US2009202429A1PendingUtilityA1
Methods for testing anti-thrombotic agents
Est. expiryJun 29, 2026(expired)· nominal 20-yr term from priority
G01N 33/566G01N 2500/02A01K 67/0278A01K 2227/105C12N 15/8509G01N 2333/70596C07K 14/755A01K 2207/15A01K 2267/0393A01K 67/0275A01K 2267/0381A01K 2217/072
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Claims
Abstract
The invention provides a transgenic non-human animal expressing von Willebrand Factor A1 protein containing at least one mutation selected from the group consisting of: 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, and any combination thereof.
Claims
exact text as granted — not AI-modified1 . An isolated mutant human von Willebrand Factor A1 protein comprising one or more selected from the group consisting of: 1263 S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, and 1479G>S, wherein each amino acid position corresponds to a position in SEQ ID NO: 6.
2 . An isolated mutant human von Willebrand Factor A1 protein having SEQ ID NO: 6, wherein the protein comprises one or more mutation(s) selected from the group consisting of: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, and a 1479G>S.
3 . An isolated mutant human von Willebrand Factor A1 protein comprising a 1326H>R mutation in an amino acid sequence of SEQ ID NO: 1.
4 . A transgenic non-human animal expressing von Willebrand Factor A1 protein containing mutation(s) at one of more amino acid position selected from the group consisting of: 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385, 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, and 1479, wherein the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6.
5 . The transgenic non-human animal of claim 4 , wherein the animal is a murine, a porcine, a canine, a feline, a rabbit, or a primate.
6 . The transgenic non-human animal of claim 4 , wherein the protein comprises a single mutation.
7 . The transgenic non-human animal of claim 4 , wherein the protein comprises two or more mutations.
8 . The transgenic non-human animal of claim 4 , wherein the protein comprises at least one mutation selected from the group consisting of: 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>I, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof.
9 . The transgenic non-human animal of claim 4 , wherein the protein comprises a 1326R>H mutation, a 1314I>V mutation, or a combination thereof.
10 . The transgenic non-human animal of claim 8 or 9 , wherein the animal is a mouse.
11 . The transgenic non-human animal of claim 8 or 9 , wherein the protein comprises SEQ ID NO: 5.
12 . The transgenic non-human animal of claim 4 , wherein the VWF protein is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the A1 domain of human VWF protein as shown in SEQ ID NO: 1.
13 . The transgenic non-human animal of claim 12 , wherein the von Willebrand Factor A1 protein of the transgenic animal contains the human A1 domain shown in SEQ ID NO: 1.
14 . The transgenic non-human animal of claim 4 , wherein the von Willebrand Factor A1 protein is partially or completely replaced with a human von Willebrand Factor A1 protein comprising SEQ ID NO: 1.
15 . The transgenic non-human animal of claim 4 , wherein the animal is a model for pre-clinical testing of compounds that expresses a mutant von Willebrand Factor (VWF) A1 protein containing one or more mutations, wherein the binding specificity of the mutant VWF-A1 protein changes from being specific for the animal platelets to being specific for human platelets.
16 . The transgenic non-human animal of claim 15 , wherein the mutant VWF-A1 protein in the animal binds to human platelets.
17 . A method for identifying a compound that modulates binding of VWF-A1 protein to GPIb-alpha protein, the method comprising:
a) providing an electronic library of test compounds; b) providing atomic coordinates listed in Table 8 for at least 10 amino acid residues for the A1 domain of the VWF protein, wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Cα atoms, of not greater than about 2.5 Å, in a computer readable format; c) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; d) performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and e) determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein,
thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein.
18 . A method for identifying a compound that modulates binding of VWF-A1 protein to GPIb-alpha protein, the method comprising:
a) providing an electronic library of test compounds; b) providing atomic coordinates listed in Table 8 in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues for the A1 domain of the VWF protein, wherein the residues comprise two or more of the following residues: Pro1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; c) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; d) performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and e) determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein,
thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein.
19 . The method of claim 17 or 18 , wherein determining comprises detecting an IC 50 of less than about 7.5 μg/ml for a test compound.
