WO2014066142A1

WO2014066142A1 - Nucleic acid regulation of growth arrest-specific protein 6 (gas6)

Info

Publication number: WO2014066142A1
Application number: PCT/US2013/065521
Authority: WO
Inventors: Christopher Rusconi; Juliana M. Layzer; Sanjoy K. Mahanty; Christopher L. DECIANTIS; Katie REDICK
Original assignee: Novamedica Limited Liability Company; Regado Biosciences Inc.
Priority date: 2012-10-24
Filing date: 2013-10-17
Publication date: 2014-05-01
Also published as: EA201690863A1

Abstract

The present disclosure generally relates to a pharmacologic system to modulate the biology of platelets based upon nucleic acid ligands that can interact with and modulate the activity of Growth Arrest-Specific gene 6 (GAS6) protein. These nucleic acid ligands are actively reversible using a modulator that inhibits the activity of the nucleic acid ligand and neutralizes its pharmacologic effect, thereby restoring GAS6 function. Also provided are compositions comprising the nucleic acid ligand, the ligand and a modulator, methods to generate the nucleic acid ligand and its modulator, methods to characterize the nucleic acid ligand, and methods of using these agents and compositions in medical therapeutic and diagnostic procedures.

Description

NUCLEIC ACID REGULATION OF

GROWTH ARREST-SPECIFIC PROTEIN 6 (GAS6)

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application

61/717,949 filed October 24, 2012 the contents of which are incorporated by reference in their entirety.

TECHNICAL FIELD

The present disclosure generally relates to nucleic acid ligands that bind to and regulate Growth Arrest-Specific protein 6 (GAS6). These nucleic acid ligands are also actively reversible using a modulator that inhibits, partially or completely, the activity of the nucleic acid ligand. The disclosure further relates to compositions comprising the nucleic acid ligand and/or a modulator as well as methods of using these agents and compositions for treating symptoms of, or preventing, GAS6-mediated diseases and disorders.

BACKGROUND

Platelets are small, anuclear blood cells which are fairly quiescent under normal conditions but which respond immediately to vascular injury in the form of adhesion, activation, aggregation, and thrombus formation. The primary function of platelets is to stop blood loss and exposure of the subendothelial matrix after tissue trauma. It is well known that damage to a blood vessel can expose extracellular matrix components to the blood, particularly molecules such as 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, platelets have more recently been implicated in a variety of other diseases and disorders including inflammation, tumor growth and metastasis, and immunological host defense. Accordingly, platelet receptor-binding proteins are attractive targets for regulation of platelet function as a means for treating symptoms of, or preventing platelet-mediated disorders. Platelet-mediated disorders include vascular diseases as well as a variety of disorders associated with high-risk diabetes. Inflammatory disorders shown to be platelet-mediated include inflammatory arthritides and scleroderma. The role of inflammation and white blood cell activity in inflammatory joint disease is well-known. More recently, the presence of platelets in synovial fluid of inflamed joints has been identified (Boilard et al., Science, 2010, 327:580-583). Moreover, platelet microparticles in joint fluid from patients suffering from inflammatory arthritis have been shown to be proinflammatory, eliciting cytokine responses from synovial fibroblasts via IL-1.

Growth Arrest-Specific protein 6 (GAS6) is a member of the vitamin K- dependent protein family that binds to the Tyro3 (a.k.a.Sky)/Axl/Mer (TAM) family of related tyrosine kinase receptors on the surface of platelets and vascular endothelium (Gould, W.R., et al., J. Throm. Haemostasis, 2004; 3:733-741; Hafizi, et al., FEBS Journal; 2006, 273:5231-5244). GAS6 is structurally related to the protein S

anticoagulation factor; the amino acid sequences of the two proteins are 43% identical. GAS6 is detected as a 70-85 kDa protein by SDS PAGE analysis. GAS6 is expressed in endothelial cells, vascular smooth muscle cells, bone marrow cells, and platelets, and soluble GAS6 has been identified in human plasma (Balough, I., et al., Arterioscler. Throm. Vase. Biol; 2005, 25: 1-7; Balough, I. ., et al., Arterioscler. Throm. Vase. Biol; 2005, 25: 1280-1286). GAS6 is involved in vascular inflammation and thrombus stability, enhancing and amplifying thrombin-, ADP- or collagen-activation of platelets, perpetuating platelet activation and stabilizing platelet aggregation (Sailer, F., et al., Blood Cells, Molecules, & Diseases; 2006, 36:373-378). Blocking GAS6 or its receptors reduces thrombus size, and impairs clot retraction (Ishimoto, et al. FEBS Letters 466 (2000) 197-199; Angelillo-Scherrer, et al., J. Clin. Invest. 2005 Feb;l 15(2):237-46). Gas6 ^{~ ~} mice exhibit reduced venous and arterial thrombosis and were protected against experimental thrombo-embolism. These mice did not, however, suffer spontaneous bleeding and had normal bleeding times after tail clipping (Angellilo-Scherrer, A., et al., Nature Medicine; 2001, 7:215-2215; Tjwa, M., et al, Blood; 2008, 111 :4096-4105). Anti-GAS6 monoclonal antibodies have been shown to have therapeutic effect, and the impact of GAS6 inhibition was shown to be due to blocking the ability of GAS6 to amplify platelet aggregation and secretion in response to known agonists (Gould, W.R., et al., J. Throm. Haemostasis, 2004; 3:733-741; Sailer, F., et al., Blood Cells, Molecules, & Diseases; 2006, 36:373-378).

GAS6 is also believed to play a role in stimulation of cell proliferation, protection against apoptosis and cancer. GAS6 is present in normal human serum tightly complexed to the Axl protein, but may be released from cells during inflammation, cardiovascular damage or carcinogenesis. The GAS6-Axl interaction protects endothelial cells against apoptosis (Hasanbasic, et al., Am. J. Physiol. Heart Circ. Physiol. 287: 1207-1213, 2004). Recently, it has been shown that the Axl and Mer receptors can be cleaved close to the cell membrane, yielding soluble molecules consisting of the extracellular parts of the receptors. Gas6 protein in human blood circulation is mostly bound to sAxl (soluble Axl) receptor. The Gas6 that is bound to sAxl is functionally inactive, i.e., unable to activate cell bound Axl (Ekman, C, et al, J. thromb. Haemost, 2010, 4: 838-.844). Patients with sepsis (Borgel, D., et. al, Critical Care Medicine, 2006, 34:219-222) and acute pancreatitis have been reported to have highly elevated Gas6 concentrations in serum (Uhara, S., et al, J. gastroenterology and hepatology, 2009, 24: 1567-1573). A novel function of human recombinant Gas6 as a chemoattractant in cultured rat and human vascular smooth muscle cells has been uncovered and shown to be mediated by Gas6-Axl interaction (Yih-Woei, C, et al, J, Biol. Chem. 1998, 273: 7123-7126). Increased expression of of Gas6 and Axl have been observed in different tumors, and reported to have increased Gas6 and sAxl in plasma from patients with renal carcinomas (Gustafsson A., etal, Clin Cancer Res, 2009, 15:4742-4749 ), and Axl and Gas6 are frequently overexpressed in human gliomas tumor vessels (Hutterer, et al., Clin. Cancer Research, 2008, 14(1): 130-138). Very recently Loges et. al, provided new evidence of the regulation and significance of Gas6/Axl activity in cancer. These authors identified a novel tumor promoting mechanism whereby tumors educate tumor -associated macrophages to produce high levels of Gas6, leading to selective induction of tumor growth and metastasis, without interfering with cancer cell survival, tumor associated inflammation, and angiogenesis (Longes, S., et.al, Blood, 2010, 115:2264-2273).

Active control and modulation of GAS6 activity can provide significant clinical benefit. Accordingly, there is a need for therapeutic and modulatable agents designed to specifically target and regulate the function of the GAS6 protein in GAS6-mediated diseases and disorders.

BRIEF SUMMARY

Described herein are nucleic acid ligands, or pharmaceutically acceptable salts thereof, which specifically bind Growth Arrest-Specific protein 6 (GAS6), as well as modulators thereof. Further provided are methods of use for these agents and

compositions containing these agents, including for use in prevention and/or treatment of diseases and disorders mediated by Growth Arrest-Specific protein 6 (GAS6).

In one aspect, a GAS6 ligand is provided, wherein the ligand comprises an isolated nucleic acid sequence. In one embodiment, at least one nucleotide of the isolated nucleic acid sequence is a ribonucleotide. In another embodiment, at least one nucleotide of the isolated nucleic acid sequences a deoxyribonucleic acid. In still another embodiment, the isolated nucleic acid sequence comprises a mixture of ribonucleotides and deoxyribonucleotides.

In one embodiment, the GAS6 ligand comprises an isolated nucleic acid sequence having a secondary structure comprising at least one stem and at least one loop.

In one embodiment, the isolated nucleic acid sequence of the GAS6 ligand 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 30 to about 45 nt in length, about 20 nt to about 35 nt in length, about 20 nt to about 30 nt in length, about 30 nt to about 35 nt in length, about 30 to about 40 nt in length, or about 35 nucleotides in length.

In one embodiment, the ligand binds to the protein GAS6. In one embodiment, the ligand binds to a particular domain of GAS6. In one embodiment, the ligand binds to a GAS6 receptor-binding domain. In another embodiment, the ligand binds to a GAS6 protein that is binding to the TAM receptor on a TAM receptor-expressing cell line.

In one embodiment, the ligand comprises an isolated nucleic acid sequence, wherein one or more of the nucleotides are modified. In a particular embodiment, a nucleotide modification is a stabilizing modification. In another particular embodiment, a nucleotide modification increase stability of the ligand in vitro and/or in vivo. In still another particular embodiment, the nucleotide modification increases bioavailability of the ligand in vivo.

In one embodiment, one or more nucleotides of the isolated nucleic acid sequence comprise a modified sugar and/or a modified base. In a particular embodiment, the modification is a 2 '-stabilizing modification. In another particular embodiment, the 2'- stabilizing modification is selected from a 2'-fluoro or 2'-OCH₃ modification on the nucleotide sugar ring.

Accordingly, in one embodiment provided is a ligand or a pharmaceutically acceptable salt thereof comprising an isolated nucleic acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to a sequence as described herein.

In one embodiment provided is a ligand or a pharmaceutically acceptable salt thereof comprising an isolated nucleic acid sequence that is at least about 80%, about 85%o, about 90%), about 95%, or about 98%> identical to a sequence selected from Table 1 , 2, 3, 4, 5, 6, 7 or 8.

In some embodiments, the isolated nucleic acid sequence of the GAS6 ligand as provided herein comprises a 2'F nucleoside.

In some embodiments, the GAS6 ligand comprises a modified phosphate backbone.

In some embodiments, the GAS6 ligand comprises a phosphorothioate.

In some embodiments, the GAS6 ligand is conjugated to a carrier. In some aspects, the carrier is a hydrophilic moiety. In other aspects, the GAS6 ligand is conjugated to polyethylene glycol.

In one embodiment, provided is a ligand or a pharmaceutically acceptable salt thereof selected from the ligands listed in Table 1 , 2, 3, 4, 5, 6, 7 or 8 below. In another embodiment provided is a ligand or a pharmaceutically acceptable salt thereof wherein the ligand is modified at its 5 ' end. In another embodiment the ligand comprises an isolated nucleic acid sequence that includes one or more mutations, deletions, or chemical modifications (e.g. modifications to ribose sugar, to the purine or pyrimidine base, insertion of a spacer, conjugation to a carrier, etc.) as described herein. In one embodiment, the ligand comprises an isolated nucleic acid sequence, wherein one or more of the nucleotides are modified. In a particular embodiment, the nucleotide modification is a stabilizing modification. In yet another embodiment, the nucleotide modification increase stability of the ligand in vitro and/or in vivo. In still another embodiment, the nucleotide modification increase bioavailability of the ligand in vivo.

In one embodiment, one or more nucleotides of the GAS6 ligand comprise a modified sugar and/or a modified base. In a particular embodiment, the modification is a 2 '-stabilizing modification. In yet another particular embodiment, the 2 '-stabilizing modification is selected from 2'-fluoro or 2'-OCH₃ modification on the nucleotide sugar ring.

In one embodiment, one or more nucleotides of the GAS6 comprise a

modification at the 2' hydroxyl position. In a particular embodiment, the modification is selected from the group consisting of 2 '-O-methyl and 2'-fluoro. In yet another embodiment, one or more nucleotides is 2 '-O-methyl cytosine, 2 '-O-methyl uridine, 2'- O-methyl adenosine or 2 '-O-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 oxyacetic acid (v), 5-methyl thiouracil, 3-(3-amino-3-N carboxypropyl) uridine (acp3U), and 2,6- diaminopurine.

In one embodiment, the GAS6 ligand comprises at least one modified sugar moiety.

In one embodiment, the GAS6 ligand comprises at least one modified phosphate backbone.

In one embodiment, the GAS6 ligand comprises an inverted thymine at its 3' end. In one embodiment, the GAS6 ligand comprises a spacer. In another

embodiment, the spacer is a glycol spacer. In another embodiment, a loop of the GAS6 ligand comprises the glycol spacer. In yet another embodiment, a loop of the GAS6 ligand consists of a glycol spacer. In still another embodiment, the glycol spacer is provided by incorporation of 9-O-Dimethoxytrityl-triethylene glycol, l-[(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 isolated nucleotide GAS6 ligand sequence and is attached to the 5' end of a second internal nucleotide adjacent to the first internal nucleotide of the isolated nucleotide GAS6 ligand sequence.

In one embodiment, the GAS6 ligand comprises an aliphatic amino linker. In another embodiment, the aliphatic amino linker is attached to the 5 ' end of the isolated nucleic acid sequence of the GAS6 ligand. In yet another embodiment, the aliphatic amino linker is attached to the 3' end of the isolated nucleic sequence of the GAS6 ligand. In still another embodiment, the aliphatic amino linker is provided by

incorporation of 6-(trifluoroacetamino)hexanol (2-cyanoethyl-N,N- diisopropyl)phosphoramidite .

In another embodiment, the GAS6 ligand comprises a hexaethylene glycol linker (6GLY) incorporated using 9-O-Dimethoxytrityl-triethylene glycol, l-[(2-cyanoethyl)- (Ν,Ν-diisopropyl)]- phosphoramidite.

