High affinity TGFβ nucleic acid ligands and inhibitors

• US 6,713,616 B2
• Filed: 02/23/2001
• Issued: 03/30/2004
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Pagratis, Nikos, Lochrie, Michael, Gold, Larry
• Primary Examiner(s): Forman, B J
• Application Number:
  US09/791,301
• Publication Number:
  US 20030064943A1
• Time in Patent Office: 1,131 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/23.1
• US Class Current: 536/23.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/547 Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/49 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• CPC Class Codes: C07K 14/50 Fibroblast growth factor [FGF]
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/1136 : against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/115 : Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/13 : Decoys
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/53 : partially self-complementary or closed
• CPC Class Codes: C12N 9/1276 : RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/37 : involving peptidase or proteinase
• CPC Class Codes: C12Q 1/6804 : Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6811 : Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/70 : involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/703 : Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/101 : incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: G01N 2333/16 : HIV-1, HIV-2
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/503 : basic FGF [bFGF]
• CPC Class Codes: G01N 2333/575 : Hormones
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/9726 : Tissue plasminogen activator
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 2333/976 : Trypsin; Chymotrypsin
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to TGFβ2. Included in the invention are specific RNA ligands to TGFβ2 identified by the SELEX method. Also included are RNA ligands that inhibit the interaction of TGFβ2 with its receptor.

Claims

1. A purified and isolated non-naturally occurring RNA ligand to transforming growth factor β

1 (TGFβ

1) wherein said ligand is selected from the group consisting of the sequences set forth in Table 22 (SEQ ID NOS;

194-215).

2. A complex comprised of a TGFβ

1 nucleic acid ligand and a non-immunogenic, high molecular weight compound or lipophilic compound, wherein said ligand is selected from the group consisting of the sequences set forth in claim 1.

3. The complex of claim 2 further comprising a linker between said ligand and said non-immunogenic, high molecular weight compound or lipophilic compound.

4. The complex of claim 2 wherein said non-immunogenic, high molecular weight compound is polyalkylene glycol.

5. The complex of claim 4 wherein said polyalkylene glycol is polyethylene glycol (PEG).

6. The complex of claim 5 wherein said PEG has a molecular weight of about between 10-80 K.

7. The complex of claim 6 wherein said PEG has a molecular weight of about 20-45 K.

8. The complex of claim 7 wherein said complex is:

LIGAND=5′

mGmGmGrUrGrCrCrUrUrUrUrGrCrCrUmAmGmGrUrUmGrUmGmArUrU rUmGrUmAmArCrCrUrUrCrUrGrCrCrCmA3′

-3′

rU (SEQ ID NO;

210)

wherein r prefix indicates unmodified ribonucleoside, m prefix indicates 2′

-O-methyl substituted ribonucleotide.

9. A complex comprised of a TGFβ

2 nucleic acid ligand, and a non-immunogenic, high molecular weight compound or lipophilic compound, wherein said ligand is selected from the group consisting of the sequences set forth in Tables 5, 7, 8, 11, 13, 14, 16-19, and FIG. 9 (SEQ ID NOS;

21-121 and 128-193).

10. The complex of claim 9 further comprising a linker between said ligand and said non-immunogenic, high molecular weight compound or lipophilic compound.

11. The complex of claim 9 wherein said non-immunogenic, high molecular weight compound is a polyalkylene glycol.

12. The complex of claim 11 wherein said polyalkylene glycol is polyethylene glycol (PEG).

13. The complex of claim 12 wherein said PEG has a molecular weight of about between 10-80 K.

14. The complex of claim 13 wherein said PEG has a molecular weight of about 20-45 K.