Patent Application
20050095227
The sarcoplasmic reticulum (SR) is an internal membrane system, which plays a critical role in the regulation of cytosolic Ca2+ concentrations and thus, excitation-contraction coupling in muscle. Contraction is mediated through the release of Ca2+ from the SR, while relaxation involves the active re-uptake of Ca2+ into the SR lumen by a Ca2+-ATPase. In cardiac muscle, the SR Ca2+-ATPase activity (SERCA2a) is under reversible regulation by phospholamban.
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 β-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 Ca2+ handling. Deficient SR Ca2+ 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 Ca2+-ATPase activity and altered Ca2+ kinetics.
In one aspect, the invention features a method of treating a subject, e.g., a subject having heart failure. The method includes introducing into the subject, e.g., into the heart of a subject, a viral delivery system that includes a therapeutic nucleic acid.
In one embodiment, the viral delivery system is an adeno-associated viral delivery system. The adeno-associated virus can be of serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9).
In one embodiment, the subject has or is at risk for heart failure, e.g. a non-ischemic cardiomyopathy, mitral valve regurgitation, ischemic cardiomyopathy, or aortic stenosis or regurgitation. In one embodiment, the subject needs an improved sarcoplasmic reticulum Ca2+ uptake in the cardiac muscle.
In one embodiment, the subject is a human. For example, the subject is between ages 18 and 65. In another embodiment, the subject is a non-human animal.
In one embodiment, the nucleic acid of the viral delivery system encodes a a protein, e.g., a heart-specific protein or a protein effective in modulating cardiac physiology.
In one embodiment, the expression of the protein encoded by the nucleic acid of the delivery system is sustained, e.g., for at least three months.
In one embodiment, the nucleic acid of the viral delivery system encodes a sarcoplasmic reticulum Ca2+ ATPase pump, e.g., SERCA2a pump. The expression of the sarcoplasmic reticulum Ca2+ ATPase pump, e.g., SERCA2a pump can be sustained, e.g., for at least one, two, three, four, or six months.
In one embodiment, the nucleic acid can produce an antisense sequence or siRNA that reduces expression of a desired protein.
In one embodiment, treating the subject ameliorates at least one symptom of heart failure.
In one embodiment, the introduction of the viral delivery system is performed without direct manipulation of the coronary vasculature. In one embodiment, at least 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, 1×1015, or 1×1016 genomes of the virus are delivered. In one embodiment, at most 1×1019 genomes of the virus are delivered.
In one embodiment, the subject is undergoing or has undergone left ventricular assist device implantation.
In one embodiment, the subject is evaluated after introducing the viral delivery system, e.g., by echocardiography or metabolic stress testing.
In one embodiment, the viral delivery system is introduced by an injection, e.g., a direct injection into the heart, e.g., a direct injection into the left ventricle surface.
In one embodiment, the viral delivery system is introduced by a percutaneous injection, e.g., retrograde from the femoral artery retrograde to the coronary arteries.
In one embodiment, introducing the viral delivery system includes restricting blood flow through coronary vessels, e.g., partially or completely, introducing the viral delivery system into the lumen of the coronary artery, and allowing the heart to pump, while the coronary vein outflow of blood is restricted. Restricting blood flow through coronary vessels can be performed, e.g., by inflation of at least one, two, or three angioplasty balloons. Restricting blood flow through coronary vessels can last, e.g., for at least one, two, three, or four minutes. Introduction of the viral delivery system into the coronary artery can be performed, e.g., by an antegrade injection through the lumen of an angioplasty balloon. The restricted coronary vessels can be: the left anterior descending artery (LAD), the distal circumflex artery (LCX), the great coronary vein (GCV), the middle cardiac vein (MCV), or the anterior interventricular vein (AIV). Introduction of the viral delivery system can be performed after ischemic preconditioning of the coronary vessels, e.g., by restricting blood flow by e.g., inflating at least one, two, or three angioplasty balloons. Ischemic preconditioning of the coronary vessels can last for at least one, two, three, or four minutes.
In one embodiment, introducing the viral delivery system includes restricting the aortic flow of blood out of the heart, e.g., partially or completely, introducing the viral delivery system into the lumen of the circulatory system, and allowing the heart to pump, e.g., against a closed system (isovolumically), while the aortic outflow of blood is restricted. Restricting the aortic flow of blood out of the heart can be performed by redirecting blood flow to the coronary arteries, e.g., to the pulmonary artery. Restricting the aortic flow of blood can be accomplished by clamping, e.g., clamping a pulmonary artery. Introducing the viral delivery system can be performed e.g., with the use of a catheter or e.g., by direct injection. Introducing the viral delivery system can be performed by a delivery into the aortic root.
In another aspect, the invention features a viral delivery system, including a nucleic acid encoding a non-viral therapeutic. In one embodiment, the viral delivery system is an adeno-associated viral delivery system. The adeno-associated virus can be of serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9). For example, the viral delivery system is a modified adeno-associated virus or a reconstituted virus or virus-like particle, e.g., that can infect cells, e.g., a myocytes, e.g., a cardiomyocyte.
In one embodiment, the nucleic acid encodes a protein, e.g., a protein effective in modulating cardiac physiology, e.g., a SERCA2a protein.
In one embodiment, the nucleic acid produces an antisense sequence, e.g., a sequence to downregulate a protein effective in modulating cardiac physiology, e.g., a SERCA2a protein.
In one embodiment, the nucleic acid produces an siRNA sequence, e.g., a sequence to downregulate a protein effective in modulating cardiac physiology, e.g., a SERCA2a protein.
In one embodiment, the nucleic acid includes a heart-specific promoter, e.g., a promoter from cardiac troponin T, alpha myosin light chain, or myosin heavy chain promoter.
In one embodiment, the nucleic acid includes at least a functional segment of a cytomegalovirus (CMV) promoter.
In another aspect, 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, (for example, that the blood flow is redirected to the coronary arteries), introducing the desired compound into the lumen of the circulatory system, e.g., into a blood vessel such as an artery (for example, such that said compound flows into the coronary arteries), allowing the heart to pump while the aortic outflow of blood is restricted (e.g., partially or completely restricted), and reestablishing the flow of blood.
In one embodiment, the compound includes a nucleic acid, which directs the expression of a peptide, e.g., a sarcoplasmic reticulum Ca2+ ATPase pump or a β galactosidase. In another embodiment, the compound includes a protein (e.g., a peptide or larger protein).
In one embodiment, the compound includes a virus vector suitable for somatic gene delivery.
In one embodiment, the compound is delivered using an adeno-associated virus, e.g., serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9). The delivery of the adeno-associated virus can include delivery of a number of genomes, e.g., at least 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, 1×1015, or 1×1016 genomes and, e.g., less than 1×1019 genomes.
In one embodiment, the subject is a human.
In one embodiment, the subject is a non-human animal.
In one embodiment, the subject has or is at risk for heart failure, e.g. a non-ischemic cardiomyopathy, mitral valve regurgitation, ischemic cardiomyopathy, or aortic stenosis or regurgitation. The method can include other features described herein.
In another aspect, the invention features a method that includes: restricting the flow of blood through coronary vessels via coronary vein blockade; and introducing a compound into the lumen of the coronary artery. For example, the compound is delivered by a viral delivery system, e.g., an adeno-associated virus delivery system (e.g., an AAV6 system). The compound can be any compound described herein, e.g., a compound that includes a nucleic acid sequence encoding SERCA2a.
The method can include allowing the heart to pump, e.g., while the coronary vein drainage is restricted; and reestablishing the flow of blood through coronary vessels, thereby allowing the said compound to flow into and be delivered to heart cells. The method can include a preconditioning step. For example, the method can include ischemic preconditioning in both the left anterior descending artery and the left circumflex artery is performed. The ischemic preconditioning can be for between 30 seconds and three minutes, e.g., about a one-minute ischemic preconditioning.
The subject is, for example, a human, e.g., a human is suffering from heart failure.
The method can include one or more of: obstructing the left anterior descending artery, obstructing the left circumflex artery, obstructing the great coronary vein, and obstructing the anterior interventricular vein. For example, the method includes obstructing the left circumflex artery and the middle cardiac vein.
The coronary vein blockade can comprise an occlusion by an angioplasty balloon. For example, the obstructions comprise partial or complete occlusions by angioplasty balloons.
The compound can be introduced, for example, by a percutaneous antegrade intracoronary transfer comprising an injection through the center lumen of the inflated angioplasty balloon in either artery. For example, blood flow can be restricted for between 30 seconds and 5 minutes, e.g., for about 2 to 4 minutes or about 3 minutes. It is possible to perform ischemic preconditioning in the left anterior descending artery and/or or the left circumflex artery.
