EP1667729A2 - Aptamere a oligonucleoside phosphorothioate et phosphorodithioate selectionne de maniere combinatoire et selon la structure pour le ciblage des facteurs de transcription ap-1 - Google Patents

Aptamere a oligonucleoside phosphorothioate et phosphorodithioate selectionne de maniere combinatoire et selon la structure pour le ciblage des facteurs de transcription ap-1

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Publication number
EP1667729A2
EP1667729A2 EP04776088A EP04776088A EP1667729A2 EP 1667729 A2 EP1667729 A2 EP 1667729A2 EP 04776088 A EP04776088 A EP 04776088A EP 04776088 A EP04776088 A EP 04776088A EP 1667729 A2 EP1667729 A2 EP 1667729A2
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European Patent Office
Prior art keywords
antigen
thioaptamer
virus
immune response
adjuvant
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EP04776088A
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German (de)
English (en)
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EP1667729A4 (fr
Inventor
David G. Gorenstein
Bruce A. Luxon
Norbert Herzog
Judith F. Aronson
David Beasley
Allan Barret
Robert E. Shope
Xian Bin Yang
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University of Texas System
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University of Texas System
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Publication of EP1667729A2 publication Critical patent/EP1667729A2/fr
Publication of EP1667729A4 publication Critical patent/EP1667729A4/fr
Withdrawn legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/13—Decoys
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/12011—Bunyaviridae
    • C12N2760/12211—Phlebovirus, e.g. Rift Valley fever virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Arenavirus Hemorrhagic Fevers such as Lassa fever, Junin, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, have several features in common with sepsis and the systemic inflammatory response syndrome, including fulminant clinical course, fever, shock, capillary leak syndrome, decreased myocardial contractility, abnormalities of coagulation and platelet function, and elevated serum levels of TNF ⁇ (Aronson et al., 1994; Cummins, 1990).
  • Arenaviruses are non-cytopathic viruses with a tropism for macrophages and other reticuloendothelial cells (Cummins, 1990; Peters et al., 1987); the pathogenesis of these diseases is believed to involve excessive production of pro-inflammatory cytokines (Aronson et al., 1995; Peters et al., 1987). Unpublished data (Bausch et al., CDC) show cytokines to be massively activated in human Lassa fever, and also confirm that Lassa virus can directly induce cytokine secretion by infecting human macrophages in vitro (Mahanty et al., CDC, unpublished).
  • Endotoxic shock results from an innate, anaphylactic response to bacterial lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • NF- B activation figures A series of intracellular signaling events, in which NF- B activation figures importantly leads to enhanced transcription of proinfiammatory mediators, including TNF ⁇ , IL-1 and inducible nitric oxide synthase, ultimately promoting vasodilatation, capillary leakiness, and myocardial suppression
  • DNA-binding activity can be detected in the nuclei of macrophages and other cell types (Boher, et al., 1997); similar observations have been made in human sepsis (Velasco et al., 1997).
  • the AP-1 transcription factor family include the dimeric basic region leucine zipper proteins that belong to the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, Fra-2) Maf (c-Maf, MafB, MafA, MafG/F/K, Nrl) and ATF/CREB (CREB, CREBP-2, ATF1, ATF2, LRF1/ATF3, ATF4, ATFa, ATF6, B-ATF, JDP1, JDP2) subfamilies which recognize either 12-0-tetradecanoylphorbol-13-acetate (TPA) response elements (5'-TGAG/CTCA-3') or cAMP response elements (CRE, 5'-TGACGTCA-3') (Chinenov and Kerppola, 2001; Shaulian and Karin, 2002).
  • TPA 12-0-tetradecanoylphorbol-13-acetate
  • CRE CRE, 5'-TGACGTCA-3'
  • the present invention is based on the recognition that thiomodified aptamers may be designed, isolated and used to manipulate transcription factors such as NFKB and AP-1 to interdict the pathogenetic sequence, or even boost early protective innate immune responses (Figure 1).
  • transcription factors such as NFKB and AP-1
  • Figure 1 To demonstrate the feasibility of using the modified thioaptamers disclosed herein at physiological concentrations, animal model systems were used that models both severe fatal disease and self-limited infection with mild disease.
  • a well-recognized and widely used guinea pig model for Lassa Fever uses the New World arenavirus Pichinde (PIC) (Peters et al., 1987) was used and adapted to study pathogenesis by comparing an attenuated variant of PIC (P2) and a closely related virulent variant derived by serial guinea pig passage (P18) (setting et al., 1981).
  • PIC New World arenavirus Pichinde
  • the present invention was used to modulate induction of CREB, a transcription factor regulated by cyclic AMP (cAMP) signaling.
  • cAMP cyclic AMP
  • the modified thioaptamers were used to modulate CREB activity and were demonstrated to modify virulent and attenuated Arenavirus infection.
  • the CREB protein is also a member of the AP-1 family of transcription factors whose targeting by XBY-S2 has provided protection for animals infected with arenavirus and flavivirus.
  • cAMP is a ubiquitous second messenger (Antoni et al., 2000) synthesized in cells by adenylyl cyclases in response to many extra-cellular stimuli.
  • the present invention provides a number of advantages due to the use of modified thioaptamers and combinatorial selection methods.
  • the present invention provides very high affinity - nM to sum-nM (> monoclonal IgMs and >non-substituted aptamers), target-specific aptamers, demonstrating single protein target binding within cellular extracts.
  • the modified thioaptamers have greater resistance to cellular or serum nuclease degradation than normal backbone aptamers, or proteases towards antibodies. Due to the increased nuclease resistance, the aptamers disclosed herein may be packaged to have indefinite shelf-life, ease of storage as lyophilized powders at room temperatures, unlike unmodified RNA or antibodies and are relatively inexpensive to produce.
  • compositions disclosed herein allow for high reproducibility in quality control, unlike diasteromeric mixtures for non-stereospecifically produced monothiophosphate aptamers, or protein production of antibodies.
  • use of bead- based thioaptamer libraries or library of libraries provides large combinatorial libraries readily selected by multicolor flow cytometry at very high speeds (10 8 /hr).
  • the location of protein binding on such a microarray may be detected using well known protein detection methods, e g , fluorescence
  • the protein for use with the invention may be protein from a crude extract or even partially purified or isolated, e g , one or more proteins isolated from a gel
  • the system and method disclosed herein may further include the use of binding the thioaptamers to beads and sorting the beads to isolate and identify proteins that have specifically bound to the thioaptamers
  • the beads when using a thioaptamer library of beads, the beads may be sorted based on protein binding, e g , based on fluorescence labeling of the aptamer and or the protein using a flow-cytometer
  • the protein may be from a cell extract, which may even be a cell extract from a virally infected or diseased cell
  • the thioaptamers are attached to beads and the beads are substantially protein-free
  • the thioaptamers When using a one-bead, one-thioaptamer (ODN) library or even a library of libraries the thioaptamers may be one or more beads that include an [S]-ODN and/or [S 2 ]-ODN combinato ⁇ al libraries
  • the ODNs may be single or double stranded and may include
  • one or more of the thioaptamers may be a library of aptamers that binds to one or more transcritption factors and includes sequences or sequence motifs for transcription factor binding, e.g., a NF- ⁇ B, a RBP-J ⁇ , an AP-1, an NF IL-6, an SP-1, a GRE, an SRE motif and/or mixtures thereof.
  • sequences or sequence motifs for transcription factor binding e.g., a NF- ⁇ B, a RBP-J ⁇ , an AP-1, an NF IL-6, an SP-1, a GRE, an SRE motif and/or mixtures thereof.