In one embodiment, the GAS6 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 GAS6 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 GAS6 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 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 GAS6 ligand is capped at the 3' end by synthesis of the ligand with inverted deoxythymidine at the 3' end of the ligand.

In one embodiment, the GAS6 ligand comprises a phosphorothioate linkage.

In one embodiment, the GAS6 ligand specifically binds to the GAS6 protein (SEQ ID NO: l).

In one embodiment, the GAS6 ligand binds to and decreases or inhibits a function of a variant of GAS6, wherein the GAS6 variant is at least about 80%, about 85%, about 90%, about 91%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the GAS6 amino acid sequence (SEQ ID NO: l or SEQ ID NO:2).

In one embodiment, binding of the GAS6 ligand to GAS6 stabilizes an active conformation of GAS6. In another embodiment, binding of the GAS6 ligand to GAS6 stabilizes an inactive conformation of GAS6.

In one embodiment, the binding of the GAS6 ligand reversible, such that the GAS6 ligand bound to GAS6 can become unbound. In another embodiment, the GAS6 ligand bound to GAS6 becomes unbound to GAS6 in the presence of a modulator.

In one aspect, a modulator which binds the GAS6 ligand is provided, wherein the modulator reverses, partially or completely, the activity of the GAS6 ligand.

In one embodiment, the GAS6 ligand inhibits intracellular signaling via GAS6. In another embodiment, the inhibiting of intracellular signaling via GAS6 using a GAS6 ligand comprises reducing generation of inositol trisphosphate or inhibiting fluctuations in intracellular calcium levels. In one embodiment, the GAS6 ligand specifically binds GAS6 and improves, ameliorates, or reduces symptoms of GAS6- mediated disorders. In another embodiment, binding of the GAS6 ligand to GAS6 results in inhibition of, or reduction of, GAS6 activity. In yet another embodiment, binding of the GAS6 ligand to GAS6 results in the inability of, or a reduction in the ability of, GAS6 to interact with its receptor. In another embodiment, binding of the GAS6 ligand to GAS6 results in an inhibition of, or reduction of, platelet activation. In still another embodiment, binding of the GAS6 ligand to GAS6 results in an inhibition of, or reduction of, platelet aggregation. In one embodiment, the GAS6 ligand inhibits the ability of GAS6 to propagate thrombus formation. In one embodiment, the GAS6 ligand reduces thrombus size. In one embodiment, the GAS6 ligand reduces clot retraction. In one embodiment, the GAS6 ligand inhibits the ability of GAS6 to interact with tumor cells. In one embodiment, the GAS6 ligand inhibits the ability of GAS6 to bind tumor cells and attract platelets to tumor sites. In one embodiment, the GAS6 ligand inhibits the ability of GAS6 to interact with tumor cells, thereby inhibiting metastasis. In one embodiment, the GAS6 ligand inhibits the ability of GAS6 to interact with tumor cells, thereby reducing the ability of tumor cells to release growth factors. In one embodiment, the GAS6 ligand inhibits the ability of GAS6 to interact with Human Immunodeficiency Virus (HIV). In one embodiment, the GAS6 ligand inhibits the ability of GAS6 to facilitateHIV

dissemination.

In one embodiment, the modulator of the GAS6 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 another embodiment, a modulator of the GAS6 ligand is provided, wherein the modulator reverses, partially or completely, the activity of the GAS6 ligand.

In one embodiment, the modulator comprises an isolated nucleic acid sequence. In another embodiment, the modulator comprises a DNA sequence, an R A 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 of the GAS6 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 the GAS6 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 the GAS6 ligand.

In one embodiment, the modulator consists of an oligonucleotide which is complementary to at least a portion of the GAS6 ligand. In another embodiment, the modulator comprises an oligonucleotide which is complementary to at least a portion of the GAS6 ligand. In another embodiment, the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a loop in the GAS6 ligand. In still another embodiment, the modulator comprises an oligonucleotide sequence which is complementary to at least a portion of a stem in the GAS6 ligand. In yet another embodiment, the modulator comprises an oligonucleotide sequence which is

complementary to at least a portion of a stem in the GAS6 ligand and to at least a portion of a loop in the GAS6 ligand.

In one embodiment, the modulator of the GAS6 ligand comprises a nucleic acid which is complementary to at least a portion of the GAS6 ligand. In another

embodiment, the modulator 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 the GAS6 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 a particular embodiment, the nucleotide comprises 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, nucleotides is a 2' fluoro cytidine, a 2' fluoro uridine, a 2' fluoro adenosine or a 2 '-fluoro guanosine.

In one embodiment, 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 -methyl guanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2 -thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 5 -methoxy cytosine, 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 at least a portion of a loop and/or a stem of the GAS6 ligand.

In one embodiment, the modulator disrupts the secondary structure of the GAS6 ligand. In another embodiment, the modulator stabilizes the secondary structure of the GAS 6 ligand.

In one embodiment, the modulator disrupts the tertiary structure of the GAS6 ligand. In another embodiment, the modulator stabilizes the secondary structure of the GAS 6 ligand.

In one embodiment, the binding of the modulator to the GAS6 ligand exposes a suicide position within the GAS6 ligand, thereby disrupting the secondary structure of the GAS6 ligand and leading to enhanced destruction of the GAS6 ligand by nucleases.

In one embodiment, binding of the modulator to a GAS6 ligand-GAS6 complex reduces or eliminates binding of the GAS6 ligand to GAS6.

In another aspect, a method of modulating the activity of a GAS6 ligand comprising an isolated nucleic acid sequence is provided.

In one embodiment, a method of modulating the activity of a GAS6 ligand comprising an isolated nucleic acid sequence is provided, comprising administering a modulator of the GAS6 ligand to a host who has been administered the GAS6 ligand. In one embodiment, the modulator can be a oligonucleotide modulator, or derivative thereof, and in certain embodiments, is complimentary to a portion of the GAS6 ligand.

In a further aspect, a method of regulating GAS6 function using a GAS6 ligand comprising an isolated nucleic acid sequence is provided.

In one embodiment, the method for regulating GAS6 function comprises administering to a host a therapeutically effective amount of a GAS6 ligand comprising an isolated nucleic acid sequence. In another embodiment, the method further comprises administering a GAS6 ligand modulator to the host.

In another aspect, a method of treating symptoms of, or ameliorating a GAS6- mediated disease or disorder is provided. That is a disease or disorder where GAS6 activity is implicated in, or results in the disease, disorder or symptoms thereof. 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 GAS6 ligand that binds to GAS6. In one

embodiment, the host is diagnosed with a platelet-mediated disease or disorder. In still another embodiment, the host is diagnosed with a cancer.

In one embodiment, the platelet-mediated disease or disorder is selected from the group consisting of cardiovascular disorders, cerebrovascular disorders, acute coronary syndromes, diabetes-related disorders, autoimmune inflammatory disorders, and cancer.

In one embodiment, the cardiovascular disorder or cerebrovascular disorder is a thrombosis, thromboembolism (venous or arterial), 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 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 cancer is a glioma.

In one embodiment, the GAS6 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 symptoms in a host in need thereof by administering a GAS6 ligand comprising an isolated nucleic acid sequence, wherein the GAS6 ligand regulates GAS6 activity is provided.

In another aspect, a method for treating symptoms in a host in need thereof by administering a GAS6 ligand comprising an isolated nucleic acid sequence, wherein the GAS6 ligand regulates platelet function is provided.

In another aspect, a method for treating symptoms in a host in need thereof by administering a GAS6 ligand comprising an isolated nucleic acid sequence, wherein the GAS6 ligand regulates a platelet-mediated disease or disorder selected from the group consisting of cardiovascular disorders, cerebrovascular disorders, acute coronary syndromes, diabetes-related disorders, autoimmune inflammatory disorders, and cancer.

In one embodiment, a therapeutically effective dose of a GAS6 ligand is administered.

In one embodiment, the therapeutically effective dose reduces or inhibits platelet activation, adhesion and/or aggregation.

In one embodiment, the therapeutically effective dose reduces or inhibits thrombus size.

In one embodiment, the therapeutically effective dose reduces or inhibits clot retraction In one aspect, a pharmaceutical composition comprising a therapeutically effective amount of a GAS6 ligand which binds GAS6 is provided.

In one aspect, a pharmaceutical composition comprising a therapeutically effective amount of a modulator, wherein the modulator regulates the activity of a GAS6 ligand which binds GAS6 is provided.

In one embodiment, the pharmaceutical composition comprises a GAS6 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 GAS6 ligand and/or a modulator which regulates the activity of the GAS6 nucleic ligand is provided.

Methods, pharmaceutical compositions and uses of the nucleic acid ligands described herein are also provided as modulatable therapeutics for use in disorders or treatment regimes requiring anti-platelet or antithrombotic therapies. In certain embodiments, the treatment is a surgical intervention. The methods can include administering the nucleic acid ligand to GAS6 to a host in need thereof, where 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. Additionally, pharmaceutical compositions are provided in which the nucleic acid ligand or its modulator are in combination with a pharmaceutically acceptable carrier. Compositions containing the modulator can be designed for administration to a host who has been given a nucleic acid ligand to allow modulation of the activity of the ligand, and thus regulate the coagulation state of the host at risk of hemorrhage.

In one aspect, a use of a ligand in the manufacture of a medicament for the regulation of GAS6 activity wherein the treatment comprises administering to a host in need thereof a therapeutically effective amount of the ligand, or a pharmaceutically acceptable salt thereof is provided.

In one aspect, a use of a ligand in the manufacture of a medicament for the treatment of a GAS6-mediated disorder wherein the treatment comprises administering to a host in need thereof a therapeutically effective amount of the ligand, or a

pharmaceutically acceptable salt thereof is provided.

In one embodiment, the GAS6-mediated disorder is a platelet-mediated disorder.

In one embodiment, the platelet-mediated disorder is selected from the group consisting of a vascular disorder, a cerebrovascular disorder, a platelet-mediated inflammatory disorder, a diabetes-related disorder, a cancer, and HIV infection.

In another aspect, a method for determining whether a GAS6 ligand inhibits binding of a GAS6 polypeptide to an Axl polypeptide, comprising: a) mixing a sample preparation of cells which overexpress a GAS6 receptor protein, and

b) measuring binding of GAS6 to the GAS6 receptor protein in the presence and in the absence of the GAS6 ligand is provided.

In one embodiment the method uses ELISA assay or flow cytometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the SELEX nucleic acid ligand selection process.

FIG. 2 shows the selection conditions for the SELEX round Sel4 performed to identify nucleic acid ligands to GAS 6. FIG. 3 shows a binding progression for Sel4

FIG. 4 shows results of an ELISA assay performed to measure inhibition by a GAS6 aptamer of GAS6-Axl interaction.

FIG. 5 shows results of a cell-based flow cytometry assay to measure inhibition by a GAS6 ligand of GAS6 binding to a cell surface GAS6 receptor protein. FIG. 6 shows results of a cell-based flow cytometry assay to measure inhibition by a GAS6 ligand of GAS6 binding to a cell surface GAS6 receptor protein.

FIG. 7 shows results of a cell-based flow cytometry assay to measure inhibition by a GAS6 ligand of GAS6 binding to a cell surface GAS6 receptor protein. FIG. 8 shows a schematic depicting the proposed secondary structure for R12Gas4-72.

FIG. 9 shows a chart depicting the conservation of sequences as percentage in Loopl and Loop3 from degernate SELEX of parent R12Gas4-72. Percentage variation in Loop 1 and Loop 3 of degenerate SELEX family, where the family is defined as all sequences with ten or fewer variations from the parent R12Gas4-72 sequence. Parent sequence is listed across the top of the figure, with each column corresponding to one base position. Percentage of each base represented within the population is indicated by row. Italics indicates bases which are present in the population at greater than 45% frequency. Bold indicates bases which are present in the population at greater than 95% frequency.

FIG. 10 shows a schematic depicting the proposed secondary structure for 16T1XM31.

FIG. 11 shows results of a cell-based flow cytometry assay to measure inhibition by a GAS6 truncated ligand of GAS6 binding to a cell surface GAS6 receptor protein.

FIG. 12 shows results of a cell-based flow cytometry assay to measure inhibition by a GAS6 truncated ligand of GAS6 binding to a cell surface GAS6 receptor protein.

FIG.13 shows a schematic depicting the proposed secondary structure of 16T1XM31 as well as regions of complementarity between the ligand and the GAS6 ligand modulators RB673-RB677.

FIG. 14 shows results of a cell-based flow cytometry assay of 16TIXM31with various concentrations of GAS6 ligand modulator RB674.

DETAILED DESCRIPTION

Nucleic acid ligands, also called "aptamers," are non-naturally occurring, single- stranded nucleic acids that adopt a specific three-dimensional shape which enables binding to a desired target molecule. For ligands which bind to peptides and proteins, association of a ligand with its target protein may lead to the inhibition of the protein's function, much like the binding of a monoclonal antibody to its target protein may lead to the inhibition of the protein's function. A unique feature of ligands is the ability to generate active control agents to them in the form of complementary oligonucleotides that hybridize to the ligand by Watson-Crick base pairing. These active control agents fundamentally change the ligands active structure, and thereby neutralize their pharmacologic activity. The present invention provides compounds, compositions and methods that include nucleic acid ligands to Growth Arrest-Specific protein 6 (GAS6), to mediate the biological function and interaction of GAS6. Additionally provided are modulators that can regulate the activity of the GAS6 nucleic acid ligands.

A. Definitions

As used herein, the following definitions shall apply unless otherwise indicated.

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 "GAS6 nucleic acid ligand" or "GAS6 ligand" or "anti-GAS6 ligand" of "nucleic acid GAS6 ligand" refers to a ligand or aptamer that specifically binds to GAS6. 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 Kj 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 Kj for the ligand with respect to the target will be 10-fold, 50- fold, 100-fold, or 200-fold less than the Ka with respect to the unrelated material or accompanying material in the environment. The nucleic acids of the ligand may be conjugated such as to a carrier group and may also include chemical modifications to one or more of the sugar, base, or phosphate moieties of one or more of the nucleotides. "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.

"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.

"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.

"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 or therapeutically effective amount is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent) of a disease state. The pharmaceutically effective dose or therapeutically effective amount 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.

