WO2019150309A1 - Modulateurs de gpr68 et leurs utilisations pour le traitement et la prévention de maladies - Google Patents

Modulateurs de gpr68 et leurs utilisations pour le traitement et la prévention de maladies Download PDF

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WO2019150309A1
WO2019150309A1 PCT/IB2019/050799 IB2019050799W WO2019150309A1 WO 2019150309 A1 WO2019150309 A1 WO 2019150309A1 IB 2019050799 W IB2019050799 W IB 2019050799W WO 2019150309 A1 WO2019150309 A1 WO 2019150309A1
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chr14
gpr68
chr
subject
gene product
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Scott HAMMACK
Bryan Laffitte
Ardem Patapoutian
Jie Xu
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Hammack Scott
Bryan Laffitte
Ardem Patapoutian
Jie Xu
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention provides methods, compositions, and therapeutic uses related to modulators of a GPR68 gene product for the treatment and prevention of diseases, such as cardiavascular diseases and liver fibrosis.
  • Mechanotransduction is the central feature of many biological systems, including the sensing of touch and pain, hearing, cardiovascular dynamics, as well as turgor pressure sensing in bacteria.
  • membrane proteins capable of sensing acute mechanical forces have been identified, including the mechanosensitive ion channels MscL and MscS in bacteria, the DEGenerin/Epithelial Na + Channel (DEG/ENaC) MEC-4, MEC-10 and Transient Receptor Potential channel TRP-4 in C.elegans, and the transient receptor potential (TRP) ion channel NOMPC in Drosophila (Driscoll and Chalfie, 1991 ; Huang and Chalfie, 1994; Levina et al., 1999; Sukharev et al., 1994; Walker et al., 2000; Yan et al., 2013).
  • Piezos are a family of mechanically-activated (MA) ion channels conserved through evolution (Coste et al., 2010). Piezos are activated by disparate forms of mechanical forces, including indentation, stretch, and fluid shear stress. Consistent with this broad sensitivity to mechanical forces, Piezos play crucial roles in various physiological settings relevant to mechanotransduction.
  • MA mechanically-activated
  • Piezol can be activated by fluid shear stress and is required for vascular development and function in mice, while Piezo2 is essential for somatosensation (Li et al., 2014; Lukacs et al., 2015; Nonomura et al., 2017; Ranade et al., 2014; Retailleau et al., 2015; Wang et al., 2016; Woo et al., 2014).
  • Transmembrane channel-like (TMC) 1 and 2 are crucial components of the mechanotransduction channel in hair cells of the mammalian inner ear (Pan et al., 2013; Xiong et al., 2012); however, whether these proteins constitute the pore-forming subunit of this complex remains unsettled.
  • Mammalian TRP channels have diverse sensory roles, though none of them are bona fide mechanically-activated channels, with the possible exception of TRPV4. While mechanical indentation and membrane stretch does not activate TRPV4, elastomeric pillar array mediated membrane stretch leads to channel activation (Rocio Servin-Vences et al., 2017).
  • GPCRs G protein- coupled receptors
  • ATR1 Angiotensin II receptor type 1
  • BDKRB2 bradykinin receptor B2
  • PTH1 R parathyroid hormone 1 receptor
  • Some embodiments disclosed herein provide methods of modulating flow- mediated dilation (FMD) response or flow-mediated outward remodeling (FMR) of small- diameter arteries in a subject in need thereof comprising administering a modulator of a GPR68 gene product to the subject.
  • the modulator is an antagonist or an agonist of the GPR68 gene product.
  • the subject being treated has an abnormal vessel dilation and constriction response.
  • the abnormal vessel dilation and constriction response is associated with endothelial dysfunction.
  • the endothelial dysfunction is associated with vascular disorder, peripheral arterial disease, heart failure, hypertension, hypercholesterolemia, diabetes, septic shock, Behcet’s disease, exposure to smoking tobacco products, exposure to air pollution, or a combination thereof.
  • the modulator of the GPR68 gene product is selected from the group consisting of a small molecule compound, an antibody, a nucleic acid molecule, a protein, and a combination thereof.
  • the nucleic acid molecule is selected from the group consisting of an antisense oligonucleotide, a guide RNA, a ribozyme, an aptamer, and a small interfering RNA.
  • the subject is a human.
  • the methods further comprise measuring the vessel dilation response of the subject.
  • the measuring the vessel dilation response comprises brachial artery ultrasound imaging (BAUI).
  • Some embodiments disclosed herein provide methods of reducing systemic vascular resistance (SVR) in a subject in need thereof comprising administering a modulator of a GPR68 gene product to the subject.
  • the modulator is an antagonist or an agonist of the GPR68 gene product.
  • the subject being treated has an abnormal SVR.
  • the abnormal SVR is associated with peripheral arterial disease, heart failure, hypertension, hypercholesterolemia, diabetes, septic shock, or a combination thereof.
  • the modulator of the GPR68 gene product is selected from the group consisting of a small molecule compound, an antibody, a nucleic acid molecule, a protein, and a combination thereof.
  • the nucleic acid molecule is selected from the group consisting of an antisense oligonucleotide, a guide RNA, a ribozyme, an aptamer, and a small interfering RNA.
  • the subject is a human.
  • the methods further comprise measuring the SVR of the subject.
  • the measuring the SVR comprises measuring blood pressure (BP), heart rate (HR), stroke volume (SV), or a combination thereof.
  • the SVR of the subject is reduced by about 10% to about 50%.
  • Some embodiments disclosed herein provide methods of treating a cardiovascular disease in a subject in need thereof comprising administering a pharmaceutical composition comprising a modulator of a GPR68 gene product to the subject.
  • the modulator is an antagonist or an agonist of the GPR68 gene product.
  • the cardiovascular disease is selected from the group consisting of congestive heart failure (CHF), peripheral artery disease, stroke, diabetic nephropathy, and renal hypertension.
  • CHF congestive heart failure
  • the CHF comprises HF with reduced ejection fraction (aka HF due to left ventricular dysfunction) or HF with preserved ejection fraction (HFpEF) (aka diastolic HF or HF with normal ejection fraction).
  • the CHF is associated with a coronary artery disease selected from myocardial infarction (heart attack), high blood pressure, atrial fibrillation, and valvular heart disease, excess alcohol use, infection, or cardiomyopathy of an unknown cause.
  • the modulator of the GPR68 gene product reduces the SVR or the left ventricle afterload of the subject. In some embodiments, the SVR or the left ventricle afterload of the subject is reduced by about 10% to about 50%.
  • the methods further comprise diagnosing the cardiovascular disease in the subject. In some embodiments, the cardiovascular disease is diagnosed based on the history of the symptoms, physical examination, echocardiography, blood test, electrocardiography, chest radiography, or a combination thereof.
  • the modulator of the GPR68 gene product is selected from the group consisting of a small molecule compound, an antibody, a nucleic acid molecule, a protein, and a combination thereof.
  • the nucleic acid molecule is selected from the group consisting of an antisense oligonucleotide, a guide RNA, a ribozyme, an aptamer, and a small interfering RNA.
  • the subject is a human.
  • the methods further comprise administering an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a b-adrenergic receptor blocker, or a diuretics to the subject.
  • ACE angiotensin converting enzyme
  • ARB angiotensin receptor blocker
  • a b-adrenergic receptor blocker a diuretics
  • Some embodiments disclosed herein provide modulators of a GPR68 gene product for use as a medicament for the treatment of a cardiovascular disease in a subject in need thereof.
  • the modulator is an antagonist or an agonist of the GPR68 gene product.
  • the cardiovascular disease is selected from the group consisting of congestive heart failure (CHF), peripheral artery disease, stroke, diabetic nephropathy, and renal hypertension.
  • CHF congestive heart failure
  • the CHF comprises HF with reduced ejection fraction (aka HF due to left ventricular dysfunction) or HF with preserved ejection fraction (HFpEF) (aka diastolic HF or HF with normal ejection fraction).
  • the CHF is associated with a coronary artery disease selected from myocardial infarction (heart attack), high blood pressure, atrial fibrillation, and valvular heart disease, excess alcohol use, infection, or cardiomyopathy of an unknown cause.
  • the modulator of the GPR68 gene product reduces the SVR or the left ventricle afterload of the subject. In some embodiments, the SVR or the left ventricle afterload of the subject is reduced by about 10% to about 50%.
  • the treatment further comprises diagnosing the cardiovascular disease in the subject. In some embodiments, the cardiovascular disease is diagnosed based on the history of the symptoms, physical examination, echocardiography, blood test, electrocardiography, chest radiography, or a combination thereof.
  • the modulator of the GPR68 gene product is selected from the group consisting of a small molecule compound, an antibody, a nucleic acid molecule, a protein, and a combination thereof.
  • the nucleic acid molecule is selected from the group consisting of an antisense oligonucleotide, a guide RNA, a ribozyme, an aptamer, and a small interfering RNA.
  • the subject is a human.
  • the treatment further comprises administering an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a b-adrenergic receptor blocker, or a diuretics to the subject.
  • ACE angiotensin converting enzyme
  • ARB angiotensin receptor blocker
  • a b-adrenergic receptor blocker a diuretics
  • Some embodiments disclosed herein provide modulators of a GPR68 gene product in the manufacture of a medicament for the treatment of a cardiovascular disease in a subject in need thereof.
  • the modulator is an antagonist or an agonist of the GPR68 gene product.
  • the cardiovascular disease is selected from the group consisting of congestive heart failure (CHF), peripheral artery disease, stroke, diabetic nephropathy, and renal hypertension.
  • CHF congestive heart failure
  • the CHF comprises HF with reduced ejection fraction (aka HF due to left ventricular dysfunction) or HF with preserved ejection fraction (HFpEF) (aka diastolic HF or HF with normal ejection fraction).
  • the CHF is associated with a coronary artery disease selected from myocardial infarction (heart attack), high blood pressure, atrial fibrillation, and valvular heart disease, excess alcohol use, infection, or cardiomyopathy of an unknown cause.
  • the modulator of the GPR68 gene product reduces the SVR or the left ventricle afterload of the subject. In some embodiments, the SVR or the left ventricle afterload of the subject is reduced by about 10% to about 50%.
  • the treatment further comprises diagnosing the cardiovascular disease in the subject. In some embodiments, the cardiovascular disease is diagnosed based on the history of the symptoms, physical examination, echocardiography, blood test, electrocardiography, chest radiography, or a combination thereof.
  • the modulator of the GPR68 gene product is selected from the group consisting of a small molecule compound, an antibody, a nucleic acid molecule, a protein, and a combination thereof.
  • the nucleic acid molecule is selected from the group consisting of an antisense oligonucleotide, a guide RNA, a ribozyme, an aptamer, and a small interfering RNA.
  • the subject is a human.
  • the treatment further comprises administering an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a b-adrenergic receptor blocker, or a diuretics to the subject.
  • ACE angiotensin converting enzyme
  • ARB angiotensin receptor blocker
  • a b-adrenergic receptor blocker a diuretics
  • Some embodiments disclosed herein provide methods of treating liver fibrosis in a subject in need thereof comprising administering a pharmaceutical composition comprising an antagonist of a GPR68 gene product to the subject.
  • the antagonist of the GPR68 gene product is selected from the group consisting of a small molecule compound, an antibody, a nucleic acid molecule, a protein, and a combination thereof.
  • the nucleic acid molecule is selected from the group consisting of an antisense oligonucleotide, a guide RNA, a ribozyme, an aptamer, and a small interfering RNA.
  • the subject is a human.
  • Some embodiments disclosed herein provide methods of identifying a modulator of a GPR68 gene product comprising: providing a population of cells expressing a GPR68 gene product; adding a library of candidate molecules to the population of cells; applying a shear stress or an acidic shock to the population of cells; measuring the calcium transient in the population of cells; and identifying a candidate molecule in a cell that shows enhanced/reduced calcium transient in the cell.
  • the population of cells comprises endothelial cells, such as mouse primary brain microvascular endothelial cells, or human microvascular endothelial cells from brain, lung, bladder, or skin.
  • the library of candidate molecules comprises a small molecule compound, an antibody, a nucleic acid molecule, or a protein.
  • the shear stress comprises disturbed flow or laminar flow.
  • the acidic shock comprises extracellular proton at pH 6.5.
  • FIGs. 1 A-1 G show construction and validation of high-throughput shear stress stimulation system.
  • FIG. 1 A is a schematic representation of the disturbed flow stimulation system.
  • FIG. 1 B is an exemplary drawing depicts the major components of the 384-well format high-throughput disturbed flow stimulation system (left) and the bubble-relief geometry of the stimulator pin (right).
  • FIG. 1 C shows a top view of HT disturbed flow system showing the acoustic transducer assembly.
  • FIG. 1 D shows a bottom view of the HT system showing the 384- pin array.
  • FIG. 1 E shows exemplary intracellular calcium levels of HeLa cells measured by FLIPR, under 6.5 Pa shear stress stimulation in the presence of 2.5 mM EGTA or untreated control.
  • FIG. 1 F shows exemplary intracellular calcium levels of HeLa cells under the same disturbed flow stimulation after the treatment of scrambled siRNA or hPIEZOI siRNA. Data is average of 48 wells from 3 trials for each condition, plotted as mean ⁇ SEM.
  • FIGs. 2A-2D show that MDA-MB-231 cells show PIEZ01 - and PIEZ02- independent shear stress-induced calcium transients.
  • FIG. 2A shows exemplary responses of various human cancer cell lines to shear stress induced by the high throughput disturbed flow system. Shear stress was applied at 2 Pa, 60 Hz, with 0.2 s on, 2s off for 40 s. ** p ⁇ 0.01 FIG.
  • FIG. 2B shows exemplary response of MDA-MB-231 cells to shear stress at 2 Pa, measured by FLIPR.
  • the shear stress was applied for 4 s at 60 Hz by the HT disturbed flow system.
  • EGTA was added to the cells 2 min prior to the onset of shear stress.
  • Thapsigargin was incubated with cells for 15 min before the shear stress stimulation.
  • FIG. 2C shows quantification of the MDA- MB-231 cells' response to shear stress in the presence of the EGTA and Thapsigargin. ** p ⁇ 0.01 .
  • FIG. 2D shows quantification of the MDA-MB-231 cells' response to shear stress 72 h after transfection of PIEZ01 and PIEZ02 siRNA. n.s., not siginificant. All results are plotted as mean ⁇ SEM.
