WO1999004636A1 - Evaluation of, delivery of, and use of agents to treat heart disorders - Google Patents
Evaluation of, delivery of, and use of agents to treat heart disorders Download PDFInfo
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- WO1999004636A1 WO1999004636A1 PCT/US1998/014968 US9814968W WO9904636A1 WO 1999004636 A1 WO1999004636 A1 WO 1999004636A1 US 9814968 W US9814968 W US 9814968W WO 9904636 A1 WO9904636 A1 WO 9904636A1
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- heart
- treatment
- phospholamban
- evaluating
- cell
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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- 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
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
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- C—CHEMISTRY; METALLURGY
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- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
Definitions
- This invention relates to methods for evaluating treatments suitable for heart disorders and to methods for delivering compounds to the heart of a subject.
- SR sarcoplasmic reticulum
- SERCA2a SR Ca--- "1" -ATPase activity
- Phospholamban is a small phosphoprotein, about 6,080 daltons in size, which is an integral element of the cardiac SR membrane. Phospholamban is phosphorylated in vivo in response to ⁇ -adrenergic agonist stimulation. In the dephosphorylated state, phospholamban inhibits SR Ca2 + -ATPase activity by decreasing the affinity of the enzyme for Ca2 + .
- Heart failure is characterized by a number of abnormalities at the cellular level in the various steps of excitation-contraction coupling of the cardiac cells.
- One of the key abnormalities in both human and experimental heart failure is a defect in SR function which is associated with abnormal intracellular Ca ⁇ "1" handling.
- Deficient SR Ca ⁇ + uptake during relaxation has been identified in failing hearts from both humans and animal models and has been associated with a decrease in the activity of SR Ca-*- "1" - ATPase activity and altered Ca ⁇ + kinetics.
- the invention features, a method of evaluating a treatment for a heart disorder.
- the method includes: providing a heart cell, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; administering the treatment to the heart cell; and evaluating the effect of the treatment on the heart cell, thereby evaluating the treatment for a heart disorder.
- the method includes evaluating the effect of the treatment on a parameter related to heart function.
- the parameter can include an assessment of contractility, Ca2 + metabolism, e.g., intracellular Cs? + concentration, SR Ca2 + ATPase activity, force generation, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- the treatment is administered in vivo, e.g., to an experimental animal.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed. Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a disorder, e.g., a heart disorder.
- the treatment is administered in vitro.
- the cell is derived from an experimental animal or a human.
- the cell can be cultured and/or immortalized.
- the nucleic acid encodes a phospholamban protein.
- the phospholamban can be from the same species that the heart cell is from or it can be from a different species.
- a mouse phospholamban can be expressed in a mouse cell or a human phospholamban can be expressed in a cell from an experimental animal.
- the nucleic acid is introduced into the heart cell by way of a vector suitable for somatic gene transfer, e.g., a viral vector, e.g., an adenoviral vector.
- a vector suitable for somatic gene transfer e.g., a viral vector, e.g., an adenoviral vector.
- the invention features, a method of evaluating a treatment for a heart disorder. The method includes: providing a heart, into some or all the cells of which has been introduced, by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; administering the treatment to the heart; and evaluating the effect of the treatment on the heart, thereby evaluating the treatment for a heart disorder.
- the method includes evaluating the effect of the treatment on a parameter related to heart function.
- the parameter can include an assessment of contractility, Ca2+ metabolism, e.g., intracellular C ⁇ + concentration, SR Ca-"- "1" ATPase activity, force generation, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- an assessment of contractility, Ca2+ metabolism e.g., intracellular C ⁇ + concentration
- SR Ca-"- "1" ATPase activity e.g., force generation, a force frequency relationship
- cardiocyte survival or apoptosis or ion channel activity e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- the treatment is administered in vivo, e.g., to an experimental animal.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed. Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a disorder, e.g., a heart disorder.
- the treatment is administered in vitro.
- the heart is derived from an experimental animal or a human.
- the nucleic acid encodes a phospholamban protein.
- the phospholamban can be from the same species that the heart is from or it can be from a different species.
- a mouse phospholamban can be expressed in a mouse heart or a human phospholamban can be expressed in the heart of an experimental animal.
- the phospholamban can be delivered to the heart using methods described herein.
- the nucleic acid is introduced into the heart by way of a vector suitable for somatic gene transfer, e.g., a viral vector, e.g., an adenoviral vector.
- a vector suitable for somatic gene transfer e.g., a viral vector, e.g., an adenoviral vector.
