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WO2014066084A1 - Nucleic acid modulators of alpha2beta1 - Google Patents

Nucleic acid modulators of alpha2beta1 Download PDF

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Publication number
WO2014066084A1
WO2014066084A1 PCT/US2013/064829 US2013064829W WO2014066084A1 WO 2014066084 A1 WO2014066084 A1 WO 2014066084A1 US 2013064829 W US2013064829 W US 2013064829W WO 2014066084 A1 WO2014066084 A1 WO 2014066084A1
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Prior art keywords
ligand
nucleic acid
modulator
ligands
binding
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PCT/US2013/064829
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French (fr)
Inventor
Christopher Rusconi
Juliana M. Layzer
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NOVAMEDICA LLC
Regado Biosciences Inc
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NOVAMEDICA LLC
Regado Biosciences Inc
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Priority to EA201690861A priority Critical patent/EA201690861A1/en
Publication of WO2014066084A1 publication Critical patent/WO2014066084A1/en
Anticipated expiration legal-status Critical
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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • 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
    • C12N15/113—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
    • 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
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • 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
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the present invention relates, in general, to a pharmacologic system comprising a nucleic acid ligand that binds to and regulates the activity of the integrin ⁇ 2 ⁇ protein. These nucleic acid ligands are also actively reversible using a modulator that inhibits the activity of the nucleic acid ligand to neutralize its pharmacologic effect and thereby restore ⁇ 2 ⁇ function.
  • the invention further relates to compositions comprising the nucleic acid ligand and/or a modulator as well as methods of using these agents and compositions in treating a 2 i-mediated diseases and disorders.
  • Platelets are small, anuclear blood cells which are fairly quiescent under normal conditions but which respond immediately to vascular injury by adhesion, activation, aggregation, and thrombus formation.
  • the primary function of platelets is to stop blood loss after tissue trauma and exposure of the subendothelial matrix. It is well known that damage to a blood vessel can expose extracellular matrix components to the blood, particularly von Willebrand factor (VWF), collagen, fibronectin, thrombospondin, and laminin. Interaction of platelets with these exposed molecules results in activation of the platelet cells.
  • VWF von Willebrand factor
  • Integrin ⁇ 2 ⁇ (also known as GPIa/IIa or VLA-2) was the first collagen receptor to be identified on platelets (Nieuwenhuis et al, 1985, Nature, 318:470-472; Santoro, 1986, Cell, 46:913-920). ⁇ 2 ⁇ is also expressed on lymphocytes, fibroblasts, endothelial cells, epithelial cells, and various cancer cells (Santoro et al, 1995, Thromb. Haemost. 74:813-821).
  • ⁇ 2 ⁇ belongs to a family of ⁇ (VLA) integrins that mediate cell adhesion to extracellular matrix (ECM) proteins, such as collagen, fibronectin and laminin (Coller et al, 1989, Blood, 74: 182-192; Elices et al, 1989, Proc. Natl. Acad. Sci. USA, 86:9906- 9910). They share a common beta chain ( ⁇ ; CD29) that is non-covalently linked to one of at least six different alpha chains (alphal-alpha6; CD49a-f).
  • VLA ⁇
  • ECM extracellular matrix
  • ⁇ 2 ⁇ function has been implicated in a number of disorders including arterial thrombosis and other cardiovascular disorders (Samaha et al., 2006, Arterioscler Thromb Vase Biol, 2006, 26:2588-2593), autoimmunity including Multiple Sclerosis (MS) and inflammation (McCall et al, 2008, 9: 139-149; Werr et al, 2000, Blood, 95: 1804-1809), cancer, including tumor angiogenesis and tumor cell metastasis (Alghisi et al, 2006, 13: 113-135; van Muijen et al, 1995, 130:105-122; Anastassiou et al, 2009, Cancer Biother, 10:287-292) and as a receptor for echovirus cell entry and infection (Bergelson et al, 1992, Science, 255: 1718-1720.
  • MS Multiple Sclerosis
  • McCall et al, 2008, 9: 139-149 Werr et al,
  • compositions targeted to the ⁇ 2 ⁇ protein are described herein for the development of treatments for a variety of disorders including cardiovascular disorders, immune disorders and cancer.
  • compositions directed to nucleic acid ligands which specifically bind ⁇ 2 ⁇ methods of treatments and methods of use of nucleic acid ligands which specifically bind ⁇ 2 ⁇ , and modulators thereof.
  • a ⁇ 2 ⁇ ligand or a pharmaceutically acceptable salt thereof, is provided, which specifically binds the extracellular domain of the ⁇ 2 ⁇ protein.
  • the ⁇ 2 ⁇ ligand specifically binds the I domain of the ⁇ 2 ⁇ protein.
  • the ligand comprises an isolated nucleic acid sequence.
  • at least one nucleotide is a ribonucleotide.
  • at least one nucleotide is a deoxyribonucleic acid.
  • the isolated nucleic acid sequence of the ⁇ 2 ⁇ ligand comprises a mixture of ribonucleotides and deoxyribonucleotides.
  • the nucleic acid ⁇ 2 ⁇ ligand comprises a secondary structure comprising at least one stem and one loop.
  • the nucleic acid ⁇ 2 ⁇ ligand sequence is about 20 nucleotides (nt) to about 50 nt in length, about 20 nt to about 45 nt in length, about 20 nt to about 40 nt in length, about 20 nt to about 35 nt in length, about 20 nt to about 30 nt in length, or about 30 nt to about 35 nt in length.
  • the nucleic acid sequence of the ⁇ 2 ⁇ ligand comprises one or more ribonucleotides, deoxyribonucleotides, or a mixture both ribonucleotides and deoxyribonucleotides .
  • one or more of the nucleotides of the nucleic acid ⁇ 2 ⁇ ligand sequence is modified.
  • the one or more nucleotides comprise a modification at the 2' hydroxyl position.
  • the modification is selected from the group consisting of 2'-0-methyl and 2'-fluoro.
  • the one or more nucleotides is 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0-methyl adenosine or 2'-0-methyl guanosine.
  • the one or more nucleotides is a 2' fluoro cytidine or a 2' fluoro uridine.
  • the one or more nucleotides comprising a modification is selected from the group consisting of 5-fluorouracil, 5-fluorocytosine, 5-bromouracil, 5- bromocytosine, 5-chlorouracil, 5-chlorocytosine, 5-iodouracil, 5-iodocytosine, 5- methylcytosine, 5-methyluracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 6-methylcytosine, N6-adenine, 7- methylgu
  • the ⁇ 2 ⁇ ligand comprises at least one modified sugar moiety.
  • the ⁇ 2 ⁇ ligand comprises at least one modified phosphate backbone.
  • the nucleic acid ⁇ 2 ⁇ ligand sequence comprises an inverted thymine at its 3 ' end.
  • the nucleic acid ⁇ 2 ⁇ ligand comprises a spacer.
  • the spacer is a glycol spacer.
  • a loop of the nucleic acid ⁇ 2 ⁇ ligand comprises the glycol spacer.
  • the glycol spacer is provided by incorporation of 9-O-Dimethoxytrityl-triethylene glycol, 1- [(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
  • the glycol spacer is attached to the 3' end of a first internal nucleotide of the nucleotide ⁇ 2 ⁇ ligand sequence and is attached to the 5' end of a second internal nucleotide adjacent to the first internal nucleotide of the nucleotide ⁇ 2 ⁇ ligand sequence.
  • the nucleic acid ⁇ 2 ⁇ ligand comprises an aliphatic amino linker.
  • the aliphatic amino linker is attached to the 5 ' end of the nucleic acid ⁇ 2 ⁇ ligand sequence.
  • the aliphatic amino linker is attached to the 3' end of the nucleic acid ⁇ 2 ⁇ ligand sequence.
  • the aliphatic amino linker is provided by incorporation of 6-
  • the nucleic acid ⁇ 2 ⁇ ligand is linked to at least one hydrophilic moiety.
  • the at least one hydrophilic moiety is a polyalkylene glycol.
  • the ⁇ 2 ⁇ ligand comprises a polyalkylene moiety attached to the 5' end and/or the 3' end of the isolated nucleic acid sequence.
  • the polyalkylene moiety is attached via a linker.
  • the linker is an aliphatic amino linker.
  • the ⁇ 2 ⁇ ligand is linked to a 40 KD polyethylene glycol (PEG) moiety using a six carbon amino linker.
  • the six carbon amino linker is attached to the PEG moiety through an amide attachment.
  • the PEG moiety is two twenty KD PEG moieties which are attached to one or more amino acids, such as lysine, which is attached via an amide bond to the six carbon amino linker.
  • the first nucleic acid ⁇ 2 ⁇ ligand comprises a
  • nucleic acid ⁇ 2 ⁇ ligand specifically binds to ⁇ 2 ⁇ (SEQ ID NO: 1). In another embodiment, the nucleic acid ⁇ 2 ⁇ ligand specifically binds to the I domain of ⁇ 2 ⁇ (SEQ ID NO:3).
  • the ⁇ 2 ⁇ ligand has a dissociation constant of about 20 nanomolar (nM) or less.
  • the ⁇ 2 ⁇ ligand has a dissociation constant which ranges from about 400 picomolar (pM) to about 10 nM.
  • the ⁇ 2 ⁇ ligand has a dissociation constant which ranges from about 100 pM to about 10 nM .
  • the nucleic acid ⁇ 2 ⁇ ligand inhibits binding of ⁇ 2 ⁇ to collagen.
  • binding of the nucleic acid ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ stabilizes an active conformation of ⁇ 2 ⁇ . In another embodiment, binding of the nucleic acid ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ stabilizes an inactive conformation of ⁇ 2 ⁇ . In yet another embodiment, binding of the nucleic acid ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ inhibits interaction between ⁇ 2 ⁇ and a collagen molecule.
  • binding of the ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ results in inhibition of, or reduction of, ⁇ 2 ⁇ activity. In yet another embodiment, binding of the ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ results in the inability of, or the reduction in ability of, ⁇ 2 ⁇ to interact with collagen. In still another embodiment, binding of the ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ expressed on the surface of a platelet results in an inhibition of, or reduction of, platelet adhesion. In still another embodiment, binding of the ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ expressed on the surface of a platelet results in an inhibition of, or reduction of, platelet activation. In still another embodiment, binding of the ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ expressed on the surface of a platelet results in an inhibition of, or reduction of, platelet aggregation.
  • the ⁇ 2 ⁇ ligand binds to and decreases or inhibits a function of a variant of ⁇ 2 ⁇ , wherein said ⁇ 2 ⁇ variant is at least 80%, 85%, 90%, 91%, 93%>, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO : 1.
  • the ⁇ 2 ⁇ ligand has a dissociation constant ("Ka") for ⁇ 2 ⁇ of less than about 100 micromolar ( ⁇ ), less than about 1 ⁇ , less than about 500 nanomolar (nM), less than about 100 nM, less than about 50 nM, less than about 1 nM, less than about 500 picomolar (pM), less than about 300 pM, less than about 250 pM, or less than about 200 about pM.
  • Ka dissociation constant
  • a modulator to the ⁇ 2 ⁇ ligand wherein the modulator reverses, partially or completely, the activity of a ⁇ 2 ⁇ ligand.
  • the modulator comprises an isolated nucleic acid sequence. In another embodiment, the modulator comprises a DNA sequence, an RNA sequence, a polypeptide sequence, or any combination thereof. In one embodiment, the modulator is a nucleic acid modulator comprising deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. In another embodiment the nucleic acid modulator comprises at least one modified deoxyribonucleotide and/or at least one modified ribonucleotide.
  • the modulator consists of an oligonucleotide which is complementary to at least a portion of the ⁇ 2 ⁇ nucleic acid ligand. In another embodiment, the modulator comprises an oligonucleotide which is complementary to at least a portion of the ⁇ 2 ⁇ nucleic acid ligand. In another embodiment, the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a loop in the ⁇ 2 ⁇ ligand. In still another embodiment, the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a stem in the ⁇ 2 ⁇ ligand.
  • the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a stem in the ⁇ 2 ⁇ ligand and to at least a portion of a loop in the ⁇ 2 ⁇ ligand.
  • the modulator of a ⁇ 2 ⁇ nucleic acid ligand is selected from the group consisting of a ribozyme, a DNAzyme, a peptide nucleic acid (PNA), a morpholino nucleic acid (MNA), and a locked nucleic acid (LNA).
  • the modulator of a ⁇ 2 ⁇ nucleic acid ligand is selected from the group consisting of a ribozyme, a DNAzyme, a peptide nucleic acid (PNA), a morpholino nucleic acid (MNA), and a locked nucleic acid (LNA), wherein the modulator specifically binds to or interacts with at least a portion of a ⁇ 2 ⁇ nucleic acid ligand.
  • the modulator is selected from the group consisting of a nucleic acid binding protein or peptide, a small molecule, an oligosaccharide, a nucleic acid binding lipid, a polymer, a nanoparticle, and a microsphere, wherein the modulator binds to or interacts with at least a portion of a ⁇ 2 ⁇ nucleic acid ligand.
  • the modulator is a nucleic acid modulator comprising deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides.
  • the nucleic acid modulator comprises at least one modified deoxyribonucleotide and/or at least one modified ribonucleotide.
  • the modulator is an oligonucleotide which is complementary to at least a portion of the ⁇ 2 ⁇ nucleic acid ligand. In another embodiment, the modulator is an oligonucleotide which is complementary to at least a portion of a loop in the ⁇ 2 ⁇ ligand.
  • the modulator comprises an isolated nucleic acid sequence, wherein the sequence is about 10 nt to about 30 nt, about 10 nt to about 25 nt, about 10 nt to about 20 nt, about 10 nt to about 15 nt, or about 15 nt to about 20 nt in length.
  • one or more of the nucleotides of the nucleic acid modulator sequence is modified.
  • the one or more nucleotides comprise a modification at the 2' hydroxyl position.
  • the modification is selected from the group consisting of 2'-0-methyl and 2'-fluoro.
  • the one or more nucleotides is 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0-methyl adenosine, 2'-0-methyl guanosine or a 2'-0-methyl thymidine.
  • the one or more nucleotides is a 2' fluoro cytidine, a 2' fluoro uridine, a 2' fluoro adenosine or a 2'-fluoro guanosine.
  • the modification of one or more nucleotides of the nucleic acid modulator comprises a modification selected from the group consisting of 5- fluorouracil, 5-fluorocytosine, 5-bromouracil, 5-bromocytosine, 5-chlorouracil, 5- chlorocytosine, 5-iodouracil, 5-iodocytosine, 5-methylcytosine, 5-methyluracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl thiouridine, 5 -carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 6-methylcytosine, N6-
  • the modulator comprises as least one modified sugar moiety. In one embodiment, the modulator comprises at least one modified phosphate backbone.
  • the modulator comprises an oligonucleotide which hybridizes at physiological conditions to a loop of the ⁇ 2 ⁇ ligand. In another embodiment, the modulator comprises an oligonucleotide which hybridizes under physiological conditions to a stem of the ⁇ 2 ⁇ ligand. In yet another embodiment, the modulator comprises an oligonucleotide which hybridizes under physiological conditions to at least a portion of a stem of the ⁇ 2 ⁇ ligand and to at least a portion of a loop of the ⁇ 2 ⁇ ligand.
  • the modulator disrupts the secondary structure of the nucleic acid ⁇ 2 ⁇ ligand. In another embodiment, the modulator stabilizes the tertiary structure of the ⁇ 2 ⁇ ligand.
  • the modulator disrupts the tertiary structure of the nucleic acid ⁇ 2 ⁇ ligand. In another embodiment, the modulator stabilizes the tertiary structure of the ⁇ 2 ⁇ ligand. In one embodiment, the binding of the modulator to the ⁇ 2 ⁇ ligand exposes a suicide position within the ⁇ 2 ⁇ ligand, thereby disrupting the secondary structure of the ⁇ 2 ⁇ ligand and leading to enhanced destruction of the nucleic acid ⁇ 2 ⁇ ligand by nucleases.
  • binding of the modulator to a ⁇ 2 ⁇ complex reduces or eliminates binding of the ⁇ 2 ⁇ ligand to ⁇ 2 ⁇ .
  • a method of modulating the activity of a ⁇ 2 ⁇ ligand is provided.
  • a method of modulating the activity of a nucleic acid ligand to ⁇ 2 ⁇ by administering a modulator of the ⁇ 2 ⁇ ligand to a host who has been administered the nucleic acid ⁇ 2 ⁇ ligand is provided.
  • the modulator can be a oligonucleotide modulator, or derivative thereof, and in certain embodiments, is complimentary to a portion of the nucleic acid ⁇ 2 ⁇ ligand.
  • a method of regulating ⁇ 2 ⁇ function using a ⁇ 2 ⁇ ligand is provided.
  • the method for regulating ⁇ 2 ⁇ function comprises administering to a host a therapeutically effective amount of a ⁇ 2 ⁇ ligand. In another embodiment, the method further comprises administering a ⁇ 2 ⁇ ligand modulator to the host.
  • the method for regulating ⁇ 2 ⁇ function in increasing ⁇ 2 ⁇ function in another embodiment, the method for regulating ⁇ 2 ⁇ function is decreasing ⁇ 2 ⁇ function.
  • a method of treating symptoms of, or ameliorating a platelet- mediated disease or disorder is provided.
  • the method comprises administering to a host in need thereof a therapeutically effective dose of a ⁇ 2 ⁇ ligand that binds to ⁇ 2 ⁇ .
  • the host is diagnosed with high-risk diabetes.
  • the host is diagnosed with a cancer at high risk of metastasis.
  • the platelet-mediated disease or disorder is selected from the group consisting of cardiovascular disorders, acute coronary syndromes, diabetes-related disorders, autoimmune inflammatory disorders, and cancer.
  • the cardiovascular disorder is a thrombosis
  • the acute coronary syndrome is due to coronary thrombosis, unstable angina or myocardial infarction.
  • the diabetes-related disorder is diabetic retinopathy, diabetic vasculopathy, atherosclerosis, ischemic stroke, peripheral vascular disease, acute renal injury or chronic renal failure.
  • the autoimmune inflammatory disorder is multiple sclerosis, scleroderma, rheumatoid arthritis, or an inflammatory autoimmune disorder selected from the group consisting of psoriatic arthritis, reactive arthritis, inflammatory bowel disease and ankylosing spondylitis.
  • the cancer is selected from lung cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, bone cancer and liver cancer.
  • the ⁇ 2 ⁇ ligand is administered by parenteral administration, intravenous injection, intradermal delivery, intra-articular delivery, intra- synovial delivery, intrathecal, intra-arterial delivery, intracardiac delivery, intramuscular delivery, subcutaneous delivery, intraorbital delivery, intracapsular delivery, intraspinal delivery, intrasternal delivery, topical delivery, transdermal patch delivery, buccal delivery, rectal delivery, delivery via vaginal or urethral suppository, peritoneal delivery, percutaneous delivery, delivery via nasal spray, delivery via surgical implant, delivery via internal surgical paint, delivery via infusion pump or delivery via catheter.
  • a method for treating a host in need thereof by administering a ⁇ 2 ⁇ ligand, wherein the ⁇ 2 ⁇ ligand regulates platelet function is provided.
  • a therapeutically effective dose of ⁇ 2 ⁇ is administered.
  • the therapeutically effective dose reduces or inhibits platelet adhesion and/or aggregation.
  • a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid ⁇ 2 ⁇ ligand which binds ⁇ 2 ⁇ is provided. In one aspect, a pharmaceutical composition comprising a therapeutically effective amount of a modulator, wherein the modulator regulates the activity of a nucleic acid ⁇ 2 ⁇ ligand which binds ⁇ 2 ⁇ , is provided.
  • the pharmaceutical composition comprises a ⁇ 2 ⁇ ligand and pharmaceutically-acceptable excipients.
  • the pharmaceutical composition is a liquid suitable for intravenous injection.
  • the pharmaceutical composition is a liquid or dispersion suitable for subcutaneous injection.
  • kits comprising a therapeutically effective amount of a ⁇ 2 ⁇ nucleic acid ligand and/or a modulator which regulates the activity of the ⁇ 2 ⁇ nucleic acid ligand is provided.
  • FIG. 1 provides a schematic of the SELEX nucleic acid ligand selection process.
  • FIG. 2 shows a full-length cDNA sequence for the ⁇ 2 ⁇ gene Accession number NM 002203.3 (SEQ ID NO:l).
  • FIGS. 3A-C shows a full-length amino acid sequence of an ⁇ 2 ⁇ protein (A) (NP 002194) (SEQ ID NO:2), the I Domain (B) (SEQ ID NO:3), and a His-tag fusion of the I Domain (C) (SEQ ID NO:4).
  • FIG. 4 illustrates binding by an I Domain to a2 antibodies.
  • FIG. 5 illustrates binding by an I Domain to collagen.
  • FIG. 6 provides a table listing the selection conditions for initial alpha2 I Domain ligand selection.
  • FIG. 7 provides a graph showing the progression of the alpha2 I Domain selection.
  • FIG. 8 provides a graph showing results of platelet adhesion in the presence of
  • FIGS. 9A-B illustrate the starting phosphoramidite for a hexaethylene glycol linker used in synthesis (A), and the hexaethylene glycol spacer when incorporated between two nucleotides of a nucleic acid ligand (B).
  • FIGS. 10A-B shows PEG moieties which may be conjugated to a nucleic acid ligand via a linker and a configuration of a conjugated moiety.
  • the present invention provides pharmaceutical compositions of nucleic acid ligands which bind to ⁇ 2 ⁇ , modulators of the ligands, and methods of use thereof for the treatment of platelet-mediated diseases and disorders. Further provided are
  • compositions comprising a ⁇ 2 ⁇ nucleic acid ligand and/or ⁇ 2 ⁇ ligand modulator.
  • nucleic acid ligand which may also referred to herein as a “ligand” or “aptamer,” is a nucleic acid that can form a tertiary structure, which allows it to interact with a target molecule.
  • ligand a nucleic acid that can form a tertiary structure, which allows it to interact with a target molecule.
  • ⁇ 2 ⁇ nucleic acid ligand or " ⁇ 2 ⁇ ligand” or
  • ligand or "nucleic acid ⁇ 2 ⁇ ligand” refers to a ligand or aptamer that specifically binds to at least a portion of ⁇ 2 ⁇ .
  • the terms refer to oligonucleotides having specific binding regions that are capable of forming complexes with an intended target molecule in a physiological environment.
  • the affinity of the binding of an ligand to a target molecule is defined in terms of the dissociation constant (Ka) of the interaction between the ligand and the target molecule.
  • Ka dissociation constant
  • the Ka of the ligand for its target is between about InM to about 100 nM.
  • the specificity of the binding is defined in terms of the comparative dissociation constant of the ligand for target as compared to the dissociation constant with respect to the ligand and other materials in the environment or unrelated molecules in general.
  • the Ka for the ligand with respect to the target will be 10-fold, 50-fold, 100-fold, or 200-fold less than the K with respect to the unrelated material or accompanying material in the environment.
  • Ligand modulator pair or "ligand modulator pair” is meant to include a specified ligand to a target molecule, and a ligand modulator that changes the secondary and/or tertiary structure of the ligand so that the ligand' s interaction with its target is modulated.
  • the modulator can be an oligonucleotide complimentary to a portion of the ligand.
  • the modulator can change the conformation of the ligand to reduce the target binding capacity of the ligand by 10% to 100%, 20% to 100%, 25%, 40%, 50%, 60%, 70%), 80%), 90%) or 100%, or any percentage in the range between 10%> and 100%) under physiological conditions.
  • a pharmaceutically acceptable agent that can bind a ligand or aptamer as described herein and modify the interaction between that ligand and its target molecule (e.g., by modifying the structure of the ligand) in a desired manner.
  • Modulate as used herein means a lessening, an increase, or some other measurable change in activity.
  • “Host” refers to a mammal and includes human and non-human mammals.
  • Examples of host include, but are not limited to mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, horses, sheep, cows, and humans.
  • “Pharmaceutically acceptable,” as used herein means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in humans.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate, reduce or improve a symptom to some extent) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize.
  • an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon the potency of the nucleic acid ligand and modulator.
  • a “stabilized nucleic acid molecule” refers to a nucleic acid molecule that is less readily degraded in vivo (e.g., via an exonuclease or endonuclease) in comparison to a non-stabilized nucleic acid molecule.
  • Stabilization can be a function of length and/or secondary structure and/or inclusion of chemical substitutions within the sugar of phosphate portions of the oligonucleotide backbone. Stabilization can be obtained by controlling, for example, secondary structure which can stabilize a molecule. For example, if the 3' end of a nucleic acid molecule is complementarily to an upstream region, that portion can fold back and form a "stem loop" structure which stabilizes the molecule.
  • binding affinity and "binding activity” are meant to refer to the tendency of a ligand molecule to bind or not to bind to a target.
  • the energetics of said interactions are significant in “binding activity” and “binding affinity” because they define the necessary concentrations of interacting partners, the rates at which these partners are capable of associating, and the relative concentrations of bound and free molecules in a solution.
  • the energetics may be characterized through, among other ways, the determination of a dissociation constant, Ka.
  • Treatment means any treatment of disease in a mammal, including: (a) protecting against the disease, that is, causing the clinical symptoms not to develop; (b) inhibiting the disease, that is, arresting, ameliorating, reducing, or suppressing the development of clinical symptoms; and/or (c) relieving the disease, that is, causing the regression of clinical symptoms. It will be understood by those skilled in the art that in human medicine, it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events.
  • prophylaxis is intended as an element of “treatment” to encompass both “preventing” and “suppressing” as defined herein.
  • protection as used herein, is meant to include “prophylaxis.”
  • the term "effective amount” means a dosage sufficient to provide treatment for the disorder or disease state being treated. This will vary depending on the patient, the disease and the treatment being effected.
  • a ⁇ 2 ⁇ nucleic acid ligand "variant" as used herein encompasses variants that perform essentially the same function as a ⁇ 2 ⁇ nucleic acid ligand and comprises substantially the same structure.
  • the ⁇ 2 ⁇ protein is expressed on the surface of platelets, lymphocytes, fibroblasts, endothelial cells, epithelial cells, and various cancer cells.
  • Biological phenomena in which ⁇ 2 ⁇ integrin function is essential include collagen-induced platelet aggregation, cell migration on collagen, stable adhesion and cell-dependent reorganization of collagen fibers. Such activity is plays an integral role in arterial thrombosis and other vascular disorders.
  • ⁇ 2 ⁇ has been shown to play a role in the recruitment of polymorphonuclear lymphocytes to inflamed tissue sites (Werr et al, 2000, Blood, 95: 1804-1809).
  • ⁇ 2 ⁇ integrin In cancer biology, the ⁇ 2 ⁇ integrin has been associated with an invasive cell phenotype and it can be a marker for aggressive melanoma. On the other hand, overexpression of ⁇ 2 ⁇ integrin in breast cancer cells restores the normal phenotype. Clearly, ⁇ 2 ⁇ is a valid target for the development of therapies for vascular disorders and inflammatory disorders, as well as cancer and metastasis.
  • ⁇ 2 ⁇ is a heterodimer.
  • the a 2 subunit is 1,181 amino acids in length, including a signal peptide of 29 amino acids, an extracellular domain 1,102 amino acids in length, a transmembrane domain and a short cytoplasmic segment 22 amino acids in length.
  • the a 2 subunit also has a 191-amino acid insert, called the I Domain.
  • the I Domain shares structural homology to I Domains of other a subunits, as well as the vWF A Domain.
  • the I Domain spanning from Serl24 to Gly 337, has been shown to contain the binding site for collagen.
  • the I Domain contains a cation binding site described as a metal ion-dependent adhesion site (MIDAS) motif which requires Mg 2+ or Mn 2+ and which is essential for collagen binding.
  • I Domain residues shown to interact with collagen include D151, S153, S155, T221, D254 and E256 (Embsleyu et al, 2000, Cell, 101 :47-56).
  • the I Domain has also been shown to interact with other ligands, including the picornavirus, echovirus 1 (Estavillo et al, 1999, J Biol Chem, 274:35921-35926).
  • Described herein are studies done to identify ⁇ 2 ⁇ ligands that bind to the ⁇ 2 ⁇ extracellular domain, and more specifically the I Domain, in order to generate pharmaceutical agents which can be useful in the treatment of, for example, vascular disorders, inflammatory disorders, autoimmune disorderscancer and certain types of viral infection.
  • Nucleic acid ligands which specifically bind the ⁇ 2 ⁇ protein are identified using the SELEX method.
  • the ligands which were initially obtained via SELEX are then fully characterized to understand the properties of the ⁇ 2 ⁇ ligands.
  • Such characterization included sequencing, sequence alignment to determine conserved sequences, secondary structure prediction, and truncations and mutation analysis to identify ligand regions most critical for the desired function of specifically binding and inhibiting ⁇ 2 ⁇ .
  • modifications were made to optimize the ligands for pharmaceutical use. Examples of these modifications include pegylation, use of a spacer within the nucleic acid ligand and selected modifications to the sugar and phosphate portion of the nucleic acid ligand. Binding assays were performed to monitor ligand function as a result of the various modifications used.
  • SELEX refers to the Systematic Evolution of Ligands by Exponential
  • the SELEX method involves selection from a mixture of candidate
  • the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity ligands to the target molecule.
  • U.S. patent No. 5,707,796 describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA.
  • U.S. Patent No. 5,763,177 describes a SELEX-based method for selecting ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule.
  • U.S. Patent No. 5,580,737 describes a method for identifying highly specific ligands able to discriminate between closely related molecules, termed Counter-SELEX.
  • 5,567,588 and 5,861,254 describe SELEX-based methods that achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
  • U.S. Patent No. 5,496,938 describes methods for obtaining improved ligands after the SELEX process has been performed.
  • U.S. Patent No. 5,705,337 describes methods for covalently linking a ligand to its target.
  • U.S. Patent No. 5,648,214 The ability to use affinity elution with a ligand to produce ligands that are targeted to a specific site on the target molecule is exemplified in U.S. Patent No. 5,780,228, which relates to the production of high affinity ligands binding to certain lectins.
  • Methods of preparing nucleic acid ligands to certain tissues, which include groups of cell types, are described in U.S. Patent No. 6,127,119.
  • the production of certain modified high affinity ligands to calf intestinal phosphatase is described in U.S. Patent No. 6,673,553.
  • U.S. Patent No. 6,716,580 describes an automated process of identifying nucleic acid ligands that includes the use of a robotic manipulators.
  • SELEX can be used to generate bivalent binding that have two or more binding domains with affinity for two or more epitopes of a protein, including a receptor.
  • the process can be used to select for nucleic acid ligands that have affinity for two or more regions of the ⁇ 2 ⁇ protein.
  • the ligand can bind to at least two portions of the C2 regions.
  • the ligand affects dimerization of the ⁇ 2 ⁇ protein, either by disrupting or stabilizing the dimeric conformation.
  • modulators can be designed to reduce binding to only one, more than one, or all epitopes that the nucleic acid ligand binds to. The modulator can, for example, interfere with binding to only a single epitope, such as a C2-1 or C2-2 region of the receptor.
  • Nucleic acid ligands specific to ⁇ 2 ⁇ may be generated by performing SELEX against short peptides which represent the extracellular domain of the molecule, using SELEX methods as described for example in U.S. Patent No. 7,087,735.
  • nucleic acid ligands specific to ⁇ 2 ⁇ can be isolated by performing SELEX on intact cells which express ⁇ 2 ⁇ , membrane fractions enriched for the protein, on purified ⁇ 2 ⁇ or its I domain, or on cell-lines specifically over-expressing the ⁇ 2 ⁇ protein or its I Domain using SELEX methods as described, for example, in U.S. Patent No. 6,730,482.
  • the SELEX process can be directed to isolate specific ⁇ 2 ⁇ nucleic acid ligands using competitive affinity elution schemes, such as those described in U.S. Patent No. 5,780,228.
  • elution of ligands bound to the protein could be accomplished by addition of sufficient amounts of an activator or binder of ⁇ 2 ⁇ such as collagen, or the peptide GFOGER (SEQ ID NO:5) (Onley et al, JBC 2000, 275:24560-24564).
  • the ⁇ 2 ⁇ extracellular domain or an isolated I domain can be a recombinantly expressed and purified protein used for a SELEX procedure.
  • the ⁇ 2 ⁇ nucleic acid ligand binds to the ⁇ 2 ⁇ protein under physiological conditions.
  • Physiological conditions are typically related to the level of salts and pH of a solution. In vitro, physiological conditions are typically replicated in a buffer including 150mM NaCl, 2mM CaCl 2 20mM HEPES, at a pH of about 7.4.
  • native, typically unactivated, platelets are used as described above to screen a population of nucleic acid ligands and provide an enriched population, which contains ligands directed to proteins found on platelets. The enriched population is then used against either a stable cell line overexpressing the desired ⁇ 2 ⁇ protein, or a cell line that has been transiently transfected with a recombinant DNA molecule that directs expression of the protein.
  • the secondary screening can be accomplished either by using a modified SELEX procedure on isolated receptors from these cells or on the whole cells either through ligand competition studies or by identifying the effects on intracellular signaling pathways.
  • nucleic acid ligands to specific ⁇ 2 ⁇ targets can be identified using an immobilized protein.
  • a purified protein can be linked to a solid matrix by a chemical linker.
  • membranes from cells over-expressing a oil can be extracted using a detergent, such as an anionic detergent (e.g. cholate), to isolate a certain fraction of the proteins and the mixture coupled to an immobilized artificial membrane.
  • an anionic detergent e.g. cholate
  • the reorganization can be accomplished by removal of the detergent, during which lipids and proteins reorganize and form a layer with the hydrocarbon chains of the immobilized artificial membrane, which is generally on a support matrix such as a bead.
  • Nucleic acid ligands isolated by these SELEX procedures specific to ⁇ 2 ⁇ or the I domain thereof, which also possess a desired functional activity can be identified by screening nucleic acid ligands for their ability to inhibit specific agonist-induced platelet function and/or intracellular signaling events elicited by ⁇ 2 ⁇ .
  • the desired nucleic acid ligands are not merely binding partners, but are inhibitors of the receptor signaling, it is possible to identify ligands having a desired function by assessing the effect of the ligand on cells expressing the ⁇ 2 ⁇ protein.
  • collagen binding by ⁇ 2 ⁇ in the presence or absence of a ⁇ 2 ⁇ nucleic acid ligand can be measured in these systems.
