WO2004035759A2 - Oligonucleotides inhibiteurs cibles sur la metalloproteinase-9 matricielle - Google Patents

Oligonucleotides inhibiteurs cibles sur la metalloproteinase-9 matricielle

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Publication number
WO2004035759A2
WO2004035759A2 PCT/US2003/033141 US0333141W WO2004035759A2 WO 2004035759 A2 WO2004035759 A2 WO 2004035759A2 US 0333141 W US0333141 W US 0333141W WO 2004035759 A2 WO2004035759 A2 WO 2004035759A2
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WIPO (PCT)
Prior art keywords
seq
oligonucleotides
nucleobases
compound
oligonucleotide
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PCT/US2003/033141
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English (en)
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WO2004035759A3 (fr
Inventor
Duane E. Ruffner
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Genta Salus Llc
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Priority to AU2003287169A priority Critical patent/AU2003287169A1/en
Publication of WO2004035759A2 publication Critical patent/WO2004035759A2/fr
Publication of WO2004035759A3 publication Critical patent/WO2004035759A3/fr

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    • 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
    • C12N15/1137—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 against enzymes
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates generally to agents and nucleic acid targets for regulation of gene expression. More specifically, the present invention relates to compounds, compositions, and methods for regulation of the expression of nucleic acids encoding matrix metalloproteinase-9 ("MMP-9").
  • MMP-9 matrix metalloproteinase-9
  • DNA is a dual-stranded polymer compound whose strands are chains of the nucleotide subunits adenine, thymine, cytosine, and guanine. These chains are held to each other by hydrogen bonds formed between complementary nucleotides on the chains.
  • Adenine (A) forms hydrogen bonds with thymine (T); and guanine (G) forms hydrogen bonds with cytosine (C).
  • Contiguous sets of three of these nucleobases in the nucleotide strands code for individual amino acids. Together, the nucleotide chains of DNA code for all of the various proteins needed to build, maintain, and fuel the human body.
  • Messenger RNA Proteins are translated from transcribed copies of DNA called messenger RNA.
  • Messenger RNA is made when DNA partially unwinds, allowing the sequence of the DNA to be copied and transcribed by cellular machinery.
  • Messenger RNA is made of the same components as DNA, with the substitution of uracil for thymine.
  • MMP-9 defective matrix metalloprotein-9
  • Antisense technology controls the production of a protein by binding a molecule to the nucleic acid coding for the protein. Generally this molecule is a short length of DNA or RNA commonly referred to as an antisense oligonucleotide. These oligonucleotides are complementary to a segment of the nucleic acid. In use, these antisense oligonucleotides are administered to a cell or tissue desired to be treated. The oligonucleotides are taken into the cell where they associate with and bind to a region of the nucleic acid encoding the protein to which they are complementary. This binding prevents normal interaction of the nucleic acid with cellular machinery such as DNA transcription enzymes or RNA translation enzymes. This prevents proper transcription of the gene or translation of the RNA.
  • Antisense is regarded by many as a powerful technology since the antisense oligonucleotides used may be carefully targeted to specific regions on the nucleic acids, thus preventing interaction of the oligonucleotides with other molecules not desired to be inhibited or activated. These specific regions of the selected nucleic acids are often referred to as target sequences. Because antisense oligonucleotides may be so carefully targeted to these target sequences, antisense oligonucleotides may be used to provide compositions, such as drugs, that have near-absolute specificity, high efficacy, low toxicity, and few side effects.
  • Such target regions, oligonucleotides, compositions including such oligonucleotides, and methods of their use are disclosed herein.
  • the present invention is directed to oligonucleotides that are targeted to nucleic acids which encode matrix metalloproteinase-9 ("MMP-9").
  • the compounds are designed to be complementary to at least a part of a target region of a nucleic acid encoding MMP- 9.
  • the compounds are configured to modulate the expression of MMP-9.
  • the invention first encompasses compounds such as oligonucleotides configured to specifically hybridize with at least a portion of a target region including SEQ ID NO: 31 to inhibit the expression of MMP-9.
  • Some of these compounds are oligonucleotides of between about 8 and about 39 nucleobases in length having at least 8 contiguous nucleobases complementary to at least a portion of SEQ ID NO: 31.
  • nucleobase refers to adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U); their post-replicationally or post-transcriptionally modified derivatives; and other bases suitable for inclusion in a nucleic acid.
  • oligonucleotides may alternatively be between about 10 and about 30 nucleobases in length. More preferably, the oligonucleotides are between about 14 and about 24 nucleobases in length.
  • the oligonucleotides include at least about 8 contiguous nucleobases complementary to nucleotides 8-23 of SEQ ID NO: 31.
  • the oligonucleotides have a sequence selected from the group comprising SEQ ID NO: 1 and SEQ ID NO: 2.
  • the oligonucleotides of the invention may include features or components such as modified intemucleotide linkages, modified sugar moieties, and modified nucleobases.
  • the oligonucleotides may also contain nucleotides from DNA or RNA.
  • the invention also encompasses compounds such as oligonucleotides configured to specifically hybridize with at least a portion of SEQ 3D NO: 32 .to inhibit the expression of MMP-9.
  • Some of these compounds are oligonucleotides of between about 8 and about 50 nucleobases in length having at least 8 contiguous nucleobases complementary to SEQ ID NO: 32. These oligonucleotides may be of between about 10 and 30 nucleobases in length. Most preferably, the oligonucleotides are between about 14 and about 24 nucleobases in length.