The method can further include opening the pericardium. The method can include other features described herein.
In other aspect the invention features a method of delivering a compound to the heart of a subject, e.g., a subject undergoing a device implantation, e.g., a left ventricular assist device implantation. The method includes introducing (e.g., injecting) the compound into at least one site of the left ventricle. The compound can be delivered using a viral delivery system; e.g., the viral delivery system is introduced. For example, the subject is a human, e.g., a human who has heart failure, e.g., non-ischemic cardiomyopathies.
In one embodiment, the method includes: identifying one or more sites in the left ventricle surface; injecting each site with a virus-containing solution (e.g., between 0.01 to 0.4 ml), below the surface, e.g., at about 5 mm below the surface.
The method can further include evaluating the effects of delivering the compound, e.g., evaluating the effect of the treatment on a parameter related to contractility, e.g., sarcoplasmic reticulum Ca2+ ATPase pump activity. The evaluation can include echocardiography and/or metabolic stress testing. The method can include tissue harvest at the time of transplantation.
In one aspect, the invention features a method of treating a subject, e.g., by treating a heart cell of the subject. The subject is a human, or a non-human animal. The method includes introducing into a heart cell, e.g., in a heart tissue, or in a heart, in vitro or in vivo, a nucleic acid which results in the expression of SERCA2a. The method allows for improving the condition of a subject having a heart disorder.
In a preferred embodiment, treating the heart cell includes modulating the ratio of phospholamban to SERCA2a in the heart cell.
In a preferred embodiment, the subject, e.g., a human or a non-human animal, is at risk for, or has, a heart disorder, e.g., heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal heart contractility, or abnormal Ca2+ metabolism.
In one embodiment, the disorder is one characterized by a deficient SR Ca2+ uptake, or one characterized by an increased SR Ca2+ uptake.
In a preferred embodiment, the heart disorder is heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal heart contractility, or abnormal Ca2+ metabolism.
In a preferred embodiment, the nucleic acid is introduced into the subject by somatic gene transfer, e.g., by catheter perfusion. In another preferred embodiment, the nucleic acid is introduced into the subject by somatic gene transfer and is not introduced into the germ line of the subject.
In a preferred embodiment, the subject is a human.
In a preferred embodiment, the nucleic acid is introduced in vitro.
In a preferred embodiment, the nucleic acid is introduced in vivo.
In another embodiment, the method further includes evaluating in the subject any of: survival, cardiac metabolism, heart contractility, heart rate, ventricular function, e.g., left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), Ca2+ metabolism, e.g., intracellular Ca2+ concentration, e.g., peak or resting [Ca2+]. SR Ca2+ ATPase activity, phosphorylation state of phospholamban, force generation, relaxation and pressure of the heart, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, sodium potassium ATPase pump activity, activity of 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, IGF-1 receptor, P13 kinase, AKT kinase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin. The evaluation can be performed before, after, or during the treatment.
In another aspect, the invention features a method of treating a subject, e.g., by treating a heart cell of the subject. The subject is a human, or a non-human animal. The method includes introducing into the subject a nucleic acid that decreases phospholamban activity. In one example, the nucleic acid encodes an antisense sequence, which is at least partially complementary to a phospholamban DNA sequence. In another example, the nucleic acid cassette can produce an siRNA.
In a preferred embodiment, the subject is at risk for, or has, a heart disorder, e.g., heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal heart contractility, or abnormal Ca2+ metabolism.
In a preferred embodiment, the heart disorder is heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal heart contractility, or abnormal Ca2+ metabolism.
In a preferred embodiment, the nucleic acid is introduced into the subject by somatic gene transfer, e.g., by catheter perfusion. In another preferred embodiment, the nucleic acid is introduced into the subject by somatic gene transfer and is not introduced into the germ line of the subject.
In a preferred embodiment, the subject is a human, e.g., a human who is at risk for, or has, heart failure.
In a preferred embodiment, the nucleic acid is introduced in vitro.
In a preferred embodiment, the nucleic acid is introduced in vivo.
In another embodiment, the method further includes evaluating in the subject any of: survival, cardiac metabolism, heart contractility, heart rate, ventricular function, e.g., left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), Ca2+ metabolism, e.g., intracellular Ca2+ concentration, e.g., peak or resting [Ca2+], SR Ca2+ ATPase activity, phosphorylation state of phospholamban, force generation, relaxation and pressure of the heart, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, sodium potassium ATPase pump activity, activity of 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, IGF-1 receptor, PI3 kinase, AKT kinase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin. The evaluation can be performed before, after, or during the treatment.
In another aspect, the invention features a method of treating a subject, e.g., by treating a heart cell of the subject. The subject is a human, or a non-human animal. The method includes introducing into the subject, e.g., the heart of the subject, a first nucleic acid which results in the expression of an antisense nucleic acid which is at least partially complementary to a phospholamban DNA sequence, and introducing into the subject a second nucleic acid which results in the expression of SERCA2a.
In a preferred embodiment, the subject, e.g., a human or a non-human animal, is at risk for, or has, a heart disorder, e.g., heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal heart contractility, or abnormal Ca2+ metabolism.
In a preferred embodiment, the heart disorder is heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal heart contractility, or abnormal Ca2+ metabolism.
In a preferred embodiment, the first and second nucleic acids are introduced into the subject by somatic gene transfer, e.g., by catheter perfusion. In another preferred embodiment, the nucleic acids are introduced into the subject by somatic gene transfer and are not introduced into the germ line of the subject.
In a preferred embodiment, the subject is a human.
In a preferred embodiment, the nucleic acids are introduced in vitro.
In a preferred embodiment, the nucleic acids re introduced in vivo.
In another embodiment, the method further includes evaluating in the subject any of: survival, cardiac metabolism, heart contractility, heart rate, ventricular function, e.g., left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), Ca2+ metabolism, e.g., intracellular Ca2+ concentration, e.g., peak or resting [Ca2+], SR Ca2+ ATPase activity, phosphorylation state of phospholamban, force generation, relaxation and pressure of the heart, a force frequency relationship, cardiocyte survival or apoptosis or ion channel activity, e.g., sodium calcium exchange, sodium channel activity, calcium channel activity, sodium potassium ATPase pump activity, activity of 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, IGF-1 receptor, P13 kinase, AKT kinase, sodium-calcium exchanger, calcium channel (L and T), calsequestrin or calreticulin. The evaluation can be performed before, after, or during the treatment.
In another aspect, 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.
In preferred embodiments, the method includes evaluating the effect of the treatment on a parameter related to heart function. The parameter, by way of example, can include an assessment of contractility, Ca2+ metabolism, e.g., intracellular Ca2+ 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.
In preferred embodiments, 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.
In preferred embodiments, the treatment is administered in vitro. In preferred embodiments the cell is derived from an experimental animal or a human. In preferred embodiments the cell can be cultured and/or immortalized.
In preferred embodiments, 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. For example, a mouse phospholamban can be expressed in a mouse cell or a human phospholamban can be expressed in a cell from an experimental animal.
In preferred embodiments, 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 or an adeno-associated vector (e.g., AAV6).
In another aspect, 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.
In preferred embodiments, the method includes evaluating the effect of the treatment on a parameter related to heart function. The parameter, by way of example, can include an assessment of contractility, Ca2+ metabolism, e.g., intracellular Ca2+ 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.
In preferred embodiments, 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.
In preferred embodiments, the treatment is administered in vitro. In preferred embodiments the heart is derived from an experimental animal or a human.
In preferred embodiments, 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. For example, 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.
In preferred embodiments, 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 or an adeno-associated vector (e.g., AAV6).
In another aspect, 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.
In preferred embodiments, the method includes evaluating the effect of the treatment on a parameter related to heart function. The parameter, by way of example, can include an assessment of contractility, Ca2+ metabolism, e.g., intracellular Ca2+ 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.
In preferred embodiments, 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.
In preferred embodiments, the treatment is administered in vitro. In preferred embodiments the heart tissue is derived from an experimental animal or a human.
In preferred embodiments, 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. For example, 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.
In preferred embodiments, 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 or an adeno-associated vector (e.g., AAV6).
In another aspect, 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.
In preferred embodiments, the method includes evaluating the effect of the treatment on a parameter related to heart function. The parameter, by way of example, can include an assessment of contractility, Ca2+ metabolism, e.g., intracellular Ca2+ 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.
In preferred embodiments, 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.
In preferred embodiments, 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. For example, a mouse phospholamban can be expressed in a mouse cell or a human phospholamban can be expressed in a cell from an experimental animal.
In preferred embodiments, 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. In further preferred embodiments 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 Ca2+ concentration can be measured in the first cell and the SR Ca2+-ATPase activity can be measured in the second cell or the intracellular Ca2+ concentration can be measured in both cells.