"Sequence identity" or "%> identity" as used herein refers to the similarity of two sequences with respect to the number of nucleotides (nt) that are identical when the sequences are aligned for maximal correspondence such as by visual inspection or by use of an algorithm such as the basic local alignment search tool (e.g. BLAST, Altshul et al, Nucleic Acids Res., 15:3389-3402, 1997).

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, ¾.

"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 GAS6 nucleic acid ligand "variant" as used herein encompasses variants that perform essentially the same function as a GAS6 nucleic acid ligand and comprises substantially the same structure. Accordingly, in one embodiment provided is a ligand or a pharmaceutically acceptable salt thereof comprising a nucleic acid sequence that is at least 80%, 85%, 90%>, or 95% identical to a sequence as described herein.

B. Growth Arrest-Specific protein 6

Growth Arrest-Specific protein 6 (GAS6) is a gamma-carboxyglutamic acid

(Gla)-containing protein 721 amino acid residues in length (UniProtKB/Swiss-Prot Accession No. Q 14393; disclosed herein as SEQ ID NO: 1) thought to be involved in the stimulation of cell proliferation, and may play a role in thrombosis. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. Munoz et al. determined the intron-exon structure of GAS6 and analyzed the gene for the presence of allelic variants that could be associated with atherothrombotic disease. Using in silico analyses, they determined the sequence of the GAS6 gene, which spans 43.8 kb of DNA and consists of 15 exons. They also identified 8 different variants that were confirmed to be SNPs. A preliminary analysis of 5 of these SNPs in a group of 110 healthy controls and 188 patients with atherothrombotic disease suggested a statistically significant difference between controls and stroke patients in the allelic distributions of one of these variants, 834+7G-A (Munoz, et al, Hum Mutat., 2004 May; 23(5):506-12).

The amino acid sequence of the GAS6 protein is presented below (SEQ ID

NO: l):

1 mapslspgpa alrrapqlll lllaaecala allpareatq flrprqrraf qvfeeakqgh

61 lerecveelc sreearevfe ndpetdyfyp ryldcinkyg spytknsgfa tcvqnlpdqc

121 tpnpcdrkgt qacqdlmgnf fclckagwgg rlcdkdvnec sqenggclqi chnkpgsfhc

181 schsgfelss dgrtcqdide cadseacgea rcknlpgsys clcdegfays sqekacrdvd

241 eclqgrceqv cvnspgsytc hcdgrgglkl sqdmdtcele agwpcprhrr dgspaarpgr

301 gaqgsrsegh ipdrrgprpw qdilpcvpfs vaksvkslyl grmfsgtpvi rlrfkrlqpt

361 rlvaefdfrt fdpegillfa gghqdstwiv lalragrlel qlryngvgrv tssgpvinhg 421 mwqtisveel arnlvikvnr davmkiavag dlfqpergly hlnltvggip fhekdlvqpi 481 nprldgcmrs wnwlngedtt iqetvkvntr mqcfsvterg sfypgsgfaf ysldymrtpl 541 dvgtestwev e vahirpaa dtgvlfalwa pdlravplsv alvdyhstkk lkkql vlav 601 ehtalalmei kvcdgqeh v tvslrdgeat levdgtrgqs evsaaqlqer lavlerhlrs 661 pvltfagglp dvpvtsapvt afyrgcmtle vnrrlldlde aaykhsdita hscppvepaa

721 a

The GAS6 protein used in the SELEX method described herein was a C-terminal His6 fusion to GAS 6 amino acids Ala49 to Ala678 (SEQ ID NO:2; below):

afqvfeeakqghlerecveelcsreearevfendpetdyfypryldcinkygspytknsgfatcvqnlpdq ctpnpcdrkgtqacqdlmgnffclckagwggrlcdkdvnecsqenggclqichnkpgsfhcschsgfelss dgrtcqdidecadseacgearcknlpgsysclcdegfayssqekacrdvdeclqgrceqvcvnspgsytch cdgrgglklsqdmdtceleagwpcprhrrdgspaarpgrgaqgsrseghipdrrgprpwqdilpcvpfsva ksvkslylgrmfsgtpvirlrfkrlqptrlvaefdfrtfdpegillfagghqdstwivlalragrlelqlr yngvgrvtssgpvinhgmwqtisveelarnlvikvnrdavmkiavagdlfqperglyhlnltvggipfhek dlvqpinprldgcmrswnwlngedttiqetvkvntrmqcfsvtergsfypgsgfafysldymrtpldvgte stwevevvahirpaadtgvlfalwapdlravplsvalvdyhstkklkkqlvvlavehtalalmeikvcdgq ehvvtvslrdgeatlevdgtrgqsevsaaqlqerlavlerhlrspvltfagglpdvpvtsa C. Development Of GAS6 Nucleic Acid Ligands

Nucleic acid ligands which specifically bind the GAS6 protein were identified using the SELEX method. The GAS6 ligands which were initially obtained via SELEX were then fully characterized to understand their properties. 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 GAS6. 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). Nucleic acid ligands specific to GAS6 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 GAS6 can be isolated by performing SELEX on purified GAS6 or fragment thereof 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 GAS6 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 GAS6, elution of ligands bound to the protein could be accomplished by addition of sufficient amounts of an extracellular domain of a TAM (Tyro 3/Axl/Mer) receptor of GAS6.

SELEX can be used 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 GAS6. In certain embodiments, the ligands affect multiple functions of GAS6 independently. 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 of the ligand to only a single epitope or region of GAS6.

GAS6 can be a recombinantly expressed and purified protein used for a SELEX procedure. In certain embodiments, the GAS6 nucleic acid ligand binds to GAS6 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, nucleic acid ligands to specific GAS6 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.

Nucleic acid ligands isolated by these SELEX procedures specific to GAS6, 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 GAS6. As the desired nucleic acid ligands are not merely binding partners, but are inhibitors of the GAS6 mediated-receptor signaling, it is possible to identify ligands having a desired function by assessing the effect of the ligand on platelet activity. This can include characterizing the effects of the ligand on various signally pathways known to be regulated by GAS6. For example, GAS6 signaling can lead to clustering of the GAS6 protein or GAS6 receptors, and to the activation of kinases to start a local signal chain of events that activates phosp ho lipase Cy2, releasing the second messengers 1,4,5-inositol trisphosphates and diacylglycerol that are responsible for raising Ca²⁺ levels and activating protein kinase C. Any of these second messenger systems or signals can be measured using methods well known to those having ordinary skill in the art.

In some embodiments, the ligand interacts with a receptor-binding domain of GAS6. The ligand can interfere with GAS6 binding to its receptor(s). In certain embodiments, the ligand can inhibit intracellular signaling via a GAS6 activated receptor, including reducing the generation of inositol trisphosphate or fluctuations in intracellular calcium levels. The ligand can affect platelet activation by thrombin, ADP, collagen or other platelet agonists whose activity is amplified by GAS6. The ligand can also affect platelet adhesion to collagen or collagen-related peptides. The ligand can affect platelet aggregation induced by thrombin, ADP, collagen or other platelet agonists whose activity is amplified by GAS6. The ligand can reduce thrombus size. The ligand can reduce clot retraction.

Ligands can also be screened for inhibition of the GAS6 interaction with TAM receptors in plate-based ELISA assays or the GAS6 interaction with TAM receptors over- expressed on cell lines using flow cytometry approaches. The specificity of a given nucleic acid ligand for GAS6 can be further distinguished by the ability of the ligand to block intracellular signaling events triggered by known activation of a TAM receptor by GAS 6.

A ligand as described herein 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. 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.

The binding affinity of the ligands with respect to the target can be defined in terms of Kj. The value of this dissociation constant can be determined directly by well- known methods, such as by radioligand binding methods described in Example 1..

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. 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.

Efficacy of a GAS6 ligand in regulating GAS6 function or treating symptoms of platelet-mediated disease depends largely upon the ability of the ligand to bind with sufficient affinity to the GAS6 protein. Accordingly, after obtaining GAS6 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 (GAS6) 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 Kj 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 (Kj) is, under ideal conditions, equivalent to Kj. However, in no event will a Ki be less than IQ. Thus, determination of Kj, in the alternative, sets a maximal value for the value of IQ. Under those circumstances where technical difficulties preclude accurate measurement of IQ, measurement of K; can conveniently be substituted to provide an upper limit for IQ. A Ki value can also be used to confirm that a ligand of the present invention binds a target. In characterizing GAS6 ligand binding properties, specificity may be analyzed using competition binding or functional assays with known GAS6 binding molecules.

Application of any of the above-described methods, alone or in combination, will give rise to a plurality of nucleic acid ligands specific to GAS6. 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 GAS6

The GAS6 ligands disclosed herein are preferably nucleic acid ligands. GAS6 ligands which specifically bind GAS6 were selected using the SELEX method, described in more detail below and in Example 1 , then modified to increase stability, affinity for GAS6 and/or the ability to regulate GAS6 activity.

Ligands isolated according to methods described herein are presented below in

Tables 1-4.

E. Modulators

In some embodiments, the nucleic acid ligands to GAS6 are reversible. In one aspect, the disclosure provides a method of modulating the activity of a nucleic acid ligand to GAS6 by administering a modulator of the GAS6 ligand to a host who has been administered the nucleic acid ligand.

Modulators can 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 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), mopholino 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 nt, 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 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 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 15 nt , 6 nt to 20 nt, 6 nt to 25 nt, 8 nt to 20 nt, 8 nt to 25 nt, 10 nt to 15 nt, 10 nt to 20 nt or 10 nt to 25 nt of the targeted ligand. In other embodiments, the modulator oligonucleotide can comprise a sequence complementary to about 6 nt, about 8 nt, about 10 nt, about 12 nt, about 14 nt, about 16 nt, about 18 nt, about 20 nt, about 22 nt, or about 25 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 disclosure 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 disclosure can be single- stranded DNA or R A 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 other embodiments, the modulator is a nucleic acid complementary to at least one stem region in the ligand. In yet another embodiments, the modulator is a nucleic acid complementary to at least one stem and one loop region in the ligand. In other embodiments, the modulator is an oligonucleotide having at least a sequence that hybridizes at physiologic conditions to the nucleic acid ligand. 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 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 disclosure. The LNA monomers are bi-cyclic compounds structurally similar to R A-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 disclosure (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 disclosure, 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 poiy- (..-lysine and pofy-L-ora ime.

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-2,3-¾z5(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 disclosure also provides methods to identify the modulators of nucleic acid GAS6 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 disclosure. 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 coagulation 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 GAS6 is first incubated with GAS6 protein or fragment thereof, or a mixture containing the GAS6 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 GAS6 protein. The ligand can be labeled, for example, by a radioactive or nonradioactive moiety, to allow detective of the ligand-GAS6 complex within the gel. When using the Gel-Shift assay to screen for ligands having GAS6 -binding activity, the complex can then be extracted from the gel and the isolated ligand analyzed to identify ligands having the desired GAS6 binding activity.

Gel shift assays can also be used to screen modulators for binding nucleic acid ligands to GAS6_, 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 GAS6 nucleic acid ligand with GAS6. For example, the GAS6-ligand 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 GAS6-ligand 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 GAS6 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 GAS6 protein or fragment could be attached to the sensor chip surface via, for example, a histidine tag. The bound GAS6 proteins are then exposed to a solution containing the potential ligand molecules. Binding of the nucleic acid ligand to the GAS6 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 GAS6 ligands. Again, the reaction mixture to which the chip- bound GAS6 protein is exposed can contain both a known GAS6 ligand with increasing amounts of modulator and the effects determined by standard BIACORE analysis of the resultant interaction between GAS6 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 a 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 the GAS6 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 GAS6 ligands 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.

GAS6 nucleic acid ligands of the present disclosure 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 from Tables 1-5 including 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).

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^*-fluoro (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) 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 some embodiments, the ligand may be substituted with a spacer using methods known to skilled artisans. Spacers include a hexaethylene glycol spacer such as (9-0- Dimethoxytrityl-triethylene glycol, 1 -[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite. Examples of other spacers include, but are not limited to 5 '-0- Dimethoxytrityl- 1 ' 2 ' Dideoxyribose-3 ' -[(2-cy anoethyl)-(N,N-diisopropyl)] - phosphoramidite; 18-O-Dimethoxytritylhexaethyleneglycol, 1 -[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite; and 12-(4,4'-Dimethoxytrityloxy)dodecyl-l-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

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'-0-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 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.

In pharmaceutical compositions the ligands can be provided in forms, such as salt forms that improve solubility or bioavailability. Suitable salts include inorganic cations such as sodium and potassium.

Any of the oligonucleotides of the disclosure 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

The GAS6 ligands can also include modifications that improve bioavailability or stability. Such modifications can include 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, small nucleic acid ligands (<20 KD) 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.

In one embodiment, the ligands or modulators can be covalently bound or otherwise attached to a non-immunogenic, high molecular weight carrier. Suitable carriers include pharmaceutically acceptable water soluble polymers such as polyethylene glycol (PEG), polyaminoamines (PAMAM); polysaccharides such as dextran, or polyoxazo lines (POZ). 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.

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 be attached to the PEG molecule through covalent bonds. For example, an oligonucleotide ligand or modulator can be bonded to a 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 GAS6 ligands or GAS6 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 GAS6 ligands or GAS6 ligand modulators to the same GAS6 protein target sequence or different GAS6 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 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 (MMT: 4-Monomethoxytrityl, OEtCN:

cyanoethoxy; Trity: triphenylmethyl):

The 5 '-thiol modified linker is used with PEG-maleimides, PEG-vinylsulfone, PEG-iodoacetamide and PEG-orthopyridyl-disulfide, for example.

The PEG carrier 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:

Additional PEG reagents that can be used to prepare stabilized compounds of the disclosure include other branched PEG N-Hydroxysuccinimide (mPEG-NHS) such as:

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):

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 reagents are: (SUNBRIGHT GL2-400GS2);

NBRIGHT GL2-400HS); and

(SUNBRIGHT GL2-400TS).

The reagents can also include non-branched Succinimidyl alpha-methylbutanoate (mPEG-SMB) of the general formula:

in which mPEG is between 10 and 30KD. In one embodiment, the reactive ester is -0-CH₂CH₂CH(CH3)-C0₂-NHS. Compounds of this structure are sold by Nektar Therapeutics.

