single-stranded oligonucleotide comprising a 5'-end sequence, an intermediate sequence and a 3'-end sequence, both ends having reverse complementarity with the intermediate sequence, intermediate sequence having loops at both ends; phosphate groups are not phosphorothioated; antiinflammatory agent
Inventors
Kunugiza, Yasuo • Tomita, Naruya • Hashimoto, Hideo • Yoshikawa, Hideki • Morishita, Ryuichi
Assignees
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Publication Number
US-7595301-B2
Publication Date
2009-09-29
Expiration Date
Abstract
Conventional oligonucleotides are opened at both ends and thereby unstable. Their stability against catabolic enzymes is increased by phosphorothioate modification, but such phosphorothioate causes toxicity. The present invention provides oligonucleotides and medicaments in which these problems are improved. That is, it provides staple oligonucleotides and medicaments containing the same as the active ingredient. Specifically, it provides transcription factor inhibitors, antisense oligonucleotides and siRNAs. More specifically, it provides agents for preventing, treating or improving inflammation, autoimmune diseases, central diseases, reperfusion injury in ischaemic diseases, worsened prognosis after organ transplantation or organ surgery, or restenosis after PTCA. Further specifically, it provides agents for preventing, treating or improving arthritis, dermatitis, nephritis, hepatitis, renal failure, cystitis, prostatitis, urethritis, ulcerative colitis, Crohn disease, chronic rheumatoid arthritis, osteoarthritis, atopic dermatitis, contact dermatitis, psoriasis, cutaneous ulcer or decubitus.
Core Innovation
The invention relates to a staple oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, as a single-stranded oligonucleotide comprising a 5′-end sequence, an intermediate sequence, and a 3′-end sequence. The 5′-end sequence has reverse complementarity with the intermediate sequence, and the 3′-end sequence has reverse complementarity with the intermediate sequence. The intermediate sequence has loops at both ends.
The staple oligonucleotide is defined such that the loops do not form an intramolecular complementary bond. The phosphate groups are not phosphorothioated, distinguishing the staple structures from phosphorothioate-terminated single-stranded oligonucleotides that can be degraded.
The disclosed staple oligonucleotides and medicaments are provided for improving stability of conventional end-open oligonucleotides without phosphorothioate toxicity. The content describes stability in synovial fluid, including that non-phosphorothioated staple decoys remain detectable while certain phosphorothioated-terminated single-stranded precursors are degraded.
Claims Coverage
The partial content includes one independent claim, which defines the staple oligonucleotide structure and the loop constraint. Overall, the independent claim contains multiple inventive structural features: reverse-complementary 5′/3′ end sequences to an intermediate region, loops at both ends, and loops that do not form intramolecular complementary bonds.
Staple oligonucleotide with reverse-complementary 5′ and 3′ ends to an intermediate sequence
The staple oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 1 and is a single-stranded oligonucleotide comprising a 5′-end sequence, an intermediate sequence, and a 3′-end sequence, where the 5′-end sequence has reverse complementarity with the intermediate sequence and the 3′-end sequence has reverse complementarity with the intermediate sequence.
Loops at both ends that do not form an intramolecular complementary bond
The intermediate sequence has loops at both ends, and the loops do not form an intramolecular complementary bond.
Phosphate groups are not phosphorothioated
The staple oligonucleotide has phosphate groups that are not phosphorothioated.
Structural formula representation indicating a non-binding site between the ends
The staple oligonucleotide is specified by a structural formula or representation in which a vertical line indicates a non-binding site located between the 5′ end and the 3′ end.
Coverage focuses on a staple oligonucleotide (SEQ ID NO: 1) with reverse-complementary 5′/3′ end sequences to an intermediate region, an intermediate region that includes loops at both ends, and a loop condition that prevents intramolecular complementary bonding; additional claim coverage specifies that phosphate groups are not phosphorothioated and includes a structural-formula representation showing a non-binding site between the ends.
Stated Advantages
Improves stability of conventional end-open oligonucleotides.
Improves stability without phosphorothioate toxicity.
Non-phosphorothioated staple decoys remain detectable in synovial fluid while certain phosphorothioated-terminated single-stranded precursors are degraded.
Documented Applications
Therapeutic use as a transcription factor inhibitor, antisense, or siRNA, particularly for inflammation and autoimmune diseases.
Anti-inflammatory and autoimmune or organ injury conditions including reperfusion injury and organ transplantation or organ surgery.
Prevention or treatment contexts including restenosis after PTCA and arthritis, as well as dermatitis, nephritis, hepatitis, renal failure, cystitis, prostatitis, urethritis, ulcerative colitis, Crohn disease, chronic rheumatoid arthritis, osteoarthritis, atopic dermatitis, contact dermatitis, psoriasis, cutaneous ulcer, and decubitus.
A staple NF-κB decoy ligated at one point reduces IL-1β in LPS-stimulated rheumatoid arthritis synovial tissue and supernatant versus controls.
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