Drug perfusion into unarrested beating heart

Abstract

To provide a method for treating a heart condition by perfusing a drug to an unarrested beating heart of a patient.SOLUTION: A closed circuit may be formed by using a first drug delivery catheter 122 positioned in a patient's right coronary artery, a second drug delivery catheter 124 positioned in the patient's left main coronary artery, a drug collection catheter positioned in a coronary sinus, the coronary artery, the coronary venous system, and an external membrane oxygenator 120 interspersed between the venous and arterial branches. A drug for treating a heart condition may be perfused through the closed circuit.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 892,164, filed August 27, 2019. No. 60 / 019,599, filed on Dec. 1, 2003, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to the treatment of cardiac disease, and in particular to the localized delivery of therapeutic agents to the heart of a patient. Regarding. [Background technology]

[0003] Despite pharmacological advances in the treatment of various cardiac conditions, e.g., heart failure, mortality and morbidity remain unacceptably high. The approach is aimed at many patients (e.g., those with advanced heart failure associated with other comorbidities) Alternative approaches, such as gene therapy and cell therapy, It is uniquely tailored to its purpose and addresses the underlying causes of the pathogenesis of many cardiac diseases. It is gaining increasing attention due to its potential effectiveness.

[0004] Nevertheless, many uncertainties remain regarding delivery, including vector efficiency, dosage, specificity, and safety. Therefore, there is a need for an effective, well-tolerated, minimally invasive and more targeted and homogenous delivery of drugs suitable for the treatment of various cardiac conditions. There is a need for further research directed towards ways to achieve this. Summary of the Invention [Problem to be solved by the invention]

[0005] Method for perfusing drugs in a patient's non-arrested, beating heart in a minimally invasive manner It is an object of the present invention to provide a method.

[0006] the patient's non-arrested, beating heart so that the perfusion fluid is isolated from the patient's systemic circulation circulating perfusion fluid (which may contain one or more of blood or drugs) through the It is an object of the present invention to provide a method for

[0007] It is an object of the present invention to provide locoregional delivery of pharmacological gene therapy.

[0008] It is therefore desirable to reduce the overall dose of drugs delivered to a patient to treat a cardiac condition. This is the object of the invention.

[0009] Risks and / or adverse immune responses to the administration of drugs suitable for the treatment of cardiac conditions It is an object of the present invention to reduce the response.

[0010] have neutralizing antibodies that would have made them poor candidates for administering pharmacological gene therapy drugs The ability to re-medicate and / or administer such drugs to patients is an advantage of the present invention. That is the purpose. [Means for solving the problem]

[0011] The above objects and others are achieved in certain embodiments by detecting an unstoppable heartbeat of a patient. This is fulfilled by the present invention, which is directed to a method for perfusing drugs in a living heart. In one embodiment, the method includes placing a first drug delivery catheter in a right coronary artery of the heart. The method includes delivering a second drug to the left main coronary artery of the heart. The method further includes placing a drug collection catheter in the coronary sinus of the heart. In some embodiments, the first drug delivery catheter, the second The two drug delivery catheters and the drug collection catheter are inserted into the coronary arteries, coronary venous system of the heart. and a membrane oxygenation device to form a closed circuit. The method involves administering a drug through a closed circuit that separates the patient's coronary circulation from the patient's systemic circulation. The method further comprises perfusing the

[0012] In some embodiments, the method further comprises applying negative pressure to the drug collection catheter. In some embodiments, the negative pressure is in the range of about -100 mmHg to 0 mmHg. .

[0013] In some embodiments, the closed circuit further applies a negative suction pressure to the drug collection catheter. blood circulated through a closed circuit via the Thebesian vein and / or One or more suction mechanisms that allow for preventing and / or minimizing drug leakage It may further include:

[0014] In some embodiments, the first drug delivery catheter, the second drug delivery catheter, or In some embodiments, one or more of the catheters or drug collection catheters are introduced percutaneously. wherein the first drug delivery catheter and / or the second drug delivery catheter are antegrade In some embodiments, the first drug delivery catheter and and / or a second drug delivery catheter is inserted into the femoral artery and / or radial aorta. In some embodiments, the device is positioned via the patient's aorta by accessing the aorta (radialis). In this technique, a drug collection catheter is placed into the coronary sinus via the patient's vena cava. In embodiments, the drug collection catheter is placed via the patient's jugular or femoral vein. In some embodiments, the membrane oxygenation device includes a collection catheter and a first disposed between the drug delivery catheter and one or more of the second drug delivery catheters. In some embodiments, the first drug delivery catheter, the second drug delivery catheter, One or more of the following may be used to reduce or prevent leakage: The balloon seals the device.

[0015] In some embodiments, the method further comprises circulating the blood through a closed circuit. In some embodiments, the blood is autologous blood, matched blood from a donor, or In some embodiments, the blood component, such as serum or plasma, is In some embodiments, one or more parameters are selected. In some embodiments, the plurality of parameters includes the presence or absence of a selected antibody. In this case, approximately 1000 mL, approximately 800 mL, approximately 600 mL, approximately 400 mL, approximately 200 mL, Approximately 100 mL, or approximately 50 mL, of blood is circulated through the closed circuit.

[0016] In some embodiments, the perfusion is for about 5 minutes to about 5 hours, about 15 minutes to about 4 hours, or about 30 minutes. In some embodiments, the treatment is carried out over a duration of about 1 hour to about 3 hours, or about 1 hour to about 2 hours. In some embodiments, the perfusion is for at least 60 minutes. mL / min to approximately 750 mL / min, approximately 150 mL / min to approximately 500 mL / min, or approximately 200 mL The procedure is carried out at a flow rate of approximately 300 mL / min to 300 mL / min.

[0017] In some embodiments, the drug is suitable for the treatment of a cardiac condition. In embodiments, the cardiac condition is heart failure. In some embodiments, the genetically determined heart disease is The heart disease is a genetically determined cardiomyopathy.

[0018] In some embodiments, the drug comprises a therapeutic polynucleotide sequence. In the method, the therapeutic polynucleotide sequence is present in one or more viral vectors. In some embodiments, the one or more viral vectors are adeno-associated viruses. virus, adenovirus, retrovirus, herpes simplex virus, bovine papilloma virus , lentiviral vector, vaccinia virus, polyoma virus, Sendai virus Orthomyxoviruses, paramyxoviruses, papovaviruses, picornaviruses, Poxviruses, alphaviruses, variations thereof, and combinations thereof is selected from the group consisting of:

[0019] In some embodiments, the viral vector is an adeno-associated virus (AAV). In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV 5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, One or more of these variations and combinations thereof.

[0020] In some embodiments, the therapeutic polynucleotide sequence comprises a protein for the treatment of a cardiac condition. Contains nucleic acid sequences encoding proteins, antisense RNA, ncRNA, or miRNA In some embodiments, the protein corresponds to a gene expressed in the human heart. In some embodiments, the protein is SERCA2, MyBPC3, MYH7, PK P2, dystrophin, FKRP, or a combination or variation thereof In some embodiments, the therapeutic polynucleotide sequence is one or more of: Includes motor.

[0021] In some embodiments, less than about 20% v / v, less than about 15% v / v, less than about less than 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v, less than about 1% v / v, less than about 0.5% v / v blood circulated through a closed circuit with a blood concentration lower than or substantially 0% v / v; In some embodiments, less than about 20% v / v leaks outside the closed circuit. Less than 15% v / v, less than about 10% v / v, less than about 5% v / v, about 4% lower than about 3% v / v, lower than about 2% v / v, lower than about 1% v / v less than about 0.5% v / v, or substantially 0% v / v, through a closed circuit. Drugs that are perfused by the blood leak outside the closed circuit.

[0022] In some embodiments, the first drug delivery catheter, the second drug delivery catheter, or or one or more of the drug collection catheters is a balloon catheter.

[0023] The above objectives and others can be achieved in certain embodiments by not stopping during perfusion. and a method for maintaining perfusion of perfusate through a closed circuit in a beating patient's heart. In some embodiments, the method further includes: In some embodiments, the method includes placing a first catheter in a cardiac pulse. Further comprising placing a second catheter in the left main coronary artery. In some embodiments, the method further includes placing a collection catheter in the coronary sinus of the heart. In one embodiment, the first catheter, the second catheter, and the collection catheter are Together with the venous system, coronary venous system, and membrane oxygenator device, they form a closed circuit through the heart. In some embodiments, the method includes administering a catheter to the heart via a first catheter and a second catheter. The perfusion fluid is introduced into the collection catheter, allowing the perfusion fluid to flow through a closed circuit. In some embodiments, the closed circuit further comprises collecting perfusate via a ventilator. isolates the patient's coronary circulation from the patient's systemic circulation.

[0024] In some embodiments, the perfusion is maintained for at least 60 minutes. Perfusion is maintained for at least 120 minutes.

[0025] In some embodiments, the method further comprises applying negative pressure to the collection catheter. , the negative pressure is in the range of approximately -100mmHg to 0mmHg.

[0026] In some embodiments, the first catheter, the second catheter, or the collecting catheter One or more of the catheters are introduced percutaneously.

[0027] In some embodiments, the membrane oxygenation device includes a collection catheter and a first drug and a second drug delivery catheter. do.

[0028] In some embodiments, the method further comprises circulating the blood through a closed circuit. The blood may be autologous blood, matched blood from a donor, or a combination thereof. In some embodiments, the volume is about 1000 mL, about 800 mL, about 600 mL, about 400 mL, Approximately 200 mL, 100 mL, or 50 mL of blood is circulated through a closed circuit. can be done.

[0029] In some embodiments, the perfusion is about 75 mL / min to about 750 mL / min, about 150 mL / min / min to about 500 mL / min, or about 200 mL / min to about 300 mL / min In some embodiments, less than about 20% v / v, less than about 15% v / v, less than about less than 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v, less than about 1% v / v, less than about 0.5% v / v blood circulated through a closed circuit with a blood concentration lower than or substantially 0% v / v; Leaks outside the closed circuit.

[0030] In some embodiments, the first catheter, the second catheter, or the collecting catheter One or more of the catheters is a balloon catheter.

[0031] The above objects and others are achieved in certain embodiments by a device fluidly connected to a patient's heart. The present invention is also directed to a system for providing local regional perfusion within the heart when In some embodiments, the system is adapted for insertion into the right coronary artery of the heart. a first catheter adapted for insertion into the left main coronary artery of the heart; a collecting catheter adapted for insertion into the coronary sinus of the heart; a first catheter a membrane-type catheter fluidly connected to the oxygen source; a second catheter; a collection catheter; and a lung artificial device; and a device for driving fluid flow through the first catheter and the second catheter. In some embodiments, the first catheter includes a pump configured to operate the first catheter. The second catheter, the collection catheter, and the membrane oxygenator device together form the first catheter. A catheter was inserted into the right coronary artery and a second catheter was inserted into the left main coronary artery and When a collection catheter is inserted into the coronary sinus, it creates a blood vessel that passes through the heart, isolated from the patient's systemic circulation. This forms a closed circuit.

