opened at the top and the stool was carefully removed. The colon was opened vertically and the stool was carefully removed with a spatula. Evaluation of colitis by recording clinical parameters such as mortality, colon length, spleen weight, and diarrhea calculated as wet / dry weight of stool (after drying at 60 ° C. for 48 hours). Performed (FIG. 2). For microscopic experiments, the cecum and whole colon were fixed in 4% neutral buffered formaldehyde.

  From FIG. 2 it is clear that there are specific and significant improvements in all measured parameters. That is, the treated animals had reduced diarrhea, had a more normal colon length, had a more normal spleen weight, and showed signs of statistically significant histological improvement.

After processing and microscopic experiment fixation, tissue sampled for microscopic experiments was prepared and specimens were taken from the cecum and central part of the colon for histological processing. If the first sample was difficult to interpret, additional specimens were taken. The specimens were embedded in paraffin, cut at a standard thickness of 5 μm, stained with hematoxylin and eosin, and examined with a light microscope.

  A veterinary pathologist with extensive experience in histopathological evaluation of DSS-induced colitis in mice was tested for colitis and evaluated for inflammation. The diagnostic histopathology is based on the standardized rating system shown in Table 1.

Histological analysis of colonic sections As outlined above, sections taken from the cecum and the central part of the colon were used for histological processing. Stained with hematoxylin and eosin. The sections were then examined with a light microscope and morphological changes were recorded. From FIG. 3, as observed in both physiological parameters and histology, a single rectal administration of the antisense compound obtained from SEQ ID NO: 3 is sufficient to dramatically reduce inflammation. Conclusions can be made (FIGS. 2 and 3).

Example 3 In vitro screening of human MMP-12 mRNA for binding sites accessible to antisense sequences The effect of antisense compounds on target nucleic acid expression is easily monitored in various cell types when the target nucleic acid is present at measurable levels. can do. For those skilled in the art, there are a number of well-established methods that can be used to determine changes in the level of expressed target (see below).

In order to identify antisense compounds that show selective binding to therapeutic human MMP-12 mRNA using antisense compounds , resulting in a decrease in the amount of MMP-12 protein, the inventors have uniquely identified An in vitro screening system was set up. Surprisingly, in the coding region SEQ ID NO: 1 ^{(GenBank (R)} Accession No. NM-002426), the present inventors have identified a number of antisense sequences showing very high inhibition.

Here next, their ability to reduce the amount of human MMP-12 mRNA by sequencing the antisense and monitoring target mRNA levels using methods well known in the art (eg PCR or Northern blot analysis). Whereas Western blots show the level of protein encoded by the target mRNA. The efficacy of the antisense sequence was arbitrarily scored as a measure of the degree of inhibition of the target sequence. Table 2 lists the antisense sequences of groups that reduce their degree of efficacy.

  Antisense sequences SEQ ID NOs: 3-6 showed that MMP-12 mRNA levels were inhibited by about 75-85% compared to controls. Antisense sequences SEQ ID NOs: 7-10 showed that MMP-12 mRNA levels were inhibited by about 65-75% compared to controls. Antisense sequences SEQ ID NOs: 11-14 showed that MMP-12 mRNA levels were inhibited by about 50-65% compared to controls.

  Although the present invention has been described with reference to preferred embodiments that constitute the best mode presently known to the inventors, without departing from the scope of the invention as set forth in the claims appended hereto, It should be understood that various changes and modifications apparent to those having ordinary skill in the art may be made.

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Figure 2 shows RT-PCR analysis of MMP-12 expression of biopsy samples from patients suffering from either ulcerative colitis (A) or Crohn's disease (B). Figure 3 shows a histogram showing four different criteria used to assess improvement in the degree of inflammation in the gastrointestinal tract after administration of an antisense compound. The histological section of the mouse colon tissue obtained in Example 2 is shown.