- the invention features, a method of evaluating a treatment for a heart disorder. The method includes: providing heart tissue into some or all of the cells of which has been introduced, by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; administering the treatment to the heart tissue; and evaluating the effect of the treatment on the heart tissue, thereby evaluating the treatment for a heart disorder.
- the method includes evaluating the effect of the treatment on a parameter related to heart function.
- the parameter can include an assessment of contractility, Ca--- 4" metabolism, e.g., intracellular Ca ⁇ "1" concentration, SR Ca- ⁇ "1" ATPase activity, force generation, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- the treatment is administered in vivo, e.g., to an experimental animal.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed.
- Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a disorder, e.g., a heart disorder.
- the treatment is administered in vitro.
- the heart tissue is derived from an experimental animal or a human.
- the nucleic acid encodes a phospholamban protein.
- the phospholamban can be from the same species that the heart tissue is from or it can be from a different species.
- a mouse phospholamban can be expressed in a mouse heart tissue or a human phospholamban can be expressed in heart tissue from an experimental animal.
- the nucleic acid is introduced into the heart tissue by way of a vector suitable for somatic gene transfer, e.g., a viral vector, e.g., an adenoviral vector.
- a vector suitable for somatic gene transfer e.g., a viral vector, e.g., an adenoviral vector.
- the invention features, a method of evaluating a treatment for a heart disorder.
- the method includes: providing a first and a second heart cell, into each of which has been introduced, by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; administering the treatment to a first heart cell, preferably in vitro; evaluating the effect of the treatment on the first heart cell; administering the treatment to a second heart cell, preferably in vivo; and evaluating the effect of the treatment on the second heart cell, thereby evaluating the treatment for a heart disorder.
- the method includes evaluating the effect of the treatment on a parameter related to heart function.
- the parameter can include an assessment of contractility, Ca ⁇ "1" metabolism, e.g., intracellular Ca ⁇ 4" concentration, SR Ca2 + ATPase activity, force generation, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- the treatment is administered in vivo, e.g., to an experimental animal.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed. Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a disorder, e.g., a heart disorder.
- the nucleic acid encodes a phospholamban protein.
- the phospholamban can be from the same species that the heart cell is from or it can be from a different species.
- a mouse phospholamban can be expressed in a mouse cell or a human phospholamban can be expressed in a cell from an experimental animal.
- the first and second cell can be from the same or different animals, can be from the same or different species, e.g., the first cell can be from a mouse and the second cell can be from a human or both cells can be human.
- the first and second cell can have the same or different genotypes.
- the evaluation of the treatment in the first cell can be the same or different from the evaluation of the treatment in the second cell, e.g., the intracellular Ca- ⁇ 4" concentration can be measured in the first cell and the SR Ca ⁇ " -ATPase activity can be measured in the second cell or the intracellular Ca- ⁇ 4" concentration can be measured in both cells.
- the invention features, a method of evaluating a treatment for a heart disorder.
- the method includes: providing a first administration of a treatment to a heart cell, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; evaluating the effect of the first administration on the heart cell; providing a second administration of a treatment to a heart cell, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; and evaluating the effect of the second administration on the heart cell, thereby evaluating a treatment for a heart disorder.
- the method includes evaluating the effect of the treatment on a parameter related to heart function.
- the parameter can include an assessment of contractility, Ca- ⁇ 4" metabolism, e.g., intracellular Ca ⁇ 4" concentration, SR Ca--- 4" ATPase activity, force generation, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- an assessment of contractility, Ca- ⁇ 4" metabolism e.g., intracellular Ca ⁇ 4" concentration, SR Ca--- 4" ATPase activity, force generation, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- the treatment is administered in vivo, e.g., to an experimental animal.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed. Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a disorder, e.g., a heart disorder.
- the nucleic acid encodes a phospholamban protein.
- the phospholamban can be from the same species that the heart cell is from or it can be from a different species.
- a mouse phospholamban can be expressed in a mouse cell or a human phospholamban can be expressed in a cell from an experimental animal.
- the first and second administration can be administered to the same or to different cells.
- the first and second administration can be administered under the same or different conditions, e.g., the first administration can consist of a relatively low level treatment, e.g., a lower concentration of a substance, and the second administration can consist of a relatively high level treatment, e.g., a higher concentration of a substance or both administrations can consist of the same level treatment.
- the invention features, a method of evaluating a treatment for a heart disorder.
- the method includes: providing a heart cell, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; administering the treatment to the heart cell; evaluating the effect of the treatment on the heart cell; providing a heart, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban; administering the treatment to the heart ; and evaluating the effect of the treatment on the heart, thereby evaluating the treatment for a heart disorder.