[0032] The above objects and others are achieved in certain embodiments by a locoregional perfusion system. a first catheter inserted into the right coronary artery of the patient's heart; a second catheter inserted into the left main coronary artery of the heart; a second catheter inserted into the coronary sinus of the heart; a collecting catheter inserted into the coronary sinus of the heart; a membrane-type artificial intestine that is fluidly connected to the oxygen source, a second catheter, a collection catheter, and an oxygen source; a pulmonary device; and a catheter to the heart via the first catheter and the second catheter. a pump configured to drive fluid flow out of the heart through the collection catheter. This is further fulfilled by the present invention, which is directed to said locoregional perfusion system. In this embodiment, the first catheter, the second catheter, the collecting catheter, and the membrane oxygenator The device is a cardiac catheter that, along with the coronary arterial and venous systems of the heart, is isolated from the patient's systemic circulation. This forms a closed circuit through

[0033] In some embodiments, the membrane oxygenator device infuses drugs into a closed circuit during perfusion. The reservoir is configured to:

[0034] In some embodiments, the pump generates a negative pressure range of about -100 mmHg to 0 mmHg. It is configured to achieve this.

[0035] In some embodiments, the first catheter, the second catheter, or the collecting catheter One or more of the first catheters are introduced percutaneously. The catheter and / or second catheter are placed via antegrade intubation. In an embodiment, a collection catheter is placed into the coronary sinus via the patient's vena cava.

[0036] The above objects and others are achieved in certain embodiments by performing any of the above-described methods. This is further met by the present invention, which is directed to a local-regional perfusion system configured to:

[0037] The above and other features of the present disclosure, its nature and various advantages, may be best understood from the following detailed description taken in conjunction with the accompanying drawings. This becomes more apparent upon consideration of the following detailed description taken in conjunction with the present invention. [Brief explanation of the drawings]

[0038] [Figure 1A] 1 depicts an exemplary locoregional perfusion system according to an embodiment of the present disclosure. [Figure 1B] 1 is a diagram of an exemplary locoregional perfusion device according to an embodiment of the present disclosure. [Figure 2] 1 is a radiograph captured during locoregional perfusion of a non-arrested porcine heart showing the position of the left main coronary artery catheter, right coronary artery catheter, and coronary sinus balloon. [Figure 3]1 is a plot of pump speed, flow rate, and pressure measured during locoregional perfusion. DETAILED DESCRIPTION OF THE INVENTION

[0039] definition As used herein, the singular forms "a," "an," and "the" refer to It includes plural references unless the context clearly indicates otherwise. So, for example, "drugs" ( References to a drug include mixtures of two or more different drugs as well as a single drug. and reference to a "viral vector" includes a single viral vector. Others include mixtures of two or more different viral vectors, and so on.

[0040] Also, as used herein, "about" is used in connection with the quantity being measured. When measurements are made, a level of care must be exercised that is commensurate with the purpose of the measurements and the accuracy of the measuring equipment. refers to normal variations in the quantity being measured, as would be expected by one of ordinary skill in the art In certain embodiments, the term "about" includes the stated number plus or minus 10%, As a result, "about 10" includes 9 to 11.

[0041] As used herein, the term "polynucleotide" refers to a has its ordinary and customary meaning in any polymeric nucleic acid, e.g., DNA or R In addition to the NA molecule, the polynucleotides include chemical derivatives known to those skilled in the art. Not only can they be used to encode proteins, but they can also be used to encode proteins using techniques known in the art. Sequences that can be used to reduce the expression of a targeted nucleic acid sequence (e.g., antisense Polynucleotides also include those that target cells of the cardiovascular system (e.g., vasculitis ...) and other cardiovascular diseases. Initiate or increase expression of a targeted nucleic acid sequence or production of a targeted protein within a target gene. Targeted nucleic acids and proteins can be used to target Nucleic acids and proteins normally found in tissues containing such naturally occurring nucleic acids or proteins. may be derivatives of proteins, naturally occurring nucleic acids not normally found in the targeted tissue, or Examples of nucleic acids include, but are not limited to, nucleic acids or proteins, or synthetic nucleic acids or proteins. The one or more polynucleotides may be linked to one or more targeted nucleic acid sequences or are used in combination to increase and / or decrease proteins, simultaneously and / or Or they may be administered sequentially.

[0042] Also, as used herein, "perfusion," "perfused," and "perfusion" are used interchangeably. "Flux" has its ordinary and accustomed meaning in the art and is not intended to be limiting. or "bolus injection" over an art-recognized period (typically less than a minute) refers to administration over a period of time (typically minutes or longer) substantially longer than the normal perfusion rate. is dependent, at least in part, on the volume administered.

[0043] Also, as used herein, an "exogenous" nucleic acid or gene refers to a nucleic acid or gene that is not present in the target cell at the time of nucleic acid transfer. In the vectors used for the purpose of Although the term refers to a gene that is not naturally found in a patient or host, it is understood that the term also refers to a gene that is naturally occurring in a patient or host. It is not intended to exclude nucleic acids that encode proteins or polypeptides.

[0044] Also, as used herein, "cardiac cells" refers to cells that are involved in the maintenance or function of the structure of the heart. Any cell of the heart involved in providing cardiac function, such as a cardiomyocyte, a cell of the cardiovascular system, or a cardiac Cardiac cells include myocardial cells (both normal and abnormal electrical activity) and cells present in the valves. (having biological properties), epithelial cells, endothelial cells, fibroblasts, cells of conductive tissue, cardiac pacemakers These include Kerr cells, as well as neurons.

[0045] Also, as used herein, "isolated," "substantially isolated," "Largely separated" and their variants are classified as coronary venous circulation, cardiac circulation, and systemic venous circulation. is a term that does not require complete or absolute separation of the pulmonary circulation, or the systemic circulation, but rather is a state in which a large part, preferably a major part or even substantially all of the specified circulation is separated. Also, as used herein, "Partially separated" means that any non-trivial portion of the specified cycle is separated. This refers to the following.

[0046] Also, as used herein, "non-naturally restricted" refers to a substance that is restricted to flow through blood vessels. Any method of restricting bodily flow, including, for example, balloon catheters, sutures, etc., but not limited to natural This does not include restrictions present in the blood vessels, such as plaque buildup (stenosis). Non-natural restrictions include, for example, For example, substantial or total isolation of the coronary circulation.

[0047] Also, as used herein, "minimally invasive" refers to a procedure that involves the heart or a device that is close to the heart. is intended to include any procedure that does not require open surgical access to the blood vessels associated with the connection. Such procedures include endoscopic means for accessing the heart, and also These include the use of catheter-based procedures that rely on access via the aorta and veins. do.

[0048] Also, as used herein, "adeno-associated virus" or "AAV" refers to , all subtypes, serotypes, and pseudotypes, as well as naturally occurring and recombinant Various AAV serotypes and strains are known in the art, including: Publicly available from sources such as ATCC and academic or commercial sources. from AAV serotypes and strains published and / or available from various databases. These sequences may be synthesized using known techniques.

[0049] Also, as used herein, "serotype" refers to the ability of a capsid to bind to a defined antiserum. and distinguished from other AAVs based on their reactivity with ATP-binding proteins. refers to at least 12 known human AAVs, including AAV1 to AAV12. Although there are several serotypes, additional serotypes continue to be discovered, and the use of newly discovered serotypes is It is expected.

[0050] Also, as used herein, "pseudotyped" AAV refers to a single serotype. Capsid proteins from different or heterologous serotypes and 5' and 3' reverse end fragments Refers to AAV containing the viral genome including the intervening repeats (ITR). V(rAAV) has the cell surface binding properties of the capsid serotype and the genetic conformance of the ITR serotype. Pseudotyped rAAVs are expected to have genetic properties similar to those of the capsid protein. As long as the VP1, VP2, and VP3 caps are of a heterologous serotype relative to the ITR serotype, AAV capsid proteins, including the capsid proteins, as well as AAV1 to AAV12 proteins It may contain ITRs from any serotype of AAV, including any primate AAV serotype. In pseudotyped rAAVs, the 5' and 3' ITRs can be the same or different. Pseudotyped rAAV can be prepared using standard techniques described in the art. It is manufactured using

[0051] Also, as used herein, a "chimeric" rAAV vector refers to a vector containing a heterologous capsid. The present invention also encompasses AAV vectors containing the encapsulated protein, i.e., rAAV vectors are those containing the encapsulated protein. The peptides may be chimeric with respect to the peptide proteins VP1, VP2, and VP3, so that VP1, VP2, and VP3 are not all from the same AAV serotype. As used herein, refers to, but is not limited to, AAV1 and AAV2. Capsid proteins VP1, VP2, and VP3 encompasses AAV serotypes and other parvovirus capsid proteins or other viral proteins or other proteins, e.g., a mixture of The chimeric rAAV contains a protein that targets the delivery of the AAV to a specific cell or tissue. As used herein, also refers to a rAA containing chimeric 5' and 3' ITRs. Contains V.

[0052] Also, as used herein, "pharmaceutically acceptable excipient or carrier" means refers to any inactive ingredient in a composition that is combined with an active agent in a formulation. Excipients that may be used include carbohydrates (e.g., glucose, sucrose, or dextran), Antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins These may include polymers, high molecular weight polymers, gel formers, or other stabilizers and additives. Other examples of pharmaceutically acceptable carriers include, but are not limited to, wetting agents, emulsifying agents, and the like. Preservatives include an agent, dispersing agent, or preservative, which acts to prevent the growth or action of microorganisms. Various preservatives are well known, for example, phenol and ascorbic acid. Examples of carriers, stabilizers or adjuvants are listed in Remington's Pharmaceutical Sciences ences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985) It is possible.

[0053] Also, as used herein, "patient" refers to any individual or group of individuals who are in need of treatment. or more than one specific clinical symptom, to be treated prophylactically against a condition, or diagnosed as having a condition to be treated, particularly humans (but also non-humans) refers to obtaining

[0054] As used herein, "subject" also encompasses the definition of the term "patient." Include and do not exclude otherwise healthy individuals.

[0055] Also, as used herein, "treatment" and "treating" refer to the treatment of a condition, e.g., This includes, for example, the administration of drugs intended to reduce the severity of heart disease or prevent the condition. nothing.

[0056] Also, as used herein, "prevention" and "preventing" refer to the prevention of a condition, e.g., Examples include avoiding the onset of heart disease.