  • FIG. 3 shows that transfecting human cell lines with PIEZ01 or PIEZ02 siRNA significantly knocks down their respective mRNA levels. Relative mRNA level of PIEZ01 and PIEZ02 in MDA-MB-231 cells transfected with PIEZ01 siRNA or PIEZ02 siRNA compared with cells transfected with scrambled siRNA (mean ⁇ SEM from 4 trials).
  • FIGs. 4A-4E show that GPR68 is necessary for shear stress-induced calcium transients in MDA-MB-231 cells.
  • FIG. 4A shows exemplary response of MDA-MB-231 cells after treatment of siRNA against the indicated candidates. ** p ⁇ 0.01
  • FIG. 4B shows
  • FIG. 4C shows exemplary response of MDA-MB-231 cells to proton stimulation at various final pH. The cells were incubated with HBSS assay buffer at pH7.4 before the addition of the HBSS with various pH values. Three trials.
  • FIG. 4D shows exemplary response of MDA-MB-231 cells to proton stimulation (final pH 6.5) after knocking down GPR68 by siRNA smartpool.
  • FIG. 4E shows exemplary response of MDA-MB-231 cells to shear stress stimulation in buffers with various pH. The cells were incubated with assay buffer at pH7.4. The pH was then changed by adding buffer with various acidity 3 min prior to the onset of shear stress stimulation by the HT disturbed flow system. All results are plotted as mean ⁇ SEM.
  • FIGs. 5A-5D show that GPR68 histidine mutant loses both acid sensitivity and shear stress sensitivity.
  • FIG. 5A shows that HEK-293T cells were transiently transfected with pcDNA5, GPR68 WT or GPR68 histidine mutant constructs. Acid or shear stress-induced calcium transients were assayed 48h after transfection. For acid stimulation, the final pH was pH6.5. For shear stress stimulation, a 4s pulse of 60 Hz disturbed flow at 2 Pa was applied to the cells. Results were plotted as mean ⁇ SEM from 3 trials.
  • FIGs. 5B-5C show assessment of pH-sensitive fluorescent dye BCECF for measurement of extracellular and intracellular pH.
  • FIG. 5D shows that upon application of shear stress, there’s no measurable change in BCECF fluorescence, indicating no changes in either extracellular or intracellular pH. Traces are plotted as mean ⁇ SEM from two trials, 16 repeats for each condition.
  • FIGs. 6A-6K show that GPR68 is sensitive to shear stress imposed by both disturbed and laminar flow in HEK-293T cells.
  • FIG. 6A shows exemplary disturbed flow shear stress-induced calcium transients in HEK-293T cells.
  • HEK-293T cells were transfected with human GPR68 or vector control 48 h before the assay. Disturbed flow was applied by the HT system at 60 Hz for 4s (arrow indicates the onset of the flow).
  • FIG. 6A shows exemplary disturbed flow shear stress-induced calcium transients in HEK-293T cells.
  • HEK-293T cells were transfected with human GPR68 or vector control 48 h before the assay. Disturbed flow was applied by the HT system at 60 Hz for 4s (arrow indicates the onset of the flow).
  • FIG. 6C shows quantification of the response of HEK- 293T cells to pH 6.5 stimulation and 2 Pa disturbed shear stress. ** p ⁇ 0.01 .
  • FIG. 6D shows quantification of the response of HEK-293T cells to 2 Pa disturbed shear stress in the presence of 20 mM Cu 2+ and 10 mM U73122. Cu 2+ and U73122was added to the assay buffer 2 min prior to the start of disturbed flow n.s., not significant. ** p ⁇ 0.01 .
  • FIG. 6E shows exemplary shear stress induced responses of HEK-293T cells transiently-transfected with various human and mouse GPCRs. ** p ⁇ 0.01 .
  • 6F shows exemplary agonist response of HEK-293T cells transiently-transfected with various human and mouse GPCRs. ** p ⁇ 0.01 .
  • the chemical activators used were: pH 6.5 for human and murine GPR68, 0.5 mM Angiotensin II for AGTR1 , 0.5 mM [Arg 8 ]-Vasopressin for AVPR1 A, 0.5 mM Bradykinin for BDKRB2, 0.5 mM Acetylcholine Chloride for CHRM5, 0.5 mM Endothelin I for EDNRA, 0.5 mM Histamine Dihydrochloride for HRH1 , 20 mM Parathyroid Hormone (1 -34) for PTHR1 , and 100 mM lactate for GPR132.
  • FIG. 6G shows representative traces of intracellular calcium levels in HEK-293T cells transfected with mouse Gpr68-IRES-eGFP upon pulsatile laminar flow stimulation.
  • Cells with GFP signal were considered transfected and the GFP-negative ones were untransfected.
  • Pulsatile laminar flow was applied at 3.4 Pa, 1 Hz for 120 s.
  • FIG. 61 shows representative traces of intracellular calcium levels in HEK-293T cells transfected with mouse Gpr68-IRES-eGFP upon steady laminar flow stimulation.
  • FIG. 6K shows exemplary responses of HEK-293T cells transfected with various putative mechanosensors to steady laminar flow at 3.4 Pa. ** p ⁇ 0.01 . All results are plotted as mean ⁇ SEM.
  • FIGs. 7A-7F show that GPR68 expression is detected in endothelial cells of small- diameter vessels.
  • FIG. 7B shows representative images of colorimetric RNAscope in situ hybridization for Gpr68 vascular endothelial cells of small diameter blood vessels in brain, pancreas and liver. Scale bars: 25 pm. Arrows indicate cells with positive signal.
  • FIG. 7B shows representative images of colorimetric RNAscope in situ hybridization for Gpr68
  • FIG. 7C shows a schematic diagram of the BAC transgenic construct that is integrated in the genome of Gpr68 eGFP reporter mice.
  • the blue A-box is the homologous sequence that was used to guide recombination and insert eGFP after the start codon of Gpr68.
  • Poly-A sequence following the eGFP blocks the transcription of the
  • FIG. 7D shows an exemplary FACS plot of primary endothelial cells isolated from the bladder of Gpr68 eGFP reporter mice. Cells that are positive for CD31 and negative for CD45 are shown, and grouped in the GFP+ and GFP- populations.
  • FIG. 7E shows exemplary normalized RNA levels (RPKM) of Gpr68 and Piezol from the RNAseq data of GFP+ and GFP- endothelial cells.
  • FIG. 7F shows representative images of GFP antibody staining in arteries of 1 st order (1 s ), 2 nd order (2 s ), 3 rd order (3 s ) superior mesenteric vessels, and the vessels in the wall of small intestine (w). Scale bars: 50 pm for 1 s vessels and 2 s vessels, 25 pm for 3 s vessels, and 10 pm for vessels in the small intestine wall. 8 groups of vessels from 4 mice were examined.
  • FIGs. 8A-8E show that GFP immunoreactivity is detected in small diameter arteries in various tissues of GPR68 eGFP reporter mice.
  • FIG. 8A shows an exemplary flow cytometry analysis of GFP intensity in various populations of leukocytes isolated from the spleen of Gpr68 eGFP reporter mice. Results were from two eGFP reporter mice (light and dark green lines) and two C57B/L6 mice (grey and black lines).
  • FIG. 8B shows exemplary relative mRNA level of Gpr68 in various populations of leukocytes isolated from the spleen of C57B/L6 mice. Results are presented as mean ⁇ SEM from 3 trials.
  • FIG. 8A shows an exemplary flow cytometry analysis of GFP intensity in various populations of leukocytes isolated from the spleen of Gpr68 eGFP reporter mice. Results were from two eGFP reporter mice (light and dark green lines) and two C57B/L6 mice (grey and black lines).
  • FIG. 8C shows representative images of antibody staining against GFP and CD31 in blood vessel of pancreas isolated from Gpr68 eGFP reporter mice. Scale bar: 25 pm.
  • FIG. 8D shows representative images of antibody staining against GFP and CD31 in blood vessel of liver a, artery; b, bile duct; v, portal vein. Scale bar: 25 pm.
  • FIG. 8E shows representative images of antibody staining against GFP and CD31 in blood vessel of bladder a, artery; v, vein. Scale bar: 50 pm. tissues from 4 mice were examined.
  • FIGs. 9A-9D show that Gpr68 is necessary for laminar flow-induced calcium transients in murine primary microvascular endothelial cells.
  • FIG. 9A shows exemplary responses of mouse primary microvascular endothelial cells (MVECs) to shear stress imposed by pulsatile laminar flow with increasing amplitudes.
  • FIG. 9A shows exemplary responses of mouse primary microvascular endothelial cells (MVECs) to shear stress imposed by pulsatile laminar flow with increasing amplitudes.
  • the MVECs were isolated from cerebrum of the Gpr68 eGFP reporter mice. Pulsatile laminar flow stimulation of different amplitudes are applied at 1
  • FIG. 9B shows exemplary responses of GFP+ and GFP- MVECs to pulsatile laminar shear stress at 4 Pa.
  • FIG. 9C shows exemplary calcium transients in primary MVECs induced by to 50 pM ATP.
  • FIGs. 10A-10F show that Gpr68 is required for flow-mediated dilation and outward remodeling in third order mesenteric arteries.
  • FIG. 10A shows exemplary flow-mediated dilation (FMD) response of third order mesenteric arteries (MAs) isolated from Gpr68 -/- mice and WT littermates. Vessels were cannulated and pre-constricted using 1 pM phenylephrine, and subjected to stepwise increases in flow rates. Left, representative recordings of vessel diameter change. Scale bar: 10 minutes. Right, quantification of FMD response in 3 rd order MAs.
  • FMD flow-mediated dilation
  • FIG. 10B shows exemplary dilation responses of third order MAs to Ogerin. Vessels were cannulated and pre-constricted using 1 mM phenylephrine, and subjected to Ogerin with increasing concentration. Left, representative recordings of vessel diameter change. Scale bar: 10 minutes. Right,
  • FIG. 10C shows a schematic representation of the surgery applied to mesenteric resistance arteries in vivo in order to increase blood flow locally.
  • Second order arteries were ligated (marked by X) on 2 branches of the mesenteric arterial bed. The arteries located between the ligated branches were thus submitted to a chronic increase in blood flow. These arteries were designed as high flow (HF) arteries. Similar mesenteric arteries located at a distance were used as control or normal flow (NF) arteries.
  • Arteries were collected 2 weeks after surgery in order to determine their diameter and thus the remodeling induced by the chronic increase in blood flow. Vessels were
  • FIGs. 11 A-11 D show that Gpr68 -/- 1 st and 2 nd order mesenteric vessels have normal response to flow and vessels of all sizes have normal response to chemical stimulations.
  • FIG. 11 C shows exemplary FMD response in 3 rd order MAs in the presence of 100 mM L-N G -Nitroarginine methyl ester (L-NAME).
  • FIG. 11 D shows exemplary dilation response of various orders of MAs to 1 mM acetylcholine and 80 mM of KCI.
  • FIG. 12 shows an exemplary dose response of Ogerin, which is a mouse Gpr68 agonist.
  • Ogerin is a mouse Gpr68 agonist.
  • HEK-293T cells were transiently transfected with mouse Gpr68.
  • Relative fluorescence from calcium indicators were acquired for 10 s, then Ogerin was added to the cells in various concentrations, while measurement continued for 180 s at 1 data point per second.
  • Maximum fluorescence was plotted as mean ⁇ SEM from 3 trials.
  • Dose response curve fitting yielded an EC50 of 0.17 mM.
  • FIGs. 13A-13D show characterization of hemodynamic and cardiac parameters and assessment of mesenteric vessels of Gpr68 -/- mice.
  • FIG. 13A shows exemplary echocardiogram assessment of Gpr68 -/- and WT heart. All the structural parameters were not significantly different between KO and WT mice.
  • FIG. 13B shows exemplary blood pressure radiotelemetry recording of baseline hemodynamic parameters of Gpr68 -/- mice and WT littermates. Each dot represents one animal.
  • FIG. 13D shows exemplary dilation response of MAs to SNP at various dosages.
  • 3 rd order MAs 5 WT and 7 KO vessels were examined.
  • FIGs. 14A-14C show exemplary assessment of flow-mediated outward remodeling of Gpr68 -/- mesenteric vessels.
  • FIGs. 15A-15B show induction of GPR68 mRNA by TGF .
  • the present invention is based, in part, on the discovery that GPR68 is a necessary component for sheer stress response. GPR68 is expressed in the endothelial cells of resistance arteries ( ⁇ 100 pm), but not that of veins of any size. GPR68 mediates shear stress-induced calcium transients in primary microvascular endothelial cells and controls flow- mediated dilation (FMD) response and flow-mediated outward remodeling (FMR) of small- diameter mesenteric arteries.
  • FMD flow- mediated dilation
  • FMR flow-mediated outward remodeling
  • modulators of a GPR68 gene product and compositions, methods and uses thereof for treating cardiovascular diseases and liver fibrosis are provided herein.
  • small molecule refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da.
  • inhibitor means to suppress an activity or function, for example, about ten percent relative to a control value. Preferably, the activity is suppressed y 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. “Inhibit,” as used herein, also means to reduce the level of a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein's expression, stability, function or activity by a measurable amount or to prevent entirely.
  • Inhibitors are compounds that, e.g., bind to, partially or totally inhibit activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.
  • modulator and“modulation” of a molecule of interest, as used herein in its various forms, is intended to encompass antagonism, agonism, partial antagonism and/or partial agonism of an activity associated with a GPR68 gene product.
  • modulators may inhibit or stimulate GPR68 expression or activity.
  • Such modulators include small molecules agonists and antagonists of GPR68, antisense molecules, ribozymes, triplex molecules, and RNAi polynucleotides, and others.
  • G protein coupled receptor 68 (GPR68, also known as Ovarian cancer G-protein coupled receptor 1 (OGR1 ), GPR12A, AI2A6) is a G q/ n-coupled receptor, and GPR68 activation may lead to the cleavage of PIP2 into IP3 and DAG by PLC, and induce calcium release from the store.
  • GPR68 may trigger several acute signaling pathways, such as activation of Ca 2+ -gated channels (OraM , Kc a 2.3, Kc a 3.1 ) or synthesis of NO by NOS or release of EDHF.
