Von Willebrand Factor (VWF) Targeting Agents and Methods of Using the Same

Abstract

Compositions and methods for preventing and treating thrombosis are provided. [Solution] Provided herein are aptamers capable of inhibiting the activity of von Willebrand factor (VWF). Pharmaceutical compositions containing these aptamers are also provided. Methods for preventing blood clot formation in a subject by administering the aptamers are provided, as are methods for treating blood clots by administering VWF-targeting agents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application 62 / 395,642, filed September 16, 2016, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government support under National Institutes of Health Grant Nos. 1U54HL112307 and 5K12NS080223-3,220901. The Government has certain rights in this invention. SEQUENCE LISTING This application was filed electronically via EFS-Web and contains an electronically submitted sequence listing in .text format. The .text file contains a sequence listing entitled "2017-09-18 5667-00413_ST25_Seq_Listing.txt", created on September 18, 2017, and is 35,071 bytes in size. The sequence listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] (Technical field) The present invention relates generally to compositions and methods for preventing and treating thrombosis. More specifically, the present invention relates to agents that target von Willebrand factor (VWF) and their use in preventing blood clot formation (antithrombotic activity) and treating and / or reducing the severity of formed blood clots (thrombolytic activity). [Background technology]

[0003] Thrombosis is the main underlying problem of many cardiovascular and cerebrovascular diseases and is also the main postoperative complication. Antithrombotic drugs have been developed over the past 25 years with the goal of reducing cerebrovascular and cardiovascular disease-related complications. However, although they reduce thrombosis in patients, these drugs pose a challenge in terms of bleeding risk due to their lack of rapid and predictable reversibility. Aptamers are single-stranded nucleic acids that adopt specific secondary and tertiary structures based on their sequence that allow specific binding to their targets. Aptamers bind to and inhibit protein targets. Aptamers are typically created by an in vitro selection process called SELEX (Systematic Exploration of Ligands by Exponential Enrichment, S ystematic E evolution of L igands by EXponential enrichment). See, e.g., Ellington AD, Szostak JW. 1990. In vitro selection of RNA molecules that bind specific ligands, Nature 346:818-22; Tuerk C, Gold L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249:505-10. Aptamers can be systematically isolated, as are virtually any protein, and a wide range of molecular modifications can be performed to optimize their pharmacological behavior for the intended use. Pegaptanib sodium (developed for the treatment of macular degeneration) was the first aptamer approved for use, and other compounds are in development. See, e.g., Wang P, Yang Y, Hong H, Zhang Y, Cai W, Fang D. 2011. Aptamers as therapeutics in cardiovascular diseases. Curr Med Chem 18:4169-74. Aptamers provide a promising and safer class of antithrombotic drugs, provided that aptamer activity can be quickly reversed using universal or rationally designed antidotes.See, for example, Rusconi CP, Scardino E, Layzer J, Pitoc GA, Ortel TL, et al.2002, RNA aptamers as reversible antagonists of coagulation factor IXa, Nature 419:90-4;WO / 2008 / 066621 A3;and WO / 2008 / 121354.

[0004] Von Willebrand factor (VWF) is a promising target for aptamer-based antithrombotic drugs. VWF is a multimeric plasma protein that binds glycoprotein IbIX, leading to platelet adhesion (the first non-overlapping step in platelet aggregation) and resulting in thrombus formation. The basic subunit is 260 kDa and is produced by endothelium and platelets. VWF is required for normal hemostatic plug formation and is the carrier protein for factor VIII. Aptamers targeting VWF have been shown to inhibit clot formation. See, for example, WO / 2008 / 066621 A3. However, there is a need in the art for novel VWF-targeting aptamers that have enhanced stability against degradation by nucleases, smaller size for ease of chemical synthesis, and extended circulation time in vivo. Summary of the Invention

[0005] Provided herein are aptamer compositions that target VWF and methods of preventing and treating blood clots using VWF-targeting agents in conjunction with antidote compositions that target such aptamer compositions. In one aspect, a plurality of aptamers are provided. The aptamers can comprise a polynucleotide, the polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3-102. See Tables 1 and 2 below.

[0006] Alternatively, an aptamer can comprise a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polynucleotide comprising from 5' to 3': a first stem forming region comprising or consisting of 2, 3, 4, or 5 nucleotides, a first loop region comprising or consisting of the nucleotide sequence AAC, a second stem forming region comprising or consisting of 3, 4, or 5 nucleotides, a second loop region comprising or consisting of the nucleotide sequence CC, a third stem forming region consisting of 2-8 nucleotides, 1-12 nucleotides, a third loop region and / or spacer sequence consisting of nucleotides, a fourth stem-forming region consisting of 2-8 nucleotides and capable of forming a stem together with the third stem-forming region, a fourth loop region comprising or consisting of the nucleotide C, a fifth stem-forming region comprising or consisting of 3, 4 or 5 nucleotides and capable of forming a stem together with the second stem-forming region, a fifth loop region comprising or consisting of the nucleotide sequence CAGA, and a sixth stem-forming region comprising or consisting of 2, 3, 4 or 5 nucleotides and capable of forming a stem together with the first stem-forming region.

[0007] In some embodiments, the aptamers described herein may not exceed 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, or 16 nucleotides in length. In some embodiments, the polynucleotide comprises unmodified nucleotides. In other embodiments, the polynucleotide comprises modified forms having at least one nucleotide base modification. Nucleotide base modifications include 2'O-methyl or 2'fluoro modifications of the nucleotide.

[0008] In some embodiments, the dissociation binding constant (Kd) of the aptamer for vWF is less than 500 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 3 nM, or less than 2 nM. In another aspect, dimers, trimers and tetramers comprising the aptamers described herein are also disclosed. In another aspect, an antidote to the aptamers described herein is provided. The antidote can comprise a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs: 103-180 (nucleotide sequences in Table 3). Alternatively, the antidote can comprise a polynucleotide having a sequence that is reverse complementary to and can hybridize with at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more nucleotides of any one of the aptamers described herein.

[0009] In yet another aspect, there is provided a pharmaceutical composition comprising any of the aptamers or antidotes described herein. The pharmaceutical composition may include a pharmaceutical carrier, excipient, or diluent (i.e., an agent) that is non-toxic at the dosages and concentrations employed to the cells or mammals to which it is exposed. In another aspect, a method of preventing blood clot formation in a subject is provided, the method can include administering to the subject any one of the aptamer compositions described herein in an amount therapeutically effective to prevent blood clot formation in the subject. In yet another aspect, a method of treating a blood clot in a subject is also provided, the method comprising administering to the subject an agent that targets VWF in an amount therapeutically effective to reduce blood clots in the subject. [Brief description of the drawings]