  • Ligands can also be screened for inhibition of platelet aggregation in platelet function assays such as Light Transmittance Aggregometry performed in platelet rich plasma and wash platelet preparations or Impedance Aggregometry performed in whole blood when using rat tail type 1 collagen as the agonist for activation. Additionally, ligands can also be screened for inhibition of platelet interaction with collagen coated surfaces in static conditions or in flowing whole blood, or FACS performed in platelet rich plasma or whole blood with activation of platelets by collagen followed by staining with markers of platelet activation and aggregation including anti-CD62P (P-Selectin), anti-PACl (activated GPIIbllla) or anti-fibrinogen
  • ⁇ 2 ⁇ ligands disclosed herein are preferably nucleic acid ligands, such as aptamers.
  • ⁇ 2 ⁇ ligands which specifically bind the ⁇ 2 ⁇ extracellular domain (ECD) or the ⁇ 2 ⁇ I domain (amino acid residues Serl24-Gly337; SEQ ID NO:2) are selected using the SELEX method, described in more detail below and in Example 3, then modified to increase stability, affinity for ⁇ 2 ⁇ and/or the ability to regulate ⁇ 2 ⁇ activity.
  • a ⁇ 2 ⁇ nucleic acid ligand of the present invention is comprised of an isolated nucleic acid sequence, which can be DNA or RNA, and which can be synthesized using modified ribo- or deoxyribonucleic acids. As described herein, if a base structure of RNA is utilized, the structure will include uridine (U) in lieu of thymidine (T) in the base sequence. In certain embodiments described herein, the sequence of nucleic acids is written as an RNA sequence. Similarly, in certain embodiments described herein, wherein the nucleic acid ligand is initially identified as a DNA molecule, the sequence of nucleic acids is written as a DNA sequence.
  • a sequence of nucleotides presented in text form as a DNA sequence inherently provides description of the corresponding RNA sequence, wherein thymines (T's) within the DNA sequence are replaced with uridines (U's) to get the corresponding RNA sequence of nucleotides.
  • a sequence presented in text form as a RNA sequence inherently provides description of the corresponding DNA sequence, wherein uridines (U's) within the RNA sequence are replaced with thymines (T's) to get the corresponding DNA sequence.
  • ⁇ 2 ⁇ nucleic acid ligands obtain via the SELEX method are sequenced and their sequences aligned. Alignment of the sequences will identify the number of unique sequences selected via the SELEX method as well as the frequency at which the unique sequences were obtained. Moreover, alignment of the selected sequences may show the presence of consensus sequences within the selected ligands.
  • Secondary structure prediction analysis is then performed for the unique ⁇ 2 ⁇ ligands. Secondary structure contributes to the functional nature of the ligand. As is well understood by the skilled artisan, the secondary structure can be described in terms of stem and loop structures as they occur in the molecule in a 5 ' to 3 ' direction. Consensus sequences identified via the alignment procedure described above are then identified as being present in a stem region, a loop region, or a combination of both stem and loop.
  • Mutational analysis of ⁇ 2 ⁇ ligands identified by SELEX can then be modified to further characterize the regions of the ligands that are required for binding or that affect binding affinity of the ligands for the ⁇ 2 ⁇ target protein. Such modifications include internal deletions, truncations of 3 ' and/or 5 ' ends and one or more point mutations.
  • one or more loops of a ⁇ 2 ⁇ nucleic acid ligand may be substituted with a spacer using methods known to skilled artisans.
  • the spacer can be a non-nucleotide spacer which provides a function analogous to the original loop such that the ⁇ 2 ⁇ ligand maintains its structure and function when the loop is substituted with the spacer.
  • substitution of a loop with a hexaethylene glycol spacer provided by incorporation of (9-O-dimethoxytrityl-triethylene glycol, l-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite (see FIGS. 9A-9B) into the ⁇ 2 ⁇ nucleic acid ligand may result in no loss of affinity for ⁇ 2 ⁇ . Accordingly, one having ordinary skill in the art would understand that a loop can be replaced with a variety of non-nucleotide spacers that are commercially available.
  • spacers include, but are not limited to those provided by incorporation of, 5'-0-dimethoxytrityl-l '2'dideoxyribose-3'-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 18-0- dimethoxytritylhexaethyleneglycol,l-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite; and 12-(4,4 ' -dimethoxytrityloxy)dodecyl- 1 - [(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite into the ⁇ 2 ⁇ nucleic acid ligand.
  • Efficacy of a ⁇ 2 ⁇ ligand in regulating ⁇ 2 ⁇ function or treating platelet-mediated disease depends largely upon the ability of the ligand to bind with sufficient affinity to the ⁇ 2 ⁇ protein. Accordingly, after obtaining ⁇ 2 ⁇ ligands through the SELEX process, each ligand is sequenced, and then may be characterized in terms of binding to the target molecule.
  • the binding affinity of the ligands herein with respect to the target ( ⁇ 2 ⁇ ) can be defined in terms of IQ.
  • the value of this dissociation constant can be determined directly by well-known methods, such as by radioligand binding methods described in Example 1.
  • ⁇ 2 ⁇ ligand binding properties specificity may be analyzed using competition binding or functional assays with known ⁇ 2 ⁇ binding molecules such as collagen, GFOGER (SEQ ID NO:5) or, for example, a monoclonal antibody shown to specifically bind ⁇ 2 ⁇ .
  • ⁇ 2 ⁇ binding molecules such as collagen, GFOGER (SEQ ID NO:5) or, for example, a monoclonal antibody shown to specifically bind ⁇ 2 ⁇ .
  • the O in the GFOGER (SEQ ID NO:5) sequence is understood by those skill in the art to be hydroxyproline.
  • the Kj of binding of the ligand to ⁇ 2 ⁇ can range from between about 1 nM to about 100 nM, from about 10 nM to about 50 nM or from about 20 nM to about 0.1 nM.
  • the of binding of a ligand to ⁇ 2 ⁇ is at least 2-fold, 3 -fold, 4-fold, 5 -fold or 10-fold less than the Kj of binding of the ligand to an unrelated protein or other accompanying material in the environment.
  • the unrelated protein could also be a protein having motifs related to those present in ⁇ 2 ⁇ , such as another Ig superfamily member or another protein including a collagen-binding domain or another platelet activation or adhesion receptor.
  • the binding activity of the ligand obtained and identified by the SELEX method can be further modified or enhanced using a variety of engineering methods.
  • the ligand interacts with the I domain of ⁇ 2 ⁇ .
  • the ligand can interfere with collagen binding of the ⁇ 2 ⁇ receptor.
  • the ligand can also stabilize or disrupt a conformation of the receptor, so that the receptor has a reduced capacity to interact with collagen.
  • the ligand can affect platelet activation by collagen or other ⁇ 2 ⁇ agonists.
  • the ligand can also affect platelet adhesion to collagen or collagen-related peptides.
  • the ligand can affect platelet aggregation induced by collagen or other ⁇ 2 ⁇ agonists.
  • the nucleic acid ligands described herein can function as actively reversible agents. These are agents or pharmaceutically active molecules that, after administration to a patient, can be directly controlled by the administration of a second agent. As described in more detail below, the second agent, referred to herein as a modulator, can shut off or fine-tune the pharmacologic activity of the ligand. As a result, the pharmacologic activity of the ligand can be reversed by means other than, for example, drug clearance.
  • the nucleic acid ligands to ⁇ 2 ⁇ are reversible.
  • the invention provides a method of modulating the activity of a nucleic acid ligand to ⁇ 2 ⁇ by administering a modulator of the ⁇ 2 ⁇ ligand to a host who has been administered the nucleic acid ligand.
  • Modulators of the present invention include any pharmaceutically acceptable agent that can bind to a nucleic acid ligand and modify the interaction between that ligand and its target molecule (e.g., by modifying the structure of the nucleic acid ligand) in a desired manner, or which degrades, metabolizes, cleaves, or otherwise chemically alters the nucleic acid ligand to modify its biological effect.
  • modulators of the present invention include: oligonucleotides, or analogues thereof, that are
  • nucleic acid ligand sequence complementary to at least a portion of the nucleic acid ligand sequence (including ribozymes or DNAzymes).
  • Other examples include peptide nucleic acids (PNA), morpholino nucleic acids (MNA), or locked nucleic acids (LNA); nucleic acid binding proteins or peptides; oligosaccharides; small molecules; or nucleic acid binding polymers, lipids, nanoparticle, or microsphere-based modulators.
  • PNA peptide nucleic acids
  • MNA morpholino nucleic acids
  • LNA locked nucleic acids
  • nucleic acid binding proteins or peptides oligosaccharides
  • small molecules small molecules
  • nucleic acid binding polymers lipids, nanoparticle, or microsphere-based modulators.
  • Modulators can be designed so as to bind a particular nucleic acid ligand with a high degree of specificity and a desired degree of affinity. Modulators can also be designed so that, upon binding, the structure of the ligand is modified to either a more or less active form. For example, the modulator can be designed such that upon binding to the targeted nucleic acid ligand, the secondary and/or tertiary structure of that ligand is altered whereby the ligand can no longer bind to its target molecule or binds to its target molecule with less affinity. Alternatively, the modulator can be designed so that, upon binding, the three dimensional structure of the ligand is altered so that the affinity of the ligand for its target molecule is enhanced.
  • the modulator can be designed so that, upon binding, a structural motif is modified such that affinity of the ligand is increased.
  • a ligand/modulator pair is designed such that binding of the modulator to a nucleic acid ligand molecule, which cannot bind to the target of interest, can result in production of a structural motif within the ligand which thereby allows the ligand to bind to its target molecule.
  • Modulators can also be designed to nonspecifically bind to a particular nucleic acid ligand or set of nucleic acid ligands with sufficient affinity to form a complex. Such modulators can generally associate with nucleic acids via charge-charge interactions. Such modulators can also simultaneously bind more than one nucleic acid ligand. The modulator can be designed so that, upon binding to one or more nucleic acid ligands, the structure of the nucleic acid ligand is not significantly changed from its active form, but rather, the modulator masks or sterically prevents association of the nucleic acid ligand with its target molecule.
  • Nucleotide modulators can be of any length that allows effective binding to the ligand molecule.
  • oligonucleotide modulations can range in length from about 10 nucleotides (nt) to about 30 nt, from about 10 nt to about 20 nt, or from about 15 nt.
  • the nucleotide modulators may be 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 n25, 26 nt, 27 nt, 28 nt, 29 nt or 30 nt in length.
  • nucleotide modulators having lengths greater than 30 nt.
  • a nucleic acid ligand as described herein possesses an active tertiary structure, which can be affected by formation of the appropriate stable secondary structure.
  • the rate of nucleation of initial basepair formation between the nucleic acid ligand and oligonucleotide modulator plays a significant role in the formation of the final stable duplex, and the rate of this step is greatly enhanced by targeting the
  • the free energy is ideally favorable to the formation of the intermolecular duplex with respect to formation of the existing intramolecular duplexes within the targeted nucleic acid ligand.
  • the modulators described herein of the invention are generally oligonucleotides which comprise a sequence complementary to at least a portion of the targeted nucleic acid ligand sequence.
  • the modulator oligonucleotide can comprise a sequence complementary to about 6 nt to 25 nt, 8 nt to 20 nt, or 10 nt to 15 nt of the targeted ligand.
  • the length of the modulator oligonucleotide can be readily optimized using techniques described herein and known to persons having ordinary skill in the art, taking into account the targeted ligand and the effect sought.
  • the oligonucleotide can be made with nucleotides bearing D or L stereochemistry, or a mixture thereof. Naturally occurring nucleosides are in the D configuration.
  • oligonucleotide modulators of the invention include a sequence complementary to at least a portion of a nucleic acid ligand, absolute complementarity is not required.
  • a sequence "complementary to at least a portion of an nucleic acid ligand," referred to herein, is a sequence having sufficient complementarity to be able to hybridize with the nucleic acid ligand. The ability to hybridize can depend on both the degree of complementarity and the length of the nucleic acid. Generally, the larger the hybridizing oligonucleotide, the more base mismatches with a target ligand it can contain and still form a stable duplex (or triplex as the case may, be).
  • oligonucleotides of the invention can be single- stranded DNA or RNA or chimeric mixtures or derivatives or modified versions thereof.
  • the modulators can include modifications in both the nucleic acid backbone and structure of individual nucleic acids.
  • the modulator is a nucleic acid complementary to at least one loop region in the ligand.
  • the modulator is an oligonucleotide having at least a sequence that hybridizes at
  • the modulator is an oligonucleotide that hybridizes under physiological conditions to a stem in the secondary structure of the ligand, or to a region that is a combination of at least part of a loop and at least part of a stem.
  • the modulator can be designed to disrupt or stabilize the secondary and/or tertiary structure of the nucleic acid ligand.
  • the modulator is designed to bind to a "suicide position" on the ligand and thereby disrupt the sequence of the ligand.
  • a suicide position is a single stranded portion of the ligand susceptible to enzymatic cleavage.
  • the suicide position becomes single stranded and labile upon binding of the modulator to the ligand and can enhance cleavage of the ligand by enzymes in the circulation, such as blood or liver endonuc leases.
  • the modulator binds to the ligand after which the ligand can no longer interact with its target.
  • a modulator sequence comprises at least one modified nucleotide.
  • a 2'-0-methyl and 2'-fluoro modification which can include 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0-methyl adenosine, 2'-0-methyl guanosine, 2' fluoro cytidine, or 2' fluoro uridine.
  • oligonucleotides e.g., 2'-0-methyl or 2'-fluoro oligonucleotides
  • oligonucleotides about 15 nucleotides in length can be used that are staggered by about 5 nucleotides on the ligand (e.g., oligonucleotides complementary to 1-15, 6-20, 11-25, etc. of ligand).
  • An empirical strategy can be particularly effective because the impact of the tertiary structure of the nucleic acid ligand on the efficiency of hybridization can be difficult to predict.
  • Assays described in the Examples that follow can be used to assess the ability of the different oligonucleotides to hybridize to a specific nucleic acid ligand, with particular emphasis on the molar excess of the oligonucleotide required to achieve complete binding of the nucleic acid ligand.
  • the ability of the different oligonucleotide modulators to increase the rate of dissociation of the nucleic acid ligand from, or association of the ligand with, its target molecule can also be determined by conducting standard kinetic studies using, for example, BIACORE assays.
  • Oligonucleotide modulators can be selected such that a 5-50 fold molar excess of oligonucleotide, or less, is required to modify the interaction between the ligand and its target molecule in the desired manner.
  • the targeted nucleic acid ligand can be modified so as to include a single-stranded tail (3' or 5') in order to promote association with an oligonucleotide modulator.
  • Suitable tails can comprise 1 to 20 nucleotides, 1 to 10 nucleotides, 1 to 5 nucleotides or 3 to 5 nucleotides.
  • Tails may also be modified (e.g., a 2'-0-methyl and 2'- fluoro modification, which can include 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0- methyl adenosine, 2'-0-methyl guanosine, 2' fluoro cytidine, or 2' fluoro uridine).
  • Tailed ligands can be tested in binding and bioassays (e.g., as described in the Examples that follow) to verify that addition of the single-stranded tail does not disrupt the active structure of the nucleic acid ligand.
  • a series of oligonucleotides (for example, 2'-0- methyl oligonucleotides) that can form, for example, 1, 2, 3, 4 or 5 base pairs with the tail sequence can be designed and tested for their ability to associate with the tailed ligand alone, as well as their ability to increase the rate of dissociation of the ligand from, or association of the ligand with, its target molecule.
  • Scrambled sequence controls can be employed to verify that the effects are due to duplex formation and not non-specific effects.
  • the modulator is a ribozyme or a DNAzyme.
  • Enzymatic nucleic acids act by first binding to a target RNA or DNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of a molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA or DNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA, thereby allowing for inactivation of RNA ligands.
  • Group I Introns are about 300 to >1000 nucleotides in size and require a U in the target sequence immediately 5' of the cleavage site and binds 4-6 nucleotides at the 5 '-side of the cleavage site.
  • Another class is RNaseP RNA (Ml RNA), which are about 290 to 400 nucleotides in size.
  • Ml RNA RNaseP RNA
  • a third class is Hammerhead Ribozymes, which are about 30 to 40 nucleotides in size. They require the target sequence UH (where H is not G) immediately 5' of the cleavage site and bind a variable number of nucleotides on both sides of the cleavage site.
  • a fourth class is the Hairpin Ribozymes, which are about 50 nucleotides in size. They require the target sequence GUC immediately 3' of the cleavage site and bind 4 nucleotides at the 5 '-side of the cleavage site and a variable number to the 3 '-side of the cleavage site.
  • a fifth group is Hepatitis Delta Virus (HDV) Ribozymes, which are about 60 nucleotides in size. DNAzymes are single-stranded, and cleave both RNA and DNA.
  • a general model for the DNAzyme has been proposed, and is known as the " 10-23" model. DNAzymes following the "10-23” model have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
  • the modulator itself is a nucleic acid ligand.
  • a first ligand is generated that binds to the desired therapeutic target.
  • a second ligand that binds to the first ligand is generated using the SELEX process described herein or another process, and modulates the interaction between the therapeutic ligand and the target.
  • the second ligand deactivates the effect of the first ligand.
  • the modulator is a PNA, MNA, LNA, or PCO based modulator.
  • Nucleobases of the oligonucleotide modulators of the invention can be connected via internucleobase linkages, e.g., peptidyl linkages (as in the case of peptide nucleic acids (PNAs); Nielsen et al. (1991) Science 254, 1497 and U.S. Pat. No.
  • oligonucleobases can also be Locked Nucleic Acids (LNAs). Nielsen et al, J Biomol Struct Dyn 17, 175 (1999); Petersen et al, J Mol Recognit 13, 44 (2000); Nielsen et al, Bioconjug Chem 11, 228 (2000).
  • PNAs are compounds that are analogous to oligonucleotides, but differ in composition.
  • the deoxyribose backbone of oligonucleotide is replaced with a peptide backbone.
  • Each subunit of the peptide backbone is attached to a naturally- occurring or non-naturally-occurring nucleobase.
  • PNA often has an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units.
  • the purine or pyrimidine bases are linked to each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid.
  • PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules.
  • the uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes.
  • Morpholino nucleic acids are so named because they are assembled from morpholino subunits, each of which contains one of the four genetic bases (adenine, cytosine, guanine, and thymine) linked to a 6-membered morpholine ring. Subunits of these four subunit types are joined in a specific order by non-ionic phosphorodiamidate intersubunit linkages to give a morpholino oligo.
  • LNA is a class of DNA analogues that possess some features that make it a prime candidate for modulators of the invention.
  • the LNA monomers are bi-cyclic compounds structurally similar to RNA-monomers. LNA share most of the chemical properties of DNA and RNA, it is water-soluble, can be separated by gel electrophoreses, ethanol precipitated etc (Tetrahedron, 54, 3607-3630 (1998)). However, introduction of LNA monomers into either DNA or RNA oligos results in high thermal stability of duplexes with complementary DNA or RNA, while, at the same time obeying the Watson-Crick base-pairing rules.
  • Pseudo-cyclic oligonucleobases can also be used as a modulator in the present invention (see U.S. Pat. No. 6,383,752).
  • PCOs contain two oligonucleotide segments attached through their 3 '-3' or 5 '-5' ends.
  • One of the segments (the "functional segment") of the PCO has some functionality (e.g., complementarity to a target RNA).
  • Another segment (the “protective segment”) is complementary to the 3'- or 5'-terminal end of the functional segment (depending on the end through which it is attached to the functional segment).
  • PCOs form intramolecular pseudo-cyclic structures in the absence of the target nucleic acids (e.g., RNA).
  • PCOs are more stable than conventional oligonucleotides because of the presence of 3 '-3' or 5 '-5' linkages and the formation of intramolecular pseudo-cyclic structures.
  • Pharmacokinetic, tissue distribution, and stability studies in mice suggest that PCOs have higher in vivo stability than and, pharmacokinetic and tissue distribution profiles similar to, those of PS-oligonucleotides in general, but rapid elimination from selected tissues.
  • the molecule When a fluorophore and quencher molecules are appropriately linked to the PCOs of the present invention, the molecule will fluoresce when it is in the linear configuration, but the fluorescence is quenched in the cyclic conformation. This feature can be used to screen PCO's as potential modulators.
  • the modulators are peptide-based modulators.
  • Peptide-based modulators of nucleic acid ligands represent an alternative molecular class of modulators to oligonucleotides or their analogues. This class of modulators are particularly useful if sufficiently active oligonucleotide modulators of a target nucleic acid ligand cannot be isolated due to the lack of sufficient single-stranded regions to promote nucleation between the target and the oligonucleotide modulator.
  • peptide modulators provide different bioavailabilities and pharmacokinetics than oligonucleotide modulators.
  • the modulator is a protamine (Oney et al., 2009, Nat. Med.
  • Protamines are soluble in water, are not coagulated by heat, and comprise arginine, alanine and serine (most also contain proline and valine and many contain glycine and isoleucine).
  • Modulators also include protamine variants (see e.g., Wakefield et al, J. Surg. Res. 63:280 (1996)) and modified forms of protamine, including those described in U.S. Publication No. 20040121443.
  • Other modulators include protamine fragments, such as those described in U.S. Patent No. 6,624,141 and U.S. Publication No. 20050101532.
  • Modulators also include, generally, peptides that modulate the activity of heparin, other glycosaminoglycans or
  • modulators are peptides that contain cationic-NH groups permitting stabilizing charge-charge interactions such as poly-L-lysine and poly-L-ornithine.
  • modulators of nucleic acid ligands can be isolated by appending a label to the target nucleic acid ligand and incubating together the labeled-target and bead- immobilized peptide library under conditions in which binding between some members of the library and the nucleic acid are favored.
  • the binding of the nucleic acid ligand to the specific peptide on a given bead causes the bead to be "colored" by the label on the nucleic acid ligand, and thus enable the identification of peptides able to bind the target by simple isolation of the bead.
  • the direct interaction between peptides isolated by such screening methods and the target nucleic acid ligand can be confirmed and quantified using any number of the binding assays described to identify modulators of nucleic acid ligands.
  • the ability of said peptides to modulate the activity of the target nucleic acid ligand can be confirmed by appropriate bioassays.
  • the modulators are oligosaccharide based modulators.
  • Oligosaccharides can interact with nucleic acids.
  • the antibiotic aminoglycosides are products of Streptomyces species and interact specifically with a diverse array of RNA molecules such as various ribozymes, RNA components of ribosomes, and HIV-1 's TAR and RRE sequences.
  • oligosaccharides can bind to nucleic acids and can be used to modulate the activity of nucleic acid ligands.
  • the modulator is a small molecule based modulator.
  • a small molecule that intercalates between the ligand and the target or otherwise disrupts or modifies the binding between the ligand and target can also be used as the therapeutic regulator.
  • Such small molecules can be identified by screening candidates in an assay that measures binding changes between the ligand and the target with and without the small molecule, or by using an in vivo or in vitro assay that measures the difference in biological effect of the ligand for the target with and without the small molecule. Once a small molecule is identified that exhibits the desired effect, techniques such as combinatorial approaches can be used to optimize the chemical structure for the desired regulatory effect.
  • the modulator is a nucleic acid binding polymer, lipid, nanoparticle or microsphere.
  • the modulator can be selected from the group consisting of: l,2-dioleoyl-sn-glycero-3- ethylphosphocholine (EDOPC); dilauroylethylphosphatidylcholine (EDLPC);
  • EDLPC/EDOPC pyridinium surfactants; dioleoylphosphatidyl-ethanolamine (DOPE); ( ⁇ )-N-(3-aminopropyl)-N,N-dimethyl-2,3-3 ⁇ 4z ' 5(dodecyloxy)- 1 -propanaminium bromide (GAP-DLRIE) plus the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE) (GAP-DLRIE/DOPE); ( ⁇ )-N,N-dimethyl-N-[2-(spermine carboxamido)ethyl]-2,3- bis(dioeyloxy-l-propaniminium petahydrochloride (DOSPA);
  • DOSPA dioleoylphosphatidyl-ethanolamine
  • DOSPA dioleoylphosphatidyl-ethanolamine
  • DOSPA dioleoylphosphatidyl-ethanolamine
  • DOSPA dioleoylphosphat
  • EDLPC dilauroylethylphosphatidylcholine
  • EMPC Ethyldimyristoyl phosphatidylcholine
  • DOTAP dilauroylethylphosphatidylcholine
  • DMRIE dilauroylethylphosphatidylcholine
  • DOGS dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide
  • DPES dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide
  • the modulator is selected from the group consisting of: chitosan; a chitosan derivative; 1 ,5 -dimethyl- 1 , 5 -diazaundecamethylene
  • polymethobromide polyoxyethylene/ polyoxypropylene block copolymers; poly-L- lysine; polyamidoamine (PAMAM); ⁇ -cyclodextrin-containing polycation (CDP); ⁇ - cyclodextrin-containing polycation (imidazole-containing variant) (CDP-Im);
  • polyphosphoramidate polymer (8kDa, 30kDa) (PPA-DPA 8k, PPA-DPA 30k);
  • polybrene spermine; PEG-block-PLL-dendrimers; polyethylenimine (PEI); mannose- PEI; transferin-PEI; linera-PEI (1PEI); gelatin; methacrylate/methacrylamide; poly(beta- amino esters); polyelectrolyte complexes (PEC); poly(vinalyamine) (PVA); Collagen; polypropylene imine (PPI); polyallylamine; polyvinylpyridine; aminoacetalized poly( vinyl alcohol); acrylic or methacrylic polymer; Newkome dendrimer;
  • polyphenylene dimethyldioctadecylammonium bromide (DAB);
  • the modulator is selected from chitosan and chitasan derivatives.
  • Chitosan derivatives include water soluble chitosan nanoparticles (such as described in US Patent No. 6,475,995; US Patent Application No. 2006/0013885;
  • chitosan may be used to aggregate and/or encapsulate ligands into a polyelectrolyte complex in vivo following injection into a host. This is based in part on interactions of the primary amines found on chitosan and the
  • Chitosan derivatives include trimethyl chitosan chloride (TMC), which can be synthesized at different degrees of quaternization; mono-carboxymethylated chitosan (MCC) which is a polyampholytic polymer; glutaraldehyde cross-linked derivative (CSGA); thiolated chitosan (Lee, et al. (2007) Pharm. Res. 24: 157-67); glycol chitosan (GC), a chitosan derivative conjugated with ethylene glycol (Lee, et al.
  • TMCC mono-carboxymethylated chitosan
  • CSGA glutaraldehyde cross-linked derivative
  • GC glycol chitosan
  • GC glycol chitosan
  • empty chitosan nanoparticles are generated and used as modulators.
  • Chitosan or chitosan derivatives of molecular weight range of 10,000 Da to >1, 000,000 Da may be used.
  • the chitosan is of 500,000 Da or less.
  • the chitosan is of 100,000 Da or less.
  • the compound is between 10,000 and 100,000 Da, between 10,000 and 90,000, between 10,000 and 80,000, between 20,000 and 70,0000, between 30,000 and 70,000, about 30,000, about 40,000, about 50,000 or about 60,000 Da.
  • chitosan polymers containing different degrees of deacetylated primary amines are used.
  • the different degrees of deacetylation alters the charge state of the polymer and thereby the binding properties of the polymer.
  • ligands may bind with and become trapped on the nanoparticle surface, or enter the nanoparticle and become encapsulated by ionic interactions.
  • the modulator is a polyphosphate polymer microsphere.
  • the modulator is a derivative of such a microsphere such as poly(L-lactide-co-ethyl-phosphite) or P(LAEG-EOP) and others, as described in US Patent No. 6,548,302.
  • Such polymers can be produced to contain a variety of functional groups as part of the polymeric backbone.
  • the polymers may contain quaternary amines with a positive charge at physiologic pH, such that they can complex or encapsulate one or more nucleic acids upon contact. In certain embodiments, the polymers do not contain positive charges.
  • the present invention also provides methods to identify the modulators of nucleic acid ⁇ 2 ⁇ ligands.
  • Modulators can be identified in general, through binding assays, molecular modeling, or in vivo or in vitro assays that measure the modification of biological function.
  • the binding of a modulator to a nucleic acid is determined by a gel shift assay.
  • the binding of a modulator to a nucleic acid ligand is determined by a BIACORE assay.
  • Standard binding assays can be used to identify and select modulators of the invention.
  • Non-limiting examples are gel shift assays and BIACORE assays. That is, test modulators can be contacted with the nucleic acid ligands to be targeted under test conditions or typical physiological conditions and a determination made as to whether the test modulator in fact binds the ligand. Test modulators that are found to bind the nucleic acid ligand can then be analyzed in an appropriate bioassay (which will vary depending on the ligand and its target molecule, for example platelet aggregometry tests) to determine if the test modulator can affect the biological effect caused by the ligand on its target molecule.
  • an appropriate bioassay which will vary depending on the ligand and its target molecule, for example platelet aggregometry tests
  • the Gel-Shift assay is a well-known technique used to assess binding capability. For example, a nucleic acid ligand to ⁇ 2 ⁇ is first incubated with ⁇ 2 ⁇ protein or fragment thereof, or a mixture containing the ⁇ 2 ⁇ protein or fragment, and then separated on a gel by electrophoresis Upon binding of the ligand to the protein, the complex will be larger in size and its migration will therefore be retarded relative to that of the free ligand which can be applied to a control lane in the gel in the absence of ⁇ 2 ⁇ protein.
  • the ligand can be labeled, for example, by a radioactive or non-radioactive moiety, to allow detective of the ligand-a 2 i complex within the gel.
  • the complex can then be extracted from the gel and the isolated ligand analyzed to identify ligands having the desired ⁇ 2 ⁇ binding activity.
  • Gel shift assays can also be used to screen modulators for binding nucleic acid ligands to ⁇ 2 ⁇ , as association of the modulator with the nucleic acid ligand retards the mobility of the nucleic acid ligand relative to that of the free ligand (see, for example, Rusconi et al, 2002, Nature, 419:90-94.).
  • modulators can be added to such an assay format and screened for their ability to block association of a ⁇ 2 ⁇ nucleic acid ligand with ⁇ 2 ⁇ .
  • the mixture can be incubated in the presence of increasing amounts of potential modulator.
  • a modulator with the desired activity will specifically reduce formation of the complex as detected by the Gel-Shift assay.
  • BIACORE technology is known to the skilled artisan as a reliable and valuable tool for identifying and analyzing macromolecular interactions, include polypeptide- nucleic acid interactions. Accordingly, one could use this technology to screen for or to identify nucleic acid aptamers or ligands which specifically bind the ⁇ 2 ⁇ protein or fragment thereof.
  • the BIACORE technology measures binding events on a sensor chip surface, so that an interactant attached to the surface determines the specificity of the analysis.
  • the ⁇ 2 ⁇ protein or fragment could be attached to the sensor chip surface via, for example, a histidine tag.
  • the bound ⁇ 2 ⁇ proteins are then exposed to a solution containing the potential ligand molecules. Binding of the nucleic acid ligand to the ⁇ 2 ⁇ protein gives an immediate change in the surface plasmon resonance (SPR) signal The signal is directly proportional to the mass of molecules that bind to the surface.
  • SPR surface plasmon resonance
  • the BIACORE could be used to identify or analyze modulators of the ⁇ 2 ⁇ ligands.
  • the reaction mixture to which the chip- bound ⁇ 2 ⁇ protein is exposed can contain both a known ⁇ 2 ⁇ ligand with increasing amounts of modulator and the effects determined by standard BIACORE analysis of the resultant interaction between ⁇ 2 ⁇ and its ligand.
  • oligonucleotide or analogue thereof, peptide, polypeptide, oligosaccharide or small molecule can bind to the ligand in a manner such that the interaction with the target is modified.
  • assays for example, electrophoretic mobility shift assays (EMSAs), titration calorimetry, scintillation proximity assays, sedimentation equilibrium assays using analytical ultracentrifugation (see for eg.
  • fluorescence polarization assays fluorescence anisotropy assays, fluorescence intensity assays, fluorescence resonance energy transfer (FRET) assays, nitrocellulose filter binding assays, ELISAs, ELONAs (see, for example, U.S. Pat. No. 5,789,163), RIAs, or equilibrium dialysis assays can be used to evaluate the ability of an agent to bind to a nucleic acid ligand.
  • Direct assays in which the interaction between the agent and the nucleic acid ligand is directly determined can be performed, or competition or displacement assays in which the ability of the agent to displace the ligand from its target can be performed (for example, see Green, Bell and Janjic, Biotechniques 30(5), 2001, p 1094 and U.S. Pat. No. 6,306,598).
  • a candidate modulating agent Once a candidate modulating agent is identified, its ability to modulate the activity of a nucleic acid ligand for its target can be confirmed in a bioassay.
  • binding assays can be used to verify that the agent is interacting directly with the ligand and can measure the affinity of said interaction.
  • mass spectrometry can be used for the identification of a modulator that binds to a nucleic acid ligand, the site(s) of interaction between the modulator and the nucleic acid ligand, and the relative binding affinity of agents for the ligand (see for example U.S. Pat. No. 6,329,146).
  • Such mass spectral methods can also be used for screening chemical mixtures or libraries, especially combinatorial libraries, for individual compounds that bind to a selected target ligand that can be used in as modulators of the ligand.
  • mass spectral techniques can be used to screen multiple target nucleic acid ligands simultaneously against, e.g. a combinatorial library of compounds.
  • mass spectral techniques can be used to identify interaction between a plurality of molecular species, especially "small" molecules and a molecular interaction site on a target ligand.
  • a modulator is a protein.
  • a nucleic acid ligand is linked to a biotin molecule.
  • a streptavadin or avidin is administered to bind to and reverse the effects of the ligand (see
  • Avidin is a tetrameric protein produced in the oviducts of birds, reptiles and amphibians which is deposited in the whites of their eggs.
  • Streptavidin is a tetrameric protein purified from the bacterium
  • the tetrameric protein contains four identical subunits
  • a modulator is a cationic molecule.
  • the ligand forms a guanine quartet (G-quartet or G-quadruplex) structure.
  • the molecules are metal chelating molecules.
  • the modulator is a porphyrin.
  • the compound is TMPyP4. See Joachimi, et.al. JACS 2007, 129,
  • the modulator has the ability to substantially bind to a nucleic acid ligand in solution at modulator concentrations of less than ten (10.0) micromolar (uM), one (1.0) micromolar (uM), preferably less than 0.1 uM, and more preferably less than 0.01 uM.
  • uM ten (10.0) micromolar
  • uM one (1.0) micromolar
  • IC 50 value a 50 percent reduction in target biological activity is observed by modulation in the presence of the target, and at 50% reduction is referred to herein as an IC 50 value.
  • the ligand is preferably inexpensive to synthesize, safe for use in a host, and stable in vivo.
  • Wild-type RNA and DNA oligonucleotides are typically not stable in vivo because of their susceptibility to degradation by nucleases. Resistance to nuclease degradation can be greatly increased by the incorporation of modifying groups at the 2'-position.