In another aspect, 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.
In preferred embodiments, the method includes evaluating the effect of the treatment on a parameter related to heart function. The parameter, by way of example, can include an assessment of contractility, Ca2+ metabolism, e.g., intracellular Ca2+ 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.
In preferred embodiments, 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.
In preferred embodiments, 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. For example, a mouse phospholamban can be expressed in a mouse cell or a human phospholamban can be expressed in a cell from an experimental animal.
In preferred embodiments 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.
In another aspect, 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.
In preferred embodiments, the method includes evaluating the effect of the treatment on a parameter related to heart function. The parameter, by way of example, can include an assessment of contractility, Ca2+ metabolism, e.g., intracellular Ca2+ 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.
In preferred embodiments, 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.
In preferred embodiments, 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. For example, 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.
In preferred embodiments 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.
In another aspect, 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.
In preferred embodiments, the compound includes: a nucleic acid which directs the expression of a peptide, e.g., a phospholamban or a SR Ca2+-ATPase and a viral vector suitable for somatic gene delivery, e.g., an adenoviral vector or an adeno-associated vector (e.g., AAV6).
In preferred embodiments, the subject is at risk for a heart disorder, e.g., heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection.
In preferred embodiments, 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.
In preferred embodiments, the method further includes restricting blood flow into the left side of the heart, e.g., by restricting the pulmonary circulation through obstruction of the pulmonary artery, so as to lessen dilution of the compound.
In preferred embodiments the method further includes opening the pericardium and introducing the compound, e.g., using a catheter.
In preferred embodiments, 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.
In preferred embodiments, the nucleic acid, which directs the expression of the peptide, is homogeneously overexpressed in the heart of the subject.
In another aspect, 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.
In another aspect, 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.
In another aspect, 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.
In another aspect, 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.
In preferred embodiments, the nucleic acid is introduced using the methods described herein.
In preferred embodiments, the phospholamban can be from the same species as the subject or it can be from a different species. For example, 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.
In preferred embodiments, 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 or an adeno-associated vector (e.g., AAV6).
In preferred embodiments, 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.
As used herein, 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. For example, such treatment can be a surgical procedure, or the administration of a therapeutic agent such as a drug, a peptide, an antibody, an ionophore and the like.
As used herein, the term “heart disorder” refers to a structural or functional abnormality of the heart that impairs its normal functioning. For example, 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 Ca2+ metabolism, and disorders characterized by arrhythmia.
As used herein, the term “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. For example, the cell can be a cardiac myocyte.
As used herein, the term “heart” refers to a heart present in a subject or to a heart which is maintained outside a subject.
As used herein, the term “heart tissue” refers to tissue which is derived from the heart of a subject.
As used herein, the term “somatic gene transfer” refers to the transfer of genes into a somatic cell as opposed to transferring genes into the germ line.
As used herein, 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.
As used herein, 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.
As used herein, the term “misexpression” refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over- or underexpression; 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 transgenic nucleic acid.
As used herein, 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.
As used, herein, 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. For example, 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.
As used herein, 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.
As used herein, the term “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.
Heart failure secondary to systolic dysfunction is a disease of epidemic proportions in the U.S. with over 5 million effected individuals. Heart failure accounts for over one million hospitalizations, 400,000 deaths, and 40 billion dollars in health care expenses each year with 5-year survival being less than 50%. Recent advances in therapy for patients with mild to moderate symptoms have improved symptoms, decreased hospitalizations and lengthened survival. However, heart failure is a progressive disease and most patients eventually develop unremitting end-stage symptoms. Some patients present either at the time of initial diagnosis or during the course of their disease with fulminant heart failure requiring immediate therapeutic intervention. Historically, transplantation has provided the primary treatment for these patients.
Complete recovery of ventricular function after heart failure is still elusive. Failing human hearts of most etiologies are characterized by abnormal intracellular Ca2+ regulation secondary to a deficiency in the SR Ca2+ ATPase (SERCA2a) pumps. Improvement of contractile function in vitro in human isolated cardiomyocytes has been achieved by reconstituting SERCA2a by gene transfer. This target may offer a new modality for the treatment of heart failure in humans. Our results showed that a percutaneous, clinically feasible method of gene transfer of SERCA2a in mitral-regurgitation-induced model of heart failure in the swine reverses contractile dysfunction. This disclosure includes clinical applications of SERCA2a gene therapy for ventricular dysfunction.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The drawings are first briefly described.
SERCA2a/Phospholamban and Heart Disorder
Somatic gene transfer, e.g., adenoviral or adeno-associated viral gene transfer, is particularly effective in mammalian myocardium both in vivo and in vitro. Gene transfer techniques can be used to ameliorate at least one symptom of a subject having heart failure or other heart disorder associated with altered SR Ca2+ physiology. In particular, adeno-associated viral systems (e.g., AAV6) can be used to provide a nucleic acids to heart cells in a subject. The viral system can deliver a gene encoding a protein that modulates heart cell activity, e.g., a gene encoding SERCA2a or other transmembrane regulator.
We observed that, with respect to heart cells, AAV6 conferred the fastest gene expression, as well as the most specific and efficient expression in the heart, compared to other AAVs. (See Example 13 and
In addition, adenoviral gene transfer of SERCA2a is both dose dependent and time dependent in rat neonatal cardiomyocytes. An adenovirus encoding phospholamban under the RSV promoter, provided a 4-fold increase in phospholamban, which was also dose dependent. 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.
The expression of 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 Ca2+ transient and impaired relaxation. Increasing levels of phospholamban relative to SERCA2a significantly altered intracellular Ca2+ handling in the isolated cardiomyocytes by prolonging the relaxation phase of the Ca2+ transient, decreasing Ca2+ release, and increasing resting Ca2+. These results show that altering the relative ratio of phospholamban to SERVA2a can account for the abnormalities in Ca2+ handling observed in failing ventricular myocardium. In addition, overexpressing SERCA2a can largely “rescue” the phenotype created by increasing the phospholamban-to-SERCA2a ratio. Restoring the normal phospholamban-to-SERCA2a ratio through somatic gene transfer can correct the abnormalities of Ca2+ handling and contraction seen in failing hearts.
Evaluation of Treatment
A treatment can be evaluated by assessing the effect of the treatment on a parameter related to contractility. For example, SR Ca2+ ATPase activity or intracellular Ca2+ 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, such as may be derived with cardiac muscle cell extracts, 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 Ca2+, which is mediated by a test therapeutic agent. Moreover, 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 Ca2+. 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. Moreover, 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. For example, 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. Methods for isolating tissue (e.g., heart tissue) and the disaggregation of tissue to obtain cells (e.g., heart cells) are described in Freshney R. I., Culture of Animal Cells, A Manual of Basic Technique, Third Edition, 1994, the contents of which are incorporated herein by reference.
Viral Vectors Suitable for Somatic Gene Transfer
Expression vectors, suitable for somatic gene transfer, can be used to express the compound, e.g., a SERCA2a gene or a phospholamban gene. Examples of such vectors include replication defective retroviral vectors, adenoviral vectors and adeno-associated viral vectors (AAVs).
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. Opin. Biotechnol. 5(6):617-625, 1994, the contents of which are incorporated herein by reference.
Adeno-associated virus is a nonpathogenic human parvovirus, capable of site-specific integration into chromosome 19. Fisher et al., Nature Medicine 3(3):306-312, 1997. Replication of the virus, however, requires a helper virus, such as an adenovirus. Fisher et al., Nature Medicine 3(3):306-312, 1997. An AAV coding region can be replaced with nonviral genes, and the modified virus can be used to infect both dividing and non-dividing cells. Xiao et al., Jo. Virol. 70(11): 8098-8108, 1996; Kaplitt et al., Ann. Thorac. Surg. 62: 1669-1676, 1996. Exemplary methods for the preparation and use of AAVs are described in Fisher et al., Nature Medicine 3(3):306-312, 1997; Xiao et al., Jo. Virol. 70(11): 8098-8108, 1996; Kaplitt et al., Ann. Thorac. Surg. 62: 1669-1676, 1996, the contents of which are incorporated herein by reference.
AAV6 is specific and confers fast expression in the heart. Example 14 demonstrates that gene transfer with AAV6 in the heart of a large animal induces excellent efficiency. Example 15 shows that AAV6.CMV.SERCA2a delivered to a pre-clinical large animal model of heart failure induces improvement in ventricular function and reverses heart failure.
Expression of Phospholamban
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.
The following examples which further illustrate the invention should not be construed as limiting.