[0010] [Figure 1]FIG. 1 shows a summary of VWF9.14 truncated forms T10 (SEQ ID NO:10), T25 (SEQ ID NO:6), T49 (SEQ ID NO:5), T59 (SEQ ID NO:4), and T79 (SEQ ID NO:3). In addition to the predicted secondary structures of these aptamers, the length (nt), Kd (nM), and Bmax (%) of these aptamers (determined by an in vitro nitrocellulose filter binding assay) are shown below these secondary structures. It was found that the aptamer could be truncated from 60 to 30 nucleotides without reducing its binding ability with VWF. Note: Nucleotides highlighted in red indicate the major deletions introduced at each subsequent innovative step. Nucleotides highlighted in yellow indicate base substitutions. VWF aptamer 9.14T79 is T59 with five uracil nucleotides at the 3' end. [Diagram 2] Figure 2 shows the predicted secondary structures of the VWF9.14T59 (SEQ ID NO: 4) and VWF9.14T79 (SEQ ID NO: 3) aptamers. The T79 aptamer contains a 3' uracil tail plus the T59 aptamer to promote antidote binding. The dots in the T59 and T79 structures indicate where the proposed antidotes VWF9.14T59-AO11 (AO11, SEQ ID NO: 113) and VWF9.14T79-AO2 (AO55, SEQ ID NO: 157) bind, respectively. [Diagram 3] Figure 3 shows the predicted secondary structure of the optimized VWF aptamer, T79 VRT7 (SEQ ID NO: 7). 2'-O-methyl modified bases are highlighted in red and 2'-fluoro modified bases are highlighted in green. The length (nt), Kd (nM), and Bmax (%) of these aptamers (determined by in vitro nitrocellulose filter binding assay) are shown below these secondary structures. [Figure 4] Figure 4 shows carotid artery blood flow tracings in a murine arterial thrombosis model in which vehicle (no aptamer / negative control) was injected prior to FeCl3 injury. The vessel was occluded approximately 4 to 5 minutes following removal of the FeCl3 patch, as measured by flow probe. [Diagram 5]Figure 5 shows carotid artery blood flow tracings in two experimental murine arterial thrombosis models in which the VWF9.14T79-VRT7 aptamer was injected at a dose of 0.375 mg / kg (upper tracing) or 0.0375 mg / kg (lower tracing) prior to FeCl3 injury. Following removal of the FeCl3 patch, the vessel remained patent for more than 60 min as measured by a flow probe. [Figure 6] Figure 6 shows carotid artery blood flow tracings in a murine arterial thrombosis model experiment in which the PEG-VWF9.14T79-VRT7 aptamer was injected at a dose of 0.375 mg / kg prior to FeCl3 injury. As measured by a flow probe, the vessel remained patent for over 60 minutes after removal of the two 7.5% FeCl3 patches. [Figure 7] Figure 7 shows a summary of percent carotid blood flow in a murine arterial thrombosis model experiment in which various doses of VWF9.14T79-VRT7 aptamer (0.009375 mg / kg to 0.375 mg / kg) were injected prior to FeCl3 injury. A negative control is shown for reference. A dose as low as 0.0375 mg / kg was sufficient to maintain more than 75% blood flow (compared to the blood flow before patch application) for 60 minutes after removal of the FeCl3 patch, as measured by a flow probe. [Figure 8] FIG. 8 is a scatter plot showing the number of clots disrupted before (PRE) and after (POST) injection of a 10-fold molar ratio of VWF9.14T79-AO2 (AO55, SEQ ID NO:157) antidote or no antidote (Neg) following injection of saline or PEG-VWF9.14T79-VRT7 (SEQ ID NO:7) aptamer (dose 0.375 mg / kg) in a murine saphenous vein bleeding model. [Figure 9A] FIG. 9A shows scatter plots of clot disruption before and after injection of a 10-fold molar ratio of VWF9.14T79-AO2 (DTRI-025, SEQ ID NO:157) antidote or no antidote (saline) following injection of saline (control) or VWF9.14T79-VRT7 aptamer (dose 0.375 mg / kg, SEQ ID NO:7) in a murine saphenous vein bleeding model. [Figure 9B]FIG. 9B shows a bar graph of clot disruption before and after injection of saline (control) or VWF9.14T79-VRT7 aptamer (dose 0.375 mg / kg, SEQ ID NO:7) followed by a 10-fold molar ratio of VWF9.14T79-AO2 (DTRI-025, SEQ ID NO:157) antidote or no antidote (saline) in a murine saphenous vein bleeding model. [Figure 10] Figure 10 shows an example of carotid artery blood flow tracings and time course in a single experiment of a combined model of arterial thrombosis and saphenous vein bleeding. The left side of the figure captures the first half of this study, which evaluates the vascular patency of the carotid artery after thrombotic challenge. The right side captures the vessel dissection, bleeding, and clot formation before and after antidote administration. [Figure 11] FIG. 11 shows the number of clots disrupted before (PRE) and after (POST) injection of VWF9.14T9-AO2 (AO55, SEQ ID NO: 157) antidote after saphenous vein hemorrhage thrombosis following injection of PEG-VWF9.14T79-VRT7 aptamer (SEQ ID NO: 7), cholesterol-VWF9.14T79-VRT7 aptamer, or elastin-like polypeptide (ELP)-VWF9.14T79-VRT7 aptamer in a combined arterial thrombosis and saphenous vein hemorrhage model. [Figure 12]Figure 12 shows the results of platelet function assay (PFA) for VWF9.14T59 (SEQ ID NO: 4) with and without antidotes VWF9.14T59-AO3, -AO10, and -AO11. The SEQ ID NOs are AO3 (SEQ ID NO: 105), AO10 (SEQ ID NO: 112), and AO11 (SEQ ID NO: 113), respectively. Also shown are the results of PFA for aptamer VWF9.14T79 (SEQ ID NO: 3) with and without antidotes VWF9.14T79-AO1 (AO43; SEQ ID NO: 145) and VWF9.14T79-AO2 (AO55; SEQ ID NO: 157). Results are shown for the aptamer VWF9.14T82 (SEQ ID NO: 78) with and without the antidotes VWF9.14T82-AO1 (AO46; SEQ ID NO: 148) and VWF9.14T82-AO2 (AO58; SEQ ID NO: 160). Results are shown for the aptamer VWF9.14T84 (SEQ ID NO: 80) with and without the antidotes VWF9.14T84-AO1 (AO48; SEQ ID NO: 150) and VWF9.14T84-AO2 (AO60; SEQ ID NO: 162). [Figure 13] FIG. 13 shows the PFA results for VWF9.14T86 (SEQ ID NO:82), VWF9.14T87 (SEQ ID NO:83), VWF9.14T89 (SEQ ID NO:85), VWF9.14T90 (SEQ ID NO:86), VWF9.14T93 (SEQ ID NO:89), VWF9.14T94 (SEQ ID NO:90), and VWF9.14T95 (SEQ ID NO:91) with and without antidotes (AO61 (SEQ ID NO:163), AO62 (SEQ ID NO:164), AO63 (SEQ ID NO:165), AO64 (SEQ ID NO:166), AO65 (SEQ ID NO:167), AO66 (SEQ ID NO:168), and AO67 (SEQ ID NO:169), respectively). [Figure 14] FIG. 14 shows the predicted secondary structure and PFA data for VWF9.14T79vrt7 (DTRI-031; (SEQ ID NO:7)) with and without various molar ratios of the antidote DTRI-038 (SEQ ID NO:180). [Figure 15]Figure 15 shows the predicted secondary structures of the 79 vrt7 / DTRI-031 (SEQ ID NO:7) and T59 vrt19 (SEQ ID NO:4 and Table 2) aptamers, as well as the sequences of the DTRI-006-DTRI-013 aptamers (see Table 2 and SEQ ID NOs:4, 98, 99, 100 and 101, respectively). DTRI-008 (SEQ ID NO:4) showed no strong effect and was similar to DTRI-006 (SEQ ID NO:4). DTRI-009 (SEQ ID NO:98) showed a comparable KD to DTRI-006, but with a lower Bmax. DTRI-013 (SEQ ID NO:101) showed a comparable KD to DTRI-006, but with a lower Bmax. [Figure 16] FIG. 16 shows the predicted secondary structure and nitrocellulose filter binding assay data for the DTRI-019 (SEQ ID NO:8) aptamer. [Figure 17] FIG. 17 shows nitrocellulose filter binding assay data for the T79VRT7 / DTRI-031 (SEQ ID NO:7) aptamer. [Figure 18] 18 is a graph showing the percentage of initial carotid flow at various time points after occlusion in mice treated with: control, rTPA (recombinant tissue plasminogen activator), anti-VWF aptamer (both VWF9.14T79VRT7 and cholesterol-VWF9.14T79-VRT7), or no perfusion. As shown in the graph, the anti-VWF aptamer (VWF9.14T79VRT7 (SEQ ID NO: 7)) had superior thrombolytic activity compared to rTPA. [Figure 19] 19 is a graph showing stroke volume following vascular injury in a murine intracranial hemorrhage model. Mice were treated with vehicle, rTPA, anti-VWF aptamer (VWF9.14T79-VRT7 (SEQ ID NO: 7)) or anti-VWF aptamer (VWF9.14T79-VRT7) and VWF antidote (VWF9.14T79-T79-AO2 (also referred to as AO55 (SEQ ID NO: 157)). [Fig. 20A-E]Figure 20 shows that aptamer 9.14T79vrt7 (SEQ ID NO: 7) inhibits platelet adhesion under high shear forces, inhibits platelet aggregation in whole blood, and prevents thrombosis in vivo. The aptamer prevented human platelet adhesion in a dose-dependent manner. Figure 20A: Buffer control showed 100% platelet adhesion. Aptamer activity was measured as a percentage of control. Figure 20B: The assay showed approximately 50% platelet adhesion at 56 nmol / L, while Figure 20C showed complete inhibition at 900 nmol / L (n=3 in each group). Figure 20D: The aptamer inhibited platelet adhesion in a dose-dependent manner, with significant inhibition (p<0.05) at doses between 56-900 nmol / L (n=3 in each group). FIG. 20E: Linear regression analysis of the dose-response curve determined that the log IC50 of the aptamer was 1.86 (72.5 nmol / L). [Figure 21] Figure 21 is a graph showing the results of PFA-100 demonstrating that T79vrt7 (SEQ ID NO:7) completely inhibits platelet aggregation. Doses of 100-400 nmol / L exceed the upper limit of the assay, with 25 nM showing significant platelet aggregation compared to control (n=4 / group) (p<0.01). [Figure 22] Figure 22 is a graph showing carotid artery thrombosis in mice treated with 9.14T79vrt7 (SEQ ID NO:7) at doses ranging from 0.009375mg / kg-3mg / kg. The figure shows that at doses as low as 0.0185mg / kg, there was carotid artery patency compared to the negative control (n=3 / group). Doses above 0.0375mg / kg were not presented for clarity, but all show vascular patency. [Figure 23A-D]Figure 23 shows that aptamer 9.14T79vrt7 / DTRI-031 (SEQ ID NO:7) exhibits superior thrombolysis in murine carotid artery occlusion compared to rTPA. Figure 23A. Aptamer-treated mice (n=8) at a dose of 0.5 mg / kg exhibit superior thrombolysis compared to both rTPA-treated mice (10 mg / kg dose, n=8) (p<0.05) and saline controls (n=8) (p<0.01). Histopathology of mouse carotid arteries showed that aptamer-treated animals (Figure 23B) had patent vessels without the presence of occlusive thrombus compared to rTPA animals (Figure 23C) and saline controls (Figure 23D) (n=8 / group). [Fig. 24A-B] Figure 24 shows that aptamer 9.14T79vrt7 / DTRI-031 (SEQ ID NO: 7) prevents platelet adhesion and aggregation in a whole thrombosis assay system in dog whole blood, recanalizes carotid artery occlusion in dogs, and shows no cerebral hemorrhage or embolism. Figure 24A: Dog whole blood incubated with 12.5 nmol / L, 18.75 nmol / L, 25 nmol / L and 100 nmol / L doses of 9.14T79vrt7 (n=5 / group) compared to negative saline control (p<0.05). Figure 24B: Still images of whole blood flowing over collagen tubules during the first 10 seconds of every minute. The fuzzy compartments observed in the control group are aggregated platelets. The dose of 9.14T79vrt7 was 100 nmol / L. [Fig.24C.DEHK] (FIG. 24C-G): Administration of 9.14T79vrt7 45 minutes after occlusion resulted in recanalization in each of the three test dogs (FIG. 24C-E) (n=5 / group) compared to rTPA (FIG. 24F) and saline control (FIG. 24G). FIG. 24H: 9.14T79vrt7 did not cause intracranial hemorrhage or thromboembolism (n=3 / group) compared to rTPA (FIG. 24I) or control (FIG. 24J). Histology of the carotid artery demonstrated recanalization of the occluded segment in 9.14T79vrt7-treated dogs (FIG. 24K) (n=3 / group) compared to rTPA (FIG. 24L) and control (FIG. 24M). [Figure 24F.GIJLM](FIG. 24C-G): Administration of 9.14T79vrt7 45 minutes after occlusion resulted in recanalization in each of the three test dogs (FIG. 24C-E) (n=5 / group) compared to rTPA (FIG. 24F) and saline control (FIG. 24G). FIG. 24H: 9.14T79vrt7 did not cause intracranial hemorrhage or thromboembolism (n=3 / group) compared to rTPA (FIG. 24I) or control (FIG. 24J). Histology of the carotid artery demonstrated recanalization of the occluded segment in 9.14T79vrt7-treated dogs (FIG. 24K) (n=3 / group) compared to rTPA (FIG. 24L) and control (FIG. 24M). [Diagram 25] FIG. 25 shows that antidote oligonucleotides negate 9.14T79vrt7 / DTRI-031 (SEQ ID NO:7) in ex vivo and murine femoral vein models. Graphs are expressed as % of normal hemostasis in mice not receiving any treatment. Untreated control groups (data not shown) were similar to saline-treated groups (n=7 / group). 9.14T79 (n=11) administered at 0.375 mg / kg showed no clot disruption compared to control and saline-treated groups (p<0.0001). Addition of antidote 5 minutes after aptamer administration and measurement of clot disruption 2 minutes thereafter showed a reversal to complete hemorrhage similar to animals not administered aptamer (n=7). Administration of AO alone did not result in an increase in thrombus compared to negative controls (data not shown) (*=statistical significance). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure is based in part on the inventors' discoveries, which relate to novel, optimized, reversible VWF-targeted aptamers useful for both preventing (antithrombotic activity) and treating (thrombolytic activity) blood clots. Compared to previous VWF-targeted aptamers, the VWF-targeted aptamers of the present disclosure have enhanced stability against nuclease degradation, are small in size allowing for easier chemical synthesis, and exhibit extended circulation time in vivo. Disclosed herein are methods for preventing and treating blood clots in a subject using VWF-targeting agents (e.g., newly discovered VWF-targeting aptamers), along with compositions of aptamers and antidotes. These compositions and methods may be useful in several applications, including, but not limited to, the prevention or treatment of blood clots (in vitro, in vivo, ex vivo) or associated with stroke, cerebrovascular thrombosis, deep vein thrombosis (DVT), pulmonary embolism (PE), atrial fibrillation, coronary artery thrombosis, intracardiac thrombosis, postoperative thrombosis, cancer-induced thrombosis, cancer-associated thrombin expression, infection, and disseminated intravascular coagulation (DIC).