  • the oligonucleotides may additionally have at least about 8 contiguous nucleobases complementary to nucleotides 4-49, 62-93, or 109-122 of SEQ ID NO: 32.
  • the oligonucleotide may have a sequence selected from the group comprising SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16.
  • the oligonucleotides of the invention may include features or components such as modified intemucleotide linkages, modified sugar moieties, and modified nucleobases.
  • the invention also includes compounds such as oligonucleotides configured to specifically hybridize with at least a portion of SEQ 3D NO: 33 to inhibit the expression of MMP-9.
  • Some of these compounds are oligonucleotides of between about 8 and about 34 nucleobases in length having at least 8 contiguous nucleobases complementary to SEQ ID NO: 33.
  • the oligonucleotides may also be between about 10 and about 30 nucleobases in length. Most preferably, the oligonucleotides may be between about 14 and about 24 nucleobases in length.
  • the oligonucleotides may additionally have at least about 8 contiguous nucleobases complementary to nucleotides 7-21 of SEQ ID NO: 33.
  • the oligonucleotides have a sequence selected from the group comprising SEQ ID NO: 17 and SEQ ID NO: 18.
  • the oligonucleotides of the invention may include features or components such as modified intemucleotide linkages, modified sugar moieties, and modified nucleobases.
  • the invention still further includes compounds such as oligonucleotides configured to specifically hybridize with at least a portion of SEQ ID NO: 34 to inhibit the expression of MMP-9.
  • Some of these compounds are oligonucleotides of between about 8 and about 29 nucleobases in length having at least 8 contiguous nucleobases complementary to SEQ ID NO: 34.
  • Others of these oligonucleotides may instead be of between about 10 and about 25 nucleobases in length. Most preferably, the oligonucleotides are between about 14 and about 24 nucleobases in length.
  • the oligonucleotides may additionally have at least about 8 contiguous nucleobases complementary to nucleotides 7-25 of SEQ ID NO: 34.
  • the oligonucleotides have a sequence selected from the group comprising SEQ ID NO: 19 and SEQ ED NO: 20.
  • the oligonucleotides of the invention may include features or components such as modified intemucleotide linkages, modified sugar moieties, and modified nucleobases.
  • the invention also includes compounds such as oligonucleotides configured to specifically hybridize with at least a portion of SEQ ID NO: 35 to inhibit the expression of MMP-9.
  • Some of these compounds are oligonucleotides of between about 8 and about 50 nucleobases in length having at least 8 contiguous nucleobases complementary to SEQ ID NO: 35.
  • Others of these oligonucleotides may instead be of between about 10 and about 34 nucleobases in length. Most preferably, the oligonucleotides are between 14 and about 24 nucleobases in length.
  • the oligonucleotides may additionally have at least about 8 contiguous nucleobases complementary to nucleotides 2-24 or 39-66 of SEQ ED NO: 35.
  • the oligonucleotides have a sequence selected from the group comprising SEQ ED NO: 21, 22, 23, 24, 25, 26, 27, and 28.
  • the oligonucleotides of the invention may include features or components such as modified intemucleotide linkages, modified sugar moieties, and modified nucleobases.
  • the invention also includes compounds such as oligonucleotides configured to specifically hybridize with at least a portion of SEQ ID NO: 36 to inhibit the expression of MMP-9.
  • oligonucleotides of between about 8 and about 27 nucleobases in length having at least about 8 contiguous nucleobases complementary to SEQ ED NO: 36.
  • the oligonucleotides may also be between about 10 and 30 nucleobases in length. More preferably, the oligonucleotides may be between about 14 and 24 nucleobases in length.
  • the oligonucleotides may additionally have at least 8 contiguous nucleobases complementary to nucleotides 6-21 of SEQ ED NO: 36.
  • the oligonucleotides have a sequence selected from the group comprising SEQ ED NO: 29 and SEQ ED NO: 30.
  • the oligonucleotides of the invention may include features or components such as modified intemucleotide linkages, modified sugar moieties, and modified nucleobases.
  • compositions such as pharmaceuticals including the oligonucleotide compounds disclosed above that are targeted to any of the target regions of the invention found in SEQ ED NOS: 31-36.
  • Such compositions may include additional components such as pharmaceutically-acceptable carriers or diluents.
  • the invention also includes methods of inhibiting the expression of MMP-9 in a cell or tissue.
  • Such methods include the step of contacting the cell or tissue with a composition made with the oligonucleotides of the invention.
  • Such oligonucleotides may include compounds comprising a segment of from about 8 to about 34 nucleobases in length of a sequence chosen from the group consisting of SEQ ED NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 39, 0, and 45.
