WO2011069487A2 - Aptamères d'adn se liant spécifiquement au récepteur soluble de l'interleukine 6 - Google Patents

Aptamères d'adn se liant spécifiquement au récepteur soluble de l'interleukine 6 Download PDF

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Publication number
WO2011069487A2
WO2011069487A2 PCT/DE2010/001413 DE2010001413W WO2011069487A2 WO 2011069487 A2 WO2011069487 A2 WO 2011069487A2 DE 2010001413 W DE2010001413 W DE 2010001413W WO 2011069487 A2 WO2011069487 A2 WO 2011069487A2
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seq
dna aptamer
dna
aptamer
receptor
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PCT/DE2010/001413
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German (de)
English (en)
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WO2011069487A3 (fr
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Ulrich Hahn
Stefan Rose-John
Cindy Meyer
Tijana Zivkovic
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Universität Hamburg
Christian-Albrechts-Universität Zu Kiel
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Publication of WO2011069487A2 publication Critical patent/WO2011069487A2/fr
Publication of WO2011069487A3 publication Critical patent/WO2011069487A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the invention relates to aptamers which specifically bind a target molecule.
  • cytokine interleukin-6 IL-6
  • IL-6 interleukin-6
  • the biological effect of cytokines on the target cells is mediated by specific, usually membrane-bound receptors.
  • the cytokine interleukin-6 (IL-6) acts on a variety of different cells and plays a central role, especially in inflammatory processes.
  • Cytokine IL-6 is known to be involved in a variety of diseases, e.g. inflammatory autoimmune diseases such as rheumatoid arthritis.
  • soluble forms also exist which consist of the extracellular domains of the membrane-like forms and also possess the ability to bind ligand, albeit at a reduced affinity to the membrane-like form. Soluble receptors can neutralize ligands, for example, by their comparatively high concentration and act in this way
  • soluble interleukin-6 receptor sIL-6R
  • the complex of IL-6 and sIL-6R causes dimerization of the membrane gpl30 (Taga, T. et al. (1989) Cell, 58, 573-581 Mackiewicz, A. et al., (1992) J. Immunol., 149, 2021-2027; Scheller and Rose-John, Med. Microbiol Immunol. (2006), 195, 173-183; Rose-John et al. , (2006), JJ Leukocyte Biol. 80, 227-235; Jones et al.
  • gpl30 is present in almost all organs, e.g. Heart, kidneys, spleen, liver, lung, placenta, and brain (Saito, M. et al., 1992, J. Immunol., 148, 4066-4071) .
  • interleukin-6 may therefore also act on cells which do not express the membrane-bound interleukin-6 receptor but the glycoprotein gp 130 (Hirota, H. et al., (1995) Proc Natl Acad., USA, 92, 4862-4866).
  • tocilizumab (Actemra®) is a monoclonal antibody currently used to treat rheumatoid arthritis. Tocilizumab (Actemra®) exerts its action via the blockade of the human IL-6 receptor, resulting in the binding of IL-6 to the receptor and a consequent
  • Other side effects include gastrointestinal perforation, hypersensitivity reactions including anaphylaxis, headache and
  • Aptamers are artificial DNA or RNA molecules that contain another molecule, e.g. a protein, specifically bind. With high specificity and affinity as well as chemical
  • aptamers have a low immunogenicity compared to antibodies.
  • aptamers may be selected by a method termed SELEX (Evolutionary Evolutionary Ligations by Exponential Enrichment) (Ellington and Szostak (1990), Nature 346, 818-822, Gopinath (2007), Anal. Bioanal Chem 387, 171-182, WO 91/19813).
  • the invention provides a DNA aptamer which specifically binds the human soluble interleukin-6 receptor and comprises the sequence GGGNGGGHGGGWGGG (SEQ ID NO: 1). However, DNA aptamers containing the sequence are excluded
  • GGGTGGGTGGGTGGGT (abbreviated (G 3 T) 4 , see SEQ ID NO: 6).
  • N is A, T, TT or TTT.
  • the soluble human interleukin-6 receptor has a molecular weight of about 50 kDa at 339 amino acids (AA).
  • the amino acid sequence of sIL-6R is shown in SEQ ID NO: 7.