  • the complex combinatorial library made by a method that includes the steps of synthesizing an aptamer bead library having a first thioaptamer and concatenating to each of the first thioaptamers a second aptamer or thioaptamer suspected of binding to, e.g., a nuclear regulatory factor.
  • the first and second thioaptamers may even be suspected of binding the same nuclear regulatory factor or a different nuclear regulatory factor.
  • Yet another embodiment of the present invention is a method of identifying a thio-modified therapeutic agent that includes mixing a sample suspected of including a DNA binding protein with a concatenated first and second thioaptamer under binding conditions and isolating the one or more DNA binding proteins that bind specifically to the concatenated aptamers.
  • Another embodiment of the present invention is a composition, adjuvant, vaccine and method of modifying an immune response that includes providing a host cell with aptamers that suppress the activity of a nuclear regulatory factor critical for activation of an immune response.
  • the immune response may be an innate immune response, a cytotoxic or a helper T cell immune response.
  • the thioaptamer modified the immune response by shifting the helper 1-type (Thl) to T helper 2-type (Th2) ratio.
  • the immune response that is modified may be to a virus, a bacteria, a fungus, a cancer, a self- antigen, a heterologous antigen, a retrovirus, a hemorraghic virus or a neuropathologic virus, e.g., West
  • the immune response that is modified may be modified in vivo, in vitro and/or ex vivo.
  • the modification of the immune response may be an increase or decrease of the immune response as measured by, e.g., antibody production, cytotoxic T cell activation, cytokine release, apoptosis, cell proliferation, cell killing, chromium release, nucleic or amino acid uptake or release and other methods known to those skilled in the immunological arts.
  • the type of helper T cell response may be modified by providing a host or target cell with one or more thioaptamers that suppress the activity of a nuclear regulatory factor critical for activation of, e.g., a helper T cell response.
  • the T cell immune response may be to, e.g., a virus, a bacteria, a fungus, a cancer, a self-antigen, a heterologous antigen, a retrovirus, a hemorraghic virus or even a neuropathologic virus.
  • the modification to the immune response may be to a challenge to the innate or the adaptive immune response.
  • the helper T cell response may be a T helper 1-type response or a T helper 2-type response.
  • Arenaviridae Lassa, Junin, Machupo, Guanarito, and Sabia viruses, which are the causative agents of Lassa fever and Argentine, Venezuelan, and Brazilian hemorrhagic fevers, respectively
  • Filoviridae Ebola and Marburg
  • Flaviviridae yellow fever, Omsk hemorrhagic fever, and Kyasanur Forest disease viruses
  • Bunyaviridae Rift Valley fever (RFV), Congo-Crimean hemorrhagic fever.
  • RSV Ring Valley fever
  • Another target viral family includes Hantaviruses.
  • the thioaptamers of the present invention may be an adjuvant that forms part of a vaccine, such as a composition that includes one or more partially thio-modified or even concatenated aptamers that modulate an immune response.
  • the thioaptamer adjuvant may also include at least one antigen.
  • the antigens for innate immune response activation may be a pathogen-associated molecular pattern antigen, e.g., a CpG molecule, a saccharide, a lectin, a polysaccharide and the like.
  • the adjuvant thioaptamer may include sequences for specific recognition and binding to nuclear regulatory factors, e.g., NF-AT, NF-KB, RBP-JK, AP-1, NF IL-6, SP-1, GRE and SRE.
  • nuclear regulatory factors e.g., NF-AT, NF-KB, RBP-JK, AP-1, NF IL-6, SP-1, GRE and SRE.
  • partially thioaptamers include one or more of the aptamers of SEQ ID NOS.: 2, 3, 4, 5, 6, 7, 8 and 9.
  • the thioaptamer may an adjuvant that includes one or more partially thioaptamers that bind to, e.g., a DNA binding protein and modulate an immune response, e.g., an innate or an adaptive immune response.
  • the adjuvant may be provided with a physiologically acceptable aqueous vehicle, in a lyophilized, a particulate or even a dissolved form with or without an antigen, e.g., the antigen described hereinabove.
  • Yet another embodiment of the present invention is a method for modifying an immune response that includes administering a composition that includes an antigen and one or more partially thio-modified aptamers or thioaptamers.
  • the modifications to the immune response include, e.g., activation or deactivation of the innate immune response and/or modifications to the type of immune response mounted (humoral versus cell-based) such as a change in the profile of helper T cell involved with or "lead” the immune response
  • the composition may also include cytokines, e g , ⁇ nterleuk ⁇ n-1 (IL-1), ⁇ nterleuk ⁇ n-2 (IL-2), ⁇ nterleuk ⁇ n-3 (IL-3), ⁇ nterleukm-4 (IL-4), ⁇ nterleuk ⁇ n-5 (IL-5), ⁇ nterleuk ⁇ n-6 (IL-6), ⁇ nterleuk ⁇ n-7 (IL-7), ⁇ nterleuk ⁇ n-8 (IL-8), ⁇ nterleuk ⁇ n-10
  • the thioaptamer (thioaptamer) and an antigen may be provided in dry form or even be disposed in a vehicle suitable for oral, intramuscular, subcutaneous, intravenous or parenteral administration, e.g., in a sterile saline solution.
  • the partially thioaptamer may be specific for AP-1, NF- ⁇ B, NF IL-6, or combinations thereof.
  • Figure 7 is a gel that shows that XBY-S2 eliminates API DNA binding activities in macrophages treated with liposomes with and without the indicated aptamers for 24 hours, wherein the nuclear extracts were analyzed by electrophoretic mobility shift assay (EMSA) with the AP-1 and NF- ⁇ B oligonucleotide probes;
  • ESA electrophoretic mobility shift assay
  • Figure 11 is a graph that shows survival curves of guinea pigs with thioaptamers for infection by arenavirus
  • Figure 12 is a graph that shows survival curves following West Nile Virus infection in guinea pigs treated with the NF- ⁇ B aptamer XBY-6, the AP-1 aptamer XBY-S2, or the liposome vehicle of animals infected by injection with lethal doses of West Nile Virus;
  • the modified nucleotide aptamer can include one or more phosphorothioate or phosphordithioate linkages selected from dATP( ⁇ S), dTTP( ⁇ S), dCTP( ⁇ S) and dGTP( ⁇ S), dATP( ⁇ S 2 ), dTTP( ⁇ S 2 ), dCTP( ⁇ S 2 ) and dGTP( ⁇ S 2 ).
  • no more than three adjacent phosphate sites of the modified nucleotide aptamer are replaced with phosphorothioate groups.
  • thioaptamers may be obtained by adding bases enzymatically using a mix of four nucleotides, wherein one or more of the nucleotides is a mix of unmodified and thiophosphate- modified nucleotides, to form a partially thiophosphate-modified thioaptamer library.
  • thioaptamers these are made by adding bases to an oligonucleotide wherein a portion of the phosphate groups are thiophosphate-modified nucleotides, and where no more than three of the four different nucleotides are substituted on the 5'-phosphate positions by 5'-thiophosphates in each synthesized oligonucleotide are thiophosphate-modified nucleotides.
  • a dosage unit for use of the aptamers and partially thioaptamers of the present invention may be a single compound or mixtures thereof with other compounds, e.g., a potentiator.
  • the compounds may be mixed together, form ionic or even covalent bonds.
  • the aptamers and partially thioaptamers of the present invention may be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
  • different dosage forms e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the aptamers and partially thioaptamers of the present invention to a patient in need of therapy that includes the aptamers and partially thioaptamers.
  • the aptamers and partially thioaptamers may also be administered as any one of known salt forms.