  • 2'-fluoro or amino groups may be incorporated into oligonucleotide pools from which ligands have been subsequently selected.
  • 2'- fluoropyrimidines were used in an in vitro transcription reaction to generate an initial oligonucleotide pool for ligand selection (see Example 1).
  • resultant ligands selected from such libraries containing 2'-hydroxyl sugars at each purine position so while more stable in vivo than a comparable RNA or DNA ligand, require additional optimization.
  • the ligands identified using the methods described herein are subsequently modified in a variety of ways to obtain a ligand which has enhanced function and stability, as well as increased feasibility for large-scale manufacturing processes.
  • the ligands and modulators can be modified or engineered to improve their desired structure, function and/or stability by a variety of means. These include, but are not limited to, substituting particular sugar residues, changing the composition and size of particular regions and/or structures in the ligand, and designing ligands that can be more effectively regulated by a modulator.
  • the design and optimization of a nucleic acid ligand involves an appreciation for the secondary structure of the ligand as well as the relationship between the secondary structure and the modulator control. Unlike conventional methods of modifying nucleic acids, the design of the ligands to a ⁇ 2 ⁇ protein may include consideration of the impact of changes to the ligand on the design of potential modulators. If a ligand is modified by truncation, for example, the corresponding modulator should be designed to control the truncated ligand.
  • the secondary structure of ligands identified through the SELEX process can be predicted by various methods known to persons having ordinary skill in the art. For example, each sequence may be analyzed using a software program such as Mfold (mfold.bioinfo.rpi.edu; see also Zuker, 2003, Nucleic Acids Res. 31 :3406-3415 and
  • ⁇ 2 ⁇ nucleic acid ligands of the present invention can be modified by varying overall ligand length as well as the lengths of the stem and loop structures.
  • ligand truncations may be generated in which a portion of the 5' and/or 3' end of a ligand is deleted from the ligand selected in the SELEX process.
  • one method used can be to heat anneal an oligonucleotide (e.g. a DNA oligonucleotide) complementary to a 5' or 3' terminal region of the ligand, then compare binding of the ligand with and without the annealed oligonucleotide.
  • This method can be performed using oligonucleotides which anneal to various lengths of the 5 ' or 3 ' ends of the ligand to determine 5 ' and 3 ' boundaries which provide a fully functional ligand.
  • the design includes decreasing the size of the ligand.
  • the size of the modulator is changed in relation to the size of the ligand.
  • guanine strings are reduced to less than four guanine, or less than three guanine, or less than two guanine or no guanines.
  • the joint effect of these changes must meet the challenge of creating a ligand that provides adequate activity but is easily neutralized by the modulator.
  • an improved ligand can also be modified so as to include a single-stranded tail (3' or 5') in order to promote association with an
  • Suitable tails can comprise 1 nt to 20 nt, preferably, 1 nt to 10 nt, 1 nt to 5 nt or 3 nt to 5 nt. It is readily understood that such tails may included modified nucleotides as described in more detail below.
  • Tailed ligands can be tested in binding and bioassays (e.g., as described below) to verify that addition of the single-stranded tail does not disrupt the active structure of the ligand.
  • a series of oligonucleotides (for example, 2'-0-methyl oligonucleotides) that can form, for example, 1 , 3 or 5 base-pairs with the tail sequence can be designed and tested for their ability to associate with the tailed ligand alone, as well as their ability to increase the rate of dissociation of the ligand from, or association of the ligand with, its target molecule.
  • Scrambled sequence controls can be employed to verify that the effects are due to duplex formation and not non-specific effects.
  • Determination of a consensus structure also facilitates engineering of ligands to identify one or more nucleotides which may enhance or decrease ligand structure and function. For example, one may more efficiently identify and test nucleotide additions, deletions and substitutions to specific stem and loop structures.
  • Knowledge of a consensus secondary structure also allows one to avoid modifications which may be detrimental to ligand structure and function. For example, certain modifications may be conserved within the consensus secondary structure, such as a 2'-fluoro within a stem or loop region. In these instances, removal of a 2'-fluoro from the stem or loop of an ligand may result in the loss of activity.
  • the ligands are nucleic acid molecules selected using the SELEX method and include truncates and substantially homologous sequences thereof.
  • a "substantially homologous" sequence is one that forms the same secondary structure by Watson-Crick base pairing within a particular molecule.
  • sequences are "substantially homologous” if they share at least 80%, 85% or more sequence identity, such as 90%>, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a specified ligand.
  • a homologous sequence can be found in any region that allows Watson- Crick binding to form the same secondary structure, regardless of sequence identity within the specific region.
  • Ligands may also be designed to have a suicide position, which allows more effective regulation by paired modulators. Upon binding of the ligand by the modulator, the suicide position becomes single stranded and labile, thereby facilitating cleavage of the ligand by enzymes naturally present in the blood, such as blood or liver endonucleases. This provides a means for effective and substantially immediate elimination of the active ligand from circulation.
  • nucleic acid ligand can increase the in vivo stability of the nucleic acid ligand or enhance or mediate the delivery of the nucleic acid ligand. Additionally, certain chemical modifications can increase the affinity of the nucleic acid ligand for its target, by stabilizing or promoting the formation of required structural elements within the nucleic acid ligand or providing additional molecular interactions with the target molecule.
  • Modifications of the ligands can include, but are not limited to, those which provide chemical groups that incorporate additional charge, polarizability,
  • Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8- position purine modifications, modifications at exocyclic amines, substitution of 4- thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like.
  • Modifications can also include 3' and 5' modifications such as capping.
  • the SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. Patent No. 5,660,985 that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. Patent No.
  • 5,580,737 describes specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH 2 ), 2'-fluoro (2'-F), and/or 2'-0-methyl (2'-OMe).
  • U.S. Patent No. 5,756,703 describes oligonucleotides containing various 2'-modified pyrimidines.
  • the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Patent Nos. 5,637,459 and 5,683,867.
  • U.S. Patent No. 5,637,459 describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH 2 ), 2 * -fiuoro (2 * -F), and/or 2 * -0-methyl (2 * -OMe).
  • the SELEX method further encompasses combining selected nucleic acid ligands with lipophilic or Non- Immunogenic, High Molecular Weight compounds in a diagnostic or therapeutic complex as described in U.S. Patent No. 6,011,020.
  • nucleic acid ligands are derived by the SELEX method
  • modifications can be pre- or post-SELEX modifications.
  • Pre-SELEX modifications can yield ligands with both specificity for its target and improved in vivo stability.
  • Post- SELEX modifications made to 2'-hydroxyl (2' -OH) nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligands.
  • the modifications of the ligand include a 3'-3' inverted phosphodiester linkage at the 3' end of the molecule, and 2' fluoro (2'-F), 2' amino (2'- NH 2 ), 2'deoxy, and/or 2' O methyl (2'-OMe) and/or 2' deoxy modification of some or all of the nucleotides.
  • the ligands described herein were initially generated via SELEX using libraries of transcripts in which the C and U residues were 2 '-fluoro substituted and the A and G residues were 2' -OH. While such modifications generate ligand molecules suitable for screening, the high 2' hydroxyl content make them unsuitable for drug development candidates due to the fact that these positions can be very sensitive to nuclease degradation in vivo, limiting the maximal concentration that can be achieved post- parenteral administration as well as their circulating half-life. Accordingly, once functional sequences are identified, such as through the SELEX method, individual residues can be tested for tolerance to substitutions by assessing the effects of these substitutions on ligand structure, function and stability.
  • the nucleic acids making up the ligand include modified sugars and/or modified bases.
  • the modifications include stabilizing modifications such as 2 '-stabilizing modifications.
  • 2'- stabilizing modifications can include 2'-fluoro, 2'deoxy or 2 '-O-methyl modifications on the sugar ring.
  • the design includes decreasing the 2'-hydroxyl content of the ligand or the modulator, or both. In another embodiment, the design includes decreasing the 2'-fluoro content of the ligand or the modulator, or both. In another embodiment, the design includes increasing the 2 '-O-methyl content of the ligand or the modulator, or both.
  • the oligonucleotide can comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-inethylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2a-thiouracil, ⁇ -D- mannosyl
  • the oligonucleotides of the presently described ligands and modulators can comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines.
  • the 2'-position of the furanose residue is substituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group.
  • the nucleic acid ligand or modulator of the invention can comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, hexose, 2'-fluororibose, 2'-0-methylribose, 2'-0-methoxyethylribose, 2'-0- propylribose, 2'-0-methylthioethylribose, 2'-0-diethylaminooxyethylribose, 2'-0-(3- aminopropyl)ribose, 2'-0-(dimethylaminopropyl)ribose, 2'-0-(methylacetamido)ribose, and 2'-0-(dimethylaminoethyloxyethyl)ribose.
  • modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose,
  • the ligand or modulator can comprise at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a
  • phosphorodithioate a phosphoramidothioate, a phosphoramidate, a phosphorodiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the ligand molecule which comprises stem and loop structures, may be further stabilized for therapeutic use by the substitution of one or more nucleic acid loop structures with a more stable loop structure.
  • FIG. 9A illustrates the starting
  • FIG. 9B illustrates the hexaethylene glycol spacer when incorporated between two nucleotides of a nucleic acid ligand.
  • the ligands can be provided in forms, such as salt forms that improve solubility or bioavailability.
  • oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from, for example, Biosearch, Applied Biosystems).
  • an automated DNA synthesizer such as are commercially available from, for example, Biosearch, Applied Biosystems.
  • ⁇ 2 ⁇ ligands can also include modifications that improve bioavailability or stability.
  • the nucleic acid ligand and/or its modulator can be covalently attached to a lipophilic compound such as cholesterol, dialkyl glycerol, or diacyl glycerol.
  • the modification is conjugation to a carrier molecule which may include, but is not limited to a hydrophilic or hydrophobic moiety.
  • a carrier molecule which may include, but is not limited to a hydrophilic or hydrophobic moiety.
  • One example is polyethylene glycol molecules conjugated to the nucleic acid sequence. Conjugation to, for example, a polymer as described below, can confine distribution to the plasma compartment and increase circulating half-life.
  • a ⁇ 2 ⁇ nucleic acid ligand or a ⁇ 2 ⁇ ligand modulator can be covalently bound or otherwise attached to a non-immunogenic, high molecular weight compound such as polyethylene glycol (PEG) or other water soluble pharmaceutically acceptable polymer including, but not limited to, polyaminoamines (PAMAM); polysaccharides such as dextran, or polyoxazo lines (POZ).
  • PEG polyethylene glycol
  • PAMAM polyaminoamines
  • POZ polyoxazo lines
  • a ⁇ 2 ⁇ nucleic acid ligand or ⁇ 2 ⁇ ligand modulator can be associated with the high molecular weight compound through covalent bonds. Where covalent attachment is employed, the high molecular weight compound may be covalently bound to a variety of positions on the ligand or modulator.
  • the ligand or the modulator can be encapsulated inside a liposome for administration to a host in need thereof.
  • the ligand or modulator is attached to polyethylene glycol (PEG).
  • PEGs polyethylene glycols
  • the ligand or modulator can attached to the PEG molecule through covalent bonds.
  • an oligonucleotide ligand or modulator can be bonded to the 5 '-thiol through a maleimide or vinyl sulfone functionality.
  • activated PEG and other activated water-soluble polymers are activated with a suitable activating group appropriate for coupling to a desired site on the therapeutic agent.
  • suitable activating group appropriate for coupling to a desired site on the therapeutic agent.
  • Representative polymeric reagents and methods for conjugating these polymers to an active agent are known in the art and further described in, e.g., Zalipsky, S., et al., "Use of Functionalized Poly(Ethylene Glycols) for Modification of
  • a water soluble polymer bearing an activated ester such as an NHS ester, e.g., mPEG- succinimidyl-a-methylbutanoate
  • an activated ester such as an NHS ester
  • mPEG- succinimidyl-a-methylbutanoate is reacted with an amine group of the active agent to thereby result in an amide linkage between the active agent and the water-soluble polymer.
  • Additional functional groups capable of reacting with reactive amino groups include, e.g., N-hydroxysuccinimidyl esters, p-nitrophenylcarbonates,
  • succinimidylcarbonates aldehydes, acetals, N-keto-piperidones, maleimides, carbonyl imidazoles, azalactones, cyclic imide thiones, isocyanates, isothiocyanates, tresyl chloride, and halogen formates, among others.
  • a plurality of ⁇ 2 ⁇ ligands or ⁇ 2 ⁇ ligand modulators can be associated with a single PEG molecule.
  • the ligands and modulators can be the same or different sequences and modifications.
  • a plurality of PEG molecules can be attached to each other.
  • one or more ⁇ 2 ⁇ ligands or ⁇ 2 ⁇ ligand modulators to the same ⁇ 2 ⁇ protein target sequence or to different ⁇ 2 ⁇ protein sequence targets can be associated with each PEG molecule.
  • multiple ligands or modulators specific for the same target are attached to PEG, there is the possibility of bringing the same targets in close proximity to each other in order to generate specific interactions between the same targets.
  • multiple ligands or modulators specific for different targets are attached to PEG, there is the possibility of bringing the distinct targets in close proximity to each other in order to generate specific interactions between the targets.
  • an amino linker such as the C6 hexylamino linker, 6-(trifluoroacetamido)hexanol (2-cyanoethyl-N,N-diisopropyl)phosphoramidite, shown in FIG. 10, can be used to add the hexylamino linker to the 5' end of the synthesized oligonucleotide.
  • Other linker phosphoramidites that may be used to add linkers to the synthesized oligonucleotides are described below:
  • the 5 '-thiol modified linker is used with PEG-maleimides, PEG-vinylsulfone, PEG- iodoacetamide and PEG-orthopyridyl-disulfide, for example.
  • the PEG can range in size from 5 to 200 KD, with typical PEGs used in pharmaceutical formulations in the 10-60 KD range. Linear chain PEGs of up to about 30 KD can be produced. For PEGs of greater than 30 KD, multiple PEGs can be attached together (multi-arm or 'branched' PEGs) to produce PEGs of the desired size.
  • the general synthesis of compounds with a branched, "mPEG2" attachment two mPEGs linked via an amino acid is described in Monfardini, et al., Bioconjugate Chem. 1995, 6:62-69. For 'branched' PEGs, i.e.
  • the PEGs or mPEGS can be linked together through an amino acid such as a lysine or they can be linked via, for example, a glycerine.
  • a glycerine For branched PEGs in which each mPEG is about 10, about 20, or about 30KD, the total mass is about 20, about 40 or about 60KD and the compound is referred to by its total mass (i.e. 40KD mPEG2 is two linked 20KD mPEGs).
  • 40KD total molecular weight PEGs that can be used as reagents in producing a PEGylated compound, include, for example, [N 2 -(monomethoxy 20K polyethylene glycol carbamoy ⁇ -N ⁇ monomethoxy 20K polyethylene glycol carbamoyl)]-lysine N-hydroxysuccinimide of the structure:
  • PEG-NHS branched PEG N-Hydroxysuccinimide
  • the branched PEGs can be linked through any appropriate reagent, such as an amino acid, and in certain embodiments
  • embodiments are linked via lysine residues or glycerine residues.
  • mPEG-SPA non-branched mPEG-Succinimidyl Propionate
  • mPEG is about 20KD or about 30KD.
  • the reactive ester is -0-CH2CH2-C02-NHS.
  • the reagents can also include a branched PEG linked through glycerol, such as the SunbrightTM series from NOF Corporation, Japan. Specific, non-limiting examples of these rea ents are:
  • the reagents can also include non-branched Succinimidyl alpha-methylbutanoate (mPEG-SMB) of the general formula: , in which mPEG is between 10 and 30KD.
  • mPEG-SMB non-branched Succinimidyl alpha-methylbutanoate
  • the reactive ester is -0-CH 2 CH 2 CH(CH 3 )-C0 2 -NHS.
  • Nektar Therapeutics catalog numbers cat#2M4K0R01.
  • PEG reagents can also include nitrophenyl carbonate linked PEGs, such as of the following structure:
  • PEGs with thiol-reactive groups that can be used with a thiol-modified linker, as
  • Branched PEGs with thiol reactive groups that can be used with a thiol-modified linker, as described above, include compounds in which the branched PEG has a total molecular weight of about 40 or 60 KD (where each mPEG is 20 or 30 KD).
  • PEG reagents can also be of the following structure:
  • PEG-maleimide pegylates thiols of the target compound in which the double bond of the maleimic ring breaks to connect with the thiol.
  • the rate of reaction is pH dependent and, in one embodiment, is carried out between pH 6 and 10, or between pH 7 and 9 or about pH 8.
  • a plurality of ⁇ 2 ⁇ ligand modulators can be associated with a single PEG molecule.
  • the modulator can be to the same or different ⁇ 2 ⁇ nucleic acid ligands.
  • a plurality of PEG molecules can be attached to each other.
  • one or more modulators to the same nucleic acid ligand or different ligands can be associated with each PEG molecule. This also results in an increase in avidity of each modulator to its target.
  • the nucleic acid ligand or its modulator can be covalently attached to a lipophilic compound such as cholesterol, dialkyl glycerol, or diacyl glycerol.
  • a lipophilic compound such as cholesterol, dialkyl glycerol, or diacyl glycerol.
  • the lipophilic compound or non-immunogenic, high molecular weight compound can be covalently bonded or associated through non-covalent interactions with a ligand or modulator(s). Attachment of the ligand or oligonucleotide modulator to lipophilic or non-immunogenic high molecular weight compounds can be done directly or with the utilization of linkers or spacers.
  • the lipophilic compound or non-immunogenic high molecular weight compound may be covalently bound to a variety of positions on the ligand or modulator, such as to an exocyclic amino group on the base, the 5-position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5 ' or 3' terminus.
  • the lipophilic compound or non-immunogenic high molecular weight compound may be attached to the ligand or modulator using, for example, a six carbon amino linker.
  • one or more phosphate groups may be included between the linker and the nucleic acid sequence.
  • Oligonucleotides of the invention can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve properties such as stability of the molecule and affinity for the intended target.
  • L is -(CH 2 )p- wherein p is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;
  • 5'Ligand3' is a ligand as described herein.
  • p is 6.
  • Methods, pharmaceutical compositions and uses of the ⁇ 2 ⁇ nucleic acid ligands described herein are provided as modulatable anti- ⁇ agents for use in disorders or treatment regimes requiring anti- ⁇ therapy.
  • the treatment is a surgical intervention.
  • the methods can include administering the ⁇ 2 ⁇ nucleic acid ligand to a host in need thereof, wherein the host is suffering from, or at risk of suffering from, an occlusive thrombotic disease or disorder of the coronary, cerebral or peripheral vascular system, an autoimmune disorder, an inflammatory disorder, a cancer or metastasis thereof, or an echovirus infection or echovirus-associated disease.
  • a method of treating symptoms of an autoimmune disorder including but not limited to, multiple sclerosis (MS).
  • MS multiple sclerosis
  • a method of treating symtpoms of an inflammatory disease including but not limited to, rheumatoid arthritis (RA) is provided.
  • RA rheumatoid arthritis
  • a method of treating symptoms of a cancer including but not limited to, lung cancer, prostate cancer, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, and colon cancer is provided. In another embodiment, a method of inhibiting or blocking metastasis of the cancer is provided.
  • a method of inhibiting or blocking an echovirus infection is provided. In another embodiment, a method of treating an echovirus-associated disease is provided.
  • the ⁇ 2 ⁇ ligand inhibits conversion of ⁇ 2 ⁇ to an active conformation.
  • a method of treating symptoms of, or preventing formation of a vascular event, in particular a thrombotic or thromboembolitic event including administering a ⁇ 2 ⁇ nucleic acid ligand of the invention to a host in need thereof.
  • the ⁇ 2 ⁇ nucleic acid ligand is provided for extended periods of time.
  • a ⁇ 2 ⁇ ligand modulator may only be used in emergency situations, for example, if treatment leads to hemorrhage, including intracranial or gastrointestinal hemorrhage.
  • the modulator is administered when emergency surgery is required for patients who have received ⁇ 2 ⁇ nucleic acid ligand treatment.
  • the modulator is administered to control the concentration of the ⁇ 2 ⁇ nucleic acid ligand and thereby the duration and intensity of treatment.
  • the ⁇ 2 ⁇ nucleic acid ligand is provided as a platelet anesthetic during a cardiopulmonary bypass procedure.
  • the ⁇ 2 ⁇ nucleic acid ligand is administered to provide a period of transition off of or on to oral anti-platelet medications, and the modulator is used to reverse the ⁇ 2 ⁇ nucleic acid ligand once therapeutic levels of the oral anti-platelet agent are established.
  • compositions can include, but are not limited to, a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • the precise nature of the composition will depend, at least in part, on the nature of the ligand and/or modulator, including any stabilizing modifications, and the route of administration.
  • Compositions containing the modulator can be designed for administration to a host who has been given a ⁇ 2 ⁇ nucleic acid ligand to allow modulation of the activity of the ligand, and thus regulate anti-platelet activity of the administered ⁇ 2 ⁇ nucleic acid ligand.
  • compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules, as liquids for oral administration; as elixirs, syrups, suppositories, gels, or in any other form used in the art, including eye drops, creams, lotions, salves, inhalants and the like.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules, as liquids for oral administration; as elixirs, syrups, suppositories, gels, or in any other form used in the art, including eye drops, creams, lotions, salves, inhalants and the like.
  • sterile formulations such as saline -based washes, by surgeons, physicians or health care workers to treat a particular area in the operating field
  • compositions comprising a ⁇ 2 ⁇ nucleic acid ligand or ⁇ 2 ⁇ ligand modulator of the present invention can be formulated at least in part by the admixture of a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier examples of such carriers and methods of formulation can be found in Remington: The Science and Practice of Pharmacy, 20 th edition (Lippincott Williams & Wilkins, 2000) and Ansel et al.,
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents, including but not limited to phosphate-buffered saline.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, EDTA, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, sodium chloride, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • compositions suitable for effective administration, such compositions will contain an effective amount of the nucleic acid ligand or modulator.
  • Such compositions can contain admixtures of more than one compound.
  • the compositions typically contain about 0.1% weight percent (wt%) to about 50 wt%, about 1 wt% to about 25 wt%, or about 5 wt% to about 20 wt% of the active agent (ligand or modulator).
  • compositions for parenteral administration including
  • subcutaneous, intramuscular or intravenous injections and infusions are provided herein.
  • aseptic suspensions and solutions are desired.
  • Isotonic preparations that generally contain suitable preservatives are employed when intravenous administration is desired.
  • the pharmaceutical compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, buffered water, saline, 0.4% saline, 0.3%> glycine, hyaluronic acid, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, buffered water, saline, 0.4% saline, 0.3%> glycine, hyaluronic acid, aqueous dextrose, glycerol, ethanol, and the like.
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • a surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • Nonionic detergents that could be included in the formulation as surfactants include, but are not limited to, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose, carboxymethyl cellulose and any of the pluronic detergents such as Pluronic F68 and/or Pluronic F127 (e.g., see Strappe et al. Eur. J. of Pharm. Biopharm., 2005, 61 : 126-133).
  • Surfactants could be present in the formulation of a protein or derivative either alone or as a mixture in different ratios.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • suitable binders include without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
  • the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • Other dispersing agents include glycerin and the like.
  • Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl ether propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
  • carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl ether propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • Active agents administered directly (e.g., alone) or in a liposomal formulation are described, for example, in U.S. Pat. No. 6,147,204.
  • the compounds of the present invention can also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amide -phenol, polyhydroxy- ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention can be coupled to soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amide -phenol, polyhydroxy- ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention can be coupled
  • biodegradable polymers useful in achieving controlled release of a drug
  • a class of biodegradable polymers useful in achieving controlled release of a drug
  • a drug for example, polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone, polyoxazolines, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • PEG polyethylene glycol
  • polylactic acid polyepsilon caprolactone
  • polyoxazolines polyhydroxy butyric acid
  • polyorthoesters polyacetals
  • polydihydro-pyrans polycyanoacrylates
  • cross-linked or amphipathic block copolymers of hydrogels for example, polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone, polyoxazolines, polyhydroxy butyric acid, polyorthoesters
  • Lipophilic compounds and non-immunogenic high molecular weight compounds with which the modulators of the invention can be formulated for use in the present invention can be prepared by any of the various techniques presently known in the art or subsequently developed.
  • they are prepared from a phospholipid, for example, distearoyl phosphatidylcholine, and may include other materials such as neutral lipids, for example, cholesterol, and also surface modifiers such as positively charged (e.g., sterylamine or aminomannose or aminomannitol derivatives of cholesterol) or negatively charged (e.g., diacetyl phosphate, phosphatidyl glycerol) compounds.
  • Multilamellar liposomes can be formed by the conventional technique, that is, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase is then added to the vessel with a swirling or vortexing motion which results in the formation of multilamellar liposome vesicles (MLVs).
  • MLVs multilamellar liposome vesicles
  • UVs Unilamellar liposome vesicles
  • UVs can then be formed by homogenization, sonication or extrusion (through filters) of MLVs.
  • UVs can be formed by detergent removal techniques.
  • the complex comprises a liposome with a targeting nucleic acid ligand(s) associated with the surface of the liposome and an encapsulated therapeutic or diagnostic agent.
  • Preformed liposomes can be modified to associate with the nucleic acid ligands.
  • a cationic liposome associates through electrostatic interactions with the nucleic acid.
  • a nucleic acid attached to a lipophilic compound, such as cholesterol can be added to preformed liposomes whereby the cholesterol becomes associated with the liposomal membrane.
  • the nucleic acid can be associated with the liposome during the formulation of the liposome.
  • a stent or medical device may be coated with a formulation comprising a ⁇ 2 ⁇ ligand or ⁇ 2 ⁇ ligand modulator according to methods known to skilled artisans.
  • kits are also envisioned.
  • the kits comprises the reagents, active agents, and materials that may be required to practice the above methods.
  • the kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of a ⁇ 2 ⁇ ligand and/or a ⁇ 2 ⁇ ligand modulator.
  • the kit may have a single container means, and/or it may have distinct container means for each compound.
  • Modes of administration of the ⁇ 2 ⁇ ligands and/or ⁇ 2 ⁇ ligand modulators of the present invention to a host include, but are not limited to, parenteral (by injection or gradual infusion over time), intravenous, intradermal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular, intraspinal, intrasternal, topical, transdermal patch, via rectal, buccal, vaginal or urethral suppository, peritoneal, percutaneous, nasal spray, surgical implant, internal surgical paint, infusion pump or via catheter.
  • the agent and carrier are administered in a slow release formulation such as an implant, bolus, microparticle, microsphere, nanoparticle or nanosphere.
  • the ⁇ 2 ⁇ nucleic acid ligand is delivered via subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps).
  • the ⁇ 2 ⁇ nucleic acid ligand is delivered via subcutaneous administration and the modulator is delivered by subcutaneous or intravenous
  • the therapeutic compositions comprising ligands and modulators of the present invention may be administered intravenously, such as by injection of a unit dose.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the host, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier or vehicle.
  • Local administration for example, to the interstitium of an affected joint, is also provided.
  • Local administration can be achieved by injection, such as from a syringe or other article of manufacture containing a injection device such as a needle.
  • the rate of administration from a syringe can be controlled by controlled pressure over desired period of time to distribute the contents of the syringe.
  • local administration can be achieved by infusion, which can be facilitated by the use of a pump or other similar device.
  • vascular tissue Representative, non-limiting approaches for topical administration to a vascular tissue are also provided and include (1) coating or impregnating a blood vessel tissue with a gel comprising a nucleic acid ligand, for delivery in vivo, e.g., by implanting the coated or impregnated vessel in place of a damaged or diseased vessel tissue segment that was removed or by-passed; (2) delivery via a catheter to a vessel in which delivery is desired; (3) pumping a composition into a vessel that is to be implanted into a patient.
  • the compounds can be introduced into cells by microinjection, or by liposome encapsulation.
  • ⁇ 2 ⁇ ligands are also provided.
  • administration of the ⁇ 2 ⁇ ligands to a subject by coating medical devices such as stents with pharmaceutical compositions containing the ligand.
  • Methods for coating to allow appropriate release and administration of the ligand are known to those having ordinary skill in the art.
  • Optimum dosing regimens for the compositions described herein can be readily established by one skilled in the art and can vary with the modulator, the patient and the effect sought.
  • the effective amount can vary according to a variety of factors such as the individual's condition, weight, sex, age and amount of nucleic acid ligand administered. Other factors include the mode of administration.
  • the compositions will be administered in dosages adjusted for body weight, e.g., dosages ranging from about 1 ⁇ g/kg body weight to about 100 mg/kg body weight. More typically, the dosages will range from about 0.1 mg/kg to about 20 mg/kg, and more typically from about 0.5 mg/kg to about 10 mg/kg, or about 1.0 to about 5.0 mg/kg, or about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 6.0 mg/kg, about 7.0 mg/kg, about 8.0 mg/kg, about 9.0 mg/kg or about 10.0 mg/kg.
  • dosages ranging from about 1 ⁇ g/kg body weight to about 100 mg/kg body weight. More typically, the dosages will range from about 0.1 mg/kg to about 20 mg/kg, and more typically from about 0.5 mg/kg to about 10 mg/kg, or about 1.0 to about 5.0 mg/kg, or about 1.0 mg/kg, about 2.0 mg/kg,
  • the dose initially provides a plasma concentration of drug about 0.002 ⁇ g/ml to about 2000 ⁇ g/ml of drug, more typically from about 2.0 ⁇ g/ml to about 400 ⁇ g/ml, and more typically from about 10 ⁇ g/ml to 200 ⁇ g/ml, or about 20 ⁇ g/ml to about 100 ⁇ g/ml drug, about 20 ⁇ g/ml, about 40 ⁇ g/ml, about 60 ⁇ g/ml, about 80 ⁇ g/ml, about 100 ⁇ g/ml, about 120 ⁇ g/ml, about 140 ⁇ g/ml, about 160 ⁇ g/ml, about 180 ⁇ g/ml, or about 200 ⁇ g/ml.
  • the ratio of modulator to ligand can be adjusted based on the desired level of inhibition of the ligand.
  • the modulator dose can be calculated based on correlation with the dose of ligand.
  • the weight-to-weight dose ratio of modulator to ligand is 1 : 1.
  • the ratio of modulator to ligand is greater than 1 : 1 such as 2: 1 or about 2: 1, 3: 1 or about 3: 1, 4: 1 or about 4: 1, 5: 1 or about 5: 1, 6: 1 or about 6: 1, 7: 1 or about 7: 1, 8: 1 or about 8: 1, 9: 1 or about 9: 1, 10: 1 or about 10: 1 or more.
  • the dose ratio of modulator to ligand is less than about 1 : 1 such as 0.9:1 or about 0.9: 1, 0.8: 1 or about 0.8: 1, 0.7: 1 or about 0.7: 1, 0.6: 1 or about 0.6:1, 0.5: 1 or about 0.5: 1, 0.45: 1 or about 0.45: 1, 0.4: 1 or about 0.4: 1, 0.35: 1 or about 0.35: 1, 0.3: 1 or about 0.3: 1, 0.25: 1 or about 0.25: 1, 0.2: 1 or about 0.2: 1, 0.15: 1 or about 0.15: 1, 0.1 : 1 or about 0.1 : 1 or less than 0.1 : 1 such as about 0.005: 1 or less.
  • the ratio is between 0.5 : 1 and 0.1 : 1 , or between 0.5 : 1 and 0.2: 1 , or between 0.5 : 1 and 0.3:1. In other embodiments, the ratio is between 1 :1 and 5:1, or between 1 : 1 and 10: 1, or between 1 : 1 and 20: 1.
  • nucleic acid ligands as disclosed herein can be administered intravenously in a single daily dose, an every other day dose, or the total daily dosage can be administered in several divided doses.
  • Ligand and/or modulator administration may be provide once per day (q.d.), twice per day (b.i.d.), three times per day (t.i.d.) or more often as needed. Thereafter, the modulator is provided by any suitable means to alter the effect of the nucleic acid ligand by administration of the modulator.
  • Nucleic acid ligands of the present invention can be administered subcutaneously twice weekly, weekly, every two weeks or monthly. In some embodiments, the ligands or modulators are administered less often than once per day. For example, ligand administration may be carried out every other day, every three days, every four days, weekly, or monthly.
  • co-administration or sequential administration of other agents can be desirable.
  • the active agents can be administered concurrently, or they each can be administered at separately staggered times.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the host to be treated, capacity of the host's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are provided.
  • the alpha2 (a2) integrin contains a -200 amino acid inserted domain referred to as the "I Domain.”
  • This protein fragment has been shown to contain the ligand binding site for collagen, as well as triple helical collagen peptide GFOGER, various snake venoms, and some inhibitory antibodies. Numerous groups have expressed recombinant human ⁇ 2 I Domain in E.coli bacteria and utilized this protein fragment to demonstrate that it is functional.
  • HT1080 cells are a human fibrosarcoma cell line known to express ⁇ 2 ⁇ 1.
  • HT1080 cells were grown in Eagle's MEM (from ATCC) + 10% FBS, passaged twice, harvested at -70% confluency, and then pelleted by standard cell biology techniques.
  • PolyA mRNA was extracted from the pelleted cells using Qiagen' s Oligotex Direct mRNA kit.
  • a QiaShredder Qiagen was utilized to homogenize the cells during the extraction process.
  • a cDNA library of HT1080 mRNA was generated with 100 ng of HT 1080 mRNA, a poly dT oligo, and Ambion's MMLV-Reverse Transcriptase following manufacturer's instructions.
  • PCR was performed on the HT1080 cDNA utilizing Pfu polymerase (Stratagene) and 5' primer CGGGATCCCCTGATTTTCAGCTCTCAGCC (SEQ ID NO: 6) and 3' primer
  • CCAAGCTTTTAACCTTCAATGCTGAAAATTTGTTCTCCTAATGTCCC (SEQ ID NO:7) by standard PCR techniques. BamHl and HindUl sites (in bold) introduced for cloning purposes in the 5' and 3' primers, respectively. A stop codon was also introduced immediately prior to the HindUl site in the 3' primer.
  • the resulting PCR was desalted with a Qiagen Qiaquick PCR spin column.
  • the cleaned PCR product was digested with BamRl and HindUl for 2.75 hours and then desalted with a second Qiagen Qiaquick PCR column.
  • pQE-80L expression vector (Qiagen) was digested with BamRl and HmdIII and gel purified by standard molecular biology techniques. The digested ⁇ 2 I Domain PCR product and digested pQE-80L vector were ligated with T4 DNA Ligase (Invitrogen) for 4 hours at room temperature and then transformed into XL-2 Ultracompetent E. coli (Stratagene/Agilent) all per manufacturer's instructions.