- the method includes evaluating the effect of the treatment on a parameter related to heart function.
- the parameter can include an assessment of contractility, Ca ⁇ " metabolism, e.g., intracellular Ca- ⁇ 4" concentration, SR Ca-"- 4" ATPase activity, force generation, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, or sodium potassium ATPase pump activity.
- the treatment is administered in vivo, e.g., to an experimental animal.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed. Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a disorder, e.g., a heart disorder.
- the nucleic acid encodes a phospholamban protein.
- the phospholamban can be from the same species that the heart cell and/or the heart is from or it can be from a different species.
- a mouse phospholamban can be expressed in a mouse heart cell and/or heart or a human phospholamban can be expressed in a heart cell and/or heart from an experimental animal.
- the treatment can be administered to the heart cell in vitro and to the heart in vivo or the treatment can be administered to the heart cell and to the heart in vitro.
- the invention features, a method of delivering a compound to the heart of a subject. The method includes: restricting the aortic flow of blood out of the heart, such that blood flow is re-directed to the coronary arteries; introducing the compound into the lumen of the circulatory system such that it flows into the coronary arteries; allowing the heart to pump while the aortic outflow of blood is restricted, thereby allowing the compound to flow into and be delivered to the heart; and reestablishing the flow of blood to the heart .
- the compound includes: a nucleic acid which directs the expression of a peptide, e.g., a phospholamban or a SR Ca ⁇ 4" -ATPase and a viral vector suitable for somatic gene delivery, e.g., an adenoviral vector.
- a heart disorder e.g., heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection.
- the subject can be a human or an experimental animal.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed. Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a disorder, e.g., a heart disorder.
- the method further includes restricting blood flow into the left side of the heart, e.g., by restricting the pulmonary circulation through obstraction of the pulmonary artery, so as to lessen dilution of the compound.
- the method further includes opening the pericardium and introducing the compound, e.g., using a catheter.
- the compound is: introduced into the lumen of the aorta, e.g., the aortic root, introduced into the coronary ostia or introduced into the lumen of the heart.
- the nucleic acid which directs the expression of the peptide, is homogeneously overexpressed in the heart of the subject.
- the invention features, a heart cell, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban.
- the heart cell can be provided as a purified preparation.
- the invention features, a heart tissue, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban.
- the heart tissue can be provided as a tissue preparation.
- the invention features, a heart, into which has been introduced by somatic gene transfer, a nucleic acid which results in the expression of phospholamban.
- the heart can be provided in a subject or ex vivo, i.e. removed from a subject.
- the invention features, a method for treating a subject at risk for a heart disorder.
- the method includes introducing into somatic heart tissue of the subject, a nucleic acid which encodes phospholamban.
- the nucleic acid is introduced using the methods described herein.
- the phospholamban can be from the same species as the subject or it can be from a different species.
- a human phospholamban can be introduced into a human heart or a human phospholamban can be can be introduced into the heart of an experimental animal.
- the nucleic acid is introduced into the heart by way of a vector suitable for somatic gene transfer, e.g., a viral vector, e.g., an adenoviral vector.
- the subject can be a human, an experimental animal, e.g., a rat or a mouse, a domestic animal, e.g., a dog, cow, sheep, pig or horse, or a non- human primate, e.g., a monkey.
- the subject can be suffering from a cardiac disorder, such as heart failure, ischemia, myocardial infarction, congestive heart failure, arrhythmia, transplant rejection and the like.
- the term "treatment” refers to a procedure (e.g., a surgical method) or the administration of a substance, e.g., a compound which is being evaluated for use in the alleviation or prevention of a heart disorder or symptoms thereof.
- a procedure e.g., a surgical method
- a therapeutic agent such as a drug, a peptide, an antibody, an ionophore and the like.
- the term "heart disorder” refers to a structural or functional abnormality of the heart, that impairs its normal functioning.
- the heart disorder can be heart failure, ischemia, myocardial infarction, congestive heart failure, arrhythmia, transplant rejection and the like.
- the term includes disorders characterized by abnormalities of contraction, abnormalities in Ca ⁇ 4" metabolism, and disorders characterized by arrhytmia.
- heart cell refers to a cell which can be: (a) part of a heart present in a subject, (b) part of a heart which is maintained in vitro, (c) part of a heart tissue, or (d) a cell which is isolated from the heart of a subject.
- the cell can be a cardiac myocyte.
