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  • 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
  • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3511—Conjugate intercalating or cleaving agent

Definitions

• the present invention is broadly concerned with oligomers capable of inhibiting expression of interleukin genes, transcription-inhibiting complexes, each composed of an oligomer bound to an interleukin gene, and corresponding methods having potential as therapies for inflammatory polyarthropathy. More particularly, in preferred embodiments, each of these oligomers binds in the parallel or antiparallel orientation to the polypurine strand of a polypurine-polypyrimidine region of the transcribed region of an interleukin gene, thereby resulting in the formation of a transcription-inhibiting triplex.
• Interleukin- 15 is a novel cytokine having biological functions similar to those of interleukin-2 even though there is no significant sequence homology between the two. Interleukin- 15 is produced by epithelial and fibroblast cell lines, and by peripheral blood monocytes. Furthermore, interleukin- 15-specif ⁇ c mRNA has been found in several normal human tissues including placenta, skeletal muscle, and kidney (Grabstein et al., 1994, Science 264:965-968).
• Interleukin- 15 induces T-cell proliferation, enhances natural killer (NK) cell cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and upregulates production of NK cell-derived cytokines including interferon- ⁇ (FIN- ⁇ ), granulocyte/macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF- ⁇ ) (Grabstein et al, 1994, Science 264:965-968; Burton et al, 1994, Proc. Natl. Acad. Sci. 91 :4935-4939; Bamford et al, 1994, Proc. Natl. Acad. Sci.
• FIN- ⁇ interferon- ⁇
• GM-CSF granulocyte/macrophage-colony-stimulating factor
• TNF- ⁇ tumor necrosis factor-
• Interleukin- 15 also costimulates proliferation and differentiation of B cells activated with anti- immunoglobulin M (anti-IgM) (Armitage et al., 1995, J. Immunol. 154:483-490), stimulates locomotion and chemotaxis of normal T cells (Wilkinson et al., 1995, J. Exp. Med.
• Rheumatoid arthritis is a destructive inflammatory polyarthropathy (Maini et al., 1995, in Mechanisms and Models in Rheumatoid Arthritis, pp. 25-26, eds. Henderson, Edwards, and Pettifer, Academic Press, London, 25-26).
• Chronic rheumatoid synovitis is characterized by the presence of activated fibroblast-like synoviocytes together with infiltration of the normally acellular synovial membrane by macrophages, T cells, and plasma cells (Duke et al., 1982, Clin. Exp. Immunol. 49:22-30).
• interleukin- 15 in rheumatoid arthritis synovial fluid are sufficient to exert chemoattractant activity on T cells in vitro, and can induce proliferation of peripheral blood and synovial T cells; furthermore, interleukin- 15 induces an inflammatory infiltrate consisting predominantly of T lymphocytes (Mclnnes et al., 1996, Nature Medicine 2:175-182).
• Therapies directed at T cells such as cyclosporin A and monoclonal antibodies against T-cell surface antigens, produce significant clinical improvement, confirming the importance of T cells in inflammatory polyarthropathy (Horneff et al, 1991, Arth. Rheum. 34:129-
• interleukin- 15 plays a significant role in T-cell recruitment and activation in inflammatory polyarthropathy.
• Oligomers i.e., oligonucleotides and oligonucleotide analogs such as protein nucleic acid
• Oligomers are reagents for inhibition of gene expression because of their high-affinity binding to specific nucleotide sequences.
• the best known strategy for causing inhibition of gene expression involves antisense oligonucleotides which bind to mRNA to inhibit its processing or translation.
• antisense oligonucleotides which bind to mRNA to inhibit its processing or translation.
• gene promoters can serve as targets for a novel, antisense strategy, namely the triplex strategy.
• This strategy employs single-stranded oligomers that bind to the major groove of a polypurine-polypyrimidine region of a double-stranded DNA to form a triplex in a sequence-specific manner. These oligomers are called triplex- forming oligonucleotides (TFO's) or TFO analogs.
• TFO's triplex- forming oligonucleotides
• a purine-rich DNA single strand is hydrogen bonded by Watson-Crick base- pairing to a pyrimidine-rich DNA single strand; the polypurine-polypyrimidine region is not necessarily a homopurine-homopyrimidine region in that the purine-rich DNA single strand may contain at least one pyrimidine residue and the pyrimidine-rich DNA single strand may contain at least one purine residue.