[0012] A plurality of aptamers are provided herein. As used herein, the term "aptamer" refers to a single-stranded oligonucleotide that specifically binds to a target molecule with high affinity. Aptamers can be created against a target molecule by screening combinatorial oligonucleotide libraries for high affinity binding to the target molecule (e.g., VWF). See, for example, Ellington and Szostak, Nature 1990; 346: 8 18-22 (1990), Tuerk and Gold, Science 249:505-10, 1990. The aptamers disclosed herein can be synthesized using methods well known in the art. For example, the disclosed aptamers can be synthesized using standard oligonucleotide synthesis techniques utilized by various commercial vendors, including Integrated DNA Technologies, Inc. (IDT), Sigma-Aldrich, Life Technologies, or Bio-Synthesis, Inc.

[0013] The aptamer can comprise a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:1 and SEQ ID NO:2 or SEQ ID NO:3-102. The aptamer can comprise a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:3-6 (nucleotide sequences T25, T49, T59, or T79 in Table 1). In some embodiments, the aptamer comprises SEQ ID NO:7 (T79vrt7 in Table 2), SEQ ID NO:8 (nucleotide sequence DTRI-019 in Table 2), or SEQ ID NO:9 (nucleotide sequence DTRI-021 in Table 2). The terms "polynucleotide," "nucleotide sequence," "polynucleotide sequence," "nucleic acid," and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide (the terms can be used interchangeably), or any fragment thereof. These phrases can refer to DNA or RNA of genomic, natural, or synthetic origin.

[0014] With respect to nucleotide sequences, the terms "sequence identity," "percent identity," and "% identity" refer to the percentage of base matches between at least two sequences aligned using a standardized algorithm. Such algorithms can insert gaps in a standardized and reproducible manner in the sequences being compared to optimize the alignment between the two sequences, thus achieving a more meaningful comparison of the two sequences. Sequence identity of nucleotide sequences can be determined as known in the art. See, for example, U.S. Patent No. 7,396,664. A commonly used and freely available suite of sequence comparison algorithms is provided by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCB). They are available from several sources, including NCBI (Bethesda, Md.), at their websites. The BLAST software suite includes a variety of sequence analysis programs, including "blastn," which aligns a known nucleotide sequence with other polynucleotides from a variety of databases. Additionally, a tool called "BLAST2 Alignments" is available that is used for pairwise direct comparison of two nucleotide sequences and is accessible in interactive format at the NCBI website.

[0015] With respect to nucleotide sequences, sequence identity is measured over the entire length of the entire nucleotide sequence defined (e.g., as defined by the specific sequences identified herein). Furthermore, sequence identity measured herein is based on the identity of the nucleotide bases of the nucleotide sequence, regardless of any further modifications to the nucleotide sequence. For example, the nucleotide sequences in the tables described herein can include modifications to the nucleotide sequence, such as 2'fluoro, 2'O-methyl, and inverted deoxythymidine (idT) modifications. These modifications are not considered in determining sequence identity. Thus, if a base is, for example, 2'fluoroadenine (or 2'O-methyl, etc.), it is understood that the base is an adenine for the purpose of determining sequence identity with another sequence. Similarly, 3'idT modifications of the nucleotide sequences in the tables described herein are also not considered in determining sequence identity.

[0016] Alternatively, an aptamer can comprise a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a polynucleotide comprising from 5' to 3': a first stem forming region comprising or consisting of 2, 3, 4, or 5 nucleotides, a first loop region comprising or consisting of the nucleotide sequence AAC, a second stem forming region comprising or consisting of 3, 4, or 5 nucleotides, a second loop region comprising or consisting of the nucleotide sequence CC, a third stem forming region comprising 2-8 nucleotides, 1-1 a third loop region or spacer sequence consisting of 2 nucleotides, a fourth stem forming region consisting of 2-8 nucleotides and capable of forming a stem together with the third stem forming region, a fourth loop region comprising or consisting of the nucleotide C, a fifth stem forming region consisting of or consisting of 3, 4 or 5 nucleotides and capable of forming a stem together with the second stem forming region, a fifth loop region comprising or consisting of the nucleotide sequence CAGA, and a sixth stem forming region consisting of or consisting of 2, 3, 4 or 5 nucleotides and capable of forming a stem together with the first stem forming region. Non-limiting examples of such aptamers are shown in Figures 1-3 and 15-16 as T25, T49, T59, T59vrt19, T79, T79vrt7, or DTRI-019.

[0017] As used herein, a "spacer sequence" can be any chemical spacer that does not interfere with the binding activity of the aptamer. For example, the spacer sequence can include, but is not limited to, a hexaethylene glycol spacer (see, e.g., DTRI-009), a C3 spacer, spacer 9, or any other suitable stable linker known to those of skill in the art that promotes and maintains proper folding and secondary structure of the aptamer. Based on the universal structures presented in, for example, Figures 1-3 and 15-16, a person skilled in the art will readily recognize that some modifications can be made to the sequence while maintaining the overall structure and possibly function of the aptamer. For example, a person skilled in the art will simply switch the first stem-forming region ACG and the sixth stem-forming region CGU to UGC and GCA or CCG and CGG (DTRI-013, SEQ ID NO: 101), respectively, and still maintain the stem structure of the aptamer. In addition, modifications of the stem-forming region can be made that change the bases in the stem region but preserve the overall pyrimidine and purine base composition, allowing the stem regions to hybridize at similar melting temperatures. A person skilled in the art will also understand that modifications made to an aptamer that destroy the universal aptamer stem-loop structure will likely result in an aptamer that cannot efficiently bind to its target. In some embodiments, an aptamer can comprise a polynucleotide having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity from 5' to 3' to a polynucleotide comprising SEQ ID NO:1 (CGAAC(U / T)GCCC(U / T)C), a variable nucleotide sequence or spacer sequence consisting of 1-18 (or any range therebetween) nucleotides, and SEQ ID NO:2 (GACGCACAGACG).

[0018] As used herein, a "variable nucleotide sequence" can be any possible nucleotide for a given length. For example, a "variable nucleotide sequence" of 5 nucleotides includes a 4-nucleotide sequence having 5 nucleotides. 5 (or 1,025) possible nucleotide sequences may be included. In some embodiments, the aptamer can be no more than 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17 or 16 nucleotides in length.

[0019] In some embodiments, the aptamer has a dissociation constant (K) of less than 150, 125, 100, 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 2.5, 2, 1, 0.5, or 0.1 nanomolar (nM) for human VWF protein. D The K of the aptamer can be D can be measured by the methodology used by the inventors in the Examples. For example, a double filter nitrocellulose filter binding assay using human VWF protein can be performed. Aptamers can comprise polynucleotides (RNA, DNA, or peptide nucleic acid (PNA)) that are unmodified or modified with at least one nucleotide base modification. For example, nucleotide base modifications of polynucleotides to protect the polynucleotide from nuclease degradation and / or to enhance the stability of the polynucleotide are well known in the art. Common nucleotide base modifications that can be used according to the present invention include, but are not limited to, deoxyribonucleotides, 2'-O-methyl bases, 2'-fluoro bases, 2' amino bases, inverted deoxythymidine bases, 5' modifications, and 3' modifications.

[0020] In some embodiments, an aptamer can comprise a polynucleotide that comprises a modified form that includes at least one nucleotide base modification selected from the group consisting of a 2' fluoro modification, a 2' O-methyl modification, a 5' modification, and a 3' modification. Exemplary 5' modifications can include, but are not limited to, inverted deoxythymidine bases, the addition of a linker sequence (e.g., C6), the addition of cholesterol, the addition of a reactive linker sequence that allows for conjugation to another moiety (e.g., PEG). Exemplary 3' modifications can include, but are not limited to, inverted deoxythymidine bases and inverted abasic residues.

[0021] In some embodiments, the aptamer can further comprise a tail nucleotide sequence at the 5' or 3' end of the polynucleotide, which cannot base pair with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more consecutive nucleotides of the polynucleotide. The tail nucleotide sequence can consist of 2-20 nucleotides or any range therebetween. As an exemplary tail nucleotide sequence, the inventors added a 5-nucleotide uracil (oligo U-tail) to the 3' end of the aptamer as a potential artificial nucleation site for antidote binding. Thus, in some embodiments, the tail nucleotide sequence can comprise the nucleotide sequence (U / T)(U / T)(U / T)(U / T)(U / T). However, it is also contemplated that other nucleotide sequences may serve as tail nucleotide sequences that are unable to base pair with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more consecutive nucleotides of the aptamer polynucleotide. In addition, tail nucleotide sequences have also been successfully added to the 5' end of aptamers without significantly affecting the activity of the aptamer.

[0022] As an additional 5' and / or 3' modification, the aptamer can include a polynucleotide that includes a 5' linker and / or a 3' linker. Common 5' and / or 3' linkers for polynucleotides are known in the art and can include peptides, amino acids, nucleic acids, as well as homofunctional or heterofunctional linkers. Particularly useful conjugation reagents that can facilitate the formation of covalent bonds with aptamers can include the use of N-hydroxysuccinimide (NHS) esters and / or maleimides or click chemistry. Exemplary 5' and / or 3' linkers for polynucleotides can include, but are not limited to, amino C3, C4, C5, C6 or C12-linkers. Aptamers can further comprise a stabilizer. As used herein, "stabilizer" refers to any substance that can enhance the stability of polynucleotide and / or extend its in vivo circulation time. Exemplary stabilizers are known in the art and can include, but are not limited to, polyethylene glycol (PEG), cholesterol, albumin, or elastin-like polypeptides.

[0023] The aptamer and stabilizer can be "linked" covalently or non-covalently. In addition, the aptamer and stabilizer can be linked using 5' and / or 3' linkers as described herein. The aptamer and stabilizer can be linked at the 5' and / or 3' ends of the aptamer. To non-covalently link the aptamer and stabilizer, the aptamer and stabilizer can be linked by a tag system. A "tag system" can include any group of agents that can bind to each other with high affinity. Several tag systems are known in the art, including, but not limited to, biotin / avidin, biotin / streptavidin, biotin / neutravidin, or digoxigenin (DIG) systems. In some embodiments, the tag system includes biotin / avidin or biotin / streptavidin. In such embodiments, the aptamer can be modified at the 5' or 3' end to include biotin, while the stabilizer can be modified to include streptavidin or avidin. Alternatively, the aptamer can be modified at the 5' or 3' end to contain streptavidin or avidin, while the stabilizer can be modified to contain biotin.

[0024] Also provided are dimers, trimers, and tetramers comprising any one of the aptamers described herein. A "dimer" refers to linking two aptamer molecules together, for example, to enhance the in vivo stability and / or circulation time of a polynucleotide. A "trimer" refers to linking three aptamer molecules together, for example, to enhance the in vivo stability and / or circulation time of a polynucleotide. A "tetramer" refers to linking four aptamer molecules together, for example, to enhance the in vivo stability and / or circulation time of a polynucleotide. The aptamer molecules can be linked together by covalent bonds, non-covalent bonds, or a combination of both. The aptamer molecules can be linked at their 5' or 3' ends. To non-covalently link the aptamers, the aptamers can be linked by a tag system or a scaffold system.