PEG reagents can also include nitrophenyl carbonate linked PEGs, such as of the following structure:

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:

in which mPEG is about 10, about 20 or about 30KD. Additionally, the structure can be branched, such as

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:

PEG-maleimide pegylates thiols of the target compound in which the double bond of the maleimic ring reacts 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 GAS6 ligand modulators can be associated with a single PEG molecule. The modulator can be to the same or different GAS6 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 disclosure 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)„OC(=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 Platelet-Mediated Disorders

Methods, pharmaceutical compositions and uses of the GAS6 nucleic acid ligands described herein are also provided as modulatable anti-platelet agents for use in disorders or treatment regimes requiring anti-platelet therapy. In certain embodiments, the treatment is a surgical intervention. The methods can include administering the GAS6 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. In one embodiment, provided is a method for treating symptoms of a GAS6-mediated disorder comprising administering to a host in need thereof a therapeutically effective amount of an ligand or a pharmaceutically acceptable salt thereof as described herein.

GAS6 is a vitamin-K dependent glycoprotein which is expressed in and secreted by many cell types, including endothelial cells, vascular smooth muscle cells, macrophages and bone marrow cells. GAS6 has also been implicated in a variety of cellular functions, such as reversible growth arrest, survival, proliferation, and inflammation. GAS6 is the endogenous ligand for the TAM family of receptor tyrosine kinases, which include the Tyro3, Axl and MerTK receptors. The binding of GAS6 to Axl induces Axl phosphorylation and activation of the PI3 kinase/ Akt pathway, which has pro-survival and antiapoptotic effects. GAS6 has also been shown to be important for phagocytosis of apoptotic cells. Additionally, studies in cell cultures have shown an effect of GAS6 and TAM activation in processes such as vascular smooth muscle survival, division and migration or endothelial cell acivation by pro-inflammatory cytokines and survival (see Hurtado et al, 2011; Cardiovasc Biol Cell Signal, 873-882).

GAS6 has been shown to enhance the formation of stable platelet

macroaggregates in response to various agonists. GAS6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo (Angelillo-Shcerrer et al, 2001, Nat Med, 7:215-221). These data suggest that GAS6 is a platelet-response amplifier that plays a significant role in thrombosis. Accordingly, it is envisioned that aptamers which bind GAS6 and inhibit activation of AxAxl and/or the other TAM receptors, may provide a novel and safe means of preventing or reducing incidence of thrombosis, thereby treating disorders related to and/or resulting from thrombosis.

The GAS6 ligands described herein may inhibit initiation of GAS6-mediated platelet activation. Such inhibition by a GAS6 ligand may in turn reduce platelet aggregation, platelet adhesion and a platelet pro-inflammatory response.

In one embodiment, the host has or is at risk of having an occlusive thrombotic disease of the coronary, cerebral and peripheral vascular systems. In certain other embodiments, the host is preparing to undergo or undergoing a surgical intervention, or has undergone a surgical intervention that puts the host at risk of an occlusive thrombotic event. In other embodiments, the host has received a vessel graft to enable hemodialysis, which is at risk of occluding due to interactions between the vessel and platelets.

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 GAS6 nucleic acid ligand to a host in need thereof.

In one embodiment, the GAS6 nucleic acid ligand is provided for extended periods of time. In this instance, a GAS6 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 GAS6 nucleic acid ligand treatment. In another embodiment, the modulator is administered to control the concentration of the GAS6 nucleic acid ligand and thereby the duration and intensity of treatment. In another embodiment, the GAS6 nucleic acid ligand is provided as a platelet anesthetic during a cardiopulmonary bypass procedure. In another embodiment, the GAS6 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 GAS6 nucleic acid ligand once therapeutic levels of the oral anti-platelet agent are established.

In one embodiment, the GAS6 mediated disorder comprises a vascular disorder. In another embodiment, the vascular disorder is selected from the group consisting of acute coronary syndromes, thrombosis, thromboembolism, thrombocytopenia, peripheral vascular disease, and transient ischemic attack.

In another embodiment, the GAS6 mediated disorder comprises a cardiovascular disorder. In one embodiment, the cardiovascular disorder is selected from the group consisting of transient ischemic attack, ischemic stroke, and embolism.

In another embodiment, the GAS6 mediated disorder comprises a cerebrovascular disorder. In one embodiment, the cerebrovascular disorder is selected from the group consisting of transient ischemic attack, ischemic stroke, and embolism.

In one embodiment, the GAS6 mediated disorder comprises a platelet-mediated inflammatory disorder. In one embodiment, the platelet-mediated inflammatory disorder selected from the group consisting of arthritis, rheumatoid arthritis, psoriatic arthritis, reactive arthritis, inflammatory bowed disease, ankylosing spondylitis, and scleroderma.

GAS6 is an activating ligand for the receptor tyrosine kinase, Axl. High Axl expression is observed in many human tumors and is associated with tumor progression mechanisms such as tumor invasion, migration, angiogensis, proliferation and adhesion, (see Holland et al, 2010, Cancer Res., 70: 1544-1554). Further, Axl expression is associated with aggressive tumor behavior, which is turn is associated with tumor dissemination and metastasis. Accordingly, Axl has been considered a strong drug target for therapeutic inhibition of cancer invasion and dissemination. Use of a GAS6 ligand as described herein, may inhibit binding of GAS6 to Axl, thereby blocking activation of Axl and the function of Axl in cancer progression. GAS6 ligands may also function by blocking Axl-mediated angiogenesis associated with tumor growth and development. Thus, GAS6 ligands may be used in the treatment of cancer or inhibition of cancer progression, or metastasis. Such cancers include, but are not limited to, lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer (e.g., glioblastoma multiforme), testicular cancer, bone cancer, liver cancer, and metastases thereof.

In one embodiment, the GAS6 mediated disorder is a diabetes-related disorder. In one embodiment, the diabetes-related disorder is selected from the group consisting of diabetic retinopathy, diabetic vasculopathy, atherosclerosis, ischemic stroke, peripheral vascular disease, acute renal injurym and chronic renal failure.

GAS6 may also play a role in adiposity (see Lijnen et al., 2011, J. Pharmacol Exp Ther., 337:457-464). Studies have shown that adipose tissue development is impaired in GAS6 deficient mice and that GAS6 is expressed during differentiation of preadipocytes into mature adipocytes in culture. Moreover, Axl expression levels have been shown to be higher in subcutaneous adipose tissue of obese human subjects as compared to lean controls. Lijnen et al. later showed that affecting GAS6 signalling using a low molecular radius Axl antagonist impares adipocyte differentiation and reduces adipose tissue devlopment in a murine model of nutritionally induced obesity. It is therefore envisioned that GAS6 ligands may be useful in the therapeutic invention of obesity or obesity related disorders such as type 2 diabetes mellitus.

TAM receptor signaling has also been implicated in regulation of the innate immune response as well as the regulation of apoptosis, specifically, the phagocytosis of apoptotic corpses. Programmed cell death and the generation of apoptotic cells are central to cellular turnover and tissue homeostasis during adulthood. Defects in apoptotic cell clearance can lead to the accumulation of intracellular components and the aberrant (sustained) exposure of nuclear antigens to the immune system (Rothlin et al, Cur Op Immunol, 2010, 22: 1-7). Studies have shown that the TAM receptor tyrosine kinases and their ligands are essential for phagocytosis of apoptotic cells. Moreover, studies suggest that such defects in the phagocytosis of apoptotic cells are functionally linked to the inability of the TAM pathway to regulate inflammatory responses in a host. Accordingly, one might hypothesize that defects in TAM signaling are associated with autoimmune disorders in humans. It is thereby envisioned that activation of the TAM signaling pathway by activating or agonist GAS6 ligands may serve as an effective therapeutic treatment of a variety of immune or inflammatory disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis. In another embodiment, activation of TAM signaling by a GAS6 ligand may be used to treat human retinal dystrophies.

The methods as described herein may be used in combination with another therapy. In one embodiment, that other therapy is aspirin. In another embodiment, that other therapy is an inhibitory of the ADP-receptor P2Y12, such as ticlopidine, clopidogrel, ticagrelor, cangrelor or elinogrel. In yet another embodiment, the other therapy is a combination of aspirin and a P2Y12 inhibitor, administered independently, or together in a formulation. In yet another embodiment, the other therapy is an inhibitor of the platelet thrombin receptor, PAR-1 or the platelet collagen receptor, GPVI.

In one embodiment, the host is preparing to undergo or undergoing a surgical intervention, or has undergone a surgical intervention that puts the host at risk of an occlusive thrombotic event. In other embodiments, the host has received a vessel graft to enable hemodialysis, which is at risk of occluding due to interactions between the vessel and platelets.

In one embodiment the therapy includes treating a host with a therapeutically effective amount of an anti-cancer or an anti-thrombotic agent.

In one embodiment, the therapy includes treating a host with a therapeutically effective amount of an anti-HIV agent selected from the group consisting of HIV antiviral agents, immunomodulators, and anti-infective agents.

H. Administration and Pharmaceutical Composition

The GAS6 nucleic acid ligands or GAS6 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 GAS6 nucleic acid ligand to allow modulation of the activity of the ligand, and thus regulate anti-platelet activity of the administered GAS6 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 GAS6 nucleic acid ligand or GAS6 ligand modulator of the present disclosure 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, 20^th edition (Lippincott Williams & Wilkins, 2000) and Ansel et al.,

Pharmaceutical Dosage Forms and Drug Delivery Systems, 6^th 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 disclosure 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 ligands 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 ligand 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 ligands 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 disclosure can be formulated for use 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 (MLVs) 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 MLVs. Unilamellar liposomes (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 disclosure, 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 GAS6 ligand or GAS6 ligand modulator according to methods known to skilled artisans. Therapeutic kits are also envisioned. The kits comprise 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 GAS6 ligand and/or a GAS6 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 GAS6 ligands and/or GAS6 ligand modulators of the present disclosure 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 GAS6 nucleic acid ligand is delivered via subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps).

In one embodiment, the GAS6 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 disclosure 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 disclosure 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 GAS6 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 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, 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.

GAS6 nucleic acid ligands 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 disclosure 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 some embodiments, 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.

In certain embodiments, the compositions of this disclosure may further comprise another therapeutic agent. When a second agent is used, the second agent may be administered either as a separate dosage form or as part of a single dosage form with the compounds or compositions. While one or more of the inventive compounds can be used in an application of monotherapy to treat a disorder, disease or symptom, they also may be used in combination therapy, in which the use of an inventive compound or composition (therapeutic agent) is combined with the use of one or more other therapeutic agents for treating the same and/or other types of disorders, symptoms and diseases. Combination therapy includes administration of the two or more therapeutic agents concurrently or sequentially. The agents may be administered in any order.

Alternatively, the multiple therapeutic agents can be combined into a single composition that can be administered to the patient. Also provided is a use of a ligand in the manufacture of a medicament for the treatment of a GAS6-mediated disorder wherein the treatment comprises administering to a host in need thereof a therapeutically effective amount of the ligand, or a

pharmaceutically acceptable salt thereof. In one embodiment, the GAS6-mediated disorder is a platelet-mediated disorder.

Also provided are methods for determining whether a GAS6 ligand inhibits the interaction of, or binding of a GAS6 polypeptide to an Axl polypeptide. In one embodiment, the method comprises: a) mixing a sample preparation of cells which overexpress a GAS6 receptor protein, and

b) measuring binding of GAS6 to the GAS6 receptor protein in the presence and in the absence of the GAS6 ligand.

In one embodiment the method uses an ELISA assay using GAS6 protein and AXL protein/anti-AXL capture antibody fixed on a solid support. The difference in binding of GAS6/AXL in the presence and absence of a GAS6 ligand is indicative of relative inhibition of GAS6/AXL interaction. In one embodiment the method uses flow cytometry. Cells overexpressing the

AXL recptor for GAS6 are provided such as, but not limited to huyman glioblastoma cell lines such as T98G or U87. The difference in binding of GAS6/AXLon the surface of the cells in the presence and absence of a GAS6 ligand is indicative of relative inhibition of GAS6/AXL interaction. The % inhibition of each sample may be calculated using the formula:

% Inhibition =100* [((max-background)-(sample-background))/ (max- background)].

Data are normalized to min and max controls from the median of histogram plots and charted for analysis. The following examples are provided to illustrate certain aspects of the present disclosure. These examples are in no way to be considered to limit the scope of the disclosure.

EXAMPLES

Example 1: Identification of Nucleic Acid Ligands to GAS6

The SELEX method was used to obtain ligands which bind GAS6 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 oligos for designing the candidate mixture are: Sel4: 5'- CATCGATGCT AGTCGTAACG ATCC (N₃₅) CGAGAACGTT ATTGTACTCC CCTA-3' (SEQ ID NO:3; N₃₅ represents 35 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 GAGTACAATA ACGTTCTCG-3 ' (SEQ ID NO:4; T7 promoter sequence is in bold), and 5'-

CATCGATGCTAGTCGTAACGATCC-3' (SEQ ID NO:5). The reaction was filled in with Exo- Klenow, stopped by addition of EDTA to a final concentration of 2mM, and extracted with PCI (phenol:chloroform:isoamyl alcohol (25:24: 1)) and then 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 40mM Tris-HCl pH 8.0, 4% PEG-8000, 12mM MgCl₂, ImM spermidine, 0.002% Triton, 5mM DTT, ImM rGTP, ImM rATP, 3mM 2'F-CTP, 3mM 2'F-UTP, 8μg/mL inorganic pyrophosphatase, 0.5μΜ 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 (lOmM Tris pH 7.5, O. lmM EDTA) washes in an Amicon 10 spin column.

The GAS6 selection started with a complex library of ~10¹⁴ 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 bound to the purified recombinant C-term His6 tagged GAS6 protein (SEQ ID NO:2). Purified histidine -tagged GAS6 protein was obtained from R&D Systems (Minneapolis, MN), Catalog No. 885-GS, and encompassed residues Ala 49-Ala678.

Initial GAS6 ligand selection was performed in binding buffer "E." All rounds of binding also utilized 0.0024% yeast tRNA. Binding buffer E consists of 20mM HEPES pH 7.4, 50mM NaCl, 2mM CaCl₂, and 0.01% BSA. Protein-RNA complexes were partitioned over a 25mm nitrocellulose disc with washing. The bound RNA was extracted off the nitrocellulose disc with incubation in PCI (25:24: 1). Filtered 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. Specific conditions for each round of Sel4 selection are shown in FIG. 2. Rounds 1-12 were performed in binding buffer E.