- heart refers to a heart present in a subject or to a heart which is maintained outside a subject.
- heart tissue refers to tissue which is derived from the heart of a subject.
- simatic gene transfer refers to the transfer of genes into a somatic cell as opposed to transferring genes into the germ line.
- the term "compound” refers to a compound, which can be delivered effectively to the heart of a subject using the methods of the invention. Such compounds can include, for example, a gene, a drug, an antibiotic, an enzyme, a chemical compound, a mixture of chemical compounds or a biological macromolecule.
- the term “subject” refers to an experimental animal, e.g., a rat or a mouse, a domestic animal, e.g., a dog, cow, sheep, pig or horse, a non-human primate, e.g., a monkey and in the case of therapeutic methods, humans. However, it is noted that human cells, tissue or hearts can be used in vitro evaluations.
- a subject can suffer from a heart disorder, such as heart failure, ischemia, myocardial infarction, congestive heart failure, arrhythmia, transplant rejection and the like.
- the experimental animal can be an animal in which a gene related to cardiac structure or function is misexpressed. Misexpression can be achieved by methods known in the art, for example, by transgenesis, including the creation of knockout animals, or by classic breeding experiments or manipulation.
- the misexpressed gene can be a gene encoding a sarcomeric protein, a gene encoding a protein which conditions cardiocyte survival or apoptosis, or a gene encoding a calcium regulatory protein.
- Sarcomeric proteins include myosin heavy chain, troponin I, troponin C, troponin T, tropomyosin, actin, myosin light chain kinase, myosin light chain 1, myosin light chain 2 or myosin light chain 3.
- Proteins which modify cardiocyte survival or apoptosis include IGF-1 receptor, PI3 kinase, AKT kinase or members of the caspase family of proteins.
- Calcium regulatory proteins include phospholamban, SR Ca2 + ATPase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin.
- the experimental animal can be an animal model for a heart disorder, such as a hypertensive mouse or rat.
- misexpression refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post- transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus. Misexpression includes any expression from a
- the term "restricting the aortic flow of blood out of the heart” refers to substantially blocking the flow of blood into the distal aorta and its branches. For example, at least 50 % of the blood flowing out of the heart is restricted, preferably 75 % and more preferably 80, 90, or 100 % of the blood is restricted from flowing out of the heart.
- the blood flow can be restricted by obstructing the aorta and the pulmonary artery, e.g., with clamps.
- the term "introducing” refers to a process by which a compound can be placed into a chamber or the lumen of the heart of a subject.
- the pericardium can be opened and the compound can be injected into the heart, e.g., using a syringe and a catheter.
- the compound can be: introduced into the lumen of the aorta, e.g., the aortic root, introduced into the coronary ostia or introduced into the lumen of the heart.
- the terms “homogeneous fashion” and “homogeneously overexpressing” are satisfied if one or more of the following requirements are met: (a) the compound contacts at least 10 %, preferably 20, 20, 40, 50, 60, 70, 80, 90 or 100 % of the cells of the heart and (b) at least 10 %, preferably 20, 20, 40, 50, 60, 70, 80, 90 or 100 % of the heart cells take up the compound.
- purified preparation refers to a preparation in which at least 50, preferably 60, 70, 80 , 90 or 100 % of the cells are heart cells into which phospholamban has been introduced by somatic gene transfer.
- the methods of the invention allow rapid and low cost development of cardiac overexpression models.
- the methods of the invention also provide ways of examining multiple genes interacting in transgenic models, testing gene therapy approaches and evaluating treatments of cardiac disorders.
- Ad.RSV.PL Ad.RSV.PL
- Ad.RSV.SERCA2a Ad.RSV.SERCA2a
- CPA cyclopiazonic acid
- Figure 4A shows intracellular Ca2 + transients and shortening in an uninfected cardiomyocyte and in a cardiomyocyte infected for 48 hours with 10 pfu/cell of Ad.RSV. ⁇ gal and stimulated at 1 Hz.
- Figure 4B shows intracellular Ca- ⁇ 4" transients and shortening in an uninfected cardiomyocyte and in a cardiomyocyte infected for 48 hours with 1, 10, and 100 pfu/cell of Ad.RSV.PL stimulated at 1 Hz.
- Figure 4C shows intracellular Ca--- 4" transients and shortening in an uninfected cardiomyocyte, a cardiomyocyte infected with 1- pfu/cell of Ad.RSV.PL, and a cardiomyocyte infected with 10 pfu/cell of Ad.RSV.PL and 10 pfu/cell of Ad.RSV.SERCA2a for 48 hours, stimulated at 1 Hz.