[0057] Also, as used herein, "condition" or "con" ditions) are medical conditions that can be treated, alleviated, or prevented by administering an effective amount of a drug to a subject. refers to a terminal condition, such as heart disease.

[0058] Also, as used herein, "effective amount" means an amount that produces a beneficial or desired effect. At levels readily detectable by methods commonly used to detect such effects In some embodiments, such an effect refers to an amount of a drug sufficient to produce Results in at least a 10% change from the basal level when no drug is administered In other embodiments, the change is at least 20%, 50%, 8% or more from basal levels. 0%, or even a higher percentage of the drug, as described below. An effective amount will depend on the age, general health, severity of the condition being treated, and the type of drug being administered. The appropriate "effective" dose in any individual case may vary from subject to subject depending on the specific drug being administered, etc. The "effectiveness" amount can be determined by reference to relevant texts and literature and / or by routine practice. This can be determined by one skilled in the art using experimental methods.

[0059] Also, as used herein, "active agent" refers to a substance or substance that is authorized by a government agency for that purpose. any therapeutic, prophylactic, or other intended effect, whether or not approved by the refers to any material that is intended to produce

[0060] The recitation of ranges of values ​​herein is intended to be a range unless otherwise indicated herein. It is merely intended to serve as a shorthand way of individually referencing each separate value that goes within. , each separate value is incorporated herein as if it were individually set forth herein. All methods described herein are also included unless otherwise indicated herein. or otherwise clearly contradicted by the context, may be used in any suitable order. Any and all examples or illustrative expressions (e.g., "for example," " The use of "etc." is intended merely to highlight certain materials and methods and is not intended to be limiting in scope. No representation in this specification imposes limitations on the practice of the disclosed materials and methods. should not be construed as indicating any non-claimed element as essential to the do not have.

[0061] Detailed Description The present invention is directed to a method of treating a cardiac condition in a minimally invasive manner, the method comprising: Isolating the patient's coronary circulation from the patient's systemic circulation and providing a fluid, e.g., a drug-containing fluid. into an isolated or substantially isolated coronary circulation of the patient. The perfusion may be performed on the patient's non-arrested, beating heart. Separation of the circulation is described in more detail below with reference to Figures 1A and 1B.

[0062] The coronary circulation provides blood supply to the tissues of the heart. There are multiple coronary arteries. Usually, The four main coronary arteries transport oxygenated blood to the heart for distribution throughout the heart tissue. Provides: left main and right coronary arteries, left anterior descending artery, and left circumflex artery. The blood flows through the coronary sinus.

[0063] The embodiments disclosed herein include a first drug delivery catheter, a second drug delivery catheter, and a including catheters, drug collection catheters, coronary arteries, coronary venous system, and external membrane oxygenator and forming a closed circuit (consisting essentially of or consisting of) a patient's blood from the patient's systemic circulation. The present disclosure contemplates isolating or substantially isolating the coronary circulation of certain In an embodiment, the closed circuit is used to connect the patient's coronary circulation to the patient's systemic circulation. and perfusing the myocardium with a suitable drug for the treatment of a cardiac condition while qualitatively isolating the myocardium. In some embodiments, the methods disclosed herein involve detecting isolated intracardiac tissue. Drugs are delivered to the entire myocardium rather than to a region. The delivered drug may be distributed homogeneously throughout the heart.

[0064] Isolating a patient's coronary circulation from the patient's systemic circulation when treating cardiac conditions is often necessary. These advantages include: (1) localized delivery of drugs, and the ability to transfer drugs to other organs; (2) increased targeted drug dose; (3) reduced overall drug dose; and (4) reducing the risk and side effects of selected patients re-medicating or administering certain therapies (e.g., For example, gene therapy using viral vectors in patients with antibodies against viral vectors The possibility of administering the drug to a patient population that was not a suitable candidate for the current treatment method is a concern. Not limited to.

[0065] FIG. 1A illustrates an exemplary local-regional perfusion (LRP) system 100 in accordance with an embodiment of the present disclosure. Depicted is an LRP system 100 shown in a closed circuit configuration with a heart 110 ( (Both forward and rearward views 110A and 110B are shown for clarity). The system 100 includes a membrane oxygenator device 120, a blood gas analysis (BGA) monitor 130, and a pressure monitor 140. The LRP system 100 is connected to the right coronary artery 11 of the heart 110. A first catheter 122 is placed in the left main coronary artery 114 of the heart 110. By placing a catheter 114 in the coronary sinus 116 of the heart and a collection catheter 126 in the coronary sinus 116 of the heart. The catheter may be assembled by a first catheter 122, a second catheter 124, and The collection catheter 126 is connected to the coronary arteries, the coronary venous system, the membrane oxygenator device 120, and one or more together with one or more optional additional components to form a closed circuit. may isolate or substantially isolate the patient's coronary circulation from the patient's systemic circulation.

[0066] The first catheter 122, the second catheter 124, and the collection catheter 126 are It may be introduced percutaneously and in a minimally invasive manner. The catheter 122 and / or the second catheter 124 may be inserted via antegrade intubation. In other embodiments, the first catheter 122 and / or the second catheter The catheter 124 may be introduced via retrograde intubation. When used for drug delivery, the first catheter 122 and the second catheter 124 is sometimes referred to herein as a "drug delivery catheter" and a collection catheter. The catheter 126 is sometimes referred to herein as a "drug collection catheter."

[0067] The first catheter 122 and / or the second catheter 124 are optionally standard A standard infusion catheter may be used, which may include a suitable guidewire and infusion pump. Each catheter may deliver perfusion fluid to the heart 110, for example, It may also contain drugs to be delivered to the heart 110 during local-regional perfusion.

[0068] The first catheter 122 and / or the second catheter 124 may be, for example, a femoral aorta. It is placed through the patient's aorta by accessing the venous and / or radial aorta. In one embodiment, the first catheter 122 accesses the femoral artery. In another embodiment, the first The catheter 122 is positioned through the patient's aorta by accessing the radial aorta. In one embodiment, the second catheter 124 is inserted into the femoral artery. In another embodiment, the second The catheter 122 is placed through the patient's aorta by accessing the radial aorta. This may be done.

[0069] The collection catheter 126 may be a balloon catheter, such that the balloon The coronary sinus 116 expands and all the blood circulating through the closed circuit is collected by the collecting catheter. The balloon catheter may be used to ensure that the blood flows through the catheter 126. a rthy® catheter, or any other catheter discussed herein as will be appreciated by those skilled in the art. It may also be any other catheter suitable for the intended purpose discussed. In this embodiment, the collection catheter 126 may be placed via the patient's vena cava. In one embodiment, the collection catheter 126 is placed via the patient's jugular vein. In another embodiment, the collection catheter 126 is placed via the patient's femoral vein. In some embodiments, the first catheter 122, the second catheter 1 24, collection catheter 126, or a combination thereof, each to help reduce leakage. The catheter may be a balloon catheter for the purpose of

[0070] The LRP system 100 may include one or more additional components, such as, but not limited to: For example, one or more pumps, one or more suction mechanisms, one or more irrigation fluids, and combinations thereof. For example, the LRP system 100 may include some In embodiments, operably connected to or part of the membrane oxygenator device 120 The pressure monitor 140 is depicted as including a pressure monitor 140, which measures coronary artery pressure. Continuous force monitoring controls the perfusion rate (i.e., flow rate) to ensure safety. The first pressure sensor 142 and the second pressure sensor 144, for example, to measure the pressure in the right coronary artery and the left main coronary artery, respectively. may be inserted with the first catheter 112 and the second catheter 114, respectively. The LRP system 100 is further depicted as including a BGA monitor. For example, before perfusion via the first catheter 122 and the second catheter 124 The gas concentration in the perfusate (e.g., For example, when the perfusate contains blood, the membrane oxygenator device 120 is operated to measure the blood flow rate. The membrane oxygenator device 120 and one or more additional components are , the collection catheter 126 and the first catheter 122 or the second catheter 124 It may be placed between one or more of them.

[0071] FIG. 1B is a diagram of a membrane oxygenator device 120. The membrane oxygenator device 120 is Oxygenates the perfusate, mixes the perfusate with other components (e.g., drugs), and removes carbon dioxide from the perfusate. and / or the first catheter 122 (in the right coronary artery 112) and / or One or more of the second catheters 124 (in the left main coronary artery 114) are filled with perfusion fluid. The membrane oxygenator device 120 may be used to pump oxygen and other substances contained in the blood. Any commercially available extracorporeal membrane oxygenator (ECM) to exchange the retained carbon dioxide O) It may be a device.

[0072] As shown in FIG. 1B, the membrane oxygenator device 120 includes various components. The components include a heat exchanger 156 (which is connected to the outlet 152 and the first catheter 122 and the second catheter 123). the perfusion fluid passes through before entering the catheter 124 of the second catheter, the delivery pump 158, and the reservoir 160. (Constituents, e.g., blood and / or drugs, are passed through collection catheter 126 via inlet 154. (to add to the perfusate returning through the 162 and 164 (e.g., for measuring pressure and / or blood gas content), and a membrane oxygenator 166. In some embodiments, the deoxygenated blood is passed through a membrane oxygenator. The oxygen-rich gas enters the oxygenator 166 and mixes with the oxygen-rich gas. The gas may be supplied from a gas blender 168, which mixes oxygen with carbon dioxide in various ratios. The gas may be mixed with hydrogen and nitrogen gas and is regulated by a gas regulator 170.

[0073] The perfusate may be blood (or its components, e.g., plasma or serum) and / or cardiac fluid. Suitable drugs and / or vehicles for the treatment of the condition, such as saline or dextrose The delivery pump 158 pumps the perfusion fluid into the first catheter. The catheter 122 and / or the second catheter 124 may be delivered to the catheter 122 and / or the second catheter 124. In the embodiment, the perfusion fluid may be contained in an IV bag or syringe and delivered by a delivery pump 15. 8 with or without the first catheter 124 and / or the second catheter 12 4 may be administered directly to

[0074] The suction mechanism is used to apply negative suction pressure to the collection catheter 126 to remove the Thebesius vein. The negative suction pressure may be approximately -15 0mmHg, approximately -100mmHg, approximately -50mmHg, approximately -20mmHg, approximately -15mm Hg, approximately -10mmHg, approximately -5mmHg, 0mmHg, or at any of these points It may be within a subrange defined by:

[0075] The blood circulated through the closed circuit can be autologous blood, matched blood from a donor, or In some embodiments, blood components, such as serum or The blood or plasma is selected according to one or more parameters. One may be the presence or absence of a selected antibody. For example, if the drug is a therapeutic nucleic acid If the viral vector is one or more viral vectors containing the sequence, the patient's own blood is screened. Screening to determine the presence of antibodies against one or more viral vectors The presence of antibodies in the patient's own blood may reduce the effectiveness of the treatment and / or May be completely inactivating and / or result in an unwanted immune response Therefore, the patient's own blood may be diluted with matched seronegative blood from a donor. and displacing or replacing the drug, thereby reducing the patient's immune response to the drug and improving the drug's effectiveness. It may be possible to enhance efficacy.