  • the amino acid sequence of the sIL-6R is identical to that of the extracellular domain of the membrane-bound interleukin-6 receptor (IL-6R), so that the aptamer according to the invention naturally also specifically binds the IL-6R. Any reference to the sIL-6R should therefore also include the IL-6R, unless expressly stated otherwise.
  • the DNA aptamer according to the invention is affine and specific for the human sIL-6 receptor and is unlikely or not immunogenic. It provides a promising means to treat or prevent diseases involving the soluble IL-6 receptor. It is also simple and inexpensive to produce, durable and storable.
  • the DNA aptamer according to the invention can be used in a variety of ways to diagnose or influence conditions or diseases of humans in which IL-6 and / or the soluble and / or the membrane-bound interleukin-6 receptor are directly or indirectly involved , It can be used, for example, to prepare a drug or diagnostic agent that can be used to diagnose, prevent and / or treat inflammatory conditions or diseases, cancers or infections.
  • diseases in which the inventive DNA aptamer Applications include rheumatoid arthritis, sepsis, asthma, Crohn's disease, multiple sclerosis, depression, breast cancer, multiple myeloma, and HIV infection.
  • the DNA aptamer according to the invention can also be used to introduce other molecules, for example peptides, proteins, oligonucleotides, dyes, diagnostic agents etc., into a cell.
  • these molecules are coupled to the DNA aptamer using techniques that are familiar to those skilled in the art.
  • the molecule coupled to the DNA aptamer according to the invention can then be introduced into the cell by means of the sIL-6R or the IL-6R and re-released intracellularly.
  • the molecules can also be biologically or medically active substances, so that the DNA aptamer can also have a function as part of a prodrug.
  • DNA aptamer an isolated single-stranded DNA (ssDNA) containing a target molecule, e.g. a protein that specifically binds.
  • a target molecule e.g. a protein that specifically binds.
  • DNA aptamer ssDNA oligonucleotides having at most 150, preferably at most 130, at most 110, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20 , at most 15 or at most 10 nucleotides understood.
  • nucleotide are here in particular the basic building blocks of nucleic acids, i. organic molecules derived from a sugar residue, usually a pentose, e.g.
  • Deoxyribose or ribose an organic base (nucleobase) and phosphoric acid.
  • the phosphoric acid is regularly linked to the sugar via an ester bond, the sugar to the nucleobase via an N-glycosidic bond.
  • the nucleobases adenine (A), cytosine (C), guanine (G) and thymine (T) regularly occur, while in ribonucleic acid (RNA) the base uracil (U) is substituted for thymine.
  • the phosphoric acid is usually present in RNA and DNA as monophosphate.
  • a "target molecule” is understood here preferably to be non-RNA and non-DNA molecules, for example proteins, peptides and glycoproteins.
  • binding is meant a bond that does not rely on the Watson-Crick pairing between nucleotides. It is preferably a non-covalent bond.
  • a DNA aptamer or a fragment thereof which binds specifically to the human sIL-6 receptor is here understood as meaning a DNA aptamer or DN A-aptamer fragment which has a related function
  • a DNA aptamer or DNA aptamer fragment which specifically binds the human sIL-6 receptor is understood as meaning a DNA aptamer or DNA aptamer fragment which has a dissociation constant of at most 1000 nM (nmol / l preferably at most 500 nM, more preferably at most 250 nM and particularly preferably at most 150 nM If reference is made here to dissociation constants, this preferably refers to average values of a one-site binding described in more detail below. Size as determined by filter binding studies As already indicated above, the term includes an hsEL-6 receptor specific binding also to the hIL-6 receptor, ie the membrane-bound human IL-6 receptor.
  • a diagnostic agent is meant herein a product that can be used in a diagnostic procedure.
  • the term also includes products, such as reagents or kits of reagents, used to determine outside the human or animal body a particular condition and / or deviation from a normal condition, e.g. a deviating sIL-6R concentration can be detected.
  • the DNA aptamer according to the invention is distinguished by the presence of four G triplets which are also characterized by an adenine, thymine or cytosine nucleotide, if appropriate, in particular in the case of the first and second G triplet viewed from the S 'direction are linked together by two or three TTiymin nucleotides.