  • Figure 1 A shows an RNA oligonucleotide of the invention (SEQ ED NO: 37) with 5' and 3' terminal hairpin loops targeted to the F9 target region of MMP-9;
  • Figure IB shows a phosphorothioate DNA oligonucleotide of the invention (SEQ ID NO: 38) with 5' and 3' terminal hairpin loops targeted to the F9 target region of MMP-9;
  • Figure 2 is a photograph of a PCR gel showing the results of an assay using the oligonucleotides of Figure 1 A (SEQ ED NO: 37) and Figure IB (SEQ ED NO: 38) to inhibit the expression of MMP-9 in Htl080 cells;
  • Figure 3 A shows an RNA oligonucleotide of the invention (SEQ ID NO: 37) with 5' and 3' terminal hairpin loops targeted to the F9 region of MMP-9;
  • Figure 3B shows an RNA oligonucleotide (SEQ ID NO: 39) targeted to the F9 region of MMP-9;
  • Figure 3C shows the result of an in vitro assay of MMP-9 inhibition by the oligonucleotide of Figure 3B (SEQ ED NO: 39) compared with the inhibition brought about by the oligonucleotide with two terminal hairpin loops of Figure 3A (SEQ ED NO: 37);
  • Figure 4A is an illustration of the F9 antisense target SEQ ID NO: 44 and the F9 RNAi target SEQ ED NO: 42 of MMP-9 on a segment of the MMP-9 gene sequence;
  • Figure 4B shows the oligonucleotides used in an assay conducted to compare their effectiveness in silencing the MMP-9 gene
  • Figure 5 is a photograph of an ethidium bromide-stained electrophoresis gel showing the results of PCR with MMP-9- and glyceraldehyde phosphate dehydrogenase- (GAPDH) specific PCR primers showing MMP-9- and GAPDH-specific PCR fragments;
  • GPDH glyceraldehyde phosphate dehydrogenase-
  • Figure 6 is a bar graph showing a plot of the ratio of the intensities of MMP-9 to GAPDH from the gel of Figure 5.
  • the present invention relates to oligomeric compounds for modulating the function of nucleic acid molecules encoding matrix metalloproteinase-9 ("MMP-9").
  • MMP-9 matrix metalloproteinase-9
  • the invention relates to oligomeric compounds such as inhibitory oligonucleotides that are configured to interact with nucleic acids encoding MMP-9.
  • the invention further includes compositions comprising such oligonucleotides, including pharmaceutical compounds, and methods for their use.
  • the invention additionally includes methods of treating diseases that respond to the modulation of MMP-9 such as multiple sclerosis and several cancers.
  • oligonucleotide and “nucleic acid” denote polynucleotides — olymers of nucleotides.
  • target nucleic acid and “nucleic acid encoding MMP-9” include polynucleotides having at least a portion of the code for MMP-9.
  • DNA encoding MMP-9 is thus included, as is RNA such as pre-mRNA and mRNA, cDNA, and hybrid nucleic acids such as artificial sequences having at least a portion of the sequence of MMP-9.
  • nucleic acid also include sequences having any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1 -methyl guanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethylura
  • oligonucleotides refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and intemucleotide (or "backbone") linkages, as well as oligonucleotides having non-naturally occurring portions with similar function.
  • inhibitory oligonucleotide denotes an oligonucleotide having a sequence that enables the oligonucleotide to interact with a selected portion of a nucleic acid.
  • the oligonucleotides of the invention may have a sequence that is substantially complementary to at least a segment of the selected portion of the nucleic acid.
  • the oligonucleotide may have a substantially sense sequence or a substantially antisense sequence.
  • Such oligonucleotides may disrupt the function of the nucleic acid by specifically hybridizing with it. Some such oligonucleotides may specifically hybridize to the selected portion of the nucleic acid.
  • the inhibitory oligonucleotide may have a sequence that is substantially identical to that of the selected portion of the nucleic acid.
  • antisense oligonucleotide is used herein to denote an oligonucleotide which is complementary to, and thus has the capacity to specifically hybridize with, a nucleic acid. This is especially used herein to refer to oligonucleotides whose binding modulates the normal activity or function of the target nucleic acid. The modulation of nucleic acid activity caused by such oligonucleotides is broadly termed "antisense" technology.
  • the inhibitory oligonucleotides of the present invention also include antisense oligonucleotides.
  • the antisense oligonucleotides of the invention may be equal in size to the entire target sequence to which they are targeted. Alternatively, such oligonucleotides may be from about 8 to about 50 nucleobases in length (i.e. from about 8 to about 50 linked nucleosides). Still further, the oligonucleotide compounds may be antisense oligonucleotides of from about 10 to about 34 nucleobases in length.
  • the antisense oligonucleotide compounds of the invention may include ribozymes, external guide sequences (EGS), oligozymes, other short catalytic RNAs, or other catalytic oligonucleotides which are configured to hybridize to the target nucleic acid and modulate its expression.
  • ribozymes external guide sequences (EGS)
  • oligozymes other short catalytic RNAs
  • catalytic oligonucleotides which are configured to hybridize to the target nucleic acid and modulate its expression.
  • complementary refers to the capacity of two nucleotides to pair precisely with each other. This is often termed “Watson-Crick pairing.” This term may also be used to refer to oligonucleotides which exhibit the ability of pairing precisely with each other. For example, if the nucleotides located at a certain position on two oligonucleotides are capable of hydrogen bonding, then the oligonucleotides are considered to be complementary to each other at that position. The oligonucleotides and the DNA or RNA themselves are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • oligonucleotide and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise paring such that stable and specific binding may occur between the oligonucleotides and the DNA or RNA target. It is understood in the art that the sequence of an inhibitory oligonucleotide compound need not be 100 percent complementary to that of its target nucleic acid to be specifically hybridizable. An inhibitory oligonucleotide compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA.
  • a sufficient degree of complementarity prevents non-specific binding of the inhibitory oligonucleotide compound to nontarget sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • a nucleoside is a combination of a base and a sugar.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of heterocyclic bases are purines and pyrimidines.