  • Aptamers and partially thioaptamers is typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and/or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices.
  • a pharmaceutically acceptable carrier or carrier materials selected based on the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the aptamers and partially thioaptamers may be formulated to provide, e.g., maximum and/or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While the aptamers and partially thioaptamers may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier.
  • the carrier may be solid or liquid, depending on the type and/or location of administration selected.
  • the aptamers and partially thioaptamers may be administered in the form of liposome delivery systems, e.g., small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles, whether charged or uncharged.
  • Liposomes may include one or more: phospholipids (e.g., cholesterol), stearylamine and/or phosphatidylcholines, mixtures thereof, and the like.
  • the oral drug components may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents, mixtures thereof, and the like.
  • Citric acid and its salts and sodium EDTA may also be included to increase stability.
  • parenteral solutions may include pharmaceutically acceptable preservatives, e.g., benzalkonium chloride, methyl- or propyl-paraben, and/or chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, relevant portions incorporated herein by reference.
  • Capsules may be prepared by filling standard two-piece hard gelatin capsules each with 10 to 500 milligrams of powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams of cellulose and 6 milligrams magnesium stearate.
  • Soft Gelatin Capsules A mixture of active ingredient is dissolved in a digestible oil such as soybean oil, cottonseed oil or olive oil. The active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 100-500 milligrams of the active ingredient. The capsules are washed and dried.
  • Tablets A large number of tablets are prepared by conventional procedures so that the dosage unit was 100-500 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 50-275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.
  • Effervescent tablets To provide an effervescent tablet appropriate amounts of, e.g., monosodium citrate and sodium bicarbonate, are blended together and then roller compacted, in the absence of water, to form flakes that are then crushed to give granulates. The granulates are then combined with the active ingredient, drug and/or salt thereof, conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.
  • active ingredient, drug and/or salt thereof conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.
  • aqueous suspension is prepared for oral administration so that each 5 ml contain 100 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S. P., and 0.025 ml of vanillin.
  • the aptamers and partially thioaptamers and, optionally, one or more potentiators may be mixed with a pharmaceutically acceptable carrier.
  • the carrier may be a solid or liquid and the type is generally chosen based on the type of administration being used.
  • the active agent may be coadministered in the form of a tablet, capsule, liposome, as an agglomerated powder, in a liquid form or as a suppository.
  • the present invention includes vaccines for both active and passive immunization.
  • Immunogenic compositions suitable for use as a vaccine, include the modified thioaptamers of the present invention.
  • the thioaptamers are prepared in a manner disclosed herein.
  • the vaccines disclosed herein are not the antigenic material, that is, they are not intended to cause an immune response, but rather, are include either alone or in combination with an antigen to "drive” or modify an immune response by altering the activity of nuclear binding proteins, including, e.g.: NF-ATs, AP-ls, NF-IL6, NF- ⁇ B, HIV reverse transcriptase, Venezuelan Equine Encephalitis nucleocapsid (using an RNA thioaptamer), HepC IRES nucleic acid, protein(s) involved in CpG-induced "innate immunity,” and the like.
  • nuclear binding proteins including, e.g.: NF-ATs, AP-ls, NF-IL6, NF- ⁇ B, HIV reverse transcriptase, Venezuelan Equine Encephalitis nucleocapsid (using an RNA thioaptamer), HepC IRES nucleic acid, protein(s) involved in CpG-induced "innate immunity,” and the like.
  • the active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
  • parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • the thioaptamer and the methods disclosed herein may be used to manipulate Thl:Th2 ratios in a variety of clinical situations.
  • a thioaptamer may be provided that inhibits Th2 activation, which may be useful in allergic diseases, malignancies and infectious diseases.
  • the thioaptamer may be used to enhance Th2 activation for treatment of autoimmune diseases and/or to improve organ transplantation.
  • the present inventors recognized that it is not possible to simply replace thiophosphates in a sequence that was selected for binding with a normal phosphate ester backbone oligonucleotide. Simple substitution was not practicable because the thiophosphates can significantly decrease (or increase) the specificity and or affinity of the selected ligand for the target. It was also recognized that thiosubstitution leads to a dramatic change in the structure of the aptamer and hence alters its overall binding affinity.
  • the sequences that were thioselected according to the present methodology, using as examples of DNA binding proteins AP-1, NF-IL6 and NF- ⁇ B, were different from those obtained by normal phosphate ester combinatorial selection.
  • the present invention takes advantage of the "stickiness" of thio- and dithio-phosphate ODN agents to enhance the affinity and specificity to a target molecule.
  • the method of selection concurrently controls and optimizes the total number of thiolated phosphates to decrease non-specific binding to non-target proteins and to enhance only the specific favorable interactions with the target.
  • the present invention permits control over phosphates that are to be thio-substituted in a specific DNA sequence, thereby permitting the selective development of aptamers that have the combined attributes of affinity, specificity and nuclease resistance.
  • a method of post-selection aptamer modification in which the therapeutic potential of the aptamer is improved by selective substitution of modified nucleotides into the aptamer oligonucleotide sequence.
  • An isolated and purified target binding aptamer is identified and the nucleotide base sequence determined.
  • Modified achiral nucleotides are substituted for one or more selected nucleotides in the sequence.
  • the substitution is obtained by chemical synthesis using dithiophosphate nucleotides.
  • the resulting aptamers have the same nucleotide base sequence as the original aptamer but, by virtue of the inclusion of modified nucleotides into selected locations in the sequences, improved nuclease resistance and affinity is obtained.
  • Phosphorodithioate analogs have been synthesized to produce an important class of sulfur-containing ohgonucleotides, the dithiophosphate S 2 -ODNs.
  • These dithioates include an internucleotide phosphodiester group with sulfur substituted for both nonlinking phosphoryl oxygens, so they are both isosteric and isopolar with the normal phosphodiester link, and are also highly nuclease resistant.
  • One group showed highly effective protection of the dithioate against degradation by endogenous nucleases after 58% backbone modification.
  • the S 2 -ODNs in contrast to the phosphoramidite- synthesized monothiophosphate (S-ODNs), are achiral about the dithiophosphate center, so problems associated with diastereomeric mixtures (Lebedev & Wickstrom, 1996) are completely avoided.
  • the S 2 - ODNs and the S-ODNs are taken up efficiently by cells, especially if encapsulated in liposomes.
  • thiosubstitution can also perturb the structure of the duplex (Cho et al., 1993) (Volk et al, 2002) although monothiophosphates substituted in the DNA strand of DNA/RNA hybrids do not appear to have dramatically altered duplex structures (Bachelin et al., 1998; Gonzalez et al., 1995).
  • the present invention uses sequence-based, structure-based and combinatorial methods to identify both sequences and thiophosphate substitution patterns to develop thioaptamers that retained the highest specificity and affinity in binding to target proteins. The use of partial thiophosphate substitution resulted in aptamer that were more stable in vivo.
  • thiophosphate aptamers may be used with the present invention.
  • a recent advance in combinatorial chemistry has been the ability to construct and screen large random sequence nucleic acid libraries for affinity to proteins or other targets (Ekland et al., 1995; Gold et al., 1997; Tian et al., 1995).
  • the aptamer nucleic acid libraries are usually selected by incubating the target (protein, nucleic acid or small molecule) with the library and then separating the non-binding species from the bound. The bound fractions may then be amplified using the polymerase chain reaction (PCR) and subsequently reincubated with the target in a second round of screening.