  • the DNA aptamer according to the invention comprises the nucleotide sequence GGGNGGGHGGGWGGGW (SEQ ID NO: 2).
  • the DNA aptamer according to the invention comprises the nucleotide sequence VGGGNGGGHGGGWGGG (SEQ ID NO: 3), VGGGNGGGHGGGWGGGW (SEQ ID NO: 4) or GGGTGGGCGGGTGGGT (SEQ ID NO: 5).
  • V A, G or C.
  • GGGTGGGTGGGTGGGTGGGT SEQ ID NO: 6
  • N, H and W are preferably not simultaneously A.
  • N, H and W are not all A at the same time.
  • N, H and W are not all A at the same time.
  • the DNA aptamer according to the invention comprises one of the sequences given in SEQ ID NO: 8-12 or SEQ ID NO: 13-17 or a fragment thereof which specifically binds the human soluble meleleukin-6 receptor.
  • One or more, possibly also all bases of the nucleotides of the DNA aptamer or of the DNA aptamer fragment can be modified. This can be beneficial to
  • the DNA aptamer of the invention may also be combined with other compounds, e.g. Cholesterol or polyethylene glycol (PEG), coupled or multimerized, for example, to increase bioavailability, reduce degradation or excretion.
  • other compounds e.g. Cholesterol or polyethylene glycol (PEG), coupled or multimerized, for example, to increase bioavailability, reduce degradation or excretion.
  • a 3'-3'-dT cap deoxythymidine
  • dT deoxythymidine
  • a fragment of a DNA aptamer according to the invention preferably comprises at least 15, 16, 17, 18, 19 or 20, more preferably at least 25, 30, 35, 40, 45, 50, 55 or 60 consecutive nucleotides of any of the sequences shown in SEQ NO: 8-12 or the sequences given in SEQ ID NO: 13-17.
  • the fragment may also be at least 70, 80, 90, 100 or 101 consecutive nucleotides.
  • a fragment comprises at least one of the sequences according to SEQ ID NO: 1-5.
  • the DNA aptamer according to the invention preferably has a sequence as given in any one of SEQ ID Nos. 18-32, i.
  • the DNA aptamer according to the invention consists of a nucleic acid having one of the sequences shown in SEQ ID NO: 18-32
  • the aptamer according to the invention binds non-competitively to the human soluble interleukin-6 receptor. This will cause the bond of
  • Interleukin 6 (IL-6) on the sDL-6R is not affected in principle.
  • the signal transduction can be modified or prevented. This can be effected, for example, by attaching the gpl30 or else the IL-6 sterically or otherwise by means of an antibody directed against the DNA aptamer or, for example, by a molecule coupled to the DNA aptamer, e.g. a peptide, nucleic acid or other compound is hindered.
  • a binding molecule e.g. an antibody directed against the aptamer. It can also be an indirect
  • a molecule coupled to the DNA aptamer e.g., biotin or an antigen
  • another molecule e.g., avidin or an antibody
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an aptamer according to the invention.
  • the pharmaceutical composition moreover preferably comprises suitable carrier material, excipients and the like.
  • the drug may also contain one or more other active ingredients.
  • the drugs may also be coupled to the DNA aptamer, i. covalently or non-covalently bound. Suitable formulations and dosage forms are known to those skilled in the art or may be routinely prepared according to the prior art.
  • aptamers according to the invention can also be bound to nanoparticles, for example, which are loaded with other active ingredients, whereby a targeted supply of the active ingredients is made possible.
  • the invention also relates to the use of a DNA aptamer according to the first aspect of the invention for the manufacture of a medicament, preferably a medicament for the treatment of a condition or disease associated with non-normal levels of the sIL-6R in body fluid, e.g. Blood, or associated with a non-normal occurrence of IL-6R, or a diagnostic agent, e.g. to diagnose a disease associated with the normal condition
  • the medicament is for treatment and the diagnostic agent is for diagnosing inflammatory conditions, cancer or infections, for example rheumatoid arthritis, sepsis, asthma, Crohn's disease, multiple sclerosis, depression, breast cancer, multiple myeloma or HIV infection.