  • Nucleotides are nucleosides that additionally include a phosphate group linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3', or 5' hydroxyl moiety of the sugar.
  • the phosphate groups link adjacent nucleosides to one another to a form a linear polymer. The ends of such linear polymers can also be joined, thus forming a circular structure.
  • the phosphate groups form the intemucleotide backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • Examples of compounds useful in this invention include oligonucleotides containing modified backbones or non-natural intemucleotide linkages.
  • Possible modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, ammoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and borano-phosphates having normal 3'- 5' linkages, as well as their 2'-5'-linked analogs.
  • modified oligonucleotides may have inverted polarity, where one or more of the intemucleotide linkages is a 3' to 3', 5' to 5', or 2' to 2' linkage. Oligonucleotides having inverted polarity may comprise a single 3' to 3' linkage at the 3 '-most intemucleotide linkage. Various salts, mixed salts and free acid forms of the modified and non-modified oligonucleotides are also included. Preferred modified oligonucleotides may have backbones not including a phosphorus atom.
  • backbones may be formed by a short chain alkyl or cycloalkyl intemucleotide linkage, or by one or more short chain heteroatomic or heterocyclic intemucleotide linkages.
  • These include oligonucleotides having morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH 2 component parts.
  • oligonucleotide analogs both the sugar and the intemucleotide linkage of the nucleotides are replaced with novel groups.
  • the base units are maintained to permit hybridization.
  • One such oligomeric compound is a peptide nucleic acid, or "PNA".
  • PNA compounds the sugar backbone of an oligonucleotide is replaced with an amide- containing backbone, such as an aminoethylglycine backbone.
  • Inhibitory compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • a "coding sequence,” or a sequence which "encodes" a particular protein is a nucleic acid sequence (or a portion thereof) which is transcribed (in the case of DNA) or translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • nucleotide sequences in a particular nucleic acid molecule For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated “upstream,” “downstream,” “3',” or “5"' relative to another sequence, it is to be understood that it is the position of the sequences in the "sense” or "coding" strand of a DNA molecule that is being referred to, as is conventional in the art.
  • Homology refers to the percent of identity between at least two oligonucleotides or polypeptides. The percent identity between the sequences from one moiety to another can be determined by techniques known in the art. Homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, Dayhoff, M.O. (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3, National biomedical Research Foundation, Washington, DC. Default parameters can be used for alignment. One alignment program is BLAST, used with default parameters. Details of these programs can be found at the following internet address: http :// ww.n cbi.nIm.gov/egi-bin/BI AST.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization; supra.
  • a “functional homologue” or a “functional equivalent” of a given polypeptide includes molecules derived from the wild-type polypeptide sequence, as well as recombinantly-produced or chemically-synthesized polypeptides which function in a manner similar to the wild-type molecule to achieve a desired result.
  • a functional homologue of MMP-9 encompasses derivatives and analogues of those polypeptides — including any single or multiple amino acid additions, substitutions and/or deletions occurring internally or at the amino or carboxy termini thereof — so long as integration activity remains.
  • “Gene transfer” or “gene delivery” refers to methods or systems for reliably inserting foreign DNA into host cells.
  • vector any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • vector includes cloning and expression vehicles, as well as viral vectors.
  • transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 3: 197.
  • exogenous DNA moieties such as a nucleotide integration vector and other nucleic acid molecules
  • a "host cell” as used herein may be either a eukaryotic or a prokaryotic cell.
  • a host cell could be a yeast cell, an insect cell, or a mammalian cell which has been transfected with an exogenous DNA sequence, and the progeny of that cell. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.
  • cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need to be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.
  • operably linked refers to an arrangement of elements wherein the components so described are configured to as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound without adding any undesired toxicological effects.
  • Gene inhibition technologies are often used to modulate functions such as DNA replication or transcription, RNA translocation to the site of translation, RNA translation, RNA splicing, and catalytic activity conducted or aided by the RNA.
  • the overall effect of such interference with the function of the target nucleic acid is modulation of the expression of MMP-9. This is brought about by the interference of the single-stranded inhibitory oligonucleotides of the invention with MMP-9 mRNA.
  • the interference of the inhibitory oligonucleotides blocks proper function of the MMP-9 mRNA, thus preventing proper expression. This interference is commonly referred to as "knockdown" of the target nucleic acid.
  • modulation can mean either an increase or a decrease in the expression of a gene.
  • inhibition is a preferred form of modulation of gene expression
  • mRNA is a preferred nucleic acid target.
  • Targeting inhibitory compounds to specific nucleic acids is generally a multistep process.
  • a nucleic acid is identified that participates in a disease state.
  • This nucleic acid is then sequenced.
  • the nucleic acid may be, for example, a cellular gene or mRNA whose expression produces a product active in the disease, or in a nucleic acid molecule from infectious agent such as a vims, bacterium, or other infectious microbe.
  • the target sequence is one encoding matrix metalloproteinase-9.
  • a next step in the targeting process involves determining potential sites on the target nucleic acid molecule which are susceptible to interaction with an inhibitory oligonucleotide. This process also involves evaluating whether targeting one specific site will modulate the expression of a nucleic acid more effectively than targeting other specific sites.