[0025] Antidotes are also provided herein and include polynucleotides having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 103-180 (nucleotide sequences in Table 3). Alternatively, antidotes can include polynucleotides having a sequence that is reverse complementary to and capable of hybridizing to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides of any one of the aptamers described herein.

[0026] Pharmaceutical compositions are provided that include any of the aptamers or antidotes described herein. Pharmaceutical compositions can include pharmaceutical carriers, excipients, or diluents (i.e., agents) that are non-toxic at the dosages and concentrations used to cells or mammals exposed thereto. Pharmaceutical formulations are often in pH-buffered aqueous solutions. Examples of pharmaceutical carriers include: buffers, such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN. TM brand name surfactants, polyethylene glycol (PEG), and PLURONICS TM Surfactants. In some embodiments, the pharmaceutical carrier can comprise a buffer containing about 20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM KCl.

[0027] Methods of preventing blood clot formation in a subject are provided. The methods can include administering to the subject any one of the aptamer compositions described herein in a therapeutically effective amount to prevent blood clot formation in the subject. "Preventing blood clot formation" can include reducing the likelihood of a blood clot, reducing the size of the blood clot, or slowing the further progression of blood clot formation. As used herein, the term "subject" refers to both human and non-human animals. The term "non-human animal" of the present disclosure includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, mice, chickens, amphibians, reptiles, etc. In some embodiments, the subject is a human patient.

[0028] A subject in need of blood clot prevention may require prevention of blood clots associated with, for example, but not limited to, stroke, cerebrovascular thrombosis, deep vein thrombosis (DVT), pulmonary embolism (PE), atrial fibrillation, coronary artery thrombosis, intracardiac thrombosis, postoperative thrombosis, cancer-induced thrombosis, cancer-associated thrombin expression, infection, disseminated intravascular coagulation (DIC) and arterial thrombosis (including cerebral arteries, coronary arteries and peripheral arteries of the head and neck, visceral arteries, arteries of the arms and legs). In some embodiments, a subject in need of blood clot prevention may suffer from atrial fibrillation or be at risk of having deep vein thrombosis, stroke, heart attack, or pulmonary embolism. Therapeutically effective amount or effective amount as used herein means the amount of a composition sufficient to effect treatment (as described above) when administered to a subject for the prevention or treatment of blood clots. The therapeutically effective amount will vary according to the formulation or composition, the disease and its severity, and the age, weight, health and responsiveness of the subject being treated.

[0029] In addition to disclosing a method for preventing blood clots in a subject, the inventors show that VWF-targeted agents can be used thrombolytically to reduce or "destroy" already formed blood clots. In the Examples, the inventors show that one of the disclosed VWF-targeted aptamers, T79vrt7 / DTRI-031, is superior to recombinant tissue plasminogen activator (rTPA) in a murine carotid artery occlusion model. Surprisingly, these results indicate that VWF-targeted agents can also be used not only to treat formed blood clots (thrombolytic activity), but also to prevent blood clot formation (antithrombotic activity) as well. Based on the use of this new VWF-targeting agent, a method of treating a blood clot in a subject is also provided. The method includes administering to the subject a VWF-targeting agent in an amount therapeutically effective to reduce blood clots in the subject. "Treating a blood clot" or "reducing a blood clot" refers to reducing the size and / or shape of the blood clot to increase blood flow at the site of the clot. A subject in need of treatment for a blood clot may require treatment for a blood clot associated with, for example, but not limited to, stroke, cerebrovascular thrombosis, deep vein thrombosis (DVT), pulmonary embolism (PE), atrial fibrillation, coronary artery thrombosis, intracardiac thrombosis, postoperative thrombosis, cancer-induced thrombosis, cancer-associated thrombin expression, infection, disseminated intravascular coagulation (DIC), and arterial thrombosis (including cerebral arteries, coronary arteries and peripheral arteries of the head and neck, visceral arteries, arteries of the arms and legs). In some embodiments, the subject in need of treatment for a blood clot suffers from deep vein thrombosis, stroke, heart attack, or pulmonary embolism.

[0030] As used herein, a "VWF targeting agent" is any agent (including, but not limited to, a polypeptide, polynucleotide, or small molecule) that can partially or completely block, inhibit, or neutralize one or more biological activities of von Willebrand factor (VWF) protein. In some embodiments, a VWF targeting agent can include an agent that can bind to the A1 domain of a VWF protein and block the binding of the VWF protein by the gp1b alpha protein. A VWF targeting agent can function in a direct or indirect manner. For example, a VWF targeting agent can directly bind to a VWF protein and thus partially or completely block, inhibit, or neutralize one or more biological activities of a VWF protein in vitro or in vivo. A VWF targeting agent can also function indirectly by (1) interacting with another molecule that can bind to VWF (e.g., activating, inducing, blocking, or inhibiting), or (2) modulating or affecting the expression (i.e., transcription or translation) of a VWF protein in a cell. The VWF protein can be any of the VWF proteins found in any mammal, including, but not limited to, humans or domesticated animals such as dogs, cats, horses, cows, pigs, mice or rats.

[0031] The VWF targeting agent may be a polypeptide, including but not limited to a peptide or an antibody. As used herein, the term "antibody" is used in the broadest sense as used in the art to refer to an antibody-based polypeptide affinity agent. For example, the antibody may include a polyclonal antibody, a monoclonal antibody, a single chain antibody, or an antibody fragment (e.g., Fab, Fab', F(ab')2, Fv fragment), a diabody, a linear antibody, a nanobody, or a multispecific antibody formed from an antibody fragment. The antibody may be a chimeric antibody, a humanized antibody, or a fully human antibody. The antibody may be any one of the five major known classes of immunoglobulins, including IgA, IgD, IgE, IgG, and IgM. In some embodiments, the VWF targeting agent may be an anti-VWF antibody that can bind to the VWF protein and thereby partially or completely block, inhibit, or neutralize one or more of the biological activities of the VWF protein. Suitable anti-VWF antibodies include, but are not limited to, caplacizumab, ALX-0681, or ALX-0081.

[0032] Peptides useful as VWF targeting agents can be identified using techniques well known in the art (eg, phage display). In some embodiments, the VWF targeting agent can be an aptamer that can bind to the VWF protein and thereby partially or completely block, inhibit, or neutralize one or more of the biological activities of the VWF protein. Suitable aptamers include, but are not limited to, those described in WO / 2008 / 066621 A3 (Sullenger et al.) and the aptamers described therein. The VWF targeting agent can also be a small molecule. A small molecule can be a chemical molecule having a molecular weight of less than about 2500 daltons, 2000 daltons, 1000 daltons, or 500 daltons.

[0033] The methods of preventing or treating blood clots described herein can further include administering to the subject an antidote in a therapeutically effective amount to neutralize the aptamer or VWF targeting agent. "Neutralizing" an aptamer or VWF targeting agent refers to reducing the antithrombotic or thrombolytic activity of the aptamer or VWF targeting agent. Antidotes that can be used in accordance with the present invention can include sequence-specific antidotes, such as those described herein and in WO / 2008 / 066621 A3. Antidotes can also include sequence-non-specific antidotes (i.e., cationic polymers), such as those described in WO / 2008 / 121354.

[0034] The compositions described herein (i.e., aptamers, antidotes, and pharmaceutical compositions) can be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transdermal, nasopharyngeal, intralesional, intratumoral, intradermal, or transmuscular absorption. Thus, the compositions can be formulated as ingestible, injectable, topical, or suppository formulations. Administration of the compositions to a subject animal can exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger amounts of the compositions is expected to achieve increased beneficial biological effects over administration of smaller amounts. Furthermore, efficacy is also expected at dosages below levels at which toxicity is observed.

[0035] It will be understood that the prescribed dosage administered in any given case will be adjusted according to the composition administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical requirements that may modify the activity of the composition and the response of the subject, as is well known to those skilled in the art.For example, the specific dose for an individual subject will depend on age, weight, general health, diet, timing and mode of administration, excretion rate, concomitant medications, and the severity of the individual disease to which treatment is applied.The dosage for a given patient can be determined using conventional considerations, for example by routine comparison of the differential activity of the compositions described herein and known drugs, for example by means of suitable conventional pharmacological protocols.

[0036] The maximum dosage for a subject is the highest dosage that does not cause undesirable or unacceptable side effects. The variables for individual treatment regimens are large and a significant dose range is expected. The route of administration will also affect dosing requirements. Dosing of the composition will prevent or treat blood clots by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to no treatment.

[0037] The compositions described herein can be administered to a subject once or more than once to effectively prevent or treat blood clots. Suitable dosage ranges are on the scale of hundreds of micrograms of active ingredient, ranging from about 0.01 to 10 mg / kg / day, preferably from about 0.1 to 1 mg / kg / day. The exact amount of active ingredient required for administration depends on the judgment of the attending physician and may be specific to each subject. It will be apparent to those skilled in the art that the therapeutically effective amount of the compositions described herein will depend, among other things, on the administration schedule, the unit dose of the drug administered, whether the composition is used in combination with other therapeutic agents, the condition and health of the recipient, and the therapeutic activity of the particular composition.

[0038] The disclosure is not limited to the specific details of the components or methods shown herein. The compositions and methods disclosed herein can be made, performed, used, accomplished, and / or formed in a variety of ways that will be apparent to one of skill in the art in light of the disclosure below. The words and terms used herein are merely for descriptive purposes and should not be construed as limiting the scope of the claims. The general indicators used in the specification and claims, such as first, second, and third, refer to various structures or method steps and are not intended to be construed as indicating any particular structure or step, or any particular order or arrangement for such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. Any and all examples or illustrative language (e.g., "such as") provided herein is intended merely to facilitate the disclosure and does not imply any limitation of the scope of the disclosure unless specifically claimed. Any language in the specification and structures shown in the drawings should not be construed as indicating that any unclaimed component is essential to the practice of the subject matter of the disclosure. Use herein of the terms "including," "comprising," or "having," or variations thereof, is intended to encompass the subsequently listed components and equivalents thereof as well as additional components. Embodiments that recite "including," "comprising," or "having" certain components are also considered to "consist essentially of" or "consist" of those certain components.

[0039] The recitation of ranges of values ​​herein is intended to serve as a shorthand method for referring to each separate value included in the range individually, unless otherwise indicated herein, and each separate value is incorporated into the description as if it were individually recited. For example, if a concentration range is stated as 1% to 50%, it is intended to expressly recite values ​​such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., herein. These are examples of what is specifically intended, and all possible combinations of values ​​between and including the lowest and highest recited values ​​should be considered to be expressly recited in this disclosure. The use of the word "about" to express a particular recited amount or range of amounts is intended to indicate that the amount includes values ​​very close to the recited amount, for example, values ​​that may or will be due to manufacturing variations, equipment and human error in making measurements, and the like. All percentages referring to amounts are by weight, unless otherwise indicated.