[0076] The various components illustrated in FIG. 1B are part of a membrane oxygenator device 120 or Although separate components are shown, this diagram is merely illustrative and does not include One or more of the following may be contained within or separate from (external to) the membrane oxygenator device 120. It should be understood that this may also be possible.

[0077] The LRP system 100 may be set up and operated as follows: (1) A collection catheter 126 is carefully placed into the coronary sinus 116 and tightly sealed, providing a venous access to the heart. (2) the first catheter 122 and the second catheter 123 allow collection of only venous (deoxygenated) blood; and a second catheter 124 is inserted in a sealed manner into the right coronary artery (RCA) and the left main coronary artery. (3) The catheter is then placed into the renal artery (RCA) using standard tubing. (4) connect the arterial and venous systems of the pulmonary device 120; The coronary arteries are perfused antegrade with oxygenated blood, and simultaneously return Deoxygenated blood is collected from the venous cardiac system via a collection catheter 126 using gentle negative pressure. (5) The blood is then directed to a reservoir 160 and then to a membrane oxygenator 166 and oxygen is supplied to the heart via a first catheter 122 and a second catheter 124. The drug (e.g., vector) is reinfused antegrade into the liver (driven by a delivery pump 160). When a thrombus is administered, it is primed with blood or plasma before being delivered to the reservoir 160 Drugs can be added to the perfusate via the The fluid can be applied via reservoir 160 during the perfusion process.

[0078] In some embodiments, the antibody content of the patient with seronegative matched blood from the donor is Autologous blood dilution may result in a reduction of adverse immune responses and / or improved drug efficacy. For example, the adverse effects of a patient's immune response may include diluting or replacing the patient's own blood. Autologous blood transfusion with seronegative matched blood from donors compared with patient immune responses in the donor. Dilution or replacement of approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60% The impact may be reduced by about 70%, about 80%, about 90%, or may be completely alleviated. The efficacy of the administered drug is determined by the efficacy of the drug in the patient without autologous blood dilution or replacement. Dilution or replacement of autologous blood with seronegative matched blood from a donor compared with the sex About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80 %, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 300%, approximately 400%, or It may be increased by approximately 500%.

[0079] In some embodiments, the blood portion of the perfusate is about 5 mL to about 5000 mL, about 50 mL L ~ approx. 2500mL, approx. 100mL ~ approx. 1000mL, approx. 150mL ~ approx. 500mL, approx. 50mL, approx. 75mL, approx. 100mL, approx. 125mL, approx. 150mL, approx. 175mL, approx. 200mL, approx. 225mL, approx. 250mL, approx. 275mL, approx. 300mL, approx. 325mL , approx. 350mL, approx. 375mL, approx. 400mL, approx. 425mL, approx. 450mL, approx. 475 mL, approx. 500mL, approx. 550mL, approx. 600mL, approx. 650mL, approx. 700mL, approx. 7 50mL, approximately 800mL, approximately 850mL, approximately 900mL, approximately 950mL, or approximately 100 It may be in the range of 0 mL.

[0080] Autologous blood versus matched blood from a donor in a blood circulation system The ratio of fluids, if necessary, is most receptive to the drug and least likely to induce immunosuppression upon drug administration. In some embodiments, the blood mixture may be adjusted to produce a response. The ratio is about 1:100 to about 100:1, about 1:80 to about 80:1, or about 1:50 to about 50:1. , about 1:30 to about 30:1, about 1:20 to about 20:1, about 1:10 to about 10:1, about 1: 8 to about 8:1, about 1:5 to about 5:1, about 1:3 to about 3:1, or about 1:2 to about 2:1 (Volume of autologous blood): (Volume of matched blood from donor) may be in this range.

[0081] The flow rate of the perfusate through the closed circuit may be adjusted to match the patient's blood flow rate. As will be appreciated by those skilled in the art, blood flow rates vary from patient to patient and for any given patient , fluctuates throughout the day. Therefore, the flow of perfusion fluid circulated through a closed circuit The flow rate may be adjusted in situ. The flow rate may be measured in a closed circuit. In certain embodiments, the flow rate is measured using a transonic probe (e.g., In some embodiments, the measurement may be performed using a clamp over the tubing. The flow rate of the perfusate at any given time during perfusion is based on the patient's mL / min. Within approximately 20%, within approximately 15%, within approximately 10%, within approximately 8%, within approximately 5% of the subject's blood flow velocity, It may be within about 3%, within about 2%, within about 1%, or within about 0.5%. The flow rate of the perfusate circulated through the tract is adjusted to avoid ischemia and / or to maintain perfusion. It is important not to deviate significantly from the patient's own blood flow rate.

[0082] An exemplary flow rate for the perfusion fluid circulated through the closed circuit is, but is not limited to, about 75 mL. / min ~ approx. 750mL / min, approx. 100mL / min ~ approx. 650mL / min, approx. 125mL / min ~ approx. 600mL / min, approx. 150mL / min ~ approx. 500mL / min, approx. 175mL / min ~ approx. 400m L / min, approx. 200mL / min ~ approx. 300mL / min, approx. 150mL / min, approx. 175mL / min, Approx. 200mL / min, Approx. 225mL / min, Approx. 250mL / min, Approx. 275mL / min, Approx. 300 The flow rate may be in the range of about 325 mL / min, about 325 mL / min, or about 350 mL / min.

[0083] The perfusion solution may be used for, but is not limited to, about 5 minutes to about 5 hours, about 15 minutes to about 4 hours, or about 30 minutes to about 3 hours. or circulated through a closed circuit for a duration ranging from about 1 hour to about 2 hours. In some embodiments, the treatment duration is in the span of days, e.g., 1 day, 2 days, 3 days, or It may be carried out over 1 day, 4 days, 5 days, 6 days, 7 days, etc.

[0084] Using the systems disclosed herein, in some embodiments, Higher doses of the drug are delivered directly to the heart than could be safely administered through systemic delivery. In some embodiments, the vasopressin may be administered to the vasopressin receptor ... The same therapeutic effect (as achieved with larger doses subjected to only partial isolation of the arterial circulation) A lower overall dose of the drug may be required to achieve a therapeutic effect, and the reason for this is There is virtually no leakage of perfusate outside the heart and / or into the Thebesian venous system. Because there is a possibility.

[0085] In some embodiments, less than about 50% v / v, less than about 40% v / v, less than about less than 30% v / v, less than about 20% v / v, less than about 15% v / v, less than about 1 less than 0% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / less than about 2% v / v, less than about 1% v / v, less than about 0.5% v / v A perfusion solution (e.g., 0% v / v) circulated through a closed circuit with a concentration lower than or essentially 0% v / v For example, blood and / or drugs leak outside the closed circuit during the perfusion process.

[0086] Perfusion fluid outside the closed circuit (compared to other methods disclosed in the art) The leakage reduction occurs within the closed circuit and within each individual component utilized in the closed circuit. This may be due to the tight seal provided.

[0087] In certain embodiments, some perfusate leakage from the closed circuit may remain. For example, the maximum is about 0.5% v / v, about 1% v / v, about 2% v / v, about 3% v / v, about 4% v / v, %v / v, approx. 5%v / v, approx. 10%v / v, approx. 15%v / v, approx. 20%v / v, approx. 30% v / v, about 40% v / v, or about 50% v / v, circulated through a closed circuit The perfusion fluid may leak outside the closed circuit. Any drugs lost through perfusion fluid leakage A volume of 1000 mg / mL of 10 ... The calculated exposure time may be from about 5 minutes to about 5 minutes in certain embodiments. time, within the range of about 15 minutes to about 4 hours, about 30 minutes to about 3 hours, about 1 hour to about 2 hours, or It may be any subrange therebetween.

[0088] therapeutic composition Drugs suitable for treating cardiac conditions (i.e., drugs included in the perfusate) are therapeutically effective. In some embodiments, the therapeutic polynucleotide may comprise a polynucleotide sequence. The sequence may encode a protein for the treatment of a cardiac condition. Proteins may be of human origin or may be derived from different species (e.g., but not limited to, mouse, feline, etc.). In some embodiments, the therapeutic polynucleoside may be derived from a mammal (such as a pig, a monkey, or a mammal). The protein encoded by the peptide sequence corresponds to a gene expressed in the human heart. good.

[0089] Exemplary proteins include, but are not limited to, SERCA2, MYBPC3, MYH7, PKP 2, MYL3, MYL2, ACTC1, TPM1, TNNT2, TNNI3, TTN, F HL1, ALPK3, dystrophin, FKRP, variants thereof, or The protein or proteins used may include one or more of the following combinations: may also be functional variants of the proteins referred to herein, It may exhibit significant amino acid sequence identity compared to the original protein. The acid identity may be at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least It may be at least about 97%, at least about 98%, or at least about 99%. In the present specification, the term "functional variant" refers to a variant of a protein that is not a naturally occurring This means that the protein is capable of partially or completely performing the function of the corresponding protein present in the protein. Functional variants of proteins include, for example, one or more amino acid substitutions, deletions, or may contain proteins that differ from their naturally occurring counterparts by addition.

[0090] Amino acid substitutions can be conservative or non-conservative. Substitutions are , i.e., amino acid residues with amino acids of similar polarity that act as functional equivalents Preferably, the amino acid residue used as a substitute is The amino acid residue to be substituted is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue can be selected from another hydrophobic residue. Aqueous residues can be substituted, or polar residues can be substituted with other polar residues having the same charge. Functionally equivalent amino acids that may be used for conservative substitutions include , for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine Contains thiamin, methionine, proline, phenylalanine, and tryptophan. Uncharged Examples of polar amino acids are serine, threonine, glutamine, asparagine, tyrosine and Examples of charged polar (basic) amino acids include histidine, arginine, and and lysine. Examples of charged polar (acidic) amino acids are aspartic acid and glutamine. Contains phosphate.

[0091] by one or more (e.g., 2, 3, 4, 5, 10, or 15) additional amino acids Proteins that differ in some way from their naturally occurring counterparts are also considered variants. The additional amino acids are present within the amino acid sequence of the original protein (i.e., as an insertion) or they may be added to one or both ends of the protein Essentially, the addition of an amino acid alters the function of a protein that occurs naturally in the subject being treated. Insertions may occur at any position provided that the insertion does not impair the ability of the polypeptide to perform its function. Furthermore, protein variants may also have one or more amino acid sequences that differ from the original polypeptide. This includes proteins with one or more amino acid deletions. Any amino acid position may be affected as long as it does not impair the ability to perform normal function.