Enrichment of the ligand libraries for GAS6 was monitored in direct binding studies utilizing radiolabeled ligand RNA from respective rounds of SELEX and soluble GAS6. Binding studies were performed with trace ³²P end-labeled RNA added to serial dilutions of GAS6 in Binding Buffer E. 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-³²Ρ-ΑΤΡ and subsequently cleaned with a Biorad MicroBio Spin P-30 spin column. End-labeled RNA was diluted to a final concentration of 2000cpm^L and heat denatured at 65°C for 5 minutes. RNA and GAS6 dilutions were equilibrated at 37°C prior to use. RNA (5μί) was added to varying concentrations of GAS6 (15μί) at 37°C and incubated together for 5 to 15 minutes. The complexed RNA/GAS6 protein mixture was then loaded over a Protran BA85

nitrocellulose membrane and 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. Results for binding of enriched ligand libraries from round 12 of the Sel4 SELEX as compared to the GAS6- na^'ive starting ligand Sel4 library are shown in FIG. 3.

Example 2: Sequencing and Identification of GAS6 Nucleic Acid

Ligands

The final PCR products representing anti-GAS6 enriched ligand libraries from

Round 12 of the SELEX experiments described in Example 1 were digested with EcoRl and BamHl, cleaned with a purification kit, and directionally cloned into linearized pUC19 vector. Bacterial colonies were streaked for single clones and 5mL overnight cultures were inoculated from single colonies. Plasmid DNA was prepared from single colonies using Invitrogen Purelink Quick Plasmid Miniprep kits. Seventy three plasmids from the Sel4 SELEX experiment were sequenced utilizing a vector primer. Individual aptamers were screened for their ability to inhibit GAS6/Axl interaction (see examples 3 and 4). The corresponding unique Sel4 DNA sequences of the full-length ligand clones which demonstrate Gas6/Axl inhibition are shown in Table 1 below, whereas sequences representing the random region are provided in Table 2.

Table 1: Full-Length Sel4 DNA Ligands Identified via SELEX

Table 2: Sel4 Random Re ion Se uences of Inhibitor A tamers

Representation of these unique Sel4 ligands as full-length RNA is shown in Table 3, and as RNA sequences indicating the sites of incorporation of the 2'-fluoropyrimidine nucleotides used in the SELEX experiment in Table 4 (f indicates a 2'-fluoropyrimidine modification and r indicates a non-modified ribonucleotide). Full-length refers to sequences resulting from the SELEX process, comprising sequences derived from both the random portion of the ligand library used in the SELEX process as well as sequences from the fixed sequence portions flanking the random region.

Table 3: Full-Length Sel4 RNA Sequences

fCrGrAfCfUrArGfCrAfUfCrGrAfUrG

TGrGrGrGrArGfUrAfCrArAfUrArAfCrGfUfUfCfUfCrG 20 fCfCfCrGfUfCrGfCfUrAfUrGfCfUfUrGrGfCfUrGfCfU

4-27- TArGrAfUfCfCrGrAfCfCfCfCrGrGrGrAfUfCrGfUfUrA

Modified fCrGrAfCfUrArGfCrAfUfCrGrAfUrG

TGrGrGrGrArGfUrAfCrArAfUrArAfCrGfUfUfCfUfCrG 21 TGrArAfUfCrGrGrGrGfUfUfCrGrArArAfCfCrGrArAfC

4-72- fUfCrGfCfUfCfCrAfCrGfCfCrGrGrGrAfUfCrGfUfUrA

Modified fCrGrAfCfUrArGfCrAfUfCrGrAfUrG

rG=2'Ribo G; rA=2' ibo A; fC=2'-Fluoro C; fU=2'-Fluoro U

Example 3: Ability of GAS6 Ligands to Inhibit GAS6/AXL Interaction: ELISA Assay

To determine if the GAS6 nucleic acids ligands were capable of inhibiting the interaction between GAS6-AXL, Sandwich ELISA assay was utilized. Briefly, the ability of RNA aptamers to block the binding of human Gas6 protein to human Axl receptor (one of the 3 TAM receptors) was determined using a modified version of previously described sandwich ELISA assay (Ekman, Stenoff and Dahlback: J Thrombosis and Haemostasis 8: 838-844; April 2010).

ELISA reagents were purchased from R&D Systems. R&D Systems methods for Duoset Gas6 ELISA (DY885) and sAxl ELISA (DY154) were closely followed and combined to create a GAS6/Axl ELISA. All steps were performed at room temperature. Assay plates were washed with 280 μΕ/well PBS 0.05%Tween20 three times between binding steps. The 96 well Corning plates #3631 were coated overnight with 100 μΕ/well anti-Axl capture antibody in PBS. The plates were washed and then blocked with BlockAce (AbD Serotec) for a minimum of 2 hours and washed again. Human Axl chimera (R&D Systems AF154) was added to anti-Axl coated wells for 20 minutes followed by a wash sequence. Meanwhile, human Gas6 protein (R&D Systems GS885) was allowed to bind biotinylated antiGas6 FAB885 in 1 ml tubes for 20 minutes. The Gas6-antiGas6 complex was transferred to microtubes containing GAS6 nucleic acid ligands and incubated 5 minutes. The aptamer-Gas6-antiGas6 complex was transferred to Axl-AntiAxl coated plates for 5 minutes. Plates were washed and Streptavidin-HRP was added. Plates were incubated 20 minutes then washed as described. R&D Systems DY999 color reactants were then applied. After 20 minutes the reaction was halted with 2N H₂SO₄ and absorbance was read at a wavelength of 540nm in a Flexstation 3 automated plate reader (Molecular Devices Corp CA, USA).

Four of the GAS6 ligands were capable of inhibiting the GAS6/AXL interaction with the rank potency of 4-72> 4-6~ 4-53 »>all other sequences. The relative inhibition is shown in Table 5 and a representative ELISA, which also includes an inactive GAS6 Ligand 4-63, is shown in FIG. 4. The full length RNA 4-63 sequence is:

GGGGAGUACAAUAACGUUCUCGCCCCUCGUGUCGCCGGUACCUAAGCGCUCGGCCAUGGA UCGUUACGACUAGCAUCGAUG (SEQ ID NO: 22)

Table 5: Gas6 Ligand Sel4 RNA Random Region Sequences and ELISA

+++ Most Inhibition of Gas6*Axl Binding

++ Moderate Inhibition of Gas6*Axl Binding

+ Some Inhibition of Gas6*Axl Binding

Example 4: Ability of GAS6 Ligands to Inhibit GAS6/AXL Interaction: Flow Cytometry Assay

Human glioblastoma cell lines T98G (ATCC # CRL-1690™) or U87 (ATCC # HTB-14™) which both overexpress the AXL receptor for GAS6, were grown in Eagles MEM (ATCC # 30-2003 with 10% FBS) to 80% confluence. Cells were harvested, spun at 150 x g for 5 minutes and resuspended in cold dilution buffer containing 0.01% BSA, 2mM CaCl₂, 150mM NaCl in 20mM HEPES pH 7.4. Initially, 25μ1 of a 4X

concentration of GAS6 aptamer clones were incubated with 25 μΐ of 4x hGAS6 protein (His tagged- R&D Cat# 885-GS) in a 1.5 mL eppendorf tube for 5 minutes at RT.

Samples were moved to ice where 50 (lxlO⁶ cells) cell suspension was added and incubated forlO minutes. Cells were spun down for 2 minutes in a microcentrifuge (100 x g) and resuspended in 100 μΐ, of dilution buffer containing 5 μΐ, of human biotinylated anti-GAS6 pAB (R&D : BAF885) or anti-polyHistidine Biotinylated MAb (R&D:

BAM050). This suspension was incubated on ice for 20 minutes, centrifuged 2 minutes in a microcentrifuge (lOOxg), and then the pellet was resuspended in 100 μΐ, of dilution buffer containing 1 μΐ_^ of Streptavidin-PE (Prozyme cat# PJRS70). The cell suspension was incubated for another 10 minutes on ice, centrifuged as described earlier, then the pellet was resuspended in 500 μΐ, of dilution buffer, and analyzed on the FL2 channel using a FACS Calibur flow cytometer.

For quantitation of the GAS6/AXL interaction, two separate dot plots (one log

SSC vs FSC and log FSC vs FL2-H) and a histogram were created on the flow viewing area. The cells were identified using the dot plot of SSC vs FSC and a region of interest 1 (Rl) around the cells was created (this is to avoid debris counted as cells). The cells in region 1 (Rl) were observed on the dot plot FSC vs FL2-H (adjusted FL2 using control). The cells were viewed on the histogram plotted counts vs FL2-H. Sigmoidal plots were generated in Graphpad Prism using normalized data of the median from each plot (the peak of the median for each histogram was obtained from the Histogram Stats results in Cell Quest Pro software version 5.2.1). FIG. 5 shows a representative histogram for 4-72 inhibition of GAS6 binding to the AXL receptor expressed on the surface of T98G cells. A dose response analysis was done using anti-HIS-SA-PE detection in FACS-Calibur. The data shown include a background control (grey line), 1.3 nM GAS6 (black line), and 1/4^Λ dilutions from a 1 μΜ stock of the 4-72 aptamer with 1.3 nM GAS6, and

demonstrate effective inhibition of GAS6 binding to the T98G samples.

FIG. 6 shows normalized percent inhibition of GAS6 binding to the AXL receptor expressed on the surface of T98G or U87 cells. Inhibition by aptamer clone 4- 72 of hGAS6 (R&D885) binding to the human tumor cells was demonstrated using anti- GAS6-SA-PE (open circles and open triangles) or anti-polyhistidine-SA-PE (closed circles and closed triangles). For each sample the % inhibition was calculated using the formula:

% Inhibition =100*[((max-background)-(sample-background))/ (max- background)].

Data are normalized to min and max controls from the median of histogram plots and charted in GraphPad Prism.

FIG. 7 shows representative histograms of inhibition of GAS6 binding to the AXL receptor expressed on the surface of U87 cells by GAS6 inhibitory aptamers as well as several non-inhibiting aptamers as controls. In each plot, the grey line represents background (no added GAS6 protein), the black line represents samples with 1.3 nM

GAS6, and the dashed line represents samples with 1 μΜ of the indicated aptamer with

1.3 nM GAS6. The greatest inhibition of binding was observed with clones 4-27 and 4-

72, with less yet significant inhibition achieved by clones 4-6 and 4-53.

Two inactive GAS6 Ligands 4-15 and 4-74 were also used as negative controls for Flow Cytometry.

The full length R A sequence of 4-15 is:

GGGG AGUAC AAUAAC GUUCUC G AAAACUGC AC ACC CGC ACUG AUC GGG AG GUGG AGC GG AUC GUUAC G ACUAGC AUCG AUG (SEQ ID NO:27)

The full length RNA sequence of 4-74 is:

GGGGAGUACAAUAACGUUCUCGGCGCCCCAUCCAAACGUGUGGGUGGGCU GACAGCGGGAUCGUUACGACUAGCAUCGAUG (SEQ ID NO:28)

As demonstrated in FIGs 5-7, aptamers 4-6, 4-27, 4-53 and 4-72 are capable of blocking GAS6 interaction in a dose dependant manner to native Axl expressed on U87 and T98 cells.

Example 5: Degenerate SELEX

The two most functionally active clones, as measured by ELISA, R12Gas4 - 72 and R12Gas4 - 53 did not demonstrate any key sequence homology nor were there closely related sequences to either found in the clones. In order to identify key nucleotides and better constrain the sequences' secondary structures a degenerate SELEX was performed in parallel for both parent sequences. The starting template oligos for the degenerate SELEX were the R12Gas4 - 72 and R12Gas4 - 53 sequences, respectively, with degeneracy introduced by design into the N35 region. The synthesis of the

R12Gas4 - 72 degenerate template oligo, named 72d, was 60% original base and 13.3% each of the remaining three bases throughout the R12Gas4 - 72 random region sequence. Likewise, the synthesis of the R12Gas4 - 53 degenerate template oligo, named 53d, was 60% original base and 13.3% each of the remaining three bases throughout the R12Gas4 - 53 random region sequence. By introducing degenerate versions of these parent sequences to the selective pressures of SELEX, one can determine broader sequence families that can elucidate secondary structure and which specific nucleotide substitutions are preferred for binding Gas6. The 72d and 53d degenerate RNA pools had relatively comparable binding as naive Sel4 starting pool for Gas6. After six rounds of selection, the binding affinity of round 6 RNA pools from the 72d and 53d degenerate selections were relatively comparable to the round 12 Sel4 RNA pool from the original selection. A second series of degenerate SELEX was performed starting with fresh 53d RNA pool, named a_53d, and demonstrated the same relative enhancement of binding to Gas6 over six rounds of selection. Specific conditions for degenerate selections 72d and 53d are shown in Table 6 A and specific conditions for degenerate selection a_53d are shown in Table 6B.

Table 6A: Degenerate Selection Conditions 72d and 53d

0.01%

Nitrocellulose 0.0024%tRNA BSA

Table 6B: Degenerate Selection Conditions 53d Repeat

0.01%

Nitrocellulose 0.0024%tRNA BSA

Fifty aptamers from round 6_72d, fifty aptamers from round 6 53d, and fifty- four aptamers from round 6a_53d were cloned and sequenced as described in Example 2. Thirty-two aptamers from across all three degenerate selections were selected for screening via FACS. These 32 sequences were selected to sample from variation on the parent sequences R12Gas4 - 72 and R12Gas4 - 53 as well as new sequences. Where a variation is here defined as having ten or fewer nucleotide base changes compared to the parent sequence within the N35 random region and a new sequence is defined as having greater than ten nucleotide base changes compared to the parent sequence within the N35 random region. Only sequences defined as being variations of R12Gas4 - 72 parent sequence were found to be active by FACS within this sampling. Sequences with fewer than 14 cumulative base substitutions from R12Gas4 - 72 parent sequence can be found in Table 7. The sequences are listed to incorporate the components of the conserved secondary structure features of R12Gas4 - 72 parent as depicted in FIG. 8.