[0040] Any reference, including any non-patent or patent document cited herein, is not an admission that it constitutes prior art. In particular, it will be understood that, unless specifically stated otherwise, the citation of any document herein is not an admission that any of such documents form part of the common general knowledge in the art in the United States or any other country. Discussion of any cited document expresses the accuracy and pertinence of what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are incorporated herein by reference in their entirety, unless otherwise indicated. In the event of any discrepancy between any definitions and / or descriptions found in the cited documents, the present disclosure will control. Unless otherwise specified or indicated by context, the terms "a," "an," and "the" mean "one or more." For example, "a protein" or "an RNA" should be interpreted as meaning "one or more proteins" or "one or more RNAs," respectively. The following examples are intended to be merely illustrative and are not meant to be limiting of the scope of the invention or the appended claims. EXAMPLES

[0041] Example 1 - Optimization of the VWF aptamer Materials and Methods Preparation and folding of RNA aptamers RNA aptamers were synthesized in-house using standard oligonucleotide synthesis methods. Prior to platelet function assays (PFA) and in vivo models, RNA-based aptamers can be "folded" in an appropriate physiological buffer, e.g., platelet binding buffer (20 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM KCl). The aptamer solution was heated to 95°C for 3 min and immediately placed on ice, followed by 5 to 10 min at room temperature.

[0042] Aptamer binding assay Affinity constant (K d K values ​​were determined using a dual-filter nitrocellulose filter binding assay (Rusconi et al., Thromb. Haemost. 84:841-848, 2000). All binding studies were performed at 37°C using binding buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl2, and 0.01% BSA). Human purified VWF (without factor VIII) was purchased from the following commercial supplier (Haematologic Technologies Inc.; Essex Junction, VT) and used in dual-filter nitrocellulose filter binding assays to determine the K values ​​of the aptamers. d Briefly, T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ 32RNA was end-labeled at the 5' end with [P]ATP (Amersham Pharmacia Biotech, Piscataway, NJ) (Fitzwater and Polisky, Methods Enzymol. 267:275-301, 1996). End-labeled RNA was diluted in binding buffer F and heat denatured at 65°C for 5 min, then equilibrated at 37°C. Direct binding was performed using a trace amount of 32 This was carried out by incubating P-RNA with various concentrations of VWF protein in binding buffer F for 5 min at 37°C. The fraction of nucleic acid-protein complex bound to the nitrocellulose membrane was quantified using a phosphorimager (Molecular Dynamics, Sunnyvale, CA). Nonspecific binding of radiolabeled nucleic acid was subtracted to leave only specific binding (Wong and Lohman, Proc. Natl. Acad. Sci. USA 90:5428-5432, 1993).

[0043] FeCl3-induced arterial thrombosis We describe an FeCl3 chemical injury model to induce arterial thrombosis in mice. Anesthesia was induced in a closed chamber with 4 to 5% isoflurane inhalation for 5-7 min before intubation. After gas induction, 27 g 1 / 2 A single IP injection of avertin / tribromoethanol (1.25%, 12.5 mg / mL) was given using a needle at a dose between 100-250 mg / kg depending on efficacy. Total volume ranged from 0.15 to 0.50 cc depending on body weight (typically for mice weighing 20-25 g). The animal was returned to the induction chamber for an additional 1-2 minutes before intubation was performed. The mouse was transferred to a dedicated intubation stand and intubated using a needle-free 20-22 gauge catheter. Once intubation was confirmed, the ventral neck was shaved and the mouse was transferred to a heated operating table. The mouse was immediately connected to a Harvard Apparatus rodent ventilator in dorsal recumbency and maintained at approximately 90-110 breaths per minute with a 70% nitrogen:30% oxygen mixture and a tidal volume of approximately 0.2 mL. Isoflurane was maintained at approximately 1-3%. Body temperature was maintained at approximately 37°C with a Physitemp TCAT-2DF.

[0044] After confirming anesthesia of the surgical surface, a midline neck incision was made in the skin and the fascia was dissected open to expose the right common carotid artery. After exposure and isolation of the right common carotid artery, the left jugular vein was exposed by dissection and three 7-0 silk ligatures were placed. A small incision was made using microsurgical scissors, hemostasis was maintained with only one 7-0 silk ligature, and a PE-10 polyethylene catheter or equivalent was placed in the vein and secured with the remaining two 7-0 silk ligatures. After confirming catheter patency with 0.9% saline, a continuous rate of 0.9% saline infusion was initiated using a Harvard Apparatus PHD2000 (or equivalent) infusion pump at a rate of 0.5-3 μL / min and maintained until the end of the study. A 0.5-PSB transit time flow probe (Transonic Systems Inc.) was placed around the carotid artery to measure blood flow. During the study, blood flow and temperature measurements were captured using LabChart software (ADInstruments). Once normal blood flow (1.0 to 3.0 mL / min) was maintained for at least 5 min, antithrombotic test drugs or controls were administered via the jugular catheter in a volume of 100 to 200 μL using a saline-based vehicle over 1 min. Aptamer drug doses ranged from 0.005 to 1.0 mg / kg.

[0045] Approximately 5 minutes after drug or antidote administration, one or two small pieces of filter paper (1 mm x 2 mm) were saturated with 2.5% to 10% FeCl3. These "patches" were then placed on the ventral + / - dorsal aspect of the exposed carotid artery close to the flow probe. The patches were left in place for 3 minutes. After removal of the patch, the corresponding area of ​​the artery was gently washed with 0.9% saline. Carotid blood transit time was measured continuously until the end of the study, which was defined as not more than 60 minutes after stable thrombus formation (i.e., approximately 0.0 mL per minute of carotid transit time) or not more than 60 minutes after application of the FeCl3 patch. Once the end point was reached, isoflurane was increased to 2-4%. Two 7-0 silk ligatures were then placed looped around the artery proximal to the site of thrombus formation. The flow probe was removed and the artery was isolated at a point approximately 5 to 8 mm distal between the silk ligatures. Arterial sections were removed for histopathological examination. Subsequently, the animals were euthanized by an overdose of anesthetic gas followed by secondary physical methods.

[0046] Saphenous vein bleeding model We describe a murine saphenous vein bleeding model for the evaluation of hemostasis. Anesthesia was induced by inhalation of 4 to 5% isoflurane in a closed chamber for 2–3 min. 1 / 2 A single IP injection of avertin / tribromoethanol (1.25%, 12.5 mg / mL) was given using a needle at a dose between 100-250 mg / kg depending on efficacy. Total volume ranged from 0.15 to 0.50 cc depending on body weight (typically for mice weighing 20-25 g). The animal was returned to the induction chamber for an additional 1-2 minutes before intubation was performed. The mouse was transferred to a dedicated intubation stand and intubated using a needleless 20-22 gauge catheter. Once intubation was confirmed, the ventral neck and medial surface of both pelvic limbs were shaved and the mouse was transferred to a heated operating table. The mouse was immediately connected in dorsal recumbency to a Harvard Apparatus rodent ventilator and maintained with a 70% nitrogen:30% oxygen mixture at approximately 90-110 breaths per minute and a tidal volume of approximately 0.2 mL. Isoflurane was maintained at approximately 1-3%. Body temperature was maintained at Physitemp The TCAT-2DF and rectal probe were maintained at approximately 37°C.

[0047] After confirmation of anesthesia of the surgical surface, a midline neck incision of the skin was made. Surgical exposure of the left jugular vein was achieved by dissection. Once the jugular vein was isolated, a PE-10 polyethylene catheter or equivalent was placed into the vein and secured using two looped 7-0 silk ligatures. Once catheter patency was confirmed with 0.9% saline, a continuous rate of 0.9% saline infusion was initiated using a Harvard Apparatus PHD2000 (or equivalent) infusion pump at a rate between 0.5-3 μL / min and maintained until the end of the study. After catheter placement was complete, the skin on the medial aspect of the left and right pelvic limbs was incised to expose the entire length of the saphenous vascular bundle (saphenous artery and vein, medial saphenous vein). The bundle was maintained with 1-2 drops of 0.9% saline to prevent drying.

[0048] Test drugs or controls were administered IV over 1 min using a saline-based vehicle in a volume of 100-200 μL via a jugular catheter. Doses of aptamer drugs ranged from 0.005 to 1.0 mg / kg. Approximately 5 to 120 min after administration of the test drug, the exposed saphenous vein was incised with a 23-26 g needle, followed by a longitudinal incision of approximately 1 to 2 mm in the distal portion of the vessel using microdissection scissors. Extravasation was gently wiped away with a fine-tipped mini-cotton swab until hemostasis occurred. The clot in the distal portion of the vessel was then removed using a 23-26 g needle, and bleeding was allowed to resume. Blood was wiped away again until hemostasis reoccurred. Clot disruption was repeated after each occurrence of hemostasis for a total of 15 to 30 min from the initial injury. Injury, clot disruption, hemostasis, and temperature measurements were captured throughout the study using LabChart software (ADInstruments). The corresponding antidote molecule was then administered IV through the jugular catheter after the test drug in a volume of 100 to 200 μL. RNA-based oligonucleotide antidotes ranged from 0.005 to 100 mg / kg. Approximately 5 min after antidote administration, the clot in the distal portion of the vessel was again removed with a 23-26 g needle to resume bleeding. Blood was wiped away until hemostasis resumed. Clot disruption was repeated after each occurrence of hemostasis for a total time of 15 to 30 min. Once the end point was reached, approximately 0.5 mL of blood was collected by cardiac puncture or withdrawn from the caudal vena cava. The animals were subsequently euthanized by an overdose of anesthetic gas followed by secondary physical methods.

[0049] PFA 100 Protocol The Platelet Function Analyzer, PFA-100 (Dade Behring, Deerfield, IL) provides a quantitative measurement of platelet function in anticoagulated whole blood (Ortel et al, Thromb.Haemost.84:93-97, 2000). Briefly, aptamers were diluted in the appropriate buffer (i.e., 150 mM NaCl, 20 mM HEPES (pH 7.4), 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2; or 150 mM NaCl, 20 mM HEPES (pH 7.4), 2 mM CaCl2; or PBS) and heat denatured. Aptamers were added to fresh whole blood at the indicated final concentrations and incubated at RT for 3-5 min, followed by analysis on the PFA-100 using a collagen / ADP test cartridge. The maximum closure time of the PFA-100 was 300 s. The detoxification activity of the aptamers was measured by mixing whole blood with the aptamers, followed by administration of the detoxifier and measurement by PFA.

[0050] result VWF9.14 variant In order to optimize the VWF9.14 aptamer, we made several VWF9.14 aptamer truncation variants and several VWF9.14 aptamer engineered variants. See, for example, Figures 1-3. The VWF9.14 aptamer truncation variants are shown in Table 1 below.