1. Construction of El-Deleted Recombinant Adenovirus Vectors
The construction of 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.βgal, which carries a nuclear localizing form of β-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. Briefly, the phospholamban cDNA was subcloned into the bacterial plasmid vector pAdRSV4, which uses the RSV 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. By use of this strategy, independent plaques were isolated, and expression of phospholamban protein was verified by immunostaining. A positive plaque was further plaque-purified, and protein expression was reconfirmed to yield the recombinant adenovirus Ad.RSV.PL. This adenovirus is structurally similar to Ad.RSV.βgal 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. et al., Methods in Molecular Biology: Gene Transfer and Expression Protocols, 1991, 109-128, the contents of which are incorporated herein by reference. The titers of stocks used for these studies were as follows: 3.1×1014 pfu/mL for Ad.RSV.PL 2.6×1010 pfu/m: for Ad.RSV.SERCA2a, and 2.7×1014 pfu/mL for Ad.RSV.βgal, with a particle-to-pfu ratio of 40:1, 42:1, and 37:1, respectively.
2. Preparation of 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. Measurements of cell shortening and cytosolic Ca2+ were performed on neonatal cardiomyocytes cultured on round, coated, glass coverslips (0.1 mm thickness, 31 mm diameter) in 35 mm culture dishes. Cells were counted using a hemocytometer. Approximately 5×105 cells were plated in each coverslip.
3. Adenoviral Infection of Isolated Cells
In three different infection experiments with increasing concentrations of Ad.RSV.βgal, the percentages of cells expressing βgal after 48 hours, by histochemical staining in 10 different high-power fields were 98.2% (multiplicity of infection, 1 pfu/cell), 99.1% (multiplicity of infection, 10 pfu/cell), and 100% (multiplicity of infection, 100 pfu/cell). In a similar manner, myocardial cells were infected with three concentrations of Ad.RSV.PL 1.0, 10. and 100 pfu/cell for 48 hours. Infection with either Ad.RSV.βgal, Ad.RSV.PL, or Ad.RSV.SERCA2a did not change the morphology of the cells. For each infection experiment with the adenovirus, one myocyte was used to measure functional parameters. As shown in
As shown in
The response to increasing stimulation frequencies in mammalian cardiomyocytes is governed by the SR. We have shown that in the uninfected cardiomyocytes, an increase in stimulation frequency did not significantly alter either peak or resting [Ca2+]. This response is typical of rat cardiomyocytes that have either a flat response to increasing frequency of stimulation or a decrease in contractile force. However, in cardiomyocytes infected with Ad.RSV.PL, there was a significantly greater increase in resting [Ca2+] and a decrease in peak [Ca2+]. These results would suggest that diminished SR Ca2+ uptake leads to a diminished CA2+ release, which becomes even more accentuated at higher frequencies of stimulation.
4. Intracellular Ca2+ Measurements and Cell Shortening Detection
Measurements of intracellular Ca2+ and cell shortening were performed as described earlier in Hajjar et al. (1997), Kang et al. (1995) and Kang et al (1996), the contents of which are incorporated herein by reference. Briefly, myocardial cells were loaded with the Ca2+ indicator fura 2 by incubating the cells in medium containing 2 μmol/L fura 2-AM (Molecular Probex) for 30 minutes. The cells were then washed with PBS and allowed to equilibrate for 10 minutes in a light-sealed temperature-controlled chamber (32° C.) mounted on a Zeiss Axlovers 10 inverted microscope (Zeiss). 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. [Ca2+] was calculated according to the following formula: [Ca2+]=Kd (R−Rmin)/(Rmax−R)D, where R is the ratio of fluorescence of the cell at 360 and 380 nm: Rmin and Rmax represent the ratios of fura 2 fluorescence in the presence of saturating amounts of Ca2+ and effectively “zero Ca2+ respectively, Kd is the dissociation constant of Ca2+ from fura 2; and D is the ratio of fluorescence of fura 2 at 380 nm in zero Ca2+ and saturating amounts of Ca2+. Unless otherwise stated, measurements of peak [Ca2+] were made at the end of diastole. High-contrast microspheres attached to the cell surface of the cardiomyocytes were imaged using a charge-coupled device video camera attached to the microscope, and motion along a selected raster line segment who quantified by a video motion detector system (IONOPTIX). As shown in
Similarly, peak [Ca2+] decreased up to 10 pfu/cell, with no further decrease at 100 pfu/cell. Coinfection with Ad.RSV.SERCA2a (multiplicity of infection of 10 pfu/cell) restored both the Ca2+ transient and the shortening to near normal levels, as shown in
Adenoviral gene transfer of phospholamban provides an attractive system for further elucidation of the effects of inhibiting SR Ca2+-ATPase on intracellular Ca2+ handling. A decrease in SRCa2+ uptake rates is expected to lead to a smaller amount of Ca2+ sequestered by the SR, resulting in a smaller amount of Ca2+ release. In neonatal cardiomyocytes, a significantly prolonged Ca2+ transient and a higher resting [Ca2+] was observed reflecting the decreased Ca24 uptake and a decrease in peak [Ca2+] levels reflecting less Ca2+ available for release. These results show that the SR Ca24-ATPase is important during relaxation by controlling the rate and amount of CA2+ sequestered and during contraction by releasing the Ca2+ that is taken up by the SR. Overexpression of both phospholamban and SERCA2a partially restored the Ca2+ transient; however, the time course of the Ca2+ transient was still prolonged in cardiomyocytes infected with both Ad.RSV.SERCA2a and Ad.RSV.PL. This finding was somewhat surprising, since the SR Ca2+-ATPase activity was restored to normal and even enhanced in cardiomyocytes infected with both Ad.RSV.SERCA2a and Ad.RSV.PL.
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 Ca2+ pump, thereby enhancing the SR Ca2+-ATPase, we were specifically interested in evaluating the effects of β-agonism on the relaxation phase of the Ca2+ transient. In the basal state, the overexpression of phospholamban significantly prolongs the Ca2+ transient. As shown in
5. Preparation of 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 500 g for 20 minutes. The resultant supernatant was centrifuged at 25,000 g for 60 minutes to pellet the SR-enriched membrane. The pellet was re-suspended in a buffer containing (mmol/L) KCl 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. Biochem., 1976, 72:248-260, the contents of which are incorporated herein by reference, using bovine scrum albumin for the standard curve (Bio-Rad).
6. Western Blot Analysis of Phospholamban and SERCA2a in SR Preparations
SDS-PAGE was performed on the isolated membranes from cell cultures under reducing conditions on a 7.5% separation gel with a 4% stacking gel in a Miniprotean II cell (Bio-Rad). Proteins were then transferred to a Hybond-ECL nitrocellulose for 2 hours. The blots were blocked in 5% nonfat milk in Tris-buffered saline for 3 hours at room temperature. For immunoreaction, the blot was incubated with 1:2500 diluted monoclonal anti-SERCA2 antibody (Affinity BioReagents) or 1:2500 diluted anti-cardiac phospholamban monoclonal IgG (UBI) for 90 minutes at room temperature. After washing, 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).
7. SR Ca2+-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. By use of a photomotor (Beckman DU 640) adjusted at a wavelength of 540 nm, oxidation of NADH (which is coupled to the SR Ca2+-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 Ca2+-ATPase was calculated as follows: Δabsorbance/6.22×protein×time (in nmol ATP/mg protein×min). The measurements were repeated at different [Ca2+] levels. The effect of the specific Ca2+-ATPase inhibitor CPA at a concentration range of 0.001 to 10 μmol/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. As shown in
The SR Ca2+-ATPase plays a key role in excitation-contraction coupling, lowering Ca2+ during relaxation in cardiomyocytes, and “loading” the SR with Ca2+ for the subsequent release and contractile activation. The Ca2+-pumping activity of this enzyme is influenced by phospholamban. In the unphosphorylated state, phospholamban inhibits the Ca2+-ATPase, whereas phosphorylation of phospholamban by cAMP-dependent protein kinase and by Ca2+ calmodulin-dependent protein kinase reverses this inhibition. Therefore, an increase in phospholamban content should decrease the affinity of the SR Ca2+ pump for Ca2+. As shown in
8. Statistical Analyses
Data were represented as mean±SEM for continuous variables. Student's test was used to compare the means of normally distributed continuous variables. Parametric one-way ANOVA techniques were used to compare normally distributed contiguous variables among uninfected groups of cells, Ad.RSV.βgal-infected cells, Ad.RSV.PL-infected cells, and Ad.RSV.SERCA2a-infected cells.