  • FIG 3A and FIG. 3B The complete amino acid sequence of a2 protein and the ⁇ 2 I Domain amino acid sequence to be expressed is displayed in FIG 3A and FIG. 3B (SEQ ID NO:3), respectively, and represents amino acids Serl24-Gly337.
  • Cloning into the pQE-80L expression vector results in the addition of several amino acids at the amino end of the a2 I Domain, specifically the addition of amino acids MRGSHHHHHHG (SEQ ID NO: 8).
  • the MRGSHHHHHHG (SEQ ID NO: 8) amino acid addition is for purification and cloning purposes.
  • FIG. 3C SEQ ID NO:4
  • the a2 I Domain/pQE-80L plasmids were transformed for protein expression into
  • BL21 DE3 RIPL E. coli cells (Stratagene/Agilent) per manufacturer's instructions.
  • a 25mL LB Ampicillin (LB A) culture was inoculated with a2 I Domain/pQE80-L BL21 DE3 RIPL transformants and grown overnight shaking at 37°C. Five mis of the overnight culture was used to inoculate 1L LB A media.
  • Cells were grown shaking at 37°C to OD 6 oo -0.7, at which point the ⁇ 2 I Domain protein expression was induced by the addition of IPTG to a final concentration of ImM.
  • Cells were grown for an additional 4 hours post induction, harvested by spinning at 3500rpm for 20 minutes at 4°C, and cell pellets were then frozen at -80C until purification.
  • the thawed cell pellets from 1L of culture were resuspended in a total of 25mL cold Lysis Buffer (50mM Tris pH 8.0, 200mM NaCl, 2mM Imidazole).
  • 25mL cold Lysis Buffer 50mM Tris pH 8.0, 200mM NaCl, 2mM Imidazole.
  • Ten mgs of lysozyme, lOOuL of 0.1M PMSF, and 20ul of 5mg/mL Leupeptin were added to the 25mL cell Lysis Buffer mixture and incubated on a rocker for 20 minutes at 4°C.
  • Deoxycholic acid was added to a final concentration of 0.08% and incubated on a rocker for 20 minutes at 4°C.
  • the cell lysis process was completed by the addition of 2.5mL
  • the His-a2 I Domain protein was eluted from the Ni-NTA beads by addition of
  • the resulting dialyzed His-a2 I Domain protein was determined to be -75-80% pure by gel electrophoresis.
  • the dialyzed fractions were pooled and repurified utilizing lower amounts of Ni-NTA Sepharose and higher binding stringency to drive purification to a higher purity.
  • Imidazole and NaCl were added back to the dialyzed protein to a final concentration of 2 mM and 250 mM, respectively.
  • 5.3mL Qiagen Ni-NTA Sepharose CL-6B was added to the protein and incubated for 40 minutes with rocking. The sample was spun at 1500rpm for 5 minutes and unbound material was decanted off.
  • wash Buffer 50mM Tris pH 8.0, 250mM NaCl, lOmM Imidazole
  • the Wash Buffer slurry was pelleted by spinning at 1500rm for 5 minutes and unbound material removed by decanting.
  • the Ni-NTA Sepharose was washed with an additional 35mL Wash Buffer for 20 minutes with rocking, spun at 1500rpm for 5 minutes, and then unbound material removed by decanting.
  • the Wash Buffer washing was repeated two more times with 20 minute incubations.
  • the His-a2 I Domain was eluted from the Ni-NTA beads by addition of lOmL Elution Buffer (50mM Tris pH 8.0, 150mM NaCl, 500mM Imidazole), rocking for 20 minutes, spinning 2500rpm for 5 minutes, and eluted material removed by pipetting.
  • Elution Buffer 50mM Tris pH 8.0, 150mM NaCl, 500mM Imidazole
  • the purified protein was utilized in ELISA based assays to determine its ability to recognize antibodies and collagen. To this end, the PBS (without cations) dialyzed ⁇ 2 I Domain protein was biotinylated.
  • Biotinylated ⁇ 2 I domain protein was diluted to a final concentration of 2nM in PBS (with cations) + 1% BSA. Blocked plates were incubated with ⁇ of 2nM ⁇ 2 I domain protein for 30 minutes with gentle shaking at room temperature. Unbound biotinylated ⁇ 2 I domain protein was removed by aspiration, and wells were washed 6x300 ⁇ PBS (with cations) + 0.05% Tween-20 with ⁇ 1 minute incubations prior to aspiration.
  • Streptavidin-HRP(50 ⁇ ) (R&D Systems) was diluted in lOmL PBS (with cations) + 1%BSA, and 100 ⁇ was incubated in each well for 20 minutes at room temperature with gentle shaking. Unbound Streptavidin-HRP was removed by aspiration, and wells were washed 6x300 ⁇ PBS (with cations) + 0.05%> Tween-20 with ⁇ 1 minute incubations prior to aspiration. Equal volumes of HRP substrate color reagents (R&D
  • H 2 0 2 and tetramethylbenzidine were mixed and 100 ⁇ added to each well.
  • the reaction was allowed to proceed for 20 minutes at RT with gentle shaking and stopped by the addition of 50 ⁇ 2N H 2 S0 4 .
  • the absorbance was read at 450 nm using a
  • FlexStation3 multi-purpose plate reader As shown in FIG. 4, the 2nM biotinylated ⁇ 2 I Domain protein was capable of recognizing a2 antibodies 12F1 and Gi9, and no binding was observed to either the control antibody or to BSA.
  • the ability of the biotinylated ⁇ 2 I Domain to bind collagen was determined with an ELISA assay.
  • the ⁇ 2 I Domain requires Mg 2+ cations to bind to collagen and this was verified in the assay as a control. Separate reactions were run with cations and without cations. BSA control reactions were performed in the presence of cations.
  • Unbound biotinylated ⁇ 2 I Domain protein was removed by aspiration, and wells were washed 6x300 ⁇ PBS (+/-cations as appropriate) + 0.05%> Tween-20. At this point, unbound ⁇ 2 I Domain protein has been removed and the presence of cations should be irrelevant.
  • Streptavidin-HRP(50uL) R&D Systems
  • Unbound Streptavidin-HRP was removed by aspiration, and wells were washed 6x300 ⁇ PBS (with cations) + 0.05%> Tween-20 with ⁇ 1 minutes incubations prior to aspiration.
  • Equal volumes of HRP substrate color reagents (R&D Systems) H 2 0 2 and tetramethylbenzidine were mixed and 100 ul added to each well. The reaction was allowed to proceed for 20 minutes at RT with gentle shaking and stopped by the addition of 50 ⁇ , 2N H 2 SO 4 .
  • the absorbance was read at 450 nm using a FlexStation3 multi-purpose plate reader.
  • the biotinylated I Domain protein is capable of binding to collagen in a concentration dependent manner. As expected, the removal of cations from the reaction drastically reduces its ability to bind collagen.
  • the SELEX method was used to obtain aptamers which bind the ⁇ 2 I Domain as described and illustrated in FIG. 1.
  • a starting candidate DNA library was generated by heat annealing and snap- cooling 1 nmole of template DNA oligo and 1.5 nmoles of 5' DNA primer oligo.
  • the sequence of the DNA template oligo for designing the candidate mixture are: 5'- TCTCGGATCC TCAGCGAGTC GTCTG(N 40 )CCGCA TCGTCCTCCC TA-3' (SEQ ID NO: 9) (N 4 o represents 40 contiguous nucleotides synthesized with equimolar quantities of A, T, G and C), the 5' primer oligo and 3' primer oligo are, respectively, 5'- GGGGGAATTC TAATACGACT CACTATAGGG AGGACGATGC GG-3' (SEQ ID NO: 10) (T7 promoter sequence is in bold), and 5'-TCTCGGATCC TCAGCGAGTC GTCTG-3'. (SEQ ID NO: 11) The reaction was filled in with Exo " Klenow, stopped by addition of EDTA to
  • isoamyl alcohol 24: 1.
  • the extract was desalted, concentrated, and unincorporated nucleotides removed with an Amicon 10 spin column.
  • the DNA template was utilized in a transcription reaction to generate a 2'-fluoropyrimidine starting library.
  • In vitro transcription conditions were 40 mM Tris-HCl pH 8.0, 4% PEG-8000, 12 mM MgCl 2 , 1 mM spermidine, 0.002% Triton, 5 mM DTT, lmM rGTP, lmM rATP, 3mM 2'F-CTP, 3mM 2'F-UTP, 8 ⁇ g/mL inorganic pyrophosphatase, 0.5 ⁇ DNA library, and Y639F mutant T7 polymerase.
  • TE 10 mM Tris pH 7.5, O.lmM EDTA
  • the ⁇ 2 I Domain selection started with a complex library of ⁇ 10 different 2'- fluoropyrimidine RNA sequences.
  • the complex RNA pool was precleared against a biotin-PEG6-His 6 peptide, immobilized on magnetic streptavidin beads.
  • binding buffer "E-M” consists of 20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM MgCl 2 , 0.015% BSA, and 0.001%tRNA. tRNA was included to reduce non-specific binding of the RNA pool to the ⁇ 2 I Domain protein. Protein-RNA complexes were partitioned over a 25 mm nitrocellulose disc with washing. The bound RNA was extracted off the nitrocellulose disc with incubation in PCI (25 :24: 1). Water was added and the aqueous phase extracted, followed by a chloroform:isoamyl alcohol (24: 1) extraction.
  • RNA was ethanol precipitated.
  • One quarter of the precipitated RNA was heat annealed to the 3 ' primer and reverse transcribed utilizing AMV RT.
  • the entire RT reaction was utilized in PCR with 5' and 3' primers and standard PCR conditions to generate DNA template for the next round of RNA generation. Selection conditions are outlined in FIG. 6.
  • Enrichment of the aptamer libraries for ⁇ 2 I Domain was monitored in direct binding studies utilizing radiolabeled aptamer RNA from respective rounds of SELEX and ⁇ 2 I Domain. Binding studies were performed with trace P 32 end-labeled RNA added to serial dilutions of ⁇ 2 I Domain in Binding Buffer E-M. To prepare radiolabeled RNAs for binding studies, one hundred picomoles of RNA was dephosphorylated with Bacterial Alkaline Phopshatase at 50°C for 1 hour. The reaction was
  • RNA was diluted to a final concentration of 2000 cpm ⁇ L and heat denatured at 65°C for 5 minutes. RNA and ⁇ 2 I Domain dilutions were equilibrated at 37°C prior to use.
  • RNA (5 ⁇ ) was added to varying concentrations of ⁇ 2 I Domain (15 ⁇ ) at 37°C and incubated together for 5 to 15 minutes.
  • the complexed RNA/a2 I Domain protein mixture was then loaded over a Protran BA85 nitrocellulose membrane, overlayed on a Genescreen Plus Nylon membrane, in a 96 well vacuum manifold system with washing.
  • the membranes were exposed to a phosphorimager screen, scanned, and quantitated with a Molecular Dynamics Storm 840 Phosphorimager. The fraction bound was calculated by dividing the counts on the nitrocellulose by the total counts and adjusting for the background.
  • the progression of the ⁇ 2 I Domain I selection is shown in FIG. 7.
  • the final PCR products representing anti-a 2 I Domain enriched ligand libraries from the SELEX experiments described in Example 3 are digested with the appropriate restriction enzymes, cleaned with a purification kit, and directionally cloned into linearized pUC19 vector. Bacterial colonies are streaked for single clones and 5 mL overnight cultures are inoculated from single colonies. Plasmid DNA is prepared from single colonies using Qiagen Plasmid Mini Prep kits. DNA sequences derived from the random region are analyzed by various methods, including alignment of the selected sequences to identify unique sequences as well as the frequency of each unique sequence.
  • Alignment also serves to identify conserved primary sequences shared by each of the selected clones.
  • the affinity of each of the anti-a 2 I Domain ligands for ⁇ 2 I Domain is determined by direct binding studies using radiolabeled trace ligand RNA and soluble a 2 I domain, per the binding methodology described above in Example 3.
  • the anti-a 2 I Domain ligands had a moderate affinity for the ⁇ 2 I domain in the buffer conditions the selection was performed (20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM MgCl 2 , 0.015% BSA, and 0.001%tRNA).
  • the anti-a 2 I Domain ligands exhibited very poor binding affinities to the ⁇ 2 1 domain under the desired physiological condition of 150mM NaCl.
  • Truncated compounds for several of the anti-a 2 I Domain ligands containing 5' and 3 ' required sequence boundaries as predicted using the methods described above are prepared and their affinity for ⁇ 2 1 Domain determined.
  • Ligands isolated from 2'-fluorpyrimidine/2'-hydroxypurine libraries which exhibit sufficient nuclease stability are selected for in vitro screening.
  • the high 2'-hydroxyl content make them unsuitable for drug development candidates due to the fact that these positions can be very sensitive to nuclease degradation in vivo, limiting the maximal concentration that can be achieved post parenteral administration as well as their circulating half-life.
  • optimization of the anti-a 2 I Domain ligands is performed by further stabilization of the backbone by substitution of 2'-0-methyl nucleotides for 2 '-hydroxyl nucleotides, or by substitution of 2'-deoxy nucleotides for 2'- hydroxyl nucleotides, with modification of the ligand backbone by phosphorothioate substitution as needed to preserve affinity for the a 2 I Domain while enhancing nuclease stability.
  • Capping of the 3' end of a ⁇ 2 I Domain ligand molecule is accomplished by synthesis of the ligand from a CPG-support loaded with inverted deoxythymidine, to create a 3 '-3' linkage at the 3 'end of the ligand. If a modification is well tolerated, it is then used in all synthetically produced modifications to the ⁇ 2 I Domain ligand.
  • ligands In addition to the extent of nuclease stabilization, distribution and half-life of ligands post parenteral administration is greatly impacted by their molecular weight. Conjugation of ligands to high molecular weight carriers, such as high molecular weight polyethylene glycol (PEG), limit the distribution of an ligand to mainly the plasma compartment, leading to higher C max per dose unit, and greatly limit renal filtration of the ligand, and thus greatly enhance the ligand's in vivo potency and circulating half-life.
  • PEG polyethylene glycol
  • anti-a 2 I Domain ligands are conjugated to a high molecular weight carrier to provide the greatest potential half-life with distribution mainly limited to the plasma compartment.
  • PEGylation of ligands can be achieved by conjugation of the PEG to a unique site on the ligand, added by incorporation of a site-specific linker to the ligand during synthesis. Therefore, the impact of linker addition and PEG conjugation to one or more ⁇ 2 I Domain ligands is assessed.
  • Example 7 Methods for Evaluating Antiplatelet Activity, Specificity of Activity, and Modulation of Activity of anti-a 2 1 Domain Ligands
  • CIPA Collagen-Induced Platelet Aggregation
  • PRP Platelet Rich Plasma Preparation
  • Human platelet-rich plasma is prepared from fresh whole blood collected in 60 ml syringes using 0.3 mM PPACK in saline (9: 1 blood: anticoagulant saline mix; Biomol Cat# PI1117) as an anticoagulant.
  • the blood is centrifuged at low speed centrifugation (250xg) in 50 ml conical tubes for 16 minutes.
  • the platelet rich plasma separated from the blood cells by centrifugation is removed using 10 ml serological pipettes and platelet poor plasma (PPP) is prepared from leftover blood by high speed centrifugation at 2200xg for 10 minutes.
  • the PPP is removed and saved for the light transmission aggregometry (LTA) blank.
  • Platelet aggregation in PRP is monitored using 450 of PRP (plus 25 of
  • Platelet aggregation is initiated by the addition of indicated concentrations of collagen (Rat Tail Collagen Type -1) to yield a percent aggregation between 70-90%, and the light transmission is continuously recorded for 4-6 min.
  • Human washed platelets are prepared essentially as described by Mustard et al. (1972; Br.J.Haematol 22, 193-204). Briefly, human blood is collected into one-sixth volume of acid/citrate/dextrose (ACD) buffer (85mM sodium citrate, 65mM citric acid, and 1 lOmM glucose), placed in a water bath at 37°C for 30 minutes then centrifuged at 250xg for 16 minutes at room temperature.
  • ACD acid/citrate/dextrose
  • HEPES-buffered Tyrode's solution 136.5 mM NaCl, 2.68 mM KC1, 1 mM MgCl 2 , 2 mM CaCl 2 , 12 mM NaHC0 3 , 0.43 mM NaH 2 P0 4 , 5.5 mM glucose, 5 mM HEPES pH 7.4, 0.35% bovine serum albumin
  • PKI 2 prostaglandin I 2
  • the platelet suspension is incubated in a 37°C water bath for 10 minutes, 5 ⁇ (final concentration) PGI 2 added and the mixture centrifuged at 1900xg for 8 minutes.
  • the resulting pellet is resuspended in 40 mL of HEPES -buffered Tyrode's solution containing 5 ⁇ (final concentration) PGI 2 and then incubated for 10 minutes in a 37°C water bath, and centrifuged at 1900xg for 8 minutes.
  • the pellet is resuspended at a density of 3 x 10 8 platelets/mL in HEPES-buffered
  • Collagen-induced WP platelet aggregation is determined by measuring the transmission of light through a 0.5 ml suspension of stirred (1200 rpm) washed platelets (425 ul washed platelets, 25 ⁇ fibrinogen, 25 ⁇ of inhibitors or controls and 25 ⁇ of collagen) in a lumi-aggregometer at 37°C (Chrono-Log Corp. Havertown, PA). The baseline of the instrument is set using 0.5 ml of Hepes-buffered Tyrode's solution. Prior to aggregation measurements, the platelet suspension is supplemented with 1 mg/ml fibrinogen.
  • Platelet aggregation is initiated by the addition of indicated concentrations of Collagen (Rat Tail Collagen Type-1) to yield a percent aggregation between 70-90%, and the light transmission is continuously recorded for at least 6 min.
  • Reat Tail Collagen Type-1 a concentration of Collagen
  • anti-a 2 1 Domain ligands are added to the platelet suspension at a concentration to yield the desired final concentration, and incubated for 3 min before addition of collagen, and the response is recorded for 4-6 min after collagen addition.
  • the potency of collagen is determined for each donor from the maximal extent of percentage aggregation obtained from a dose response curve using 2X serial dilution of 4 ⁇ g/ml of collagen in saline, and a challenge concentration is determined.
  • the ability of anti-a 2 1 Domain ligands to inhibit CIPA is tested in both WP and PRP preparations as described above, using a broad range of anti-a 2 I Domain ligand concentrations (2 ⁇ - 7.8 nM).
  • the collagen coated plate is then blocked by completely filling the outlet well (1ml) with PBS +5%BSA w/v and perfusing the solution into the channel at 5 dyn/cm 2 for 15 min. The flow is stopped and the plate incubated for an additional 10 minutes at room temp.
  • the blood is drawn from healthy volunteers into PPACK (0.3 mM) anticoagulant into 60 mL syringes using a 19 3/4 gauge needle.
  • the blood is immediately fluorescently labeled with 4 ⁇ Calcein-AM (Invitrogen P/N C3100 MP) for 1 hr at 37°C (Calcein-AM is added to the blood very gently by inverting the tube a few times to mix and the blood is used within 3.5 hrs of draw).
  • 20 ⁇ 1 of 40 ⁇ Sel2 pool, R7-24 or R7I-43 RNA was added to 200 ⁇ 1 of Calcein labeled blood and incubated at room temperature for 5 minutes.
  • the flow experiment is initiated by adding 200 of labeled blood on top of the outlet well and perfusion begun immediately using 5 dyn/cm 2 whole blood flow settings at 37°C using BiofluxTM software.
  • the data fluorescence images of platelet aggregates
  • the data is collected using a time lapse fluorescence inverted microscope (Zeiss 200M Axiovert Microscope attached to an Axiocam Charged-Coupled Device camera and Axiovision software) every 6 seconds for a total duration of 6 minutes.
  • the tagged image file (tiff) formatted images are used to calculate fluorescence intensity using Bioflux MontageTM software and then the data is exported to Microsoft Excel and plotted using Graphpad
  • Ligand-coated wells are blocked by incubation with 200 ⁇ of blocking buffer (0.05 M Tris-HCl, 0.14 M NaCl, 5% BSA, pH 7.4) for 30 min.
  • the wells are washed 3 times with 200 ⁇ of adhesion buffer, then 50 ⁇ of platelet suspension (10 7 platelets) are added to each well and left for 1 h.
  • the wells are emptied and washed 3 times with 200 ⁇ of adhesion buffer to remove non-adherent platelets.
  • Adherent platelets are lysed by incubation for 1 h with 150 ⁇ per well of lysis buffer (0.07 M tri-sodium citrate, 0.3 M citric acid, 0.1% Triton X-100 (v/v), 5 mM /?-nitrophenyl phosphate). The reaction is terminated by the addition of 100 ⁇ of 2 M NaOH to each well. Adhesion is measured colorimetrically as the absorbance of the /?-nitrophenol product at 405 nm in a Maxline Emax microplate reader (Molecular Devices Ltd., Crawley, UK).
  • TIP A TRAP Induced Platelet Aggregation
  • AAIPA Arachidonic Acid Induced Platelet Aggregation
  • RIP A Ristocetin Induced Platelet Aggregation
  • ADP is used as a specific agonist of the P2Yi 2 and P2Yi receptors, TRAP as an agonist of PAR- 1, Arachidonic Acid as an agonist of the thromboxane A2 (TXA2) receptor and Ristocetin as an agonist of the vWF-GPlba interaction.
  • the potency of each agonist is determined from the maximal extent of percentage aggregation obtained from a dose response curve for the respective agonist, and a challenge concentration is determined to target EC70-90 % for each respective agonist.
  • a challenge concentration of 5 ⁇ ADP is typically used to stimulate platelet aggregation, and for evaluating their potential interaction with PAR-1, a challenge concentration of 2.5 ⁇ TFLLRN (TRAP) is typically used to stimulate platelet aggregation (the specific challenge agonist concentration for each experiment is determined based on the agonist dose response curve for each donor).
  • ADP and TRAP - induced platelet aggregation is determined in WP preparations as described above.
  • a challenge concentration of 1.0-2.0 mg/mL Ristocetin (Sigma Cat# R7752) is used to stimulate platelet aggregation (the specific challenge agonist concentration for each experiment is determined based on the agonist dose response curve for each donor). Ristocetin-induced platelet aggregation is determined in PRP preparations as described above.
  • HIP1 antibody to GPlba (or isotype IgG control, Axxora Bio; 25 ⁇ g/ml final) is used as a positive control to demonstrate that inhibition of the target receptor GPlba is detectable in the assays.
  • Collagen-induced platelet aggregation was carried out in WP and PRP preparations as described above.
  • anti-a 2 1 Domain ligand concentration are tested at their IC 95 _ioo (the ligand concentration necessary to inhibit by 95-100% the aggregation elicited by a given concentration of challenge agonist).
  • an anti-a 2 I Domain ligand molecule and a modulator designed to bind the ligand molecule are added to a total volume 4 ml of WP suspension at 37°C (order and timing of addition as described above for evaluating nucleic acid modulators), 450 ⁇ , aliquots of the WP suspension mixture are removed at indicated time points (0, 0.16, 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 hrs), and collagen-induced platelet aggregation performed.

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Abstract

Compositions are provided comprising the nucleic acid ligand and/or a modulator as well as methods of using these agents and compositions in treating α2β1-mediated diseases and disorders. Also provided is a pharmacologic system comprising a nucleic acid ligand that binds to and regulates the activity of the integrin α2β1 protein. These nucleic acid ligands are also actively reversible using a modulator that inhibits the activity of the nucleic acid ligand to neutralize its pharmacologic effect and thereby restore α2β1 function.

Description

NUCLEIC ACID MODULATORS OF ALPHA2BETA1
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application 61/717,944 filed October 24, 2012 the contents of which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates, in general, to a pharmacologic system comprising a nucleic acid ligand that binds to and regulates the activity of the integrin α2βι protein. These nucleic acid ligands are also actively reversible using a modulator that inhibits the activity of the nucleic acid ligand to neutralize its pharmacologic effect and thereby restore α2βι function. The invention further relates to compositions comprising the nucleic acid ligand and/or a modulator as well as methods of using these agents and compositions in treating a2 i-mediated diseases and disorders. BACKGROUND OF THE INVENTION
Platelets are small, anuclear blood cells which are fairly quiescent under normal conditions but which respond immediately to vascular injury by adhesion, activation, aggregation, and thrombus formation. The primary function of platelets is to stop blood loss after tissue trauma and exposure of the subendothelial matrix. It is well known that damage to a blood vessel can expose extracellular matrix components to the blood, particularly von Willebrand factor (VWF), collagen, fibronectin, thrombospondin, and laminin. Interaction of platelets with these exposed molecules results in activation of the platelet cells.
While platelets have long been recognized as having a predominant role in hemostasis and thrombosis, it is becoming increasingly recognized that platelets may also play a significant role in a variety of other disorders such as inflammation, tumor growth and metastasis, and immunological host defense. Accordingly, platelet receptor proteins are attractive targets for regulation of platelet function as a means of treating or preventing platelet-mediated disorders. Integrin α2βι (also known as GPIa/IIa or VLA-2) was the first collagen receptor to be identified on platelets (Nieuwenhuis et al, 1985, Nature, 318:470-472; Santoro, 1986, Cell, 46:913-920). α2βι is also expressed on lymphocytes, fibroblasts, endothelial cells, epithelial cells, and various cancer cells (Santoro et al, 1995, Thromb. Haemost. 74:813-821).
α2βι belongs to a family of βΐ (VLA) integrins that mediate cell adhesion to extracellular matrix (ECM) proteins, such as collagen, fibronectin and laminin (Coller et al, 1989, Blood, 74: 182-192; Elices et al, 1989, Proc. Natl. Acad. Sci. USA, 86:9906- 9910). They share a common beta chain (βΐ; CD29) that is non-covalently linked to one of at least six different alpha chains (alphal-alpha6; CD49a-f).
α2βι function has been implicated in a number of disorders including arterial thrombosis and other cardiovascular disorders (Samaha et al., 2006, Arterioscler Thromb Vase Biol, 2006, 26:2588-2593), autoimmunity including Multiple Sclerosis (MS) and inflammation (McCall et al, 2008, 9: 139-149; Werr et al, 2000, Blood, 95: 1804-1809), cancer, including tumor angiogenesis and tumor cell metastasis (Alghisi et al, 2006, 13: 113-135; van Muijen et al, 1995, 130:105-122; Anastassiou et al, 2009, Cancer Biother, 10:287-292) and as a receptor for echovirus cell entry and infection (Bergelson et al, 1992, Science, 255: 1718-1720.
Therapeutic compositions targeted to the α2βι protein are described herein for the development of treatments for a variety of disorders including cardiovascular disorders, immune disorders and cancer.
SUMMARY OF THE INVENTION
Described herein are compositions directed to nucleic acid ligands which specifically bind α2βι, methods of treatments and methods of use of nucleic acid ligands which specifically bind α2βι, and modulators thereof.
In one aspect, a α2βι ligand, or a pharmaceutically acceptable salt thereof, is provided, which specifically binds the extracellular domain of the α2βι protein. In one embodiment, the α2βι ligand specifically binds the I domain of the α2βι protein.
In one embodiment, the ligand comprises an isolated nucleic acid sequence. In another embodiment, at least one nucleotide is a ribonucleotide. In another embodiment, at least one nucleotide is a deoxyribonucleic acid. In still another embodiment, the isolated nucleic acid sequence of the α2βι ligand comprises a mixture of ribonucleotides and deoxyribonucleotides.
In one embodiment, the nucleic acid α2βι ligand comprises a secondary structure comprising at least one stem and one loop.
In one embodiment, the nucleic acid α2βι ligand sequence is about 20 nucleotides (nt) to about 50 nt in length, about 20 nt to about 45 nt in length, about 20 nt to about 40 nt in length, about 20 nt to about 35 nt in length, about 20 nt to about 30 nt in length, or about 30 nt to about 35 nt in length.
In one embodiment, the nucleic acid sequence of the α2βι ligand comprises one or more ribonucleotides, deoxyribonucleotides, or a mixture both ribonucleotides and deoxyribonucleotides .
In one embodiment, one or more of the nucleotides of the nucleic acid α2βι ligand sequence is modified. In another embodiment, the one or more nucleotides comprise a modification at the 2' hydroxyl position. In another embodiment, the modification is selected from the group consisting of 2'-0-methyl and 2'-fluoro. In yet another embodiment, the one or more nucleotides is 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0-methyl adenosine or 2'-0-methyl guanosine. In still another embodiment, the one or more nucleotides is a 2' fluoro cytidine or a 2' fluoro uridine.
In one embodiment, the one or more nucleotides comprising a modification is selected from the group consisting of 5-fluorouracil, 5-fluorocytosine, 5-bromouracil, 5- bromocytosine, 5-chlorouracil, 5-chlorocytosine, 5-iodouracil, 5-iodocytosine, 5- methylcytosine, 5-methyluracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 6-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 5- methoxycytosine, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), butoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil, 5 -methyl thiouracil, 3-(3-amino-3-N carboxypropyl) uridine (acp3U), and 2,6-diaminopurine.
In one embodiment, the α2βι ligand comprises at least one modified sugar moiety.
In one embodiment, the α2βι ligand comprises at least one modified phosphate backbone.
In one embodiment, the nucleic acid α2βι ligand sequence comprises an inverted thymine at its 3 ' end.
In one embodiment, the nucleic acid α2βι ligand comprises a spacer. In another embodiment, the spacer is a glycol spacer. In another embodiment, a loop of the nucleic acid α2βι ligand comprises the glycol spacer. In still another embodiment, the glycol spacer is provided by incorporation of 9-O-Dimethoxytrityl-triethylene glycol, 1- [(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. In yet another embodiment, the glycol spacer is attached to the 3' end of a first internal nucleotide of the nucleotide α2βι ligand sequence and is attached to the 5' end of a second internal nucleotide adjacent to the first internal nucleotide of the nucleotide α2βι ligand sequence.
In one embodiment, the nucleic acid α2βι ligand comprises an aliphatic amino linker. In another embodiment, the aliphatic amino linker is attached to the 5 ' end of the nucleic acid α2βι ligand sequence. In yet another embodiment, the aliphatic amino linker is attached to the 3' end of the nucleic acid α2βι ligand sequence. In still another embodiment, the aliphatic amino linker is provided by incorporation of 6-
(trifluoroacetamino)hexanol (2-cyanoethyl-N,N-diisopropyl)phosphoramidite.
In one embodiment, the nucleic acid α2βι ligand is linked to at least one hydrophilic moiety. In another embodiment, the at least one hydrophilic moiety is a polyalkylene glycol.
In one embodiment, the α2βι ligand comprises a polyalkylene moiety attached to the 5' end and/or the 3' end of the isolated nucleic acid sequence. In another
embodiment, the polyalkylene moiety is attached via a linker. In yet another
embodiment, the linker is an aliphatic amino linker.
In one embodiment, the α2βι ligand is linked to a 40 KD polyethylene glycol (PEG) moiety using a six carbon amino linker. In a another embodiment, the six carbon amino linker is attached to the PEG moiety through an amide attachment. In a yet another embodiment, the PEG moiety is two twenty KD PEG moieties which are attached to one or more amino acids, such as lysine, which is attached via an amide bond to the six carbon amino linker.
In one embodiment, the first nucleic acid α2βι ligand comprises a
phosphorothioate linkage.
In one embodiment, the nucleic acid α2βι ligand specifically binds to α2βι (SEQ ID NO: 1). In another embodiment, the nucleic acid α2βι ligand specifically binds to the I domain of α2βι (SEQ ID NO:3).
In one embodiment, the α2βι ligand has a dissociation constant of about 20 nanomolar (nM) or less.
In one embodiment, the α2βι ligand has a dissociation constant which ranges from about 400 picomolar (pM) to about 10 nM.
In one embodiment, the α2βι ligand has a dissociation constant which ranges from about 100 pM to about 10 nM .
In one embodiment, the nucleic acid α2βι ligand inhibits binding of α2βι to collagen.
In one embodiment, binding of the nucleic acid α2βι ligand to α2βι stabilizes an active conformation of α2βι. In another embodiment, binding of the nucleic acid α2βι ligand to α2βι stabilizes an inactive conformation of α2βι. In yet another embodiment, binding of the nucleic acid α2βι ligand to α2βι inhibits interaction between α2βι and a collagen molecule.
In one embodiment, binding of the α2βι ligand to α2βι results in inhibition of, or reduction of, α2βι activity. In yet another embodiment, binding of the α2βι ligand to α2βι results in the inability of, or the reduction in ability of, α2βι to interact with collagen. In still another embodiment, binding of the α2βι ligand to α2βι expressed on the surface of a platelet results in an inhibition of, or reduction of, platelet adhesion. In still another embodiment, binding of the α2βι ligand to α2βι expressed on the surface of a platelet results in an inhibition of, or reduction of, platelet activation. In still another embodiment, binding of the α2βι ligand to α2βι expressed on the surface of a platelet results in an inhibition of, or reduction of, platelet aggregation.
In one embodiment, the α2βι ligand binds to and decreases or inhibits a function of a variant of α2βι, wherein said α2βι variant is at least 80%, 85%, 90%, 91%, 93%>, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO : 1.
In one embodiment the α2βι ligand has a dissociation constant ("Ka") for α2βι of less than about 100 micromolar (μΜ), less than about 1 μΜ, less than about 500 nanomolar (nM), less than about 100 nM, less than about 50 nM, less than about 1 nM, less than about 500 picomolar (pM), less than about 300 pM, less than about 250 pM, or less than about 200 about pM.
In a second aspect, a modulator to the α2βι ligand is provided, wherein the modulator reverses, partially or completely, the activity of a α2βι ligand.
In one embodiment, the modulator comprises an isolated nucleic acid sequence. In another embodiment, the modulator comprises a DNA sequence, an RNA sequence, a polypeptide sequence, or any combination thereof. In one embodiment, the modulator is a nucleic acid modulator comprising deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. In another embodiment the nucleic acid modulator comprises at least one modified deoxyribonucleotide and/or at least one modified ribonucleotide.
In one embodiment, the modulator consists of an oligonucleotide which is complementary to at least a portion of the α2βι nucleic acid ligand. In another embodiment, the modulator comprises an oligonucleotide which is complementary to at least a portion of the α2βι nucleic acid ligand. In another embodiment, the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a loop in the α2βι ligand. In still another embodiment, the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a stem in the α2βι ligand. In yet another embodiment, the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a stem in the α2βι ligand and to at least a portion of a loop in the α2βι ligand. In one embodiment, the modulator of a α2βι nucleic acid ligand is selected from the group consisting of a ribozyme, a DNAzyme, a peptide nucleic acid (PNA), a morpholino nucleic acid (MNA), and a locked nucleic acid (LNA).
