Materials and Methods for Intracardiac Gene Delivery

Abstract

Provided herein are materials, methods, and devices for the targeted delivery of pharmaceutical agents. In particular, provided herein are materials, methods, and devices for the targeted delivery of pharmaceutical agents to the atria or ventricles of the heart.

Description

Technical Field

[0001] Matters Relating to Priority This application claims priority to U.S. Provisional Patent Application No. 62 / 884,012, filed Aug. 7, 2019; U.S. Provisional Patent Application No. 62 / 942,516, filed Dec. 2, 2019; U.S. Provisional Patent Application No. 62 / 947,737, filed Dec. 13, 2019; and U.S. Provisional Patent Application No. 62 / 961,514, filed Jan. 15, 2020, the entire contents of each of which are incorporated herein by reference.

[0002] Field The present disclosure relates to targeted gene delivery. In particular, materials, methods, and devices for targeted intracardiac gene delivery are provided herein.

Background Art

[0003] Background Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting more than 4 million Americans. It is also a major cause of stroke. The annual medical cost of AF in the United States exceeds $6 billion. Thus, the diagnosis and management of AF represent an important and challenging aspect of cardiovascular medicine.

[0004] Gene therapy may be a viable option for the treatment of disorders such as AF. However, systemic gene delivery often results in sub-therapeutic concentrations of the gene in the target organ. In addition, systemic delivery has the risk of unwanted gene expression in organs distant from the target area and can have significant side effects. However, direct local gene therapy of the heart often fails due to insufficient transfer of the gene into cardiomyocytes.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, there is a need for a novel method for gene-based therapy that is safe and effective for the treatment of heart disorders such as AF.

Means for Solving the Problem

[0006] Overview Materials, methods, and devices for the targeted delivery of agents are provided herein. In some aspects, methods for delivering an agent to a subject are provided herein. The method includes delivering the agent to an area of the subject's coronary vasculature and electroporating the subject's target coronary tissue. In some aspects, methods for treating a heart disorder in a subject are provided herein. A method for treating a heart disorder in a subject includes delivering an agent to an area of the subject's coronary vasculature and electroporating the subject's target coronary tissue. The heart disorder may be cardiac arrhythmia, congestive heart failure, or coronary artery disease.

[0007] In some aspects, the area of the coronary vasculature is different from the target coronary tissue. For example, the area of the coronary vasculature may be the aortic root, coronary artery, or coronary sinus. The target coronary tissue may be the left atrium, right atrium, left ventricle, or right ventricle.

[0008] In some aspects, electroporation is performed before delivery of the agent to the area of the subject's coronary vasculature, simultaneously with delivery of the agent, and / or after delivery of the agent. Electroporation may be performed by epicardial or endocardial electroporation.

[0009] In some aspects, the agent includes a therapeutic agent for the treatment of a heart disorder in the subject. For example, the agent may include nucleotides, oligonucleotides, proteins, peptides, small molecules, or macromolecules.

Brief Description of the Drawings

[0010]

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DETAILED DESCRIPTION OF THE INVENTION

[0011] Definitions Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the embodiments described herein, but some preferred methods, compositions, devices, and materials are described herein. However, prior to the description of the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein and may vary according to conventional experimentation and optimization. Also, it should be understood that the terms used in this description are for the purpose of describing only specific variations or embodiments and are not intended to limit the scope of the embodiments described herein.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the description including definitions will control. Accordingly, the following definitions apply in the context of the embodiments described herein.

[0013] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nanocarrier” refers to one or more nanocarriers and equivalents thereof known to those of skill in the art.

[0014] As used herein, the term “about,” when referring to a value, includes variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from a particular amount, which variations are appropriate for carrying out the methods of the present disclosure.

[0015] As used herein, the term "comprise" and its linguistic variants indicate the presence of the recited features, elements, method steps, etc., without excluding the presence of additional features, elements, method steps, etc. Conversely, the term "consisting of" and its linguistic variants indicate the presence of the features, elements, method steps, etc., and the exclusion of any features, elements, method steps, etc. not recited, except for impurities normally included. The phrase "consisting essentially of" indicates the features, elements, method steps, etc., and any additional features, elements, method steps, etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open phrase "comprising". Such embodiments encompass a plurality of closed "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such phrases.

[0016] As used herein, the term "coronary vasculature" refers to the blood vessels that carry the coronary circulation and supply blood to the heart muscle (myocardium). The term "blood vessel" includes both arteries and veins. The "coronary arteries" supply oxygenated blood to the myocardium, and the "cardiac veins" drain the once-oxygenated blood.

[0017] The term "gene therapy" has its ordinary meaning in the art. Briefly, "gene therapy" refers to the transfer of a gene of interest (e.g., DNA or RNA) into host cells and / or tissues. The gene of interest typically encodes a product for which in vivo production is desired. The gene of interest may also include various regulatory elements such as transcriptional promoters. It should be noted that the end result of gene therapy need not necessarily include a cure, but may instead include reducing the severity of one or more symptoms of the disease.