- Figure 7 A is a graph showing the response of intracellular Ca2 + transients to increasing frequency of stimulation in an uninfected cardiomyocyte.
- Figure 7B is a graph showing the response of intracellular Ca ⁇ " transients to increasing frequency of stimulation in a cardiomyocyte infected with 10 pfu/cell of Ad.RSV.PL.
- Figure 7C is a graph showing the response of intracellular Ca- ⁇ 4" transients to increasing frequency of stimulation in a cardiomyocyte infected with 10 pfu/cell of Ad.RSV.PL and 10 pfu/cell of Ad.RS V. SERC A2a for 48 hours.
- Figure 8 shows intracavitary pressure tracings from rats 48 hours after cardiac gene transfer with either Ad.EGFP (left) or Ad.PL (right).
- the pressure tracing of the Ad.PL transduced hearts displays a markedly prolonged relaxation and reduced pressure development.
- Figure 9 is a drawing showing the somatic gene delivery method.
- Figure 10A is a graph demonstrating that infection of neonatal cardiac myocytes with the construct Ad.asPL increased the contraction amplitude and significantly shortened the time course of the contraction.
- Figure 1 OB is a graph demonstrating that adenovirus-mediated gene transfer of the antisense cDNA for phospholamban results in a modification of intracellular calcium handling. Phospholamban and Heart Disorder
- somatic gene transfer e.g., adenoviral gene transfer
- SERCA2a is both dose dependent and time dependent in rat neonatal cardiomyocytes.
- the smaller size of phospholamban compared with SERCA2a (6 kD in its monomer form compared with 110 kD) may explain, at least in part, the more effective protein expression by Ad.RSV.PL than by Ad.RSV.SERCA2a under similar conditions. Nevertheless, using these recombinant adenoviruses, significant overexpression of phospholamban and SERCA2a was achieved, individually and in combination. Co- infection with both Ad.RSV.PL and Ad.RSV.SERCA2a mediated overexpression of both SERCA2a and phospholamban that was the same as the expression from infection with either Ad.RSV.PL or Ad.RSV.SERCA2a alone.
- the ability to simultaneously manipulate expression of multiple proteins in the context of primary myocytes is an advantage of somatic gene transfer for the study of interacting components of complex systems.
- phospholamban relative to SERCA2a is altered in a number of disease states. In hypothyroidism phospholamban levels are increased, whereas in hyperthyroidism phospholamban levels are decreased. An increased ratio of phospholamban to SERCA2a is an important characteristic of both human and experimental heart failure. Both experimental and human heart failure are characterized by a prolonged Ca + transient and impaired relaxation. Increasing levels of phospholamban relative to SERCA2a significantly altered intracellular Ca 2+ handling in the isolated cardiomyocytes by prolonging the relaxation phase of the Ca 2+ transient, decreasing Ca 2+ release, and increasing resting Ca 2+ .
- a treatment can be evaluated by assessing the effect of the treatment on a parameter related to contractility. For example, SR Ca- ⁇ 4" ATPase activity or intracellular Ca- ⁇ 4" concentration can be measured, using the methods described above. Furthermore, force generation by hearts or heart tissue can be measured using methods described in Strauss et al., Am. J. Physiol., 262:1437-45, 1992, the contents of which are incorporated herein by reference. In many drug screening programs which test libraries of therapeutic agents and natural extracts, high throughput assays are desirable in order to maximize the number of therapeutic agents surveyed in a given period of time.
- Assays which are performed in cell-free systems are preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of the parameter being measured, e.g., the intracellular levels of Ca--- 4" , which is mediated by a test therapeutic agent.
- the effects of cellular toxicity and/or bioavailability of the test therapeutic agent can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the therapeutic agent on the parameter being measured, e.g., the intracellular levels of Ca--- 4" . It is often desirable to screen candidate treatments in two stages, wherein the first stage is performed in vitro, and the second stage is performed in vivo.
- the efficacy of a test therapeutic agent can be assessed by generating dose response curves from data obtained using various concentrations of the test therapeutic agent.
- a control assay can also be performed to provide a baseline for comparison. In the control assay, the heart cell is incubated in the absence of a test agent. Propagation of Heart Cells
- a heart cell culture can be obtained by allowing heart cells to migrate out of fragments of heart tissue adhering to a suitable substrate (e.g., a culture dish) or by disaggregating the tissue, e.g., mechanically or enzymatically to produce a suspension of heart cells.