[0092] Finally, variants of cardiac sarcomere proteins also contain structural modifications, e.g., modified amino acids. Modified amino acids refer to proteins that differ from naturally occurring proteins by the presence of certain amino acids. natural processes, such as processing or post-translational modification, or processes known in the art. Typical amino acid modifications are amino acids modified by any of the following chemical modification processes: The cleavage of ATP is mediated by phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosaminylation, and glycosylation. Dethiolation, acylation, branching, ADP-ribosylation, cross-linking, disulfide bridge formation, Formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization , and / or phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids , or covalently or non-covalently attached to a lipid.

[0093] The therapeutic polynucleotide sequence encoding the target protein can be used in a gene therapy vector, i.e., other sequences required to provide for expression of the exogenous nucleic acid, e.g., a promoter, Kozak sequence, and near the poly A signal, including translation and termination codons, It may be administered to the subject to be treated in the form of a nucleic acid construct containing the coding sequence. .

[0094] For example, the gene therapy vector may be part of a mammalian expression system. Expression systems and expression constructs are commercially available, and several mammalian expression systems are available. There are many different manufacturers that distribute these compounds and that can be used in the present invention, such as: Plasmid or viral vector-based systems, e.g., LENTI-Smart (Trademark) (InvivoGen), GenScript (Trademark) Expression vectors, pAdVAntage(TM) (Promega), ViraPow er(trademark) Lentiviral, Adenoviral Expression Systems (Invitrogen), and adeno-associated virus expression system (Cell Biolabs).

[0095] The gene therapy vector for expressing the exogenous therapeutic polynucleotide sequence of the present invention comprises: For example, an exogenous therapeutic polynucleotide sequence may be introduced into a cell, and the protein encoded by the nucleic acid may be transfected into the cell. a suitable viral or non-viral expression vector to be introduced for subsequent expression of the protein; The expression vector can be an episomal vector, i.e., a vector that is expressed within the host cell. They can be either autonomously replicating in the host or integrated vectors, i.e., stably integrated into the genome of the cell. Expression in the host cell can be constitutive or regulated. (e.g., inducible).

[0096] In certain embodiments, the gene therapy vector is a viral expression vector. Viral vectors for use in the invention may be used to transform a virus into a different virus without destroying its infectivity. A viral genome in which portions of the native sequence have been deleted to allow for the introduction of a seed polynucleotide. Due to specific interactions between viral components and host cell receptors, Viral vectors are highly suitable for efficient transfer of genes into target cells. Suitable viral vectors for facilitating gene transfer into mammalian cells include those from different viruses. viruses, e.g., AAV, adenovirus, retrovirus, herpes simplex virus, bovine Papillomavirus, lentivirus, vaccinia virus, polyomavirus, Sen Diviruses, Orthomyxoviruses, Paramyxoviruses, Papovaviruses, Picorna Viruses, poxviruses, alphaviruses, or any suitable vector for gene therapy Other viral shuttles, variations of these, and combinations of these This can be done.

[0097] An "adenoviral expression vector" or "adenovirus" is defined as (a) a vector capable of expressing a therapeutic polynucleotide; (b) to support packaging of the nucleic acid sequence construct; and / or for ultimate expression of tissue- and / or cell-specific constructs cloned into The term "adenoviral vector" is intended to include constructs containing sufficient adenoviral sequences to In one embodiment, the expression vector comprises a genetically engineered form of adenovirus. Genetic organization of adenovirus, a 36 kilobase (kb) linear double-stranded DNA virus. Knowledge of the structure allows replacement of large segments of adenoviral DNA with foreign sequences up to 7 kb. Let's say.

[0098] Adenovirus propagation and manipulation is known to those skilled in the art and can be carried out in vitro. This group of viruses exhibits a wide host range in vitro and in vivo. For example, 10 per mL 9 ~10 11 Plaque-forming units can be obtained, resulting in highly infectious The adenovirus life cycle does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and therefore It has low genotoxicity to host cells. No side effects have been reported in studies, and its efficacy as an in vivo gene transfer vector is unclear. They have demonstrated safety and / or therapeutic potential.

[0099] Retroviruses (also called "retroviral vectors") carry these genes. They can integrate into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and and gene delivery vectors due to their ability to be packaged in specialized cell lines. may be selected as

[0100] The retroviral genome contains three genes, gag, pol, and env. These encode capsid proteins, polymerase enzymes, and envelope components, respectively. The sequence found upstream of the gag gene regulates the packaging of the genome into virions. Two long terminal repeat (LTR) sequences are located at the 5′ end of the viral genome. These contain strong promoter and enhancer sequences. , which are also required for integration into the host cell genome.

[0101] To construct a retroviral vector, a nucleic acid encoding a gene of interest is introduced into a inserted into the viral genome at the location of specific viral sequences, producing a replication-defective virus. To produce virions, the gag, pol, and / or env genes are Packaging cell lines containing the nucleotide sequence but lacking the LTR and / or packaging components are also provided. Constructed containing cDNA along with retroviral LTR and packaging sequences. When a recombinant plasmid is introduced into this cell line (e.g., by calcium phosphate precipitation) The packaging sequence allows the RNA transcripts of the recombinant plasmid to be packaged into viral particles. The recombinant retrovirus is then secreted into the culture medium. They are then collected, optionally concentrated, and used for gene transfer. Vectors are capable of infecting a wide variety of cell types. Constant expression requires the division of host cells.

[0102] The retrovirus can be from any subfamily, e.g., murine sarcoma Viruses, bovine leukemia virus, Rous sarcoma virus, murine leukemia virus, mink cell vectors from alveolar focus-inducing viruses, reticuloendotheliosis viruses, or avian leukosis viruses The skilled artisan can use portions from different retroviruses, e.g., LT The R, tRNA binding site, and packaging signal are combined to form a recombinant retrovirus. These retroviruses are then typically transformed into transduction-competent vectors. It is used to produce retroviral vector particles. For this purpose, the vector The retrovirus is introduced into a suitable packaging cell line. By incorporating a ribosomal enzyme into retroviral particles, the retroviral particles can be integrated into the host cell DNA in a site-specific manner. It can be built for inclusion.

[0103] Herpes simplex virus (HSV) is neurotropic and therefore has potential in the treatment of nervous system disorders. Furthermore, they can be integrated into host cell chromosomes, and other host cell HS establishes latent infection in non-dividing neuronal cells without altering vacuolar metabolism The ability of V, together with the presence of a promoter active during latency, makes HSV an attractive vector. Although much attention has focused on the neurotropic applications of HSV, this vector has also Given its broad host range, it can be exploited for other tissues.

[0104] Another factor that makes HSV an attractive vector is the size and organization of its genome. Because the vector is large, the integration of multiple genes or expression cassettes is possible compared to other smaller viral vectors. In addition, various performances (time, strength, etc.) are available. It is more efficient than in other systems due to the availability of different viral regulatory sequences that control It is possible to control expression to a large extent. It is also beneficial to have messages that are encoded, making gene manipulation even easier. It is a point.

[0105] HSV is also relatively easy to manipulate and can be grown to high titers. Therefore, the volume required to achieve a sufficient multiplicity of infection (MOI) and Delivery is less problematic, both in terms of reduced need for repeated dosing and reduced need for repeated dosing. Non-pathogenic variants of HSV have been developed for use in the context of gene therapy. It is readily available.

[0106] Lentiviruses are complex retroviruses that encode the common retroviral genes gag, In addition to pol and env, it contains other genes with regulatory or structural functions Higher complexity allows the virus to complete its life cycle, such as during latent infection. Some examples of lentiviruses include those that target human immunodeficiency Viruses (HIV-1, HIV-2) and simian immunodeficiency virus (SIV) By attenuating the HIV pathogenic genes through doubling, lentiviral vectors For example, the genes env, vif, vpr, vpu, and nef have been deleted. Therefore, the vector is considered biologically safe.

[0107] Lentiviral vectors can be plasmid-based or virus-based and can deliver foreign nucleic acid It contains the necessary sequences for integration, selection, and transfer of the nucleic acid into the host cell. The gag, pol and env genes of the vector of interest are also Therefore, the relevant gene is inserted into the selected vector. The vector is cloned into a vector and then used to transform a target cell of interest.

[0108] Vaccinia virus vectors are advantageous due to their ease of construction, the relatively high levels of expression they can produce, and their It is widely used due to its broad host range and large capacity to carry DNA. Seniors have a linear, double-stranded DNA genome of approximately 186 kb that exhibits a marked "AT" preference. Approximately 10.5 kb of inverted terminal repeats flank the genome. Most of the essential genes are It appears to map within the central region that is most highly conserved among poxviruses. The estimated number of open reading frames in vaccinia virus is 150-20 0. Both strands are coding, but there is a large overlap in the reading frames. -Rap is not common.

[0109] At least 25 kb can be inserted into the vaccinia virus genome. The cytidine vector is inserted into the viral thymidine kinase gene via homologous recombination The vector containing the transgene is selected based on the tk phenotype. The inclusion of a non-translated leader sequence from the vector results in higher levels of expression than conventional vectors. The transgene contributes 10% or more of the protein of infected cells within 24 hours. Accumulates quickly.

[0110] Empty capsids of papovaviruses, such as mouse polyomavirus, are used for gene transfer. The use of empty polyoma DNA has attracted attention as a possible vector for It was first described when purified empty capsids were incubated in a cell-free system. The DNA in the new particles was protected from the action of pancreatic DNase. It was used to transfect rat FIII cells with transforming polyoma DNA fragments. The capsid and reconstituted particles contain polyoma capsid antigens VP1, VP2, and V It consists of all three P3s.

[0111] AAVs are parvoviruses that belong to the Dependovirus genus. They are small, non- It is an enveloped, single-stranded DNA virus that requires a helper virus to replicate. A helper virus (e.g., adenovirus) is required to form functionally intact AAV virions. Coinfection with other viruses (such as flu, flu virus, herpes virus, or vaccinia virus) is required. In vitro, in the absence of helper virus superinfection, AAV remains latent. In the latent state, the viral genome exists in an episomal form, but the infectious virus No ribonuclease is produced. Subsequent infection with a helper virus "rescues" the genome. The genome is then replicated and packaged into a viral capsid, which Infectious virions are reconstituted. Recent data indicate that α- and β-glucan exist primarily as large episomal concatemers. In one embodiment, the gene therapy vector used herein is an AAV vector. AAV vectors are purified, replication-incompetent, pseudotyped vectors. It may also be an rAAV particle.