  • the invention relates to the use of the sequence (G 3 T) 4 (SEQ ID NO: 6), the sequence of SEQ ID NO: 11 or SEQ ID NO: 16
  • Inflammatory conditions or cancer preferably for the treatment of rheumatoid arthritis, sepsis, asthma, Crohn's disease, multiple sclerosis, depression, breast cancer or multiple myeloma.
  • Figure 1 Binding of aptamers 13-5, 13-11, 13-15, 13-27 and 13-32 to the target molecule sIL-6R.
  • the proportion of bound DNA aptamer (in%) was determined at defined sIL-6R concentrations in filter binding studies and plotted as a function of the sIL-6R concentration (logarithmic plot).
  • the measurement points and error bars shown result from the averages and standard deviations of three independent of each other Studies.
  • the dissociation constants were calculated using a one-site binding model.
  • Fig. 2 The fusion protein Hyper-IL-6. Schematically shown is the hyper IL-6 fusion construct consisting of the N- and C-terminal truncated sIL-6R (AS 113-323) and IL 6, which are linked together via a flexible linker. Linker amino acids are given in single letter code.
  • Fig. 3 Filter binding studies to analyze the specificity of the interaction between the aptamers 13-15, 13-27 and 13-32 and the target molecule sIL-6R.
  • Aptamers 13-15, 13-27 and 13-32 were exposed to increasing concentrations of the sIL-6R (see Figure 3B above) and the control proteins CEACAM1 (A) and IL6 (A and B).
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • Ellington and Szostak (1990) Nature 346, 818-822, Gopinath (2007), Anal., Bioanal. Chem 387, 171-182, WO 91/19813
  • steps of iterative cycles (1) incubation of a nucleic acid library with the target molecule, (2) separation of binding from non-binding nucleic acids, and (3) amplification of binding species, i. a
  • sIL-6R the amino acid sequence of sIL-6R see SEQ ID NO: 7
  • IL-6R the soluble version of the membrane-bound mterleukin-6 receptor
  • To immobilize the sIL-6R was biotinylated and subsequently immobilized via a biotin-streptavidin interaction on the surface of streptavidin-coupled Dynabeads ®.
  • the protein-coupled particles were used for aptamer selection. After separation non- binding nucleic acids by magnetic separation and washing steps, binding DNA species were eluted by heating and amplified by means of a PCR reaction. After strand separation followed the next round of selection.
  • the dsDNA was denatured by NaOH addition and the aptamer strand was was coupled from the complementary template strand with the help of magnetic Dynabeads ® to which streptavidin separately.
  • the corresponding reverse primer was previously biotinylated.
  • the resulting ssDNA was neutralized by a defined amount of HCl. After 13 rounds, the
  • the ssDNA output library Dl had the following basic structure:
  • sequence of ssDNA library Dl was characterized by a randomized region of 60 nucleotides flanked by 21 and 20 nucleotide constant regions at both the 3 'and 5' ends. The constant regions were used for attachment of the PCR primer.
  • Selection buffer B (lx PBS (0.137 M NaCl, 2.7 mM KCl, 6.5 mM Na 2 HP0 4 , 15 mM KH 2 PO 4 ); 3 mM MgCl 2 ; pH 7.4) for 30 min at room temperature (RT ).
  • the DNA molecules binding to the target were separated from non-binding molecules by magnetic separation, the supernatant discarded, and the DNA-target complexes washed once with 100 ⁇ l of selection buffer B (see above).
  • the elution of binding nucleic acids was carried out after careful resuspension in 55 ⁇ aqua dest. under heat at 80 ° C for 3 min. This eluate was subjected directly to a PCR without further purification steps.
  • the success of the PCR was monitored by PAGE (10% native polyacrylamide (PAA) gel).
  • the number of washing steps after separation of the non-binding DNA molecules was increased up to 12 with increasing number of selection rounds. After thirteen rounds, the
  • the sequences given include both the 60 nucleotide randomized region and the flanking 5 'and 3' primer regions. The number before the hyphen indicates the selection round (13).
  • the aptamer. 13-27 includes the known sequence motif (G 3 T) 4 .