In one embodiment, the modulator of a α2βι nucleic acid ligand is selected from the group consisting of a ribozyme, a DNAzyme, a peptide nucleic acid (PNA), a morpholino nucleic acid (MNA), and a locked nucleic acid (LNA), wherein the modulator specifically binds to or interacts with at least a portion of a α2βι nucleic acid ligand.
In one embodiment, the modulator is selected from the group consisting of a nucleic acid binding protein or peptide, a small molecule, an oligosaccharide, a nucleic acid binding lipid, a polymer, a nanoparticle, and a microsphere, wherein the modulator binds to or interacts with at least a portion of a α2βι nucleic acid ligand.
In one embodiment, the modulator is a nucleic acid modulator comprising deoxyribonucleotides, ribonucleotides, or a mixture of deoxyribonucleotides and ribonucleotides. In another embodiment the nucleic acid modulator comprises at least one modified deoxyribonucleotide and/or at least one modified ribonucleotide.
In one embodiment, the modulator is an oligonucleotide which is complementary to at least a portion of the α2βι nucleic acid ligand. In another embodiment, the modulator is an oligonucleotide which is complementary to at least a portion of a loop in the α2βι ligand.
In one embodiment, the modulator comprises an isolated nucleic acid sequence, wherein the sequence is about 10 nt to about 30 nt, about 10 nt to about 25 nt, about 10 nt to about 20 nt, about 10 nt to about 15 nt, or about 15 nt to about 20 nt in length.
In one embodiment, one or more of the nucleotides of the nucleic acid modulator sequence is modified. In another embodiment, the one or more nucleotides comprise a modification at the 2' hydroxyl position. In another embodiment, the modification is selected from the group consisting of 2'-0-methyl and 2'-fluoro. In yet another embodiment, the one or more nucleotides is 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0-methyl adenosine, 2'-0-methyl guanosine or a 2'-0-methyl thymidine. In still another embodiment, the one or more nucleotides is a 2' fluoro cytidine, a 2' fluoro uridine, a 2' fluoro adenosine or a 2'-fluoro guanosine. In one embodiment, the modification of one or more nucleotides of the nucleic acid modulator comprises a modification selected from the group consisting of 5- fluorouracil, 5-fluorocytosine, 5-bromouracil, 5-bromocytosine, 5-chlorouracil, 5- chlorocytosine, 5-iodouracil, 5-iodocytosine, 5-methylcytosine, 5-methyluracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl thiouridine, 5 -carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 6-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5 -methoxyaminomethyl-2 -thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 5- methoxycytosine, 2-methylthio-N6-isopentenyladenine, uracil oxyacetic acid (v), butoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5 -oxyacetic acid methylester, uracil oxyacetic acid (v), 5-methyl thiouracil, 3-(3-amino-3-N carboxypropyl)uridine (acp3U), and 2,6- diaminopurine.
In one embodiment, the modulator comprises as least one modified sugar moiety. In one embodiment, the modulator comprises at least one modified phosphate backbone.
In one embodiment, the modulator comprises an oligonucleotide which hybridizes at physiological conditions to a loop of the α2βι ligand. In another embodiment, the modulator comprises an oligonucleotide which hybridizes under physiological conditions to a stem of the α2βι ligand. In yet another embodiment, the modulator comprises an oligonucleotide which hybridizes under physiological conditions to at least a portion of a stem of the α2βι ligand and to at least a portion of a loop of the α2βι ligand.
In one embodiment, the modulator disrupts the secondary structure of the nucleic acid α2βι ligand. In another embodiment, the modulator stabilizes the tertiary structure of the α2βι ligand.
In one embodiment, the modulator disrupts the tertiary structure of the nucleic acid α2βι ligand. In another embodiment, the modulator stabilizes the tertiary structure of the α2βι ligand. In one embodiment, the binding of the modulator to the α2βι ligand exposes a suicide position within the α2βι ligand, thereby disrupting the secondary structure of the α2βι ligand and leading to enhanced destruction of the nucleic acid α2βι ligand by nucleases.
In one embodiment, binding of the modulator to a α2βι
Figure imgf000011_0001
complex reduces or eliminates binding of the α2βι ligand to α2βι .
In another aspect, a method of modulating the activity of a α2βι ligand is provided.
In one embodiment, a method of modulating the activity of a nucleic acid ligand to α2βι by administering a modulator of the α2βι ligand to a host who has been administered the nucleic acid α2βι ligand is provided. In one embodiment, the modulator can be a oligonucleotide modulator, or derivative thereof, and in certain embodiments, is complimentary to a portion of the nucleic acid α2βι ligand.
In a further aspect, a method of regulating α2βι function using a α2βι ligand is provided.
In one embodiment, the method for regulating α2βι function comprises administering to a host a therapeutically effective amount of a α2βι ligand. In another embodiment, the method further comprises administering a α2βι ligand modulator to the host.
In one embodiment, the method for regulating α2βι function in increasing α2βι function. In another embodiment, the method for regulating α2βι function is decreasing α2βι function.
In another aspect, a method of treating symptoms of, or ameliorating a platelet- mediated disease or disorder is provided.
In one embodiment, the method comprises administering to a host in need thereof a therapeutically effective dose of a α2βι ligand that binds to α2βι . In another embodiment, the host is diagnosed with high-risk diabetes. In still another embodiment, the host is diagnosed with a cancer at high risk of metastasis. In one embodiment, the platelet-mediated disease or disorder is selected from the group consisting of cardiovascular disorders, acute coronary syndromes, diabetes-related disorders, autoimmune inflammatory disorders, and cancer.
In one embodiment, the cardiovascular disorder is a thrombosis,
thromboembolism, or transient ischemia attack (TIA). In another embodiment, the acute coronary syndrome is due to coronary thrombosis, unstable angina or myocardial infarction. In still another embodiment, the diabetes-related disorder is diabetic retinopathy, diabetic vasculopathy, atherosclerosis, ischemic stroke, peripheral vascular disease, acute renal injury or chronic renal failure. In another embodiment, the autoimmune inflammatory disorder is multiple sclerosis, scleroderma, rheumatoid arthritis, or an inflammatory autoimmune disorder selected from the group consisting of psoriatic arthritis, reactive arthritis, inflammatory bowel disease and ankylosing spondylitis. In one embodiment, the cancer is selected from lung cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, bone cancer and liver cancer.
In one embodiment, the α2βι ligand is administered by parenteral administration, intravenous injection, intradermal delivery, intra-articular delivery, intra- synovial delivery, intrathecal, intra-arterial delivery, intracardiac delivery, intramuscular delivery, subcutaneous delivery, intraorbital delivery, intracapsular delivery, intraspinal delivery, intrasternal delivery, topical delivery, transdermal patch delivery, buccal delivery, rectal delivery, delivery via vaginal or urethral suppository, peritoneal delivery, percutaneous delivery, delivery via nasal spray, delivery via surgical implant, delivery via internal surgical paint, delivery via infusion pump or delivery via catheter.
In another aspect, a method for treating a host in need thereof by administering a α2βι ligand, wherein the α2βι ligand regulates platelet function is provided.
In one embodiment, a therapeutically effective dose of α2βι is administered.
In one embodiment, the therapeutically effective dose reduces or inhibits platelet adhesion and/or aggregation.
In one aspect, a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid α2βι ligand which binds α2βι is provided. In one aspect, a pharmaceutical composition comprising a therapeutically effective amount of a modulator, wherein the modulator regulates the activity of a nucleic acid α2βι ligand which binds α2βι, is provided.
In one embodiment, the pharmaceutical composition comprises a α2βι ligand and pharmaceutically-acceptable excipients. In another embodiment, the pharmaceutical composition is a liquid suitable for intravenous injection. In yet another embodiment, the pharmaceutical composition is a liquid or dispersion suitable for subcutaneous injection.
In one aspect, a kit comprising a therapeutically effective amount of a α2βι nucleic acid ligand and/or a modulator which regulates the activity of the α2βι nucleic acid ligand is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a schematic of the SELEX nucleic acid ligand selection process. FIG. 2 shows a full-length cDNA sequence for the α2βι gene Accession number NM 002203.3 (SEQ ID NO:l).
FIGS. 3A-C shows a full-length amino acid sequence of an α2βι protein (A) (NP 002194) (SEQ ID NO:2), the I Domain (B) (SEQ ID NO:3), and a His-tag fusion of the I Domain (C) (SEQ ID NO:4).
FIG. 4 illustrates binding by an I Domain to a2 antibodies.
FIG. 5 illustrates binding by an I Domain to collagen.
FIG. 6 provides a table listing the selection conditions for initial alpha2 I Domain ligand selection.
FIG. 7 provides a graph showing the progression of the alpha2 I Domain selection.
FIG. 8 provides a graph showing results of platelet adhesion in the presence of
R A ligand.
FIGS. 9A-B illustrate the starting phosphoramidite for a hexaethylene glycol linker used in synthesis (A), and the hexaethylene glycol spacer when incorporated between two nucleotides of a nucleic acid ligand (B).
FIGS. 10A-B shows PEG moieties which may be conjugated to a nucleic acid ligand via a linker and a configuration of a conjugated moiety. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides pharmaceutical compositions of nucleic acid ligands which bind to α2βι, modulators of the ligands, and methods of use thereof for the treatment of platelet-mediated diseases and disorders. Further provided are
pharmaceutical formulations comprising a α2βι nucleic acid ligand and/or α2βι ligand modulator.
A. Definitions
The term "about", as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass variations of ±20% or ±10%, ±5%), ±1%), or ±0.1%) from the specified amount, as such variations are appropriate to perform the disclosed method.
A "nucleic acid ligand," which may also referred to herein as a "ligand" or "aptamer," is a nucleic acid that can form a tertiary structure, which allows it to interact with a target molecule. A "α2βι nucleic acid ligand" or "α2βι ligand" or
Figure imgf000014_0001
ligand" or "nucleic acid α2βι ligand" refers to a ligand or aptamer that specifically binds to at least a portion of α2βι. The terms refer to oligonucleotides having specific binding regions that are capable of forming complexes with an intended target molecule in a physiological environment. The affinity of the binding of an ligand to a target molecule is defined in terms of the dissociation constant (Ka) of the interaction between the ligand and the target molecule. Typically, the Ka of the ligand for its target is between about InM to about 100 nM. The specificity of the binding is defined in terms of the comparative dissociation constant of the ligand for target as compared to the dissociation constant with respect to the ligand and other materials in the environment or unrelated molecules in general. Typically, the Ka for the ligand with respect to the target will be 10-fold, 50-fold, 100-fold, or 200-fold less than the K with respect to the unrelated material or accompanying material in the environment.
"Ligand modulator pair" or "ligand modulator pair" is meant to include a specified ligand to a target molecule, and a ligand modulator that changes the secondary and/or tertiary structure of the ligand so that the ligand' s interaction with its target is modulated. The modulator can be an oligonucleotide complimentary to a portion of the ligand. The modulator can change the conformation of the ligand to reduce the target binding capacity of the ligand by 10% to 100%, 20% to 100%, 25%, 40%, 50%, 60%, 70%), 80%), 90%) or 100%, or any percentage in the range between 10%> and 100%) under physiological conditions.
"Modulator," "antidote," "regulator" or "control agent" refer to any
pharmaceutically acceptable agent that can bind a ligand or aptamer as described herein and modify the interaction between that ligand and its target molecule (e.g., by modifying the structure of the ligand) in a desired manner.
"Modulate" as used herein means a lessening, an increase, or some other measurable change in activity.
"Host" refers to a mammal and includes human and non-human mammals.
Examples of host include, but are not limited to mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, horses, sheep, cows, and humans.
"Pharmaceutically acceptable," as used herein means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in humans.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate, reduce or improve a symptom to some extent) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon the potency of the nucleic acid ligand and modulator.
A "stabilized nucleic acid molecule" refers to a nucleic acid molecule that is less readily degraded in vivo (e.g., via an exonuclease or endonuclease) in comparison to a non-stabilized nucleic acid molecule. Stabilization can be a function of length and/or secondary structure and/or inclusion of chemical substitutions within the sugar of phosphate portions of the oligonucleotide backbone. Stabilization can be obtained by controlling, for example, secondary structure which can stabilize a molecule. For example, if the 3' end of a nucleic acid molecule is complementarily to an upstream region, that portion can fold back and form a "stem loop" structure which stabilizes the molecule.
The terms "binding affinity" and "binding activity" are meant to refer to the tendency of a ligand molecule to bind or not to bind to a target. The energetics of said interactions are significant in "binding activity" and "binding affinity" because they define the necessary concentrations of interacting partners, the rates at which these partners are capable of associating, and the relative concentrations of bound and free molecules in a solution. The energetics may be characterized through, among other ways, the determination of a dissociation constant, Ka.
"Treatment" or "treating" as used herein means any treatment of disease in a mammal, including: (a) protecting against the disease, that is, causing the clinical symptoms not to develop; (b) inhibiting the disease, that is, arresting, ameliorating, reducing, or suppressing the development of clinical symptoms; and/or (c) relieving the disease, that is, causing the regression of clinical symptoms. It will be understood by those skilled in the art that in human medicine, it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, as used herein the term "prophylaxis" is intended as an element of "treatment" to encompass both "preventing" and "suppressing" as defined herein. The term "protection," as used herein, is meant to include "prophylaxis."
The term "effective amount" means a dosage sufficient to provide treatment for the disorder or disease state being treated. This will vary depending on the patient, the disease and the treatment being effected.
A α2βι nucleic acid ligand "variant" as used herein encompasses variants that perform essentially the same function as a α2βι nucleic acid ligand and comprises substantially the same structure.
B. α2βι
The α2βι protein is expressed on the surface of platelets, lymphocytes, fibroblasts, endothelial cells, epithelial cells, and various cancer cells. Biological phenomena in which α2βι integrin function is essential include collagen-induced platelet aggregation, cell migration on collagen, stable adhesion and cell-dependent reorganization of collagen fibers. Such activity is plays an integral role in arterial thrombosis and other vascular disorders. α2βι has been shown to play a role in the recruitment of polymorphonuclear lymphocytes to inflamed tissue sites (Werr et al, 2000, Blood, 95: 1804-1809). In cancer biology, the α2βι integrin has been associated with an invasive cell phenotype and it can be a marker for aggressive melanoma. On the other hand, overexpression of α2βι integrin in breast cancer cells restores the normal phenotype. Clearly, α2βι is a valid target for the development of therapies for vascular disorders and inflammatory disorders, as well as cancer and metastasis.
α2βι is a heterodimer. The a2 subunit is 1,181 amino acids in length, including a signal peptide of 29 amino acids, an extracellular domain 1,102 amino acids in length, a transmembrane domain and a short cytoplasmic segment 22 amino acids in length. The a2 subunit also has a 191-amino acid insert, called the I Domain. The I Domain shares structural homology to I Domains of other a subunits, as well as the vWF A Domain.
The I Domain, spanning from Serl24 to Gly 337, has been shown to contain the binding site for collagen. The I Domain contains a cation binding site described as a metal ion-dependent adhesion site (MIDAS) motif which requires Mg2+ or Mn2+ and which is essential for collagen binding. I Domain residues shown to interact with collagen include D151, S153, S155, T221, D254 and E256 (Embsleyu et al, 2000, Cell, 101 :47-56). The I Domain has also been shown to interact with other ligands, including the picornavirus, echovirus 1 (Estavillo et al, 1999, J Biol Chem, 274:35921-35926).
Described herein are studies done to identify α2βι ligands that bind to the α2βι extracellular domain, and more specifically the I Domain, in order to generate pharmaceutical agents which can be useful in the treatment of, for example, vascular disorders, inflammatory disorders, autoimmune disorderscancer and certain types of viral infection.
C. Development Of Nucleic Acid Ligands to α2βι
Nucleic acid ligands which specifically bind the α2βι protein are identified using the SELEX method. The ligands which were initially obtained via SELEX are then fully characterized to understand the properties of the α2βι ligands. Such characterization included sequencing, sequence alignment to determine conserved sequences, secondary structure prediction, and truncations and mutation analysis to identify ligand regions most critical for the desired function of specifically binding and inhibiting α2βι. After identifying optimal ligand sequence and secondary structures, modifications were made to optimize the ligands for pharmaceutical use. Examples of these modifications include pegylation, use of a spacer within the nucleic acid ligand and selected modifications to the sugar and phosphate portion of the nucleic acid ligand. Binding assays were performed to monitor ligand function as a result of the various modifications used.
SELEX refers to the Systematic Evolution of Ligands by Exponential
Enrichment. This method allows the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. The SELEX method is described in, for example, U.S. Patent Nos. 5,475,096; and 5,270,163 (see also WO 91/19813).
The SELEX method involves selection from a mixture of candidate
oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, such as mixtures comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent No. 5,707,796 describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Patent No. 5,763,177 describes a SELEX-based method for selecting ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Patent No. 5,580,737 describes a method for identifying highly specific ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. Patent Nos. 5,567,588 and 5,861,254 describe SELEX-based methods that achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Patent No. 5,496,938, describes methods for obtaining improved ligands after the SELEX process has been performed. U.S. Patent No. 5,705,337, describes methods for covalently linking a ligand to its target.
The feasibility of identifying nucleic acid ligands to small peptides in solution was demonstrated in U.S. Patent No. 5,648,214. The ability to use affinity elution with a ligand to produce ligands that are targeted to a specific site on the target molecule is exemplified in U.S. Patent No. 5,780,228, which relates to the production of high affinity ligands binding to certain lectins. Methods of preparing nucleic acid ligands to certain tissues, which include groups of cell types, are described in U.S. Patent No. 6,127,119. The production of certain modified high affinity ligands to calf intestinal phosphatase is described in U.S. Patent No. 6,673,553. U.S. Patent No. 6,716,580 describes an automated process of identifying nucleic acid ligands that includes the use of a robotic manipulators.
SELEX can be used to generate bivalent binding that have two or more binding domains with affinity for two or more epitopes of a protein, including a receptor.
Specifically, in one embodiment, the process can be used to select for nucleic acid ligands that have affinity for two or more regions of the α2βι protein. For example, in certain embodiments, the ligand can bind to at least two portions of the C2 regions. In certain embodiments, the ligand affects dimerization of the α2βι protein, either by disrupting or stabilizing the dimeric conformation. In these embodiments, modulators can be designed to reduce binding to only one, more than one, or all epitopes that the nucleic acid ligand binds to. The modulator can, for example, interfere with binding to only a single epitope, such as a C2-1 or C2-2 region of the receptor.
Nucleic acid ligands specific to α2βι may be generated by performing SELEX against short peptides which represent the extracellular domain of the molecule, using SELEX methods as described for example in U.S. Patent No. 7,087,735. Alternatively nucleic acid ligands specific to α2βι can be isolated by performing SELEX on intact cells which express α2βι, membrane fractions enriched for the protein, on purified α2βι or its I domain, or on cell-lines specifically over-expressing the α2βι protein or its I Domain using SELEX methods as described, for example, in U.S. Patent No. 6,730,482.
Additionally, the SELEX process can be directed to isolate specific α2βι nucleic acid ligands using competitive affinity elution schemes, such as those described in U.S. Patent No. 5,780,228. For example, to isolate nucleic acid ligands specific to α2βι, elution of ligands bound to the protein could be accomplished by addition of sufficient amounts of an activator or binder of α2βι such as collagen, or the peptide GFOGER (SEQ ID NO:5) (Onley et al, JBC 2000, 275:24560-24564).
The α2βι extracellular domain or an isolated I domain can be a recombinantly expressed and purified protein used for a SELEX procedure. In certain embodiments, the α2βι nucleic acid ligand binds to the α2βι protein under physiological conditions.
Physiological conditions are typically related to the level of salts and pH of a solution. In vitro, physiological conditions are typically replicated in a buffer including 150mM NaCl, 2mM CaCl2 20mM HEPES, at a pH of about 7.4. In certain embodiments, native, typically unactivated, platelets are used as described above to screen a population of nucleic acid ligands and provide an enriched population, which contains ligands directed to proteins found on platelets. The enriched population is then used against either a stable cell line overexpressing the desired α2βι protein, or a cell line that has been transiently transfected with a recombinant DNA molecule that directs expression of the protein. The secondary screening can be accomplished either by using a modified SELEX procedure on isolated receptors from these cells or on the whole cells either through ligand competition studies or by identifying the effects on intracellular signaling pathways.
In certain embodiments, nucleic acid ligands to specific α2βι targets can be identified using an immobilized protein. In some of these embodiments, a purified protein can be linked to a solid matrix by a chemical linker. In other embodiments, membranes from cells over-expressing a oilcan be extracted using a detergent, such as an anionic detergent (e.g. cholate), to isolate a certain fraction of the proteins and the mixture coupled to an immobilized artificial membrane. Generally, it is thought that the reorganization can be accomplished by removal of the detergent, during which lipids and proteins reorganize and form a layer with the hydrocarbon chains of the immobilized artificial membrane, which is generally on a support matrix such as a bead.
Nucleic acid ligands isolated by these SELEX procedures specific to α2βι or the I domain thereof, which also possess a desired functional activity can be identified by screening nucleic acid ligands for their ability to inhibit specific agonist-induced platelet function and/or intracellular signaling events elicited by α2βι. As the desired nucleic acid ligands are not merely binding partners, but are inhibitors of the receptor signaling, it is possible to identify ligands having a desired function by assessing the effect of the ligand on cells expressing the α2βι protein.
In addition, collagen binding by α2βι in the presence or absence of a α2βι nucleic acid ligand, can be measured in these systems.
Ligands can also be screened for inhibition of platelet aggregation in platelet function assays such as Light Transmittance Aggregometry performed in platelet rich plasma and wash platelet preparations or Impedance Aggregometry performed in whole blood when using rat tail type 1 collagen as the agonist for activation. Additionally, ligands can also be screened for inhibition of platelet interaction with collagen coated surfaces in static conditions or in flowing whole blood, or FACS performed in platelet rich plasma or whole blood with activation of platelets by collagen followed by staining with markers of platelet activation and aggregation including anti-CD62P (P-Selectin), anti-PACl (activated GPIIbllla) or anti-fibrinogen
Application of any of the above-described methods, alone or in combination, will give rise to a plurality of nucleic acid ligands specific to α2βι. Upon identification of a nucleic acid ligand with the desired inhibitory properties, modulators of this ligand can be identified as described below. D. Nucleic Acid Ligands to α2βι
The α2βι ligands disclosed herein are preferably nucleic acid ligands, such as aptamers. α2βι ligands which specifically bind the α2βι extracellular domain (ECD) or the α2βι I domain (amino acid residues Serl24-Gly337; SEQ ID NO:2) are selected using the SELEX method, described in more detail below and in Example 3, then modified to increase stability, affinity for α2βι and/or the ability to regulate α2βι activity. A α2βι nucleic acid ligand of the present invention is comprised of an isolated nucleic acid sequence, which can be DNA or RNA, and which can be synthesized using modified ribo- or deoxyribonucleic acids. As described herein, if a base structure of RNA is utilized, the structure will include uridine (U) in lieu of thymidine (T) in the base sequence. In certain embodiments described herein, the sequence of nucleic acids is written as an RNA sequence. Similarly, in certain embodiments described herein, wherein the nucleic acid ligand is initially identified as a DNA molecule, the sequence of nucleic acids is written as a DNA sequence. It is understood that a sequence of nucleotides presented in text form as a DNA sequence inherently provides description of the corresponding RNA sequence, wherein thymines (T's) within the DNA sequence are replaced with uridines (U's) to get the corresponding RNA sequence of nucleotides. Similarly, it is understood that a sequence presented in text form as a RNA sequence inherently provides description of the corresponding DNA sequence, wherein uridines (U's) within the RNA sequence are replaced with thymines (T's) to get the corresponding DNA sequence.
Several α2βι nucleic acid ligands obtain via the SELEX method are sequenced and their sequences aligned. Alignment of the sequences will identify the number of unique sequences selected via the SELEX method as well as the frequency at which the unique sequences were obtained. Moreover, alignment of the selected sequences may show the presence of consensus sequences within the selected ligands.
Secondary structure prediction analysis is then performed for the unique α2βι ligands. Secondary structure contributes to the functional nature of the ligand. As is well understood by the skilled artisan, the secondary structure can be described in terms of stem and loop structures as they occur in the molecule in a 5 ' to 3 ' direction. Consensus sequences identified via the alignment procedure described above are then identified as being present in a stem region, a loop region, or a combination of both stem and loop.
Mutational analysis of α2βι ligands identified by SELEX can then be modified to further characterize the regions of the ligands that are required for binding or that affect binding affinity of the ligands for the α2βι target protein. Such modifications include internal deletions, truncations of 3 ' and/or 5 ' ends and one or more point mutations. In some embodiments, one or more loops of a α2βι nucleic acid ligand may be substituted with a spacer using methods known to skilled artisans. The spacer can be a non-nucleotide spacer which provides a function analogous to the original loop such that the α2βι ligand maintains its structure and function when the loop is substituted with the spacer. For example, substitution of a loop with a hexaethylene glycol spacer provided by incorporation of (9-O-dimethoxytrityl-triethylene glycol, l-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite (see FIGS. 9A-9B) into the α2βι nucleic acid ligand may result in no loss of affinity for α2βι. Accordingly, one having ordinary skill in the art would understand that a loop can be replaced with a variety of non-nucleotide spacers that are commercially available. Examples of such spacers include, but are not limited to those provided by incorporation of, 5'-0-dimethoxytrityl-l '2'dideoxyribose-3'-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 18-0- dimethoxytritylhexaethyleneglycol,l-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite; and 12-(4,4 ' -dimethoxytrityloxy)dodecyl- 1 - [(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite into the α2βι nucleic acid ligand.
Efficacy of a α2βι ligand in regulating α2βι function or treating platelet-mediated disease depends largely upon the ability of the ligand to bind with sufficient affinity to the α2βι protein. Accordingly, after obtaining α2βι ligands through the SELEX process, each ligand is sequenced, and then may be characterized in terms of binding to the target molecule. The binding affinity of the ligands herein with respect to the target (α2βι) can be defined in terms of IQ. The value of this dissociation constant can be determined directly by well-known methods, such as by radioligand binding methods described in Example 1. It has been observed, however, that for some small oligonucleotides, direct determination of Ka is sometimes difficult, and can lead to misleadingly high results. Under these circumstances, a competitive binding assay for the target molecule or other candidate substance can be conducted with respect to substances known to bind the target or candidate. The value of the concentration at which 50% inhibition occurs (Ki) is, under ideal conditions, equivalent to Kj. However, in no event will a Ki be less than Kj. Thus, determination of K, in the alternative, sets a maximal value for the value of Ka. Under those circumstances where technical difficulties preclude accurate measurement of IQ, measurement of K can conveniently be substituted to provide an upper limit for Kj. A value can also be used to confirm that a ligand of the present invention binds a target. In characterizing α2βι ligand binding properties, specificity may be analyzed using competition binding or functional assays with known α2βι binding molecules such as collagen, GFOGER (SEQ ID NO:5) or, for example, a monoclonal antibody shown to specifically bind α2βι. The O in the GFOGER (SEQ ID NO:5) sequence is understood by those skill in the art to be hydroxyproline.
In some embodiments, the Kj of binding of the ligand to α2βι can range from between about 1 nM to about 100 nM, from about 10 nM to about 50 nM or from about 20 nM to about 0.1 nM. In other embodiments, the of binding of a ligand to α2βι is at least 2-fold, 3 -fold, 4-fold, 5 -fold or 10-fold less than the Kj of binding of the ligand to an unrelated protein or other accompanying material in the environment. The unrelated protein could also be a protein having motifs related to those present in α2βι, such as another Ig superfamily member or another protein including a collagen-binding domain or another platelet activation or adhesion receptor.
As will be discussed in greater detail below, the binding activity of the ligand obtained and identified by the SELEX method can be further modified or enhanced using a variety of engineering methods.
In some embodiments, the ligand interacts with the I domain of α2βι. The ligand can interfere with collagen binding of the α2βι receptor. The ligand can also stabilize or disrupt a conformation of the receptor, so that the receptor has a reduced capacity to interact with collagen. The ligand can affect platelet activation by collagen or other α2βι agonists. The ligand can also affect platelet adhesion to collagen or collagen-related peptides. The ligand can affect platelet aggregation induced by collagen or other α2βι agonists.
The nucleic acid ligands described herein can function as actively reversible agents. These are agents or pharmaceutically active molecules that, after administration to a patient, can be directly controlled by the administration of a second agent. As described in more detail below, the second agent, referred to herein as a modulator, can shut off or fine-tune the pharmacologic activity of the ligand. As a result, the pharmacologic activity of the ligand can be reversed by means other than, for example, drug clearance.
E. Modulators
In some embodiments, the nucleic acid ligands to α2βι are reversible. In one aspect, the invention provides a method of modulating the activity of a nucleic acid ligand to α2βι by administering a modulator of the α2βι ligand to a host who has been administered the nucleic acid ligand.
Modulators of the present invention include any pharmaceutically acceptable agent that can bind to a nucleic acid ligand and modify the interaction between that ligand and its target molecule (e.g., by modifying the structure of the nucleic acid ligand) in a desired manner, or which degrades, metabolizes, cleaves, or otherwise chemically alters the nucleic acid ligand to modify its biological effect. Examples of modulators of the present invention include: oligonucleotides, or analogues thereof, that are
complementary to at least a portion of the nucleic acid ligand sequence (including ribozymes or DNAzymes). Other examples include peptide nucleic acids (PNA), morpholino nucleic acids (MNA), or locked nucleic acids (LNA); nucleic acid binding proteins or peptides; oligosaccharides; small molecules; or nucleic acid binding polymers, lipids, nanoparticle, or microsphere-based modulators.
Modulators can be designed so as to bind a particular nucleic acid ligand with a high degree of specificity and a desired degree of affinity. Modulators can also be designed so that, upon binding, the structure of the ligand is modified to either a more or less active form. For example, the modulator can be designed such that upon binding to the targeted nucleic acid ligand, the secondary and/or tertiary structure of that ligand is altered whereby the ligand can no longer bind to its target molecule or binds to its target molecule with less affinity. Alternatively, the modulator can be designed so that, upon binding, the three dimensional structure of the ligand is altered so that the affinity of the ligand for its target molecule is enhanced. That is, the modulator can be designed so that, upon binding, a structural motif is modified such that affinity of the ligand is increased. In another embodiment, a ligand/modulator pair is designed such that binding of the modulator to a nucleic acid ligand molecule, which cannot bind to the target of interest, can result in production of a structural motif within the ligand which thereby allows the ligand to bind to its target molecule.
Modulators can also be designed to nonspecifically bind to a particular nucleic acid ligand or set of nucleic acid ligands with sufficient affinity to form a complex. Such modulators can generally associate with nucleic acids via charge-charge interactions. Such modulators can also simultaneously bind more than one nucleic acid ligand. The modulator can be designed so that, upon binding to one or more nucleic acid ligands, the structure of the nucleic acid ligand is not significantly changed from its active form, but rather, the modulator masks or sterically prevents association of the nucleic acid ligand with its target molecule.
Nucleotide modulators can be of any length that allows effective binding to the ligand molecule. For example, oligonucleotide modulations can range in length from about 10 nucleotides (nt) to about 30 nt, from about 10 nt to about 20 nt, or from about 15 nt. The nucleotide modulators may be 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 n25, 26 nt, 27 nt, 28 nt, 29 nt or 30 nt in length. One having ordinary skill in the art can also envision nucleotide modulators having lengths greater than 30 nt.
A nucleic acid ligand as described herein possesses an active tertiary structure, which can be affected by formation of the appropriate stable secondary structure.
Therefore, while the mechanism of formation of a duplex between a complementary oligonucleotide modulator of the invention and a nucleic acid ligand is similar to formation of a duplex between two short linear oligoribonucleotides, both the rules for designing such interactions and the kinetics of formation of such a product can be impacted by the intramolecular ligand structure.
The rate of nucleation of initial basepair formation between the nucleic acid ligand and oligonucleotide modulator plays a significant role in the formation of the final stable duplex, and the rate of this step is greatly enhanced by targeting the
oligonucleotide modulator to single-stranded loops and/or single-stranded 3' or 5' tails present in the nucleic acid ligand. For the optimal formation of the intermolecular duplex to occur, the free energy is ideally favorable to the formation of the intermolecular duplex with respect to formation of the existing intramolecular duplexes within the targeted nucleic acid ligand.
The modulators described herein of the invention are generally oligonucleotides which comprise a sequence complementary to at least a portion of the targeted nucleic acid ligand sequence. For example, the modulator oligonucleotide can comprise a sequence complementary to about 6 nt to 25 nt, 8 nt to 20 nt, or 10 nt to 15 nt of the targeted ligand. The length of the modulator oligonucleotide can be readily optimized using techniques described herein and known to persons having ordinary skill in the art, taking into account the targeted ligand and the effect sought. The oligonucleotide can be made with nucleotides bearing D or L stereochemistry, or a mixture thereof. Naturally occurring nucleosides are in the D configuration.
While the oligonucleotide modulators of the invention include a sequence complementary to at least a portion of a nucleic acid ligand, absolute complementarity is not required. A sequence "complementary to at least a portion of an nucleic acid ligand," referred to herein, is a sequence having sufficient complementarity to be able to hybridize with the nucleic acid ligand. The ability to hybridize can depend on both the degree of complementarity and the length of the nucleic acid. Generally, the larger the hybridizing oligonucleotide, the more base mismatches with a target ligand it can contain and still form a stable duplex (or triplex as the case may, be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. The oligonucleotides of the invention can be single- stranded DNA or RNA or chimeric mixtures or derivatives or modified versions thereof.
The modulators can include modifications in both the nucleic acid backbone and structure of individual nucleic acids. In certain embodiments, the modulator is a nucleic acid complementary to at least one loop region in the ligand. In some embodiments, the modulator is an oligonucleotide having at least a sequence that hybridizes at
physiological conditions to at least one loop in the two dimensional structure of the ligand. In other embodiments, the modulator is an oligonucleotide that hybridizes under physiological conditions to a stem in the secondary structure of the ligand, or to a region that is a combination of at least part of a loop and at least part of a stem. Depending on the desired function of the modulator, the modulator can be designed to disrupt or stabilize the secondary and/or tertiary structure of the nucleic acid ligand.
In some embodiments, the modulator is designed to bind to a "suicide position" on the ligand and thereby disrupt the sequence of the ligand. A suicide position is a single stranded portion of the ligand susceptible to enzymatic cleavage. In one exemplary embodiment, the suicide position becomes single stranded and labile upon binding of the modulator to the ligand and can enhance cleavage of the ligand by enzymes in the circulation, such as blood or liver endonuc leases. In certain
embodiments, the modulator binds to the ligand after which the ligand can no longer interact with its target.