  • PCR polymerase chain reaction
  • agents selected from combinatorial RNA and DNA libraries have previously always had normal phosphate ester backbones, and so would generally be unsuitable as drugs or diagnostics agents that are exposed to serum or cell supernatants because of their nuclease susceptibility.
  • the effect of substitution of nuclease-resistant thiophosphates cannot be predicted, since the sulfur substitution can lead to significantly decreased (or increased) binding to a specific protein (Milligan & Uhlenbeck, 1989).
  • the present invention have described the combinatorial selection of phosphorothioate oligonucleotide aptamers from random or high-sequence-diversity libraries, based on tight binding to the target (e.g. a protein or nucleic acid) of interest, relevant portions of which are incorporated herein by reference.
  • target e.g. a protein or nucleic acid
  • An in vitro selection approach for RNA thioaptamers has also been described Ellington and co-workers (Jhaveri et al., 1998).
  • a combinatorial library was created by PCR, using an appropriate dNTP( ⁇ S) in the Taq polymerization step.
  • a combinatorial thiophosphate duplex and single stranded (ss) libraries was screened successfully for binding to a number of different protein and nucleic acid targets, including NF-IL6, NF- ⁇ B, HIV reverse transcriptase, Venezuelan Equine Encephalitis nucleocapsid (using an RNA thioaptamer), HepC IRES nucleic acid, and others, including a protein involved in CpG-induced "innate immunity.”
  • a filter binding method was used that was modified to minimize non-specific binding of the S-ODNs to the nitrocellulose filters.
  • the wild-type CK-1 duplex sequence contains 3 tandem repeats of a 14-mer NF-KB consensus-like sequence (5' -CCA GGA GAT TCC ACC CAG GAG ATT CCA CCC AGG AGA TTC CAC 3') (SEQ ID NO.: 11).
  • S-ODN CK-1 monothioate aptamers were made because it was unlikely that the phosphodiester form is appropriate for therapeutics or diagnostics because of its short half-life in cells, cell extracts and serum. The phosphorothioate and dithioate internucleoside modifications are therefore needed.
  • the CK-1 aptamer inhibited p65/p65 and p50/p50 equally; confirming that S-ODNs with large numbers of phosphorothioate linkages are "sticky" and tend to bind proteins non-specifically.
  • the present inventors also found that if the number of phosphorothioate linkages is decreased to only 2-4, specificity can be restored, but binding is not enhanced. Therefore, the original publications described only the specificity of the phosphodiester ohgonucleotides and did not address the problem of altered specificity of the phosphorothioates.
  • Binding studies were conducted using a chemiluminescent EMSA, which uses a biotinylated thioaptamer.
  • the biotinylated thioaptamer binds tightly to p50; the sequences are different from those obtained for in vitro combinatorial selection against p65 homodimers (Table 2).
  • the chemically synthesized phosphorothioate aptamers are a diastereomeric mixture of both Rp and Sp configurations.
  • S 2 -ODN CK-14 dithioate aptamers were also isolated.
  • the CK-14 14-mer duplex was also synthesized with some strategically placed dithioate linkages (both of the non-bridging oxygens are replaced by sulfurs).
  • strategic dithioate linkage ODNs have exhibit significant differences, as they have altered binding specificity, and lack the extreme "stickiness" of the fully thioated aptamer. With an increasing number of dithioate substitutions in the same sequence, binding by the S 2 -ODN increases dramatically (data not shown).
  • the band did not co-migrate with either the p50/p50 or p50/p65 bands, but the change in the altered chemical structure changes the mobility of the ODN. Only one major band is seen, however, even though the lysate contains at least two major distinguishable NF- ⁇ B complexes (p50 homodimers and p50/p65 heterodimers).
  • Phosphorodithioate and phosphorothioate aptamers via split synthesis combinatorial selection.
  • the identification of specific S-ODN and S 2 -ODN thioaptamers that bind proteins based upon in vitro combinatorial selection methods is limited to substrates only accepted by polymerases required for reamplification of selected libraries by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Another disadvantage of using the polymerization of substituted nucleoside 5'-triphosphates into ODN aptamers are the restrictions on the choice of P-chirality by the enzymatic stereospecificity.
  • a one-bead one-oligonucleotide (one-ODN) (e.g., O-ODN, S- ODN, S 2 -ODN, both DNA or RNA) may be used in conjunction with combinatorial library selection methodology used to identifying a specific oligonucleotide aptamer that binds to specific proteins or other molecules (Yang, et al., 2002; Gorenstein, et al., U.S. patent applied).
  • the method may use S 2 -ODN reagents with sulfurs replacing both of the non-bridging phosphate oxygens that are isosteric and isopolar with the normal phosphorodiester and are particularly advantageous for binding and screening.
  • S 2 -ODNs are achiral about the dithiophosphate center, which eliminated problems associated with diastereomeric mixtures generally obtained for the chemically synthesized S-ODN.
  • the dsDNA thioaptamer library beads were screened for the ability to bind the NF- ⁇ B p50/p50 dimer labeled with the Alexa Fluor 488 dye (Molecular Probes). After initial binding of protein, the beads were thoroughly washed with PBS with 0.1% Tween 20 to minimize nonspecific binding. Typically, a few positive beads were intensely stained when viewed by fluorescence, while the majority of the beads remained unstained as (data not shown). With the aid of a micropipette coupled to a micromanipulator, the intensely stained beads were retrieved. Only highly positive beads from several thousand were found using this method. As described below, multicolor flow cytometry and cell/bead sorting was used to automate the selection process to select the tightest binding thioaptamer-protein complexes.
  • the S- ODN, 5'-CtGTGAGtCGACTgAtGaCGGt-3' (SEQ ID NO.: 7) (small letters represent location of 3'- mono thiophosphates) was synthesized independently on the non-cleavable linker bead support, hybridized with its complementary ODN and then mixed again with the p50/p50 protein labeled with the Alexa Fluor 488 dye.
  • the fluorescence intensity of all of the beads viewed under the fluorescence microscope was qualitatively similar to the intensity of the selected bead containing this sequence within the combinatorial library.
  • the beads were screened against a target protein labeled with a fluorescent dye, the beads have also been screened directly against cell extracts as well.
  • the binding of the NF- ⁇ B to a specific sequence can be detected using a primary anti-NF- ⁇ B antibody such as anti-P50 (Rabbit IgG antibody, Santa Cruz Biotechnology, Inc.) followed by a secondary antibody conjugated with Alexa Fluor 488 (goat anti-rabbit IgG from Molecular Probes).
  • Beads that included the XBY-6 oligonucleotide were screened against WI- 38 VA13, an SV40 virus-transformed human fibroblastic cell line extract by similar fluorescent microscopy.
  • Flow cytometry sorting of thioaptamer bead-based library The present inventors have also demonstrated the successful application of high throughput/multi-color flow cytometry and bead sorting to screen aptamer bead libraries for those beads which bind to, e.g., a target protein (Gorenstein, et al., patent pending, 2002). Modifications were made to a custom-built flow cytometer to make it more amenable to bead identification and isolation. For example, bead fluorescence and forward scatter were the two parameters chosen for real-time characterization of each aptamer bead passing the first sort point of the custom-built flow cytometer/sorter. Other scanning and sorting parameters may be used to select, isolate, view, designate, characeterize, etc. the beads through a flow cytometer.
  • "positive" beads intain thioaptamer-bound target protein, the target protein was fluorescent-labelled with Alexa 488 dye) were easily sorted from negative beads.
  • Flow cytometry may be used to replace, e.g., visual fluorescence microscope identification of beads containing bound target protein and the need to isolate the individual "positive” beads with the micromanipulator described previously.