  • Activation of GPR68 may also activate KCNK channels and lead to the hyperpolarization of the cells.
  • Activation of GPR68 may lead to hyperpolarization of smooth muscles surrounding the vessels, causing them to relax and therefore dilate the vessels.
  • GPR69 mRNA was also found to be induced by pro-fibrotic stimuli (e.g. TGFp) in primary hepatic stellate cells from rat and human.
  • A“GPR68 gene product” may be an RNA, such as an mRNA, or a polypeptide, of a GPR68 gene.
  • the human GPR68 protein is found in GENBANKTM Accession No: NP 001 171 147.1 , and has the amino acid sequence set forth below (SEQ ID NO: 1 ):
  • mouse GPR68 protein is found in GENBANKTM Accession No: NP 780702.1 , and has the amino acid sequence set forth below (SEQ ID NO: 2):
  • the rat GPR68 protein is found in GENBANKTM Accession No: NP 001 101519.1 , and has the amino acid sequence set forth below (SEQ ID NO: 3):
  • the cynomolgus monkey GPR68 protein is found in GENBANKTM Accession No: XP_005562082.1 , and has the amino acid sequence set forth below (SEQ ID NO: 4):
  • the human GPR68 gene is found on chromosome 14 (14q32.1 1 ) in GENBANKTM Accession No: NG 052988.1 .
  • transcript variant 1 of human GPR68 mRNA is found in GENBANKTM
  • transcript variant 2 of human GPR68 mRNA is found in GENBANKTM
  • transcript variant 3 of human GPR68 mRNA is found in GENBANKTM
  • transcript variant 1 of mouse GPR68 mRNA is found in GENBANKTM
  • the transcript variant 3 of mouse GPR68 mRNA is found in GENBANKTM Accession No: NM 001 177674.1 , and has the nucleotide sequence set forth below (SEQ ID NO: 10):
  • the rat GPR68 mRNA is found in GENBANKTM Accession No: NM 001 108049.1 , and has the nucleotide sequence set forth below (SEQ ID NO: 1 1 ):
  • transcript variant X1 of cynomolgus monkey GPR68 mRNA is found in GENBANKTM Accession No: XM 005562026.2, and has the nucleotide sequence set forth below (SEQ ID NO: 12):
  • transcript variant X2 of cynomolgus monkey GPR68 mRNA is found in
  • transcript variant X4 of cynomolgus monkey GPR68 mRNA is found in GENBANKTM Accession No: XM 015454130.1 , and has the nucleotide sequence set forth below (SEQ ID NO: 15):
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • nucleic acid or“polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form.
  • nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g ., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ai., Nucleic Acid Res. 19:5081 (1991 ); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et ai., Mol. Cell. Probes 8:91 -98 (1994)).
  • polypeptide “peptide,” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are“silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (/.e., gaps) as compared to the reference sequence (e.g., a polypeptide), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequences.
  • Two sequences are“substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein.
  • the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence.
  • identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence.
  • shorter amino acid sequences e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • A“comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001 .
  • polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • the terms“subject,”“patient,” and“individual” interchangeably refer to a mammal, for example, a human or a non-human primate mammal.
  • the mammal can also be a laboratory mammal, e.g., mouse, rat, rabbit, hamster.
  • the mammal can be an agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid) or domestic mammal (e.g., canine, feline).
  • the terms“treat,”“treating,” or“treatment” of any disease or disorder refer in one embodiment, to ameliorating the disease or disorder (i.e ., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • “treat,”“treating,” or“treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “treat,”“treating,” or“treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • “treat,”“treating,” or“treatment” refers to preventing or delaying the onset or development or progression of a disease or disorder.
  • the terms“therapeutically acceptable amount” or“therapeutically effective dose” interchangeably refer to an amount sufficient to effect the desired result (i.e., a reduction in inflammation, inhibition of pain, prevention of inflammation, inhibition or prevention of inflammatory response). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.
  • a“prophylactically effective dosage,” and a“therapeutically effective dosage,” of a GPR68 modulator can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with a cardiovascular disease or liver fibrosis.
  • the phrase“consisting essentially of” refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any inactive carrier or excipients for the intended purpose of the methods or compositions. In some embodiments, the phrase“consisting essentially of” expressly excludes the inclusion of one or more additional active agents other than a GPR68 modulator. In some embodiments, the phrase“consisting essentially of” expressly excludes the inclusion of more additional active agents other than a GPR68 modulator and a second co-administered agent.
  • Some embodiments of the present invention provides a modulator, e.g., an antagonist or an agonist, of a GPR68 gene product.
  • a GPR68 polypeptide is used to refer collectively to all naturally occurring isoforms of a GPR68 protein of any species, or a variant thereof.
  • a “GPR68 polypeptide” can be a human GPR68 polypeptide, a mouse GPR68 popypeptide, a rat GPR68 polypeptide, a cyno GPR68 polypeptide, or a variant thereof.
  • a GPR68 polypeptide can be a protein having the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof.
  • a GPR68 variant can differ from a naturally occurring GPR68 protein by, for example, a modification (e.g., substitution, deletion, or insertion) of one or more amino acid residues in the naturally occurring GPR68 protein, but retains the biological activities of GPR68, e.g., endothelial shear stress sensor or mechanosensor activity.
  • the GPR68 variant can have one or more conservative or nonconservative amino acid substitution.
  • A“conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the GPR68 variant includes one or more mutations (e.g., substitutions (e.g., conservative substitutions or substitutions), insertions, or deletions) of non- essential amino acids relative to a naturally occurring GPR68 protein.
  • A“non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of GPR68 protein without abolishing or more preferably, without substantially altering a biological activity, such as endothelial shear stress sensor or mechanosensor activity, whereas changing an“essential” amino acid residue results in a substantial loss of biological activity.
  • a GPR68 variant may have at least one, two, three, or four, and no more than 10,
  • mutations e.g., substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) relative to a naturally occurring GPR68 protein.
  • substitutions e.g., conservative substitutions or substitutions of non-essential amino acids
  • insertions or deletions
  • a GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a naturally occurring GPR68 protein.
  • a human GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 1 .
  • a mouse GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 2.
  • a rat GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO:
  • a cyno GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 4.
  • the modulator of a GPR68 gene product may decrease or increase the level of a GPR68 gene product. It will be understood by one skilled in the art, based upon the disclosure provided herein, that a decrease or increase in the level of a GPR68 gene product encompasses the decrease or increase in the expression, including DNA transcription, mRNA translation, mRNA stability, protein stability or any all of their combinations. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that a decrease or increase in the level of a GPR68 gene product includes a decrease or increase in the activity of GPR68, e.g., endothelial shear stress sensor or mechanosensor activity.
  • decrease or increase in the level or activity of GPR68 includes, but is not limited to, decreasing or increasing the amount of polypeptide of GPR68, and decreasing or increasing transcription, translation, or both, of a nucleic acid encoding GPR68; and it also includes decreasing or increasing any activity of GPR68, e.g., endothelial shear stress sensor or mechanosensor activity.
  • the modulator of a GPR68 gene product may reduce flow-mediated dilation (FMD) response and/or flow-mediated outward remodeling (FMR) of small-diameter arteries, also known as resistance arteries, in a subject.
  • FMD flow-mediated dilation
  • FMR flow-mediated outward remodeling
  • the modulatoer of a GPR68 gene product e.g., an agonist or antagonist of a GPR68 gene product
  • the modulator of a GPR68 gene product may reduce the systemic vascular resistance (SVR) and/or the left ventricle afterload of a subject.
  • the modulator of a GPR68 gene product e.g., an agonist or antagonist of a GPR68 gene product
  • the GPR68 modulator is a small molecule.
  • a small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.
  • a small molecule modulator of the invention comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.
  • Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are methods of making the libraries.
  • the methods may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single
  • an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles.
  • the shape and rigidity of the core determines the orientation of the building blocks in shape space.
  • the libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores.
  • the modulator is a small molecule
  • a small molecule antagonist or agonist may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.
  • the GPR68 modulator is a small molecule compound named Ogerin.
  • the expression of GPR68 may be inhibited by an RNA interference (RNAi) molecule, e.g., siRNA, shRNA, etc.
  • RNAi molecules comprise an antisense strand and a sense strand.
  • the antisense and sense strand can be two physically separated strands, or can be components of a single strand or molecule, e.g., they are linked a loop of nucleotides or other linker.
  • RNAi RNA interference
  • the antisense and sense strand can be two physically separated strands, or can be components of a single strand or molecule, e.g., they are linked a loop of nucleotides or other linker.
  • a non limiting example of the former is a siRNA; a non-limiting example of the latter is a shRNA.
  • Some RNAi agents for GPR68 are available commercially, e.g., GPR68 TRC lentiviral shRNA from Dharmacon.
  • RNA interference molecules can be determined using computer software programs and the gene of GPR68.
  • RNAi agent refers to an siRNA (short inhibitory RNA), shRNA (short or small hairpin RNA), iRNA (interference RNA) agent, RNAi (RNA interference) agent, dsRNA (double-stranded RNA), microRNA, and the like, which specifically binds to the GPR68 mRNA and which mediates the targeted cleavage of the RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • siRNA short inhibitory RNA
  • shRNA short or small hairpin RNA
  • iRNA interference RNA
  • RNAi RNA interference agent
  • dsRNA double-stranded RNA
  • microRNA and the like, which specifically binds to the GPR68 mRNA and which mediates the targeted cleavage of the RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • the RNAi agent is an oligonucleotide composition that activates the RISC complex/pathway.
  • the RNAi agent comprises an antisense strand sequence (antisense oligonucleotide).
  • the RNAi comprises a single strand. This single-stranded RNAi agent
  • oligonucleotide or polynucleotide can comprise the sense or antisense strand, as described by Sioud 2005 J. Mol. Bioi. 348:1079-1090, and references therein.
  • the disclosure encompasses RNAi agents with a single strand comprising either the sense or antisense strand of an RNAi agent described herein.
  • the use of the RNAi agent to GPR68 results in a decrease of GPR68 post-translational modification, production, expression, level, stability and/or activity, e.g., a "knock-down" or "knock-out" of the GPR68 target gene or target sequence.
  • the GPR68 antagonist is molecule capable of mediating RNA interference against a GPR68 mRNA selected from the group consisting of SEQ ID NOs: 5-15.
  • RNA interference-inducing molecules include but are not limited to siRNA, dsRNA, stRNA, shRNA, microRNAi (mRNAi)/microRNA (miRNA), antisense oligonucleotides etc. and modified versions thereof, where the RNA interference molecule silences the gene expression of GPR68.
  • An anti-sense oligonucleic acid, or a nucleic acid analogue for example but are not limited to DNA, RNA, peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), or locked nucleic acid (LNA) and the like.
  • RNA interference is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting expression of the target gene.
  • mRNA messenger RNA
  • dsRNA double stranded RNA
  • RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs.
  • siRNAs are incorporated into a protein complex (termed“RNA induced silencing complex,” or“RISC”) that recognizes and cleaves target mRNAs.
  • RISC protein complex
  • RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes.
  • “inhibition of target gene expression” includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene as compared to a situation wherein no RNA interference has been induced.
  • the decrease can be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an RNA interfering agent.
  • siRNA Short interfering RNA
  • small interfering RNA is defined as an agent which functions to inhibit expression of a target gene, e.g., by RNAi.
  • a siRNA can be chemically synthesized, can be produced by in vitro transcription, or can be produced within a host cell.
  • siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21 , 22, or 23 nucleotides in length, and can contain a 3' and/or 5' overhang on each strand having a length of about 0, 1 , 2, 3, 4, or 5 nucleotides.
  • the length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.
  • the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
  • PTGS post-transcriptional gene silencing
  • siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).
  • shRNAs small hairpin (also called stem loop) RNAs
  • these shRNAs are composed of a short (e.g., about 19 to about 25 nucleotide) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand.
  • the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
  • shRNAs can be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA April; 9(4):493-501 , incorporated by reference herein in its entirety).
  • the target gene or sequence of the RNA interfering agent can be a cellular gene or genomic sequence, e.g. a GPR68 gene.
  • An siRNA can be substantially homologous to the target gene or genomic sequence, or a fragment thereof.
  • the term “homologous” is defined as being substantially identical, sufficiently complementary, or similar to the target mRNA, or a fragment thereof, to effect RNA interference of the target.
  • RNA suitable for inhibiting or interfering with the expression of a target sequence includes RNA derivatives and analogs.
  • the siRNA is identical to its target.
  • the siRNA preferably targets only one sequence.
  • Each of the RNA interfering agents, such as siRNAs can be screened for potential off-target effects by, for example, expression profiling. Such methods are known to one skilled in the art and are described, for example, in Jackson et al, Nature Biotechnology 6:635-637, 2003.
  • expression profiling one can also screen the potential target sequences for similar sequences in the sequence databases to identify potential sequences which can have off-target effects. For example, according to Jackson et al. (Id.) 15, or perhaps as few as 1 1 contiguous nucleotides of sequence identity are sufficient to direct silencing of non-targeted transcripts. Therefore, one can initially screen the proposed siRNAs to avoid potential off-target silencing using the sequence identity analysis by any known sequence comparison methods, such as BLAST.
  • siRNA molecules need not be limited to those molecules containing only RNA, but, for example, further encompasses chemically modified nucleotides and non-nucleotides, and also include molecules wherein a ribose sugar molecule is substituted for another sugar molecule or a molecule which performs a similar function. Moreover, a non-natural linkage between nucleotide residues can be used, such as a phosphorothioate linkage. For example, siRNA containing D-arabinofuranosyl structures in place of the naturally-occurring D- ribonucleosides found in RNA can be used in RNAi molecules according to the present invention (U.S. Pat. No. 5,177,196).
  • RNA molecules containing the o- linkage between the sugar and the heterocyclic base of the nucleoside which confers nuclease resistance and tight complementary strand binding to the oligonucleotides molecules similar to the oligonucleotides containing 2'-0-methyl ribose, arabinose and particularly D-arabinose (U.S. Pat. No. 5,177,196).
  • the RNA strand can be derivatized with a reactive functional group of a reporter group, such as a fluorophore.
  • a reporter group such as a fluorophore.
  • Particularly useful derivatives are modified at a terminus or termini of an RNA strand, typically the 3' terminus of the sense strand.