In some embodiments, a modulator sequence comprises at least one modified nucleotide. For example, a 2'-0-methyl and 2'-fluoro modification, which can include 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0-methyl adenosine, 2'-0-methyl guanosine, 2' fluoro cytidine, or 2' fluoro uridine.
Various strategies can be used to determine the optimal site within a nucleic acid ligand for binding by an oligonucleotide modulator. An empirical strategy can be used in which complimentary oligonucleotides are "walked" around the nucleic acid ligand. In accordance with this approach, oligonucleotides (e.g., 2'-0-methyl or 2'-fluoro oligonucleotides) about 15 nucleotides in length can be used that are staggered by about 5 nucleotides on the ligand (e.g., oligonucleotides complementary to 1-15, 6-20, 11-25, etc. of ligand). An empirical strategy can be particularly effective because the impact of the tertiary structure of the nucleic acid ligand on the efficiency of hybridization can be difficult to predict.
Assays described in the Examples that follow can be used to assess the ability of the different oligonucleotides to hybridize to a specific nucleic acid ligand, with particular emphasis on the molar excess of the oligonucleotide required to achieve complete binding of the nucleic acid ligand. The ability of the different oligonucleotide modulators to increase the rate of dissociation of the nucleic acid ligand from, or association of the ligand with, its target molecule can also be determined by conducting standard kinetic studies using, for example, BIACORE assays. Oligonucleotide modulators can be selected such that a 5-50 fold molar excess of oligonucleotide, or less, is required to modify the interaction between the ligand and its target molecule in the desired manner.
Alternatively, the targeted nucleic acid ligand can be modified so as to include a single-stranded tail (3' or 5') in order to promote association with an oligonucleotide modulator. Suitable tails can comprise 1 to 20 nucleotides, 1 to 10 nucleotides, 1 to 5 nucleotides or 3 to 5 nucleotides. Tails may also be modified (e.g., a 2'-0-methyl and 2'- fluoro modification, which can include 2'-0-methyl cytosine, 2'-0-methyl uridine, 2'-0- methyl adenosine, 2'-0-methyl guanosine, 2' fluoro cytidine, or 2' fluoro uridine).
Tailed ligands can be tested in binding and bioassays (e.g., as described in the Examples that follow) to verify that addition of the single-stranded tail does not disrupt the active structure of the nucleic acid ligand. A series of oligonucleotides (for example, 2'-0- methyl oligonucleotides) that can form, for example, 1, 2, 3, 4 or 5 base pairs with the tail sequence can be designed and tested for their ability to associate with the tailed ligand alone, as well as their ability to increase the rate of dissociation of the ligand from, or association of the ligand with, its target molecule. Scrambled sequence controls can be employed to verify that the effects are due to duplex formation and not non-specific effects.
In another embodiment, the modulator is a ribozyme or a DNAzyme. Enzymatic nucleic acids act by first binding to a target RNA or DNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of a molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA or DNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA, thereby allowing for inactivation of RNA ligands. There are at least five classes of ribozymes that each display a different type of specificity. For example, Group I Introns are about 300 to >1000 nucleotides in size and require a U in the target sequence immediately 5' of the cleavage site and binds 4-6 nucleotides at the 5 '-side of the cleavage site. Another class is RNaseP RNA (Ml RNA), which are about 290 to 400 nucleotides in size. A third class is Hammerhead Ribozymes, which are about 30 to 40 nucleotides in size. They require the target sequence UH (where H is not G) immediately 5' of the cleavage site and bind a variable number of nucleotides on both sides of the cleavage site. A fourth class is the Hairpin Ribozymes, which are about 50 nucleotides in size. They require the target sequence GUC immediately 3' of the cleavage site and bind 4 nucleotides at the 5 '-side of the cleavage site and a variable number to the 3 '-side of the cleavage site. A fifth group is Hepatitis Delta Virus (HDV) Ribozymes, which are about 60 nucleotides in size. DNAzymes are single-stranded, and cleave both RNA and DNA. A general model for the DNAzyme has been proposed, and is known as the " 10-23" model. DNAzymes following the "10-23" model have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
In another embodiment, the modulator itself is a nucleic acid ligand. In this embodiment, a first ligand is generated that binds to the desired therapeutic target. In a second step, a second ligand that binds to the first ligand is generated using the SELEX process described herein or another process, and modulates the interaction between the therapeutic ligand and the target. In one embodiment, the second ligand deactivates the effect of the first ligand.
In another exemplary embodiment, the modulator is a PNA, MNA, LNA, or PCO based modulator. Nucleobases of the oligonucleotide modulators of the invention can be connected via internucleobase linkages, e.g., peptidyl linkages (as in the case of peptide nucleic acids (PNAs); Nielsen et al. (1991) Science 254, 1497 and U.S. Pat. No.
5,539,082) and morpholino linkages (Qin et al., Antisense Nucleic Acid Drug Dev. 10, 11 (2000); Summerton, Antisense Nucleic Acid Drug Dev. 7, 187 (1997); Summerton et al, Antisense Nucleic Acid Drug Dev. 7, 63 (1997); Taylor et al, J Biol Chem. 271, 17445 (1996); Partridge et al, Antisense Nucleic Acid Drug Dev. 6, 169 (1996)), or by any other natural or modified linkage. The oligonucleobases can also be Locked Nucleic Acids (LNAs). Nielsen et al, J Biomol Struct Dyn 17, 175 (1999); Petersen et al, J Mol Recognit 13, 44 (2000); Nielsen et al, Bioconjug Chem 11, 228 (2000).
PNAs are compounds that are analogous to oligonucleotides, but differ in composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced with a peptide backbone. Each subunit of the peptide backbone is attached to a naturally- occurring or non-naturally-occurring nucleobase. PNA often has an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units. The purine or pyrimidine bases are linked to each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid. PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules. The uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes.
Morpholino nucleic acids are so named because they are assembled from morpholino subunits, each of which contains one of the four genetic bases (adenine, cytosine, guanine, and thymine) linked to a 6-membered morpholine ring. Subunits of these four subunit types are joined in a specific order by non-ionic phosphorodiamidate intersubunit linkages to give a morpholino oligo.
LNA is a class of DNA analogues that possess some features that make it a prime candidate for modulators of the invention. The LNA monomers are bi-cyclic compounds structurally similar to RNA-monomers. LNA share most of the chemical properties of DNA and RNA, it is water-soluble, can be separated by gel electrophoreses, ethanol precipitated etc (Tetrahedron, 54, 3607-3630 (1998)). However, introduction of LNA monomers into either DNA or RNA oligos results in high thermal stability of duplexes with complementary DNA or RNA, while, at the same time obeying the Watson-Crick base-pairing rules.
Pseudo-cyclic oligonucleobases (PCOs) can also be used as a modulator in the present invention (see U.S. Pat. No. 6,383,752). PCOs contain two oligonucleotide segments attached through their 3 '-3' or 5 '-5' ends. One of the segments (the "functional segment") of the PCO has some functionality (e.g., complementarity to a target RNA). Another segment (the "protective segment") is complementary to the 3'- or 5'-terminal end of the functional segment (depending on the end through which it is attached to the functional segment). As a result of complementarity between the functional and protective segment segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target nucleic acids (e.g., RNA). PCOs are more stable than conventional oligonucleotides because of the presence of 3 '-3' or 5 '-5' linkages and the formation of intramolecular pseudo-cyclic structures. Pharmacokinetic, tissue distribution, and stability studies in mice suggest that PCOs have higher in vivo stability than and, pharmacokinetic and tissue distribution profiles similar to, those of PS-oligonucleotides in general, but rapid elimination from selected tissues. When a fluorophore and quencher molecules are appropriately linked to the PCOs of the present invention, the molecule will fluoresce when it is in the linear configuration, but the fluorescence is quenched in the cyclic conformation. This feature can be used to screen PCO's as potential modulators.
In another exemplary embodiment, the modulators are peptide-based modulators. Peptide-based modulators of nucleic acid ligands represent an alternative molecular class of modulators to oligonucleotides or their analogues. This class of modulators are particularly useful if sufficiently active oligonucleotide modulators of a target nucleic acid ligand cannot be isolated due to the lack of sufficient single-stranded regions to promote nucleation between the target and the oligonucleotide modulator. In addition, peptide modulators provide different bioavailabilities and pharmacokinetics than oligonucleotide modulators. In one exemplary embodiment the modulator is a protamine (Oney et al., 2009, Nat. Med. 15: 1224-1228). Protamines are soluble in water, are not coagulated by heat, and comprise arginine, alanine and serine (most also contain proline and valine and many contain glycine and isoleucine). Modulators also include protamine variants (see e.g., Wakefield et al, J. Surg. Res. 63:280 (1996)) and modified forms of protamine, including those described in U.S. Publication No. 20040121443. Other modulators include protamine fragments, such as those described in U.S. Patent No. 6,624,141 and U.S. Publication No. 20050101532. Modulators also include, generally, peptides that modulate the activity of heparin, other glycosaminoglycans or
proteoglycans (see, for example, U.S. Patent No. 5,919,761). In one exemplary embodiment, modulators are peptides that contain cationic-NH groups permitting stabilizing charge-charge interactions such as poly-L-lysine and poly-L-ornithine.
Several strategies to isolate peptides capable of binding to and thereby modulating the activity of a target nucleic acid ligand are available. For example, encoded peptide combinatorial libraries immobilized on beads have been described, and have been demonstrated to contain peptides able to bind viral RNA sequences and disrupt the interaction between the viral RNA and a viral regulatory protein that specifically binds said RNA (Hwang et al. Proc. Natl. Acad. Sci USA, 1999, 96: 12997). Using such libraries, modulators of nucleic acid ligands can be isolated by appending a label to the target nucleic acid ligand and incubating together the labeled-target and bead- immobilized peptide library under conditions in which binding between some members of the library and the nucleic acid are favored. The binding of the nucleic acid ligand to the specific peptide on a given bead causes the bead to be "colored" by the label on the nucleic acid ligand, and thus enable the identification of peptides able to bind the target by simple isolation of the bead. The direct interaction between peptides isolated by such screening methods and the target nucleic acid ligand can be confirmed and quantified using any number of the binding assays described to identify modulators of nucleic acid ligands. The ability of said peptides to modulate the activity of the target nucleic acid ligand can be confirmed by appropriate bioassays.
In an additional embodiment, the modulators are oligosaccharide based modulators. Oligosaccharides can interact with nucleic acids. For example, the antibiotic aminoglycosides are products of Streptomyces species and interact specifically with a diverse array of RNA molecules such as various ribozymes, RNA components of ribosomes, and HIV-1 's TAR and RRE sequences. Thus oligosaccharides can bind to nucleic acids and can be used to modulate the activity of nucleic acid ligands.
In another embodiment, the modulator is a small molecule based modulator. A small molecule that intercalates between the ligand and the target or otherwise disrupts or modifies the binding between the ligand and target can also be used as the therapeutic regulator. Such small molecules can be identified by screening candidates in an assay that measures binding changes between the ligand and the target with and without the small molecule, or by using an in vivo or in vitro assay that measures the difference in biological effect of the ligand for the target with and without the small molecule. Once a small molecule is identified that exhibits the desired effect, techniques such as combinatorial approaches can be used to optimize the chemical structure for the desired regulatory effect.
In a further exemplary embodiment, the modulator is a nucleic acid binding polymer, lipid, nanoparticle or microsphere. In further non-limiting examples, the modulator can be selected from the group consisting of: l,2-dioleoyl-sn-glycero-3- ethylphosphocholine (EDOPC); dilauroylethylphosphatidylcholine (EDLPC);
EDLPC/EDOPC; pyridinium surfactants; dioleoylphosphatidyl-ethanolamine (DOPE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-¾z'5(dodecyloxy)- 1 -propanaminium bromide (GAP-DLRIE) plus the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE) (GAP-DLRIE/DOPE); (±)-N,N-dimethyl-N-[2-(spermine carboxamido)ethyl]-2,3- bis(dioeyloxy-l-propaniminium petahydrochloride (DOSPA);
dilauroylethylphosphatidylcholine (EDLPC); Ethyldimyristoyl phosphatidylcholine (EDMPC); (±)-N,N,N-trimethyl-2,3-bis(z-octadec-9-ene-oyloxy)- 1 -propanaminium chloride (DOTAP); (±)-N-2-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)- 1 - propanaminium bromide (DMRIE); (±)-N,N,N-trimethyl-2,3-bis(z-octadec-9-enyloxy)-l- propanaminium chloride (DOTMA); 5-carboxyspermylglycine dioctadecyl-amide (DOGS); dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES); 1 ,3 dioleoyloxy-2-(6-carboxyspermyl)-propyl-amid (DOSPER); tetramethyltetrapalmitoyl spermine (TMTPS); (tetramethyltetraoleyl spermine (TMTOS); tetramethlytetralauryl spermine (TMTLS); tetramethyltetramyristyl spermine (TMTMS); tetramethyldioleyl spermine (TMDOS); diphytanoylphosphatidyl-ethanolamine (DPhPE); and (±)-N-(3- aminopropyl)-N,N-dimethyl-2,3-¾z5(dodecyloxy)-l -propanaminium bromide (GAP- DLRIE).
In other embodiments, the modulator is selected from the group consisting of: chitosan; a chitosan derivative; 1 ,5 -dimethyl- 1 , 5 -diazaundecamethylene
polymethobromide; polyoxyethylene/ polyoxypropylene block copolymers; poly-L- lysine; polyamidoamine (PAMAM); β-cyclodextrin-containing polycation (CDP); β- cyclodextrin-containing polycation (imidazole-containing variant) (CDP-Im);
polyphosphoramidate polymer (8kDa, 30kDa) (PPA-DPA 8k, PPA-DPA 30k);
polybrene; spermine; PEG-block-PLL-dendrimers; polyethylenimine (PEI); mannose- PEI; transferin-PEI; linera-PEI (1PEI); gelatin; methacrylate/methacrylamide; poly(beta- amino esters); polyelectrolyte complexes (PEC); poly(vinalyamine) (PVA); Collagen; polypropylene imine (PPI); polyallylamine; polyvinylpyridine; aminoacetalized poly( vinyl alcohol); acrylic or methacrylic polymer; Newkome dendrimer;
polyphenylene; dimethyldioctadecylammonium bromide (DAB);
cetyltrimethylammonium bromide (CTAB); albumin; acid-treated gelatin; polylysine; polyornithine; polyarginine; DEAE-cellulose; DEAE-dextran; and poly(N,N- dimethylaminoethylmethacrylate); and polypropylamine (POP AM). In one embodiment, the modulator is selected from chitosan and chitasan derivatives. Chitosan derivatives include water soluble chitosan nanoparticles (such as described in US Patent No. 6,475,995; US Patent Application No. 2006/0013885;
Limpeanchob et al, (2006) Efficacy and Toxicity of Amphotericin B-Chitosan
Nanoparticles; Nareusan University Journal 14(2):27-34). Given the polycationic nature of the chitosan polymer (essentially a very large polyamine polymer composed of repeating glucosamine monomers), chitosan may be used to aggregate and/or encapsulate ligands into a polyelectrolyte complex in vivo following injection into a host. This is based in part on interactions of the primary amines found on chitosan and the
phosphodiester backbone of the ligand.
In certain embodiments, the primary amines on the chitosan polymer can be substantially modified to alter the water solubility and charge state. Chitosan derivatives include trimethyl chitosan chloride (TMC), which can be synthesized at different degrees of quaternization; mono-carboxymethylated chitosan (MCC) which is a polyampholytic polymer; glutaraldehyde cross-linked derivative (CSGA); thiolated chitosan (Lee, et al. (2007) Pharm. Res. 24: 157-67); glycol chitosan (GC), a chitosan derivative conjugated with ethylene glycol (Lee, et al. (2007) Int J Pharm.); [N-(2-carboxybenzyl)chitosan (CBCS) (Lin, et al. (2007) Carbohydr Res. 342(l):87-95); a beta-cyclodextrin-chitosan polymer (Venter, et al. (2006) Int J Pharm. 313(l-2):36-42); O-carboxymethylchitosan; N,0-carboxymethyl chitosan; or a chitosan chemically modified by introducing xanthate group onto its backbone.
In one embodiment, empty chitosan nanoparticles are generated and used as modulators. Chitosan or chitosan derivatives of molecular weight range of 10,000 Da to >1, 000,000 Da may be used. In certain embodiments, the chitosan is of 500,000 Da or less. In certain embodiments, the chitosan is of 100,000 Da or less. In some
embodiments, the compound is between 10,000 and 100,000 Da, between 10,000 and 90,000, between 10,000 and 80,000, between 20,000 and 70,0000, between 30,000 and 70,000, about 30,000, about 40,000, about 50,000 or about 60,000 Da.
In some embodiments, chitosan polymers containing different degrees of deacetylated primary amines are used. In these embodiments, the different degrees of deacetylation alters the charge state of the polymer and thereby the binding properties of the polymer. Upon contact of the chitosan nanoparticle with ligands in the host, ligands may bind with and become trapped on the nanoparticle surface, or enter the nanoparticle and become encapsulated by ionic interactions.
In another embodiment, the modulator is a polyphosphate polymer microsphere. In certain embodiments, the modulator is a derivative of such a microsphere such as poly(L-lactide-co-ethyl-phosphite) or P(LAEG-EOP) and others, as described in US Patent No. 6,548,302. Such polymers can be produced to contain a variety of functional groups as part of the polymeric backbone. In one example, the polymers may contain quaternary amines with a positive charge at physiologic pH, such that they can complex or encapsulate one or more nucleic acids upon contact. In certain embodiments, the polymers do not contain positive charges.
The present invention also provides methods to identify the modulators of nucleic acid α2βι ligands. Modulators can be identified in general, through binding assays, molecular modeling, or in vivo or in vitro assays that measure the modification of biological function. In one embodiment, the binding of a modulator to a nucleic acid is determined by a gel shift assay. In another embodiment, the binding of a modulator to a nucleic acid ligand is determined by a BIACORE assay.
Standard binding assays can be used to identify and select modulators of the invention. Non-limiting examples are gel shift assays and BIACORE assays. That is, test modulators can be contacted with the nucleic acid ligands to be targeted under test conditions or typical physiological conditions and a determination made as to whether the test modulator in fact binds the ligand. Test modulators that are found to bind the nucleic acid ligand can then be analyzed in an appropriate bioassay (which will vary depending on the ligand and its target molecule, for example platelet aggregometry tests) to determine if the test modulator can affect the biological effect caused by the ligand on its target molecule.
The Gel-Shift assay is a well-known technique used to assess binding capability. For example, a nucleic acid ligand to α2βι is first incubated with α2βι protein or fragment thereof, or a mixture containing the α2βι protein or fragment, and then separated on a gel by electrophoresis Upon binding of the ligand to the protein, the complex will be larger in size and its migration will therefore be retarded relative to that of the free ligand which can be applied to a control lane in the gel in the absence of α2βι protein. The ligand can be labeled, for example, by a radioactive or non-radioactive moiety, to allow detective of the ligand-a2 i complex within the gel. When using the Gel-Shift assay to screen for ligands having -binding activity, the complex can then be extracted from the gel and the isolated ligand analyzed to identify ligands having the desired α2βι binding activity.
Gel shift assays can also be used to screen modulators for binding nucleic acid ligands to α2βι, as association of the modulator with the nucleic acid ligand retards the mobility of the nucleic acid ligand relative to that of the free ligand (see, for example, Rusconi et al, 2002, Nature, 419:90-94.).
Additionally, modulators can be added to such an assay format and screened for their ability to block association of a α2βι nucleic acid ligand with α2βι. For example, the
Figure imgf000037_0001
mixture can be incubated in the presence of increasing amounts of potential modulator. A modulator with the desired activity will specifically reduce formation of the
Figure imgf000037_0002
complex as detected by the Gel-Shift assay.
BIACORE technology is known to the skilled artisan as a reliable and valuable tool for identifying and analyzing macromolecular interactions, include polypeptide- nucleic acid interactions. Accordingly, one could use this technology to screen for or to identify nucleic acid aptamers or ligands which specifically bind the α2βι protein or fragment thereof. The BIACORE technology measures binding events on a sensor chip surface, so that an interactant attached to the surface determines the specificity of the analysis. In other words, the α2βι protein or fragment could be attached to the sensor chip surface via, for example, a histidine tag. The bound α2βι proteins are then exposed to a solution containing the potential ligand molecules. Binding of the nucleic acid ligand to the α2βι protein gives an immediate change in the surface plasmon resonance (SPR) signal The signal is directly proportional to the mass of molecules that bind to the surface.
As described for the gel-shift assay, the BIACORE could be used to identify or analyze modulators of the α2βι ligands. Again, the reaction mixture to which the chip- bound α2βι protein is exposed can contain both a known α2βι ligand with increasing amounts of modulator and the effects determined by standard BIACORE analysis of the resultant interaction between α2βι and its ligand.
There are a number of other assays that can determine whether an oligonucleotide or analogue thereof, peptide, polypeptide, oligosaccharide or small molecule can bind to the ligand in a manner such that the interaction with the target is modified. For example, electrophoretic mobility shift assays (EMSAs), titration calorimetry, scintillation proximity assays, sedimentation equilibrium assays using analytical ultracentrifugation (see for eg. www.cores.utah.edu/interaction), fluorescence polarization assays, fluorescence anisotropy assays, fluorescence intensity assays, fluorescence resonance energy transfer (FRET) assays, nitrocellulose filter binding assays, ELISAs, ELONAs (see, for example, U.S. Pat. No. 5,789,163), RIAs, or equilibrium dialysis assays can be used to evaluate the ability of an agent to bind to a nucleic acid ligand. Direct assays in which the interaction between the agent and the nucleic acid ligand is directly determined can be performed, or competition or displacement assays in which the ability of the agent to displace the ligand from its target can be performed (for example, see Green, Bell and Janjic, Biotechniques 30(5), 2001, p 1094 and U.S. Pat. No. 6,306,598). Once a candidate modulating agent is identified, its ability to modulate the activity of a nucleic acid ligand for its target can be confirmed in a bioassay. Alternatively, if an agent is identified that can modulate the interaction of a ligand with its target, such binding assays can be used to verify that the agent is interacting directly with the ligand and can measure the affinity of said interaction.
In another embodiment, mass spectrometry can be used for the identification of a modulator that binds to a nucleic acid ligand, the site(s) of interaction between the modulator and the nucleic acid ligand, and the relative binding affinity of agents for the ligand (see for example U.S. Pat. No. 6,329,146). Such mass spectral methods can also be used for screening chemical mixtures or libraries, especially combinatorial libraries, for individual compounds that bind to a selected target ligand that can be used in as modulators of the ligand. Furthermore, mass spectral techniques can be used to screen multiple target nucleic acid ligands simultaneously against, e.g. a combinatorial library of compounds. Moreover, mass spectral techniques can be used to identify interaction between a plurality of molecular species, especially "small" molecules and a molecular interaction site on a target ligand.
In vivo or in vitro assays that evaluate the effectiveness of a modulator in modifying the interaction between a nucleic acid ligand and a target are specific for the disorder being treated. There are ample standard assays for biological properties that are well known and can be used. Examples of biological assays are provided in the patents cited in this application that describe certain nucleic acid ligands for specific applications.
In some embodiments, a modulator is a protein. For example, in certain embodiments, a nucleic acid ligand is linked to a biotin molecule. In those instances, a streptavadin or avidin is administered to bind to and reverse the effects of the ligand (see
Savi et. al. J Thrombosis and Haemostasis, 6: 1697-1706). Avidin is a tetrameric protein produced in the oviducts of birds, reptiles and amphibians which is deposited in the whites of their eggs. Streptavidin is a tetrameric protein purified from the bacterium
Streptomyces avidinii. The tetrameric protein contains four identical subunits
(homotetramer) each of which can bind to biotin (Vitamin B7, vitamin H) with a high degree of affinity and specificity.
In certain embodiments, a modulator is a cationic molecule. In certain
embodiments, the ligand forms a guanine quartet (G-quartet or G-quadruplex) structure.
These structures are bound by cationic molecules. In certain embodiments, the molecules are metal chelating molecules. In some embodiments, the modulator is a porphyrin. In some embodiments, the compound is TMPyP4. See Joachimi, et.al. JACS 2007, 129,
3036-3037 and Toro, et.al. Analytical Biochemistry 2008, Aug 1, 379 (1) 8-15.
In one embodiment, the modulator has the ability to substantially bind to a nucleic acid ligand in solution at modulator concentrations of less than ten (10.0) micromolar (uM), one (1.0) micromolar (uM), preferably less than 0.1 uM, and more preferably less than 0.01 uM. By "substantially" is meant that at least a 50 percent reduction in target biological activity is observed by modulation in the presence of the target, and at 50% reduction is referred to herein as an IC50 value.
F. Optimizing Ligands and Modulators
In order for a ligand to be suitable for use as a therapeutic, the ligand is preferably inexpensive to synthesize, safe for use in a host, and stable in vivo. Wild-type RNA and DNA oligonucleotides are typically not stable in vivo because of their susceptibility to degradation by nucleases. Resistance to nuclease degradation can be greatly increased by the incorporation of modifying groups at the 2'-position.
2'-fluoro or amino groups may be incorporated into oligonucleotide pools from which ligands have been subsequently selected. In the present disclosure, 2'- fluoropyrimidines were used in an in vitro transcription reaction to generate an initial oligonucleotide pool for ligand selection (see Example 1). However, resultant ligands selected from such libraries containing 2'-hydroxyl sugars at each purine position, so while more stable in vivo than a comparable RNA or DNA ligand, require additional optimization. Accordingly, the ligands identified using the methods described herein are subsequently modified in a variety of ways to obtain a ligand which has enhanced function and stability, as well as increased feasibility for large-scale manufacturing processes.
After initial identification of the ligands (via e.g., SELEX) and the modulators (e.g., design based on sequence complementarity), the ligands and modulators can be modified or engineered to improve their desired structure, function and/or stability by a variety of means. These include, but are not limited to, substituting particular sugar residues, changing the composition and size of particular regions and/or structures in the ligand, and designing ligands that can be more effectively regulated by a modulator.
The design and optimization of a nucleic acid ligand involves an appreciation for the secondary structure of the ligand as well as the relationship between the secondary structure and the modulator control. Unlike conventional methods of modifying nucleic acids, the design of the ligands to a α2βι protein may include consideration of the impact of changes to the ligand on the design of potential modulators. If a ligand is modified by truncation, for example, the corresponding modulator should be designed to control the truncated ligand.
The secondary structure of ligands identified through the SELEX process can be predicted by various methods known to persons having ordinary skill in the art. For example, each sequence may be analyzed using a software program such as Mfold (mfold.bioinfo.rpi.edu; see also Zuker, 2003, Nucleic Acids Res. 31 :3406-3415 and
Mathews, et al, 1999, J. Mol. Biol. 288:911-940). Subsequently, comparative sequence analysis of the various selected sequences can be used to align the sequences based upon conserved consensus secondary structural elements to arrive at a predicted secondary consensus structure for α2βι. An analysis such as that described above allows one to design and test variants of the sequences obtained through SELEX to generate ligands with enhanced function and stability.
α2βι nucleic acid ligands of the present invention can be modified by varying overall ligand length as well as the lengths of the stem and loop structures. For example, ligand truncations may be generated in which a portion of the 5' and/or 3' end of a ligand is deleted from the ligand selected in the SELEX process. To determine the extent of truncations which are tolerated by a ligand, one method used can be to heat anneal an oligonucleotide (e.g. a DNA oligonucleotide) complementary to a 5' or 3' terminal region of the ligand, then compare binding of the ligand with and without the annealed oligonucleotide. If no significant binding difference is observed between the ligand with and the ligand without the annealed oligonucleotide, this suggests that the annealed portion of the ligand is dispensable for binding of the ligand to the target protein. This method can be performed using oligonucleotides which anneal to various lengths of the 5 ' or 3 ' ends of the ligand to determine 5 ' and 3 ' boundaries which provide a fully functional ligand.
In another embodiment, the design includes decreasing the size of the ligand. In another embodiment, the size of the modulator is changed in relation to the size of the ligand. In yet another embodiment, guanine strings are reduced to less than four guanine, or less than three guanine, or less than two guanine or no guanines. However, the joint effect of these changes must meet the challenge of creating a ligand that provides adequate activity but is easily neutralized by the modulator.
For targeting of a modulator, an improved ligand can also be modified so as to include a single-stranded tail (3' or 5') in order to promote association with an
oligonucleotide modulator. Suitable tails can comprise 1 nt to 20 nt, preferably, 1 nt to 10 nt, 1 nt to 5 nt or 3 nt to 5 nt. It is readily understood that such tails may included modified nucleotides as described in more detail below.
Tailed ligands can be tested in binding and bioassays (e.g., as described below) to verify that addition of the single-stranded tail does not disrupt the active structure of the ligand. A series of oligonucleotides (for example, 2'-0-methyl oligonucleotides) that can form, for example, 1 , 3 or 5 base-pairs with the tail sequence can be designed and tested for their ability to associate with the tailed ligand alone, as well as their ability to increase the rate of dissociation of the ligand from, or association of the ligand with, its target molecule. Scrambled sequence controls can be employed to verify that the effects are due to duplex formation and not non-specific effects.
Determination of a consensus structure also facilitates engineering of ligands to identify one or more nucleotides which may enhance or decrease ligand structure and function. For example, one may more efficiently identify and test nucleotide additions, deletions and substitutions to specific stem and loop structures.
Knowledge of a consensus secondary structure also allows one to avoid modifications which may be detrimental to ligand structure and function. For example, certain modifications may be conserved within the consensus secondary structure, such as a 2'-fluoro within a stem or loop region. In these instances, removal of a 2'-fluoro from the stem or loop of an ligand may result in the loss of activity.
In certain embodiments, the ligands are nucleic acid molecules selected using the SELEX method and include truncates and substantially homologous sequences thereof. As used herein, in the context of homologous regions, a "substantially homologous" sequence is one that forms the same secondary structure by Watson-Crick base pairing within a particular molecule. In certain embodiments, sequences are "substantially homologous" if they share at least 80%, 85% or more sequence identity, such as 90%>, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a specified ligand. In the context of a nucleic acid ligand of a specified length, such as 50 or less nucleotides, a homologous sequence can be found in any region that allows Watson- Crick binding to form the same secondary structure, regardless of sequence identity within the specific region.
Ligands may also be designed to have a suicide position, which allows more effective regulation by paired modulators. Upon binding of the ligand by the modulator, the suicide position becomes single stranded and labile, thereby facilitating cleavage of the ligand by enzymes naturally present in the blood, such as blood or liver endonucleases. This provides a means for effective and substantially immediate elimination of the active ligand from circulation.
Chemical Modifications
One problem encountered in the therapeutic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid ligand can increase the in vivo stability of the nucleic acid ligand or enhance or mediate the delivery of the nucleic acid ligand. Additionally, certain chemical modifications can increase the affinity of the nucleic acid ligand for its target, by stabilizing or promoting the formation of required structural elements within the nucleic acid ligand or providing additional molecular interactions with the target molecule.
Modifications of the ligands can include, but are not limited to, those which provide chemical groups that incorporate additional charge, polarizability,
hydrophobicity, hydrogen bonding, electrostatic interactions, and functionality to the nucleic acid ligand bases or to the ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8- position purine modifications, modifications at exocyclic amines, substitution of 4- thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like.
Modifications can also include 3' and 5' modifications such as capping.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. Patent No. 5,660,985 that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. Patent No. 5,580,737 describes specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-0-methyl (2'-OMe). U.S. Patent No. 5,756,703, describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Patent Nos. 5,637,459 and 5,683,867. U.S. Patent No. 5,637,459 describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH 2), 2*-fiuoro (2*-F), and/or 2*-0-methyl (2*-OMe). The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic or Non- Immunogenic, High Molecular Weight compounds in a diagnostic or therapeutic complex as described in U.S. Patent No. 6,011,020.
Where the nucleic acid ligands are derived by the SELEX method, the
modifications can be pre- or post-SELEX modifications. Pre-SELEX modifications can yield ligands with both specificity for its target and improved in vivo stability. Post- SELEX modifications made to 2'-hydroxyl (2' -OH) nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligands. In one embodiment, the modifications of the ligand include a 3'-3' inverted phosphodiester linkage at the 3' end of the molecule, and 2' fluoro (2'-F), 2' amino (2'- NH2), 2'deoxy, and/or 2' O methyl (2'-OMe) and/or 2' deoxy modification of some or all of the nucleotides.
The ligands described herein were initially generated via SELEX using libraries of transcripts in which the C and U residues were 2 '-fluoro substituted and the A and G residues were 2' -OH. While such modifications generate ligand molecules suitable for screening, the high 2' hydroxyl content make them unsuitable for drug development candidates due to the fact that these positions can be very sensitive to nuclease degradation in vivo, limiting the maximal concentration that can be achieved post- parenteral administration as well as their circulating half-life. Accordingly, once functional sequences are identified, such as through the SELEX method, individual residues can be tested for tolerance to substitutions by assessing the effects of these substitutions on ligand structure, function and stability.
In certain embodiments, the nucleic acids making up the ligand include modified sugars and/or modified bases. In certain embodiments, the modifications include stabilizing modifications such as 2 '-stabilizing modifications. In one embodiment, 2'- stabilizing modifications can include 2'-fluoro, 2'deoxy or 2 '-O-methyl modifications on the sugar ring.
In one embodiment, the design includes decreasing the 2'-hydroxyl content of the ligand or the modulator, or both. In another embodiment, the design includes decreasing the 2'-fluoro content of the ligand or the modulator, or both. In another embodiment, the design includes increasing the 2 '-O-methyl content of the ligand or the modulator, or both.
The oligonucleotide can comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-inethylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2a-thiouracil, β-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-inethylthio-N6- isopentenyladenine, uracil oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil oxyacetic acid (v), 5-methyl thiouracil, 3-(3-amino-3-N carboxypropyl) and 2,6-diaminopurine.
The oligonucleotides of the presently described ligands and modulators can comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines. In one embodiment, the 2'-position of the furanose residue is substituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group. In another embodiment, the nucleic acid ligand or modulator of the invention can comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, hexose, 2'-fluororibose, 2'-0-methylribose, 2'-0-methoxyethylribose, 2'-0- propylribose, 2'-0-methylthioethylribose, 2'-0-diethylaminooxyethylribose, 2'-0-(3- aminopropyl)ribose, 2'-0-(dimethylaminopropyl)ribose, 2'-0-(methylacetamido)ribose, and 2'-0-(dimethylaminoethyloxyethyl)ribose.