The degenerate selection analysis revealed a conserved secondary structure for the family of sequences related to R12Gas4 - 72 parent; inclusive of those sequences which have ten or fewer variations from the parent.

The conserved secondary structure of the degenerate family of sequences consists of two stem regions and three loop regions. Stem 1 being of minimally six basepairs in length and consisting of the proposed 5 ' terminus and 3 ' terminus of the active domain of the aptamer. On the 5' side of the aptamer, Stem 1 is adjacent to Loop 1, consisting of approximately five nucleotides. On the 3' side of the aptamer, Stem 1 is adjacent to Loop 3, consisting of approximately eight nucleotides. Loops 1 and 3 also feed into Stem 2 which is approximately seven basepairs in length. Stem 2 is capped by Loop 2 which is approximately between four and six nucleotides in length.

Sequence analysis of the degenerate SELEX clones from the R12Gas4 - 72 parent family show the sequence of Stem l, 5'- U C U C G G - 3', is largely due to the first five bases 5'- U C U C G -3' being contributed from the fixed 5' region of the Sel 4 library as well as the last three bases 5'- A G A -3' of its complement being contributed by the fixed 3' region of the Sel4 library. While, there is 100% conservation of the final G, at position six of Stem 1 , even though it is derived from the first degenerate position in the Sel4 library, subsequent experiments demonstrated this basepair can be deleted and activity retained. Likewise, the Stem 1 complement, 5'- C C G A G A -3', is largely dictated by the 3 ' fixed region of the library and by basepairing with the 5 ' fixed region in classic Watson-Crick pairs. Of note the 5' C of this 3' side of Stem 1 is universally conserved along with its complement G as detailed above. The secondary structure of Stem 2 is highly conserved along with multiple instances of dual mutations that conserve the base pairing along the stem, specifically at positions three, four, five, six, and seven. Of note, the G-U wobble and its neighboring G-C at the base of Stem 2, positions two and one, respectively, is conserved in all but two of the 84 clones that make up this cohort. There are multiple instances of the apical end of Stem 2 unpairing one base pair and contributing to Loop 2 becoming a hexaloop instead of a tetraloop; this seems to be well tolerated and maintains FACS activity in both sequences tested that contain this feature.

Loop 1 and Loop 3 are highly conserved as can be seen in FIG.9, which demonstrates variation in sequence at each base position as a percentage with the parent sequence listed along the top. The only position with lower than 95% conservation within Loopl and Loop 3 is base seven of Loop 3 where there is an almost equal preference for either a C or G; in contrast A is seen in only one sequence and U is not represented at all.

Loop 2 composition from degenerate SELEX sequences tolerates substitution at all four base positions, seen both individually and concurrently. Loop 2 also seems to tolerate expanding from a four base tetraloop to a six base hexaloop when a base or both bases in the apical base pair from Stem 2 are substituted such that they no longer form a Watson-Crick pair.

Five functionally active degenerate sequences which highlight some of the above key features were selected for more in depth analysis: R6a_53d_l ("dl"), R6_53d_4 ("d4"), R6a_53d_16 ("dl6"), R6_53d_35 ("d35"), and R6a_53d_50 ("d50") as well as R6_72d_5 ("72") which is identical to the parent sequence. Clone d50 was selected because it only has one base changed compared to parent 72, and it is at the only position that has less than 95% conservation within Loops 1 and 3 of the above population. Clone d50's only substitution has a G in place of the parent C at position seven of Loop 3. This G substitution is seen in a slight majority of all sequences in the population. Three of the other sequences also incorporate this substitution, so d50 was selected to characterize this substitution in isolation. Clone d4 has the same G for C substitution at position seven of Loop 3 as well as changing the first three bases of Loop 2, such that Loop 2 becomes 5'- U G C C -3'. Clone dl6 has the same G for C substitution at position seven of Loop 3 as well as changing the first and fourth bases of Loop 2, such that Loop 2 becomes 5'- G A A A -3'. In this manner, Loop 2 is now a classic GNRA tetraloop (defined where G is G, N is any base, R is G or A, and A is A), which may offer some further thermodynamic stability. When examining both d4 and dl6, all four base positions of Loop 2 have experienced substitution. Clone dl has the same G for C substitution at position seven of Loop 3 as well as a conserved basepair substitution where position five of Stem 2 is changed from U to C along with its complement being changed from A to G, going from a U-A basepair to a C-G basepair. Clone dl has the key feature that position seven of Stem 2 is changed from G to A, which is no longer able to pair with its former complement C, reducing Stem 2 to six basepairs and contributing to Loop 2 becoming a hexaloop. Clone d35 only has a conserved basepair substitution where position three of Stem 2 is changed from G to C along with its complement being changed from C to G, going from a G-C basepair to a C-G basepair, conserving the structure of the stem.

Table 7: Degenerate Sel4 RNA Structure Defined Sequences and FACS

R6 53d 29 UCUCGGAAUCGGGCUUCGAAUCCGGAGUCGCUCCACGCCGGGA 4 1 ND 53

R6a 53d 46 UCUCGGAAUCGGGCUUCGAGACCGAAGUCGCUCCAGGCCGGGA 4 1 + 54

R6 53d 30 UCUCGGAAUCGGGGUUCCUAACAGAACUCGCUCCAGGCCGGGA 4 1 ND 55

R6 53d 13 UCUCGGAAUCGGGGUUCAACACUGAACUCGCUCCAGGCCGGGA 4 1 ND 5 6

R6a 53d 13 UCUCGGAAUCGGGGUUCGAUGCUGAACUCGCUCCAGGCCGGGA 4 1 ND 5 7

R6a 53d 7 UCUCGGAAUCGGGGUUCGUGACUGAACUCGCUCCAGGCCGGGA 4 1 + 5 8

R6 53d 4 UCUCGGAAUCGGGGUUCGUGCCCGAACUCGCUCCAGGCCGGGA 4 1 + 5 9

R6 53d 26 UCUCGGAAUCGGGGUUGGGAACNCAACUCGCUCCACGCCGGGA 4 1 ND 60

R6 53d 18 UCUCGGAAUCGGGGUUUGGAAACGAACUCGCUCCAGGCCGGGA 4 1 ND 61

R6 72d 49 UCUCGGAAUCGGGGUUCAAAAUCGAAGUCGCUCCAGGCCGGGA 4 1 ND 62

R6a 53d 8 UCUCGGAAUCGGGCUUCGAANGCGAAGUCGCUCCAGGCCGGGA 5 1 ND 63

R6a 53d 14 UCUCGGAAUCGGGCUUCGUAUCCGAAGUCGCUCCAGGCCGGGA 5 1 ND 64

R6 53d 37 UCUCGGAAUCGGGGUACUCAAUCGAACUCGCUCCAGGCCGGGA 5 1 ND 65

R6a 53d 1 UCUCGGAAUCGGGGUCCAAAUCCGGACUCGCUCCAGGCCGGGA 5 1 + 6 6

R6a 53d 29 UCUCGGAAUCGGGGUCCCUAACCGGACUCGCUCCAGGCCGGGA 5 1 ND 67

R6 53d 36 UCUCGGAAUCGGGGUCCUUAGCCGGACUCGCUCCACGCCGGGA 5 1 ND 68

R6 72d 7 UCUCGGAAUCGGGGUUCAAUGGCGAACUCGCUCCAGGCCGGGA 5 1 ND 6 9

R6 53d 46 UCUCGGAAUCGGGGUUCCAGAGAGAACUCGCUCCAGGCCGGGA 5 1 ND 7 0

R6 53d 6 UCUCGGAAUCGGGGUUCCAUGACGAACUCGCUCCAGGCCGGGA 5 1 ND 7 1

R6 53d 44 UCUCGGAAUCGGGAUUCGUAAUCGGAUUCGCUCCAGGCCGGGA 6 1 ND 72

R6a 53d 54 UCUCGGAAUCGGGCUUCGUUUCCGAAGUCGCUCCAGGCCGGGA 6 1 ND 73

R6 53d 43 UCUCGGAAUCGGGGUACCUUACCGUACUCGCUCCAGGCCGGGA 6 1 + 74

R6a 53d 38 UCUCGGAAUCGGGGUGCUUAACUGCACUCGCUCCAGGCCGGGA 6 1 ND 75

R6 53d 25 UCUCGGAAUCGGGGUUCCAAUGAGCACUCGCUCCAGGCCGGGA 6 1 ND 7 6

R6 53d 41 UCUCGGAAUCGGGGUUCCGCGCGGAACUCGCUCCAGGCCGGGA 6 1 ND 7 7

R6a 53d 31 UCUCGGAAUCGGGGUUCCUCUCGGAACUCGCUCCAGGCCGGGA 6 1 ND 7 8

R6 53d 1 UCUCGGAAUCGGGGUUCGGGNUCGGACUCGCUCCAGGCCGGGA 6 1 ND 7 9

R6 53d 34 UCUCGGAAUCGGGCUUCAGAAAUGAAGUCGCUCCAGGCCGGGA 7 1 ND 8 0

R6 53d 20 UCUCGGAAUCGGGCUUCAGUUCCGAAGUCGCUCCAGGCCGGGA 7 1 ND 8 1

R6a 53d 6 UCUCGGAAUCGGGCUUCGCUUGCGAAGUCGCUCCAGGCCGGGA 7 1 ND 82

R6 53d 22 UCUCGGAAUCGGGGUCCAUAAACGGACUCGCUCCACCCUGGGA 7 1 ND 83

R6 53d 49 UCUCGGAAUCGGGCCUCGACAUUGAGGUCGCUCCAGGCCGGGA 8 1 ND 84

R6a 53d 17 UCUCGGAAUCGGGCUCCCUAAACGGAGUCGCUCCAGGCCGGGA 8 1 ND 85

R6 53d 28 UCUCGGAAUCGGGGUACCUGGCGGUACUCGCUCCAGGCCGGGA 8 1 ND 8 6

R5 72d 20 UCUCGGAAGCGGGGGUGGGACCCCAACUCUCUCCCCGCCGGGGA 9 1 ND 8 7

R6 53d 33 UCUCGGAAUCGGGGUUCCUGGCGCAGCUCGCUCCCCCCCGGGA 9 1 ND 8 8

R6 53d 7 UCUCGGAAUCGGCUUUCUAACUCUUACUCUUUCCUCGCCGGGA 10 1 ND 8 9

R6 53d 39 UCUCGGAGCCCCGGUACAAAACCGUGUUCGCUCCAGGCCGGGA 10 1 ND 90

R5 72d 17 UCUCGGAGUGGGGGGUGGAAUCCCAGCUCGCUCCCCCCCGGGGA 10 1 ND 91

R6 53d 47 UCUCUGAUCGGGGAUCCUAGCCCAAGUCGCGCCCCCCCGGGA 11 1 - 92

R6 53d 16 UCUCUGAUCGCGGUACCAAACGCAACACGCGCCGCCCCGGGA 11 1 ND 93

R6 53d 3 UCUCGGAUUCGCGGUUCCAAGCCCGUCUCCCCUCACGCGCGGA 12 1 ND 94

R6 53d 48 UCUCAGACCGCGGAAAGACAUCGAGCACGGACCACGCCGGGA 13 1 ND 95

R6 53d 8 UCUCCAAUCUUUCUUACAAACCGUACUCUCUUCAUGACCGGA 13 1 ND 9 6

R6 53d 50 UCUCGGAAGCAAACUUCCCCUCCGAACACUCUCCUCACCGGGA 13 1 ND 97

R6 53d 11 UCUCGGAAUCCCUGAUCCAUACCCAGGUCCCCCCUCCCCGGGA 13 1 ND 98 R6 53d 14 UCUCGGUCUCGGCGUGUGCUCCCGAACUGCCUCUACACCCGGA 13 1 ND 99

R6 53d 24 UCUCGGUGCCCCGGUUCAAGACCGUGCUCGCGCCAAUCAGGGA 13 1 ND 100

R6 53d 2 UCUCGUAACCGUGCCUCUACUCCCACCUCCCUCCAUGACGGGA 13 1 ND 101

R6 53d 31 UCUCUGAUCGCGCUUGGCAACCGAAGUCAUGUCGCCCCGGGA 13 1 - 102

R6 53d 45 UCUCUGAUCGGGUCUCACAGCCGAUCCGGCUGCCCCCCGGGA 13 1 ND 103

R6 53d 19 UCUCGGAAGUGGCGUAUCGACCCUAAUCAGCUCCUCCCCGGGA 14 1 ND 104

All sequences related to R12Gas4_72 with 14 or fewer nucleotide substitutions.

Sorted by variation from parent sequence ("delta") and then sequence

composition, number of clones containing sequence indicated as "repeats."

FACS results "+" represents a shift, "-" represents no shift, "ND" means FACS data has not been determined. Where G=2'Ribo G; A=2'Ribo A; C=2'Fluoro C;

U=2'Fluoro U.

Example 6: Truncation and Mutation Probing of anti-Gas6 Ligand Sequences

Truncated versions of all five of the above listed clones were generated in vitro with the addition of the consensus initiation sequence for T7 RNA polymerase

"GGGAGA" to the 5' end of each clone. The resultant full length clones and

corresponding truncate are as follows: dl and 1T1, d4 and 4T1, dl6 and 16T1, d35 and

35T1, d50 and 50T1, and for the parent 72 and 72T22. Truncation sequences are listed in Table 8. All six of these truncates were found to have moderate FACS activity with

72T22 and 16T1 being slightly stronger than the rest. Strong FACS activity was regained by extending Stem 1 by adding "UCUCCC" to the 3' end of the truncated sequences to generate 72T22X and 16T1X, respectively.

A series of mutant compounds for 72T22 and 16T1X containing nucleotide

substitutions were generated and their ability to block Gas6/Axl interaction was measured with FACS activity in comparison to their non-mutant parent. Mutation sequences are listed in Table 9. Mutations at positions one and two of Loop 1 to UU were shown to kill FACS activity (72T22M1). Mutations at positions three and four of Loop 1 to AG were shown to kill FACS activity (72T22M2). Mutational analysis supports the importance of conservation of sequence in Loop 1, consistent with the degenerate SELEX results.