[0112] AAV has not been associated with any known human disease and is generally not considered pathogenic. AAVs are not transduced into non-dividing cells and do not appear to alter the physiological properties of the host cell upon integration. They are able to infect a wide range of host cells, including bacteria, and are capable of infecting cells from different species. They are rapidly cleared or inactivated by both cellular and humoral responses. In contrast to some vectors that are commonly used, AAV vectors have a variety of in vivo It has been shown to induce sustained transgene expression in tissues. Persistence of recombinant AAV-mediated transgenes in non-dividing cells is comparable to that of native AAV viruses. The absence of a gene and the ITR-associated ability of the vector to form episomal concatemers can be attributed.

[0113] AAV has a high frequency of persistence as episomal concatemers and is a novel vector containing cardiomyocytes. For the first time, they are able to infect non-dividing cells, making them suitable for use in, for example, tissue culture and in vivo The cell types of the present invention are useful for gene delivery to mammalian cells in It is an attractive vector system for use in transduction.

[0114] Typically, rAAV contains a gene of interest flanked by two AAV terminal repeats. Plasmids containing and / or wild-type AAV coding sequences without terminal repeats It is produced by cotransfecting an expression plasmid containing The cells also contain the adenoviral genes required for AAV helper function. Infect and / or transfect with adenovirus and / or plasmids rAAV stocks produced in this manner are contaminated with adenovirus. , which can be done (e.g., by cesium chloride density centrifugation or column chromatography) Alternatively, the AAV coding region must be physically separated from the rAAV particle. Adenoviral vectors containing AAV coding regions and / or AAV coding regions Alternatively, cell lines containing some or all of the adenovirus helper genes can be used. Cell lines that carry rAAV DNA as an integrated provirus can also be used. It can be used.

[0115] Multiple serotypes of AAV exist in nature, with at least 12 serotypes (AAV1 to AAV1 2) Despite the high degree of homology, different serotypes have different tissue tropisms. Upon transfection, AAV causes little, if any, damage in the host. Therefore, AAV is highly suitable for gene therapy approaches. Suitable.

[0116] In some embodiments, the present disclosure provides AAV1, AAV2, AAV3, AAV4, AA V5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 , ANC AAV, chimeric AAV derived from these, variations thereof, and and is more suitable for high efficiency transduction in the tissue of interest. and directed to a drug containing an AAV vector that is one or more of the combinations of In certain embodiments, the gene therapy vector is an AAV serotype 1 vector. In certain embodiments, the gene therapy vector is an AAV serotype 2 vector. In certain embodiments, the gene therapy vector is an AAV serotype 3 vector. In certain embodiments, the gene therapy vector is an AAV serotype 4 vector. In certain embodiments, the gene therapy vector is an AAV serotype 5 vector. In certain embodiments, the gene therapy vector is an AAV serotype 6 vector. In certain embodiments, the gene therapy vector is an AAV serotype 7 vector. In certain embodiments, the gene therapy vector is an AAV serotype 8 vector. In certain embodiments, the gene therapy vector is an AAV serotype 9 vector. In certain embodiments, the gene therapy vector is an AAV serotype 10 vector. In certain embodiments, the gene therapy vector is an AAV serotype 11 vector. In certain embodiments, the gene therapy vector is an AAV serotype 12 vector. It is a vector.

[0117] A suitable dose of AAV for humans is approximately 1 x 10 8 vg / kg ~ approx. 3×10 14 vg / kg, approximately 1 x 10 8 vg / kg, approximately 1×10 9 vg / kg, approximately 1×10 10 vg / kg, Approximately 1×10 11 vg / kg, approximately 1×10 12 vg / kg, approximately 1×10 13 vg / kg, or or about 1 x 10 14 The total amount of viral particles or DRPs can be in the range of vg / kg. , about, at least, at least about, at most, or at most about, 5×10 15 vg / kg , 4×10 15 vg / kg, 3 × 10 15vg / kg、2×10 15 vg / kg、1×1 0 15 vg / kg、9×10 14 vg / kg、8×10 14 vg / kg、7×10 14 v g / kg、6×10 14 vg / kg、5×10 14 vg / kg、4×10 14 vg / kg 、3×10 14 vg / kg、2×10 14 vg / kg、1×10 14 vg / kg、9×1 0 13 vg / kg、8×10 13 vg / kg、7×10 13 vg / kg、6×10 13 v g / kg、5×10 13 vg / kg、4×10 13 vg / kg、3×10 13 vg / kg 、2×10 13 vg / kg、1×10 13 vg / kg、9×10 12 vg / kg、8×1 0 12 vg / kg、7×10 12 vg / kg、6×10 12 vg / kg、5×10 12 v g / kg、4×10 12 vg / kg、3×10 12 vg / kg、2×10 12 vg / kg 、1×10 12 vg / kg、9×10 11 vg / kg、8×10 11 vg / kg、7×1 0 11 vg / kg、6×10 11 vg / kg、5×10 11 vg / kg、4×10 11 v g / kg、3×10 11 vg / kg、2×10 11vg / kg, 1 × 10 11 vg / kg , 9×10 10 vg / kg, 8 × 10 10 vg / kg, 7 × 10 10 vg / kg, 6×1 0 10 vg / kg, 5 × 10 10 vg / kg, 4 × 10 10 vg / kg, 3 × 10 10 v g / kg, 2 x 10 10 vg / kg, 1 × 10 10 vg / kg, 9 × 10 9 vg / kg, 8×10 9 vg / kg, 7 × 10 9 vg / kg, 6 × 10 9 vg / kg, 5 × 10 9 vg / kg, 4 × 10 9 vg / kg, 3 × 10 9 vg / kg, 2 × 10 9 vg / kg, 1×1 0 9 vg / kg, 9 × 10 8 vg / kg, 8 × 10 8 vg / kg, 7 × 10 8 vg / kg , 6×10 8 vg / kg, 5 × 10 8 vg / kg, 4 × 10 8 vg / kg, 3 × 10 8 v g / kg, 2 x 10 8 vg / kg, or 1 × 10 8 vg / kg, or The dosages listed above fall within the range defined by any two of the values ​​of It is measured in units of vg per kg.

[0118] Using the systems and methods disclosed herein, in some embodiments: Higher doses of drugs are delivered directly to the heart than could otherwise be safely administered through systemic delivery. and can be administered only to the heart because of the outside of the heart and / or the Thebesian vein. This is because there is virtually no leakage of perfusate into the system. However, AAV toxicity can also result in systemic effects, such as hepatotoxicity, platelet activation and loss, and It is believed that all of these toxicities and others may be due to complement activation and loss. Reduced, even minimal, through localized regional perfusion fluid application as described in the methods and systems disclosed herein. Therefore, approximately 5 × 10 per kg of cardiac tissue 15 vg Doses of up to 100 mg / kg of cardiac tissue may be well tolerated. The AAV dose to the heart, expressed as vg per 1000 mg, is about 2 to about 200 mg, about 5 to about 150 mg, and about 6 to about 100 mg. , or a factor of about 10 to about 100, or any subrange therein, The dose given may be exceeded.

[0119] In addition to viral vectors, non-viral expression constructs can also be used to deliver target proteins to the patient's cells. or a functional variant or fragment thereof. Non-viral expression vectors that allow in vivo expression of proteins in target cells may also be used. Examples of vectors include plasmids, modified RNA, cDNA, and antisense oligomers. , DNA-lipid complexes, nanoparticles, exosomes, any other suitable for gene therapy These include non-viral shuttles, variations thereof, and combinations thereof.

[0120] In addition to viral and non-viral expression vectors, nuclease systems are also , enter the patient's cells and encode the target protein or a functional variant or fragment thereof. vectors and / or electroporation systems to introduce the gene Exemplary nuclease systems include those that utilize non- Limited, clustered regularly interspaced sho rt palindromic repeats (CRISPR), DNA cleavage enzymes (e.g. For example, Cas9, meganucleases, TALENs, zinc finger nucleases, Any other nuclease system suitable for gene therapy, variations thereof, and and combinations thereof. For example, in one embodiment, The virus vector (e.g., AAV) is used for nucleases (e.g., CRISPR). Other viral vectors (e.g., AAV) may be used that contain DNA cleaving enzymes (e.g., Cas9), thereby acting as both a nuclease and a DNA cleaver. The enzyme is introduced into the target cell.

[0121] It is used to deliver therapeutic polynucleotide sequences encoding therapeutic genes into cells. Other vector delivery systems that can be used are receptor-mediated delivery vehicles. These are Utilizing the selective uptake of macromolecules by receptor-mediated endocytosis in eukaryotic cells Due to the cell type-specific distribution of various receptors, delivery can be highly specific. A gene targeting vehicle mediated by ATP has two components: a cell receptor-specific ligand and DNA binding. It may also contain an agent.

[0122] Suitable methods for the transfer of non-viral vectors into target cells include, for example, lipofection. The precipitation method, calcium phosphate coprecipitation method, DEAE dextran method, and microglass tube method Direct DNA transfer methods using tubes, ultrasound, and electroporation Prior to the introduction of the vector, the cardiomyocytes are permeabilized with a permeabilizing agent, such as phosphatidylcholine, Streptolysin, sodium caprate, decanoylcarnitine, tartaric acid, lysolecithin Exosomes may also be treated with cephalosporins, cephalosporins, and Triton X-100. It may be used to transfer avian DNA or AAV capsid DNA.

[0123] The gene therapy vectors of the present invention comprise a nucleic acid sequence operably linked to a nucleic acid sequence encoding a target protein. The promoter sequence may be a promoter sequence that ensures compact and strong expression. Preferably, the promoter is one that controls the gene therapy vector. In some embodiments, the expression of a target protein in the myocardium of a patient treated with the In the method, the gene therapy vector is operably linked to a nucleic acid sequence encoding a target protein. As used herein, "cardiac specific promoter" includes a cardiac specific promoter linked to the promoter. A "specific promoter" is a promoter whose activity in cardiac cells is greater than that in any other non-cardiac cell type. Preferably, the promoter used in the vector of the present invention is A cardiac-specific promoter suitable for expression in cardiac cells has activity that is reduced compared to its activity in non-cardiac cell types. at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 2 times It has 5-fold, or at least 50-fold, higher activity in cardiac cells.

[0124] The cardiac-specific promoter may be a selected human promoter or a selected human promoter. At least about 80%, at least about 90%, at least about 95%, at least about 90% for the motor at least about 96%, at least about 97%, at least about 98%, or at least about 99% The promoter may also be a promoter containing a functionally equivalent sequence having at least 100% sequence identity. An exemplary non-limiting promoter that may be used is the cardiac troponin T promoter (TNNT 2) Other non-limiting examples of promoters include the alpha myosin heavy chain promoter tar, myosin light chain 2v promoter, alpha myosin heavy chain promoter, alpha- Cardiac actin promoter, alpha-tropomyosin promoter, cardiac troponin C Promoter, cardiac troponin I promoter, cardiac myosin-binding protein C promoter and sarcoplasmic / endoplasmic reticulum Ca 2+ -ATPase (SERCA) promoter ( For example, isoform 2 of this promoter (SERCA2) can be mentioned.