9. Adenoviral Somatic Gene Transfer
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 μl 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
The expression pattern seen after direct injection is localized, whereas the catheter-based technique is essentially homogeneous. The pressure tracing of the Ad.PL transduced hearts displayed a markedly prolonged relaxation and reduced pressure development as shown in
10. Gene Transfer of the Sarcoplasmic Reticulum Calcium ATPase Improves Left Ventricular Function in Aortic-Banded Rats in Transition to Failure
In human and experimental models of heart failure, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) activity has been shown to be significantly decreased. In this example, the ability of SERCA2a expression to improve ventricular function in heart failure was investigated by creating an ascending aortic constriction in 10 rats. After 20-24 weeks, during the transition from left ventricular hypertrophy to failure, 200 μl of a solution containing 5×109 plaque forming units of replication-deficient adenovirus carrying SERCA2a (Ad.SERCA) (n=4) or the reporter gene β-galctosidase (Ad.βgal) (n=6) were injected intracoronary via the catheter-based technique described supra. Two days after the procedure, the rats underwent open chest measurement of left ventricular pressure. Heart rate (HR), left ventricular end-diastolic pressure (LVEDP), and left ventricular systolic pressure (LVSP) were measured. Peak+dP/dt and −dP/dt were calculated. As shown in Table 2, the magnitudes of peak +dP/dt and −dP/dt which are indices of systolic and diastolic function were markedly increased in hearts transduced with the SERCA2a carrying adenovirus. Therefore, this example indicates that overexpression of SERCA2a in a rat model of pressure-overload hypertrophy in transition to failure improved left ventricular systolic and diastolic function.
#p < 0.05 compared to Ad.βgal
11. Gene Transfer of Antisense of Phospholamban Improves Contractility in Isolated Cardiomyocytes in Rat and Human
A. 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. In this Example, an adenovirus carrying the full length antisense cDNA of phospholamban (Ad.asPL) was constructed using the methods described above. This construct was then used to infect neonatal cardiac myocytes as described in Example 3. As indicated in
B. Since human heart failure is mainly due to coronary artery disease or is idiopathic in nature, we ablated phosholamban by antisense strategies using adenoviral gene transfer in isolated ventricular cardiac myocytes from eight patients with end-stage heart failure of various etiologies (idiopathic, ischemic and hypertrophic). The co-expression of green fluorescent protein GFP allowed us to identify the cells that were infected and expressing the transgene after 48 hours.
Following isolation, failing human cardiomyocytes were infected with an adenovirus carrying antisense phospholamban. Forty-eight hours after infection, a cardiomyocyte is visualized with white light and at 510 nm with single excitation peak at 490 nm of blue light. Co-expression of GFP demonstrated visually the ablation of phospholamban in the cell. Recordings were performed from cardiomyocytes isolated from a donor nonfailing heart and from a failing heart infected with either an adenovirus expressing green fluorescent protein, Ad.GFP or carrying the antisense of phospholamban, Ad.asPL, stimulated at 1 Hz at 37° C. The failing cell had a characteristic decrease in contraction and prolonged relaxation along with a prolonged Ca2+ transient. Ablation of phospholamban in the failing cardiomyocyte normalized these parameters. Ablation of phospholamban in failing cardiomyocytes induced a faster contraction velocity (15.4±2.7 vs 6.9±2% shortening/sec, p=0.008 )and enhanced relaxation velocity (18.6±4.4 vs 6.6±3.7, p=0.01).
These results show that regardless of etiology, in human heart failure, improving calcium cycling by decreasing phospholamban inhibition to SERCA2a, restores contractility in failing ventricular cells of different etiologies. These findings also extend previous results that overexpression of SERCA2a improves contractile function in human failing cardiac myocytes. Finally, these findings underscore the importance of validating experimental results from murine models in relevant human tissues.
12. Gene Transfer of the Sarcoplasmic Reticulum Calcium ATPase Improves Survival in Aortic-Banded Rats in Transition to Failure
Pharmacological agents that increase contractility have been repeatedly shown to worsen survival in patients with congestive heart failure and to increase the energetic requirements on the heart (O'Connor et el. (1999). Am Heart J 138(1 Pt 1):78-86). Since the heart performs uninterrupted biochemical and mechanical work, it requires a continuous supply of energy in the form of ATP by mostly oxidative metabolism under normal conditions with major energy reserve molecule represented by phosphocreatine (PCr). In the normal heart, although the majority (60%) of the energy consumption is due to cross-bridge cycling, relaxation requires an energy expenditure of 15% to remove Ca2+ from the cytoplasm. This high level of free energy |ΔGp| required by the SERCA2a reaction is directly related to the magnitude of the Ca2+ gradient across the SR (Tian et al. (1998) Am J Physiol 275(6 Pt 2):H2064-71). Failing hearts have a reduced ratio PCr/ATP in human as well as in animal models of heart failure so that less energy reserve is available for the cellular processes. This decrease in energy reserve has been shown to be by itself a predictor of mortality in patients with dilated cardiomyopathy (Neubauer et al. (1997) Circulation 96(7):2190-6).
In this Example, unlike other pharmacologic agents that increase inotropy, reconstitution of normal levels of SERCA2a by adenoviral gene transfer improves contractile performance as well as survival in aortic banded rats with developed heart failure without adversely affecting energetics possibly by reducing the intracellular diastolic Ca2+ overload.
Experimental Protocols for Examples 1-13
A. Construction of Recombinant Adenoviruses
We constructed an adenovirus containing SERCA2a and GFP controlled by separate CMV promoters (Ad.SERCA2a). An adenovirus containing both β-galactosidase and GFP controlled by separate CMV promoters (Ad.βgal-GFP) was used as control as described earlier (Haq et al. (2000) J Cell Biol 151(1):117-130). The titer of stocks used for these studies measured by plaque assays were: 3×1011 pfu/ml for Ad.βgal-GFP and 1.8×1011 pfu/ml for Ad.SERCA2a with a particle/pfu ratio of 8:1 and 18:1 respectively (viral particles/ml determined using the relationship one absorbance unit at 260 nm is equal to 1012 viral particles/ml). These recombinant adenoviruses were tested for the absence of wild-type virus by PCR of the early transcriptional unit E1.
B. Aortic Banding
Four-week old Sprague Dawley rats (70-80 g) were obtained from Taconic Farms. After 2-3 days of acclimatization, the rats were anesthetized with intraperitoneal pentobarbital (65 mg/kg) and placed on a ventilator. A suprasternal incision was made exposing the aortic root and a tantalum clip with an internal diameter of 0.58 mm (Weck, Inc.) was placed on the ascending aorta. Animals in the sham group underwent a similar procedure without insertion of a clip. The supraclavicular incision was then closed and the rats were transferred back to their cages. The supraclavicular approach was performed because during gene delivery a thoracotomy is necessary and by not opening the thorax during the initial aortic banding avoids adhesions when gene delivery is performed thereby decreasing the morbidity of the procedure.
Animals were initially divided into two groups: one group of 45 animals with aortic banding and a second group of 42 animals which were sham-operated. Three animals did not survive the initial operation in the aortic banding group and 2 animals did not survive in the sham-operated group. In the animals which were aortic banded we waited 26-28 weeks for the animals to develop left ventricular dilatation prior to cardiac gene transfer. In this last group as well as in the sham-operated group, fourteen animals did not undergo gene transfer and were followed longitudinally. The rest of the animals underwent adenoviral gene transfer with either Ad.SERCA2a or Ad.bgal-GFP.
C. 31P NMR Measurements
NMR Spectroscopy
Stable energetic state in rat hearts was confirmed from 31p NMR signals of phosphocreatine, ATP, and inorganic phosphate as described in Lewandowski et al. ((1995) American J Physiol 269(1 Pt 2):H160-8). NMR data was collected on a Bruker 400 MHz spectrometer interfaced to a 9.4 tesla, vertical bore, superconducting magnet. 31P spectra were obtained from isolated hearts perfused within a broad-band, 20 mm NMR probe (Bruker Instruments). 31P-NMR spectra were acquired in 128 scans using a 161 MHz, 45° excitation pulse, a 1.8s repetition time, 35 ppm sweep width, and 8 K data set. Post processing of the summed free induction decay (FID's) NMR data included 20 Hz line broadening, Fourier transformation, and phase correction. Peak assignments were referenced to the well established resonance signal of PCr at 0 ppm, with identification and assignment of the α, β, and γ phosphate signals of ATP. Signal intensity was determined using NMR-dedicated data analysis.