  • the flow-sorted "positive” beads can then be subjected to, e.g., one-bead PCR to identify the thioaptamer that binds the target protein.
  • Fluorescence sorting was also used to demonstrate the use of the one-bead, one-ODN:protein system using dual color sorting.
  • the Ig ⁇ B dsDNA consensus sequences were immobilized onto 15-20 micron polystyrene microspheres.
  • the DNA bound beads were then incubated with purified p50 and p65 proteins, respectively.
  • DNA transcription factor complexes were detected with primary antibodies specific for the p50 and p65 proteins followed by an additional incubation with Alexa 488- conjugated secondary antibody for p50 and PE- conjugated secondary antibody for p65.
  • the beads were viewed by fluorescent microscopy and then analyzed on the MCU's HiReCS system.
  • TLR9 acts as a receptor for unmethylated CpG dinucleotides in the bacterial DNA.
  • Human and mouse TLR9 share an overall amino-acid identity of 75.5%. TLR9 is highly expressed in spleen (Krieg, 2002).
  • the immunostimulatory properties of bacterial DNA appears to be related to short six base sequences called CpG motifs that have the general structure of two 5' purines, an unmethylated CpG motif, and two 3' pyrimidines (Krieg, 2002). Though such sequences rarely appear in mammalian DNA due to CpG suppression and methylation of cytosine nucleotides, they are relatively abundant in bacterial DNA, occurring at the expected frequency (1 in 16) and in unmethylated form.
  • pathogenic Arenaviruses appear to block or modify immunoregulatory cell signaling pathways (Peters & Zaki, 2002, Solomon and Vaughn, 2002; Fennewald et al., 2002).
  • Using the present invention it was possible to disrupt Arenavirus and Flavivirus cell signals that contribute to immune evasion and pathogenesis.
  • thioaptamers it was demonstrated that the thio-modified aptamers of the present invention could be used to counteract viral induced cellular perturbations and protect the infected host.
  • viruses have developed ingenious strategies to counteract the host defenses that normally control viral replication and spread. Similarly, viral strategies modify the cellular environment to promote viral macromolecular synthesis and viral replication. This highly ordered interation often has the unfortunate consequence of inducing disease in the host.
  • Viruses have evolved mechanisms to interfere with major histocompatibility complex antigen presentation, block apoptosis, disrupt complement cascades and modulate multiple cytokine networks (Lalani & McFadden, 1999; Ploegh, 1998). Viruses have targeted cell-signaling pathways involved in cytokine and chemokine signaling, the regulation of apoptosis, and the cell cycle.
  • Influencing key transcription factors that regulate pro or anti-inflammatory cytokines is an efficient means by which viruses could cripple multiple immune responses (Powell et al, 1996; Tait et al, 2000).
  • the strategies employed by the smaller, less genetically complex viruses are equally elegant, and often even more of an enigma.
  • Pichinde infection of guinea pigs is particularly suited to studies on the immunomodulation by virus infection.
  • the differential effect of virus infection was identified as including a profound effect on the transcription factors NF- ⁇ B and RBP-J ⁇ .
  • FIG. 2 is a graph that shows that polyl/C is an effective inducer of the proinflammatory cytokine TNF- ⁇ .
  • Infection with P2 and PI 8 also alter the expression of this and other inflammatory cytokines.
  • P2 and PI 8 induced equally cytokines such as IL-6; which are moderately different in their induction of TNF- ⁇ and substantially different in IL-12 induction ( Figure 3).
  • XBY-6 SEQ ID NO.: 1 targeting NF- ⁇ B p50 homodimers and XBY-S2 targeting AP-1, both with six dithio residues.
  • XBY-S2 SEQ ID NO.: 2 is demonstrated to bind specifically to AP-1 proteins in pre-B cell nuclear extracts (70Z/3) and to human recombinant c-jun protein dimers (AP-1).
  • supershift analyses indicate that XBY-S2 binds to several members of the AP-1 protein family including JunD, CREB and possibly ATF2, and c-Jun.
  • the XBY-6 thioaptamer binds specifically to the NF- ⁇ B p50 (or pl05) homodimer (Figure 6). Macrophage cultures were treated with XBY-S2 and XBY-6 and nuclear extracts were produced to assay the effects of these thioaptamers on the DNA binding activities of the transcription factors to which they are targeted. In Figure 7, macrophage cultures were treated with liposomes, and liposome containing the indicated thioaptamers overnight and nuclear extracts produced and assayed using the indicated ohgonucleotides. The XBY-S2 thioaptamer efficiently eliminated transcription factor binding to the AP-1 oligonucleotide. In contrast, treatment with XBY-6 resulted in an increase in the NF- ⁇ B DNA binding activity.
  • XBY-6 thioaptamer and Pichinde virus Based on the preliminary results obtained with XBY-6 thioaptamer and Pichinde virus, it was determined if XBY-6 or XBY-S2 would have any antiviral activity against flaviviruses.
  • the thioaptamers (10 ⁇ g) were delivered IP in Tfx50 liposomes and administered in two doses (one day before and 90 minutes before virus challenge).
  • mice of which 6 would be sampled
  • virus challenge 100 LD 50 virus.
  • Table 6 the initial results were reproducible. Both control groups (PBS and liposomes) succumbed to challenge with WN virus while the thioaptamer-treated mice survived and remained healthy.
  • the proportion of mice treated with XBY-S2 thioaptamer who survived challenge was the same in both studies (80%) while XBY-6 treatment protected 50% of mice in the second study as compared to 80% of mice in the first study. These differences were not statistically significant given the small sample sizes.
  • the thioaptamer may be inducing localized interferon (or other mediators of the innate immune response) that inhibits replication of the virus since the thioaptamer includes double-stranded DNA while double-stranded RNA is known to be an efficient inducer of interferon.
  • Study 2 Female 3-4 week-old NIH Swiss mice were given aptamers at one day before and 90 minutes before administration of 100 LD50 WN virus strain USA99b by the ip route. Group # surviving [%] AST(days ⁇ SD) PBS only 0/10 [0] 8.3 ⁇ 0.8 Liposomes only 0/10 [0] 7.7 ⁇ 1.1 XBY-S2 8/10 [80] 8.5+0.7 XBY-6 5/10 [50] 8.0 ⁇ 0.7
  • Figure 10 is a graph that shows survival curves following Pichinde PI 8 infection in guinea pigs treated with the NF-KB aptamer, XBY-6, the scrambled control, B92, or vehicle, MT, of animals infected by injection of 1000 pfu of Pichinde P18 at day 0, treatment consisted of intraperitoneal injections at days 0, l and 2;
  • duplex aptamers were the dithioate 14-mers XBY-6 (C12-XBY-6), the normal phosphate backbone 22-mer NF- ⁇ B binding site with the C12 5'-amino linker (C12-Ig ⁇ B) or a non-specific, non-covalently linked duplex (polydldC) as a control. These aptamers were spotted individually onto spots of a preactivated ProteinChip Array (PS20) in 2 ⁇ l of 25 mM NaHC ⁇ 3 (pH 9) and incubated overnight at room temperature and high humidity.
  • PS20 ProteinChip Array
  • Figure 14 shows that the XBY-6 thioaptamer can also capture recombinant p50 (MW ⁇ 46,200) on gel beads to which the 5'amino-C12 linked XBY-6 is coupled to 20 ul (1: 1) AminoLink® Plus Coupling gel (Pierce, Immunoprecipitation kit, cat # 45335).
  • p50 MW ⁇ 46,200
  • AminoLink® Plus Coupling gel Pierce, Immunoprecipitation kit, cat # 45335.