[0051] Table 1: Truncated aptamers A=2'OH adenine; C=2'fluorocytosine; G=guanine; U=2'fluorouracil idT = inverted deoxythymidine at the 3' end (sequence lengths and sequence numbers below do not include idT); NB = no binding; ND = not determined

[0052] TIFF0007675990000001.tif217169 TIFF0007675990000002.tif217169 TIFF0007675990000003.tif209168 TIFF0007675990000004.tif148169 TIFF0007675990000005.tif154170

[0053] The engineered variants of the VWF9.14 aptamer that were generated are shown in Table 2 below. Table 2: Modified truncated forms of the VWF9.14 aptamer Table Legend : All sequences are 5' to 3' oriented. Length does not include reverse deoxythymidine. fU = 2' fluorouracil; fA = 2' fluoroadenine; fC = 2' fluorocytosine; mA = 2'O-methyladenine; mC = 2'O-methylcytosine; mG = 2'O-methylguanine; mU = 2'O-methyluracil; fG = 2'fluoroguanine; rG = 2'riboguanine; rA = 2' riboadenine; idT = inverted deoxythymidine at the 3' end; (C6L) = hexylamino linker; (6GLY) = hexaethylene glycol linker (incorporated using 9-O-dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); cholesterol = cholesterol triethylene glycol amidite incorporated at the 5' end; NB = no binding; ND = not determined.

[0054] TIFF0007675990000006.tif146167 TIFF0007675990000007.tif186165 TIFF0007675990000008.tif210167 TIFF0007675990000009.tif219165 TIFF0007675990000010.tif207166 TIFF0007675990000011.tif212165 TIFF0007675990000012.tif153167 TIFF0007675990000013.tif179167 TIFF0007675990000014.tif161167 TIFF0007675990000015.tif165165 TIFF0007675990000016.tif175169 TIFF0007675990000017.tif193163 TIFF0007675990000018.tif84168

[0055] In addition to generating truncated and engineered variants of the VWF9.14 aptamer, we generated several antidote sequences that target these variants, which are shown in Table 3 below.

[0056] Table 3: Antidote sequences All sequences are shown in the 5' to 3' direction; lengths do not include inverted deoxythymidines; mG = 2'O-methyl G; mA = 2'O-methyl A; mC = 2'O-methyl C; mU = 2'O-methyl U; idT = reverse deoxythymidine

[0057] TIFF0007675990000019.tif140164 TIFF0007675990000020.tif159166 TIFF0007675990000021.tif155166 TIFF0007675990000022.tif129166

[0058] Binding Tests To determine the binding affinity of the VWF9.14 aptamer variants to the VWF protein, we performed binding assays with several variants. The binding data are summarized in Tables 1-2 above. See also Figures 1 and 16-17.

[0059] PFA analysis of VWF9.14 aptamer variants As shown in Figures 12 and 13, we performed platelet function assays (PFA) for VWF9.14T59 with or without antidotes VWF9.14T59-AO3, -AO10, and -AO11 (AO3, AO10, and AO11, respectively). In addition, the results of PFA for aptamer VWF9.14T79 with or without antidotes VWF9.14T79-AO1 (AO43) and VWF9.14T79-AO2 (AO55) are also shown. The results for aptamer VWF9.14T82 with or without antidotes VWF9.14T82-AO1 (AO46) and VWF9.14T82-AO2 (AO58) are also shown. Results are shown for the aptamer VWF9.14T84 performed with or without the antidotes VWF9.14T84-AO1 (AO48) and VWF9.14T84-AO2 (AO60). These results show that the antithrombotic activity of some VWF9.14 aptamers can be restored using the intended antidotes.

[0060] In vivo testing of VWF9.14 aptamer variants Several of the VWF9.14 aptamer variants were tested in a murine arterial thrombosis model and a murine saphenous vein bleeding model. See Figure 4-11. In the murine arterial thrombosis model, the left jugular vein was cannulated, the right carotid artery was exposed, and a flow probe was placed. Five minutes later, mice were injected iv with the indicated vehicle and a 10% FeCl3 patch was placed on the carotid artery for three minutes. Carotid blood flow was then monitored and recorded with the probe for one hour. In the murine arterial thrombosis model, injection of vehicle (no aptamer / negative control) prior to FeCl3 injury resulted in occlusion of the vessel approximately two minutes after removal of the FeCl3 patch (see Figure 4). On the other hand, by injecting the T79vrt7 / DTRI-031 or PEG-VWF9.14T79-VRT7 / DTRI-031 aptamers at a dose of 0.375 mg / kg prior to FeCl3 injury in this model, the vessel remained patent for 60 min after removal of the FeCl3 patch (see Figures 5 and 7). These results indicate that the aptamers have potent antithrombotic activity.

[0061] VWF9.14T79-VRT7 aptamer was also injected into the model prior to FeCl3 injury at a dose of 0.0375 mg / kg, and the vessel remained patent for more than 60 minutes after removal of the FeCl3 patch (see FIG. 5). This result indicates that VWF9.14T79-VRT7 aptamer can be administered at lower doses and still exhibit potent antithrombotic activity. A dose-ranging study is shown in FIG. 7. In the murine saphenous vein bleeding model, mice were cannulated in the left jugular vein to expose the right medial saphenous vein prior to injection of the aptamer at the indicated doses. Five minutes after aptamer injection, the saphenous vein was incised and bleeding was monitored for 15 minutes. Antidote was then injected to neutralize the aptamer action, and bleeding was monitored for an additional 20 minutes. The activity of the PEG-VWF9.14T79-VRT7 aptamer was successfully neutralized by injection of the VWF9.14T79-AO2 (AO55) antidote (see Figure 8). For example, as shown in Figure 8, clot destruction was increased after injection of the VWF9.14T9-AO2 (AO55) antidote, indicating that the activity of the aptamer could be neutralized.

[0062] Furthermore, we tested the PEG-VWF9.14T79-VRT7 aptamer, cholesterol-VWF9.14T79-VRT7 aptamer, elastin-like polypeptide (ELP)-VWF9.14T79-VRT7 aptamer and VWF9.14T79-AO2 (AO55) antidote in a combination of murine arterial thrombosis and saphenous vein bleeding. In this model, the left jugular vein was cannulated, the right carotid artery was exposed and a Transonic flow probe was placed in the animal. Five minutes after the probe was placed, the aptamer was injected at the indicated dose. After another five minutes, two 7.5% FeCl3 patches were placed on the right carotid artery for three minutes before the patches were removed. Thirty minutes after removal, the saphenous vein was exposed and incised, and bleeding was monitored for 15 minutes. The antidote was then injected and clot formation was monitored for an additional 15 minutes (see Figures 10-11). In this model, after initially demonstrating the potent antithrombotic activity of each aptamer, the VWF9.14T79-AO2 (AO55) antidote was still able to successfully reverse the activity of the aptamer.

[0063] Example 2 - Thrombolytic activity of VWF aptamers For thrombolytic activity, the T79VRT7 aptamer was also tested in a murine carotid artery occlusion model and a murine intracranial hemorrhage model. See Figures 18-19.

[0064] Murine carotid artery occlusion thrombolysis model We utilized a murine carotid artery occlusion model in which adult C57BL / 6J mice (Jackson Laboratory, Bar Harbor, ME) (18-24 g) were intubated and the left jugular vein was exposed. We then exposed the right common carotid artery and placed a Transonic flow probe (Transonic Systems Incorporated, Ithaca, NY) surrounding the artery. Blood flow was measured for 5 min to obtain a stable baseline. Thrombosis was then induced by applying 10% ferric chloride-soaked Whatman paper to the vessel. The time to occlusion was then recorded. Twenty min after occlusion, saline (negative control), anti-VWF aptamer (VWF9.14T79VRT7) at a dose of 0.5 mg / kg, or recombinant tissue plasminogen activator (rTPA) at a dose of 10 mg / kg were injected intravenously. Reperfusion was monitored using a Doppler flow probe to determine the time for perfusion to be reestablished. If no recanalization occurred after 60 min, the experiment was terminated. Animals were then sacrificed and the brain and common carotid arteries were harvested for analysis. As shown in Figure 18, in a murine carotid artery occlusion model, the percentage of initial carotid flow was strongly increased over time with the VWF T79VRT7 aptamer versus rTPA, control, or no perfusion, thus indicating that the VWF T79VRT7 aptamer had superior thrombolytic activity compared to rTPA.

[0065] Murine intracranial hemorrhage model We anesthetized adult C57BL / 6J mice (18-24 g) and exposed the left jugular vein and right common carotid artery. We then injected saline (negative control), a 0.5 mg / kg dose of anti-VWF aptamer (VWF9.14T79-VRT7), a 10 mg / kg dose of rTPA, or an anti-VWF aptamer (0.5 mg / kg) and a 2.5 mg / kg dose of a matched antidote oligonucleotide (VWF9.14T79-AO2 (also called AO55)) 5 min after the aptamer. To induce intracranial hemorrhage, a silicone-coated 6-0 nylon filament was inserted into the carotid artery and advanced until it punctured the internal carotid artery (ICA) terminus to induce subarachnoid hemorrhage (SAH). To assess infarct and hemorrhage volumes, magnetic resonance imaging (MRI) was performed with a 9.4 Tesla MRI (Bruker Biospin, Billerica, MA) 90 min after induction of intracranial hemorrhage. FIG. 19 shows a graph presenting stroke volume following vascular injury in a murine intracranial hemorrhage model in mice treated with vehicle, rTPA, anti-VWF aptamer (VWF9.14T79-VRT7), or anti-VWF aptamer (VWF9.14T79-VRT7), and VWF antidote (VWF9.14T79-AO2 (also called AO55)).

[0066] Example 3 - Further in vivo testing with VWF aptamers Ischemic stroke is the leading cause of death and disability in Western societies 1 Approved thrombolytic stroke treatments using recombinant tissue plasminogen activator (rTPA) are limited by several significant limitations. First, there is a significant risk of hemorrhagic conversion resulting in part from the inability to reverse rTPA activity. Second, the short therapeutic window of rTPA renders >90% of stroke patients ineligible for treatment. 2,3 Finally, rTPA only achieves approximately 30% recanalization, and reocclusion commonly occurs after primary thrombolysis, resulting in reduced early neurological improvement. Von Willebrand factor (VWF) is a glycoprotein involved in key events in platelet thrombus formation. VWF interacts with glycoprotein Ib alpha-IX-V complex on the platelet surface and induces platelet adhesion to the vascular wall. 4 Following this, glycoprotein IIb / IIIa (gpIIb / IIIa) is activated and binds fibrinogen, resulting in thrombus formation. von Willebrand disease (VWD) can be both a qualitative and quantitative reduction of von Willebrand factor. Type I VWD (the major form of VWF disease) presents with light bleeding after dental procedures or during menstruation and no incidental bleeding. 5 Furthermore, type I VWD patients are protected from cerebrovascular and cardiovascular events. 6 VWF is therefore a promising target in arterial thrombosis that could be used beyond conventional treatments.

[0067] Aptamers are single-stranded oligonucleotides that have potential advantages over other classes of therapeutic agents: they bind to their targets with high affinity and specificity. 7 They can be chemically modified to customize their bioavailability, can be chemically synthesized on a large scale, and, of greatest relevance to our application, can be rapidly reversible. 8-11 . In this example, we show that the aptamer 9.14T79vrt7 inhibits platelet adhesion under high shear stress in a dose-dependent manner and prevents platelet aggregation. The aptamer prevents thrombus formation in a murine carotid artery injury model. The aptamer also exhibits superior thrombolytic activity in both murine and canine arterial occlusion models compared to rTPA, and this was achieved without inducing intracranial hemorrhage or spilling the occluding clot into the brain. Finally, antidote oligonucleotides designed to counteract 9.14T79vrt7 neutralized the antiplatelet activity of the aptamer within 2 minutes in both human blood and a murine hemorrhage model. The studies herein suggest that the 9.14T79vrt7 aptamer represents a novel and potentially safe approach for the treatment of ischemic stroke and other acute thrombotic events.