Isolated, Perfused Rat Heart Preparation:
Hearts were retrograde perfused from a 100 cm hydrostatic perfusion column with modified Krebs-Henseleit buffer (116 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM NaH2PO4, and 25 mM NaHCO3, equilibrated with 95% O2/5% CO2 at 37° C.) that contained 5 mM glucose in a 2 liter reservoir. A polyethylene catheter was inserted into the pulmonary artery allowing collection of coronary effluent for measurement of oxygen consumption with a blood-gas analysis machine. Hearts spontaneously beat, contracting against a fluid-filled intraventricular balloon connected to a pressure transducer and inflated to an end diastolic pressure of 5 mm Hg. The isolated hearts were placed in a borosilicate glass vial. A 10-15 ml volume of coronary effluent bathed the heart. Temperature was maintained at 37° C. with both perfusate temperature and a thermal control unit interfaced to the NMR system.
D. Serial Echocardiographic Assessment
After eighteen weeks of banding, serial echocardiograms were performed on a weekly basis. Animals were anesthetized with pentobarbital 40 mg/kg intra-peritoneally, and the anterior chest shaved. Transthoracic M-mode and two-dimensional echocardiography was performed with a Hewlett-Packard Sonos 5500 imaging system (Andover, Mass.) with a 12 MHz broadband transducer. A mid-papillary level left ventricular short axis view was used and the images were stored digitally. Measurements of posterior wall thickness, left ventricular diastolic dimension and fractional shortening were performed off-line. The epicardial surface of the anterior wall was not reliably visualized in all animals. Gene transfer was performed in all animals within 3 days of detection of a drop in fractional shortening of >25% compared to the fractional shortening at 18 weeks post-banding. In the sham operated rats, gene delivery was performed at 27 weeks.
E. Adenoviral Delivery Protocol
The group of animals subjected to aortic banding were further subdivided in three additional groups of sixteen, twelve, and fourteen receiving respectively Ad.SERCA2a, Ad.bgal-GFP, or no adenovirus. The group of sham-operated animals was also subdivided into three groups of fourteen, twelve, and fourteen Ad.SERCA2a, Ad.bgal-GFP, or no adenovirus. The adenoviral delivery system has been described in Miyamoto et al. ((2000) Proc Natl Acad Sci USA 97(2):793-8). Briefly, after anesthetizing the rats and performing a thoracotomy, a 22 G catheter containing 200 ml of adenoviral solution (1010 pfu) was advanced from the apex of the left ventricle to the aortic root. The aorta and main pulmonary artery were clamped for 20 seconds distal to the site of the catheter and the solution injected, then the chest was closed, the animals were extubated and transferred back to their cages.
F. Measurements of Left Ventricular Volume & Elastance
Prior to euthanasia, rats in the different treatment groups were anesthetized with 65 mg/kg of pentobarbital and mechanically ventilated. After thoracotomy, a small incision was then made in the apex of the left ventricle and a 1.4 French high fidelity pressure transducer (Millar Instruments, Tex.) introduced into the left ventricle. Pressure measurements were digitized at 1.0 kHz and stored for further analysis using commercially available software (Sonolab, Sonometrics Co., Alberta, Canada) and four 0.7 mm piezoelectric crystals (Sonometrics Co., Canada) were placed over the surface of the left ventricle along the short axis of the ventricle at the level of the mitral valve and at the apex of the left ventricle to measure the inter-crystal distances. The left ventricular volume was derived using a mathematical model using CARDIOSOFT (Sonometrics Co., Canada). Left ventricular pressure-volume loops were generated under different loading conditions by clamping the inferior vena cava. The end-systolic pressure-volume relationship was obtained by producing a series of pressure dimension loops over a range of loading conditions and connecting the upper left hand corners of the individual pressure-dimension loops to generate the maximal slope.
G. Western Blot Analysis
SDS-PAGE was performed on the tissue lysate under reducing conditions on 7.5% separation gels with a 4% stacking gel in a Miniprotean II cell (BIORAD). Proteins were then transferred to a Hybond-ECL nitrocellulose for 2 hours and blocked in 5% nonfat milk for 3 hours. For immunoreaction, the blots were incubated with 1:2,500 diluted monoclonal antibodies to either SERCA2a (MA3-919; Affinity BioReagents, CO), or 1: 1,000 diluted anti-calsequestrin (MA3-913; Affinity Bioreagents) for 90 minutes at room temperature. After washing, the blots were exposed for 1 hour to HRP coniugated anti mouse antibody for chemo-luminescent detection.
H. SR Ca2+ ATPase Activity
SR Ca2+ ATPase activity assays were carried out based on a Pyruvate/NADH coupled reactions as previously described (Miyamoto, supra). Using a photometer (Beckman DU 640) adjusted at a wavelength of 340 nm, oxidation of NADH (which is coupled to the SR Ca2+-ATPase) was assessed at 37° C. in triplicates at different [Ca2+]. The reaction was carried out in a volume of 1 ml. Ca2+-ATPase activity was calculated as: Δ Absorbence/(6.22×protein×time) in nmol ATP/(mg protein×min).
I. Statistics
All values are presented as mean±sd. A two-factor ANOVA was performed to compare the different hemodynamic parameters among the different groups. For the echocardiography data, where the variables were examined at various intervals, ANOVA with repeated measures was performed. Comparison of survival in the different groups of animals was analyzed by a log-rank test with the Kaplan-Meier method. Statistical significance was accepted at the level of p<0.05.
Effect on Survival
Characterization of Animals
Following 18 weeks of aortic banding, the animals showed echocardiographic signs of left ventricular hypertrophy including an increase in wall thickness (both posterior and septal), an increase in posterior wall thickness, a decrease in left ventricular dimensions and an increase in fractional shortening as shown in Table 3. Of note at that time the animals showed no clinical signs of heart failure. After 26-27 weeks of banding, these animals had uniformly 1) small pericardial effusions, 2) pleural effusions, 3) an increase in lung weight, 4) ascites, and 5) dyspnea at rest all indicative signs of developed heart failure. Echocardiographically, LV end-diastolic dimensions increased and fractional shortening decreased.
PW: posterior wall thickness during diastole
LVDD: Left ventricular Diameter during diastole
LVSD: Left ventricular Systolic Diameter during Systole
FS: Fractional shortening
*p < 0.0005 vs Aortic banding (27 weeks)
‡p < 0.005,
{circumflex over ( )}p < 0.005,
#p < 0.05 vs control
Cardiac Gene Transfer & SERCA2a Expression
We first examined the expression of SERCA2a 28 days following adenoviral gene transfer. There was a decrease in SERCA2a in failing rats compared to sham operated rats. The protein expression of SERCA2a was decreased in failing rat left ventricles when compared to SERCA2a levels of sham left ventricles. Adenoviral gene transfer of SERCA2a in failing hearts increased SERCA2a protein expression restoring it to levels observed in the nonfailing hearts. The protein levels were normalized to calsequestrin which did not change among the different groups. To evaluate whether other tissues are infected we histologically examined sections of aorta, liver, and lung following infection with the cardiac specific Ad.SERCA2a. There was no evidence of SERCA2a expression in the aorta, in the liver and lungs. In the infected rat hearts there was no evidence of disruption of normal myocardial architecture or collagen deposition.
Thus, we restored SERCA2a protein to normal levels in failing hearts. In addition, we showed that the expression of SERCA2a to normal levels was sustained for up to four weeks. This seemed somewhat surprising since first generation adenoviruses induce transient expression peaking at 7-10 days and disappearing after 10 days 23. However, endogenous turnover of SERCA2a is about 14-15 days in young rats and longer in older rats 24 which would explain the sustained levels of SERCA2a.
SR Ca2+ ATPase Activity
We measured SR ATPase activity at a calcium concentration of 10 mM in 1) sham+Ad.bgal-GFP 2) failing+Ad.bgal-GFP, and 3) failing+Ad.SERCA2a. As shown in
SERCA2a Expression and Cardiac Energetics
Representative 31P-NNR spectra obtained from three groups of rats: 1) sham+Ad.bgal-GFP, 2) failing+Ad.bgal-GFP, 3) failing+Ad.SERCA2a are shown in
Thus, restoring SERCA2a levels to normal induced an improvement in the creatine phosphate to ATP ratio. The findings of improved cardiac energetics in developed heart failure was somewhat surprising since overexpression of SERCA2a would be anticipated to increase ATP hydrolysis thereby driving creatine phosphate down. Indeed, this increase in ATP hydrolysis is consistent with our observation of reduced PCr/ATP in the group of sham-operated hearts that were overexpressing SERCA2a. These results are also consistent with previous results showing that PCr/ATP was decreased in the phospholamban-deficient hearts relative to the wild-type hearts (Chu et al. (1996) Circ Res 79(6):1064-76). In heart failure, however, elevated calcium levels would increase energy demand. Furthermore, the thermodynamic reserve for the SR Ca2+-ATPase reaction is limited and in order to maintain the normal Ca2+ gradient, the SR Ca2+-ATPase reaction requires a |ΔGp| of at least 52 kJ/mol, 85-90% of it from ATP. Therefore, of all the ATPase reactions in cardiac myocytes, the SR Ca2+-ATPase reaction is the most vulnerable to a decrease in |ΔGp|.