  • 3 ⁇ g of C12-XBY-6 was coupled overnight at 4°C following the kit protocol.
  • 6 ⁇ g of p50 in IX EMSA buffer with polydldC was added to the gel and incubated for 2 hrs with shaking at room temperature. The gel was washed to remove nonspecifically bound proteins, followed by one quick rinse with water.
  • Protein bound to the gel was extracted with 5 ⁇ l of organic solvent (50% AcN and 0.01% TFA) with shaking for 20 min. All of the extracts were spotted onto NP20 ProteinChips, dried, followed by addition of saturated SPA and read on the Ciphergen PBSII MS (top two spectra). After extraction, 1 ⁇ l of the gel was loaded onto NP20 chip (bottom two spectra). Proteins still bound to the gel was analyzed using saturated SPA on the PBSII. Once again it was found that p50 can be identified by SELDI, both in the extract and retained directly on the beads.
  • Figure 15 shows the capture of nuclear extracts onto Ciphergen's PS20 ProteinChip Arrays: Either 0.5 ⁇ g of C12-XBY-6, 0.25 pm of C12-Ig ⁇ B or 0.5 ⁇ g of poly dldC were incubated on PS20 chip overnight. The chips were blocked with 7 mg/ml BSA in PBS/0.1% Tween-20. Following blocking, 49 ⁇ g of nuclear extract in optimized EMSA buffer were incubated on each spot for 2 hr with shaking. Each spot was washed with PBS/0.1% Triton three times, followed by one quick wash with water. Proteins bound on each spot were analyzed using saturated SPA on the PBSII. These results indicate that a protein was bound with a MW -105,591, which may represent pl05, the precursor to p50 or the p50/p50 homodimer.
  • thioaptamer combinatorial libraries were created that cover appropriate sequence space relative to the targeted protein.
  • duplex thioaptamers were create that have a significant population of sequences similar to the consensus sequence.
  • the complexity of the library can be as large as 10 14 different sequences and thus can cover all sequence space for a small ( ⁇ 22 nt) duplex.
  • complexity is limited to the number of different beads - 10 6 -10 8 , depending on their size.
  • a bead- based library of libraries of thioaptamers is made in which as many as 10 6 different thioaptamers are attached to a single bead and thus have a total complexity of as many as 10 12 -10 14 sequences in the library of library.
  • a library of libraries was prepared on a 1 ⁇ mole scale of polystyrene beads (60-70 ⁇ m).
  • the downstream and upstream primers 5'-d(GGATCCGGTGGTCTG)-3' (SEQ ID NO.: 26) and 5'-d(CCTACTCGCGAATTC)-3' (SEQ ID NO.: 27) were synthesized in parallel on a two-column DNA synthesizer (Expedite 8909, Applied Biosystems). Following the 5'-primer, the sequences programmed on the synthesizer for the combinatorial library were 5'-AT*GN*GA*AT*TT*NC*CA 3' (SEQ ID NO.: 28) on column 1 and 5'- GG*AG*NG*CN*CA*GG*AC -3' (SEQ ID NO.: 29) on column 2.
  • the 3'-primer sequence completed the 44-mer programmed on the synthesizer.
  • a "split and pool” was used at each position indicated by an asterisk in order to synthesize the combinatorial region for the library of libraries.
  • the letter N indicates a mixture of four bases (A, C, G and T). Five of the beads were randomly selected from the library and "one bead one PCR" was run, cloned and sequenced. The results listed below indicated the successful construction of the library of libraries.
  • E45-2-2 5'-GG AG GA CA TT GC AC-3' (SEQ ID NO.: 31)
  • E45-2-4 5'-GG AG GA CC TT CC AC-3' (SEQ ID NO.: 32)
  • E45-2-5 5'-GG AG GA CC TT GC AC-3' (SEQ ID NO.: 33)
  • E45-2-11 5'-GG AG GA CN TT TC AC-3' (SEQ ID NO.: 34)
  • E45-2-12 5'-GG AG GA CC TT TC AC-3' (SEQ ID NO.: 35)
  • E45-3-1 5'-GG GA TG GT CA GG AC-3' (SEQ ID NO.: 36)
  • E45-3-3 5'-GG GC GG AT CA GG AC-3' (SEQ ID NO.: 37)
  • E45-3-5 5'-GG GA AG AT CA GG AC-3' (SEQ ID NO.: 38)
  • E45-3-6 5'-GG GG TG AT CA GG AC-3' (SEQ ID NO.: 39)
  • E45-6-1 5'-GG AG CG GT GT CC AC-3' (SEQ ID NO.: 41)
  • E45-6-3 5'-GG AG CG GT TT GC CA-3' (SEQ ID NO.: 43)
  • E45-6-10 5'-GG AG CG AT TT CC CA-3' (SEQ ID NO.: 44)
  • E45-7-2 5'-AT AG NG CC CA GG AC-3' (SEQ ID NO.: 47)
  • E45-7-5 5'-AT AG GG CG CA GG AC-3' (SEQ ID NO.: 48)
  • E45-8-2 5'-GG AG AG CA CA TC AC-3' (SEQ ID NO.: 50)
  • E45-8-4 5'-GG AG CG CG CA GC AC-3' (SEQ ID NO.: 52)
  • E45-8-5 5'-GG AG GG CT CA GC AC-3' (SEQ ID NO.: 53)
  • E45-8-6 5'-GG AG AG CA CA AC AC-3' (SEQ ID NO.: 54)
  • E45-8-10 5'-GG AG CG CG CA TC AC-3' (SEQ ID NO.: 55)
  • High quality one-bead one-oligo libraries were contracted by join two pieces of DNA based on an enzymatic ligation reaction or using highly active phosphorothioate towards 5'-iodo groups on the ODN.
  • Standard phosphoramidite chemistry was used for synthesis of 5' monophosphate ODN (5'- P(o)CCAGGAGATTCCAC-GGATCCGGTGGTCTGT-bead) (SEQ ID NO.: 57).
  • the fully protected ODN with the non-cleavable linker beads were treated with concentrated ammonia at 37°C for 21 hours to remove the protecting groups while allowing the ODN to remain attached to the beads.
  • a selected single bead was mixed with the following components: 3 ⁇ l of 40 ⁇ M 15 mer oligonucleotide (5'- CCTACTCGCGAATTC-3', (SEQ ID NO.: 58) 3 ⁇ l of 10 X ligation buffer, 3 ⁇ l of DMSO, 2 ⁇ l of T4 RNA ligase and 19 ⁇ l of ddH 2 0.
  • the reaction was performed at 5 °C for 17 hrs. The supernatant was removed carefully and washed with water.
  • the single bead PCR reaction was run under established conditions. The PCR products were analyzed on a 15% native polyacrylamide gel.
  • the PCR product was cloned using the TA Cloning procedure (Invitrogen) and sequenced on an ABI Prism 310 Genetic Analyzer (Applied Biosystems).
  • the desired sequence 5'-CCTACTCGCGAATTC-
  • the ligation reaction allows longer random regions of aptamers to be synthesized on the beads with higher yield since a primer region does not have to be stepwise synthesized onto the bead sequence.
  • the beads were screened for the ability to bind the appropriate protein (such as the various NF- ⁇ B dimers or API dimers) labeled with the Alexa Fluor 488 dye (Molecular Probes) or by binding fluorophor labeled antibodies as previously described.
  • the beads are sorted using a multicolor flow cytometry and cell/bead sorting to visualize and sort the protein-bound thioaptamer beads and select the tightest binding thioaptamer-protein complexes as shown in Figure 6.
  • the most intensely stained beads will be retrieved.