[0125] Vectors useful in the present invention may have a variety of transduction efficiencies. Viral or non-viral vectors reach approximately 10% of the cells in the targeted vascular territory, approximately 2% 0%, approx. 30%, approx. 40%, approx. 50%, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 7 5%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 100%; Transduction can be performed with more than one vector (including viruses). or non-viral, or a combination thereof) can be used simultaneously or sequentially. This can be used to transfer more than one polynucleotide and / or one More cell types can be targeted. Multiple vectors or multiple agents can be used. If multiple transduction / transfection efficiencies are used, more than one may result. It is possible.

[0126] The pharmaceutical composition containing the gene therapy vector may be administered either as a liquid solution or as a suspension. The pharmaceutical compositions of the present invention may be prepared in a commonly used pharmaceutically acceptable excipient. In particular, the compositions may contain pharmaceutically acceptable carriers, such as diluents and carriers. Carriers include, for example, water, saline, Ringer's solution, or dextrose solution. In addition, the pharmaceutical composition may also contain emulsifiers, pH buffering agents, stabilizers, dyes, etc. Good too.

[0127] In certain embodiments, the pharmaceutical composition comprises a therapeutically effective gene dose, The compound can prevent or treat cardiomyopathy in a subject without being toxic to the subject. The prevention or treatment of cardiomyopathy involves administering a dose that is sufficient to treat the phenotypic characteristics associated with cardiomyopathy. Such changes may be evaluated as changes in the function of the immune system to prevent or treat cardiomyopathy. Therefore, a therapeutically effective gene dose is typically less than physiologically and when administered in a composition acceptable to humans, the composition induces a pathogenic cardiac phenotype in a treated subject. is sufficient to improve or prevent

[0128] Cardiac conditions that can be treated by the methods disclosed herein include, but are not limited to, genetically determined cardiac disease (e.g., genetically determined cardiomyopathy), arrhythmic cardiac disease, heart failure, Ischemia, arrhythmia, myocardial infarction, congestive heart failure, graft rejection, abnormal cardiac contractions, non-ischemic cardiomyopathy , mitral regurgitation, aortic stenosis or regurgitation, abnormal Ca 2+ Metabolism, congenital heart disease, The present invention may include one or more of the following: primary or secondary cardiac tumors, and combinations thereof. stomach. [Example]

[0129] Illustrative Examples The following examples are included to aid in the understanding of the present disclosure and, of course, are not intended to be limiting unless otherwise specified. and should not be construed as limiting the embodiments specifically described and claimed. Including all known or later-developed equivalent replacements within the knowledge of the artisan. Such variations of the embodiments and changes in formulation or experimental design are also contemplated. Minor variations such as these should be considered to fall within the scope of the embodiments incorporated herein. do.

[0130] [Example 1] Feasibility study of locoregional perfusion in three pigs The procedure was successfully performed in three pigs (sus scrofa domestica) for 60 minutes. Feasibility of the established LRP system. Two pigs underwent thoracotomy for research purposes. In the third pig, no thoracotomy was performed, although all catheters were introduced percutaneously. First, the entire LRP procedure was performed percutaneously.

[0131] Utilizing the LRP system 100 shown in and described with reference to FIGS. 1A and 1B LRP was performed on three animals. No technical problems were observed in any of the three animals. The LRP procedure could be maintained without issue, with the heart beating spontaneously for 60 minutes. During LRP, all animals (n = 3) were hemodynamically stable without any need for inotropic agents. Cardiac function after LRP was unremarkable and baseline was stable in all animals. Total coronary artery occlusion was tolerated: in animal 1, the LCA was completely In animal 2, the RCA was not completely occluded (the leakage was considered mild), and in animal 3, the RCA was not completely occluded. In animal 3, both coronary arteries were occluded. Ta.

[0132] Due to the variable anatomy of pigs, tight occlusion of the coronary sinus (CS) is technically difficult. In contrast to humans, in pigs, the azygos vein directly enters the coronary sinus, which is difficult to achieve. Complete occlusion was achieved in animal 3. A partial occlusion was achieved (using a Reliant balloon) in animals 1 and 2. Achieved (ProPledge catheter). Range: 166 mL / min to 244 mL / min The flow rate during the 60-minute LRP procedure was achieved within the intended use and The present invention is not limited to the use in the LRP system according to the embodiment shown. List any auxiliary devices used.

[0133] Figure 2 shows a radiograph of a typical LRP in situ device, with coronary catheter The catheter is indicated by an arrow and the coronary sinus balloon (collection catheter) is indicated by a triangle. is shown.

[0134] Mean values ​​of LRP parameters (pump speed, flow, and pressure) for three animals are summarized in Figure 3 (bars represent standard deviation).

[0135] The table below illustrates their intended use and applications in an LRP system according to an embodiment of the present disclosure. Including use.

[0136] Figure 2 shows a radiograph of a typical LRP in situ device, with coronary catheter The catheter is indicated by an arrow and the coronary sinus balloon (collection catheter) is indicated by a triangle. is shown.

[0137] Mean values ​​of LRP parameters (pump speed, flow, and pressure) for three animals are summarized in Figure 3 (bars represent standard deviation).

[0138] [Table 1]

[0139] [Table 2]

[0140] [Example 2] Safety study of a locoregional perfusion system in two pigs LR using a percutaneous approach for 60 minutes in two pigs (sus scrofa domestica) by performing the procedure and following the animals for 24 hours after the procedure while keeping them under anesthesia. The safety of the LRP system was established. After a 24-hour period, the animals were sacrificed and their hearts were Macroscopic and microscopic examination of the heart was performed. Additionally, blood biomarkers were obtained and cardiac tissue The damage was assessed.

[0141] In both experiments, the LRP was successfully performed without any serious adverse effects. Animal 5 experienced a technical issue and the tubing connection on the pump head failed after 10 minutes. The LRP was immediately discontinued and all catheters were removed and deflated. The head was immediately replaced and the LRP system was reconnected, deflated, and restarted. The animals were hemodynamically stable and no serious adverse effects were observed. It was then maintained for 60 minutes, demonstrating safety and efficacy even with minor equipment malfunctions.

[0142] The animals remained in a stable condition throughout the procedure, including the initiation, resumption, and 24 hours after the LRP. The patient was hemodynamically stable without any need for inotropic agents.

[0143] In both animals, the left main and right coronary arteries were completely occluded, and the coronary sinus was partially occluded. Achieving a mean LRP flow of 173 mL / min while still occluding (leakage was moderate) Postoperative and 24-hour cardiac function was unremarkable and comparable to baseline. Cardiac biomarkers (myoglobin and troponin) were significantly elevated during the LRP procedure. There was only a slight increase in the blood pressure at and immediately after the 24-hour follow-up, but the blood pressure immediately returned to baseline during the 24-hour follow-up. The continuous hemodynamic stability of the animals throughout the entire procedure, Besides the absence of adverse effects and only a minor and transient increase in cardiac biomarkers, Considering the ECG changes are only transient with immediate normalization over 24 hours, the LRP procedure Table 3 shows the animals4 and animals used in the safety studies. Flow and pressure characteristics over a 60-minute LRP procedure in Subject 5 are compiled.

[0144] [Table 3]

[0145] The hearts of Animals 4 and 5 were examined macroscopically after sacrifice.

[0146] The heart weight of Animal 4 was 312 grams. No gross pathology was observed, especially myocardial ischemia. There were no signs of pulmonary embolism or myocardial infarction. A localized hematoma was observed in the posterior aspect of the heart of Animal 4. was observed in the right coronary artery segment, which was most likely due to wire injury during the procedure. expensive.

[0147] The heart weight of Animal 5 was 293 grams. No gross pathology was observed, particularly myocardial ischemia. There were no signs of pulmonary embolism or myocardial infarction. A localized hematoma was observed in the posterior aspect of the heart of Animal 5. This was observed in the distal right coronary artery and distal left circumflex artery segments, which may have been due to wire scarring during the procedure. Most likely due to harm.

[0148] To confirm the biochemical integrity of cardiac tissue, serum cardiac biomarkers were obtained and analyzed. These are summarized in Table 4. Creatine kinase (CK) levels remained stable during the LRP procedure. However, most likely due to the animals lying in a supine position, Myoglobin levels remained stable during the LRP procedure, and There was only a minimal increase afterward, but still within the reference level for animal 4 and animal 5. Troponin T was only slightly higher than the reference level for the LRP procedure. The blood pressure increased minimally during the follow-up period, peaked at 60 minutes, and then returned to baseline values ​​during follow-up. It decreased.

[0149] [Table 4]

[0150] In the preceding description, numerous specific details, examples, and embodiments are set forth in order to provide a thorough understanding of the present invention. For example, specific materials, dimensions, process parameters, etc. are described. The materials, materials, or features may be combined in any suitable manner in one or more embodiments. The word "example" or "exemplary" means something that serves as an example, instance, or illustration. "Example" or "exemplary" is used herein to mean Any aspect or design described herein is not intended to be preferred or advantageous over other aspects or designs. Rather, the use of the words "example" or "exemplary" are simply intended to present concepts in a concrete manner. where the term "or" is used, it shall mean an inclusive "or" and not an exclusive "or." That is, unless otherwise specified or clear from the context, Therefore, "X contains A or B" means any of the natural inclusive permutations. That is, X contains A, or X contains B, or X contains A and B. If both are included, then "X contains A or B" is satisfied under either of the above cases. Throughout this specification, the terms "embodiment," "particular embodiment," or "one implementation" may be used. References to "embodiments" may be used to refer to specific configurations, structures, or features described in connection with an embodiment. It is therefore intended that the entire specification be construed as including at least one embodiment. The terms "embodiment," "a particular embodiment," or "one implementation" may be used in various places throughout the specification. Appearances of the phrase "in an embodiment" do not necessarily all refer to the same embodiment.

[0151] The present invention has been described with reference to specific exemplary embodiments thereof. As such, it should be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those described will become apparent to those skilled in the art and are set forth in the accompanying claims. It is intended to fall within the scope of the claims. [Explanation of symbols]

[0152] 100 LRP System 110A forward view 110B Rear view 112 Right coronary artery 114 Left main coronary artery 116 Coronary sinus 120 Membrane oxygenator device 122 First Catheter 124 Second Catheter 126 Collecting catheter 130 Blood Gas Analysis (BGA) Monitor 140 Pressure Monitor 142 First pressure sensor 144 Second Pressure Sensor 152 Exit 154 Entrance 156 Heat exchanger 158 Delivery Pump 160 reservoir 162 sensors 164 sensors 166 Membrane oxygenator 168 Gas Blender 170 Gas regulator

Claims

1. 1. A method of perfusing a drug in a non-arrested, beating heart of a patient, comprising: placing a first drug delivery catheter in a right coronary artery of the heart; placing a second drug delivery catheter in the left main coronary artery of the heart; placing a drug collection catheter in the coronary sinus of the heart, the drug collection catheter, the second drug delivery catheter, and the drug collection catheter Together with the coronary arteries of the heart, the coronary venous system of said heart, and the membrane oxygenator device, form a closed circuit. said disposing, and perfusing the drug through the closed circuit, wherein the closed circuit comprises: isolating the patient's coronary circulation from the patient's systemic circulation, said perfusing A method comprising:

2. The method of claim 1 , further comprising applying negative pressure to the drug collection catheter.

3. 3. The method of claim 2, wherein the negative pressure is in the range of about -100 mmHg to 0 mmHg.