  • this targeting process may be conducted according to a variety of methods know in the art. Additionally, it may be conducted using the method outlined in United States Patent Application number 09/647,344, filed December 4, 2000, entitled Directed Antisense Libraries, which is incorporated herein in its entirety. That application discloses a procedure that allows the construction of directed antisense libraries.
  • These libraries contain all overlapping fragments spanning the entire length of the gene of interest. Transcription in vitro or in vivo of a DNA fragment produces an inhibitory RNA targeted to the site on the RNA transcript that is encoded by the DNA fragment. Transcription of the entire DNA fragment library produces all antisense RNA molecules targeting all positions on the RNA target. Expression of this library in mammalian cells allows the identification of effective target sites on the nucleic acid for use in antisense-mediated gene inhibition.
  • the directed libraries generated above may be assayed for their ability to modulate the expression of target change in vivo in cultured cells.
  • the antisense library is transduced into a suitable cell line expressing the gene of interest.
  • transfection conditions may be chosen such that generally only one member of the library is taken up by each individual host cell. The result is a group of cells, each expressing a different inhibitory molecule targeted to a different site on the RNA transcript of the gene of interest. All target sites are present in the entire cell population produced.
  • cell clones may be identified in which the expression of the target RNA has been reduced or eliminated.
  • clones possess an inhibitory oligonucleotide targeted to a site on the RNA transcript which all allows effective modulation of the gene's activity.
  • the plasmid containing the effective inhibitory oligonucleotide may be recovered, and its sequence identified.
  • oligonucleotides are chosen which are sufficiently complementary to the target to give the desired effect. This determination is made by evaluating which oligonucleotides hybridize sufficiently well and with sufficient specificity to the given target site.
  • hybridization is defined as hydrogen bonding, which may be Watson-Crick, Hoogstein, or reversed Hoogstein hydrogen bonding between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • the invention includes inhibitory oligonucleotides directed to six different target regions found on nucleic acids encoding matrix metalloproteinase-9 to modulate the function of the nucleic acid. Each of these target regions contains many possible inhibitory oligonucleotides, each of which falls within the scope of this invention.
  • the invention further includes specific inhibitory oligonucleotides identified from among the potential inhibitory oligonucleotides for each target sequence, which are presently preferred for the practice of this invention.
  • a first target region of the MMP-9 nucleic acids is found in SEQ ED NO: 31. This sequence is about 39 nucleobases in length. Oligonucleotides complementary to a portion of the target region of SEQ ID No.
  • inhibitory oligonucleotides which may modulate the expression of MMP-9. These oligonucleotides may preferably be between about 8 to about 39 bases in length, more preferably about 10 to about 30 nucleobases in length, and most preferably about 14 to about 25 nucleobases in length. Such oligonucleotides which are complementary to SEQ ED NO. 31 include but are not limited to, SEQ ED NOS: 1 and 2, which are useful as inhibitory oligonucleotides in the practice of the invention. The invention further includes oligonucleotides comprising at least 8 nucleobases of SEQ ID NOS: 1 and 2 for use as inhibitory oligonucleotides. A second target region of the MMP-9 nucleic acids is found in SEQ ID NO: 32.
  • Oligonucleotides complementary to a portion of the target region of SEQ ED NO: 32 are inhibitory oligonucleotides which may modulate the expression of MMP-9. These oligonucleotides may preferably be about 8 to about 34 nucleobases in length, more preferably about 10 to about 30 nucleobases in length, and most preferably about 14 to about 24 nucleobases in length.
  • Such oligonucleotides which are complementary to SEQ. ED. NO: 32 include, but are not limited to, SEQ ED NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, which are useful as inhibitory oligonucleotides in the practice of the invention.
  • the invention further includes oligonucleotides comprising at least 8 nucleobases of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 for use as inhibitory oligonucleotides.
  • a third target region of the MMP-9 nucleic acids is found in SEQ ID NO: 33. This sequence is about 34 nucleobases in length. Oligonucleotides complementary to a portion of the target region of SEQ ED NO: 33 are inhibitory oligonucleotides which may modulate the expression of MMP-9. These oligonucleotides may preferably be between about 8 to about 34 nucleobases in length, more preferably about 10 to about 30 nucleobases in length, and most preferably about 14 to about 24 nucleobases in length.
  • Such oligonucleotides which are complementary to SEQ ID NO: 33 include, but are not limited to, SEQ ED NOS: 17, 18, which are useful as inhibitory oligonucleotides in the practice of the invention.
  • the invention further includes oligonucleotides comprising least 8 nucleobases of SEQ ED NOS: 17, 18 for use as inhibitory oligonucleotides.
  • the fourth target region of the MMP-9 nucleic acids is found in SEQ ED NO: 34.
  • This sequence is about 29 nucleobases in length.
  • Oligonucleotides complementary to a portion of the target region of SEQ ED NO: 34 are inhibitory oligonucleotides which may modulate the expression of MMP-9. These oligonucleotides may preferably be of between about 8 to about 29 nucleobases in length, more preferably about 10 and about 25 nucleobases in length, and most preferably about 14 to about 20 nucleobases in length.
  • Such oligonucleotides which are complementary to SEQ ID NOS: 34 include, but are not limited to, SEQ ED NO: 19, and 20, which are useful as inhibitory oligonucleotides in the practice of the invention.
  • the invention further includes oligonucleotides including, but not limited to 39, 40, and 45.