Mutations at positions one and two of Loop 2 to UU were well tolerated in regard to

FACS activity (72T22M3). Mutations at positions three and four of Loop 2 to UG were well tolerated in regard to FACS activity (72T22M4). Mutational analysis supports the tolerance to substitution within the sequence in Loop 2, consistent with the degenerate

SELEX results. Mutations at positions one and two of Loop 3 to CG were shown to kill

FACS activity (72T22M5), (72T22XM1), and (16T1XM1). Mutations at positions three and four of Loop 3 to AG were shown to kill FACS activity (72T22M6), (72T22XM2), and (16T1XM2). Mutations at positions five and six of Loop 3 to GU were shown to kill FACS activity (72T22M7). Single nucleotide deletions in Loop 3 at position eight (16T1XM34), position six (16T1XM35), and position one (16T1XM36), also all result in loss of all FACS activity. Mutational and deletion analysis supports the importance of conservation of sequence in positions one through six and position eight in Loop 3, consistent with the degenerate SELEX results.

Table 8: R12Gas4-72 Truncates

16T1XM31 GGGAGAU CGG AAUCG GG UCG GAAA CGA UC GCUCCAGG CCG AUCUCCC +++ 135

16T1XM32 GGGAGAU GG AAUCG GG UCG GAAA CGA UC GCUCCAGG CC AUCUCCC +++ 136

FACS results "+++" represents a strong shift, "++" represents a moderate shift,

"+" represents a weak shift, "-" represents no shift. Where G=2'Ribo G;

A=2'Ribo A; C=2'Fluoro C; U=2'Fluoro U and an underscore "_" represents a

deleted nucleotide.

Sequences for the 16T1X family of truncations contain spaces in the sequence to align structural features of stems and loops into columns within the table.

Table 9: R12Gas4-72 Mutants

FACS results "+++" represents a strong shift, "++" represents a moderate shift,

"+" represents a weak shift, "-" represents no shift. Where G=2'Ribo G;

A=2'Ribo A; C=2'Fluoro C; U=2'Fluoro U and an underscore "_" represents a

deleted nucleotide.

Sequences for the mutants contain spaces in the sequence to align structural

features of stems and loops into columns within the table.

Stem 2 position six was mutated to A along with its complement changed to U to conserve the stem structure (16T1XM4), changing from a C-G to A-U basepair, FACS activity was preserved. Stem 2 can tolerate mutations that maintain its stem secondary structure inclusive of positions three through seven. The G-U wobble pair at position two of Stem 2 was highly conserved in the degenerate SELEX results and was identified for further studies with mutational analysis. The G-U was mutated to a G-C Watson-Crick basepair (16T1XM5), with resultant FACS inactivity. The G-U was mutated to a A-U Watson-Crick basepair (16T1XM12), likewise, with resultant FACS inactivity; see FIG. 11 for representative FACS results. Even flipping the G-U wobble to a U-G wobble (16T1XM20), killed FACS activity. The results of the degenerate SELEX as well as these three mutants are consistent with the requirement of a G-U wobble pair at position two in Stem 2 for functional activity.

The G-C basepair at position one of Stem 2 was highly conserved in degenerate SELEX results and was further investigated. The G-C basepair was dual mutated to a C- G basepair (16T1XM19), which should thermodynamically be very similar in regards to conserving the secondary structure of the stem, however this was found to be functionally inactive on FACS. The same mutation in a later truncate (16T1XM25) likewise is inactive on FACS. Furthermore, flipping both basepairs simultaneously in positions one and two such that they become C and U with their complements G and G (16T1XM33) also killed FACS activity. The sequence at the base of Stem 2 needs to be maintained as G G in positions one and two with their complements U and C, such that the initial two basepairs of Stem 2 are G-C Watson-Crick basepair in position one and G-U wobble at position two, otherwise FACS activity is lost.

Attention was shifted towards truncation of both Stem 1 and Stem 2 in the new parent 16T1X. Stem 1 is a 12 basepair stem consisting of the sequence 5'- G G G A G A U C U C G G -3' (SEQ ID NO: 158) and the complementary 5'- C C G G G A U C U C C C -3' (SEQ ID NO: 159). Truncations 16T1XM6 through Mi l are progressively larger basepair deletions taken out of Stem 1 of 16T1X. Deletion of U in position nine and its wobble complement G of Stem 1 (16T1XM6) maintains complete FACS activity - This U-G wobble is not required for functional activity. Deletion of CU in positions eight and nine and their complement GG of Stem 1 (16T1XM7) maintains strong FACS activity. Deletion of CUC in positions eight, nine, and ten and their complement GGG of Stem 1 (16T1XM8) maintains strong FACS activity. Deletion of UCUC in positions seven, eight, nine, and ten and their complement GGGA of Stem 1 (16T1XM9) maintains strong

FACS activity. Deletion of UCUCG in positions seven, eight, nine, ten, and eleven and their complement CGGGA of Stem 1 (16T1XM10) demonstrates strong FACS activity. Deletion of AUCUCG in positions six, seven, eight, nine, ten, and eleven and their complement CGGGAU of Stem 1 (16T1XM11) demonstrates only moderate FACS activity. Functional activity is wholly maintained even after the deletion of four or five basepairs from Stem 1, but is diminished with the deletion of six basepairs.

Truncations 16T1XM13 through Ml 8 investigate Stem 2 by individually deleting one basepair at a time, starting proximal to Loops 1 and 3 and ending towards apical Loop 2. Stem 2 is herein written as 5'- G G G U U C G -3' and its complement 5'- C G A A C U C -3'; including a conserved G-U wobble basepair. Deletion of G in position one and its complement C of Stem 2 (16T1XM13) results in loss of FACS activity.

Deletion of G in position two and its complement wobble base U of Stem 2 (16T1XM14) results in loss of FACS activity; further supporting the necessity of the G-U wobble pair for functional activity. Deletion of G in position three and its complement C of Stem 2 (16T1XM15) maintains strong FACS activity. Deletion of U in either equivalent position four or five, and its complement A of Stem 2 (16T1XM16) maintains strong FACS activity. Deletion of C in position six and its complement G of Stem 2 (16T1XM17) maintains strong FACS activity. Deletion of G in position seven and its complement C at the apical terminus of Stem 2 (16T1XM18) maintains strong FACS activity.

Combining some of the above individual basepair deletions in Stem 2 provides further information. Deletion of GU in positions three and four and their complement

AC of Stem 2 (16T1XM21) maintains strong FACS activity. Deletion of GU in positions three and four and their complement AC combined with deletion of G in position seven and its complement C at the apical terminus of Stem 2 (16T1XM22) results in weak FACS activity. Deletion of GU in positions three and four and their complement AC combined with deletion of C in position six and its complement G of Stem 2

(16T1XM27), results in weak FACS activity. Deletion of GUU in positions three, four, and five and their complement AAC of Stem 2 (16T1XM28) kills FACS activity.

Having demonstrated that truncation is tolerated to some extent in both Stem 1 and Stem 2 individually; combinations including truncations from both stems

simultaneously were next attempted. Deletion of UCUC in positions seven, eight, nine, and ten and their complement GGGA of Stem 1 combined with deletion of GU in positions three and four and their complement AC of Stem 2 (16T1XM23) results in only moderate to strong FACS activity. Deletion of UCUC in positions seven, eight, nine, and ten and their complement GGGA of Stem 1 combined with deletion of GU in positions three and four and their complement AC combined with deletion of G in position seven and its complement C at the apical terminus of Stem 2 (16T1XM24) results in weak

FACS activity. Deletion of UCUCG in positions seven, eight, nine, ten, and eleven and their complement CGGGA of Stem 1 combined with deletion of GU in positions three and four and their complement AC of Stem 2 (16T1XM26) results in moderate to strong FACS activity. Deletion of U in position nine and its wobble complement G of Stem 1 combined with deletion of GU in positions three and four and their complement AC of Stem 2 (16T1XM30) maintains strong FACS activity. Deletion of CU in positions eight and nine and their complement GG of Stem 1 combined with deletion of GU in positions three and four and their complement AC of Stem 2 (16T1XM31) maintains strong FACS activity. The proposed secondary structure of the 47 nucleotide 16T1XM31 is depicted in FIG. 10. Deletion of CUC in positions eight, nine, and ten and their complement GGG of Stem 1 combined with deletion of GU in positions three and four and their

complement AC of Stem 2 (16T1XM32) maintains strong FACS activity; but slightly less strong than 16T1XM31. After this screening it appears that 16T1XM31 is the shortest truncate that has FACS activity comparable to its full length counterpart; see FIG. 11 and FIG. 12 for representative FACS results. The minimally active aptamer motif for binding to and inhibiting GAS6 as informed by degenerate SELEX, and truncation and mutation studies can be represented by 16T1XM31. Consisting of a Stem 1 of minimally four basepairs in length, preferably of between four and fourteen basepairs, more preferably between six and twelve basepairs in length. In this embodiment Stem 1 is ten basepairs in length with sequence 5'- G G G A G A U C G G - 3' (SEQ ID

NO: 160) and the complementary 5'- C C G A U C U C C C -3' (SEQ ID NO: 161). Stem 1 contains the 5 ' terminus and 3 ' terminus of the oligonucleotide chain. The primary requirement of Stem 1 is its secondary structure as a stem, with wide variation in sequence tolerated at all positions. Stem 1 on the 5' side of the aptamer leads into Loopl , which is minimally five bases in length, in this embodiment written as 5'- A A U C G -3'. Degenerate SELEX demonstrated conservation of greater than 95% at all five positions of Loop 1.

Loop 1 on the 5 ' side of the aptamer and Loop 3 on the 3 ' side of the aptamer lead into Stem 2. Stem 2 consists of minimally four basepairs in length, preferably of between four and fourteen basepairs, more preferably between five and twelve basepairs in length. In this embodiment Stem 2 is five basepairs in length with sequence written as 5'- G G U C G -3' and its complement 5'- C G A U C -3'. The results of the degenerate SELEX as well as mutational analysis are consistent with the requirement of a G-U wobble pair at position two in Stem 2 for functional activity. The G-C basepair at position one of Stem 2, adjacent to Loops 1 and 3, was highly conserved in degenerate SELEX and mutational analysis and is consistent with its requirement for functional activity. In one embodiment the sequence at the base of Stem 2 is G G in positions one and two with their

complements U and C, such that the initial two basepairs of Stem 2 are G-C Watson- Crick basepair in position one and G-U wobble at position two. Variations in the identity of the basepairs at other positions within Stem 2 seem to be very well tolerated as seen in degenerate SELEX results and mutational analysis.

Apical Loop 2 is found atop Stem 2 and is minimally three bases in length, preferably of between three and ten bases in length, more preferably between four and six bases in length. Degenerate SELEX results show tolerance for substitution at any and all positions within Loop2, which is further supported by mutational analysis. Loop 2 is a simple linker region and can be of sequence 5'- G A A A -3' as in this embodiment, or tolerate a wide variety of substitutions in other embodiments. Loop 2 may tolerate substitution with a non-nucleotide linker domain.

Loop 3 connects Stem 2 and Stem 1 on the 3 ' side of the aptamer. Loop 3 is minimally eight bases in length. In this embodiment Loop 3 has sequence 5'- G C U C C A G G -3'. Degenerate SELEX demonstrated conservation of greater than 95% at positions one through six as well as position eight. Positions one through six are also shown to be intolerant of substitution through mutational analysis, resulting in loss of function. Position seven in Loop 3 in one embodiment can be G, and in another embodiment can be C; substitution with A or U is not well tolerated. Therefore, Loop 3 can be adequately defined by the consensus sequence 5'- G C U C C A G/C G -3'. Example 7: Further Truncation and Optimization of the 2' Sugar Modification of anti-Gas6 Ligand

Ligands isolated from 2'-fluorpyrimidine/2'-hydroxypurine libraries exhibit sufficient nuclease stability for in vivo screening. However, the high 2'hydroxyl content makes 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, we sought to optimize the anti-GAS6 ligands by further stabilization of the backbone by substitution of 2'0-methyl nucleotides for 2'-hydroxyl nucleotides (Table 10). Additional substitutions of 2'0-methyl nucleotides for 2'-fluoro nucleotides were also made to further improve stability, reduce cost of manufacture, and reduce the level of potential impurities that can arise during heating of 2'-fluorouridine- containing oligonucleotides during the manufacturing process. Finally, "capping" of the 3' end, which prevents exonuclease degradation of oligonucleotides, was also attempted to further enhance in vivo stability.

Capping of the 3' end of 16T1XM31 was accomplished by synthesis of the ligand from a CPG-support loaded with inverted deoxythymidine, to create a 3 '-3' linkage (RB665) at the 3' end of the ligand. This modification was well tolerated, and was therefore used in all synthetically produced modifications to this ligand. RB665 has the sequence:

rGrGrGrArGrAfUfCrGrGrArAfUfCrGrGrGfUfCrGrGrArArAfCrGrAfUfCrGfCfUfCfCr ArGrGfCfCrGrAfUfCfUfCfCfCiT (SEQ ID NO: 162),

where "r" represents a ribonucleic acid, "f" represents a 2'-fluoro nucleotide, and "iT" represents the inverted deoxythymidine. Table 10 Modified Gas6 Ligands

DMSLIBRARYO 1 :21817722.1 91

rG=2'Ribo G; rA=2'Ribo A; fC=2'-Fluoro C; fU=2'-Fluoro U; mG=2 ' -O-methyl G; mA=2' -O-methyl A; mC=2 ' -O-methyl C; mU=2 ' -O-methyl U; iT= inverted

deoxythymidine; (6GLY)= hexaethylene glycol linker (incorporated using 9-0- Dimethoxytrityl-triethylene glycol, 1- [ ( 2-cyanoethyl ) - (N, N-diisopropyl ) ] - phosphoramidite )

FACS results "+++" represents a strong shift, "++" represents a moderate shift, "+" represents a weak shift, "-" represents no shift; "ND" means FACS data has not been determined.

The "RB ID" is a unique identifier that refers to the ligand having the sequence with specific modifications noted in the column "Modified Sequences." Sequences are listed in a 5' -3' direction

Loop 2 showed no apparent conservation in length or sequence composition in degenerate SELEX or mutational analysis, but rather, may have served a role as a linker that keeps the two halves of the molecule attached through a flexible structure. This would predict that a nucleotide composition of Loop 2 is not required for functional binding to GAS6. Consistent with this prediction, substitution of Loop 2 with a hexaethylene glycol spacer (RB678) results in no significant loss of FACS activity, as compared to the parent ligand (RB665). Subsequently, RB678 served as the parent compound for further optimization of the anti-Gas6 ligand.