The ligand or modulator can comprise at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphorodiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
The ligand molecule, which comprises stem and loop structures, may be further stabilized for therapeutic use by the substitution of one or more nucleic acid loop structures with a more stable loop structure. FIG. 9A illustrates the starting
phosphoramidite for a hexaethylene glycol linker used in synthesis. FIG. 9B illustrates the hexaethylene glycol spacer when incorporated between two nucleotides of a nucleic acid ligand.
In pharmaceutical compositions the ligands can be provided in forms, such as salt forms that improve solubility or bioavailability.
Any of the oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from, for example, Biosearch, Applied Biosystems).
Ligands and modifiers are described herein using abbreviations readily understood by a skilled artisan and noted as follows: "rA" is 2ΌΗ A or adenosine; "A" is a 2'-deoxy A or 2'-deoxyadenosine; "mA" is 2'-0-methyl A or 2'-methoxy-2'- deoxyadenine; "rG" is 2'-OH G or guanosine; "G" is a 2'-deoxy G or 2'-deoxyguanosine; "mG" is 2'-0-methyl G or 2'-methoxy-2'deoxyguanosine; "fC" is 2'-fluoro C or 2'- fluoro-2'deoxycytidine; "mC" is 2'-0-methyl C or methoxy-2'-deoxycytidine; "fU" is 2'- fluoro U or 2'-fluoro-uridine; "mU" is 2'-0-methyl U or 2'-methoxy-uridine; and "iT" is inverted 2Ή T, (C6L) is a hexylamino linker; (6GLY) is a hexaethylene glycol spacer; (PEG40KGL2-NOF) is an approximately 40kDa Branched PEG (SUNBRIGHT™ product No. GL2-400GS2), (6FAM) is 6-carboxyfluorescein; (s) is a phosphorothioate linkage between two nucleotides.
Coupling to a Carrier
α2βι ligands can also include modifications that improve bioavailability or stability. In one embodiment, the nucleic acid ligand and/or its modulator can be covalently attached to a lipophilic compound such as cholesterol, dialkyl glycerol, or diacyl glycerol. In another embodiment, the modification is conjugation to a carrier molecule which may include, but is not limited to a hydrophilic or hydrophobic moiety. One example is polyethylene glycol molecules conjugated to the nucleic acid sequence. Conjugation to, for example, a polymer as described below, can confine distribution to the plasma compartment and increase circulating half-life.
Sugar modifications, as described above, can ensure stability but they do not guarantee adequate pharmacokinetics for nucleic acid ligands to be therapeutically active. In healthy individuals, ligands are cleared from plasma within minutes of IV injection, probably through renal excretion. Keeping intact ligands in the blood from hours to days after injection has been accomplished by conjugating them to larger macromolecules such as polyethylene glycol (PEG). Ligand plasma clearance has also been decreased by embedding them in liposomes.
Therefore, in one embodiment, a α2βι nucleic acid ligand or a α2βι ligand modulator can be covalently bound or otherwise attached to a non-immunogenic, high molecular weight compound such as polyethylene glycol (PEG) or other water soluble pharmaceutically acceptable polymer including, but not limited to, polyaminoamines (PAMAM); polysaccharides such as dextran, or polyoxazo lines (POZ). A α2βι nucleic acid ligand or α2βι ligand modulator can be associated with the high molecular weight compound through covalent bonds. Where covalent attachment is employed, the high molecular weight compound may be covalently bound to a variety of positions on the ligand or modulator. In some embodiments, the ligand or the modulator can be encapsulated inside a liposome for administration to a host in need thereof.
In one embodiment, the ligand or modulator is attached to polyethylene glycol (PEG). Polyethylene glycols (PEGs) can be conjugated to biologically active compounds to serve as "inert" carriers to potentially (1) prolong the half- life of the compound in the circulation, (2) alter the pattern of distribution of the compound and/or (3) camouflage the compound, thereby reducing its immunogenic potential and protecting it from enzymatic degradation. The ligand or modulator can attached to the PEG molecule through covalent bonds. For example, an oligonucleotide ligand or modulator can be bonded to the 5 '-thiol through a maleimide or vinyl sulfone functionality.
Typically, activated PEG and other activated water-soluble polymers are activated with a suitable activating group appropriate for coupling to a desired site on the therapeutic agent. Representative polymeric reagents and methods for conjugating these polymers to an active agent are known in the art and further described in, e.g., Zalipsky, S., et al., "Use of Functionalized Poly(Ethylene Glycols) for Modification of
Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical
Applications, J. M. Harris, Plenus Press, New York (1992); and in Zalipsky, Advanced Drug Reviews, 1995, 16: 157-182. Such reagents are also commercially available.
For example, in one approach for preparing an amide-linked conjugate, a water soluble polymer bearing an activated ester such as an NHS ester, e.g., mPEG- succinimidyl-a-methylbutanoate, is reacted with an amine group of the active agent to thereby result in an amide linkage between the active agent and the water-soluble polymer. Additional functional groups capable of reacting with reactive amino groups include, e.g., N-hydroxysuccinimidyl esters, p-nitrophenylcarbonates,
succinimidylcarbonates, aldehydes, acetals, N-keto-piperidones, maleimides, carbonyl imidazoles, azalactones, cyclic imide thiones, isocyanates, isothiocyanates, tresyl chloride, and halogen formates, among others.
In one embodiment, a plurality of α2βι ligands or α2βι ligand modulators can be associated with a single PEG molecule. The ligands and modulators can be the same or different sequences and modifications. In yet a further embodiment, a plurality of PEG molecules can be attached to each other. In this embodiment, one or more α2βι ligands or α2βι ligand modulators to the same α2βι protein target sequence or to different α2βι protein sequence targets can be associated with each PEG molecule. In embodiments where multiple ligands or modulators specific for the same target are attached to PEG, there is the possibility of bringing the same targets in close proximity to each other in order to generate specific interactions between the same targets. Where multiple ligands or modulators specific for different targets are attached to PEG, there is the possibility of bringing the distinct targets in close proximity to each other in order to generate specific interactions between the targets.
While a variety of linkers and methods for conjugation of hydrophilic moieties such as PEG molecules are well known to persons in the art, several embodiments are provided below. In one embodiment, an amino linker, such as the C6 hexylamino linker, 6-(trifluoroacetamido)hexanol (2-cyanoethyl-N,N-diisopropyl)phosphoramidite, shown in FIG. 10, can be used to add the hexylamino linker to the 5' end of the synthesized oligonucleotide. Other linker phosphoramidites that may be used to add linkers to the synthesized oligonucleotides are described below:
TFA-amino C4 CED phosphoramidite (available from ChemGenes, cat# CLP-1453) of the structure:
Figure imgf000049_0001
5'-amino modifier C3 TFA (available from Glen Research cat# 10-1923-90) of the
Figure imgf000049_0002
5'-Amino-Modifier C3-TFA
5 '-amino modifier 5 (available from Glen Research cat# 10-1905-90) of the structure: MMT
OEtCN
MMT: 4-Monomethoxytrityl
5 '-Amino Modifier 5,
5 '-amino modifier C12 (available from Glen Research cat# 10-1912-90) of the structure:
Figure imgf000050_0001
MMT: 4-Monomethoxytrityl
5'-Amino-Modifier CI 2,
5'thiol-modifier C6 (available from Glen Research cat# 10-1926-90) of the structure:
Figure imgf000050_0002
The 5 '-thiol modified linker is used with PEG-maleimides, PEG-vinylsulfone, PEG- iodoacetamide and PEG-orthopyridyl-disulfide, for example.
The PEG can range in size from 5 to 200 KD, with typical PEGs used in pharmaceutical formulations in the 10-60 KD range. Linear chain PEGs of up to about 30 KD can be produced. For PEGs of greater than 30 KD, multiple PEGs can be attached together (multi-arm or 'branched' PEGs) to produce PEGs of the desired size. The general synthesis of compounds with a branched, "mPEG2" attachment (two mPEGs linked via an amino acid) is described in Monfardini, et al., Bioconjugate Chem. 1995, 6:62-69. For 'branched' PEGs, i.e. compounds that include more than one PEG or mPEG linked to a common reactive group, the PEGs or mPEGS can be linked together through an amino acid such as a lysine or they can be linked via, for example, a glycerine. For branched PEGs in which each mPEG is about 10, about 20, or about 30KD, the total mass is about 20, about 40 or about 60KD and the compound is referred to by its total mass (i.e. 40KD mPEG2 is two linked 20KD mPEGs). 40KD total molecular weight PEGs, that can be used as reagents in producing a PEGylated compound, include, for example, [N2-(monomethoxy 20K polyethylene glycol carbamoy^-N^monomethoxy 20K polyethylene glycol carbamoyl)]-lysine N-hydroxysuccinimide of the structure:
Figure imgf000051_0001
Additional PEG reagents that can be used to prepare stabilized compounds of the invention include other branched PEG N-Hydroxysuccinimide (mPEG-NHS) of the general formula:
Figure imgf000051_0002
Figure imgf000051_0003
with a 40KD or 60KD total molecular weight (where each mPEG is about 20 or about 30KD). As described above, the branched PEGs can be linked through any appropriate reagent, such as an amino acid, and in certain
embodiments are linked via lysine residues or glycerine residues.
They can also include non-branched mPEG-Succinimidyl Propionate (mPEG- SPA), of the general formula:
Figure imgf000052_0001
, in which mPEG is about 20KD or about 30KD. In a specific embodiment, the reactive ester is -0-CH2CH2-C02-NHS.
The reagents can also include a branched PEG linked through glycerol, such as the Sunbright™ series from NOF Corporation, Japan. Specific, non-limiting examples of these rea ents are:
(SUNBRIGHT GL2-400GS2);
Figure imgf000052_0002
(SUNBRIGHT GL2-400HS); and
Figure imgf000052_0003
The reagents can also include non-branched Succinimidyl alpha-methylbutanoate (mPEG-SMB) of the general formula:
Figure imgf000053_0001
, in which mPEG is between 10 and 30KD. In a subembodiment, the reactive ester is -0-CH2CH2CH(CH3)-C02-NHS. Compounds of this structure are sold by Nektar Therapeutics as catalog numbers cat#2M4K0R01.
PEG reagents can also include nitrophenyl carbonate linked PEGs, such as of the following structure:
Figure imgf000053_0002
. Compounds of this structure are commercially available, for example from Sunbio, Inc. Compounds including nitrophenyl carbonate can be conjugated to primary amine containing linkers. In this reaction, the O- nitrophenyl serves as the leaving group, leaving a structure [mPEG]n-NH-CO-NH-linker- ligand.
PEGs with thiol-reactive groups that can be used with a thiol-modified linker, as
described above, include compounds of the general structure
Figure imgf000053_0003
which mPEG is about 10, about 20 or about 30KD. Additionally, the structure branched, such as
Figure imgf000054_0001
in which each mPEG is about 10, about 20, or about 30KD and the total mass is about 20, about 40, or about 60KD. Branched PEGs with thiol reactive groups that can be used with a thiol-modified linker, as described above, include compounds in which the branched PEG has a total molecular weight of about 40 or 60 KD (where each mPEG is 20 or 30 KD). PEG reagents can also be of the following structure:
Figure imgf000054_0002
. PEG-maleimide pegylates thiols of the target compound in which the double bond of the maleimic ring breaks to connect with the thiol. The rate of reaction is pH dependent and, in one embodiment, is carried out between pH 6 and 10, or between pH 7 and 9 or about pH 8.
In one embodiment, a plurality of α2βι ligand modulators can be associated with a single PEG molecule. The modulator can be to the same or different α2βι nucleic acid ligands. In embodiments where there are multiple modulators to the same ligand, there is an increase in avidity due to multiple binding interactions with the ligand. In yet a further embodiment, a plurality of PEG molecules can be attached to each other. In this embodiment, one or more modulators to the same nucleic acid ligand or different ligands can be associated with each PEG molecule. This also results in an increase in avidity of each modulator to its target.
In one embodiment, the nucleic acid ligand or its modulator can be covalently attached to a lipophilic compound such as cholesterol, dialkyl glycerol, or diacyl glycerol. The lipophilic compound or non-immunogenic, high molecular weight compound can be covalently bonded or associated through non-covalent interactions with a ligand or modulator(s). Attachment of the ligand or oligonucleotide modulator to lipophilic or non-immunogenic high molecular weight compounds can be done directly or with the utilization of linkers or spacers.
In embodiments where direct covalent attachment is employed, the lipophilic compound or non-immunogenic high molecular weight compound may be covalently bound to a variety of positions on the ligand or modulator, such as to an exocyclic amino group on the base, the 5-position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5 ' or 3' terminus.
In embodiments where the ligand or modulator is attached to a lipophilic, or a non-immunogenic high molecular weight compound through a linker or spacer, the lipophilic compound or non-immunogenic high molecular weight compound may be attached to the ligand or modulator using, for example, a six carbon amino linker.
In another embodiment, one or more phosphate groups may be included between the linker and the nucleic acid sequence.
Additional suitable linkers and spacers for attaching the ligand or modulator to a lipophilic compound or to a non-immunogenic high molecular weight compound are described in U.S. Patent No. 7,531 ,524, incorporated herein by reference.
Oligonucleotides of the invention can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve properties such as stability of the molecule and affinity for the intended target.
In one embodiment, provided is a ligand or a salt thereof of the Formula
H3C(OCH2CH2)nOC(=0)NH-L-OP(0)20-5 'Ligand3 ' wherein n is 400 to 600;
L is -(CH2)p- wherein p is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and
5'Ligand3' is a ligand as described herein.
In one embodiment, p is 6. G. Methods to Treat α2βι -Mediated Disorders
Methods, pharmaceutical compositions and uses of the α2βι nucleic acid ligands described herein are provided as modulatable anti-α^βι agents for use in disorders or treatment regimes requiring anti-α^βι therapy. In certain embodiments, the treatment is a surgical intervention. The methods can include administering the α2βι nucleic acid ligand to a host in need thereof, wherein the host is suffering from, or at risk of suffering from, an occlusive thrombotic disease or disorder of the coronary, cerebral or peripheral vascular system, an autoimmune disorder, an inflammatory disorder, a cancer or metastasis thereof, or an echovirus infection or echovirus-associated disease.
In one embodiment, a method of treating symptoms of an autoimmune disorder, including but not limited to, multiple sclerosis (MS), is provided.
In one embodiment, a method of treating symtpoms of an inflammatory disease, including but not limited to, rheumatoid arthritis (RA) is provided.
In one embodiment, a method of treating symptoms of a cancer, including but not limited to, lung cancer, prostate cancer, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, and colon cancer is provided. In another embodiment, a method of inhibiting or blocking metastasis of the cancer is provided.
In one embodiment, a method of inhibiting or blocking an echovirus infection is provided. In another embodiment, a method of treating an echovirus-associated disease is provided.
In one embodiment, the α2βι ligand inhibits conversion of α2βι to an active conformation.
In certain embodiments a method of treating symptoms of, or preventing formation of a vascular event, in particular a thrombotic or thromboembolitic event is provided including administering a α2βι nucleic acid ligand of the invention to a host in need thereof.
In one embodiment, the α2βι nucleic acid ligand is provided for extended periods of time. In this instance, a α2βι ligand modulator may only be used in emergency situations, for example, if treatment leads to hemorrhage, including intracranial or gastrointestinal hemorrhage. In another embodiment, the modulator is administered when emergency surgery is required for patients who have received α2βι nucleic acid ligand treatment. In another embodiment, the modulator is administered to control the concentration of the α2βι nucleic acid ligand and thereby the duration and intensity of treatment. In another embodiment, the α2βι nucleic acid ligand is provided as a platelet anesthetic during a cardiopulmonary bypass procedure. In another embodiment, the α2βι nucleic acid ligand is administered to provide a period of transition off of or on to oral anti-platelet medications, and the modulator is used to reverse the α2βι nucleic acid ligand once therapeutic levels of the oral anti-platelet agent are established.
H. Pharmaceutical Compositions
The α2βι nucleic acid ligands or α2βι ligand modulators taught herein can be formulated into pharmaceutical compositions that can include, but are not limited to, a pharmaceutically acceptable carrier, diluent or excipient. The precise nature of the composition will depend, at least in part, on the nature of the ligand and/or modulator, including any stabilizing modifications, and the route of administration. Compositions containing the modulator can be designed for administration to a host who has been given a α2βι nucleic acid ligand to allow modulation of the activity of the ligand, and thus regulate anti-platelet activity of the administered α2βι nucleic acid ligand.
The design and preparation of pharmaceutical or pharmacological compositions will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules, as liquids for oral administration; as elixirs, syrups, suppositories, gels, or in any other form used in the art, including eye drops, creams, lotions, salves, inhalants and the like. The use of sterile formulations, such as saline -based washes, by surgeons, physicians or health care workers to treat a particular area in the operating field may also be particularly useful. Compositions can also be formulated for delivery via microdevice, microparticle or sponge.
Pharmaceutically useful compositions comprising a α2βι nucleic acid ligand or α2βι ligand modulator of the present invention can be formulated at least in part by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation can be found in Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins, 2000) and Ansel et al.,
Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th Ed. (Media, Pa. : Williams & Wilkins, 1995).
Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents, including but not limited to phosphate-buffered saline. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, EDTA, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, sodium chloride, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the nucleic acid ligand or modulator. Such compositions can contain admixtures of more than one compound. The compositions typically contain about 0.1% weight percent (wt%) to about 50 wt%, about 1 wt% to about 25 wt%, or about 5 wt% to about 20 wt% of the active agent (ligand or modulator).
Pharmaceutical compositions for parenteral administration, including
subcutaneous, intramuscular or intravenous injections and infusions are provided herein. For parenteral administration, aseptic suspensions and solutions are desired. Isotonic preparations that generally contain suitable preservatives are employed when intravenous administration is desired. The pharmaceutical compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. The active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, buffered water, saline, 0.4% saline, 0.3%> glycine, hyaluronic acid, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension. Additionally, solid forms suitable for dissolving in liquid prior to injection can be formulated.
To aid dissolution of an agent into an aqueous environment, a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. Nonionic detergents that could be included in the formulation as surfactants include, but are not limited to, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose, carboxymethyl cellulose and any of the pluronic detergents such as Pluronic F68 and/or Pluronic F127 (e.g., see Strappe et al. Eur. J. of Pharm. Biopharm., 2005, 61 : 126-133). Surfactants could be present in the formulation of a protein or derivative either alone or as a mixture in different ratios.
For oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
For liquid forms used in oral administration, the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents that can be employed include glycerin and the like.
Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl ether propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Active agents administered directly (e.g., alone) or in a liposomal formulation are described, for example, in U.S. Pat. No. 6,147,204.
The compounds of the present invention can also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amide -phenol, polyhydroxy- ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention can be coupled
(preferably via a covalent linkage) to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone, polyoxazolines, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels. Cholesterol and similar molecules can be linked to the nucleic acid ligands to increase and prolong bioavailability.
Lipophilic compounds and non-immunogenic high molecular weight compounds with which the modulators of the invention can be formulated for use in the present invention and can be prepared by any of the various techniques presently known in the art or subsequently developed. Typically, they are prepared from a phospholipid, for example, distearoyl phosphatidylcholine, and may include other materials such as neutral lipids, for example, cholesterol, and also surface modifiers such as positively charged (e.g., sterylamine or aminomannose or aminomannitol derivatives of cholesterol) or negatively charged (e.g., diacetyl phosphate, phosphatidyl glycerol) compounds.
Multilamellar liposomes can be formed by the conventional technique, that is, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase is then added to the vessel with a swirling or vortexing motion which results in the formation of multilamellar liposome vesicles (MLVs). Unilamellar liposome vesicles (UVs) can then be formed by homogenization, sonication or extrusion (through filters) of MLVs. In addition, UVs can be formed by detergent removal techniques. In certain embodiments of this invention, the complex comprises a liposome with a targeting nucleic acid ligand(s) associated with the surface of the liposome and an encapsulated therapeutic or diagnostic agent. Preformed liposomes can be modified to associate with the nucleic acid ligands. For example, a cationic liposome associates through electrostatic interactions with the nucleic acid. Alternatively, a nucleic acid attached to a lipophilic compound, such as cholesterol, can be added to preformed liposomes whereby the cholesterol becomes associated with the liposomal membrane. Alternatively, the nucleic acid can be associated with the liposome during the formulation of the liposome.
In another embodiment, a stent or medical device may be coated with a formulation comprising a α2βι ligand or α2βι ligand modulator according to methods known to skilled artisans.
Therapeutic kits are also envisioned. The kits comprises the reagents, active agents, and materials that may be required to practice the above methods. The kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of a α2βι ligand and/or a α2βι ligand modulator. The kit may have a single container means, and/or it may have distinct container means for each compound.
I. Methods for Administration
Modes of administration of the α2βι ligands and/or α2βι ligand modulators of the present invention to a host include, but are not limited to, parenteral (by injection or gradual infusion over time), intravenous, intradermal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular, intraspinal, intrasternal, topical, transdermal patch, via rectal, buccal, vaginal or urethral suppository, peritoneal, percutaneous, nasal spray, surgical implant, internal surgical paint, infusion pump or via catheter. In one embodiment, the agent and carrier are administered in a slow release formulation such as an implant, bolus, microparticle, microsphere, nanoparticle or nanosphere. In one embodiment, the α2βι nucleic acid ligand is delivered via subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps).
In one embodiment, the α2βι nucleic acid ligand is delivered via subcutaneous administration and the modulator is delivered by subcutaneous or intravenous
administration.
The therapeutic compositions comprising ligands and modulators of the present invention may be administered intravenously, such as by injection of a unit dose. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the host, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier or vehicle.
Additionally, one approach for parenteral administration employs the
implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained.
Local administration, for example, to the interstitium of an affected joint, is also provided. Local administration can be achieved by injection, such as from a syringe or other article of manufacture containing a injection device such as a needle. The rate of administration from a syringe can be controlled by controlled pressure over desired period of time to distribute the contents of the syringe. In another example, local administration can be achieved by infusion, which can be facilitated by the use of a pump or other similar device.
Representative, non-limiting approaches for topical administration to a vascular tissue are also provided and include (1) coating or impregnating a blood vessel tissue with a gel comprising a nucleic acid ligand, for delivery in vivo, e.g., by implanting the coated or impregnated vessel in place of a damaged or diseased vessel tissue segment that was removed or by-passed; (2) delivery via a catheter to a vessel in which delivery is desired; (3) pumping a composition into a vessel that is to be implanted into a patient. Alternatively, the compounds can be introduced into cells by microinjection, or by liposome encapsulation.
Also provided is administration of the α2βι ligands to a subject by coating medical devices such as stents with pharmaceutical compositions containing the ligand. Methods for coating to allow appropriate release and administration of the ligand are known to those having ordinary skill in the art.
Optimum dosing regimens for the compositions described herein can be readily established by one skilled in the art and can vary with the modulator, the patient and the effect sought. The effective amount can vary according to a variety of factors such as the individual's condition, weight, sex, age and amount of nucleic acid ligand administered. Other factors include the mode of administration.
Generally, the compositions will be administered in dosages adjusted for body weight, e.g., dosages ranging from about 1 μg/kg body weight to about 100 mg/kg body weight. More typically, the dosages will range from about 0.1 mg/kg to about 20 mg/kg, and more typically from about 0.5 mg/kg to about 10 mg/kg, or about 1.0 to about 5.0 mg/kg, or about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 6.0 mg/kg, about 7.0 mg/kg, about 8.0 mg/kg, about 9.0 mg/kg or about 10.0 mg/kg. Typically, the dose initially provides a plasma concentration of drug about 0.002 μg/ml to about 2000 μg/ml of drug, more typically from about 2.0 μg/ml to about 400 μg/ml, and more typically from about 10 μg/ml to 200 μg/ml, or about 20 μg/ml to about 100 μg/ml drug, about 20 μg/ml, about 40 μg/ml, about 60 μg/ml, about 80 μg/ml, about 100 μg/ml, about 120 μg/ml, about 140 μg/ml, about 160 μg/ml, about 180 μg/ml, or about 200 μg/ml.
When administering a modulator to a host which has already been administered the ligand, the ratio of modulator to ligand can be adjusted based on the desired level of inhibition of the ligand. The modulator dose can be calculated based on correlation with the dose of ligand. In one embodiment, the weight-to-weight dose ratio of modulator to ligand is 1 : 1. In other embodiments, the ratio of modulator to ligand is greater than 1 : 1 such as 2: 1 or about 2: 1, 3: 1 or about 3: 1, 4: 1 or about 4: 1, 5: 1 or about 5: 1, 6: 1 or about 6: 1, 7: 1 or about 7: 1, 8: 1 or about 8: 1, 9: 1 or about 9: 1, 10: 1 or about 10: 1 or more. In other embodiments, the dose ratio of modulator to ligand is less than about 1 : 1 such as 0.9:1 or about 0.9: 1, 0.8: 1 or about 0.8: 1, 0.7: 1 or about 0.7: 1, 0.6: 1 or about 0.6:1, 0.5: 1 or about 0.5: 1, 0.45: 1 or about 0.45: 1, 0.4: 1 or about 0.4: 1, 0.35: 1 or about 0.35: 1, 0.3: 1 or about 0.3: 1, 0.25: 1 or about 0.25: 1, 0.2: 1 or about 0.2: 1, 0.15: 1 or about 0.15: 1, 0.1 : 1 or about 0.1 : 1 or less than 0.1 : 1 such as about 0.005: 1 or less. In some embodiments, the ratio is between 0.5 : 1 and 0.1 : 1 , or between 0.5 : 1 and 0.2: 1 , or between 0.5 : 1 and 0.3:1. In other embodiments, the ratio is between 1 :1 and 5:1, or between 1 : 1 and 10: 1, or between 1 : 1 and 20: 1.
α2βι nucleic acid ligands as disclosed herein can be administered intravenously in a single daily dose, an every other day dose, or the total daily dosage can be administered in several divided doses. Ligand and/or modulator administration may be provide once per day (q.d.), twice per day (b.i.d.), three times per day (t.i.d.) or more often as needed. Thereafter, the modulator is provided by any suitable means to alter the effect of the nucleic acid ligand by administration of the modulator. Nucleic acid ligands of the present invention can be administered subcutaneously twice weekly, weekly, every two weeks or monthly. In some embodiments, the ligands or modulators are administered less often than once per day. For example, ligand administration may be carried out every other day, every three days, every four days, weekly, or monthly.
In one embodiment, co-administration or sequential administration of other agents can be desirable. For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.
The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the host to be treated, capacity of the host's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are provided. EXAMPLES
Example 1: Cloning, Expression, and Purification of Human Alpha2 I Domain Protein Fragment
The alpha2 (a2) integrin contains a -200 amino acid inserted domain referred to as the "I Domain." This protein fragment has been shown to contain the ligand binding site for collagen, as well as triple helical collagen peptide GFOGER, various snake venoms, and some inhibitory antibodies. Numerous groups have expressed recombinant human α2 I Domain in E.coli bacteria and utilized this protein fragment to demonstrate that it is functional.
Cloning:
The human a2 integrin sequence is available as accession number NM 002203.3 (FIG. 2; SEQ ID NO: l). HT1080 cells (from ATCC) are a human fibrosarcoma cell line known to express α2β1. HT1080 cells were grown in Eagle's MEM (from ATCC) + 10% FBS, passaged twice, harvested at -70% confluency, and then pelleted by standard cell biology techniques. PolyA mRNA was extracted from the pelleted cells using Qiagen' s Oligotex Direct mRNA kit. A QiaShredder (Qiagen) was utilized to homogenize the cells during the extraction process.
A cDNA library of HT1080 mRNA was generated with 100 ng of HT 1080 mRNA, a poly dT oligo, and Ambion's MMLV-Reverse Transcriptase following manufacturer's instructions. PCR was performed on the HT1080 cDNA utilizing Pfu polymerase (Stratagene) and 5' primer CGGGATCCCCTGATTTTCAGCTCTCAGCC (SEQ ID NO: 6) and 3' primer
CCAAGCTTTTAACCTTCAATGCTGAAAATTTGTTCTCCTAATGTCCC (SEQ ID NO:7) by standard PCR techniques. BamHl and HindUl sites (in bold) introduced for cloning purposes in the 5' and 3' primers, respectively. A stop codon was also introduced immediately prior to the HindUl site in the 3' primer. The resulting PCR was desalted with a Qiagen Qiaquick PCR spin column. The cleaned PCR product was digested with BamRl and HindUl for 2.75 hours and then desalted with a second Qiagen Qiaquick PCR column. pQE-80L expression vector (Qiagen) was digested with BamRl and HmdIII and gel purified by standard molecular biology techniques. The digested α2 I Domain PCR product and digested pQE-80L vector were ligated with T4 DNA Ligase (Invitrogen) for 4 hours at room temperature and then transformed into XL-2 Ultracompetent E. coli (Stratagene/Agilent) all per manufacturer's instructions.
Transformants were streaked for singles, overnight cultures grown, and DNA isolated with Qiagen DNA Plasmid Mini Kit. Plasmids were sequenced to verify the desired sequence was cloned.
Expression:
The complete amino acid sequence of a2 protein and the α2 I Domain amino acid sequence to be expressed is displayed in FIG 3A and FIG. 3B (SEQ ID NO:3), respectively, and represents amino acids Serl24-Gly337. Cloning into the pQE-80L expression vector results in the addition of several amino acids at the amino end of the a2 I Domain, specifically the addition of amino acids MRGSHHHHHHG (SEQ ID NO: 8). The MRGSHHHHHHG (SEQ ID NO: 8) amino acid addition is for purification and cloning purposes. (FIG. 3C; SEQ ID NO:4)
The a2 I Domain/pQE-80L plasmids were transformed for protein expression into
BL21 DE3 RIPL E. coli cells (Stratagene/Agilent) per manufacturer's instructions. A 25mL LB Ampicillin (LB A) culture was inoculated with a2 I Domain/pQE80-L BL21 DE3 RIPL transformants and grown overnight shaking at 37°C. Five mis of the overnight culture was used to inoculate 1L LB A media. Cells were grown shaking at 37°C to OD6oo -0.7, at which point the α2 I Domain protein expression was induced by the addition of IPTG to a final concentration of ImM. Cells were grown for an additional 4 hours post induction, harvested by spinning at 3500rpm for 20 minutes at 4°C, and cell pellets were then frozen at -80C until purification.
Purification:
The thawed cell pellets from 1L of culture were resuspended in a total of 25mL cold Lysis Buffer (50mM Tris pH 8.0, 200mM NaCl, 2mM Imidazole). Ten mgs of lysozyme, lOOuL of 0.1M PMSF, and 20ul of 5mg/mL Leupeptin were added to the 25mL cell Lysis Buffer mixture and incubated on a rocker for 20 minutes at 4°C.
Deoxycholic acid was added to a final concentration of 0.08% and incubated on a rocker for 20 minutes at 4°C. The cell lysis process was completed by the addition of 2.5mL
Promega Fast Break Lysis Buffer and then incubated at room temperature for 10 minutes. All remaining steps in the protein purification were carried out at 4°C and all buffers were kept at 4°C. The lysed cells were spun at 12000-14000 rpm for 70 minutes to clear the lysate. The cleared lysate was decanted into a fresh tube and volume adjusted to 24mL with lysis buffer.
25 mL Qiagen Ni-NTA Sepharose CL-6B slurry was added to the cleared lysate and incubated for 40 minutes with rocking. The His-a2 I Domain bound Ni-NTA beads were pelleted by spinning at 1500 rpm for 5 minutes. The unbound protein was decanted off and 35 mL Lysis Buffer was added to the Ni-NTA pellet. The his-a2 I Domain bound Ni-NTA Sepharose was washed with Lysis Buffer for 5 minutes with rocking, spun at 1500 rpm for 5 minutes, and then unbound material removed by decanting. The Lysis Buffer washing was repeated two more times as described above.
After the last Lysis Buffer wash, 35 mL of Wash Buffer (50 mM Tris pH 8.0, 250 mM NaCl, 10 mM Imidazole) was added to the His-a2 I Domain bound Ni-NTA
Sepharose pellet and incubated for 35 minutes with rocking. The Wash Buffer slurry was pelleted by spinning at 1500 rpm for 5 minutes and unbound material removed by decanting. The Ni-NTA Sepharose was washed with an additional 35mL Wash Buffer for 5 minutes with rocking, spun at 1500 rpm for 5 minutes, and then unbound material removed by decanting. The Wash Buffer washing was repeated three more times as described above.
The His-a2 I Domain protein was eluted from the Ni-NTA beads by addition of
10 mL Elution Buffer (50 mM Tris pH 8.0, 150 mM NaCl, 500 mM Imidazole), rocking for 20 minutes, spinning 2500 rpm for 5 minutes, and eluted material removed by pipetting. This process was repeated two more times and the three elutions were then pooled. The pooled elutions were dialysed against 1.5 L Storage Buffer (35 mM Tris pH 7.5, 150 mM NaCl, 0.025% Tween-20) or PBS (without cations) using a Thermo-Fisher Slide-A-Lyzer G2 3.5K MWCO cassette for 16 hours, then dialyzed against a fresh 1.5 L of appropriate buffer for an additional 6.5 hours. The dialyzed protein was run on a 10- 20% Tris SDS PAGE gradient gel (Invitrogen) and stained with Bio-Rad Bio-Safe Coomassie Stain to determine purity.
The resulting dialyzed His-a2 I Domain protein was determined to be -75-80% pure by gel electrophoresis. The dialyzed fractions were pooled and repurified utilizing lower amounts of Ni-NTA Sepharose and higher binding stringency to drive purification to a higher purity. Imidazole and NaCl were added back to the dialyzed protein to a final concentration of 2 mM and 250 mM, respectively. 5.3mL Qiagen Ni-NTA Sepharose CL-6B was added to the protein and incubated for 40 minutes with rocking. The sample was spun at 1500rpm for 5 minutes and unbound material was decanted off. 35mL of Wash Buffer (50mM Tris pH 8.0, 250mM NaCl, lOmM Imidazole) was added to the his- α2 I Domain bound Ni-NTA Sepharose pellet and incubated for 20 minutes with rocking. The Wash Buffer slurry was pelleted by spinning at 1500rm for 5 minutes and unbound material removed by decanting. The Ni-NTA Sepharose was washed with an additional 35mL Wash Buffer for 20 minutes with rocking, spun at 1500rpm for 5 minutes, and then unbound material removed by decanting. The Wash Buffer washing was repeated two more times with 20 minute incubations.