  • the invention further includes oligonucleotides comprising at least 8 nucleobases of SEQ ED NOS: 19, 20, 39, 40, and 45 for use as inhibitory oligonucleotides.
  • a fifth target region of the MMP-9 nucleic acids is found in SEQ ED NO: 35. This sequence is about 67 nucleobases in length. Oligonucleotides complementary to a portion of the target region of SEQ ED NO: 35 are inhibitory oligonucleotides which may modulate the expression of MMP-9. These oligonucleotides may preferably be between about 8 to about 50 nucleobases and length, more preferably about 10 to about 34 nucleobases in length, and most preferably about 14 to about 20 nucleobases in length.
  • Such oligonucleotides which are complementary to SEQ ED NO: 35 include, but are not limited to, SEQ ID NOS: 21, 22, 23, 24, 25, 26, 27, and 28, which are useful as inhibitory oligonucleotides in the practice of the invention.
  • the invention further includes oligonucleotides comprising least 8 nucleobases of SEQ ED NOS: 21, 22, 23, 24, 25, 26, 27, and 28, for use as inhibitory oligonucleotides.
  • a sixth target region of the MMP-9 nucleic acids is found in SEQ ID NO: 36. This sequence is about 27 nucleobases in length. Oligonucleotides complementary to a portion of the target region of SEQ ED NO: 36 are inhibitory oligonucleotides which may modulate the expression of MMP-9. These oligonucleotides may preferably be between about 8 to about 27 nucleobases in length, more preferably between about 10 and about 25 nucleobases in length and most preferably about 14 and about 24 nucleobases in length.
  • Such oligonucleotides which are complementary to SEQ ED NO: 36 include, but are not limited to, SEQ ED NOS: 29, and 30, which are useful as inhibitory oligonucleotides in the practice of the invention.
  • the invention further includes oligonucleotides comprising a least 8 nucleobases of SEQ ED NOS: 29, 30, for use as inhibitory oligonucleotides.
  • the oligonucleotides of the invention may be incorporated into pharmaceutical compositions.
  • the oligonucleotides may additionally be used in methods to modulate the expression of MMP-9 in a cell or tissue. Such methods may use the oligonucleotides alone, or they may use the pharmaceutical compositions of the invention.
  • Inhibitory oligonucleotide compounds are commonly used as research reagents and in diagnostics. Due to their ability to inhibit gene expression with specificity, such oligonucleotides are often used by those of ordinary skill to elucidate the function of particular genes. Inhibitory oligonucleotide compounds are also used to distinguish the functions of various members of a biological pathway. The specificity and sensitivity of inhibitory oligonucleotide technology is also harnessed by those skilled in the art for therapeutic uses. One type of this technology is antisense oligonucleotides. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man.
  • inhibitory oligonucleotides can be configured to be useful in treatment regimes for treatment of cells, tissues, and animals, especially humans.
  • inhibitory compounds of this invention may be conveniently and routinely made through techniques such as solid phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems, (Foster City, Calif). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • the invention encompasses any pharmaceutically acceptable salts, esters, salts of such esters, or any other compounds which, upon administration to an animal such as a human, are capable of providing (directly or indirectly) the biologically active inhibitory oligonucleotides of the invention. Accordingly, for example, the disclosure is also drawn to prodrugs, and other bioequivalents.
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, topical and other formulations, for assisting in uptake, distribution and/or absorption.
  • the inhibitory compounds of the present invention can be used as diagnostics, therapeutics, prophylaxis, and as research reagents and kits.
  • an animal such as a human, having a disease or disorder which can be treated by modulating the expression of MMP-9 is treated by administering inhibitory compounds in accordance with this invention.
  • the compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of the inhibitory compound to a suitable pharmaceutically acceptable diluent or carrier.
  • the inhibitory compounds and methods of the invention may also be useful to prevent or delay infection, inflammation, or tumor formation, for example.
  • the inhibitory compounds of the invention are useful for research and diagnostics because these compounds hybridize to nucleic acids encoding MMP-9.
  • Hybridization of the inhibitory oligonucleotides of the invention with a nucleic acid encoding MMP-9 can be detected by means known in the art. Such means may include conjugating an enzyme to the oligonucleotides, radiolabeling the oligonucleotide, or using any other suitable detection means. Kits using such detection means for detecting the level of MMP-9 present in a sample may also be prepared.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers; and tracheal, intranasal, epidermal, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, as well as intracranial (e.g., intrathecal or intraventricular) administration. Oligonucleotides with at least one 2'-O- methoxyethyl modification are believed to be particularly useful for oral administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • Conventional pharmaceutical carriers, aqueous bases, powder bases or oil bases, thickeners and the like may be necessary or desirable.
  • Topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes may be neutral, negative, and cationic.
  • Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, such as cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, including cationic lipids.
  • C Examples Example 1: In a first example, the oligonucleotides of Figures 1 A (SEQ ID NO: 37) and IB
  • the F9 RNA of Figure 1 A is an all-RNA oligonucleotide targeted against MMP-9 mRNA by its central 14-nucleotide targeting sequence.
  • the F9 psDNA of Figure IB is identical to F9 RNA except that it is composed of deoxynucleotides instead of ribonucleotides. Specifically, thymidines replace the uridines found in F9 RNA, and in addition, phosphorothioate linkages replace all of the phosphodiester linkages found in the F9 RNA.