  • the 2'-hydroxyl at the 3' terminus can be readily and selectively derivatized with a variety of groups.
  • siRNA and miRNA molecules having various“tails” covalently attached to either their 3'- or to their 5'-ends, or to both, are also known in the art and can be used to stabilize the siRNA and miRNA molecules delivered using the methods of the present invention.
  • intercalating groups, various kinds of reporter groups and lipophilic groups attached to the 3' or 5' ends of the RNA molecules are well known to one skilled in the art and are useful according to the methods of the present invention.
  • Descriptions of syntheses of 3'-cholesterol or 3'-acridine modified oligonucleotides applicable to preparation of modified RNA molecules useful according to the present invention can be found, for example, in the articles: Gamper, H. B., Reed, M.
  • siRNAs useful for the methods described herein include siRNA molecules of about 15 to about 40 or about 15 to about 28 nucleotides in length, which are homologous to the GPR68 gene.
  • the GPR68 targeting siRNA molecules have a length of about 19 to about 25 nucleotides. More preferably, the targeting siRNA molecules have a length of about 19, 20, 21 , or 22 nucleotides.
  • the targeting siRNA molecules can also comprise a 3' hydroxyl group.
  • the targeting siRNA molecules can be single-stranded or double stranded; such molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3'). In specific
  • the RNA molecule is double stranded and either blunt ended or comprises overhanging ends.
  • At least one strand of the GPR68 targeting RNA molecule has a 3' overhang from about 0 to about 6 nucleotides (e.g., pyrimidine nucleotides, purine nucleotides) in length.
  • the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length.
  • the targeting RNA molecule is double stranded— one strand has a 3' overhang and the other strand can be blunt-ended or have an overhang.
  • the length of the overhangs can be the same or different for each strand.
  • the RNA of the present invention comprises about 19, 20, 21 , or 22 nucleotides which are paired and which have overhangs of from about 1 to about 3, particularly about 2, nucleotides on both 3' ends of the RNA.
  • the 3' overhangs can be stabilized against degradation.
  • the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine 2 nucleotide 3' overhangs by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi.
  • the absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium.
  • the RNAi agent comprises a modification that causes the RNAi agent to have increased stability in a biological sample or environment.
  • the RNAi agent comprises at least one sugar backbone modification (e.g., phosphorothioate linkage) or at least one 2' -modified nucleotide.
  • sugar backbone modification e.g., phosphorothioate linkage
  • 2' -modified nucleotide e.g., phosphorothioate linkage
  • the RNAi agent comprises: at least one 5'-uridine-adenine-3' (5'-ua-3') dinucleotide, wherein the uridine is a 2'-modified nucleotide; at least one 5'-uridine-5 guanine-3' (5' -ug-3') dinucleotide, wherein the 5'-uridine is a 2' -modified nucleotide; at least one 5'-cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or at least one 5'-uridine-uridine-3' (5' -uu-3 ') dinucleotide, wherein the 5' -uridine is a 2'-modified nucleotide.
  • dinucleotide motifs are particularly prone to serum nuclease degradation (e.g. RNase A).
  • Chemical modification at the 2'-position of the first pyrimidine nucleotide in the motif prevents or slows down such cleavage.
  • This modification recipe is also known under the term 'endo light'.
  • the RNAi agent comprises a 2'-modification selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2’-0-methyl, 2’-0-methoxyethyl (2’-0-M0E), 2’- O-aminopropyl (2’-0-AP), 2’-0-dimethylaminoethyl (2’-0-DMA0E), 2’-0-dimethylaminopropyl (2’-0-DMAP), 2’-0-dimethylaminoethyloxyethyl (2’-0-DMAE0E), and 2’-0-N-methylacetamido (2’-0-NMA).
  • 2'-deoxy, 2'-deoxy-2'-fluoro 2’-0-methyl, 2’-0-methoxyethyl (2’-0-M0E), 2’- O-aminopropyl (2’-0-AP), 2’-0-dimethylaminoethyl (2’-0-DMA0
  • all pyrimidines are 2'-0-methyl-modified nucleosides.
  • one or more nucleotides can be modified, or substituted with DNA, or a nucleotide substitute such as a peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), unlocked nucleic acid (UNA).
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • TAA threose nucleic acid
  • GNA glycol nucleic acid
  • ANA arabinose nucleic acid
  • FANA 2 ' -fluoroarabinose nucleic acid
  • the sense and/or antisense strand can terminate at the 3’ end with a phosphate or modified internucleoside linker, and further comprise, in 5’ to 3’ order: a spacer, a second phosphate or modified internucleoside linker, and a 3’ end cap.
  • modified internucleoside linker is selected from phosphorothioate,
  • R 3 is selected from 0-, S-, NH 2 , BH 3 , CH 3 , Ci-e alkyl, Ce-io aryl, Ci- 6 alkoxy and C 6 -io aryl-oxy, wherein C alkyl and C 6 -io aryl are unsubstituted or optionally independently substituted with 1 to 3 groups independently selected from halo, hydroxyl and NH 2 ; and R 4 is selected from O, S, NH, and CH 2 .
  • the spacer can be a sugar, alkyl, cycloakyl, ribitol or other type of abasic nucleotide, 2’-deoxy-ribitol, diribitol, 2’- methoxyethoxy-ribitol (ribitol with 2’-MOE), C 3 -e alkyl, or 4-methoxybutane-1 ,3-diol (5300).
  • the 3’ end cap can be selected from any of various 3’ end caps described herein or known in the art.
  • one or more phosphates can be replaced by a modified internucleoside linker.
  • the RNAi agent comprises at least one blunt end.
  • the RNAi agent comprises an overhang having 1 nt to 4 nt.
  • the RNAi agent comprises an overhang at the 3'-end of the antisense strand of the RNAi agent.
  • the RNAi agent is ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.
  • the composition further comprises a second RNAi agent to a GPR68 gene product.
  • RNAi agents include: the shRNAs to a GPR68 gene product disclosed herein (particularly those having a target sequence of any of SEQ ID NOs: 5-15, and the complementary sequence thereof, or a target sequence comprising 15 contiguous nt of a GPR68 target sequence thereof). Additional RNAi agents to GPR68 can be prepared, or are known in the art.
  • RNAi agent to GPR68 may be recited to target a particular GPR68 sequence, indicating that the recited sequence may be comprised in the sequence of the sense or anti-sense strand of the RNAi agent; or, in some cases, a sequence of at least 15 contiguous nt of this sequence may be comprised in the sequence of the sense or anti-sense strand. It is also understood that some of the target sequences are presented as DNA, but the RNAi agents targeting these sequences can be RNA, or any nucleotide, modified nucleotide or substitute disclosed herein.
  • the RNAi agent to GPR68 includes any shRNA used in the experiments described herein, namely GPR68 sh1 , sh2, and sh3 (shRNAI , shRNA2 and shRNA3), whose GPR68 target sequences are presented below:
  • the RNAi agent to GPR68 includes shRNAs that are commercially available, e.g., SHCLNG-NM 003485, SHCLND-NM 003485, SHCLNV- NM 003485, SHCLNG-NMJ 75493, SHCLND-NMJ 75493, SHCLNV-NMJ 75493 (Sigma- Aldrich); sc-75186-SH, sc-75186-V (Santa Cruz); VGH5518-200225097, VGH5518-200225475, VGH5518-200226483, VGH5518-200227207, VGH5518-200228644, VGH5518-200228799, VGH5518-200229262, VGH5518-200230237, VGH5518-200260949 (Dharmacon).
  • shRNAs that are commercially available, e.g., SHCLNG-NM 003485, SHCLND-NM 003485, SHCLNV- NM 003485, SHCLNG
  • the RNAi agent to GPR68 includes siRNAs that are commercially available, e.g., NM 003485, NM_001 108049, NM_175493 (Sigma-Aldrich); sc-75186 (Santa Cruz); E-005591 - 00-0005, EQ-005591 -00-0002, A-005591 -15-0005, A-005591 -16-0005, A-005591 -17-0005, A- 005591 -18-0005, EU-005591 -00-0002, L-005591 -00-0005, LQ-005591 -00-0002, J-005591 -07- 0002, J-005591 -08-0002, J-005591 -097-0002, J-005591 -10-0002, LU-005591 -00-0002, M- 005591 -02-0005, MQ-005591 -02-0002, D-0055
  • RNAi agents of the present invention can be delivered or introduced (e.g., to a cell in vitro or to a subject) by any means known in the art.
  • iRNA "Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be "introduced into a cell," wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism.
  • iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781 which are hereby
  • RNAi agent Delivery of RNAi agent to tissue is a problem both because the material must reach the target organ and must also enter the cytoplasm of target cells. RNA cannot penetrate cellular membranes, so systemic delivery of naked RNAi agent is unlikely to be successful. RNA is quickly degraded by RNAse activity in serum. For these reasons, other mechanisms to deliver RNAi agent to target cells has been devised. Methods known in the art include but are not limited to: viral delivery (retrovirus, adenovirus, lentivirus, baculovirus, AAV); liposomes
  • RNAi agents Lipofectamine, cationic DOTAP, neutral DOPC
  • nanoparticles cationic polymer, PEI
  • tkRNAi bacterial delivery
  • LNA chemical modification
  • Liposomes have been used previously for drug delivery (e.g., delivery of a chemotherapeutic).
  • Liposomes e.g., cationic liposomes
  • a process of making liposomes is also described in W004/002453AI.
  • neutral lipids have been incorporated into cationic liposomes (e.g., Farhood et al. 1995).
  • Cationic liposomes have been used to deliver RNAi agent to various cell types (Sioud and Sorensen 2003; U.S. Patent Application
  • SNALP refers to a stable nucleic acid-lipid particle.
  • a SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed.
  • SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in
  • liver cells can be efficiently transfected by injection of the siRNA into a mammal's circulatory system.
  • RNAi agent delivery A variety of molecules have been used for cell-specific RNAi agent delivery.
  • the nucleic acid-condensing property of protamine has been combined with specific antibodies to deliver siRNAs (Song et al. 2005 Nat Biotch. 23: 709-717).
  • the self-assembly PEGylated polycation polyethylenimine has also been used to condense and protect siRNAs (Schiffelers et al. 2004 Nucl. Acids Res. 32: 49, 141 -1 10).
  • siRNA-containing nanoparticles were then successfully delivered to integrin overexpressing tumor neovasculature (Hu-Lieskovan et al. 2005 Cancer Res. 65: 8984-8992).
  • RNAi agents of the present invention can be delivered via, for example, Lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles; double-stranded RNA binding motifs (dsRBMs); or via modification of the RNAi agent (e.g., covalent attachment to the dsRNA).
  • LNP Lipid nanoparticles
  • NL neutral liposomes
  • dsRBMs double-stranded RNA binding motifs
  • modification of the RNAi agent e.g., covalent attachment to the dsRNA
  • Lipid nanoparticles are self-assembling cationic lipid based systems. These can comprise, for example, a neutral lipid (the liposome base); a cationic lipid (for siRNA loading); cholesterol (for stabilizing the liposomes); and PEG-lipid (for stabilizing the formulation, charge shielding and extended circulation in the bloodstream).
  • the cationic lipid can comprise, for example, a headgroup, a linker, a tail and a cholesterol tail.
  • the LNP can have, for example, good tumor delivery, extended circulation in the blood, small particles (e.g., less than 100 nm), and stability in the tumor microenvironment (which has low pH and is hypoxic).
  • Neutral liposomes are non-cationic lipid based particles.
  • Polymer nanoparticles are self-assembling polymer-based particles.
  • Double-stranded RNA binding motifs are self-assembling RNA binding proteins, which will need modifications.
  • GPR68 Aptamers are self-assembling RNA binding proteins, which will need modifications.
  • the modulator of a GPR68 gene product may be an aptamer, including for example a protein aptamer or a polynucleotidal aptamer.
  • the aptamer inhibits or enhances the expression, activity, or both of a GPR68 gene product.
  • an apatmer is a nucleic acid or oligonucleotide molecule that binds to a specific molecular target, such as a GPR68 gene product comprising a sequence set forth in SEQ ID NOs: 1 -15.
  • aptamers are obtained from an in vitro evolutionary process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which selects target-specific aptamer sequences from combinatorial libraries of single stranded oligonucleotide templates comprising randomized sequences.
  • aptamer compositions are double-stranded or single-stranded, and in various embodiments include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer include modified or non-natural nucleotides, for example nucleotides that have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide is replaced by 2'-F or 2'-NH2), which in some instances, improves a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • individual aptamers having the same nucleotide sequence differ in their secondary structure.
  • the aptamers of the invention are conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • aptamers are specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • a method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules is known to those of skill in the art and is described in U.S. Pat. No. 5,270,163.
  • the method known as SELEX (Selective Evolution of Ligands by Exponential Enrichment) involves selection from a mixture of candidate oligonucleotides from a library comprising a large sequence variations (e.g. about 1015) and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX method includes the steps of contacting the mixture with the desired target, partitioning unbound nucleic acids from those nucleic acids which have bound to the target molecule, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
  • CRISPR or“CRISPR to GPR68” or“CRISPR to modulate GPR68” and the like is meant a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats.
  • Cas as used herein, is meant a CRISPR-associated protein.
  • CRISPR/Cas system is meant a system derived from CRISPR and Cas which can be used to silence, enhance or mutate the GPR68 gene.
  • CRISPR/Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea (Grissa et al. 2007. BMC Bioinformatics 8: 172). This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity (Barrangou et al. 2007. Science 315: 1709-1712; Marragini et al. 2008 Science 322: 1843-1845).
  • the CRISPR/Cas system has been modified for use in gene editing (silencing, enhancing or changing specific genes) in eukaryotes such as mice or primates (Wiedenheft et al. 2012. Nature 482: 331 -8). This is accomplished by introducing into the eukaryotic cell a plasmid containing a specifically designed CRISPR and one or more appropriate Cas.
  • the CRISPR sequence sometimes called a CRISPR locus, comprises alternating repeats and spacers.
  • the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in the GPR68 CRISPR/Cas system, the spacers are derived from the GPR68 gene sequence.
  • the repeats generally show some dyad symmetry, and may form a secondary structure such as a hairpin, and may or may not be palindromic.
  • RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These processed RNAs comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level (Horvath et al. 2010. Science 327: 167-170; Makarova et al. 2006 Biology Direct 1 : 7). The spacers thus serve as templates for RNA molecules, analogously to siRNAs (Pennisi 2013. Science 341 : 833-836).
  • CasA proteins form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains (Brouns et al. 2008. Science 321 : 960-964).
  • Cas6 processes the CRISPR transcript.
  • the CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2.
  • the Cmr (Cas RAMP module) proteins in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs.
  • a simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing (Pennisi 2013. Science 341 : 833-836).
  • the CRISPR/Cas system can thus be used to edit the GPR68 gene (adding or deleting a basepair), e.g., repairing a damaged GPR68 gene (e.g., if the damage to GPR68 results in high or low post-translational modification, production, expression, level, stability or activity of GPR68), or introducing a premature stop which thus decreases expression of an over-expressed GPR68.
  • the CRISPR/Cas system can alternatively be used like RNA interference, turning off the GPR68 gene in a reversible fashion.
  • the RNA can guide the Cas protein to the GPR68 promoter, sterically blocking RNA polymerases.
  • GPR68-inhibitory CRISPR system can include a guide RNA (gRNA) comprising a GPR68-targeting domain, i.e., a nucleotide sequence that is
  • RNA- directed nuclease e.g., cpf 1 or Cas molecule, e.g., Cas9 molecule.
  • RNA-directed nuclease e.g., cpf 1 or Cas molecule, e.g., Cas9 molecule
  • PAM Protospacer Adjacent Motif
  • a PAM sequence is a sequence in the target nucleic acid.
  • cleavage of the target nucleic acid occurs upstream from the PAM sequence.
  • RNA-directed nuclease molecules, e.g., cpf 1 or Cas molecules, e.g., Cas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences).
  • RNA-directed nucleases e.g., cpf 1 or Cas molecules, e.g., Cas9 molecules
  • target sequences e.g., target sequences adjacent, e.g., immediately upstream, to the PAM sequence
  • gRNA molecules comprising targeting domains capable of hybridizing to said target sequences and a tracr sequence that binds to said RNA-directed nuclease, e.g., cpf1 or Cas molecule, e.g., Cas9 molecule.
  • the CRISPR system comprises a gRNA molecule and a Cas9 molecule from S. pyogenes.
  • a Cas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence.
  • a gRNA molecule useful with S. pyogenes-based CRISPR systems may include a GPR68-targeting sequence described in Table 1 , e.g., any of SEQ ID NOs: 21 -678, and a tracr sequence known to interact with S. Pyogenes (see, e.g., Mali el ai, SCIENCE 2013; 339(6121 ): 823- 826).
  • the CRISPR system comprises a gRNA molecule and a Cas9 molecule from S. thermophilus.
  • a gRNA molecule useful with S. thermophilus-based CRISPR systems may include a GPR68- targeting sequence, and a tracr sequence known to interact with S.
  • thermophilus see, e.g., Horvath et al., SCIENCE 2010; 327(5962): 167- 170, and Deveau et al., J BACTERIOL 2008; 190(4): 1390- 1400).
  • the CRISPR system comprises a gRNA molecule and a Cas9 molecule from S. aureus.
  • a gRNA molecule useful with S. aureus-based CRISPR systems may include a GPR68-targeting sequence, and a tracr sequence known to interact with S. aureus (see, e.g., Ran F. et al., NATURE, vol. 520, 2015, pp. 186-191 ).
  • the CRISPR system comprises a gRNA molecule and a RNA-directed nuclease, e.g., cpf 1 molecule, e.g., a cpf 1 molecule from Lachnospiraceae bacterium or a cpf 1 molecule from Acidaminococcus sp.
  • a RNA-directed nuclease e.g., cpf 1 molecule, e.g., a cpf 1 molecule from Lachnospiraceae bacterium or a cpf 1 molecule from Acidaminococcus sp.
  • a gRNA molecule useful with cpf 1 -based CRISPR systems may include a GPR68-targeting sequence, and a tracr sequence which interacts with cpf 1 (see, e.g., Zetsche B. et al., CELL, vol. 163:3, Oct. 2015, 759-771 ).

Abstract

La présente invention concerne de nouvelles compositions, des procédés et des utilisations thérapeutiques associés à des modulateurs d'un produit génique GPR68. Certains modes de réalisation de la présente invention concernent des procédés de modulation d'une réponse de dilatation médiée par flux (FMD) ou d'un remodelage extérieur médié par flux (FMR) d'artères de petit diamètre, ou de réduction de la résistance vasculaire systémique (SVR) chez un sujet en ayant besoin, comprenant l'administration d'un modulateur d'un produit génique GPR68 au sujet. Certains modes de réalisation de la présente invention concernent des procédés de traitement d'une maladie cardiovasculaire chez un sujet en ayant besoin, comprenant l'administration d'une composition pharmaceutique comprenant un modulateur d'un produit génique GPR68 au sujet.
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CN113373170A (zh) * 2021-04-29 2021-09-10 江西农业大学 一种pFnCpfAb/pCrAb双质粒***及其应用
WO2023285878A1 (fr) 2021-07-13 2023-01-19 Aviation-Ophthalmology Procédés de détection, de traitement et de prévention de maladies, troubles et états oculaires induits par gpr68

Citations (92)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0171496A2 (fr) 1984-08-15 1986-02-19 Research Development Corporation of Japan Procédé pour la production d'un anticorps monoclonal chimérique
EP0173494A2 (fr) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Récepteurs chimériques par liaison et expression de l'ADN
WO1986001533A1 (fr) 1984-09-03 1986-03-13 Celltech Limited Production d'anticorps chimeriques
EP0184187A2 (fr) 1984-12-04 1986-06-11 Teijin Limited Chaîne lourde d'immunoglobuline chimère souris-humaine et chimère de l'ADN codant celle-ci
WO1986005803A1 (fr) 1985-03-30 1986-10-09 Marc Ballivet Procede d'obtention d'adn, arn, peptides, polypeptides ou proteines, par une technique de recombinaison d'adn
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
WO1988006630A1 (fr) 1987-03-02 1988-09-07 Genex Corporation Procede de preparation de molecules de liaison
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1989006283A1 (fr) 1988-01-11 1989-07-13 Ingene (International Genetic Engineering, Inc.) Nouveau vecteur de plasmide avec sequence a fonction de signal pour pectate lyase
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
WO1990004036A1 (fr) 1988-10-12 1990-04-19 Medical Research Council Production d'anticorps a partir d'animaux transgeniques
EP0368684A1 (fr) 1988-11-11 1990-05-16 Medical Research Council Clonage de séquences d'immunoglobulines de domaines variables.
EP0371998A1 (fr) 1987-07-24 1990-06-13 Int Genetic Eng Ensemble modulaire de genes d'anticorps, anticorps ainsi prepares et utilisation.
US4939666A (en) 1987-09-02 1990-07-03 Genex Corporation Incremental macromolecule construction methods
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1990014430A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Nouveau procede d'exploitation du repertoire immunologique
WO1990014443A1 (fr) 1989-05-16 1990-11-29 Huse William D Coexpression de recepteurs heteromeres
WO1990014424A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Procede d'isolement de recepteurs presentant une specificite preselectionnee
US5030453A (en) 1983-03-24 1991-07-09 The Liposome Company, Inc. Stable plurilamellar vesicles
US5032401A (en) 1989-06-15 1991-07-16 Alpha Beta Technology Glucan drug delivery system and adjuvant
WO1991010741A1 (fr) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation d'anticorps xenogeniques
WO1991017271A1 (fr) 1990-05-01 1991-11-14 Affymax Technologies N.V. Procedes de triage de banques d'adn recombine
WO1991018980A1 (fr) 1990-06-01 1991-12-12 Cetus Corporation Compositions et procedes d'identification de molecules biologiquement actives
WO1991019818A1 (fr) 1990-06-20 1991-12-26 Affymax Technologies N.V. Banque de peptides et systemes de triage
WO1992005258A1 (fr) 1990-09-20 1992-04-02 La Trobe University Gene encodant une enzyme de l'orge
WO1992014843A1 (fr) 1991-02-21 1992-09-03 Gilead Sciences, Inc. Aptamere specifique de biomolecules et procede de production
WO1992016221A1 (fr) 1991-03-15 1992-10-01 Synergen, Inc. Pegylation de polypeptides
WO1992018619A1 (fr) 1991-04-10 1992-10-29 The Scripps Research Institute Banques de recepteurs heterodimeres utilisant des phagemides
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
WO1993008278A1 (fr) 1991-10-16 1993-04-29 Affymax Technologies N.V. Banque de peptides et procede de depistage
WO1993012227A1 (fr) 1991-12-17 1993-06-24 Genpharm International, Inc. Animaux transgeniques non humains capables de produire des anticorps heterologues
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
GB2272440A (en) 1990-08-29 1994-05-18 Genpharm Int Transgenic non-human animals capable of producing heterologous antibodies
WO1995016027A1 (fr) 1993-12-06 1995-06-15 Bioinvent International Ab Procede de selection de bacteriophages specifiques
WO1996007754A1 (fr) 1994-09-02 1996-03-14 The Scripps Research Institute Procedes de production de banques d'anticorps au moyen de chaines legeres d'immunoglobuline universelles ou rendues alleatoires
WO1996013583A2 (fr) 1994-10-20 1996-05-09 Morphosys Gesellschaft Für Proteinoptimierung Mbh Hetero-association ciblee de proteines recombinees et de complexes fonctionnels
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
WO1996019256A1 (fr) 1994-12-22 1996-06-27 Advanced Cardiovascular Systems, Inc. Catheter a ballon a longueur variable
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5565362A (en) 1987-02-17 1996-10-15 Pharming B.V. DNA sequences to target proteins to the mammary gland for efficient secretion
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5595898A (en) 1985-11-01 1997-01-21 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
WO1997008320A1 (fr) 1995-08-18 1997-03-06 Morphosys Gesellschaft Für Proteinoptimierung Mbh Banques de proteines/(poly)peptides
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5627052A (en) 1990-08-02 1997-05-06 B.R. Centre, Ltd. Methods for the production of proteins with a desired function
US5643768A (en) 1989-10-05 1997-07-01 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
US5656730A (en) 1995-04-07 1997-08-12 Enzon, Inc. Stabilized monomeric protein compositions
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
EP0814259A1 (fr) 1996-06-19 1997-12-29 Motorenfabrik Hatz GmbH & Co. KG Dispositif de démarrage à froid
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US5763733A (en) 1994-10-13 1998-06-09 Enzon, Inc. Antigen-binding fusion proteins
US5766886A (en) 1991-12-13 1998-06-16 Xoma Corporation Modified antibody variable domains
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US5856456A (en) 1992-11-20 1999-01-05 Enzon, Inc. Linker for linked fusion polypeptides
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5886793A (en) 1995-04-04 1999-03-23 Oki Data Corporation Facsimile machine adapted to reduce risk of data loss
US5955341A (en) 1991-04-10 1999-09-21 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
US5962016A (en) 1997-01-31 1999-10-05 Depotech Corporation Multivesicular liposomes utilizing neutral lipids to modify in vivo release
US5969108A (en) 1990-07-10 1999-10-19 Medical Research Council Methods for producing members of specific binding pairs
US6060248A (en) 1997-02-24 2000-05-09 Tm Technologies, Inc. Process for selecting anti-sense oligonucleotides
US6162963A (en) 1990-01-12 2000-12-19 Abgenix, Inc. Generation of Xenogenetic antibodies
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
US6291160B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
US6291159B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
WO2002100435A1 (fr) 2001-06-11 2002-12-19 Centre Hospitalier Universitaire De Montreal Compositions et methodes permettant de favoriser le transfert d'acide nucleique dans des cellules
US20030012812A1 (en) 1996-10-04 2003-01-16 Mar Tormo Inhibition of bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
WO2003015757A1 (fr) 2001-08-16 2003-02-27 The Trustees Of The University Of Pennsylvania Synthese et utilisation de reactifs pour ameliorer la lipofection d'adn et/ou les therapies par medicaments et promedicaments a liberation lente
WO2004002453A1 (fr) 2002-06-28 2004-01-08 Protiva Biotherapeutics Ltd. Appareil liposomal et procedes de fabrication
US6680068B2 (en) 2000-07-06 2004-01-20 The General Hospital Corporation Drug delivery formulations and targeting
WO2004029213A2 (fr) 2002-09-28 2004-04-08 Massachusetts Institute Of Technology Compositions et methodes de transmission d'arn interferant court et d'arn interferant court en forme d'epingle a cheveux
US20040208921A1 (en) 2003-01-14 2004-10-21 Ho Rodney J. Y. Lipid-drug formulations and methods for targeted delivery of lipid-drug complexes to lymphoid tissues
US20050281781A1 (en) 2004-06-16 2005-12-22 Ostroff Gary R Drug delivery product and methods
US20060240093A1 (en) 2003-07-16 2006-10-26 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
US20070135372A1 (en) 2005-11-02 2007-06-14 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
WO2009082817A1 (fr) 2007-12-27 2009-07-09 Protiva Biotherapeutics, Inc. Silençage de l'expression de la polo-like kinase à l'aide d'un arn interférent
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
EP2765197A1 (fr) * 2013-02-11 2014-08-13 ETH Zurich Capteur de pH de concepteur en tant que commande transgène universelle
US9102989B2 (en) 2002-05-23 2015-08-11 Cognis Ip Management Gmbh Non-revertible β-oxidation blocked Candida tropicalis
US9107149B2 (en) 2010-04-07 2015-08-11 Lenovo (Singapore) Pte Ltd Wireless portable computer capable of autonomously adjusting load of wireless base station
US9401234B2 (en) 2013-03-22 2016-07-26 Polytronics Technology Corp. Over-current protection device
US20160340674A1 (en) * 2014-02-10 2016-11-24 University Of Rochester Compositions and Methods to Inhibit EZH2 for the Treatment of Cardiovascular Diseases

Patent Citations (126)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5030453A (en) 1983-03-24 1991-07-09 The Liposome Company, Inc. Stable plurilamellar vesicles
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0171496A2 (fr) 1984-08-15 1986-02-19 Research Development Corporation of Japan Procédé pour la production d'un anticorps monoclonal chimérique
EP0173494A2 (fr) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Récepteurs chimériques par liaison et expression de l'ADN
WO1986001533A1 (fr) 1984-09-03 1986-03-13 Celltech Limited Production d'anticorps chimeriques
EP0184187A2 (fr) 1984-12-04 1986-06-11 Teijin Limited Chaîne lourde d'immunoglobuline chimère souris-humaine et chimère de l'ADN codant celle-ci
US5817483A (en) 1985-03-30 1998-10-06 Stuart Kauffman Process for the production of stochastically-generated peptides,polypeptides or proteins having a predetermined property
US5814476A (en) 1985-03-30 1998-09-29 Stuart Kauffman Process for the production of stochastically-generated transcription or translation products
WO1986005803A1 (fr) 1985-03-30 1986-10-09 Marc Ballivet Procede d'obtention d'adn, arn, peptides, polypeptides ou proteines, par une technique de recombinaison d'adn
US5723323A (en) 1985-03-30 1998-03-03 Kauffman; Stuart Alan Method of identifying a stochastically-generated peptide, polypeptide, or protein having ligand binding property and compositions thereof
US5976862A (en) 1985-03-30 1999-11-02 Ixsys Corporation Process for obtaining DNA, RNA, peptides, polypeptides, or proteins, by recombinant DNA technique
US5824514A (en) 1985-03-30 1998-10-20 Stuart A. Kauffman Process for the production of expression vectors comprising at least one stochastic sequence of polynucleotides
EP0590689A2 (fr) 1985-03-30 1994-04-06 BALLIVET, Marc Procédé d'obtention d'ADN, ARN, peptides, polypeptides ou protéines, par une technique de recombinaison d'ADN
EP0229046A1 (fr) 1985-03-30 1987-07-22 Marc Ballivet Procede d'obtention d'adn, arn, peptides, polypeptides ou proteines, par une technique de recombinaison d'adn.
