Nucleic acid ligand complexes

• US 6,011,020 A
• Filed: 05/04/1995
• Issued: 01/04/2000
• 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: Schmidt, Paul G., Janjic, Nebojsa, Gold, Larry
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/434,465
• Time in Patent Office: 1,706 Days
• Field of Search: 536/22.1, 536/24.3, 536/24.31, 435/6, 435/91.2, 935/77, 514/44, 424/450, 424/1.21, 424/1.73
• 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: 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: 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 15/115 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 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 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin G01N2333/665, corticotropin G01N2333/695
• 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

This invention discloses a method for preparing a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipophilic compound or non-immunogenic, high molecular weight compound by identifying a nucleic acid ligand by SELEX methodology and associating the nucleic acid ligand with a lipophilic compound or a non-immunogenic, high molecular weight compound. The invention further discloses complexes comprising one or more nucleic acid ligands in association with a lipophilic compound or non-immunogenic, high molecular weight compound.

Claims

1. A therapeutic or diagnostic Complex comprised of a Nucleic Acid Ligand and a polyethylene glycol (PEG), wherein said Nucleic Acid Ligand is covalently linked to said PEG, and wherein said Nucleic Acid Ligand has a specific binding affinity for a Target Molecule, said Target Molecule being a three dimensional chemical structure other than a polynucleotide that binds to said Nucleic Acid Ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said Nucleic Acid Ligand is not a Nucleic Acid having the known physiological function of being bound by the Target Molecule, wherein said PEG is at least about 1000 Daltons, and wherein said Complex has improved pharmacokinetic properties relative to the pharmacokinetic properties of the Nucleic Acid Ligand alone.

2. The Complex of claim 1 wherein said Nucleic Acid Ligand is identified as a ligand of a Target Molecule from a Candidate Mixture of Nucleic Acids according to the method comprising:

a) contacting the Candidate Mixture with the Target Molecule, wherein the Nucleic Acids having an increased affinity to the Target Molecule 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 of said Target Molecule is identified.

3. The Complex of claim 2 wherein said method further comprises:

d) repeating steps b) and c).

4. The Complex of claim 1 comprising a Lipid Construct.

5. The Complex of claim 4 wherein said Lipid Construct is a Lipid Bilayer Vesicle.

6. The Complex of claim 5 wherein said Lipid Bilayer Vesicle is a Liposome.

7. The Complex of claim 1 wherein said Nucleic Acid Ligand targets the Complex to a preselected location.

8. The Complex of claim 1 wherein the Target Molecule of said Nucleic Acid Ligand is an intercellular Target Molecule.

9. The Complex of claim 1 wherein the Target Molecule of said Nucleic Acid Ligand is an intracellular Target Molecule.

10. The Complex of claim 9 wherein said Nucleic Acid Ligand has enhanced cellular uptake relative to the Nucleic Acid Ligand alone.

11. The Complex of claim 1 further comprising an additional therapeutic or diagnostic agent.

12. The Complex of claim 11 wherein said additional therapeutic or diagnostic agent is associated with said polyethylene glycol.

13. The Complex of claim 12 wherein said additional therapeutic or diagnostic agent is covalently associated with said polyethylene glycol.

14. A method for improving the pharmacokinetic properties of a Nucleic Acid Ligand comprising covalently linking said Nucleic Acid Ligand to a PEG, wherein said Nucleic Acid Ligand has a specific binding affinity for a Target Molecule, said Target Molecule being a three dimensional chemical structure other than a polynucleotide that binds to said Nucleic Acid Ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said Nucleic Acid Ligand is not a Nucleic Acid having the known physiological function of being bound by the Target Molecule, and wherein said PEG is at least about 1000 Daltons.

15. The method of claim 14 wherein said Nucleic Acid Ligand is identified from a Candidate Mixture of Nucleic Acids, said Nucleic Acid Ligand being a ligand of said Target Molecule, by the method comprising:

a) contacting the Candidate Mixture with the Target Molecule, wherein Nucleic Acids having an increased affinity to the Target Molecule 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 Nucleic Acid Ligands of said Target Molecule are identified.

16. The method of claim 14 wherein said Nucleic Acid Ligand is linked to a Lipid Construct.

17. The method of claim 16 wherein said Lipid Construct is a Lipid Bilayer Vesicle.

18. The method of claim 17 wherein said Lipid Bilayer Vesicle is a Liposome.

19. The method of claim 14 wherein said Nucleic Acid Ligand targets to a preselected location.

20. The method of claim 15 wherein said Target Molecule is an intercellular Target Molecule.

21. The method of claim 15 wherein said Target Molecule is an intracellular Target Molecule.

22. The method of claim 14 which further comprises attaching an additional therapeutic or diagnostic agent with said Nucleic Acid Ligand.

23. The method of claim 22 wherein said additional therapeutic or diagnostic agent is attached to said PEG.

24. The method of claim 23 wherein said additional therapeutic or diagnostic agent is covalently attached to said PEG.