  • the F9 RNA of Figure 1A (SEQ ED NO: 37) and the F9 psDNA of Figure IB (SEQ ID NO: 38) both include 5' and 3' terminal hairpin loops, each having a stem of four sets of paired nucleotides and a loop of four unpaired nucleotides. Further, as noted, in these oligonucleotides, the hairpin loops are linked to the targeting sequence by linker sequences. In these specific oligonucleotides, the linker sequences are 5 nucleotides long on the 5' end and 6 nucleotides long on the 3' end.
  • these oligonucleotides were used to treat Htl080 cells.
  • a first set of Htl080 cells received no oligonucleotide, and thus acted as a control.
  • Second and third sets received F9 RNA (SEQ ED NO: 37) or F9 psDNA (SEQ ED NO: 38), respectively.
  • the oligonucleotides were added to the media of the Htl080 cell culture at a concentration of 1 micromolar in the presence of a transfection reagent.
  • Suitable transfection agents include commercially available transfection reagents. Suitable transfection agents further include those taught in U.S. Patent Application No.
  • MMP-9 mRNA expression levels were examined by RT-PCR as described in Wong, et al, Monitoring MMP and TIMP mRNA expression by RT-PCR, Methods Mol. Biol, 151 :305-20 (2001).
  • the polyA mRNA obtained above was reverse transcribed using AMV reverse transcriptase according to the manufacturer's instructions (Promega, Inc, Madison, Wisconsin).
  • the polyA mRNA was then subjected to PCR amplification using Taq DNA polymerase (Promega, Inc, Madison, Wisconsin).
  • the PCR amplification was conducted using PCR primers specific to MMP-9 and to glyceraldehyde phosphate dehydrogenase (GAPDH).
  • the PCR reactions were then loaded onto 1.8% agarose gels, which were then electrophoresced. The gels were then stained with ethidium bromide to yield the gel shown in Figure 2.
  • Figure 2 is a photograph of the gel containing the results of the PCR amplification of the polyA obtained from the cell culture without oligonucleotides, the culture exposed to F9 RNA (SEQ ED NO: 37), and the culture exposed to F9 psDNA (SEQ ED NO: 38), respectfully, as labeled along the upper axis of the gel.
  • the MMP-9 and GAPDH-specific PCR fragments are designated along the vertical axis of the gel.
  • the gel shows no inhibition of MMP-9 mRNA expression in the control sample, while in the sample exposed to F9 RNA (SEQ ED NO: 37), expression of MMP-9 was nearly completely inhibited. With respect to the sample exposed to F9 psDNA (SEQ ED NO: 38), much less inhibition was observed.
  • Figure 3A shows the F9 RNA oligonucleotide with terminal hairpins (SEQ ED NO: 37) used in Example 1.
  • Figure 3B shows a 14-nucleotide antisense F9 RNA oligonucleotide (SEQ ED NO: 39) such as is commonly used in antisense applications. Both of these oligonucleotides are synthetic molecules.
  • the Matrigel invasion assay is a standard assay representative of the in vivo invasion of the lining of blood vessels by cancer cells and activated T cells.
  • the Matrigel invasion assay is a standard assay representative of the in vivo invasion of the lining of blood vessels by cancer cells and activated T cells.
  • about 250,000 HT1080 human fibroblast sarcoma cells cultured in 1ml EMEM+10%FBS were plated in 6 wells of a 12 well plate and incubated overnight at 37°C in 5% CO . Following incubation, the plates were washed with DPBS. Next, 400 ⁇ l of serum-free EMEM media was added to the plates.
  • Control wells and testing wells each received equal amounts of Lipofectamine solution (Invitrogen).
  • the control wells received only the transfection agent Lipofectamine 2000.
  • the testing wells received a solution of Lipofectamine 2000 and l ⁇ M F9 RNA or l ⁇ M F9 RNA with terminal hairpins
  • the samples were incubated for 6 hours at 37°C in 5% CO 2 . Following this, the media were aspirated and replaced with EMEM media containing 10%FBS. The samples were then incubated for another 48 hours at 37°C in 5% CO 2 .
  • HT1080 cells were treated with a variety of oligonucleotides directed against the F9 target site of MMP-9 mRNA.
  • This target site is shown in Figure 4A, which indicates both the antisense (SEQ ED NO: 44) and sense (SEQ ED NO: 43) strands of the target sites.
  • This assay allows further evaluation of the efficacy of the terminal-hairpin oligonucleotides of the invention in comparison with other oligonucleotides used in other gene-silencing technologies such as antisense and RNA interference.
  • the oligonucleotides used are shown in Figure 4B.
  • the first oligonucleotide shown is the F9 RNA oligonucleotide (SEQ ED NO: 37) with terminal hairpins of the invention.
  • This oligonucleotide is an all RNA oligonucleotide comprised of a central 14-base targeting sequence that targets the F9 antisense target site connected to 5' and 3' tem inal hairpin loops by 5- and 6-base single- stranded non-complementary linker sequences, respectively.
  • the second oligonucleotide is an RNA inhibitory oligonucleotide with 2 * -O-methyl linkages (SEQ ID NO: 45).
  • the third oligonucleotide is a F9 psDNA (SEQ ED NO: 38) having an identical targeting sequence to the F9 RNA with terminal hairpins which has a phosphorothioate DNA backbone instead of the RNA backbone of the F9 RNA oligonucleotide.
  • the fourth oligonucleotide is a F9 psDNA 14 & phosphodiester oligonucleotide (SEQ ED NO: 40).