US5618920A (en) 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5698417A (en) 1985-11-01 1997-12-16 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US6204023B1 (en) 1985-11-01 2001-03-20 Xoma Ltd. Modular assembly of antibody genes, antibodies prepared thereby and use
US5698435A (en) 1985-11-01 1997-12-16 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5595898A (en) 1985-11-01 1997-01-21 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5693493A (en) 1985-11-01 1997-12-02 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US5455030A (en) 1986-09-02 1995-10-03 Enzon Labs, Inc. Immunotheraphy using single chain polypeptide binding molecules
US5518889A (en) 1986-09-02 1996-05-21 Enzon Labs Inc. Immunoassay methods using single polypeptide chain binding molecules
US5534621A (en) 1986-09-02 1996-07-09 Enzon Labs, Inc. Immunoaffinity purification methods using single polypeptide chain binding molecules
US5763192A (en) 1986-11-20 1998-06-09 Ixsys, Incorporated Process for obtaining DNA, RNA, peptides, polypeptides, or protein, by recombinant DNA technique
US5994616A (en) 1987-02-17 1999-11-30 Pharming B.V. Targeted synthesis of protein in mammary gland of a non-human transgenic mammal
US5565362A (en) 1987-02-17 1996-10-15 Pharming B.V. DNA sequences to target proteins to the mammary gland for efficient secretion
WO1988006630A1 (fr) 1987-03-02 1988-09-07 Genex Corporation Procede de preparation de molecules de liaison
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
EP0371998A1 (fr) 1987-07-24 1990-06-13 Int Genetic Eng Ensemble modulaire de genes d'anticorps, anticorps ainsi prepares et utilisation.
EP0550400A2 (fr) 1987-07-24 1993-07-07 Xoma Corporation Ensemble modulaire de gènes d'anticorps, anticorps ainsi préparés et leurs utilisations
US4939666A (en) 1987-09-02 1990-07-03 Genex Corporation Incremental macromolecule construction methods
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1989006283A1 (fr) 1988-01-11 1989-07-13 Ingene (International Genetic Engineering, Inc.) Nouveau vecteur de plasmide avec sequence a fonction de signal pour pectate lyase
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5837500A (en) 1988-09-02 1998-11-17 Dyax, Corp. Directed evolution of novel binding proteins
US5403484A (en) 1988-09-02 1995-04-04 Protein Engineering Corporation Viruses expressing chimeric binding proteins
US5571698A (en) 1988-09-02 1996-11-05 Protein Engineering Corporation Directed evolution of novel binding proteins
EP0438474A1 (fr) 1988-10-12 1991-07-31 Medical Res Council Production d'anticorps a partir d'animaux transgeniques.
WO1990004036A1 (fr) 1988-10-12 1990-04-19 Medical Research Council Production d'anticorps a partir d'animaux transgeniques
EP0368684A1 (fr) 1988-11-11 1990-05-16 Medical Research Council Clonage de séquences d'immunoglobulines de domaines variables.
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US6291160B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
WO1990014430A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Nouveau procede d'exploitation du repertoire immunologique
WO1990014424A1 (fr) 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Procede d'isolement de recepteurs presentant une specificite preselectionnee
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
WO1990014443A1 (fr) 1989-05-16 1990-11-29 Huse William D Coexpression de recepteurs heteromeres
US6291159B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
US5032401A (en) 1989-06-15 1991-07-16 Alpha Beta Technology Glucan drug delivery system and adjuvant
US5607677A (en) 1989-06-15 1997-03-04 Alpha-Beta Technology, Inc. Glucan drug delivery system and adjuvant
US5658754A (en) 1989-10-05 1997-08-19 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
US5643768A (en) 1989-10-05 1997-07-01 Optein, Inc. Cell-free synthesis and isolation of novel genes and polypeptides
WO1991010741A1 (fr) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation d'anticorps xenogeniques
US6162963A (en) 1990-01-12 2000-12-19 Abgenix, Inc. Generation of Xenogenetic antibodies
WO1991017271A1 (fr) 1990-05-01 1991-11-14 Affymax Technologies N.V. Procedes de triage de banques d'adn recombine
US5580717A (en) 1990-05-01 1996-12-03 Affymax Technologies N.V. Recombinant library screening methods
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
WO1991018980A1 (fr) 1990-06-01 1991-12-12 Cetus Corporation Compositions et procedes d'identification de molecules biologiquement actives
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
WO1991019818A1 (fr) 1990-06-20 1991-12-26 Affymax Technologies N.V. Banque de peptides et systemes de triage
US5969108A (en) 1990-07-10 1999-10-19 Medical Research Council Methods for producing members of specific binding pairs
US5627052A (en) 1990-08-02 1997-05-06 B.R. Centre, Ltd. Methods for the production of proteins with a desired function
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
GB2272440A (en) 1990-08-29 1994-05-18 Genpharm Int Transgenic non-human animals capable of producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
WO1992005258A1 (fr) 1990-09-20 1992-04-02 La Trobe University Gene encodant une enzyme de l'orge
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
WO1992014843A1 (fr) 1991-02-21 1992-09-03 Gilead Sciences, Inc. Aptamere specifique de biomolecules et procede de production
WO1992016221A1 (fr) 1991-03-15 1992-10-01 Synergen, Inc. Pegylation de polypeptides
US6235469B1 (en) 1991-04-10 2001-05-22 The Scripps Research Institute Hetrodimeric receptor libraries using phagemids
US5955341A (en) 1991-04-10 1999-09-21 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
US5658727A (en) 1991-04-10 1997-08-19 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
US5759817A (en) 1991-04-10 1998-06-02 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
WO1992018619A1 (fr) 1991-04-10 1992-10-29 The Scripps Research Institute Banques de recepteurs heterodimeres utilisant des phagemides
WO1993008278A1 (fr) 1991-10-16 1993-04-29 Affymax Technologies N.V. Banque de peptides et procede de depistage
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5766886A (en) 1991-12-13 1998-06-16 Xoma Corporation Modified antibody variable domains
WO1993012227A1 (fr) 1991-12-17 1993-06-24 Genpharm International, Inc. Animaux transgeniques non humains capables de produire des anticorps heterologues
US5856456A (en) 1992-11-20 1999-01-05 Enzon, Inc. Linker for linked fusion polypeptides
WO1995016027A1 (fr) 1993-12-06 1995-06-15 Bioinvent International Ab Procede de selection de bacteriophages specifiques
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US5849992A (en) 1993-12-20 1998-12-15 Genzyme Transgenics Corporation Transgenic production of antibodies in milk
WO1996007754A1 (fr) 1994-09-02 1996-03-14 The Scripps Research Institute Procedes de production de banques d'anticorps au moyen de chaines legeres d'immunoglobuline universelles ou rendues alleatoires
US5767260A (en) 1994-10-13 1998-06-16 Enzon Inc. Antigen-binding fusion proteins
US5763733A (en) 1994-10-13 1998-06-09 Enzon, Inc. Antigen-binding fusion proteins
WO1996013583A2 (fr) 1994-10-20 1996-05-09 Morphosys Gesellschaft Für Proteinoptimierung Mbh Hetero-association ciblee de proteines recombinees et de complexes fonctionnels
WO1996019256A1 (fr) 1994-12-22 1996-06-27 Advanced Cardiovascular Systems, Inc. Catheter a ballon a longueur variable
US5886793A (en) 1995-04-04 1999-03-23 Oki Data Corporation Facsimile machine adapted to reduce risk of data loss
US5656730A (en) 1995-04-07 1997-08-12 Enzon, Inc. Stabilized monomeric protein compositions
WO1997008320A1 (fr) 1995-08-18 1997-03-06 Morphosys Gesellschaft Für Proteinoptimierung Mbh Banques de proteines/(poly)peptides
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
EP0814259A1 (fr) 1996-06-19 1997-12-29 Motorenfabrik Hatz GmbH & Co. KG Dispositif de démarrage à froid
US20030012812A1 (en) 1996-10-04 2003-01-16 Mar Tormo Inhibition of bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US5962016A (en) 1997-01-31 1999-10-05 Depotech Corporation Multivesicular liposomes utilizing neutral lipids to modify in vivo release
US6060248A (en) 1997-02-24 2000-05-09 Tm Technologies, Inc. Process for selecting anti-sense oligonucleotides
US6680068B2 (en) 2000-07-06 2004-01-20 The General Hospital Corporation Drug delivery formulations and targeting
WO2002100435A1 (fr) 2001-06-11 2002-12-19 Centre Hospitalier Universitaire De Montreal Compositions et methodes permettant de favoriser le transfert d'acide nucleique dans des cellules
WO2003015757A1 (fr) 2001-08-16 2003-02-27 The Trustees Of The University Of Pennsylvania Synthese et utilisation de reactifs pour ameliorer la lipofection d'adn et/ou les therapies par medicaments et promedicaments a liberation lente
US9102989B2 (en) 2002-05-23 2015-08-11 Cognis Ip Management Gmbh Non-revertible β-oxidation blocked Candida tropicalis
WO2004002453A1 (fr) 2002-06-28 2004-01-08 Protiva Biotherapeutics Ltd. Appareil liposomal et procedes de fabrication
WO2004029213A2 (fr) 2002-09-28 2004-04-08 Massachusetts Institute Of Technology Compositions et methodes de transmission d'arn interferant court et d'arn interferant court en forme d'epingle a cheveux
US20040208921A1 (en) 2003-01-14 2004-10-21 Ho Rodney J. Y. Lipid-drug formulations and methods for targeted delivery of lipid-drug complexes to lymphoid tissues
US20060240093A1 (en) 2003-07-16 2006-10-26 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
US20050281781A1 (en) 2004-06-16 2005-12-22 Ostroff Gary R Drug delivery product and methods
US20070135372A1 (en) 2005-11-02 2007-06-14 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
WO2009082817A1 (fr) 2007-12-27 2009-07-09 Protiva Biotherapeutics, Inc. Silençage de l'expression de la polo-like kinase à l'aide d'un arn interférent
US9107149B2 (en) 2010-04-07 2015-08-11 Lenovo (Singapore) Pte Ltd Wireless portable computer capable of autonomously adjusting load of wireless base station
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
EP2765197A1 (fr) * 2013-02-11 2014-08-13 ETH Zurich Capteur de pH de concepteur en tant que commande transgène universelle
US9401234B2 (en) 2013-03-22 2016-07-26 Polytronics Technology Corp. Over-current protection device
US20160340674A1 (en) * 2014-02-10 2016-11-24 University Of Rochester Compositions and Methods to Inhibit EZH2 for the Treatment of Cardiovascular Diseases

Non-Patent Citations (242)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Immunology", 1994, JOHN WILEY & SONS, INC.
"Current Protocols in Protein Science", 1997, JOHN WILEY & SONS
"GENBANK", Database accession no. NG 052988.1
"GENBANK", Database accession no. NM 001108049.1
"GENBANK", Database accession no. NM 001177673.1
"GENBANK", Database accession no. NM 001177674.1
"GENBANK", Database accession no. NM 001177676.1
"GENBANK", Database accession no. NM 001348437.1
"GENBANK", Database accession no. NM 003485.3
"GENBANK", Database accession no. NM_175493.4
"GENBANK", Database accession no. NP 001101519.1
"GENBANK", Database accession no. NP 001171147.1
"GENBANK", Database accession no. NP 780702.1
"GENBANK", Database accession no. XM 005562025.2
"GENBANK", Database accession no. XM 005562026.2
"GENBANK", Database accession no. XM 005562028.2
"GENBANK", Database accession no. XM 015454130.1
"GENBANK", Database accession no. XP 005562082.1
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT WILLIAMS & WILKINS
"Sustained and Controlled Release Drug Delivery Systems", 1978, MARCEL DEKKER, INC.
AL-LAZIKANI ET AL., J.MOL.BIOL., vol. 273, 1997, pages 927 - 748
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUC. ACIDS RES., vol. 25, 1977, pages 3389 - 3402
AOKI, H ET AL: "Proton-sensing ovarian cancer G protein-coupled receptor 1 on dendritic cells is required for airway responses in a murine asthma model", PLOS ONE, vol. 8, 2013, pages e79985
AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1995
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1987, JOHN WILEY & SONS, INC.