Effects of SERCA2a Overexpression on LV Volumes and Elastance
To determine left ventricular function, pressure-ventricular analysis was performed in a subset of animals. LV volumes were significantly increased in the failing rats (0.64±0.05 vs. 0.35±0.03 ml, p<0.02). Overexpression of SERCA2a normalized LV dimensions (0.46±0.07 ml) in the failing hearts (
Effect on Morphological Parameters
As shown in table 4, the failing hearts had a significant increase in heart mass when normalized to either tibial length or to body mass. Tibial length which was used as an index of growth independent of body weight was uniformly constant across the different groups. Body mass was also not significantly different across the different groups. Overexpression of SERCA2a in the failing heart did not have a significant effect on left ventricular mass whether normalized to tibial length or body mass.
HW: heart weight
BW: Body weight
TL: Tibial length
*p < 0.05 compared to Sham + Ad.GFP
Survival Following Gene Transfer
Herein, we show that restoration of SERCA2a expression by cardiac gene transfer in vivo improves not only contractile function but also survival and cardiac energetics. In addition, cardiac gene transfer of SERCA2a induced a reversal of adverse remodeling in the failing hearts.
In this model of heart failure SERCA2a overexpression improved parameters of inotropy and normalized contractile reserve. These effects translate into an inotropic intervention. However, other inotropic interventions have been shown clinically to increase mortality in chronic heart failure in numerous trials (Stevenson (1998) New England Journal of Medicine 339(25):1848-50). There are, however, significant differences between increasing inotropy with pharmacological agents that usually increase cAMP and enhancing inotropy with the overexpression of SERCA2a. Unlike agents that increase cAMP, thereby increasing intracellular Ca2+, reconstituting normal SERCA2a levels decreases diastolic intracellular Ca2+ by increasing uptake into the SR and enhancing Ca2+ release. Beyond the contractile benefits of lowering diastolic Ca2+, it has been shown that sustained elevations of resting Ca2+ lead to activation of serine-threonine phosphatases including calcineurin inducing hypertrophy and cell death in cells (Lim (1999) Nature Medicine 5(3):246-7). Therefore a decrease in diastolic Ca2+ may in effect decrease the stimulation of phosphatases and reduce the pro-apoptotic and pro-hypertrophy signaling. Heart failure is associated with an increased incidence of ventricular arrhythmias and triggered activity is a probable mechanism of arrhythmogenesis in heart failure. The increase in intracellular calcium secondary to SERCA2a downregulation increases the arrhythmogenic potential. Preventing an increase in intracellular calcium by overexpression of SERCA2a prevents the induction of triggered activity. Furthermore, improvement in energetics is another important finding in these examples which may have a direct influence on survival.
Our results demonstrate that restoring SERCA2a expression can improve not only systolic and diastolic performance in failing hearts but also survival and cardiac energetics. Furthermore, SERCA 2a normalization halts the adverse remodeling that occurs with congestive heart failure.
We tested the ability of different serotypes of AAV to deliver an exogenous gene to the heart. Using the cross-clamping technique described below, we injected rat hearts with 1012 genomes of different AAV subtypes (1-6) carrying beta galactosidase under the CMV promoter. Rats were anesthetized with intraperitoneal pentobarbital and placed on a ventilator. The chest was entered from 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 the pulmonary artery were identified. A 22 G catheter containing 200 μl of adenovirus was advanced from the apex of the left ventricle to the aortic root. The aorta and the pulmonary arteries were clamped distal to the site of the catheter and the solution injected. The clamp was maintained for 10 seconds, while the heart pumped against a closed system (isovolumically). This allows the solution that contains the adenovirus to circulate down the coronary arteries and perfuse the heart, without direct manipulation of the coronaries. After 40 seconds, the clamp on the aorta and the pulmonary artery was released. After removal of air and blood, the chest was closed, animals were extubated, and transferred back to their cages. Three to four rats were used at each time point.
We measured the expression of beta galactosidase in the ventricle (via X-gal activity) at various time intervals after AAV injections. We found that AAV6 has some surprising and unexpected properties relative to other AAVs. As shown in
Percutaneous antegrade intracoronary gene transfer with concomitant coronary vein blockade (CVB) was performed in both sheep and swine models. Using these large animal models we have developed a new technique of gene transfer. The left anterior descending artery (LAD) or the left circumflex artery (LCX) was cannulated and occluded with a standard angioplasty balloon. One-minute ischemic preconditioning in both the LAD and the LCX distribution (by blockade of the LAD and the LCX) was performed to allow increased viral dwell time in this model. Following the preconditioning protocol, the great coronary vein (GCV) or one of its branches was cannulated and temporarily occluded with a standard wedge balloon catheter. CVB was performed globally, implying occlusion of the proximal GCV and thus occluding venous drainage in both the LAD and LCX distribution, or selectively, in which case the anterior interventricular vein (AIV) was occluded during LAD delivery and similarly, the ostium of the middle cardiac vein (MCV) was occluded during LCX delivery. With both the arterial and the venous balloons inflated, percutaneous antegrade intracoronary gene transfer was performed by injection through the center lumen of the inflated angioplasty balloon with an adeno-associated virus carrying β-galactosidase (AAV6.β-gal) (n=5).
Twelve weeks following gene transfer with AAV6.CMV.βgal, myocardial sections of 10 μm were obtained from the septal, anterior, left lateral, posterior, and right ventricular walls. These sections were fixed with a phosphate-buffered solution (PBS), containing 0.5% glutaraldehyde for 30 minutes, and then in PBS with 30% sucrose for 30 minutes. The sections were then incubated overnight in a solution containing 5-bromo-4-chloro-3-indolyl α-D-galactopyranoside (X-gal). The results are shown in
This study was carried out according to the Guidelines for the Care and Use of Laboratory Animals, approved by the Massachusetts General Hospital, Subcommittee on Research Animal Care. In a first set of experiments, 22 normal pigs underwent creation of mitral valve regurgitation (MVR). A carotid approach was used to insert a percutaneous biotome through an 8 Fr sheath. The biotome was advanced in a retrograde fashion through the aortic valve and through the left ventricular cavity towards the posterior wall. The cordae of the posterior papillary muscle were cut to create mitral valve regurgitation. The advancement and the positioning of the catheter and the cordae were performed under 2D echocardiographic monitoring. Color Doppler echocardiography was used to quantify the degree of MVR for inter-animal homogeneity of injury. Serial echocardiograms were performed in anesthetized animals. Transthoracic M-mode and 2D echocardiography was performed using a General Electric ultrasound system with a 3-MHz transducer. A mid-papillary level LV short-axis view was used, and measurements of posterior wall thickness, LV systolic and diastolic dimension, and fractional shortening were collected at baseline just before of MVR creation, one month after the MVR creation just before gene delivery and just before sacrifice.
Three months following the MVR creation, 19 pigs had survived and underwent gene transfer of either AAV6.CMV.SERCA2a or AAV6.CMV.bgal. A right femoral approach was used to advance a 50 cm 8 Fr modified AL1 (Cordis Corporation, Miami, Fla.) in the coronary sinus. An 110 cm 5 Fr wedge-balloon (Allow International Inc, Reading, Pa.) was advanced via the GCV to the AIV over a 0.025 inch guidewire (Terumo Corporation, Tokyo, Japan). Coronary venous pressure was monitored during the catheter manipulation. The wedge balloon was inflated until coronary venous occlusion was confirmed both by angiography and a rise in the coronary venous pressure. Coronary angiography was performed before gene delivery following 100 μg nitroglycerin injection. A 9 mm length, 3.5 mm Maverik (Boston Scientific Scimed Inc, Natick, Mass.) over the wire balloon was advanced over a 0.014 inch guidewire (Guidant Corporation Temecula, Calif.) into the LAD after the first diagonal arterial branch. The coronary balloon was inflated incrementally, until complete occlusion was confirmed by angiography. A similar procedure was performed in the distal circumflex artery, proximal to the bifurcation of second obtuse marginal artery. The coronary balloon was inflated incrementally, until complete occlusion was confirmed by angiography. The AIV, and similarly the GCV at the entrance of the middle cardiac vein, were occluded during LAD and LCX delivery, respectively. With both the arterial and the venous balloons inflated (total 3 minutes), and following infusion of intracoronary adenosine (25 μg), gene transfer was performed by anterograde injection through the center lumen of the angioplasty balloon with adenoviral solution (1 ml of ˜1014 genomes in each coronary).