[0068] Materials and Methods Synthesis of aptamer truncated, modified, and antidote oligonucleotides (AO) Aptamer truncations were transcribed or synthesized in-house. Briefly, RNA aptamer truncations (T10, T21, and T22) were transcribed using T7 RNA polymerase. Aptamer truncations, variants, and antidotes were synthesized using a MerMade 6 / 12 oligonucleotide synthesizer (BioAutomation, Irving, TX). Secondary structures were predicted using software that predicts RNA secondary structure (Mfold by M. Zuker).

[0069] Preparation and folding of RNA aptamers RNA-based aptamers can be "folded" in appropriate physiological buffers prior to platelet function assays (PFA) and in vivo models 13 The aptamer solution was heated to 95°C for 3 min, immediately placed on ice for 3 min, and then allowed to come to room temperature for approximately 5 to 10 min.

[0070] Human whole blood test Human blood was collected by venipuncture from healthy volunteers after written informed consent was obtained. Blood collection was performed according to protocols approved by the Institutional Review Boards of both the Durham Veterans Administration Medical Center and Duke University Medical Center.

[0071] Platelet adhesion analysis Venaflux TMA microfluidics system (Cellix, Dublin, Ireland) measures platelet adhesion on collagen surfaces. Human blood was collected by venipuncture from healthy volunteers into hirudin tubes. Aliquots containing 300 μL of whole blood treated with aptamer or platelet binding buffer alone were flowed through the collagen-coated microchannel at 60 dynes for 3 min. The channel was subsequently washed with saline for 3 min to wash away RBCs and unbound platelets. Bound platelets were imaged using Venaflux imaging software and Image Pro Plus, and the surface area covered was calculated. Aptamers were incubated at 95°C for 3 min, placed on ice for 3 min, followed by incubation at room temperature for 10 min. After cooling, aptamers were kept on ice until use. The total surface area covered by bound platelets in treated blood was expressed as a percentage of the total coverage of negative control blood. Statistical significance was determined by analysis of variance. IC of aptamers 50 was calculated using a fitted linear regression curve.

[0072] Total Thrombus Formation Analysis System (T-TAS) Clot formation was assessed in both human and canine whole blood using the T-TAS (Zacrox, Fujimori Kogyo Co. Ltd., Tokyo, Japan). 15 Blood was collected in a hirudin tube in the PL chip (the PL tube contains 25 capillary channels coated with collagen type 1). Blood flow across the chip was maintained at a flow rate of 14 μL / min. Platelet aggregation was measured as a function of the total pressure (kPa) required to maintain the flow rate. A camera was also used to observe platelet activity across the collagen-coated channels.

[0073] Murine in vivo test Investigators who performed the surgery or analyzed the carotid flow and imaging data were blinded to the treatment groups. All in vivo experiments were approved by the Duke University Institutional Animal Care and Use Committee and the Ohio State University Institutional Animal Care and Use Committee. Furthermore, the committees adhere to the NIH guidelines for the care and use of laboratory animals.

[0074] Carotid Artery Occlusion and Thrombosis Murine carotid artery occlusion studies were performed in male or female C57BL / 6 mice (8 weeks old) obtained from a commercial supplier (Jackson Laboratory). Thrombosis / occlusion was achieved using Whatman filter paper soaked in FeCl3. Twenty minutes after carotid artery occlusion, procedures were initiated. Eight-week-old male or female C57BL / 6 mice were obtained from a commercial supplier (Jackson Laboratory). Animals were anesthetized with ketamine (55 mg / kg) and xylazine (15 mg / kg). Animals were intubated (Harvard Apparatus mouse ventilator, Holliston, MA) through a midline abdominal incision, and the common carotid artery was isolated. A baseline of carotid artery flow was obtained using a Doppler flow probe (Transonic Systems Inc., Ithaca, NY). 10% ferric chloride soaked Whatman filter paper was placed in the vessel for 3 minutes. Twenty minutes after carotid artery occlusion, procedures were initiated. Animals were treated with control (platelet binding buffer), VWF aptamer, TPA, aptamer / antidote, TPA / VWF aptamer, or no perfusion via intravenous saphenous infusion (Harvard Apparatus PHD 2000 Infusion Pump, Holliston, MA). Carotid flow was monitored for an additional 90 min to assess reperfusion. Heart rate, EKG (ADInstruments PowerLab 4 / 35 EKG monitoring system, Sydney, Australia), and blood pressure (Kent Scientific CODA non-invasive BP measurement system, Torrington, CT) were monitored throughout the procedure. Histological analysis was performed on the carotid arteries.

[0075] Femoral venous bleeding A murine femoral vein hemorrhage model was performed in 8-week-old male or female C57BL / 6 mice obtained from a commercial supplier (Jackson Laboratory) to assess the reversibility of antidote oligonucleotides. 16The ventral hair of both hind limbs was removed. Mice were then placed supine on a temperature and ECG monitor platform. All four limbs were gently restrained. The ventral skin of the left and right hind limbs was incised to expose the entire length of the saphenous neurovascular bundle. The bundle was covered with normal saline to prevent drying. The left saphenous vein was cannulated for drug administration. To assess hemostasis, the right saphenous vein was punctured with a 23G needle and then the vessel was dissected by longitudinal incision of the distal part of the vessel. Blood was gently wiped away until hemostasis occurred. The clot was then removed to resume bleeding, and blood was wiped away again until hemostasis occurred again. Clot destruction was repeated for 30 min after each hemostasis event. Two parameters were measured: 1) the number of times hemostasis occurred in 30 min, and 2) the time required for each hemostasis event.

[0076] Canine carotid artery occlusion and thrombosis Canine carotid artery occlusion studies were performed in adult male and female beagles (7-11 kg). Carotid artery occlusion was induced with FeCl3 and allowed to stabilize for 45 min before treatment was initiated. Dogs were anesthetized and intubated. Catheters were obtained in the right femoral artery and vein. The right carotid artery was exposed and carotid flow baselines were obtained using a Doppler flow probe. Thrombosis was induced for 15 min using a 50% ferric chloride patch and the clot was allowed to stabilize for 45 min. Dogs were then infused intravenously with vehicle, 0.9 mg / kg TPA or 0.5 mg / kg VWF aptamer. Aptamer and vehicle were administered as a bolus, while rTPA was administered by standard clinical protocol with a 10% bolus followed by a 45 min infusion of the remaining drug. Carotid flow was monitored for 120 min. Blood flow transit times were monitored throughout the experiment with a flow probe distal to the site of thrombosis. Carotid angiography clearly demonstrated baseline patency, thrombotic occlusion, and recanalization. Platelet inhibition was assessed by periodic blood sampling (Platelet Function Analyzer 100). At the end of the experiment, the brain and carotid artery of each animal were collected and embedded for histological analysis.

[0077] statistical analysis Values ​​are expressed as mean ± SD, and statistical analysis was performed using multiple t-tests, chi-square analysis, and two-way analysis of variance where appropriate.

[0078] result The optimized VWF aptamer 9.14T79 binds to and inhibits VWF activity in vitro and ex vivo To generate future clinically tractable VWF aptamers, we designed and tested a series of VWF aptamer derivatives derived from the 2' fluoro-pyrimidine modified RNA aptamer VWF9.14. 12,13 See Example 1. This effort yielded a lead VWF aptamer, T59, which retains high affinity binding and inhibitory activity and is 30 nucleotides in length. To improve nuclease resistance and further optimize the composition, we then systematically replaced the 2'O-methyl and / or 2'fluoro moieties with T25 and T59 truncated forms. Nearly 90 truncated forms were synthesized and tested in vitro. The fully optimized aptamer 9.14T79vrt7 is 35 nucleotides and a 60-mer (K d =18.4 nmol / L, B max = 51%) compared to the dissociation constant (K d )=11.2nmol / L, B max =56% binds to VWF (see Tables 1 and 2 above and Figures 1-3).

[0079] To evaluate the inhibitory effect of the aptamer on platelet adhesion, human whole blood samples were treated with two-fold dilutions of the aptamer starting at 900 nmol / L and down to 14 nmol / L and tested by measuring platelet adhesion under high shear stress. The aptamer prevented platelet adhesion to collagen surfaces in a dose-dependent manner (Figures 20-22). Near complete inhibition of platelet adhesion was achieved at doses from 225 to 900 nmol / L (Figures 20C and 20D), and intermediate inhibition was achieved at doses from 56 to 112 nmol / L (Figures 20B and 20D). Log-dose-response data adjustment yielded a logIC of 1.9 (72.6 nmol / L). 50 This resulted in the calculation of (Figure 20E). The effect of the aptamer on platelet aggregation was measured ex vivo in a PFA-100 human whole blood assay. The VWF aptamer completely inhibited platelet aggregation at doses above 100 nmol / L in this system, where platelet clot formation and closure times exceeded 300 seconds, which was the upper limit of the assay (Figure 21). Thus, the VWF aptamer 9.14T79vrt7 prevents both platelet adhesion and aggregation ex vivo.

[0080] 9.14T79 exhibits enhanced thrombolysis compared with recombinant tissue plasminogen activator (rTPA) in a murine carotid artery occlusion model The thrombolytic activity of the aptamer was then evaluated using a murine carotid artery occlusion model. Twenty minutes after stable carotid artery occlusion, animals were administered aptamer 9.14T79, saline control, or rTPA. The dose of rTPA used in this experiment was 10 mg / kg (11-fold higher than 0.9 mg / kg, the dose used to treat humans with ischemic stroke within 3-4.5 hours of the last known occurrence), because this is the dose reported to be effective in recanalization in a murine model of arterial thrombosis. 17 The aptamer was administered at 0.5 mg / kg and showed significantly greater recanalization (n=8 / group) compared to rTPA (p<0.05) and buffer controls (p<0.01), as shown in FIG. 23A. Histological analysis of the carotid arteries of each group roughly correlated with the degree of recanalization measured by the flow probe (FIGS. 23B, 23C, and 23D). Examination of transverse sections of the affected carotid arteries of the buffer control group showed complete occlusion in all animals examined (n=8) (FIG. 23D). Vascular sections from rTPA-treated mice showed near-complete thrombosis and occlusion of the carotid artery (n=8) (FIG. 23C). Finally, histology of carotid artery sections from VWF aptamer-treated mice showed complete patency in six samples and evidence of only one small clot in two sections of previously occluded vessels (n=8) (p=0.01) (Figure 23B).