4. the first drug delivery catheter, the second drug delivery catheter, or the drug collection 10. The method of claim 1, wherein one or more of the catheters are introduced percutaneously. The method described in paragraph .

5. The first drug delivery catheter and / or the second drug delivery catheter are antegrade.

10. The method of any one of the preceding claims, wherein the catheter is placed via intubation.

6. the first drug delivery catheter and / or the second drug delivery catheter are Transaortic access to the aorta and / or radial aorta of the patient.

10. The method of any one of the preceding claims, wherein

7. The drug collection catheter is placed into the coronary sinus via the patient's vena cava.

10. The method according to claim 9, wherein the

8. wherein the drug collection catheter is placed via the patient's jugular or femoral vein. The method according to any one of claims 1 to 4.

9. The membrane oxygenation device includes the collection catheter and the first drug delivery catheter. and one or more of said first and second drug delivery catheters.

10. The method according to claim 9 , wherein

10. 3. The method of claim 1, further comprising circulating blood through the closed circuit.

1. The method according to claim 1.

11. The blood comprises autologous blood, matched blood from a donor, or a combination thereof. The method according to claim 10.

12. A blood component, e.g., serum or plasma, is selected according to one or more parameters. wherein the one or more parameters include the presence or absence of a selected antibody. The method according to any one of claims 10 to 11.

13. Approximately 1000mL, approximately 800mL, approximately 600mL, approximately 400mL, approximately 200mL, approximately 10 100 mL, or about 50 mL of blood is circulated through the closed circuit.

13. The method of any one of 0 to 12.

14. The perfusion is for about 5 minutes to about 5 hours, about 15 minutes to about 4 hours, about 30 minutes to about 3 hours, or about 10. The method of claim 9, wherein the heating is carried out for a duration of from 1 hour to about 2 hours. method.

15. 10. The method of any one of the preceding claims, wherein the perfusion is performed for at least 60 minutes. 。

16. The perfusion is from about 75 mL / min to about 750 mL / min, from about 150 mL / min to about 500 mL / min 200 mL / min to 300 mL / min, or at a flow rate of about 200 mL / min to about 300 mL / min. The method according to any one of claims 1 to 4.

17. 10. Any one of the preceding claims, wherein the drug is suitable for the treatment of a cardiac condition. The method described in paragraph .

18. 18. The method of claim 17, wherein the cardiac condition is heart failure.

19. 18. The method of claim 17, wherein the cardiac condition is a genetically determined cardiac disease.

20. 10. The method of claim 1, wherein the genetically determined heart disease is a genetically determined cardiomyopathy.

9. The method according to claim 9.

21. 10. The method of claim 1, wherein the drug comprises a therapeutic polynucleotide sequence. How to do it.

22. the therapeutic polynucleotide sequence is present in one or more viral vectors; 22. The method of claim 21.

23. The one or more viral vectors may be an adeno-associated virus, an adenovirus, a leukemia virus, or a leukemia virus. Torovirus, herpes simplex virus, bovine papillomavirus, lentivirus vectors -, vaccinia virus, polyoma virus, Sendai virus, orthomyxovirus viruses, paramyxoviruses, papovaviruses, picornaviruses, poxviruses, and albicans and a combination thereof.

23. The method of claim 22.

24. 24. The method of claim 23, wherein the viral vector is an adeno-associated virus (AAV). Law.

25. The AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AA V7, AAV8, AAV9, AAV10, AAV11, AAV12, and variations thereof 25. The method of claim 24, wherein the method is one or more of:

26. the therapeutic polynucleotide sequence is a nucleic acid encoding a protein for the treatment of a cardiac condition The method of any one of claims 21 to 25, comprising the sequence

27. 27. The method of claim 26, wherein the protein corresponds to a gene expressed in the human heart. Law.

28. The protein is SERCA2, MyBPC3, MYH7, PKP2, dystrophy one or more of: PG, FKRP, or combinations or variations thereof The method of claim 27.

29. Any of claims 21 to 28, wherein the therapeutic polynucleotide sequence comprises a promoter.

10. The method according to claim 1.

30. less than about 20% v / v, less than about 15% v / v, less than about 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v less than about 1% v / v, less than about 0.5% v / v, or substantially 0% v / v of the blood circulated through the closed circuit, 10. The method of any one of the preceding claims, wherein the

31. less than about 20% v / v, less than about 15% v / v, less than about 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v less than about 1% v / v, less than about 0.5% v / v, or substantially 0% v / v of the drug perfused through the closed circuit, 10. The method of any one of the preceding claims, wherein the

32. the first drug delivery catheter, the second drug delivery catheter, or the drug collection Any of the preceding claims, wherein one or more of the catheters is a balloon catheter. The method according to any one of claims 1 to 5.

33. 1. A method for maintaining perfusion of a perfusion fluid through a closed circuit in a patient's heart, said method comprising: the patient is not stopped and is beating during the perfusion, and the method further comprises: placing a first catheter in a right coronary artery of the heart; placing a second catheter in the left main coronary artery of the heart; placing a collection catheter in the coronary sinus of the heart, the first catheter , the second catheter, and the collection catheter are connected to the coronary arteries, the coronary venous system, and the membrane-type and forming, together with an artificial lung device, the closed circuit through the heart. , and The perfusion fluid is introduced into the heart via the first catheter and the second catheter. and causing the perfusion fluid to flow through the closed circuit by inserting the collection catheter. and collecting the perfusion fluid via a closed loop, the closed loop being connected to the patient's systemic circulation. isolating the patient's coronary circulation from the flowing and collecting A method comprising:

34. 34. The method of claim 33, wherein the perfusion is maintained for at least 60 minutes.

35. 35. The method of claim 34, wherein the perfusion is maintained for at least 120 minutes.

36. further comprising applying a negative pressure to the collection catheter, wherein the negative pressure is about -100 mmH The method of any one of claims 33 to 35, wherein the pressure is in the range of 0 mmHg to 0 mmHg.

37. one of the first catheter, the second catheter, or the collection catheter 37. The method of any one of claims 33 to 36, wherein one or more are introduced percutaneously.

38. The membrane oxygenation device includes the collection catheter and the first drug delivery catheter. and one or more of the second drug delivery catheters. Item 38. The method according to any one of Items 33 to 37.

39. The method further comprises circulating blood through the closed circuit, wherein the blood is autologous blood, any of claims 33 to 38, comprising matched blood from a donor, or a combination thereof.

10. The method according to claim 1.

40. Approximately 1000mL, approximately 800mL, approximately 600mL, approximately 400mL, approximately 200mL, approximately 10 3. 0 mL, or about 50 mL of blood is circulated through the closed circuit.

9. The method according to claim 9.

41. The perfusion is from about 75 mL / min to about 750 mL / min, from about 150 mL / min to about 500 mL / min 41. The method of claim 33, wherein the flow rate is about 200 mL / min to about 300 mL / min.

10. The method according to any one of claims 1 to 9.

42. less than about 20% v / v, less than about 15% v / v, less than about 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v less than about 1% v / v, less than about 0.5% v / v, or substantially 0% v / v of the blood circulated through the closed circuit, The method of any one of claims 33 to 41, wherein the liquid leaks into the

43. one of the first catheter, the second catheter, or the collection catheter The method according to any one of claims 33 to 42, wherein one or more of the catheters is a balloon catheter. Law.

44. SYSTEM FOR PROVIDING LOCAL REGIONAL PERFUSION WITHIN A PATIENT'S HEART WHEN FLUIDLY CONNECTED TO THE HEART - Patent application And, a first catheter adapted for insertion into a right coronary artery of the heart; a second catheter adapted for insertion into the left main coronary artery of the heart; a collection catheter adapted for insertion into the coronary sinus of the heart; the first catheter, the second catheter, the collection catheter, and the oxygen supply a membrane oxygenation device fluidly connected to a source, the first catheter, the second catheter The catheter, the collection catheter, and the membrane oxygenator device together form the The first catheter is inserted into the right coronary artery, and the second catheter is inserted into the left main coronary artery. and the collection catheter is inserted into the coronary sinus. The membrane oxygenator device forms a closed circuit through the heart, isolated from the systemic circulation. and to drive fluid flow through the first catheter and the second catheter. Configured pump Including, the system.

45. 1. A locoregional perfusion system comprising: a first catheter inserted into the right coronary artery of the patient's heart; a second catheter inserted into the left main coronary artery of the heart; a collection catheter inserted into the coronary sinus of the heart; and the first catheter, the second catheter, the collection catheter, and the oxygen supply a membrane oxygenation device fluidly connected to a source, the first catheter, the second catheter catheter, the collection catheter, and the membrane oxygenator device are connected to the coronary arteries and a closed circuit through the heart, separated from the patient's systemic circulation, along with the venous and coronary venous systems The membrane oxygenator device, and collection into the heart via the first catheter and the second catheter. a pump configured to drive fluid flow out of the heart through a collection catheter.

1. A local-regional perfusion system comprising:

46. The membrane oxygenator device is configured to infuse a drug into the closed circuit during perfusion.

46. ​​The locoregional irrigation device of claim 44 or claim 45, comprising an enclosed reservoir. Flow system.

47. the pump is configured to generate a negative pressure range of approximately -100 mmHg to 0 mmHg; The local-regional perfusion system according to any one of claims 44 to 46,

48. the first drug delivery catheter, the second drug delivery catheter, or the drug collection 48. Any of claims 44 to 47, wherein one or more of the catheters are introduced percutaneously.

10. The local-regional perfusion system according to claim 1.

49. The first drug delivery catheter and / or the second drug delivery catheter are antegrade.

49. The local-regional perfusion system according to claim 44, wherein the system is placed via an intubation method.

50. the drug collection catheter is placed into the coronary sinus via the patient's vena cava.

50. The local-regional perfusion system according to any one of claims 44 to 49.

51. A method according to claim 44 or claim 45, adapted to perform any of the methods according to claims 1 to 43.

46. ​​The local-regional perfusion system of claim 45.

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