  • Dissociation constants were carried out by means of filter binding studies. For this purpose, a constant amount ( ⁇ 0.2 nM) of radiolabeled DNA (the labeling was carried out by incorporation of ⁇ - [ 32 ⁇ ] - ⁇ (0, 1 ⁇ / ⁇ , 100 nM) using the T4 polynucleotide kinase) with increasing concentrations (up to 1000 nM) of protein for 45 min at 37 ° C incubated. Unless otherwise stated, the lx selection buffer B (lx PBS (0.137 M NaCl, 2.7mM KCl, 6.5mM Na 2 HP0 4 , 15mM KH 2 PO 4 ); 3mM MgCl 2 ; pH 7.4).
  • a nitrocellulose membrane was pretreated for 15 minutes with KOH to reduce unspecific DNA binding, clamped in a 96-well-dot blot apparatus (Schleicher & Schull) and washed twice with 200 ⁇ l of selection buffer B under vacuum.
  • the reactions were filtered through the nitrocellulose membrane. DNA molecules binding to the protein were retained on the membrane. Non-binding DNA molecules were removed by washing four times with lx selection buffer B.
  • the nitrocellulose membrane was dried and the amounts of radioactively labeled substances remaining on the membrane were quantified using a phosphorimager (BioRad). The evaluation of the data obtained was carried out using the software Quantity One ® (BioRad). For the plot of binding curves the program GraphPad Prism® (GraphPad Software, Inc., USA) was used. The dissociation constant (K ⁇ j) was determined according to a one-site-binding model based on the following equation:
  • aptamers 13-15 SEQ ID NO: 15
  • 13-27 SEQ ID NO: 16
  • filter binding studies were carried out, again the binding to SIL-6R, but also the binding to Hyper-IL6 was investigated.
  • Hyper-IL-6 is a bioactive fusion protein of IL-6 and slL-6R, both of which
  • Interaction partners are connected to each other via a flexible polypeptide linker.
  • This linker is composed of the 16 N-terminal non-helical amino acids of IL-6 and thirteen other amino acids, predominantly glycine and serine.
  • the N-terminal immunoglobulin-like domain Dl and the C-terminus of the sIL-6R have been omitted, since both are not suitable for the
  • the aptamers 13-15, 13-27 and 13-32 filter binding studies with two control proteins, IL6 and
  • CEACAMl a cell adhesion molecule that carries a C-terminal His tag analogously to sIL-6R. No binding of the aptamer to IL-6 or CEACAM1 could be detected (see Fig. 3). In contrast, a markedly concentration-dependent binding of the aptamers to the sIL-6R was observed (see Fig. 3 B, top).
  • CTTCAACGTACTCCCGGGGGTTTGGGTGGGTGGGTATCGGGAGTAGGCCGCAACTG CCTG 13-15_SeqB 35 nucleotides, see SEQ DD NO: 18
  • GGGTTGGGTGGGTGGGT The sequence of fragment 13-27_SeqD corresponds to the known DNA aptamer (G 3 T) 4 .
  • Target protein was Hyper-IL6.
  • variants 13-27_SeqD-T4A, 13-27_SeqD-T8A, 13-27_SeqD-T12A, 13-27_SeqD-T16A and 13-27_SeqD-A thymine bases have been replaced by adenine bases, with variants 13-27_SeqD-T4A, 13 -27_SeqD-T8A, 13-27_SeqD-T12A and 13-27_SeqD-T16A each a single TA exchange at different locations, ie at positions 4, 8, 12 and 16, respectively, whereas in variant 13-27_SeqD-A all thymidine nucleotides were exchanged for adenine nucleotides.
  • IQ dissociation constants
  • BMAX proportion of maximal bound DNA
  • the fragment 13-27_SeqD-A which was provided at the positions 4, 8, 12 and 16 respectively adenine instead of thyrnine nucleotides, showed a comparatively bad tie up.
  • the fragment 13-27_SeqD + TlT16 also tied badly.
  • the separation of the aptamer-protein mixtures was carried out via a 5% native polyacrylamide ⁇ AA) gel by means of polyacrylamide gel electrophoresis (PAGE). Due to their size, aptamer-protein complexes formed under native conditions have a delayed migration behavior in the gel. The complexes therefore migrate more slowly than the corresponding free DNA molecules and are therefore detectable as so-called "shift" in the gel.
