Nucleic acid ligands to the prostate specific membrane antigen

a prostate specific and nucleic acid ligand technology, applied in the direction of group 3/13 element organic compounds, peptides, drug compositions, etc., can solve the problems of limited use of proteins as drugs and reagents, and no reported rna aptamers to membrane bound tumor antigens, etc., to inhibit native psma enzymatic activity and high affinity

Inactive Publication Date: 2005-07-21
GILEAD SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of proteins as drugs and reagents is often limited by the activity of proteases, the size of the protein, transport and the ability of an organism to make antibodies against that protein.
Despite the success of this technique, however, there are no reported RNA aptamers to membrane bound tumor antigens.

Method used

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  • Nucleic acid ligands to the prostate specific membrane antigen
  • Nucleic acid ligands to the prostate specific membrane antigen
  • Nucleic acid ligands to the prostate specific membrane antigen

Examples

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example 1

Use of SELEX to Obtain Nucleic Acid Ligands to PSMA

Materials and Methods

[0087] Cloning PSMA cDNA from LNCaP.

[0088] First strand cDNA was synthesized from 2 μg total LNCaP RNA using Superscript II RNase H Reverse Transcriptase (Life Technologies, Inc). Primers homologous to PSMA cDNA bases 134-152 and 2551-2567, flanking the entire full-length PSMA coding region, were used for PCR amplification. Amplification was performed using high fidelity Pfu DNA Polymerase (Stratagene, La Jolla, Calif.). The isolated product was ligated into the pCR-2.1 vector (Invitrogen Corporation, Carlsbad, Calif.). One successful clone, pFULPSM-1, was sequenced and found identical to Genbank accession number M99487. This clone represents the coding region for the full length PSMA protein

[0089] Preparation of Recombinant PSMA Expressing Baculovirus.

[0090] Primers containing restriction enzyme cut sites were designed to overlap the sequence of the entire extracellular portion of PSMA plus a linking glyc...

example 2

Determination of Minimal Size of Aptamers A10 and A9

[0112] To determine minimal aptamer sequences, a series of 3′ and 5′ truncations were tested for IC50. Five nucleotides could be removed from the 3′ end of aptamer xPSM-A9 (SEQ ID NO:5) with retention of activity, yielding aptamer A9-1 (SEQ ID NO:6). It was found that at least 15 nucleotides could be deleted from the 3′ end of aptamer xPSM-A10 (SEQ ID NO:15) with retention of activity, yielding aptamer A10-3 (SEQ ID NO:18). This 18.5 kD aptamer retains the ability to inhibit xPSM NAALADase activity with a KI of 20.5 nM. (FIG. 8). The shorter A10-3 could not survive 5′-truncation.

TABLE 1RoundSignal / Noise13.4226.8315.8425.75125.065688.0

[0113]

TABLE 2Reaction mixtures for reverse transcription (RT), QPCR and Transcription5X RT mixQPCR mixTranscription mix2.5 μL 10X RT buffer 10 μL 10X SQ buffer40 μL 5X nucleotides  1 μL 25 mM dNTP′S  1 μL 3′ primer 100 μM40 μL 5X Ribomax  1 μL H2O0.5 μL 5′ primer 100 μM10 μL guanosine 100 mM0.5 μL A...

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Abstract

Methods are provided for generating nucleic acid ligands of Prostate Specific Membrane Antigen (PSMA). The methods of the invention use the SELEX method for the isolation of nucleic acid ligands. The invention also includes nucleic acid ligands to PSMA, and methods and compositions for the treatment and diagnosis of disease using the nucleic acid ligands.

Description

RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 09 / 978,969, filed Oct. 16, 2001, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60 / 278,830, filed Mar. 26, 2001 “Nucleic Acid Ligands to the Prostate Specific Membrane Antigen” and which also claims the benefit of U.S. Provisional Patent Application Ser. No. 60 / 240,781, filed Oct. 16, 2000, entitled “Nucleic Acid Ligands to the Prostate Specific Membrane Antigen”. Each of the aforementioned applications is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] Described herein are high affinity nucleic acid ligands to Prostate Specific Membrane Antigen (PSMA). Also described herein are methods for identifying and preparing high affinity nucleic acid ligands to PSMA. The method used herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Further disclosed...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09A61K47/48A61K51/00A61P35/00C07K14/705C07K17/02C12N15/115C12Q1/68
CPCC07K14/705C12Q1/6886C12N2310/322C12N15/115A61P35/00
Inventor LUPOLD, SHAWNLIN, YUNHICKE, BRIANCOFFEY, DONALD
Owner GILEAD SCI INC
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