Systematic evolution of ligands by exponential enrichment: tissue SELEX

• US 6,013,443 A
• Filed: 08/05/1997
• Issued: 01/11/2000
• Est. Priority Date: 05/03/1995
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Gold, Larry, Heilig, Joseph S.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/906,955
• Time in Patent Office: 889 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/24.31, 536/25.4, 935/77, 935/78, 530/412, 530/413
• US Class Current: 435/6.11
• 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: 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 2310/18 acting by a non-sequence specific mechanism other than C12N2310/16 or C12N2310/17
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

Abstract

This invention discloses high-affinity oligonucleotide ligands to complex tissue targets, specifically nucleic acid ligands having the ability to bind to complex tissue targets, and the methods for obtaining such ligands. Tissue targets comprise cells, subcellular components, aggregates or cells, collections of cells, and higher ordered structures. Specifically, nucleic acid ligands to red blood cells ghosts, endothelia of the blood brain and CSF-blood barriers, glioblastomas, and lymphomas are described.

Claims

1. A method for purifying or identifying a transporter molecule of a blood brain barrier or cerebral spinal fluid (CSF)-blood barrier tissue comprising:

a) identifying a nucleic acid ligand to an epitope of said transporter molecule by the method comprising;

i) preparing a candidate mixture of nucleic acids;

ii) contacting said candidate mixture of nucleic acids with a blood brain barrier endothelial cell or a CSF-blood barrier epithelial cell, wherein nucleic acids having an increased affinity to said epitope of said transporter molecule relative to the candidate mixture and capable of being transported across said barrier cell may be partitioned from the remainder of the candidate mixture;

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

iv) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said epitope of said transporter molecule and capable of transport across said barrier cell, whereby a nucleic acid ligand to said epitope of said transporter molecule may be identified;

b) purifying said transporter molecule of said blood brain barrier or CSF-blood barrier tissue away from the remainder of said tissue on the basis of affinity between said epitope and said nucleic acid ligand; and

c) optionally identifying said transporter molecule.

2. The method of claim 1 wherein said transporter molecule is selected from the group consisting of a protein, a lipid and a carbohydrate.

3. A purified transporter molecule identified according to the method of claim 1.

4. The purified transporter molecule of claim 1 which is selected from the group consisting of a protein, a lipid and a carbohydrate.