  • the inventors concentrated on the NFKB and AP-1 dimers, but these methods may be applied by to other proteins involved in the immune response.
  • Multicolor flow cytometry was capable of sorting at speeds of 10 8 beads per hour or viewed in terms of assays for thioaptamers binding to target proteins, 10 8 assays per hour.
  • High throughput sorting (HTS) of homo- and heterodimers to thioaptamers by multi-color flow cytometry using multi-color flow cytometry HTS may be used to select thioaptamers that bind preferentially to heterodimers of proteins.
  • one monomer is tagged fluorescently (A) with a dye (cy3 for example) and a different monomer (B) with another dye (cy5 for example). Both proteins are mixed together and allowed to bind to the bead thioaptamer library.
  • two-color flow cytometry is used to compare cy3/cy5 color levels of each bead.
  • beads that have high cy3 levels and low cy5 levels are selected. Conversely, high cy5/low cy3 indicates a thioaptamer sequence with selectivity for the B.B dimer. For heterodimers, beads are selected for cy3/cy5 levels close to 1.
  • SELDI MS may be used to determine which proteins have been bound to selected combinatory thioaptamer beads and also used with single bead PCR to identify which bead(s) in the combinatorial library have bound to protein(s).
  • More than 2 dyes and multi-color flow cytometry may be used to select various multimers.
  • NF- KB at least 3 of the 5 different monomeric forms of the protein are combined, each with a different fluorphor and use 3-color flow cytometry to select thioaptamers that have high affinity and selectivity to homodimers A.A, B.B, CC and various heterodimeric forms from the libraries.
  • detectable markers e.g., fluorochromes
  • 5-color flow cytometry may be used.
  • Sequencing may also be performed directly on the bead.
  • Each individually selected bead is washed thoroughly with 8 M urea (pH 7.2) to remove the protein and directly used for "one-bead one-PCR" amplification using the 5' and 3' end primers (Yang, et al. 2002).
  • the PCR products are TA cloned and sequenced as previously described to create hybrid thioaptamers with normal phosphate, monothiophosphate, and dithiophosphate mixed backbones as well, keeping the total thiophosphate backbone below 80% to minimize "non-specific" sticking.
  • the current approach demonstrated in the above examples requires a different nucleotide sequence to identify a backbone modification.
  • Thioaptamer libraries were also created that only differ in the position of phosphate or dithioate but not in its base sequence. It has been shown that the positions of thiophosphates in a mixed backbone S-ODN can be determined by reaction of the S-ODN with iodoethanol followed by base catalyzed cleavage of the thiophosphate triester. This approach was used to identifying the location of monothio- and dithiophosphate linkages, independent of base sequence.
  • thioaptamer bead-based hts of the host and pathogen proteome may be used with the thioselection technology (both enzymatic [S]-ODN and synthetic [S]-ODN /[S 2 ]-ODN) to develop thioaptamers targeting very important proteins (e.g., NF- ⁇ B and AP-1) to identify promising therapeutic leads.
  • thioaptamer bead-based HTS of the proteomes with specialized high-throughput multicolor flow cytometry/bead sorting in conjunction with SELDITM mass-spectrometric methods to identify potential new therapeutic targets both of proteins involved in the immune response to BT viruses as well as viral proteins.
  • Thioaptamers may be identified to inhibit the differentially expressed proteins in host-pathogen interactions as well as underlying immune response processes and so ameliorate cytopathological immune responses resulting in shock or to enhance "innate immunity" to help mount a more effective immune response.
  • Mass spectrometric protein detection technology can be used to identify bound proteins using HTS of thioaptamer beads. This approach has significant advantages, since MS is more sensitive than fluorescent imaging and will be very useful for low-abundance proteins. In addition, if more than one protein binds to a given thioaptamer bead, then it will be possible to identify and quantify these proteins by SELDI. This is particularly helpful for identifying non-covalent dimers such as NF- ⁇ B or AP-1 (there are 22 different monomeric forms of AP-1 and thus in principle 100's of different combinations of dimers possible).
  • non-covalent dimers such as NF- ⁇ B or AP-1 (there are 22 different monomeric forms of AP-1 and thus in principle 100's of different combinations of dimers possible).
  • Thioaptamer proteomic arrays were used to demonstrate the use of ProteinChip array technology (e.g., Ciphergen) for protein identification of modified thioaptamer beads or surfaces.
  • SELDI MS combines the well-established principles of solid-phase extraction and time-of-flight mass spectrometry in a process known as surface enhanced laser desorption/ionization time-of-flight mass spectrometry.
  • ProteinChip Arrays may be customized by covalently attaching affinity reagents such as the modified thioaptamers to the spot surface. If the biological marker to be detected is known and thioaptamer affinity reagents are available, affinity surfaces can be designed to make use of this specific thioaptamer-protein interaction. Also, because SELDI uses mass spectrometric detection, several assays can be multiplexed easily by taking advantage of the unique masses of each bound protein.
  • High-throughput screening (HTS) of thioaptamer libraries by flow cytometry and SELDI Bead-based methods were used to identify both thioaptamer sequences and binding proteins in parallel, without the need to select one thioaptamer for each purified protein.
  • a number of [S]-ODN or [S 2 ]-ODN combinatorial libraries are synthesized, each containing 10 6 to 10 9 different, but related members (or a library of library with up to 10 14 sequences).
  • the solid-phase split synthesis described herein may be used to create thioaptamer-bound bead libraries (one bead, one sequence or one library) as above.
  • Each library can be sufficiently different to provide high affinity and selectivity to a small number of cellular proteins (such as AP-1 or NF- ⁇ B-type sequences).
  • One or more of the thioaptamer library beads are incubated with cellular extracts, washed thoroughly to remove weakly bound proteins and the bound proteins visualized by direct fluorescent staining with cy3, cy5, SYPRO Ruby, or other newer dyes for high sensitivity (sub-nanogram). Fluorescently stained beads can be sorted in the high-speed cell/bead sorter for the top 10 2 or more beads which have the highest amount of bound protein.
  • the thioaptamer sequence on the bead can be determined by the PCR "one bead sequencing" method disclosed herein.
  • a random library of "sticky beads” is selected and an extract containing the complete proteome to identify both the thioaptamer sequence on the single beads and the protein(s) bound.
  • HTS of combinatorial libraries to protein mixtures Besides using cell extracts, known mixtures of hundreds of commercially available proteins (cytokines, transcription factors, etc.) may be applied to the mixture of thioaptamer bead libraries. HTS cell/bead sorting is used followed by MS identification of bound proteins. This involves direct SELDI determination of the protein or peptide fragmentation methods followed by MS identification of bound proteins.
  • thioaptamers rather than beads with proteins or monoclonal antibodies attached to them is that proteolysis and MS peptide identification is not complicated by proteolysis of bait proteins or Mab's.
  • the thioaptamer-based multi-color flow cytometry HTS may also be used for targeting differentially expressed proteins within the host and pathogen proteomes, combined with MS detection (SELDI).
  • the thioaptamer bead-based combinatorial library can be used in conjunction with fluorescent tagging of proteins followed by SELDI MS to identify proteins differentially expressed in control vs. virus infected cells.
  • a combinatorial library (or a combinatorial library of libraries) of thioaptamer beads may be synthesized, each bead with a single thioaptamer sequence (or a combinatorial library of thioaptamer sequences on each bead).
  • Up to 10 8 beads can be created with a single thioaptamer sequence on each bead.
  • Cell extracts of a sample such as uninfected cells is labeled fluorescently with a dye (cy3 for example) as carried out previously and a virus-infected cell extract is then labeled fluorescently with another dye (cy5 for example). Both cell extracts are mixed together and allowed to bind to the bead thioaptamer library.