BABCOOK ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 7843 - 7848
BARRANGOU ET AL., SCIENCE, vol. 315, 2007, pages 1709 - 1712
BATZER ET AL., NUCLEIC ACID RES., vol. 19, 1991, pages 5081
BEBBINGTON ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 169 - 175
BERZOFSKY ET AL.: "Fundamental Immunology", 1984, RAVEN PRESS, article "Antibody-Antigen Interactions"
BITINAITE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 10570 - 5
BOCH ET AL., SCIENCE, vol. 326, 2009, pages 1509 - 12
BOCH, NATURE BIOTECH., vol. 29, 2011, pages 135 - 6
BOLOTIN ET AL., MICROBIOL., vol. 151, 2005, pages 2551 - 2561
BORLAUG B A; REDFIELD M M, CIRCULATION, vol. 123, no. 18, 10 May 2011 (2011-05-10), pages 2006 - 13
BOSHART ET AL., CELL, vol. 41, 1985, pages 521 - 530
BOWIE ET AL., SCIENCE, vol. 247, 1990, pages 306 - 1310
BRODY, E. N.; L. GOLD, J. BIOTECHNOL., vol. 74, 2000, pages 5 - 13
BROUNS ET AL., SCIENCE, vol. 321, 2008, pages 960 - 964
BURNS, M.E.; PUGH, E.N., JR.: "Lessons from photoreceptors: turning off g-protein signaling in living cells", PHYSIOLOGY, vol. 25, 2010, pages 72 - 84, XP055266715
CAILLON, A ET AL: "The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production", CARDIOVASCULAR RESEARCH, vol. 112, 2016, pages 515 - 525
CAPELLAS ET AL., BIOTECHNOL. BIOENG., vol. 56, no. 4, 1997, pages 456 - 463
CAPLEN ET AL., PROC. NATL. ACAD. SCI. (USA, vol. 98, 2001, pages 9742 - 9747
CARROLL ET AL., GENETICS SOCIETY OF AMERICA, vol. 188, 2011, pages 773 - 782
CARTER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 89, 1992, pages 4285
CATHOMEN ET AL., MOL. THER., vol. 16, pages 1200 - 7
CERMAK ET AL., NUCL. ACIDS RES., vol. 39, 2011, pages e82
CHACHISVILIS, M.; ZHANG, Y.L.; FRANGOS, J.A.: "G protein-coupled receptors sense fluid shear stress in endothelial cells", PNAS, vol. 103, 2006, pages 15463 - 15468
CHEN, P ET AL: "Gpr132 sensing of lactate mediates tumor-macrophage interplay to promote breast cancer metastasis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 114, 2017, pages 580 - 585
CHERYL DE VALLIÈRE ET AL: "The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment", AMERICAN JOURNAL OF PHYSIOLOGY - GASTROINTESTINAL AND LIVER PHYSIOLOGY, vol. 309, no. 6, 15 September 2015 (2015-09-15), US, pages G475 - G490, XP055584461, ISSN: 0193-1857, DOI: 10.1152/ajpgi.00408.2014 *
CHIU, J.J.; CHIEN, S.: "Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives", PHYSIOLOGICAL REVIEWS, vol. 91, 2011, pages 327 - 387
CHOTHIA ET AL., J. MOL. BIOL., vol. 227, 1992, pages 799 - 817
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 877 - 883
CHOTHIA; LESK, J. MOL. BIOL., vol. 196, 1987, pages 901
CHOTHIA; LESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHUANG, H.; JAN, Y.N.; JAN, L.Y.: "Regulation of IRK3 inward rectifier K+ channel by m1 acetylcholine receptor and intracellular magnesium", CELL, vol. 89, 1997, pages 1121 - 1132
CHUANG, H.H.; NEUHAUSSER, W.M.; JULIUS, D.: "The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel", NEURON, vol. 43, 2004, pages 859 - 869
COBURN, G.; CULLEN, B., J. OF VIROLOGY, vol. 76, no. 18, 2002, pages 9225
COLLIGAN ET AL.: "Current Protocols in Protein Science", 1997, JOHN WILEY & SONS
COLLIGAN, CURRENT PROTOCOLS IN IMMUNOLOGY
CONRAD ET AL., PLANT MOL. BIOL., vol. 38, 1998, pages 101 - 109
COSTE, B ET AL: "Piezol and Piezo2 are essential components of distinct mechanically activated cation channels", SCIENCE, vol. 330, 2010, pages 55 - 60, XP055102456
CRAMER ET AL., CURR. TOP. MICROBOL. IMMUNOL., vol. 240, 1999, pages 95 - 118
CREIGHTON, PROTEINS, 1984
CULLEN ET AL., MOLEC. CELL. BIOL., vol. 5, 1985, pages 438 - 447
DAVIES, P.F.: "Flow-mediated endothelial mechanotransduction", PHYSIOLOGICAL REVIEWS, vol. 75, 1995, pages 519 - 560
DEANFIELD, J.E.; HALCOX, J.P.; RABELINK, T.J.: "Endothelial function and dysfunction: testing and clinical relevance", CIRCULATION, vol. 115, 2007, pages 1285 - 1295
DEDMAN, A ET AL: "The mechano-gated K(2P) channel TREK-1", EUROPEAN BIOPHYSICS JOURNAL : EBJ, vol. 38, 2009, pages 293 - 303, XP019705280
DELMAS, P.; HAO, J.; RODAT-DESPOIX, L.: "Molecular mechanisms of mechanotransduction in mammalian sensory neurons", NATURE REVIEWS NEUROSCIENCE, vol. 12, 2011, pages 139 - 153
DEVEAU ET AL., J BACTERIOL, vol. 190, no. 4, 2008, pages 1390 - 1400
DEVROE ET AL., BMC BIOTECHNOL., vol. 21, 2002, pages 15
DOYON ET AL., NATURE METHODS, vol. 8, 2010, pages 74 - 79
DRISCOLL, M.; CHALFIE, M.: "The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration", NATURE, vol. 349, 1991, pages 588 - 593
D'SOUZA, C.A ET AL: "OGR1/GPR68 Modulates the Severity of Experimental Autoimmune Encephalomyelitis and Regulates Nitric Oxide Production by Macrophages", PLOS ONE, vol. 11, 2016, pages e0148439
DUMONT, O.; ET AL: "Alteration in flow (shear stress)-induced remodelling in rat resistance arteries with aging: improvement by a treatment with hydralazine", CARDIOVASCULAR RESEARCH, vol. 77, 2008, pages 600 - 608
ELLIOTT ET AL., LANCET, vol. 344, 1994, pages 1125 - 1127
EREN ET AL., IMMUNOL., vol. 93, 1998, pages 154 - 161
ERNSTROM, G.G.; CHALFIE, M.: "Genetics of sensory mechanotransduction", ANNUAL REVIEW OF GENETICS, vol. 36, 2002, pages 411 - 453
FISCH ET AL., BIOCONJUGATE CHEM., vol. 3, 1992, pages 147 - 153
FISCHER ET AL., BIOTECHNOL. APPL. BIOCHEM., vol. 30, October 1999 (1999-10-01), pages 99 - 108
FISHWALD ET AL., NAT BIOTECHNOL, vol. 14, no. 7, 1996, pages 845 - 851
FREIDJA, M.L.; ET AL: "The AGE-breaker ALT-711 restores high blood flow-dependent remodeling in mesenteric resistance arteries in a rat model of type 2 diabetes", DIABETES, vol. 61, 2012, pages 1562 - 1572
GAMPER, H. B ET AL: "Facile Preparation and Exonuclease Stability of 3'-Modified Oligodeoxynucleotides", NUCLEIC ACIDS RES., vol. 21, 1993, pages 145 - 150
GEIBLER ET AL., PLOS ONE, vol. 6, 2011, pages e19509
GONG, S ET AL: "A gene expression atlas of the central nervous system based on bacterial artificial chromosomes", NATURE, vol. 425, 2003, pages 917 - 925, XP008142847
GRAY ET AL., J. IMM. METH., vol. 182, 1995, pages 155 - 163
GREEN ET AL., NATURE GENETICS, vol. 7, 1994, pages 13 - 21
GRGIC, I.; KAISTHA, B.P.; HOYER, J.; KOHLER, R.: "Endothelial Ca+-activated K+ channels in normal and impaired EDHF-dilator responses--relevance to cardiovascular pathologies and drug discovery", BRITISH JOURNAL OF PHARMACOLOGY, vol. 157, 2009, pages 509 - 526
GRISSA ET AL., BMC BIOINFORMATICS, vol. 8, 2007, pages 172
GUO ET AL., J. MOL. BIOL., vol. 200, 2010, pages 96
GUO ET AL., J. MOL. BIOL., vol. 400, 2010, pages 96
H SAXENA ET AL: "The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH : Acid-induced contraction of smooth muscle by OGR1", BRITISH JOURNAL OF PHARMACOLOGY, vol. 166, no. 3, 1 June 2012 (2012-06-01), UK, pages 981 - 990, XP055584473, ISSN: 0007-1188, DOI: 10.1111/j.1476-5381.2011.01807.x *
HAFT ET AL., PLOS COMPUT. BIOL., vol. 1, 2005, pages e60
HANES ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, May 1997 (1997-05-01), pages 4937 - 4942
HANES ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, November 1998 (1998-11-01), pages 14130 - 14135
HAO, J.; ET AL: "Kv1.1 channels act as mechanical brake in the senses of touch and pain", NEURON, vol. 77, 2013, pages 899 - 914, XP028851483
HARLOW; LANE: "antibodies, a Laboratory Manual", 1989, COLD SPRING HARBOR
HARLOW; LANE: "Using Antibodies, A Laboratory Manual", 1998
HENIKOFF; HENIKOFF, PROC. NATL. ACAD. SCI. USA, vol. 89, 1989, pages 10915
HENRION, D ET AL: "Impaired flow-induced dilation in mesenteric resistance arteries from mice lacking vimentin", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 100, 1997, pages 2909 - 2914, XP002434195
HERMANSON, G. T.: "Bioconjugate Techniques", 1996, ACADEMIC PRESS
HOCKEMEYER ET AL., NATURE BIOTECH., vol. 29, 2011, pages 731 - 734
HOOD ET AL., ADV. EXP. MED. BIOL., vol. 464, 1999, pages 127 - 147
HORVATH ET AL., SCIENCE, vol. 327, 2010, pages 167 - 170
HORVATH ET AL., SCIENCE, vol. 327, no. 5962, 2010, pages 167 - 170
HUANG, M.; CHALFIE, M.: "Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans", NATURE, vol. 367, 1994, pages 467 - 470
HUANG, X.P. ET AL: "Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65", NATURE, vol. 527, 2015, pages 477 - 483
HU-LIESKOVAN ET AL., CANCER RES., vol. 65, 2005, pages 8984 - 8992
ISHIDA, T.; TAKAHASHI, M.; CORSON, M.A.; BERK, B.C.: "Fluid shear stress-mediated signal transduction: how do endothelial cells transduce mechanical force into biological responses?", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 811, 1997, pages 12 - 23
ITOH ET AL., BIOORG. CHEM., vol. 24, no. 1, 1996, pages 59 - 68
JAALOUK, D.E.; LAMMERDING, J.: "Mechanotransduction gone awry", NATURE REVIEWS MOLECULAR CELL BIOLOGY, vol. 10, 2009, pages 63 - 73
JACKSON ET AL., NATURE BIOTECHNOLOGY, vol. 6, 2003, pages 635 - 637
JIE XU ET AL: "GPR68 Senses Flow and Is Essential for Vascular Physiology", CELL, vol. 173, no. 3, 1 April 2018 (2018-04-01), AMSTERDAM, NL, pages 762 - 775.e16, XP055584454, ISSN: 0092-8674, DOI: 10.1016/j.cell.2018.03.076 *
JIN-PENG LIU ET AL: "Ovarian cancer G protein-coupled receptor 1-dependent and -independent vascular actions to acidic pH in human aortic smooth muscle cells", AMERICAN JOURNAL OF PHYSIOLOGY: HEART AND CIRCULATORY PHYSIOLOGY, vol. 299, no. 3, 1 September 2010 (2010-09-01), US, pages H731 - H742, XP055584478, ISSN: 0363-6135, DOI: 10.1152/ajpheart.00977.2009 *
JOHNSON ET AL., NUCLEIC ACIDS RES., vol. 29, 2001, pages 205 - 206
JONAK ET AL.: "Progress Biotech", vol. 5, 1988, ELSEVIER SCIENCE PUBLISHERS, article "In Vitro Immunization in Hybridoma Technology"
JONES ET AL., NATURE, vol. 321, 1986, pages 522
JOYNER, M.J.; CASEY, D.P.: "Regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs", PHYSIOLOGICAL REVIEWS, vol. 95, 2015, pages 549 - 601
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1983, U.S. DEPT. HEALTH
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, NIH
KALL, L.; KROGH, A.; SONNHAMMER, E.L.: "A combined transmembrane topology and signal peptide prediction method", JOURNAL OF MOLECULAR BIOLOGY, vol. 338, 2004, pages 1027 - 1036, XP004504149
KALL, L.; KROGH, A.; SONNHAMMER, E.L.: "Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server", NUCLEIC ACIDS RESEARCH, vol. 35, 2007, pages W429 - 432
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5787
KENNY ET AL., BIO/TECHNOL., vol. 13, 1995, pages 787 - 790
KIM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, pages 1156 - 1160
KNAPPIK ET AL., J. MOL. BIOL., vol. 296, 2000, pages 57 - 86
KUBY, JANIS: "Immunology", 1992, W. H. FREEMAN AND COMPANY
KUBY: "Immunology", 2000, W.H. FREEMAN & CO.