  • PAGE polyacrylamide gel electrophoresis
  • Equal amounts of [ 32 P] radiolabelled DNA were incubated with increasing amounts of protein (0-1000 nM Hyper-IL6) in lx selection buffer B (see above) for 45 min at 37 ° C.
  • Samples were spiked with 6x DNA sample buffer (50% (w / v) sucrose, 1% (w / v) SDS, 0.1% (w / v) orange G) and native PAGE using a 5% PAA gels (lx TBE (89mM Tris-HCl, 89mM boric acid, 2mM EDTA); Acrylamide: bisacrylamide 37.5: 1 5% (w / v); TEMED (, N, NN , -tetamethyl-e-lecl amine ) 0.1% (v / v); APS (ammonium persulfate) 0.1% (w / v)).
  • the electrophoretic separation was carried out at 80 V and 4 ° C in lx TBE. After completion of the electrophoresis
  • Fragment of DNA aptamer fragment of the DNA aptamer
  • Variant of a fragment of DNA aptamer 13-27 variant of a fragment of DNA aptamer 13-27
  • DNA aptamer DNA aptamer
  • randomized region plus 5 'and 3' primer regions randomized region plus 5 'and 3' primer regions

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Abstract

L'invention concerne des aptamères se liant spécifiquement à une molécule cible. Le but de l'invention est de fournir des agents diagnostiques et/ou thérapeutiques destinés à être utilisés pour le dépistage et/ou la prophylaxie ou le traitement de processus inflammatoires, de maladies cancéreuses et d'infections. A cet effet, un aptamère d'ADN selon l'invention se lie spécifiquement au récepteur soluble humain de l'interleukine 6 (sIL-6R) et comprend la séquence GGGNGGGHGGGWGGG, à l'exception toutefois de la séquence GGGTGGGTGGGTGGGT, dans laquelle N = A, T, TT ou TTT, H = A, C ou T et W = A ou T.
PCT/DE2010/001413 2009-12-07 2010-12-06 Aptamères d'adn se liant spécifiquement au récepteur soluble de l'interleukine 6 WO2011069487A2 (fr)

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DE102009056945A DE102009056945A1 (de) 2009-12-07 2009-12-07 DNA-Aptamere, die den löslichen Interleukin-6-Rezeptor spezifisch binden
DE102009056945.6 2009-12-07

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019813A1 (fr) 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5567604A (en) * 1993-04-23 1996-10-22 Aronex Pharmaceuticals, Inc. Anti-viral guanosine-rich oligonucleotides
US6423493B1 (en) * 1998-10-26 2002-07-23 Board Of Regents The University Of Texas System Combinatorial selection of oligonucleotide aptamers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019813A1 (fr) 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ELLINGTON; SZOSTAK, NATURE, vol. 346, 1990, pages 818 - 822
FISCHER, M. ET AL.: "I. A bioactive designer cytokine for,human hematopoietic progenitor cell expansion", NAT BIOTECHNOL, vol. 15, no. 2, 1997, pages 142 - 5, XP002047603, DOI: doi:10.1038/nbt0297-142
GOPINATH, ANAL. BIOANAL. CHEM., vol. 387, 2007, pages 171 - 182
HIROTA, H. ET AL., PROC. NATL. ACAD. SCI. U.S.A, vol. 92, 1995, pages 4862 - 4866
JONES ET AL., THE FASEB JOURNAL, vol. 15, 2001, pages 43 - 57
MACKIEWICZ, A. ET AL., J. IMMUNOL., vol. 149, 1992, pages 2021 - 2027
OSBORNE ET AL., CURR. OPIN. CHEM. BIOL., vol. 1, 1997, pages 5 - 9
ROSE-JOHN ET AL., J. J. LEUKOCYTE BIOL., vol. 80, 2006, pages 227 - 235
SAITO, M. ET AL., J. IMMUNOL., vol. 148, 1992, pages 4066 - 4071
SCHELLER; ROSE-JOHN, MED. MICROBIOL. IMMUNOL., vol. 195, 2006, pages 173 - 183
TAGA, T. ET AL., CELL, vol. 58, 1989, pages 573 - 581

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