High affinity RNA ligands of basic fibroblast growth factor

• US 6,759,392 B1
• Filed: 05/16/1995
• Issued: 07/06/2004
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Janjic, Nebojsa, Gold, Larry
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US08/442,423
• Time in Patent Office: 3,339 Days
• Field of Search: 435/6, 435/91.2, 514/44
• US Class Current: 514/44.00R
• 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: 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: 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 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• 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 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• 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 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5041 : involving analysis of members of signalling pathways
• 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 nucleic acid ligand solutions to basic fibroblast growth factor (bFGF). Included in the invention are nucleic acid ligands to bFGF which are inhibitors of bFGF and 2′-amino-modified RNA ligands to bFGF.

Claims

1. A method for inhibiting angiogenesis comprising administering a pharmaceutically effective amount of a nucleic acid ligand to bFGF, wherein said ligand has an increased affinity to bFGF relative to a total candidate nucleic acid mixture.

2. The method of claim 1 wherein said nucleic acid bFGF ligand is identified according to a method comprising the steps of:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture with bFGF, wherein nucleic acids having an increased affinity to bFGF relative to the total candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acids from the remainder of the nucleic acids in the candidate mixture; and

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for sequences with increased affinity to bFGF, whereby said nucleic acid ligan is identified.

3. The method of claim 2 wherein said ligand is a 2′

-NH₂-modified ligand selected from the group consisting of SEQ. ID. NOS. 101-165.

4. The method of claim 1 wherein the ligand is a 2′

-NH₂-modified sequence that is substantially homologous to and has substantially the same ability to bind bFGF as the ligands set forth in SEQ. ID. NOS. 101-165.

5. The method of claim 1 wherein the ligand is a 2′

-NH₂-modified sequence that has substantially the same structure as and substantially the same ability to bind bFGF as the ligands set forth in SEQ. ID NOS. 101-165.

6. The method of claim 1 wherein the nucleic acid bFGF ligand is a non-modified sequence selected from the group consisting of SEQ. ID. NOS. 8-22 and 24-69.

7. The method of claim 1 wherein the nucleic acid bFGF ligand is a non-modified sequence that is substantially homologous to and has substantially the same ability to bind bFGF as the ligands set forth in SEQ. ID NOS. 8-22 and 24-69.

8. The method of claim 1 wherein the nucleic acid bFGF ligand is a non-modified sequence that has substantially the same structure as and substantially the same ability to bind bFGF as the ligands set forth in SEQ. ID. NOS. 8-22 and 24-69.