20 . The method of claim 17 or 18 , further comprising:
f) obtaining or synthesizing a compound; g) contacting VWF-A1 protein with the compound under a condition suitable for GPIb-alpha-VWF-A1 binding; and h) determining whether the compound modulates GPIb-alpha-VWF-A1 protein binding using a diagnostic assay.
21 . The method of claim 20 , wherein contacting comprises perfusing platelets into a flow chamber at a shear flow rate of at least 100 s −1 , wherein mutant murine VWF-A1 protein is immobilized on a bottom surface of the chamber.
22 . The method of claim 20 , wherein contacting comprises perfusing platelets into the transgenic non-human animal of claim 4 .
23 . The method of claim 21 , wherein contacting occurs sequentially.
24 . The method of claim 21 , wherein the perfusing of platelets occurs prior to administration of the compound.
25 . The method of claim 21 , wherein the platelets are human platelets.
26 . The method of claim 21 , wherein the platelets are not murine platelets.
27 . The method of claim 20 , wherein the determining comprises detecting an increase or decrease in the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold.
28 . The method of claim 20 , wherein the determining comprises detecting an increase or decrease of platelet adhesion to a surface expressing VWF-A1 protein.
29 . The method of claim 20 , wherein the determining comprises detecting an increase or decrease in a stabilization of an interaction between VWF-A1 protein and GPIb-alpha protein.
30 . The method of claim 20 , wherein the determining comprises detecting thrombosis formation.
31 . The method of claim 20 , wherein the determining comprises identifying an occurrence of an abnormal thrombotic event in the subject.
32 . The method of claim 31 , wherein an abnormal thrombotic event comprises abnormal bleeding, abnormal clotting, death, or a combination thereof.
33 . The method of claim 20 , wherein the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof.
34 . The method of claim 21 , wherein perfusing platelets is followed by perfusion of a labeled agent.
35 . The method of claim 34 , wherein the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand.
36 . The method of claim 34 , wherein the agent targets a platelet receptor, a VWF protein, or a portion thereof.
37 . A nucleic acid encoding the protein of any of claims 1 - 3 .
38 . A vector encoding the nucleic acid of claim 37 .
39 . An animal expressing the protein of any of claims 1 - 3 .
40 . A method for treating von Willebrand Disease (VWD) in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound that promotes platelet adhesion in the subject, wherein the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold, thereby administration of the compound increases blood coagulation in the subject.
41 . The method of claim 40 , wherein coagulation is measured by a coagulation factor assay, an ex-vivo flow chamber assay, or a combination thereof.
42 . A method for rapidly detecting an internal vascular injury site in a subject, the method comprising:
a) administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; b) tracking a deposition of the labeled targeted molecular imaging agent in the subject; and c) identifying the site of a thrombus formation in the subject by imaging the targeted molecular imaging agent,
thereby the deposition of the targeted molecular imaging agent at the internal vascular injury site is indicative of internal bleeding within a subject.
43 . The method of claim 42 , wherein the targeted molecular imaging agent is administered by subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; by oral, nasal, or topical delivery; or a combination thereof.
44 . The method of claim 42 , wherein the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof.
45 . The method of claim 44 , wherein the nanoparticle comprises a perfluorocarbon.
46 . The method of claim 44 , wherein the nanoparticle is coupled to an antibody, a small molecule, a peptide, or a receptor trap.
47 . The method of claim 42 , wherein the targeted molecular imaging agent specifically binds to a platelet receptor, or a VWF protein, or a portion thereof.
48 . The method of claim 42 , wherein the targeted molecular imaging agent has a T 1/2 of at least 30 minutes.
49 . The method of claim 42 , wherein imaging comprises a PET scan, MRI, IR scan, ultrasound, nuclear imaging, or a combination thereof.
50 . The method of claim 42 , wherein the subject is further administered a thrombotic compound.
51 . The method of claim 50 , wherein the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold.
52 . A method for determining whether platelet function or morphology in a subject is abnormal, the method comprising:
a) affixing a molecule comprising a murine VWF-A1 domain to a surface of a flow chamber, wherein the domain comprises at least one mutation at a position selected from the group consisting of 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>I, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof, where the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6; b) perfusing through the flow chamber a volume of blood or plasma from a subject at a shear flow rate of at least about 100 s −1 ; c) perfusing a targeted molecular imaging agent into the flow chamber; and d) comparing the flow rate of the blood or plasma from the subject as compared to a normal flow rate, so as to determine whether the subject's platelet function or morphology is abnormal.