KUMARAN ET AL., PROTEIN SCI., vol. 6, no. 10, 1997, pages 2233 - 2241
KUNIN ET AL., GENOME BIOL., vol. 8, 2007, pages R61
LEFRANC, M.-P. ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
LEFRANC, M.P., NUCLEIC ACIDS RES., vol. 29, 2001, pages 207 - 209
LEFRANC, M.-P., THE IMMUNOLOGIST, vol. 7, 1999, pages 132 - 136
LEVINA, N ET AL: "Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity", THE EMBO JOURNAL, vol. 18, 1999, pages 1730 - 1737, XP055005994
LEVY, B.I ET AL: "Impaired tissue perfusion: a pathology common to hypertension, obesity, and diabetes mellitus", CIRCULATION, vol. 118, 2008, pages 968 - 976
LI, H.; DURBIN, R.: "Fast and accurate short read alignment with Burrows-Wheeler transform", BIOINFORMATICS, vol. 25, 2009, pages 1754 - 1760, XP055287430
LI, H.; HANDSAKER ET AL: "The Sequence Alignment/Map format and SAMtools", BIOINFORMATICS, vol. 25, 2009, pages 2078 - 2079, XP055229864
LI, H.; WANG, D ET AL: "Abnormalities in osteoclastogenesis and decreased tumorigenesis in mice deficient for ovarian cancer G protein-coupled receptor 1", PLOS ONE, vol. 4, 2009, pages e5705
LI, J.; HOU, ET AL: "Piezol integration of vascular architecture with physiological force", NATURE, vol. 515, 2014, pages 279 - 282, XP055544773
LIEDTKE, W ET AL: "Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor", CELL, vol. 103, 2000, pages 525 - 535, XP002178632
LINDAUER, U ET AL: "Cerebrovascular vasodilation to extraluminal acidosis occurs via combined activation of ATP-sensitive and Ca2+-activated potassium channels", JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM, vol. 23, 2003, pages 1227 - 1238
LIVAK, K.J.; SCHMITTGEN, T.D.: "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method", METHODS, vol. 25, 2001, pages 402 - 408
LONBERG ET AL., INT REV IMMUNOL, vol. 13, no. 1, 1995, pages 65 - 93
LONBERG ET AL., NATURE, vol. 368, 1994, pages 856 - 859
LOUFRANI, L ET AL: "Flow (shear stress)-induced endothelium-dependent dilation is altered in mice lacking the gene encoding for dystrophin", CIRCULATION, vol. 103, 2001, pages 864 - 870
LUDWIG M-G ET AL: "Proton-sensing G-protein-coupled receptors", NATURE, MACMILLAN JOURNALS LTD, LONDON, vol. 425, no. 6953, 4 September 2003 (2003-09-04), pages 93 - 98, XP002303706, ISSN: 0028-0836, DOI: 10.1038/NATURE01905 *
LUDWIG, M.G ET AL: "Proton-sensing G-protein-coupled receptors", NATURE, vol. 425, 2003, pages 93 - 98, XP002303706
LUKACS, V ET AL: "Impaired PIEZ01 function in patients with a novel autosomal recessive congenital lymphatic dysplasia", NATURE COMMUNICATIONS, vol. 6, 2015, pages 8329
LYNCH, F ET AL: "Effects of diabetes and hypertension on structure and distensibilty of human small coronary arteries", JOURNAL OF HYPERTENSION, vol. 30, 2012, pages 384 - 389
MA ET AL., PLANT PHYSIOL., vol. 109, 1995, pages 341 - 6
MA ET AL., TRENDS BIOTECHNOL., vol. 13, 1995, pages 522 - 7
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MAKAROVA ET AL., BIOLOGY DIRECT, vol. 1, 2006, pages 7
MALI, SCIENCE, vol. 339, no. 6121, 2013, pages 823 - 826
MARRAGINI ET AL., SCIENCE, vol. 322, 2008, pages 1843 - 1845
MARTIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 9268 - 9272
MARTIN, METHODS ENZYMOL., vol. 203, 1991, pages 121 - 153
MARTINDALE: "Martindale: The Extra Pharmacopoeia", 1996, AMER PHARMACEUTICAL ASSN
MCCAFFREY ET AL., NATURE, vol. 414, pages 34 - 39
MEDEROS Y SCHNITZLER, M ET AL: "Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction", THE EMBO JOURNAL, vol. 27, 2008, pages 3092 - 3103
MELCHIOR, B.; FRANGOS, J.A.: "Galphaq/11 -mediated intracellular calcium responses to retrograde flow in endothelial cells", AMERICAN JOURNAL OF PHYSIOLOGY CELL PHYSIOLOGY, vol. 303, 2012, pages C467 - 473
MENDEZ ET AL., NATURE GENETICS, vol. 15, 1997, pages 146 - 156
MENDOZA, S.A ET AL: "TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress", AMERICAN JOURNAL OF PHYSIOLOGY HEART AND CIRCULATORY PHYSIOLOGY, vol. 298, 2010, pages H466 - 476
MILLER ET AL., NATURE BIOTECH., vol. 29, 2011, pages 143 - 8
MOJICA ET AL., J. MOL. EVOL., vol. 60, 2005, pages 174 - 182
MORONI, L.; SELMI, C.; ANGELINI, C.; MERONI, P.L.: "Evaluation of Endothelial Function by Flow-Mediated Dilation: a Comprehensive Review in Rheumatic Disease", ARCHIVUM IMMUNOLOGIAE ET THERAPIAE EXPERIMENTALIS, 2017
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
MORRISON; OI, ADV. IMMUNOL., vol. 44, 1988, pages 65 - 92
MOSCOU ET AL., SCIENCE, vol. 326, 2009, pages 3501
MURPHY ET AL., BIOCHEM. J., vol. 227, 1991, pages 277 - 279
NAVONE, S.E.; ET AL: "Isolation and expansion of human and mouse brain microvascular endothelial cells", NATURE PROTOCOLS, vol. 8, 2013, pages 1680 - 1693
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443
NGUYEN ET AL., MICROBIOL. IMMUNOL., vol. 41, 1997, pages 901 - 907
NIEBAUER, J.; COOKE, J.P.: "Cardiovascular effects of exercise: role of endothelial shear stress", JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, vol. 28, 1996, pages 1652 - 1660
NIEMINEN ET AL.: "Executive summary of the guidelines on the diagnosis and treatment of acute heart failure: the Task Force on Acute Heart Failureof the European Society of Cardiology", EUR HEART J, vol. 26, 2005, pages 384 - 416
NONOMURA, K ET AL: "Piezo2 senses airway stretch and mediates lung inflation-induced apnoea", NATURE, vol. 541, 2017, pages 176 - 181
OHTSUKA ET AL., J. BIOL. CHEM., vol. 260, 1985, pages 2605 - 2608
OVCHARENKO D: "Efficient delivery of siRNAs to human primary cells", AMBION TECHNOTES, vol. 10, no. 5, 2003, pages 15 - 16
PADLAN, MOLEC. IMMUN., vol. 28, 1991, pages 489 - 498
PADLAN, MOLEC. IMMUN., vol. 31, no. 3, 1994, pages 169 - 217
PAN, B ET AL: "TMC1 and TMC2 are components of the mechanotransduction channel in hair cells of the mammalian inner ear", NEURON, vol. 79, 2013, pages 504 - 515, XP028691181
PARRY, D.A ET AL: "Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta", AMERICAN JOURNAL OF HUMAN GENETICS, vol. 99, 2016, pages 984 - 990
PATHAN, A.R.; RUSCH, N.J.: "Two-pore domain K(+) channels: evidence for TWIK-2 in blood pressure regulation", HYPERTENSION, vol. 58, 2011, pages 539 - 541
PAUL: "Fundamental Immunology", 1993
PEARSON; LIPMAN, PROC. NAT'L. ACAD. SCI. USA, vol. 85, 1988, pages 2444
PENNISI, SCIENCE, vol. 341, 2013, pages 833 - 836
POREDOS, P.; JEZOVNIK, M.K.: "Testing endothelial function and its clinical relevance", JOURNAL OF ATHEROSCLEROSIS AND THROMBOSIS, vol. 20, 2013, pages 1 - 8
POURCEL ET AL., MICROBIOL., vol. 151, 2005, pages 653 - 663
POWELL ET AL., BIOTECHNOL., vol. 8, 1990, pages 333 - 337
PRESTA ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623
RAMKHELAWON, B.; RIVAS, D.; LEHOUX, S.: "Shear stress activates extracellular signal-regulated kinase 1/2 via the angiotensin II type 1 receptor", FASEB JOURNAL : OFFICIAL PUBLICATION OF THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, vol. 27, 2013, pages 3008 - 3016
RAN F. ET AL., NATURE, vol. 520, 2015, pages 186 - 191
RANADE, S.S ET AL: "Piezol, a mechanically activated ion channel, is required for vascular development in mice", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 111, 2014, pages 10347 - 10352
REED, M. W ET AL: "Acridine and Cholesterol-Derivatized Solid Supports for Improved Synthesis of 3'-Modified Oligonucleotides", BIOCONJUGATE CHEM., vol. 2, 1991, pages 217 - 225, XP002484280
REES: "Protein Structure Prediction", 1996, OXFORD UNIVERSITY PRESS, pages: 141 - 172
RETAILLEAU, K ET AL: "Piezol in Smooth Muscle Cells Is Involved in Hypertension-Dependent Arterial Remodeling", CELL REPORTS, vol. 13, 2015, pages 1161 - 1171
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323
ROCIO SERVIN-VENCES, M.; MORONI, M.; LEWIN, G.R.; POOLE, K.: "Direct measurement of TRPV4 and PIEZ01 activity reveals multiple mechanotransduction pathways in chondrocytes", ELIFE, vol. 6, 2017
RODE, B ET AL: "Piezol channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance", NATURE COMMUNICATIONS, vol. 8, 2017, pages 350
ROSSOLINI ET AL., MOL. CELL. PROBES, vol. 8, 1994, pages 91 - 98
RUIZ ET AL., NUCLEIC ACIDS RES., vol. 28, 2000, pages 219 - 221
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR
SANDHU ET AL., CRIT. REV. BIOTECHNOL., vol. 16, 1996, pages 95 - 118
SCHIFFELERS ET AL., NUCL. ACIDS RES., vol. 32, no. 49, 2004, pages 141 - 110
SCHWADERER, A.L.; SCHWARTZ, G.J.: "Back to basics: acidosis and alkalosis", PEDIATRICS IN REVIEW / AMERICAN ACADEMY OF PEDIATRICS, vol. 25, 2004, pages 350 - 357
SILVESTRE, J.S.; SMADJA, D.M.; LEVY, B.I.: "Postischemic revascularization: from cellular and molecular mechanisms to clinical applications", PHYSIOLOGICAL REVIEWS, vol. 93, 2013, pages 1743 - 1802
SIMS ET AL., J. IMMUNOL., vol. 151, 1993, pages 2296
SIOUD, J. MOL. BIOI., vol. 348, 2005, pages 1079 - 1090
SMITH; WATERMAN, ADV. APPL. MATH., vol. 2, 1970, pages 482c
SONG ET AL., NAT BIOTCH., vol. 23, 2005, pages 709 - 717
SONG ET AL., NAT MED., 2003
STEENBAKKERS ET AL., MOLEC. BIOL. REPORTS, vol. 19, 1994, pages 125 - 134
STERN ET AL., TRENDS. GENET., vol. 28, 2010, pages 335 - 340
STEWART ET AL., RNA, vol. 9, no. 4, April 2003 (2003-04-01), pages 493 - 501
STORCH, U ET AL: "G protein-mediated stretch reception", AMERICAN JOURNAL OF PHYSIOLOGY HEART AND CIRCULATORY PHYSIOLOGY, vol. 302, 2012, pages H1241 - 1249
SUKHAREV, S.I ET AL: "A Large-Conductance Mechanosensitive Channel in E. Coli Encoded by Mscl Alone", NATURE, vol. 368, 1994, pages 265 - 268
SWEETMAN: "Martindale: The Complete Drug Reference", 2005, PHARMACEUTICAL PRESS
SYEDA, R ET AL: "Piezol Channels Are Inherently Mechanosensitive", CELL REPORTS, vol. 17, 2016, pages 1739 - 1746
SZCZEPEK ET AL., NATURE BIOTECH., vol. 25, 2007, pages 786 - 793
TAYLOR ET AL., INT. IMMUNOL., vol. 6, no. 4, 1994, pages 579 - 591
TAYLOR ET AL., NUCLEIC ACIDS RESEARCH, vol. 20, no. 23, 1992, pages 6287 - 6295
TUAILLON ET AL., PROC NATL ACAD SCI USA, vol. 90, no. 8, 1993, pages 3720 - 3724
TUTTLE, J.L ET AL: "Impaired collateral artery development in spontaneously hypertensive rats", MICROCIRCULATION, vol. 9, 2002, pages 343 - 351
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1536
WALKER, R.G.; WILLINGHAM, A.T.; ZUKER, C.S.: "A Drosophila mechanosensory transduction channel", SCIENCE, vol. 287, 2000, pages 2229 - 2234
WANG, S ET AL: "Endothelial cation channel PIEZ01 controls blood pressure by mediating flow-induced ATP release", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 126, 2016, pages 4527 - 4536
WEN ET AL., J. IMMUNOL., vol. 17, 1987, pages 887 - 892
WERLEN ET AL., BIOCONJUGATE CHEM., vol. 5, 1994, pages 411 - 417
WHITELAM ET AL., BIOCHEM. SOC. TRANS., vol. 22, 1994, pages 940 - 944
WIEDENHEFT ET AL., NATURE, vol. 482, 2012, pages 331 - 8
WOO, S.H ET AL: "Piezo2 is required for Merkel-cell mechanotransduction", NATURE, vol. 509, 2014, pages 622 - 626, XP055357974
WOOD ET AL., SCIENCE, vol. 333, 2011, pages 307
XIA ET AL., NAT. BIOTECHNOL., vol. 20, 2002
XIONG, W ET AL: "TMHS is an integral component of the mechanotransduction machinery of cochlear hair cells", CELL, vol. 151, 2012, pages 1283 - 1295, XP028960562
YAN, L.; SINGH, L.S.; ZHANG, L.; XU, Y.: "Role of OGR1 in myeloid-derived cells in prostate cancer", ONCOGENE, vol. 33, 2014, pages 157 - 164
YAN, Z ET AL: "Drosophila NOMPC is a mechanotransduction channel subunit for gentle-touch sensation", NATURE, vol. 493, 2013, pages 221 - 225
YANG ET AL., J BIOL CHEM., vol. 281, no. 33, 18 August 2006 (2006-08-18), pages 23598 - 605
ZETSCHE B. ET AL., CELL, vol. 163, 3 October 2015 (2015-10-03), pages 759 - 771
ZHANG ET AL., NATURE BIOTECH., vol. 29, 2011, pages 149 - 53
ZHANG, Y.L ET AL: "Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells", AMERICAN JOURNAL OF PHYSIOLOGY CELL PHYSIOLOGY, vol. 296, 2009, pages C1391 - 1399

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