- a suitable substrate e.g., a culture dish
- the enzymes trypsin, collagenase, elastase, hyaluronidase, DNase, pronase, dispase, or various combinations thereof can be used. Trypsin and pronase give the most complete disaggregation but may damage the cells. Collagenase and dispase give a less complete dissagregation but are less harmful.
- tissue e.g., heart tissue
- cells e.g., heart cells
- Expression vectors suitable for somatic gene transfer, can be used to express the compound, e.g., the phospholamban gene.
- examples of such vectors include replication defective retroviral vectors, adenoviral vectors and adeno-associated viral vectors.
- Adenoviral vectors suitable for use by the methods of the invention include (Ad.RSV.lacZ), which includes the Rous sarcoma virus promoter and the lacZ reporter gene as well as (Ad.CMV.lacZ), which includes the cytomegalovirus promoter and the lacZ reporter gene.
- Methods for the preparation and use of viral vectors are described in WO 96/13597, WO 96/33281, WO 97/15679, and Trapnell et al., Curr.
- the nucleic acid which results in the overexpression of phospholamban can be derived from the natural phospholamban gene including all the introns and exons, it can be a cDNA molecule derived from the natural gene (Fujji et al., J. Biol. Chem. 266:11669-11675, 1991, the contents of which are incorporated herein by reference) or a chemically synthesized cDNA molecule.
- the nucleic acid encoding the phospholamban protein can be under the control of the naturally occurring promoter or any other promoter that drives a high level expression of the phospholamban gene.
- Ad.RSV.SERCA2a has been described in detail by Hajjar et al., Circulation, 95: 423-429, 1997, the contents of which are incorporated herein by reference.
- Ad.RSV. ⁇ gal which carries a nuclear localizing form of ⁇ -galactosidase, is described in Dong et al., J. Biol. Chem. 27:29969-77, 1996, the contents of which are incorporated herein by reference.
- the rabbit phospholamban cDNA is described in Lylton J. MacLennan D.H., J. Biol. Chem., 1988, 263:15024-15031, the contents of which are incorporated herein by reference.
- the phospholamban cDNA was subcloned into the bacterial plasmid vector pAdRSV4, which uses the RS V long terminal repeat as a promoter and the SV40 polyadenylation signal and contains map units with adenovirus sequences from 0 to 1 and from 9 to 16.
- the position and orientation of the phospholamban cDNA were confirmed by restriction enzyme digestion and by polymerase chain reaction.
- the plasmid vector containing phospholamban (pAd.RSV-PL) was then cotransfected into 293 cells with PJM17.
- the homologous recombinants between pAd.RSV.PL and pJM17 contain the phospholamban cDNA substituted for El .
- Ad.RSV.PL This adenovirus is structurally similar to Ad.RSV. ⁇ gal and to Ad.RSV.SERCA2a- described in Dong O. et al., J. Biol. Chem, 1996, 271 :29969-29977, 1976.
- the recombinant viruses were prepared as high-titer stocks by propagation in 293 cells as described in Graham, F.L.
- Neonatal Cardiomyocytes Spontaneously beating cardiomyocytes were prepared from 1 to 2 day old rats and cultured in P-10 medium (GIBCO,BRL) in the presence of 5% fetal calf serum and 10% horse serum for 3 days as described previously in Kang J.X. et al., Proc. Natl. Acad. Sci. U.S.A., 1995, 92:3097-4001 and Kang J.X. and Leaf A., Euro. J. Pharmacol, 1996, 297:97-106, the contents of which are incorporated herein by reference.
- cardiomyocytes infected with Ad.RSV.PL (multiplicity of infection of 10 pfu/cell) exhibited a significant increase in resting Ca + not evident in uninfected cells.
- coinfection with Ad.RSV.SERCA2a (multiplicity of infection of 10 pfu/cell) restored the frequency response to normal.
- the response to increasing stimulation frequencies in mammalian cardiomyocytes is governed by the SR.
- the coverslip was superfused with a HEPES-buffered solution at a rate of 20 mL/h.
- Cells were stimulated at different frequencies (0.1 to 2.0 Hz) using an external stimulator (Grass Instruments).
- a dual excitation spectrofluorometer (IONOPTIX) was used to record fluorescence emissions (505 nm) elicited from exciting wavelengths of 360 and 380 nm.
- Adenoviral gene transfer of phospholamban provides an attractive system for further elucidation of the effects of inhibiting SR Ca 2+ -ATPase on intracellular Ca 2+ handling.