[0081] 9.14T79vrt7 exhibits dose-dependent platelet inhibition in canine whole blood To evaluate platelet thrombus formation under high shear and to initiate aptamer assessment in a larger animal model, we developed a total thrombosis assay system (T-TAS) (Fujimori Kogyo Co., Yokohama, Japan). 18 VWF aptamers were tested in 9.14T79vrt7. 9.14T79vrt7 inhibited canine platelet aggregation and maintained hemodynamic pressure at doses between 18.75-100 nmol / L (n=5 / group) (p<0.05 when compared to buffer). At the dose of 100 nmol / L, there was complete inhibition of platelet adhesion and aggregation (Figure 24B). Each figure shows the first 10 seconds from 1 to 5 minutes. The white hazy areas seen at 3 to 5 minutes in the buffer control are platelets adhered to the horizontal capillary channel. The panel of aptamers showed no such platelet accumulation, indicating that 9.14T79vrt7 is a potent inhibitor of canine platelet function under shear stress in vitro.

[0082] 9.14T79vrt7 demonstrates recanalization in a canine carotid artery occlusion model A canine cerebrovascular thrombosis model was used to confirm the murine results in a clinically relevant large animal. Arterial occlusion was established and sustained for an additional 45 min before treatment. Animals were administered 0.5 mg / kg 9.14T79vrt7 intravenously as a bolus or 0.9 mg / kg rTPA by standard clinical protocol of a 10% injection followed by the remaining 90% infusion over 45 min. All carotid arteries of three dogs receiving 9.14T79vrt7 recanalized 5 to 15 min after administration (Figures 24F, 24G, respectively). In contrast, animals treated with rTPA or saline control did not show recanalization after treatment (n=3 / group). To determine the safety of 9.14T79vrt7, intracerebral bleeding and clotting were evaluated in these dogs. 9.14T79vrt7 did not induce intracranial bleeding, and carotid recanalization did not result in cerebral thrombotic occlusion in any of these three animals (Figure 24H-J). Brain histology in both the 9.14T79vrt7 and rTPA groups was identical to the control saline-treated group. The lack of cerebral thrombotic occlusion in the aptamer group was reassuring, as carotid histology showed essentially complete recanalization of the vessel (Figure 24K). The top carotid section is the area of ​​vessel injury where occlusion occurred, while the bottom section is from the patent portion adjacent to the diagnostic catheter. In sharp contrast, both rTPA and saline-treated control group animals contained thrombi that continued to occlude the injured carotid artery, consistent with the inability of these approaches to regenerate blood flow (Figures 24L and 24M, respectively).

[0083] Antidote oligonucleotides can rapidly reverse the antiplatelet activity of 9.14T79vrt7 in vitro and in vivo To neutralize 9.14T79vrt7 activity when necessary, we generated antidote oligonucleotides (AOs, also called VWF9.14T79-AO2 or AO55). None of the antidotes initially tested could neutralize the 30-nucleotide aptamer T59. This is probably because once T59 is tightly bound to VWF, the good nucleation site of the aptamer is inaccessible to the antidotes. Therefore, we added a 5-nucleotide uracil (oligo-U tail) to the 3'-end of the molecule as an artificial nucleation site and tested a 16-nucleotide antidote complementary to this tail and the 3'-end of the aptamer. This antidote oligonucleotide (AO) neutralized the antiplatelet activity of the aptamer at a low ratio of 2:1 against 9.14T79vrt7 within 2 min in vitro (Figure 14) (n=2 / group). Ability of antidotes to neutralize antiplatelet aptamers in a murine femoral vein hemorrhage model 16The aptamer-induced thrombosis was evaluated by . Untreated control mice showed 12 ± 3 disruptions, similar to 17 ± 3 disruptions in the saline group (n = 7) (p > 0.05). 9.14T79vrt7 administered at a dose of 0.375 mg / kg did not result in clot disruption, which was highly significant compared to untreated controls and saline-treated animals (n = 11) (p < 0.0001). Administration of 9.14T79vrt7 followed by the addition of antidote oligonucleotide showed 16 ± 9 disruptions, similar to animals that did not receive the aptamer (n = 7). This data was expressed as % of normal thrombosis (Figure 25). Administration of the antidote alone did not result in an increase or decrease in clot disruption (data not shown). Thus, the antidote is able to rapidly reverse any hemorrhage associated with aptamer-mediated inhibition of VWF.

[0084] Consideration Traditionally, the vast majority of patients with ischemic stroke have no acute treatment options. rTPA causes bleeding, is time-limited, and cannot be reversed. Our study shows that antidote-controlled VWF inhibitors could provide a potent yet safe therapeutic option for these patients. 9.14T79vrt7 is a full-length aptamer 12 and exhibiting improved binding affinity compared to monoclonal antibodies 7,19 The aptamer 9.14T79vrt7 prevented adhesion of human platelets to collagen surfaces (Figures 20-22) as well as under high shear stress (Figure 24).

[0085] In vivo, 9.14T79vrt7 maintains arterial patency and transit time greater than 75% at doses as low as 0.0188 mg / kg (Figure 22). Compared to both the negative control and intravenous rTPA, 9.14T79vrt7 exhibited superior thrombolytic activity in a murine carotid artery occlusion model (Figure 23). The dose of IV rTPA used is 10-fold higher than that used clinically, primarily because this is the dose required to achieve thrombolysis in mice. 17Sixty minutes after drug administration, the rTPA group achieved 25% of the transit time compared with 75% of pre-injury flow in the aptamer-treated group. This effect persisted for over 100 minutes after drug administration. The aptamer was infused over 5 minutes, while rTPA was infused over 45 minutes due to the bleeding risk associated with rTPA.

[0086] At first glance, the idea of ​​drugs targeting endothelial and platelet factors to break up formed arterial thrombi is not intuitive. However, a growing body of literature supports the "degrading" activity of VWF inhibitors. In vitro studies have shown that high fluid shear stress and irregular vascular surfaces allow VWF to work its way through the thick bundles and meshwork that span the vascular lumen, binding platelets together and occluding the artery. 20 Therefore, anti-VWF therapy could have an impact on arterial occlusion. This hypothesis is supported by our observation that even in large vessels in dogs, the VWF aptamer 9.14T79vrt7 can result in recanalization of occluded vessels (Figure 24).

[0087] The major class of parenteral antiplatelet agents in clinical use are the glycoprotein IIb / IIIa (gpIIb / IIIa) inhibitors (Abciximab, Eptifibatide, and Tirofiban). These agents have significantly improved outcomes in acute coronary syndromes (ACS) and percutaneous coronary intervention (PCI). 21 However, when tested in acute ischemic stroke, they significantly increased intracranial hemorrhage without improving morbidity or mortality. 22 Therefore, we developed an antidote oligonucleotide that can easily neutralize VWF aptamer activity in the event of bleeding. This antidote completely neutralized 9.14T7vrt79 activity at a molar ratio of aptamer to antidote as low as 1:2 (Figure 14). The persistent reversibility of the aptamer was demonstrated in vivo in a venous bleeding model (Figures 9A and 9B). 13,16At a dose of 0.375 mg / kg of the aptamer, no coagulation occurred, whereas administration of a 10-fold molar excess of the antidote oligonucleotide neutralized the aptamer and restored normal hemostasis. The ability to completely and rapidly neutralize such a potent antiplatelet agent with a compatible antidote is a significant step in the development of safer and more potent parenteral antiplatelet agents for the treatment of thrombosis and especially acute ischemic stroke. References

[0088] TIFF0007675990000023.tif60170 TIFF0007675990000024.tif219166 TIFF0007675990000025.tif151170

Claims

1. An aptamer, comprising: From 5' to 3', (a) a polynucleotide having at least 90% sequence identity to SEQ ID NO:1, comprising a first stem-forming region comprising 3 nucleotides, a first loop region comprising the nucleotide sequence AAC, a second stem-forming region comprising 3 nucleotides, a second loop region comprising the nucleotide sequence CC, and a third stem-forming region consisting of 2-8 nucleotides; (b) a third loop region consisting of 4-6 nucleotides, and (c) a polynucleotide having at least 90% sequence identity to SEQ ID NO:2, comprising a fourth stem-forming region consisting of 2-8 nucleotides and capable of forming a stem together with the third stem-forming region, a fourth loop region comprising the nucleotide C, a fifth stem-forming region comprising 3 nucleotides and capable of forming a stem together with the second stem-forming region, a fifth loop region comprising the nucleotide sequence CAGA, and a sixth stem-forming region comprising 3 nucleotides and capable of forming a stem together with the first stem-forming region; A polynucleotide comprising: wherein the polynucleotide has at least 95% sequence identity to SEQ ID NO: 3 or 4, and includes an unmodified form or a modified form containing at least one nucleotide base modification; The aptamer, wherein the aptamer does not exceed 44 nucleotides in length.

2. The aptamer of claim 1 , wherein the polynucleotide consists of SEQ ID NO: 3 or 4.

3. The aptamer of claim 2, wherein the polynucleotide consists of SEQ ID NO: 7, 8 or 9.

4. An antidote comprising a polynucleotide having at least 95% sequence identity with any one of SEQ ID NOs: 103-180 and capable of hybridizing to at least 8 nucleotides of an aptamer described in any one of claims 1 to 3.

5. 5. The antidote of claim 4, wherein the polynucleotide consists of SEQ ID NO:157 and the antidote does not exceed 24 nucleotides in length.

6. A pharmaceutical composition comprising a pharmaceutical carrier and any one of the aptamers of claims 1-3 or the antidote of claims 4-5.

7. The pharmaceutical carrier is 20 mM Hepes, pH 7.4; 150 mM NaCl; 1 mM CaCl 2 1 mM MgCl 2 7. The pharmaceutical composition of claim 6, comprising: 5 mM KCl or buffered saline.

8. A pharmaceutical composition for use in a method for preventing blood clot formation in a subject animal, the pharmaceutical composition comprising an aptamer described in any one of claims 1 to 3 in a therapeutically effective amount for preventing blood clot formation in the subject animal.

9. A pharmaceutical composition for use in a method for treating blood clots in a subject animal, the pharmaceutical composition comprising an aptamer described in any one of claims 1 to 3 in a therapeutically effective amount for reducing blood clots in the subject animal.

10. The pharmaceutical composition of any one of claims 8 to 9, wherein the subject suffers from atrial fibrillation or is at risk of having deep vein thrombosis, stroke, heart attack, or pulmonary embolism.

11. The pharmaceutical composition according to any one of claims 8 to 10, wherein the target animal is a mammal.

12. The pharmaceutical composition of claim 11, wherein the mammal is a human.

13. The pharmaceutical composition of any one of claims 8 to 12, wherein the method further comprises administering to the subject animal a composition comprising at least one of the antidotes of any one of claims 4 to 5 to neutralize the action of the aptamer.

14. A pharmaceutical composition for use in neutralizing an aptamer in a subject animal, comprising an antidote described in any one of claims 4 to 5 in a therapeutically effective amount for neutralizing the aptamer in the subject animal.

15. 15. The pharmaceutical composition of claim 14, wherein the subject suffers from atrial fibrillation or has had a deep vein thrombosis, stroke, heart attack, or pulmonary embolism.

16. The pharmaceutical composition according to claim 14 or 15, wherein the subject animal is a mammal.

17. 17. The pharmaceutical composition of claim 16, wherein the mammal is a human.

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