The His-a2 I Domain was eluted from the Ni-NTA beads by addition of lOmL Elution Buffer (50mM Tris pH 8.0, 150mM NaCl, 500mM Imidazole), rocking for 20 minutes, spinning 2500rpm for 5 minutes, and eluted material removed by pipetting.
This process was repeated two more times and the three elutions were then pooled. The pooled elutions were dialysed against 1.5L Storage Buffer (35mM Tris pH 7.5, 150mM NaCl, 0.025% Tween-20) or PBS (without cations) using a Thermo-Fisher Slide-A-Lyzer G2 3.5K MWCO cassette for 16 hours, then dialyzed against a fresh 1.5L of appropriate buffer for an additional 5 hours. The dialyzed protein was run on a 10-20% Tris SDS Page gradient gel (Invitrogen) and stained with Bio-Rad Bio-Safe Coomassie Stain and determined to be -95% pure. The protein concentrations were determined with Pierce BCA protein Reagent per manufacturer's instructions. Example 2: Binding of Purified α2 I Domain protein
The purified protein was utilized in ELISA based assays to determine its ability to recognize antibodies and collagen. To this end, the PBS (without cations) dialyzed α2 I Domain protein was biotinylated.
To obtain biotinylated α2 I Domain, four 700 μΐ reactions each containing -5.18 nmoles of α2 I Domain protein and 259 nmoles of resuspended Sulpho-NHS-Biotin (Thermo Fisher) were incubated on ice for 3 hours. Each of the four reactions were desalted and unincorporated Sulpho-NHS-Biotin removed by spinning over a PBS (without cations) pre-equilibrated Zeba Spin column (Thermo Fisher) per manufacturer's instructions. The four cleaned samples were pooled and protein concentration was determined with Pierce BCA Protein Reagent per manufacturer's instructions.
Biotinylated a2 1 Domain Binding to Antibodies:
Costar 3631 -96 well plates were coated overnight at 4°C with 100 μΐ of 10 μg/ml antibodies or 100 μΐ of 1% BSA per well. Monoclonal antibodies used in this ELISA were mouse anti-human a2 12F1 (Becton Dickenson), mouse anti-human a2 Gi9
(Abeam), and as a non-specific antibody, mouse anti-human PAR-1 (Becton Dickenson). After overnight absorption onto the plate, unbound material was removed with aspiration, and the wells were washed with 3x300 μΐ PBS (with cations) + 0.05% Tween-20. Each well was blocked with 300 μΐ 0.5X BLOCK ACE (Fisher) diluted from IX BLOCK ACE 1 : 1 in PBS (with cations) for 2 hours at room temperature. The blocking reagent was removed by aspiration and wells were washed with 3x300 μΐ PBS (with cations) + 0.05% Tween-20 with a two minute incubation after the last wash before aspiration.
Biotinylated α2 I domain protein was diluted to a final concentration of 2nM in PBS (with cations) + 1% BSA. Blocked plates were incubated with ΙΟΟμΙ of 2nM α2 I domain protein for 30 minutes with gentle shaking at room temperature. Unbound biotinylated α2 I domain protein was removed by aspiration, and wells were washed 6x300 μΐ PBS (with cations) + 0.05% Tween-20 with ~1 minute incubations prior to aspiration. Streptavidin-HRP(50 μΐ) (R&D Systems) was diluted in lOmL PBS (with cations) + 1%BSA, and 100 μΐ was incubated in each well for 20 minutes at room temperature with gentle shaking. Unbound Streptavidin-HRP was removed by aspiration, and wells were washed 6x300 μΐ PBS (with cations) + 0.05%> Tween-20 with ~1 minute incubations prior to aspiration. Equal volumes of HRP substrate color reagents (R&D
Systems) H202 and tetramethylbenzidine were mixed and 100 μΐ added to each well. The reaction was allowed to proceed for 20 minutes at RT with gentle shaking and stopped by the addition of 50 μΐ 2N H2S04. The absorbance was read at 450 nm using a
FlexStation3 multi-purpose plate reader. As shown in FIG. 4, the 2nM biotinylated α2 I Domain protein was capable of recognizing a2 antibodies 12F1 and Gi9, and no binding was observed to either the control antibody or to BSA.
Biotinylated a2 1 Domain Binding to Collagen:
The ability of the biotinylated α2 I Domain to bind collagen was determined with an ELISA assay. The α2 I Domain requires Mg2+ cations to bind to collagen and this was verified in the assay as a control. Separate reactions were run with cations and without cations. BSA control reactions were performed in the presence of cations.
Costar 3631 -96 well plates were coated overnight at 4°C with 100 μΐ of
100μg/ml human placenta Collagen I (Becton Dickenson) diluted in PBS (+/- cations) or 1% BSA. After overnight absorption onto the plate, unbound material was removed with aspiration, and the wells were washed with 3x300 μΐ PBS (+/- cations as appropriate) + 0.05% Tween-20. Each well was blocked with 300μ1 0.5X BLOCK ACE diluted from IX BLOCK ACE 1 : 1 in PBS (+/-cations as appropriate) for 2.5 hours at room
temperature. The blocking reagent was removed by aspiration and wells were washed with 3x300 μΐ PBS (+/- cations as appropriate) + 0.05%> Tween-20. Serial dilutions of biotinylated α2 I domain protein were made in either PBS (with cations) + 2mM MgCl2 or PBS (without cations) + lOmM EDTA starting at 400 nM protein. Wells were then incubated with 100 ul of serially diluted α2 I Domain protein for 1.25 hours with gentle shaking at room temperature. Unbound biotinylated α2 I Domain protein was removed by aspiration, and wells were washed 6x300 μΐ PBS (+/-cations as appropriate) + 0.05%> Tween-20. At this point, unbound α2 I Domain protein has been removed and the presence of cations should be irrelevant. Streptavidin-HRP(50uL) (R&D Systems) was diluted in 10 mL PBS (with cations) + 1%BSA and 100 μΐ was incubated in wells for 20 minutes at room temperature with gentle shaking. Unbound Streptavidin-HRP was removed by aspiration, and wells were washed 6x300 μΐ PBS (with cations) + 0.05%> Tween-20 with ~1 minutes incubations prior to aspiration. Equal volumes of HRP substrate color reagents (R&D Systems) H202 and tetramethylbenzidine were mixed and 100 ul added to each well. The reaction was allowed to proceed for 20 minutes at RT with gentle shaking and stopped by the addition of 50 μΐ, 2N H2SO4. The absorbance was read at 450 nm using a FlexStation3 multi-purpose plate reader.
As shown in FIG. 5, the biotinylated I Domain protein is capable of binding to collagen in a concentration dependent manner. As expected, the removal of cations from the reaction drastically reduces its ability to bind collagen.
Example 3: Identification of Nucleic Acid Ligands to Alpha2 I Domain
The SELEX method was used to obtain aptamers which bind the α2 I Domain as described and illustrated in FIG. 1.
A starting candidate DNA library was generated by heat annealing and snap- cooling 1 nmole of template DNA oligo and 1.5 nmoles of 5' DNA primer oligo. The sequence of the DNA template oligo for designing the candidate mixture are: 5'- TCTCGGATCC TCAGCGAGTC GTCTG(N40)CCGCA TCGTCCTCCC TA-3' (SEQ ID NO: 9) (N4o represents 40 contiguous nucleotides synthesized with equimolar quantities of A, T, G and C), the 5' primer oligo and 3' primer oligo are, respectively, 5'- GGGGGAATTC TAATACGACT CACTATAGGG AGGACGATGC GG-3' (SEQ ID NO: 10) (T7 promoter sequence is in bold), and 5'-TCTCGGATCC TCAGCGAGTC GTCTG-3'. (SEQ ID NO: 11) The reaction was filled in with Exo" Klenow, stopped by addition of EDTA to a final concentration of 2 mM, and extracted with PCI
(phenol:chloroform:isoamyl alcohol (25 :24: 1)) and then extracted with chloroform:
isoamyl alcohol (24: 1). The extract was desalted, concentrated, and unincorporated nucleotides removed with an Amicon 10 spin column. The DNA template was utilized in a transcription reaction to generate a 2'-fluoropyrimidine starting library. In vitro transcription conditions were 40 mM Tris-HCl pH 8.0, 4% PEG-8000, 12 mM MgCl2, 1 mM spermidine, 0.002% Triton, 5 mM DTT, lmM rGTP, lmM rATP, 3mM 2'F-CTP, 3mM 2'F-UTP, 8μg/mL inorganic pyrophosphatase, 0.5 μΜ DNA library, and Y639F mutant T7 polymerase. Transcriptions were incubated overnight at 37°C, DNase treated, chloroform:isoamyl alcohol (24: 1) extracted twice, concentrated with an Amicon 10 spin column, and gel purified on a 12% denaturing PAGE gel. RNA was eluted out of the gel, and buffer exchanged and concentrated with TE (10 mM Tris pH 7.5, O.lmM EDTA) washes in an Amicon 10 spin column. The α2 I Domain selection started with a complex library of ~10 different 2'- fluoropyrimidine RNA sequences. The complex RNA pool was precleared against a biotin-PEG6-His6 peptide, immobilized on magnetic streptavidin beads. The precleared RNA was precleared against nitrocellulose, and then bound to the purified recombinant N-term His6 tagged α2 I Domain protein (SEQ ID NO:4).
Initial α2 I Domain ligand selection was performed in binding buffer "E-M" at 37°C. Binding buffer E-M consists of 20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM MgCl2, 0.015% BSA, and 0.001%tRNA. tRNA was included to reduce non-specific binding of the RNA pool to the α2 I Domain protein. Protein-RNA complexes were partitioned over a 25 mm nitrocellulose disc with washing. The bound RNA was extracted off the nitrocellulose disc with incubation in PCI (25 :24: 1). Water was added and the aqueous phase extracted, followed by a chloroform:isoamyl alcohol (24: 1) extraction. The resultant bound RNA was ethanol precipitated. One quarter of the precipitated RNA was heat annealed to the 3 ' primer and reverse transcribed utilizing AMV RT. The entire RT reaction was utilized in PCR with 5' and 3' primers and standard PCR conditions to generate DNA template for the next round of RNA generation. Selection conditions are outlined in FIG. 6.
Enrichment of the aptamer libraries for α2 I Domain was monitored in direct binding studies utilizing radiolabeled aptamer RNA from respective rounds of SELEX and α2 I Domain. Binding studies were performed with trace P32 end-labeled RNA added to serial dilutions of α2 I Domain in Binding Buffer E-M. To prepare radiolabeled RNAs for binding studies, one hundred picomoles of RNA was dephosphorylated with Bacterial Alkaline Phopshatase at 50°C for 1 hour. The reaction was
phenol:chloroform:isoamyl alcohol (25:24: 1) extracted, chloroform: isoamyl alcohol (24: 1) extracted, and ethanol precipitated. Three pmoles of dephophorylated RNA was end labeled with T4 Poylnucleotide Kinase with supplied buffer, and 20μΟ of γ-Ρ32-ΑΤΡ and subsequently cleaned with a Biorad MicroSpin P-30 spin column. End-labeled RNA was diluted to a final concentration of 2000 cpm^L and heat denatured at 65°C for 5 minutes. RNA and α2 I Domain dilutions were equilibrated at 37°C prior to use. RNA (5 μί) was added to varying concentrations of α2 I Domain (15 μί) at 37°C and incubated together for 5 to 15 minutes. The complexed RNA/a2 I Domain protein mixture was then loaded over a Protran BA85 nitrocellulose membrane, overlayed on a Genescreen Plus Nylon membrane, in a 96 well vacuum manifold system with washing. The membranes were exposed to a phosphorimager screen, scanned, and quantitated with a Molecular Dynamics Storm 840 Phosphorimager. The fraction bound was calculated by dividing the counts on the nitrocellulose by the total counts and adjusting for the background. The progression of the α2 I Domain I selection is shown in FIG. 7.
Example 4: Sequencing and Identification of a Structural Family of α2 1 Domain Nucleic Acid Ligands
The final PCR products representing anti-a2 I Domain enriched ligand libraries from the SELEX experiments described in Example 3 are digested with the appropriate restriction enzymes, cleaned with a purification kit, and directionally cloned into linearized pUC19 vector. Bacterial colonies are streaked for single clones and 5 mL overnight cultures are inoculated from single colonies. Plasmid DNA is prepared from single colonies using Qiagen Plasmid Mini Prep kits. DNA sequences derived from the random region are analyzed by various methods, including alignment of the selected sequences to identify unique sequences as well as the frequency of each unique sequence.
The cloning of R7 revealed that the round was highly focused with two sequences being present out of the 59 obtained sequence reads. The DNA sequences of the random region with their relative frequencies are shown in Table 1 below.
Table 1 : Sequences from R7 with % Frequency
Figure imgf000073_0001
Alignment also serves to identify conserved primary sequences shared by each of the selected clones.
Screening of sequences for potential secondary structure is conducted utilizing the mfold server (mfold.bioinfo.rpi.edu). A description of these methods is found on the server site as well as in M. Zuker (2003) "Mfold web server for nucleic acid folding and hybridization prediction." Nucleic Acids Res. 31 (13), 3406-15 and D.H. Mathews, et al. (1999) "Expanded Sequence Dependence of Thermodynamic Parameters Improves Prediction of R A Secondary Structure" J. Mol. Biol. 288, 911-940. Subsequently, comparative sequence analysis of the unique sequences is used to align the sequences based upon conserved consensus secondary structural elements to arrive at the predicted secondary structure of the anti-a2 I Domain ligands.
The affinity of each of the anti-a2 I Domain ligands for α2 I Domain is determined by direct binding studies using radiolabeled trace ligand RNA and soluble a2 I domain, per the binding methodology described above in Example 3. The anti-a2 I Domain ligands had a moderate affinity for the α2 I domain in the buffer conditions the selection was performed (20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM MgCl2, 0.015% BSA, and 0.001%tRNA). However, the anti-a2 I Domain ligands exhibited very poor binding affinities to the α21 domain under the desired physiological condition of 150mM NaCl.
Example 5: Truncation and Mutational Probing of anti-a2 I Domain Ligand Structure
Identification of conserved secondary structures of the anti-a2 I Domain ligands according to methods described above allows reliable predictions as to the minimal sequence required to form the structure and to bind with high affinity to a2. Initial experiments are aimed at defining the 5' and 3' boundary sequence requirements for maintaining an ligand having the desired structure and function.
Truncated compounds for several of the anti-a2 I Domain ligands containing 5' and 3 ' required sequence boundaries as predicted using the methods described above are prepared and their affinity for α21 Domain determined.
Example 6: Further Truncation and Optimization of the 2' Sugar Modification and Phosphodiester Backbone of Anti-a2 1 Domain Ligand
Ligands isolated from 2'-fluorpyrimidine/2'-hydroxypurine libraries which exhibit sufficient nuclease stability are selected for in vitro screening. However, the high 2'-hydroxyl content make them unsuitable for drug development candidates due to the fact that these positions can be very sensitive to nuclease degradation in vivo, limiting the maximal concentration that can be achieved post parenteral administration as well as their circulating half-life. Therefore, optimization of the anti-a2 I Domain ligands is performed by further stabilization of the backbone by substitution of 2'-0-methyl nucleotides for 2 '-hydroxyl nucleotides, or by substitution of 2'-deoxy nucleotides for 2'- hydroxyl nucleotides, with modification of the ligand backbone by phosphorothioate substitution as needed to preserve affinity for the a2I Domain while enhancing nuclease stability. Additional substitutions of 2'-0-methyl nucleotides for 2'-fluoro nucleotides are also made to further improve stability, reduce cost of manufacturing, and reduce the level of potential impurities that can arise during heating of 2'-fluorouridine-containing oligonucleotides during manufacturing processes. Finally, "capping" of the 5' and 3' ends, which prevents exonuclease degradation of oligonucleotides, is also performed to further enhance in vivo stability.
Capping of the 3' end of a α2 I Domain ligand molecule is accomplished by synthesis of the ligand from a CPG-support loaded with inverted deoxythymidine, to create a 3 '-3' linkage at the 3 'end of the ligand. If a modification is well tolerated, it is then used in all synthetically produced modifications to the α2 I Domain ligand.
In addition to the extent of nuclease stabilization, distribution and half-life of ligands post parenteral administration is greatly impacted by their molecular weight. Conjugation of ligands to high molecular weight carriers, such as high molecular weight polyethylene glycol (PEG), limit the distribution of an ligand to mainly the plasma compartment, leading to higher Cmax per dose unit, and greatly limit renal filtration of the ligand, and thus greatly enhance the ligand's in vivo potency and circulating half-life. Given that a control agent may be administered to finely tune the potency and half-life of an anti-a2 I Domain ligand, anti-a2 I Domain ligands are conjugated to a high molecular weight carrier to provide the greatest potential half-life with distribution mainly limited to the plasma compartment. PEGylation of ligands can be achieved by conjugation of the PEG to a unique site on the ligand, added by incorporation of a site-specific linker to the ligand during synthesis. Therefore, the impact of linker addition and PEG conjugation to one or more α2 I Domain ligands is assessed.
Example 7: Methods for Evaluating Antiplatelet Activity, Specificity of Activity, and Modulation of Activity of anti-a2 1 Domain Ligands
A. Collagen-Induced Platelet Aggregation (CIPA) Assay in PRP and WP 1. Platelet Rich Plasma Preparation (PRP) and Aggregation Studies:
Human platelet-rich plasma (PRP) is prepared from fresh whole blood collected in 60 ml syringes using 0.3 mM PPACK in saline (9: 1 blood: anticoagulant saline mix; Biomol Cat# PI1117) as an anticoagulant. The blood is centrifuged at low speed centrifugation (250xg) in 50 ml conical tubes for 16 minutes. The platelet rich plasma separated from the blood cells by centrifugation is removed using 10 ml serological pipettes and platelet poor plasma (PPP) is prepared from leftover blood by high speed centrifugation at 2200xg for 10 minutes. The PPP is removed and saved for the light transmission aggregometry (LTA) blank.
Platelet aggregation in PRP is monitored using 450 of PRP (plus 25
Figure imgf000076_0001
of
Saline) at 37°C (stirred at 1200 rpm) in a Chrono-Log (Havertown, PA) lumi- aggregometer for 6 minutes. Aggregation is initiated using 25 μΐ, of collagen as agonist. 500 μΐ, of platelet-poor plasma (PPP) is used as baseline in the aggregometer. For screening anti-a2 I Domain ligands for the ability to block CIPA, 450 of PRP is incubated with 25 μί of solution containing the anti-a2 I Domain ligand, at a
concentration to yield the desired final concentration, for 3 minutes at 37°C in the aggregometer cell with constant stirring at 1200 rpm before addition of the agonist collagen. Platelet aggregation is initiated by the addition of indicated concentrations of collagen (Rat Tail Collagen Type -1) to yield a percent aggregation between 70-90%, and the light transmission is continuously recorded for 4-6 min.
2. Washed Platelet Preparation (WP) and Aggregation Studies:
Human washed platelets are prepared essentially as described by Mustard et al. (1972; Br.J.Haematol 22, 193-204). Briefly, human blood is collected into one-sixth volume of acid/citrate/dextrose (ACD) buffer (85mM sodium citrate, 65mM citric acid, and 1 lOmM glucose), placed in a water bath at 37°C for 30 minutes then centrifuged at 250xg for 16 minutes at room temperature. Platelet-rich plasma is removed and centrifuged at 2200xg for 13 minutes at room temperature then resuspended in 40 mL of HEPES-buffered Tyrode's solution (136.5 mM NaCl, 2.68 mM KC1, 1 mM MgCl2, 2 mM CaCl2, 12 mM NaHC03, 0.43 mM NaH2P04, 5.5 mM glucose, 5 mM HEPES pH 7.4, 0.35% bovine serum albumin) containing lOU/mL heparin and 5 μΜ (final concentration) prostaglandin I2 (PGI2). The platelet suspension is incubated in a 37°C water bath for 10 minutes, 5 μΜ (final concentration) PGI2 added and the mixture centrifuged at 1900xg for 8 minutes. The resulting pellet is resuspended in 40 mL of HEPES -buffered Tyrode's solution containing 5 μΜ (final concentration) PGI2 and then incubated for 10 minutes in a 37°C water bath, and centrifuged at 1900xg for 8 minutes. The pellet is resuspended at a density of 3 x 108 platelets/mL in HEPES-buffered
Tyrode's solution containing O. lU/mL potato apyrase and incubated in a 37°C water bath for 1 hr prior to use in aggregometry studies.
Collagen-induced WP platelet aggregation is determined by measuring the transmission of light through a 0.5 ml suspension of stirred (1200 rpm) washed platelets (425 ul washed platelets, 25 μΐ fibrinogen, 25 μΐ of inhibitors or controls and 25 μΐ of collagen) in a lumi-aggregometer at 37°C (Chrono-Log Corp. Havertown, PA). The baseline of the instrument is set using 0.5 ml of Hepes-buffered Tyrode's solution. Prior to aggregation measurements, the platelet suspension is supplemented with 1 mg/ml fibrinogen. Platelet aggregation is initiated by the addition of indicated concentrations of Collagen (Rat Tail Collagen Type-1) to yield a percent aggregation between 70-90%, and the light transmission is continuously recorded for at least 6 min. For screening anti-a21 Domain ligands or controls (various mutants of ligands) for the ability to block CIPA, anti-a21 Domain ligands are added to the platelet suspension at a concentration to yield the desired final concentration, and incubated for 3 min before addition of collagen, and the response is recorded for 4-6 min after collagen addition.
The potency of collagen is determined for each donor from the maximal extent of percentage aggregation obtained from a dose response curve using 2X serial dilution of 4 μg/ml of collagen in saline, and a challenge concentration is determined. The ability of anti-a21 Domain ligands to inhibit CIPA is tested in both WP and PRP preparations as described above, using a broad range of anti-a2I Domain ligand concentrations (2 μΜ - 7.8 nM).
B. In vitro Flow Based Platelet Adhesion Assay in Whole Blood for anti-cc2 I Domain Ligand activity using Bioflux™ 200 (Fluxion Biosciences, Inc.,)
I. Preparation of the test plate with collagen coating: For the flow experiments, Bio flux 48 well plates (P/N 0009-0013) are routinely used. The plates are primed with 0.02 M acetic acid for 5 min at 5 dyn/cm2, and then 100-200 μ§/ι 1 of diluted Rat Tail Collagen Type 1 in 0.02 M acetic acid is perfused from the inlet well for 10 min at 5 dyn/cm2. The flow is stopped and the plate incubated at room temp for 1 hour. The collagen is washed with PBS at 5 dyn/cm2 for 10 min. The collagen coated plate is then blocked by completely filling the outlet well (1ml) with PBS +5%BSA w/v and perfusing the solution into the channel at 5 dyn/cm2 for 15 min. The flow is stopped and the plate incubated for an additional 10 minutes at room temp.
Excess PBS+BSA is removed from all wells and the plate is kept at room temperature for same day use or kept at 4°C in PBS+BSA (up to two weeks).
2. Whole blood preparation for perfusion and the flow experiment:
The blood is drawn from healthy volunteers into PPACK (0.3 mM) anticoagulant into 60 mL syringes using a 193/4 gauge needle. The blood is immediately fluorescently labeled with 4μΜ Calcein-AM (Invitrogen P/N C3100 MP) for 1 hr at 37°C (Calcein-AM is added to the blood very gently by inverting the tube a few times to mix and the blood is used within 3.5 hrs of draw). 20μ1 of 40μΜ Sel2 pool, R7-24 or R7I-43 RNA was added to 200μ1 of Calcein labeled blood and incubated at room temperature for 5 minutes. The flow experiment is initiated by adding 200
Figure imgf000078_0001
of labeled blood on top of the outlet well and perfusion begun immediately using 5 dyn/cm2 whole blood flow settings at 37°C using Bioflux™ software. The data (fluorescence images of platelet aggregates) is collected using a time lapse fluorescence inverted microscope (Zeiss 200M Axiovert Microscope attached to an Axiocam Charged-Coupled Device camera and Axiovision software) every 6 seconds for a total duration of 6 minutes. . The tagged image file (tiff) formatted images are used to calculate fluorescence intensity using Bioflux Montage™ software and then the data is exported to Microsoft Excel and plotted using Graphpad
Prism. Data is normalized to the fluorescent signal observed in the control chamber at the time at which the control chamber is occluded by fluorescent platelet aggregates (defined as the maximum platelet response). As can be seen in Fig. 8 no reduction of platelet adhesion due to the presence of RNA ligand was observed under these conditions.
C. Static Adhesion Assay in the Presence of Anti-cc21 Domain Ligands 96-Well plates are coated with 100 μΐ per well of Rat Tail Collagen Type I or the peptide GFOGER-GPP at 10 μ^πιΐ in 0.01 m acetic acid for 1 h at 20 °C. Platelet-rich plasma is prepared from fresh whole blood after 2 spins for 1 min at 1200 x g. 10% (v/v) of ACD buffer (39 mM citric acid, 75 mM tri-sodium citrate-2H20, 135 mM D-glucose, pH 4.5) and prostaglandin El (100 ng/ml final concentration) are added, and the platelet- rich plasma is spun for 6 min at 700 x g. The platelet pellet is resuspended in 6 ml of buffer (5.5 mM D-glucose, 128 mM NaCl, 4.26 mM Na2HP04-2H20, 7.46 mM
NaH2P04-2H20, 4.77 mM tri-sodium citrate-2H20, 2.35 mM citric acid, 0.35% bovine serum albumin (BSA), pH 6.5). Prostaglandin El is added as before, and the platelets are spun for 6 min at 700 x g. Platelets are resuspended to 2 x 108 platelets/ml in adhesion buffer (0.05 m Tris-HCl, 0.14 M NaCl, 0.1% BSA, pH 7.4) and treated as appropriate with MgCl2, CaCl2, or EGTA and allowed to rest for 15 min at room temperature.
Ligand-coated wells are blocked by incubation with 200 μΐ of blocking buffer (0.05 M Tris-HCl, 0.14 M NaCl, 5% BSA, pH 7.4) for 30 min. The wells are washed 3 times with 200 μΐ of adhesion buffer, then 50 μΐ of platelet suspension (107 platelets) are added to each well and left for 1 h. The wells are emptied and washed 3 times with 200 μΐ of adhesion buffer to remove non-adherent platelets. Adherent platelets are lysed by incubation for 1 h with 150 μΐ per well of lysis buffer (0.07 M tri-sodium citrate, 0.3 M citric acid, 0.1% Triton X-100 (v/v), 5 mM /?-nitrophenyl phosphate). The reaction is terminated by the addition of 100 μΐ of 2 M NaOH to each well. Adhesion is measured colorimetrically as the absorbance of the /?-nitrophenol product at 405 nm in a Maxline Emax microplate reader (Molecular Devices Ltd., Crawley, UK).
D. ADP Induced Platelet Aggregation (AIPA) Assay, TRAP Induced Platelet Aggregation (TIP A), Arachidonic Acid Induced Platelet Aggregation (AAIPA) Assay, and Ristocetin Induced Platelet Aggregation (RIP A) Assay for Evaluation of anti-cc21 Domain Ligand Specificity for α2βι The specificity of the anti-a21 Domain ligands for α2βι is determined by assessing their effect on induction of platelet aggregation by agonists whose function is mediated through other well-characterized platelet receptors. For these studies, human PRP and WP preparations are used as needed to assess the activity of the various agonists. ADP is used as a specific agonist of the P2Yi2 and P2Yi receptors, TRAP as an agonist of PAR- 1, Arachidonic Acid as an agonist of the thromboxane A2 (TXA2) receptor and Ristocetin as an agonist of the vWF-GPlba interaction. The potency of each agonist (ADP, TRAP, Arachidonic Acid, and Ristocetin) is determined from the maximal extent of percentage aggregation obtained from a dose response curve for the respective agonist, and a challenge concentration is determined to target EC70-90 % for each respective agonist.
1. Specificity Determination of the a/?tz-a21 Domain Nucleic Acid Ligands by AIPA and TIP A in WP.
For evaluating the potential interaction of anti-a21 Domain ligands with P2Yi2 and P2Yi, a challenge concentration of 5 μΜ ADP is typically used to stimulate platelet aggregation, and for evaluating their potential interaction with PAR-1, a challenge concentration of 2.5μΜ TFLLRN (TRAP) is typically used to stimulate platelet aggregation (the specific challenge agonist concentration for each experiment is determined based on the agonist dose response curve for each donor). ADP and TRAP - induced platelet aggregation is determined in WP preparations as described above.
Specific inhibitors for each receptor are used as positive controls to demonstrate that inhibition of the target receptor is detectable in the assays. SCH79797 (Tocris
Biosciences) is used as the positive control for PAR-1 antagonism, and INS50589 (Inspire Pharmaceuticals) is used as the positive control for P2Yi2 antagonism.
2. Specificity Determination of the α21 Domain Nucleic Acid Ligands by RIP A in
PRP:
For evaluating the potential interaction of anti-a21 Domain ligands with vWF or GPlba, a challenge concentration of 1.0-2.0 mg/mL Ristocetin (Sigma Cat# R7752) is used to stimulate platelet aggregation (the specific challenge agonist concentration for each experiment is determined based on the agonist dose response curve for each donor). Ristocetin-induced platelet aggregation is determined in PRP preparations as described above. HIP1 antibody to GPlba (or isotype IgG control, Axxora Bio; 25 μg/ml final) is used as a positive control to demonstrate that inhibition of the target receptor GPlba is detectable in the assays.
3. Specificity Determination of the α2 I Domain Nucleic Acid Ligands by AAIPA in PRP: For evaluating the potential for interaction of the anti-a21 Domain ligands with TXA2 receptor, a challenge concentration of 0.25-0.5 mg/ml Arachidonic Acid (Helena Biosciences; Cat# 5364) is used to stimulate platelet aggregation (the specific challenge agonist concentration for each experiment is determined based on the agonist dose response curve for each donor). Arachidonic Acid-induced platelet aggregation is determined in PRP preparations as described above.
F. Collagen and CRP-Induced Platelet Aggregation Assays for Testing of Nucleic Acid Modulators
Figure imgf000081_0001
Domain Ligands in WP and PRP:
Collagen-induced platelet aggregation was carried out in WP and PRP preparations as described above. For evaluation of the ability of nucleic acid modulators to reverse the inhibition of platelet aggregation by anti-a21 Domain ligands, anti-a21 Domain ligand concentration are tested at their IC95_ioo (the ligand concentration necessary to inhibit by 95-100% the aggregation elicited by a given concentration of challenge agonist). Platelet aggregation studies are performed as described above, except that after initial incubation of the platelet preparation with the anti-a2 i ligand, varying amounts of modulator are added, targeting a molar excess of modulator to ligand ranging from 8: 1 to 0.5: 1, and incubated together for 10 minutes before addition of agonist. E. Durability Study of anti-cc21 Domain Ligand Reversal by Nucleic Acid Modulators in WP:
For evaluating the durability of reversal of anti-a21 Domain activity by nucleic acid modulators, an anti-a2 I Domain ligand molecule and a modulator designed to bind the ligand molecule are added to a total volume 4 ml of WP suspension at 37°C (order and timing of addition as described above for evaluating nucleic acid modulators), 450 μΐ, aliquots of the WP suspension mixture are removed at indicated time points (0, 0.16, 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 hrs), and collagen-induced platelet aggregation performed. To demonstrate the activity of the WP suspension, activity of the ligand, and lack of interference by the modulator over the duration of this incubation, separate incubations are conducted over the 3.5 hr time period in which buffer alone, ligand alone or modulator alone are added to WP suspensions, and collagen-induced platelet aggregation determined. All references provided herein are incorporated by reference herein in their entirety. The invention is not to be understood as restricted to the details described herein, since these may be modified within the scope of the appended claims without departing from the spirit and scope of the invention.

Claims

CLAIMS We claim:
1. A nucleic acid ligand that binds α2βι, or a pharmaceutically acceptable salt thereof, wherein said ligand comprises a nucleic acid sequence and wherein said nucleic acid sequence forms at least one stem structure and at least one loop structure.
2. The ligand of claim 1, wherein the nucleic acid sequence comprises a 2' modified nucleotide.
3. The ligand of claim 1, wherein the ligand comprises a modified phosphate backbone.
4. The ligand of claim 1, wherein the ligand is conjugated to a carrier.
5. The ligand of claim 4, wherein the carrier is a hydrophilic moiety.
6. The ligand of claim 5, wherein the hydrophilic moiety is polyethylene glycol.
7. A modulator capable of binding to the nucleic acid ligand of any of claims 1-6 that upon binding modifies the interaction between the ligand and a target molecule to which the ligand is capable of binding.
8. The modulator of claim 7 wherein the modulator is an oligonucleotide or analog thereof that is complementary to at least a portion of a sequence of the nucleoic acid ligand.
9. The modulator of claim 7 wherein the modulator is selected from peptide nucleic acids (PNA), morpholino nucleic acids (MNA), or locked nucleic acids (LNA); nucleic acid binding proteins or peptides; oligosaccharides; small molecules; or nucleic acid binding polymers, lipids, nanoparticle, or microsphere-based modulators.
10. A pharmaceutical composition suitable for effective administration comprising an effective amount of ligand or modulator according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof.
11. A method for treating symptoms of a a2Pi-mediated disorder comprising
administering to a host in need thereof a therapeutically effective amount of the ligand according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.
12. The method of claim 11, wherein the a2 i-mediated disorder is a vascular disorder, inflammatory disorder , viral infection, autoimmune disorder, a cancer or a metastatic cancer.
13. The method of claim 11 wherein the ligand is administered by parenteral (by injection or gradual infusion over time), intravenous, intradermal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular, intraspinal, intrasternal, topical, transdermal patch, via rectal, buccal, vaginal or urethral suppository, peritoneal, percutaneous, nasal spray, surgical implant, internal surgical paint, infusion pump or via catheter routes.
14. The method of claim 13 wherein , the α2βι nucleic acid ligand is delivered via subcutaneous administration and the modulator is delivered by subcutaneous or intravenous administration.
PCT/US2013/064829 2012-10-24 2013-10-14 Nucleic acid modulators of alpha2beta1 Ceased WO2014066084A1 (en)

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Citations (3)

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WO2005106042A2 (en) * 2004-04-22 2005-11-10 Regado Biosciences, Inc. Improved modulators of coagulation factors
US20060177443A1 (en) * 2000-02-25 2006-08-10 Immunex Corporation Integrin antagonists

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US20060177443A1 (en) * 2000-02-25 2006-08-10 Immunex Corporation Integrin antagonists
US20050069910A1 (en) * 2001-06-29 2005-03-31 Turner John V. Nucleic acid ligands to complex targets
WO2005106042A2 (en) * 2004-04-22 2005-11-10 Regado Biosciences, Inc. Improved modulators of coagulation factors

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