Investigation was directed at further truncating Stem 1 which previously has been shown to be tolerant of truncation down to at least seven base pairs in length

(16T1XM10) with retention of full FACS activity. RB678 like its parents (RB665) and (16T1XM31) has a Stem 1 of ten base pairs in length. Truncations RB679 through RB682 are progressively larger base pair deletions taken off of the terminal end of Stem 1 of RB678. Deletion of G in position one and its complement C of Stem 1 (RB679) maintains strong FACS activity. Deletion of GG in position one and two and their complement CC of Stem 1 (RB680) maintains strong FACS activity. Deletion of GGG in position one, two and three and their complement CCC of Stem 1 (RB681) maintains strong FACS activity. Deletion of GGGA in position one, two, three and four and their complement UCCC of Stem 1 (RB682) also maintains strong FACS activity.

Truncations RB683 through RB686 are a similar run of deletions starting at the other end of Stem 1 of RB678. Deletion of G in position nine and its complement C of Stem 1 (RB683) maintains strong FACS activity. Deletion of CG in positions eight and nine and their complement CG of Stem 1 (RB684) maintains strong FACS activity.

Deletion of UCG in positions seven, eight and nine and their complement CGA of Stem 1 (RB685) maintains strong FACS activity. Deletion of UCGG in positions seven, eight, nine and ten and their complement CCGA of Stem 1 (RB686) also maintains strong FACS activity.

The deletion of four base pairs from Stem 1 of parent RB678 are functionally well tolerated in FACS, because of this, RB682 is made the parent of future optimization attempts. Deletion of G in position five and its complement C of Stem 1 (RB692) demonstrates moderate FACS activity. Deletion of CG in positions four and five and their complement GC of Stem 1 (RB693) has only moderate FACS activity.

Truncations RB696 through RB698 investigate Stem 2 in parent RB682 by individually deleting one base pair at a time, starting at position three and ending towards apical Loop 2. Deletion of U in position three and its complement A of Stem 2 (RB696) results in loss of FACS activity. Deletion of C in position four and its complement G of Stem 2 (RB697) results in loss of FACS activity. Deletion of G in position five and its complement C of Stem 2 (RB698) also results in loss of FACS activity.

A negative control mutant of parent RB682 was designed by mutating positions three and four of Loop 3 to G and A, respectively (RB699). Mutant RB699 is

functionally inactive in FACS.

Having truncated the anti-GAS6 ligand to 36 nucleotides plus the hexaethylene glycol spacer (RB682) we turned our attention to introducing 2'-0-methyl nucleotides. RB682 has the sequence:

rGrAfUfCrGrGrArAfUfCrGrGrGfUfCrG(6GLY)fCrGrAfUfCrGfCfUfCfCrArGrGfCfCr GrAfUfCiT (SEQ ID NO: 167)

where "r" represents a ribonucleic acid, "f" represents a 2'-fluoro nucleotide, and "iT" represents the inverted deoxythymidine, and (6GLY) represents the hexaethylene glycol spacer.

Stem 1 has been shown to be very tolerant of mutational substitutions and multiple sequences as long as the stability of the stem structure has been maintained, indicating it could be a good candidate for targeted 2'-0-methyl nucleotide substitutions. An initial pass of making modifications of two base pairs at a time in Stem 1 of RB682 was attempted. Modification of the first two base pairs, G and A in positions one and two and their complement U and C, within Stem 1 to base pairs containing 2'O-methyl sugars (RB702) resulted in full FACS activity. Modification of the middle two base pairs, U and C in positions three and four and their complement G and A, within Stem 1 to base pairs containing 2'0-methyl sugars (RB703) resulted in full FACS activity. Modification of the final two base pairs, G and G in positions five and six and their complement C and C, within Stem 1 to base pairs containing 2'0-methyl sugars (RB704) likewise, resulted in full FACS activity. A composite sequence (RB705) containing a combination of the modifications is RB702, RB703 and RB704 was synthesized and tested. RB705, in which all six base pairs have been modified to contain 2'-0-methyl sugars, also results in full FACS activity.

RB705, with the fully 2'-0-methyl modified Stem 1, served as the parent compound for exploring tolerance of 2'0-methyl modified sugars in Stem 2 introduced one base pair at a time in RB723 through RB727. Modification of the first base pair, G in position one and its complement C, within Stem 2 to a base pair containing 2'0-methyl sugars (RB723) resulted in total loss of FACS activity. Modification of the wobble base pair, G in position two and its complement U, within Stem 2 to a base pair containing 2'0-methyl sugars (RB724) resulted in moderate FACS activity. Modification of the C in position four and its complement G, within Stem 2 to a base pair containing 2'0- methyl sugars (RB726) resulted in full FACS activity. Modification of the last base pair, G in position five and its complement C, within Stem 2 to a base pair containing 2'0- methyl sugars (RB727) also resulted in full FACS activity.

Example 8: Nucleic Acid Modulators of anti-Gas6 Ligands

Ligands encode the information necessary to design nucleic acid modulators, or control agents, for them based upon complementary Watson-Crick base pairing rules. The effectiveness of a given control agent is dependent upon several factors, including accessibility of the targeted region of the ligand for nucleation with the control agent, as well as the absence of or limited internal secondary structure within the control agent, which would require denaturation prior to full-duplex formation with the ligand. To define regions of the anti-Gas6 ligands that would be preferred regions for association with nucleic acid modulators, a series of control agents (see Table 11 and Fig 13) were designed for 16T1XM31. Table 11: Modulators of Gas6 Ligands

SEQ ID NOs refer to the unmodified versions of the ligands described in the column titled, "Modified Sequence"; Sequences are listed in a 5 '-3' direction

mG=2'-0-methyl G; niA=2'-0-methyl A; mC=2'-0-methyl C; mU=2'-0-methyl U

In order to test the effectiveness of all five control agents (RB673-677) a gel-shift assay was employed. In this assay, 16T1XM31 was radiolabeled with P³² so as to render it traceable and spiked into unlabeled 16T1XM31 with either buffer or various molar ratios of one of the five control agents. More specifically, the molar concentration of 16T1XM31 was kept constant at 125 nM while a range of control agent (1 uM to 62.5 nM) was added to the reaction resulting in a molar ratio range of 1 :8 to 1 :0.5. Reactions were incubated for 15 minutes at 37C, loaded on a 16% native polyacrylamide gel and allowed to run for 3hrs. Calcium Chloride (2 mM) was present in the gel, running buffer and all the reactions. The gel was then taken down, wrapped in Saran wrap, exposed to a phosphorimager screen for 1 hour in a light-tight cassette and scanned by a Storm 840. The resulting scan showed the position of 16T1XM31 alone, and 16T1XM31 plus varying molar amounts of control agent shifted upward due to hybridization of the ligand with the control agent. The results of the minimum molar ratio of control agent needed to disrupt native folding of 16T1XM31 are indicated in Table 11. The secondary structure of anti-Gas6 ligand, 16T1XM31, is capable of being efficiently modulated by the use of control agents. These results are consistent with data collected in FACS (see below).

Example 9: Methods for Evaluating the Ability of Control Agents to Modulate GAS6 Ligands Ability to Inhibit GAS6/AXL Interaction: Flow Cytometry Assay Human glioblastoma cell line U87 (ATCC # HTB-14™), which overexpresses the

AXL receptor for GAS6, were grown in Eagles MEM (ATCC # 30-2003 with 10% FBS) to 80% confluence. Cells were harvested, spun at 150 x g for 5 minutes and resuspended in cold dilution buffer containing 0.01% BSA, 2mM CaCl₂, 150mM NaCl in 20mM HEPES pH 7.4. Initially, 12.5μ1 of an 8X concentration (2 μΜ) of GAS6 aptamer 16T1XM31 was incubated with 12.5 μΐ of an 8x hGAS6 protein (4 μg/mL) (His tagged- R&D Cat# 885-GS) in a 1.5 mL eppendorf tube for 5 minutes at 37°C. 25μ1 of a 4X concentration of Control Agent (Ι μΜ to 8 μΜ) was added and incubated for 5 minutes at 37°C. Samples were moved to ice where 50 μΐ, (lxlO⁶ cells) cell suspension was added and incubated for 10 minutes. Cells were spun down for 2.5 minutes in a microcentrifuge (100 x g) and resuspended in 100 μΐ, of dilution buffer containing ΙΟμΙ, of anti- polyHistidine Biotinylated MAb (R&D: BAM050). This suspension was incubated on ice for 20 minutes, centrifuged 2 minutes in a microcentrifuge (lOOxg), and then the pellet was resuspended in 100 μΐ, of dilution buffer containing 1 μΕ of Streptavidin-PE

(Prozyme cat# PJRS70). The cell suspension was incubated for another 10 minutes on ice, centrifuged as described earlier, then the pellet was resuspended in 600 μΐ, of cold dilution buffer, and analyzed on the FL2 channel using a FACS Calibur flow cytometer.

For quantitation of the GAS6/AXL interaction, two separate dot plots (one log SSC vs FSC and log FSC vs FL2-H) and a histogram were created on the flow viewing area. The cells were identified using the dot plot of SSC vs FSC and a region of interest 1 (Rl) around the cells was created (this is to avoid debris counted as cells). The cells in region 1 (Rl) were observed on the dot plot FSC vs FL2-H (adjusted FL2 using control). The cells were viewed on the histogram plotted counts vs FL2-H.

Five control agents, RB673-RB677, were tested under these conditions.

Consistent with the gel shift results, RB673-RB676 were capable of modulating the blocking of binding by 16T1XM31 to GAS6 on the cell surface by. A representative flow cytometry histogram for RB674 is shown in FIG. 14. RB677 was not capable of modulating the activity of 16T1XM31 under the conditions tested. This is consistent with the gel shift data which demonstrated a >8 fold molar excess of RB677 was needed to hybridize to 16T1XM31 before a shift was evident.

Claims

CLAIMS We claim:

1. A nucleic acid ligand that binds GAS6, 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 forms a secondary structure having a first stem, a first loop, a second stem, a second loop and a third loop.

3. The ligand of claim 1, wherein the nucleic acid sequence comprises at least one 2' modified nucleotide.

4. The ligand of claim 1, wherein the ligand comprises a modified phosphate backbone.

5. The ligand of claim 1, wherein the ligand comprises an inverted deoxythymidine cap.

6. The ligand of claim 1, wherein the ligand comprises a hexaethylene glycol linker.

7. The ligand of claim 1, wherein the ligand is conjugated to a carrier.

8. The ligand of claim 7, wherein the carrier is a hydrophilic moiety.

9. The ligand of claim 8, wherein the hydrophilic moiety is polyethylene glycol.

10. A pharmaceutical composition comprising a ligand according to any one of claims 1- 9, or a pharmaceutically acceptable salt thereof.

11. A modulator which binds specifically the ligand of any one of claims 1-9.

12. The modulator of claim 11 wherein the modulator 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).

13. The modulator of claim 11 wherein the modulator reverses, partially or completely, the activity of a GAS6 ligand.

14. The modulator of claim 11 wherein the modulator is selected from the group consisting of a DNA sequence, an R A 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.

15. The modulator of claim 11, wherein the nucleic acid sequence comprises a 2' modified nucleotide.

16. The modulator of claim 11, wherein the ligand comprises a modified phosphate backbone.

17. A pharmaceutical composition comprising a modulator according to any one of claims 11-16, or a pharmaceutically acceptable salt thereof.

18. A method for regulating GAS6 activity comprising administering to a host in need thereof an effective amount of the ligand according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof.

19. A method for treating symptoms of a GAS6-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 9, or a pharmaceutically acceptable salt thereof.

20. The method of claim 19, wherein the GAS6-mediated disorder is a platelet-mediated disorder.

21. The method of claim 20, wherein the platelet-mediated disorder is selected from the group consisting of a vascular disorder, a cerebrovascular disorder, a platelet-mediated inflammatory disorder, a diabetes-related disorder, a cancer, and HIV infection.

22. The method of claim 21, wherein the vascular disorder is selected from the group consisting of acute coronary syndromes, thrombosis, thromboembolism,

thrombocytophenia, peripheral vascular disease, sepsis and transient ischemic attack.

23. The method of claim 21, wherein the cerebrovascular disorder is selected from the group consisting of transient ischemic attack, ischemic stroke, and embolism.

24. The method of claim 21, wherein the platelet-mediated inflammatory disorder is selected from the group consisting of arthritis, rheumatoid arthritis, psoriatic arthritis, reactive arthritis, inflammatory bowed disease, ankylosing spondylitis, and scleroderma.

25. The method of claim 21, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, ovarian cancer, testicular cancer, pancreatic cancer, brain cancer, bone cancer, liver cancer, glioma, or a metastatis thereof.

26. The method of claim 21, wherein the diabetes-related disorder is selected from the group consisting of diabetic retinopathy, diabetic vasculopathy, atherosclerosis, ischemic stroke, peripheral vascular disease, acute renal injury and chronic renal failure.

27. The method of claim 19, wherein the GAS6 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.

28. Use of a ligand of any of claims 1-9 in the manufacture of a medicament for the treatment of a GAS6-mediated disorder wherein the treatment comprises administering to a host in need thereof a therapeutically effective amount of the ligand, or a

pharmaceutically acceptable salt thereof.

29. The use of claim 28, wherein the GAS6-mediated disorder is a platelet-mediated disorder.

30. The use of claim 29, wherein the platelet-mediated disorder is selected from the group consisting of a vascular disorder, a cerebrovascular disorder, a platelet-mediated inflammatory disorder, a diabetes-related disorder, a cancer, and HIV infection.

31. A method for determining whether a GAS6 ligand inhibits binding of a GAS6 polypeptide to an Axl polypeptide, comprising: a) mixing a sample preparation of cells which overexpress a GAS6 receptor protein, and

32. The method of claim 31 wherein the method uses an ELISA assay or flow cytometry.

33. The method of claim 31 wherein a difference in binding of GAS6/AXL in the presence and absence of a GAS6 ligand is indicative of relative inhibition of GAS6/AXL interaction.