- a decrease in SRCa 2+ uptake rates is expected to lead to a smaller amount of Ca 2+ sequestered by the SR, resulting in a smaller amount of Ca 2+ release.
- a significantly prolonged Ca 2+ transient and a higher resting (Ca 2+ ) was observed reflecting the decreased Ca 24 uptake and a decrease in peak (Ca + ) levels reflecting less Ca + available for release.
- Phospholamban has been shown to play a key role in modulating the response of agents that increase cAMP levels in cardiomyocytes. Since phosphorylation of phospholamban reduces the inhibition to the SR Ca 2+ pump, thereby enhancing the SR Ca 2+ - ATPase, we were specifically interested in evaluating the effects of ⁇ -agonism on the relaxation phase of the Ca 2+ transient. In the basal state, the overexpression of phospholamban significantly prolongs the Ca 2+ transient. As shown in Figure 6, at maximal isoproterenol stimulation, the time course of the Ca 2+ transients in the uninfected cardiomyocytes and the cardiomyocytes infected with Ad. RSV.PL were decreased to the same level.
- SR Membranes from Isolated Rat Cardiomyocytes To isolate SR membrane from cultured cardiomyocyes, a procedure modified from Harigaya et al., Circ. Res., 1969, 25:781-794, as well as, Wienzek et al., 1992, 23:1149-1163, the contents of which are incorporated herein by reference, was used. Briefly, isolated neonatal cardiomyocytes were suspended in a buffer containing (mmol/L) sucrose 500, phenylmerhyisulfonyl fluoride 1 and PIPES 20, at pH 7.4. The cardiomyocytes were then disrupted with a homogenizer. The homogenates were centrifuged at 500g for 20 minutes.
- the resultant supernatant was centrifuged at 25,000g for 60 minutes to pellet the SR-enriched membrane.
- the pellet was resuspended in a buffer containing (mmol/L) KC1 600, sucrose 30, and PIPES 20, frozen in liquid nitrogen, and stored at -70°C. Protein concentration was determined in these preparations by a modified Bradford procedure, described in Bradford et al., Anal.
- the blots were incubated in a solution containing peroxidase-labeled goat anti-mouse IgG (dilution, 1 :1000) for 90 minutes at room temperature. The blot was then incubated in a chemiluminescence system and exposed to an X-OMAT x-ray film (Fuji Films) for 1 minute. The densities of the bands were evaluated using NIH Image. Normalization was performed by dividing densitometric units of each membrane preparation by the protein amounts in each of these preparations. Serial dilution of the membrane preparations revealed a linear relationship between amounts of protein and the densities of the SERCA2a immunoreactive hands (data not shown). 6. SR Ca 2+ -ATPase Activity
- SR Ca2 + -ATPase activity assays were carried out according to Chu A. et al., Methods Enzymol, 1988, 157:36-46, the contents of which are incorporated herein by reference, on the basis of pyruvate NADH-coupled reactions.
- a photomotor Beckman DU 640 adjusted at a wavelength of 540 nm, oxidation of NADH (which is coupled to the SR Ca--- 4" - ATPase) was assessed at 37°C in the membrane preparations by the difference of the total absorbance and basal absorbance.
- the reaction was carried out in a volume of 1 mL. All experiments were carried out in triplicate.
- the activity of the Ca-"- + -ATPase was calculated as follows: ⁇ absorbance/6.22 x protein x time (in nmol ATP/mg protein x min). The measurements were repeated at different [Ca--- 4" ] levels.
- the effect of the specific Ca-"- + -ATPase inhibitor CPA at a concentration range of 0.001 to 10 ⁇ mol/L was also studied in those preparations, as described in Schwinger et al., Circulation, 1995, 92:3220-3228 and Baudet et al., Circ. Res., 1993, 73:813-819, the contents of which are incorporated herein by reference.
- CPA inhibited the SR Ca-"- + -ATPase activity in a dose-dependent fashion in all three membrane preparations (uninfected, Ad.RSV.PL, and d.RSV.PL+Ad.RSV.SERCA2a).
- the SR Ca 2+ - ATPase plays a key role in excitation-contraction coupling, lowering Ca + during relaxation in cardiomyocytes, and "loading" the SR with Ca 2+ for the subsequent release and contractile activation.
- the Ca + -pumping activity of this enzyme is influenced by phospholamban. In the unphosphorylated state, phospholamban inhibits the Ca 2+ - ATPase, whereas phosphorylation of phospholamban by cAMP-dependent protein kinase and by Ca 2+ calmodulin-dependent protein kinase reverses this inhibition. Therefore, an increase in phospholamban content should decrease the affinity of the SR Ca 2+ pump for Ca 2+ .
