High-affinity oligonucleotide ligands to vascular endothelial growth factor (VEGF)

• US 6,696,252 B2
• Filed: 05/18/2001
• Issued: 02/24/2004
• 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): Zitomer, Stephanie W.
• Application Number:
  US09/860,474
• Publication Number:
  US 20030032785A1
• Time in Patent Office: 1,012 Days
• Field of Search: 435/6, 435/91.2, 435/196, 530/350, 436/501, 536/22.1
• US Class Current: 435/6.12
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• 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 14/47 from mammals
• CPC Class Codes: C12N 15/1048 SELEX
• 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: 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 2333/8125 Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/974 : Thrombin
• 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 ligands to vascular endothelial growth factor (VEGF). Included in the invention are specific RNA ligands to VEGF identified by the SELEX method.

Claims

1. A method of inhibiting binding of vascular endothelial growth factor (VEGF) to its receptor, comprising:

a) providing a nucleic acid ligand to VEGF; and

b) contacting the nucleic acid ligand with said VEGF under conditions where a VEGF;

nucleic acid binding pair is formed, whereby binding of VEGF to its receptor is inhibited.

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

a) preparing a candidate mixture of nucleic acids;

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

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

d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby a VEGF nucleic acid ligand may be identified.

3. The method of claim 1, wherein said VEGF nucleic acid ligand is a ribonucleic acid.

4. The method of claim 1, wherein said VEGF nucleic acid ligand is a deoxyribonucleic acid.

5. The method of claim 1, wherein said VEGF nucleic acid ligand comprises modified nucleotides.

6. The method of claim 5, wherein said VEGF ligand is comprised of 2′

-amino (2′

-NH₂) modified nucleotides and 2′

-O-methyl (2′

-OMe) modified nucleotides.

7. The method of claim 1, wherein said VEGF nucleic acid ligand is modified with a moiety that decreases the activity of endonucleases or exonucleases on the nucleic acid ligand relative to the unmodified nucleic acid ligand, without adversely affecting the binding affinity of said nucleic acid ligand.

8. The method of claim 7, wherein said moiety comprises a phosphorothioate nucleotide.

9. The method of claim 1, wherein said VEGF nucleic acid ligand is modified by a method comprising substituting nucleotide residues, wherein said residues may be chemically modified, and wherein the modification decreases the activity of endonucleases or exonucleases relative to the unmodified nucleic acid ligand, without adversely affecting the binding affinity of said nucleic acid ligand.