  • oligonucleotides each contain a substantially similar 14-base targeting sequence targeting the oligonucleotide to the F9 antisense target site, albeit possibly through a variety of mechanisms.
  • These oligonucleotides differ, however, in the composition of their backbones, RNA versus 2'-O-methyl, phosphorothioate DNA and phosphodiester DNA, respectively.
  • the next oligonucleotide is an oligonucleotide configured to silence the MMP-9 gene using RNA-interference methods (SEQ ID NO: 41, SEQ ED NO: 46).
  • This F9- targeted siRNA is a double-stranded RNA duplex that targets the F9 siRNA target site.
  • the F9 siRNA target sequence is 19 nucleotides long instead of 14 nucleotides long. This longer sequence is reported to be optimal for siRNA.
  • the final oligonucleotide is a F9 RNA invert (SEQ ED NO: 42) used as a control. This oligonucleotide includes an RNA backbone but uses the F9 antisense target sequence encoded in an inverted, and thus non- complementary, orientation.
  • HT1080 cells were plated in 6-well culture plates at a density of 300,000 to 500,000 cells per well. Following plating, the cell culture media was removed from the wells and replaced with serum-free media (EMEM). Each of the oligonucleotides was complexed with Lipofectamine 2000 (Invitrogen), as per the manufacturer's instructions, using 20 microliters of Lipofectamine per nanomole of oligonucleotide.
  • the complexed oligonucleotides were added to the cells at a final concentration of 1 ⁇ M for all except the siRNA (SEQ ID NO: 41, SEQ ID NO: 46), which was added at a final concentration of 0.25 ⁇ M.
  • siRNA SEQ ID NO: 41, SEQ ID NO: 46
  • an equivalent amount of LF was added to one well in the absence of any oligonucleotide.
  • the cells were cultured for 6 hours, after which the media was removed and replaced with serum-containing EMEM. The cells were then cultured for an additional 42 hours, after which the cells were harvested.
  • PolyA mRNA was isolated from the harvested cells using the PolyA Tract System 1000 (Promega, Inc, Madison, Wisconsin). MMP-9 mRNA expression levels were examined by RT-PCR as described in Wong et al., Monitoring MMP and TIMP mRNA expression by RT-PCR, Methods Mol. Biol, 151:305-20 (2001). According to this method, polyA mRNA was reverse-transcribed using AMV reverse transcriptase according to the manufacturer's instructions (Promega, Inc, Madison, Wisconsin). The polyA mRNA was then subjected to PCR amplification using Taq DNA polymerase (Promega, Inc, Madison, Wisconsin). The PCR amplification was conducted using PCR primers specific to MMP-9 and to glyceraldehyde phosphate dehydrogenase (GAPDH).
  • GPDH glyceraldehyde phosphate dehydrogenase
  • the products of the PCR reactions were then loaded onto 1.8% agarose gels, which were electrophoresced.
  • the gels were then stained with ethidium bromide, producing the gel shown in Figure 5.
  • the MMP-9 and GAPDH-specific PCR fragments are designated along the right vertical axis of the gel, and the oligonucleotides used in each sample are designated along the horizontal axis of the gel in alignment with the associated lane on the gel.
  • MMP-9 mRNA expression is significantly inhibited, relative to GAPDH, by the F9 RNA oligonucleotides with terminal hairpins (SEQ ED NO: 37), the phosphorothioate DNA oligonucleotides with terminal hairpins (SEQ ED NO: 38), the 14-nucleotide phosphorothioate DNA antisense oligonucleotide (SEQ ED NO: 40), and to a lesser extent by the F9-targeted siRNA oligonucleotide (SEQ ED NO: 41, SEQ ED NO: 46).
  • the control RNA invert (SEQ ED NO: 42) and remaining oligonucleotides are no more effective than Lipofectamine 2000 alone.

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Abstract

La présente invention concerne des oligonucléotides inhibiteurs qui sont ciblés sur six régions cibles spécifiques se trouvant sur des acides nucléiques codant la métalloprotéine-9 matricielle. Ces oligonucléotides inhibiteurs ont en général une longueur comprise entre environ 8 et environ 50 nucléotides. Les oligonucléotides préférés sont présentés. Les oligonucléotides selon la présente invention peuvent être incorporés dans des compositions telles que des compositions pharmaceutiques et peuvent également être utilisés dans des méthodes d'inhibition de l'expression de la métalloprotéinase-9 matricielle dans une cellule ou dans un tissu.
PCT/US2003/033141 2002-10-18 2003-10-17 Oligonucleotides inhibiteurs cibles sur la metalloproteinase-9 matricielle WO2004035759A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999013886A1 (fr) * 1997-09-17 1999-03-25 East Carolina University Acides nucleiques s'hybridant a des cibles multiples, leur preparation, compositions, formulation, trousses et applications

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999013886A1 (fr) * 1997-09-17 1999-03-25 East Carolina University Acides nucleiques s'hybridant a des cibles multiples, leur preparation, compositions, formulation, trousses et applications

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRANCH A.: 'A good antisense molecule is hard to find' TIBS vol. 23, 1998, pages 45 - 50 *
JEN ET AL.: 'Suppression of gene expression by targeted disruption of messanger RNA: available options and current strategies' STEM CELLS vol. 18, 2000, pages 307 - 319 *

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