  • two-color flow cytometry is used to compare cy3/cy5 color levels of each bead. If cy3/cy5 level differs significantly (> 2-fold) from 1, then the bead was captured.
  • SELDI MS will be used to characterize the bound target further.
  • SELDI MS can be used to determine which proteins have been bound to selected combinatory thioaptamer libraries and also used with single bead PCR to identify which bead(s) in the combinatorial library have bound to protein(s).
  • Ciphergen's ProteinChip epoxy modified surfaces may be used to covalently attach 5'-amino-linker thioaptamers to beads.
  • HTS of thioaptamers targeting differentially expressed proteins in the proteome in virus infected cells relative to treated cells (“High Throughput Pharmacoproteomics").
  • three-color thioaptamer library bead sorting is used.
  • a combinatorial library (or a combinatorial library of libraries) of thioaptamer beads is synthesized, each bead with a single thioaptamer sequence (or a combinatorial library of thioaptamer sequences on each bead). Up to 10 8 beads with a single thioaptamer sequence on each bead (or 10 14 sequences on the library of libraries) are made.
  • Uninfected cell extracts are labeled fluorescently with a cy3 for example.
  • a virus- infected cell extract (or any disease cell extract such as cancerous cells) is labeled fluorescently with cy5, and then a thioaptamer therapeutic treated, virus infected (or other disease) cell culture is labeled with a third dye.
  • the three proteome cell extracts are mixed together in equal total protein quantities and allowed to bind to the bead thioaptamer library (or library of libraries). Three-color flow cytometry is used to compare cy3/cy5/dye 3 color levels of each bead. If cy3/cy5 level differs from 1 (uninfected vs.
  • a complex of combinatorial libraries are created in which multiple transcription factor-like sequences with varying thiophosphate substitution patterns are concatenated in a single long sequence so that it can bind to multiple transcription factors such as NF- ⁇ B, AP-1, SP-1, GRE, SRE, etc., requiring a thioaptamer sequence of at least 20-40-mers.
  • These embodiments provide an attractive approach to defining therapeutic strategies in which multiple proteins can be targeted with multiple thioaptamers.
  • Such a combination (adjuvant) of drug therapeutics is needed to improve immune responses in cancer, AIDS, etc.
  • Mammalian protein signaling pathways are often redundant so that if one pathway is affected, another can take over control. By perturbing multiple, highly interwoven pathways, a greater opportunity to modulate the immune response network is made available.
  • HTS High-throughput screening
  • thioaptamer beads using high-speed multicolor flow cytometry/cell sorting is used.
  • more than 10 10 beads could be screened within a single day, and specific bead subpopulations could be sorted for subsequent proteomics analysis.
  • This group also has considerable experience in HTS of cells and bacteria (as well as beads) for subsequent molecular characterizations by PCR and gene expression microarray analysis.
  • Advanced HTS technologies may be used for large library screening and functional genomics.
  • Single-cell (or bead) sorting of rare subpopulations may be used to isolate single beads from combinatorial libraries.
  • a special high speed sorter uses a unique two-stage signal processing system, configured in hardware as a single layer neural network, which allows for sophisticated cell or bead classifications based on multivariate statistics or learning through neural networks.
  • a 6-color high-speed flow cytometer/cell sorter is configured in hardware and software as a single-layer neural network that can also be used to generate real-time sort decisions on the basis of multivariate statistical classification functions. While it can perform the usual two-way sorts it is commonly used in "straight-ahead" sorting mode to allow for extremely high sort recovery and purity at high throughput rates or to efficiently sort single cells for cloning or for subsequent molecular characterizations by PCR.
  • sorted "positive" beads can be subjected to SELDI-MS analysis to confirm the identity of the proteins bound to the thioaptamer beads of the present invention (via MALDI MS molecular ion characterization).
  • SELDI-MS may be used to identify this event through the detection of multiple molecular ions.
  • LC/MS/MS Liquid Chromatography/Tandem Mass Spectrometry
  • Peptides are subsequently eluted from the RF column directly into the MS, either for mass fingerprinting, or for MS/MS sequence analysis.
  • This LC tandem MS procedure is very useful for small amounts (femtomol) of complex.
  • Yet another procedure is tandem LC/tandem MS.
  • the proteomes can be either human, GP, hampster or mouse - human and mouse genome databases are available.
  • 2D gel electrophoresis 2D PAGE can be conducted essentially as first described by (O'Farrell, 1975). High-throughput may be employed Pharmacia's IPGphor multiple sample IEF device or the first dimension, and Biorad's multiple gel SDS-PAGE systems (Protean Plus and Criterion dodeca cells) for the second. Gels will be stained with either SYPRO Ruby for high sensitivity (sub-nanogram) or Coomassie Blue when less sensitivity is required. Image analysis of gels will be achieved with a Perkin Elmer (PE) ProEXPRESS Proteomic Imaging System using Nonlinear's Progenesis imaging software.
  • PE Perkin Elmer
  • a Genomic Solutions' robotics recently purchased is utilized for protein spot picking and for sample trypsin hydrolysis (Proteomic Protein Picker), and sample clean-up, and sample application to MALDI plates (ProPrep 4 Block System).
  • Mass fingerprinting for protein identification may use an Applied Biosystems (AB) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) Voyager DE STR MS. Proteins will be identified with the Voyager's Prospector software. De novo sequencing and analysis of posttranslational modifications can be achieved by electrospray (ESI) MS/MS (capillary LC nanoflow option).
  • MALDI matrix-assisted laser desorption/ionization
  • TOF time-of-flight
  • Isotope-coded affinity tags Some differential protein expression use isotope-coded affinity tags (ICATs) for quantitative analysis of complex protein mixtures (Gygi et al., 1999). In this procedure, there is an option to fractionate proteins before to proteolysis decreases the complexity of proteins analyzed.
  • TNF Tumor Necrosis Factor
  • Dyer R. B. and Herzog, N. K. (1995). Isolation of Intact Nuclei for Nuclear Extract Preparation from Fragile B-Lymphocyte Cell Lines. Biotechniques 19, 192-195.
  • CRE Cyclic AMP Response Element-
  • AP-1 AP-1 directed Transcription by CRE-transcription Factor Decoy Oligonucleotide
  • RNA. 1, 317-326 Tian, Y., Adya, N., Wagner, S., Giam, C.Z., Green, M.R., & Ellington, A.D. (1995) "Dissecting proteimprotein interactions between transcription factors with an RNA aptamer," RNA. 1, 317-326.

Abstract

L'invention concerne une composition et des procédés de fabrication et d'utilisation d'une banque combinatoire pour identifier les thioaptamères modifiés qui se lient à des facteurs de transcription tels que IL-6, NF-λB, AP-1 et analogue, et agissent sur la réponse immunitaire d'un animal hôte. L'invention concerne aussi une composition et des méthodes de traitement d'infections virales, ainsi que des vaccins et des adjuvants vaccinaux qui modifient les réponses immunitaires de l'hôte.
EP04776088A 2003-05-23 2004-05-20 Aptamere a oligonucleoside phosphorothioate et phosphorodithioate selectionne de maniere combinatoire et selon la structure pour le ciblage des facteurs de transcription ap-1 Withdrawn EP1667729A4 (fr)

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US7910523B2 (en) * 2003-05-23 2011-03-22 Board Of Regents, The University Of Texas System Structure based and combinatorially selected oligonucleoside phosphorothioate and phosphorodithioate aptamer targeting AP-1 transcription factors
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