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

• US 5,811,533 A
• Filed: 05/19/1995
• Issued: 09/22/1998
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Janjic, Nebojsa, Gold, Larry
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/447,169
• Time in Patent Office: 1,222 Days
• Field of Search: 536/23.1, 536/25.4, 435/6, 435/91.2, 435/77, 435/78
• US Class Current: 536/23.1
• 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: 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: 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 2333/96436 : 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 33/531 : 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 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 nucleic acid ligand to vascular endothelial growth factor (VEGF) identified according to the method comprising:

a) contacting a candidate mixture of nucleic acids 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;

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

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

10. The nucleic acid ligand of claim 1 wherein the method further comprises modifying the nucleic acid ligand wherein said modifying comprises adding 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.

11. The nucleic acid ligand of claim 10 wherein said moiety comprises a phosphorothioate nucleotide.

12. The nucleic acid ligand of claim 1 wherein the method further comprises modifying the nucleic acid ligand wherein said modifying comprises 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.

13. The nucleic acid of claim 12 wherein said chemically modified residue comprises a 2'"'"'-O-Methyl (2'"'"'OMe) nucleotide.

2. A purified and isolated non-naturally occurring nucleic acid ligand to VEGF.

3. The nucleic acid ligand of claim 2 which is a ribonucleic acid.

4. The RNA ligand of claim 3 wherein said ligand is selected from the group consisting of the sequences set forth in FIG. 2.

5. The RNA ligand of claim 3 wherein said ligand has been chemically modified at the ribose and/or phosphate and/or base positions.

6. The modified RNA ligand of claim 5 wherein said ligand is comprised of 2'"'"'-amino (2'"'"'-NH₂) modified nucleotides.

7. The modified RNA ligand of claim 6 wherein said ligand is selected from the group consisting of the sequences set forth in FIG. 9 and Table 4.

8. The nucleic acid ligand of claim 2 which is a deoxyribonucleic acid.

9. The DNA ligand of claim 8 wherein said ligand is selected from the group consisting of the sequences set forth in Table 8.

18. The modified RNA ligand of claim 5 wherein said ligand is comprised of a 2'"'"'-O-Methyl (2'"'"'-OMe) modified nucleotide.

14. A modified nucleic acid ligand of VEGF, wherein said ligand is modified to comprise the addition of a moiety to a nucleic acid ligand that decreases the activity of endonucleases or exonucleases on the nucleic acid ligand, without adversely affecting the binding affinity of said nucleic acid ligand.

15. The nucleic acid ligand of claim 14 wherein said moiety comprises a phosphorothioate nucleotide.

16. A modified nucleic acid ligand of VEGF, wherein the modification comprises the substitution of nucleotide residues of the nucleic acid ligand, wherein said residues may be chemically modified, and wherein said modification 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.

17. The nucleic acid ligand of claim 16, wherein said chemically modified residue comprises a 2'"'"'-O-Methyl (2'"'"'-OMe) nucleotide.