Three months following gene transfer of SERCA2a, there was a significant improvement in the parameters of contractility and complete reversal of heart failure, as shown in Table 5.
An adeno-associated viral vector type 6 expressing SERCA2a driven by the cytomegaolvirus CMV promoter (AAV-CMV-SERCA2a) can be given by direct intracardiac injection to patients undergoing left ventricular assist device (LVAD), implantation. Based on the doses used in pigs during intracoronary injection (e.g., described in Examples 14 and 15) and in primates using direct injection of AAV2-CMV-sTNFR, two doses of AAV6-CMV-SERC2a can be given to patients undergoing LVAD placement for decompensated heart failure with non-ischemic cardiomyopathies (no coronary artery lesion ≧50%) as a bridge to transplant. The study can test, inter alia, whether: (a) the vector induces an inflammatory response, and (b) the expression of SERCA2a is persistent.
After placement of the LVAD and prior to the discontinuation of the bypass, two regions on the surface of the left ventricle can be identified, marked with sutures and then injected with AAV6-CMV-SERCA2a using a method identical to that used in the non-human primate studies. Each region can consist of a square with nine injection sites, with 1 cm between sites. Each site can be injected with 0.1 ml of virus-containing solution, ˜5 mm below the surface. One of the grids can be injected with a dose of 5×1012 particles/ml and the other with a dose of 5×1011 particles/ml. The two regions can be separated by at least 2 cm. The chest can then be closed according to standard procedures. Each patient can be followed between implantation and transplantation. Serum samples can be obtained weekly to measure CPK, troponin I, and troponin T, as markers of myocytolysis and/or toxicity. In addition, we can measure routine chemistries including BUN, Creatinine, liver function tests and hematologic profile, including sedimentation rate weekly. All participating LVAD patients can be treated according to standard protocols. Patients can be seen routinely by their physicians who can pay special attention to the development of fever, infection, arrhythmia or any unexpected finding that might be attributable to the gene therapy.
At the time of LVAD placement, the core tissue sample can be removed for further analysis. At the time of cardiac transplantation, the recipient's heart can be removed after cold cardioplegia. After placement in a cold cardioplegia solution, the injection sites can be identified and core samples obtained from each site. After dividing the samples into four equal pieces, two samples can be frozen in liquid nitrogen, one sample can be placed in formalin for histologic analysis, and one sample can be placed in OTC for subsequent immunohistochemical analysis. These samples can then be analyzed for SERCA2a expression, the presence or absence of inflammation, and routine histopathology. Samples can also be obtained from regions between the squares to assess the amount of regional spread of AAV expression.
Studies can be performed simultaneously on samples obtained from the core samples obtained at the time of LVAD placement, the two AAV-CMV-SERCA2a treatment sites, and the regions of the heart distant from the treatment sites. We can assess differences in histopathology (including cellular infiltrates, cell death, and cardiomyocyte width). The expression of genes defining cardiac function (SERCA2a, phospholamban, L-type Ca2+ channel Cav1.3, sodium-calcium exchanger NCX, atrial natriuretic factor, α- and β-myosin heavy chain) can be assessed by both Northern and Western blot analyses. The expression of pro-inflammatory cytokines (TNFα, IL-1β, and IL-6) can be assessed at the protein and/or transcript level, using ELISA and RPA analyses. The expression of collagens can also be determined by Northern blot analysis of collagen proα1 (I) and (III) transcripts, total and soluble collagen, and immunohistochemical staining for type I and type III collagens. The primary comparisons can be made between injected and untreated areas of the heart; however, comparisons can also be made with the pre-LVAD myocardial sample. These comparisons can provide an important marker of the potentially beneficial changes that occur in the failing heart during LVAD support.
In addition to providing information about the effectiveness of SERCA2a in altering the cellular phenotype in LVAD-supported hearts, we can also gain important information regarding the safety of AAV vectors and SERCA2a overexpression in the human heart, as well as demonstrate the persistence of AAV-SERCA2a expression.
All patients can be recruited from those patients undergoing LVAD implantation. The major cause of death in these patients includes thromboembolic complications and device failure; however, the majority of these deaths occur within the first 30 days of LVAD implantation.
Postoperative cardiac function can be followed by serial echocardiography, and metabolic stress testing, according to the following timetable:
For subjects in whom routine screening suggests the potential for weaning from the device, the following protocol can be followed. Three months after LVAD implantation, each patient can undergo assessment as defined below. The protocol can have three steps: 1) assessment of cardiac function, using echocardiographically derived variables; 2) measurement of cardiac hemodynamics including cardiac output, left ventricular filling pressure, and pulmonary artery pressures and heart rate using a pulmonary artery catheter (Swann-Ganz catheter); and 3) assessment of functional capacity using an exercise stress test with measurements of respiratory gas exchange. Each of these pieces of information can provide an endpoint for this study; however, the utility of the specific intervention, i.e., SERCA2a therapy, can require demonstrating an ability to wean patients with a greater level of efficacy.
Prior to weaning, patients should meet the following criteria:
The VAD flow can be reduced for a one-week period. Protocols for flow reduction are pump-dependent.
In patients who have tolerated one week of partial weaning, we can assess echocardiographic left ventricular function. While on LVAD support, patients LV function can be assessed during decreasing mechanical circulatory support and can include simultaneous measurements of: arterial blood pressure (peripheral), respiratory rate, electrocardiographic signals, and left ventricular cross sectional area (as a surrogate for volume). Echocardiogarphic automated border detection (Agilent Technologies, Sonos 5500) can be used from mid-ventricular short axis plane. All signals can be recorded on a computer using a customized analog/digital acquisition system (DATAQ Instruments, Akron, Ohio; model DI-220) and software (DATAQ Instruments; WINDAq vs 1.92) for post-processing.
Physiologic data can be acquired at baseline with full LVAD support, followed by periods of decreases in LVAD support to 1:2 fixed rate setting. An initial bolus of heparin anticoagulation can be administered prior to the initiation of the study to minimize the risk of thrombus formation. Blood pressure and symptomatic assessment of the patient can be done throughout the protocol. The weaning protocol can be aborted if the patient becomes hypotensive (systolic BP<85 mmHg) or experiences signs of lightheadedness, dyspnea, or other symptoms of hypoperfusion. If the patient tolerates the initial decrease in pump rate, LVAD support can be further decreased as follows: Novascor systems (Baxter-Edwards) can be decreased to a minimum of 30 bpm, Heartmate systems (Thermocardiosystems) can be turned down to a minimum of 20 bpm, while Thoratec (Thoratec, Inc.) drivelines can be disconnected from the drive console and re-connected to hand bulbs. These hand bulbs can be compressed once every six seconds to prevent blood stasis and thrombus formation.
Analysis of the echocardiographic data can be performed using custom-written subroutines, calculating LV end diastolic area, end systolic area, fractional area change and systolic and diastolic pressures. Baseline values can be compared to parameters calculated from 1:2 fixed rate studies and off-pump (hand bulb) studies. All acquired data can then be analyzed using an analysis of variance for repeated measure.
3. Hemodynamic Assessment of Left Ventricular Function:
The patients who maintain a left ventricular ejection fraction of at least 40% during the echocardiographic assessment can be taken to the cardiac catheterization laboratory, where a pulmonary artery catheter can be placed under fluoroscopic control. Measurement can be made within one week of the echo measurements. With settings optimized as described for the echocardiographic protocol, right heart catheterization pressures can be measured, including cardiac output, left ventricular filling pressure, pulmonary artery pressure and heart rate. All hemodynamic measurements should be within the normal range.
4. Measurement of Functional Capacity.
Once echocardiographic function and pulmonary artery hemodynamics have been obtained, the VAD will be turned off and patients will again undergo symptom-limited exercise on a treadmill using a Modified Bruce Protocol and simultaneous pulmonary gas-exchange measurements. If patients maintain a VO2 max of greater than 15, they will be identified as being candidates for VAD explantation.
Similarly designed studies can be used to evaluate other methods of introducing viral delivery systems into the heart, e.g., methods of delivering such systems to the coronary arteries, and any other method described herein.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims:
This application is a continuation-in-part of U.S. patent application Ser. No. 09/789,894, filed Feb. 21, 2001, which is a continuation-in-part of U.S. patent application Ser. No. 09/119,092, filed Jul. 20, 1998, which claims the benefit of a previously filed Provisional Application No. 60/053,356 filed Jul. 22, 1997, all of which are hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 60053356 | Jul 1997 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09789894 | Feb 2001 | US |
| Child | 10914829 | Aug 2004 | US |
| Parent | 09119092 | Jul 1998 | US |
| Child | 09789894 | Feb 2001 | US |