- Rats and mice were anesthetized with intraperitoneal pentobarbital and placed on a ventilator.
- the chest was entered form the left side through the third intercostal space.
- the pericardium was opened and a 7-0 suture placed at the apex of the left ventricle.
- the aorta and pulmonary artery were identified.
- a 22 G catheter containing 200 ⁇ l of adenovirus was advanced from the apex of the left ventricle to the aortic root.
- the aorta and pulmonary artery were clamped distal to the site of the catheter and the adenovirus solution was injected as shown in Figure 9.
- the clamp was maintained for 10 seconds while the heart was pumping against a closed system (isovolumically).
- Delayed cardiac relaxation in failing hearts is attributed to a reduced activity of the Sarcoplasmic Reticulum Calcium ATPase.
- Phospholamban inhibits SERCA2a activity and is, therefore, a potential target to improve cardiac function.
- Ad.asPL adenovirus carrying the full length antisense cDNA of phospholamban
- adenovirus-mediated gene transfer of the antisense cDNA for phospholamban also resulted in a modification of intracellular calcium handling and shortening in myocardial cells (see Figure 10B) indicating that such vectors can be used for increasing the contractility of myocardial cells in heart failure.
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CA002297328A CA2297328A1 (en) | 1997-07-22 | 1998-07-20 | Evaluation of, delivery of, and use of agents to treat heart disorders |
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WO2000063426A2 (en) * | 1999-04-15 | 2000-10-26 | Devgen Nv | Compound screening methods |
WO2002097128A2 (en) * | 2001-05-30 | 2002-12-05 | Medigene Ag | Method for identifying substances directed against myocardial cell hypertrophy |
US7291604B2 (en) | 2003-09-03 | 2007-11-06 | The General Hospital Corporation | Methods of treating restenosis |
-
1998
- 1998-07-20 WO PCT/US1998/014968 patent/WO1999004636A1/en not_active Application Discontinuation
- 1998-07-20 EP EP98934656A patent/EP1009236A4/en not_active Withdrawn
- 1998-07-20 CA CA002297328A patent/CA2297328A1/en not_active Abandoned
Non-Patent Citations (4)
Title |
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FUJII J., ET AL.: "STRUCTURE OF THE RABBIT PHOSPHOLAMBAN GENE, CLONING OF THE HUMAN CDNA, AND ASSIGNMENT OF THE GENE TO HUMAN CHROMOSOME 6.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 266., no. 18., 25 June 1991 (1991-06-25), US, pages 11669 - 11675., XP002914767, ISSN: 0021-9258 * |
HAJJAR R. J., ET AL.: "EFFECTS OF ADENOVIRAL GENE TRANSFER OF PHOSPHOLAMBAN ON LATRACELLULAR CALCIUM HOMEOSTATIS IN ISOLATED MYOCYTES.", CIRCULATION, LIPPINCOTT WILLIAMS & WILKINS, US, vol. 94., no. 08., 15 October 1996 (1996-10-15), US, pages I - 159., XP002914769, ISSN: 0009-7322 * |
KOCH W. J., ET AL.: "TRANSGENIC MANIPULATION OF MYOCARDIAL G PROTEIN-COUPLED RECEPTORS AND RECEPTOR KINASES.", CIRCULATION RESEARCH., GRUNE AND STRATTON, BALTIMORE, US, vol. 78., no. 04., 1 April 1996 (1996-04-01), US, pages 511 - 516., XP002914768, ISSN: 0009-7330 * |
See also references of EP1009236A4 * |
Cited By (6)
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WO2000063426A2 (en) * | 1999-04-15 | 2000-10-26 | Devgen Nv | Compound screening methods |
WO2000063426A3 (en) * | 1999-04-15 | 2001-02-08 | Devgen Nv | Compound screening methods |
WO2002097128A2 (en) * | 2001-05-30 | 2002-12-05 | Medigene Ag | Method for identifying substances directed against myocardial cell hypertrophy |
WO2002097128A3 (en) * | 2001-05-30 | 2003-09-12 | Medigene Ag | Method for identifying substances directed against myocardial cell hypertrophy |
US7291604B2 (en) | 2003-09-03 | 2007-11-06 | The General Hospital Corporation | Methods of treating restenosis |
US8133878B1 (en) | 2003-09-03 | 2012-03-13 | The General Hospital Corporation | Methods for treating restenosis |
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