53 . The method of claim 52 , wherein the affixing comprises (i) affixing an antibody which specifically binds VWF-A1 domain, and (ii) perfusing murine mutant VWF-A1 protein in the flow chamber at a shear flow rate of at least 100 s −1 .
54 . The method of claim 52 , wherein the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof.
55 . The method of claim 52 , wherein the targeted molecular imaging agent binds to a platelet receptor, a platelet ligand, or any region of a VWF protein or a portion thereof.
56 . The method of claim 52 , wherein the targeted molecular imaging agent comprises horseradish peroxidase (HRP) coupled to an antibody directed at VWF-A1.
57 . The method of claim 52 , wherein the comparing comprises a platelet adhesion assay, fluorescence imaging, a chromogenic indicator assay, a microscopy morphology analysis, or any combination thereof.
58 . The method of claim 52 , wherein platelets bound to VWF-A1 are less than about 500 cells/mm 2 .
59 . The method of claim 58 , wherein the platelets are substantially spherical.
60 . The method of claim 40 , 42 , or 52 , wherein the subject is a human, a canine, a feline, a murine, a porcine, an equine, or a bovine.
61 . The method of claim 52 , wherein the VWF molecule is an antibody, a peptide, or a Fab fragment directed to a VWF polypeptide or a portion thereof.
62 . A method for producing mutant von Willebrand Factor A1 protein that specifically binds human platelets, the method comprising:
(a) providing an animal expressing a mutant von Willebrand Factor A1 protein, wherein the mutation causes the platelet binding specificity of the animal von Willebrand Factor A1 protein to change to be specific for human platelets; and (b) harvesting the mutant animal von Willebrand Factor A1 so as to produce von Willebrand Factor A1 protein that specifically binds human platelets.
63 . The method of claim 62 , wherein the mutant animal von Willebrand Factor A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>1,1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof.
64 . A method for testing efficacy and toxicity of a gene therapy vector, the method comprising:
a) introducing a gene therapy vector into the animal of claim 4 , allowing sufficient time for expression of the vector; b) perfusing platelets from a subject into the animal under a condition suitable for GPIb-alpha-VWF-A1 protein binding; and c) determining whether or not a thrombotic event occurs in the animal.
65 . The method of claim 64 , wherein the vector comprises a nucleic acid encoding a platelet receptor polypeptide, a platelet ligand polypeptide, or a VWF polypeptide, or a portion thereof.
66 . The method of claim 64 , wherein the subject is a human, a dog, a cat, a horse, a pig, or a primate.
67 . The method of claim 64 , wherein the platelets are not murine platelets.
68 . The method of claim 64 , wherein the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof.
69 . The method of claim 64 , wherein the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof.
70 . The method of claim 64 , wherein perfusing platelets is followed by perfusion of a labeled agent.
71 . The method of claim 70 , wherein the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand.
72 . The method of claim 71 , wherein the agent targets a platelet receptor, a VWF protein, or a portion thereof.
73 . A method for calibrating an aggregometry device or a device for measuring clot formation or retraction, the method comprising:
a) providing hematologic data obtained from a subject, wherein blood or platelets from the subject is assessed by the device; b) determining whether or not a thrombotic event occurs in the animal of claim 4 , wherein the animal is perfused with a sample of blood or platelets from the subject; and c) correlating data obtained from (b) with the data obtained in (a) so as to calibrate the device, wherein a certain data obtained from the device is indicative of the corresponding thrombotic outcome determined in the animal of claim 4 .
74 . The method of claim 73 , wherein the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof.
75 . The method of claim 22 , wherein contacting occurs sequentially.
76 . The method of claim 22 , wherein the perfusing of platelets occurs prior to administration of the compound.
77 . The method of claim 22 , wherein the platelets are human platelets.
78 . The method of claim 22 , wherein the platelets are not murine platelets.
79 . The method of claim 22 , wherein perfusing platelets is followed by perfusion of a labeled agent.
80 . The method of claim 79 , wherein the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand.
81 . The method of claim 79 , wherein the agent targets a platelet receptor, a VWF protein, or a portion thereof.Join the waitlist — get patent alerts
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