Claims (33)

  From 8 to 50 nucleobases in length targeting a nucleic acid molecule encoding MMP-12, characterized by specifically hybridizing and inhibiting translation of metalloproteinase 12 (MMP-12) protein A compound.   2. The compound of claim 1, wherein the target sequence is SEQ ID NO: 1 or a functionally equivalent homologue thereof.   The compound of claim 1, wherein the target sequence is SEQ ID NO: 2, or a functionally equivalent homologue thereof.   2. The compound of claim 1, wherein the compound is an antisense oligonucleotide complementary to mRNA.   The compound of claim 1, wherein the oligonucleotide is a DNA molecule.   The compound of claim 1, wherein the oligonucleotide is an RNA molecule.   The compound of claim 4, wherein the antisense oligonucleotide has a sequence selected from the group consisting of SEQ ID NOs: 3-14.   The compound according to claim 4, wherein the oligonucleotide is RNAi comprising at least 8 nucleotide portions of a sequence selected from the group consisting of SEQ ID NOs: 3 to 14 and having a total length of 25 or less nucleotides.   An antisense oligo that is a compound consisting of 8 to 50 nucleobases targeting a nucleic acid molecule encoding MMP-12, which specifically hybridizes in mammals and inhibits translation of MMP-12 A nucleotide, which is chemically modified by substituting a non-crosslinked oxygen atom of the antisense nucleic acid backbone with a component selected from the group consisting of methane phosphate, methyl phosphate, and phosphorothioate. The above compound.   10. The compound of claim 9, wherein the substitution occurs at one or more nucleotides selected from the 3 'end or the 5' end or both.   10. The compound of claim 9, wherein the substitution occurs at one or more nucleotides at any position along the length of the oligonucleotide.   An antisense oligonucleotide composed of DNA or RNA, or an analog or mimic of DNA or RNA, including, but not limited to, methylphosphonate, N3 ′ → P5′-phospho 12. Any of claims 1-11, including loamidate, morpholino, peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinosyl nucleic acid (ANA), fluoro-arabinosyl nucleic acid (FANA) methoxy-ethyl nucleic acid (MOE). A compound according to one paragraph.   2. The compound of claim 1, which is an antisense oligonucleotide that is a homopolymer or heteropolymer comprising DNA or RNA, or analogs or mimic combinations of DNA or RNA.   9. A compound according to any one of claims 2 to 8, wherein the oligonucleotide comprises at least one modified sugar component nucleobase.   15. A compound according to claim 14, wherein the modified sugar component is a 2'-O-methoxyethyl sugar component.   A composition comprising a compound according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier or diluent.   The composition of claim 16, wherein the composition further comprises a colloidal dispersion.   A method of inhibiting the translation of MMP-12 in a cell or tissue, wherein the cell or tissue is contacted with the compound according to any one of claims 1 to 15, thereby inhibiting the translation of MMP-12.   A method of inhibiting translation of MMP-12 in a cell or tissue, wherein the cell or tissue is contacted with the composition according to any one of claims 16 to 17, thereby inhibiting translation of MMP-12.   20. The method of claim 18 or 19, wherein inhibition of MMP-12 expression suppresses MMP-12 dependent processes in a human subject. 21. The method of claim 20, wherein the MMP-12 dependent process is one of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, rheumatoid arthritis, psoriasis, emphysema and asthma.   A method for preventing, alleviating or treating an MMP-12-dependent disorder in a patient, wherein MMP-12 expression is suppressed in one or more cells of a human patient.   A method for preventing, alleviating or treating an MMP-12 dependent disorder in a patient, characterized in that MMP-12 levels are suppressed in one or more cells of a human patient.   24. The method of claim 22 or 23, wherein the MMP-12 dependent disorder is one of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, rheumatoid arthritis, psoriasis, emphysema and asthma.   A recombinant nucleotide sequence comprising a compound according to any one of claims 1-15.   A recombinant expression vector comprising the recombinant nucleotide sequence of claim 25.   27. The recombinant expression vector of claim 26, wherein the vector is of eukaryotic or prokaryotic origin.   28. A method of inhibiting expression of MMP-12 in a cell or tissue, wherein the cell or tissue is contacted in vivo or in vitro with a recombinant nucleotide sequence expressed with the recombinant vector of claim 27.   A recombinant host cell produced by the method of claim 28.   A transgenic non-human animal having at least one sequence according to claim 7 operably inserted into at least one cell.   32. The transgenic animal of claim 30, wherein at least one functionally inserted sequence is overexpressed.   18. A method of inhibiting expression of MMP-12 in a cell or tissue, wherein the composition of claim 16 or 17 is administered to a human with a pharmaceutically acceptable carrier at a therapeutically effective dose.   A method of diagnosing inflammatory bowel disease in a human subject comprising screening for the presence or absence of MMP-12 expression, wherein MMP-12 expression is an indicator of inflammatory bowel disease.

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