• the present invention provides novel therapies for inflammatory polyarthopathy associated with rheumatoid arthritis and eosinophilic inflammation associated with chronic asthma.
• these therapies expression of interleukin genes is inhibited, resulting in suppression of T-cell recruitment and activation, and a concomitant alleviation of inflammatory polyarthopathy or eosinophilic inflammation.
• a sequence-specific oligomer is introduced into a cell resulting in the production of a transcription-inhibiting complex composed of the oligomer bound to the interleukin gene.
• oligomers include oligonucleotides (e.g., phosphodiester, phosphorothioate, methylphosphonate, and methylphosphonothioate oligonucleotides) and oligonucleotide analogs [e.g., protein nucleic acid, morpholino, methylene (methylimino) linkage, boronated, and pteridine oligomers].
• Oligonucleotide analogs can be linked at either their 5' or 3' ends to intercalators (e.g., psoralen and acridine derivatives). Oligomers can be formulated into pharmaceutically acceptable preparations (e.g., injectable preparations, sprays, ointments, creams, gels, tablets, and perfusions).
• intercalators e.g., psoralen and acridine derivatives.
• Oligomers can be formulated into pharmaceutically acceptable preparations (e.g., injectable preparations, sprays, ointments, creams, gels, tablets, and perfusions).
• the oligomer is a phosphorothioate oligodeoxynucleotide having a length of at least about 5 nucleotides, preferably from about 5 to 50 nucleotides; this oligonucleotide preferably binds in the antiparallel orientation to the polypurine strand of a polypurine-polypyrimidine region of the transcribed region of the interleukin- 15 gene.
• Figure 1 is a photograph of an autoradiogram of a gel illustrating the results of a gel mobility shift analysis of oligonucleotide-directed triplex formation on a target DNA specific to the transcribed region of the interleukin- 15 gene; sequence ID No. 1 was end-labeled and incubated with an excess of unlabeled Sequence ID No. 3; each reaction mixture was either incubated overnight at 22°C and included 0.1 ⁇ g (lane 1), 0.2 ⁇ g (lane 2), 0.4 ⁇ g (lane 3), 0.8 ⁇ g (lane 4), or 1.6 ⁇ gs (lanes 5 and 6) of Sequence ID No.
• Oligonucleotides were synthesized on an Applied Biosystems 392 DNA synthesizer. Double-stranded oligonucleotides were prepared by mixing equal amounts of complementary single strands in the presence of 0.25 M NaCl. The mixture was heated to 80°C for 5 min, incubated at 55°C for 30 min, and then at 42°C for 30 min. Oligonucleotides were gel purified on a 10% polyacrylamide gel, electroeluted, and precipitated with ethanol. Table 1 describes the double-stranded oligonucleotides (i.e., target DNAs) of the present invention:
• Table 2 describes the single-stranded oligonucleotides (i.e., TFO's) of the present invention:
• Sequence ID No. 1 was end-labeled with [ 32 P]ATP using T 4 polynucleotide kinase, and was purified through a Sephadex G50 column. Approximately 30,000 cpm was incubated with 0 to 1.6 ⁇ g of Sequence ID No. 3 in a 10 ⁇ l reaction mixture including 20 mM Tris-HCl (pH 7.4), 20 mM MgCl 2 , 2.5 mM spermidine, 10% sucrose, and 0.25 mg/ml bovine serum albumin. Incubation was conducted overnight at 22°C or 37°C, or was conducted for 3 h at 22°C.
• Sequence ID No. 3 Forms a Triplex with Sequence ID No. 1 :
• the 21 -bp long polypyrimidine sequence of the transcribed region of the human interleukin- 15 gene occurring at nucleotide positions 184 to 205 is a rare structure; such long stretches of all C's and T's occur infrequently in other genes. It was hypothesized that a single-stranded oligonucleotide with a sequence complementary to the polypyrimidine sequence of the interleukin- 15 gene would be able to form a triplex with this structure. In order to demonstrate triplex formation with this target sequence, gel mobility shift assays were performed. The detection of a triplex structure in this electrophoresis system is based on the observation that triplex DNA migrates more slowly in a polyacrylamide gel relative to duplex DNA due to the reduction of DNA charge that is likely to accompany triplex formation.

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• 1996
  • 1996-10-25 US US08/740,215 patent/US5874566A/en not_active Expired - Fee Related
• 1997
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