[0018] As used herein, the term "subject" refers to any animal including, but not limited to, insects, humans, non-human primates, vertebrates, cows, horses, cats, dogs, pigs, rodents, etc. The terms "subject" and "patient" may be used interchangeably. The subject may be at any stage of development (e.g., embryo, fetus, infant, neonate, child, adult, etc.). The subject may be male or female.

[0019] As used herein, the terms "treat", "treatment", and "treating" refer to reducing or alleviating the severity of a particular symptom, disease state (e.g., cardiovascular disorder), or the amount of those symptoms in a subject currently experiencing or suffering from that symptom or disease state. This term does not necessarily indicate complete treatment (e.g., complete elimination of the symptom, disease, or its symptoms). "Treatment" encompasses any administration or application of a therapy or technique for a disease (e.g., in a mammal including a human), and includes suppression of the disease, prevention of its onset, alleviation of the disease, causing regression, or restoring a lost, missing, or defective function, or stimulating an inefficient process.

[0020] Detailed Description In some embodiments, provided herein are devices and methods for targeted delivery of an agent (e.g., a nucleic acid, a gene therapy agent, etc.) to a subject. The method includes delivering an agent to a region of the subject's coronary vasculature and electroporating the subject's target coronary tissue. Electroporation of the subject's target coronary tissue results in delivery of the targeted agent to (or within) the target coronary tissue.

[0021] In some embodiments, the region of the coronary vasculature is different from the target coronary tissue. For example, the region of the coronary vasculature may be an artery or vein suitable for injecting the agent, and the target coronary tissue may be a different tissue if local distribution of the agent is intended to occur.

[0022] The coronary vascular system region and the target coronary artery tissue can be selected for the delivery of a targeted agent to the cardiovascular system. Such methods can be useful for the treatment of heart disorders including atrial fibrillation. Atrial fibrillation (AF) is the most common cardiac arrhythmia. It affects more than 3 million Americans and is a major cause of stroke. AF is primarily an age-related disease and is rapidly spreading in the elderly population. Unfortunately, current treatments for AF, both pharmacology-based and ablation-based, are not optimal in patients suffering from persistent AF. Part of the reason for this is that current treatments do not target the underlying molecular mechanisms that cause AF.

[0023] In some embodiments, provided herein is an approach for the treatment of AF that targets one or more molecular mechanisms underlying the development of the AF disease state. In some embodiments, the devices and methods herein target the mechanisms underlying AF through the delivery of an agent. In certain embodiments, the devices and methods herein target the mechanisms underlying AF through the delivery of a nucleic acid. Specifically, the embodiments, devices and methods herein target the mechanisms underlying AF through the delivery of a nucleic acid gene therapy agent.

[0024] In some embodiments, provided herein are devices and methods for the targeted delivery of agents to the heart. In some embodiments, the devices and methods disclosed herein can be used for the treatment of heart disorders. For example, provided herein are devices and methods for the targeted delivery of agents to the atrium, such as agents for the treatment of atrial fibrillation. As another example, provided herein are devices and methods for the targeted delivery of agents to the ventricle, such as agents for the treatment of ventricular arrhythmia disorders. In some embodiments, the devices herein include injection and electroporation techniques (such as array-based electroporation) for the accurate and targeted delivery of agents to a desired tissue (e.g., atrium, ventricle). In some embodiments, the device enables the delivery of one or more agents (e.g., nucleic acids (e.g., transgenes)) into a desired tissue in an accurate amount to avoid potential toxicity.

[0025] In some embodiments, the devices and methods herein use electroporation or sonoporation to achieve gene delivery to an intended tissue (e.g., atrium, ventricle, etc.). Many of the embodiments herein are described in connection with electroporation; however, any such embodiment may also find use of sonoporation or other techniques for achieving the uptake of therapeutic agents (e.g., nucleic acid therapeutics) into cells or tissues. Both viral vectors and non-viral vectors can be used for cardiac gene delivery. Viruses may be advantageous vectors due to long-term gene expression. However, viral vectors have the potential for off-target effects. Thus, non-viral delivery approaches (e.g., plasmids, cosmids, etc.) can also be used according to the methods described herein.

[0026] In some embodiments, the use of electroporation or sonoporation to deliver a drug to a targeted tissue localizes gene expression to the site of electroporation / sonoporation, thereby substantially eliminating the potential for off-target effects. Plasmid DNA is rapidly degraded in the blood and has no mechanism to transfect other cells after IV injection. In some embodiments, these advantages also obviate the need for an organ- or tissue-specific promoter (e.g., a heart-specific promoter). Furthermore, physical methods such as electroporation can be significantly enhanced, even in viral gene transfection (e.g., intracardiac). In some embodiments, the devices and methods herein utilize non-viral drug (e.g., nucleic acid (e.g., transgene)) delivery that facilitates electroporation.

[0027] In some embodiments, provided herein is a method of treating a cardiac disorder in a subject, comprising delivering a drug to a region of the subject's coronary vasculature and electropermeabilizing a target coronary artery tissue of the subject. In some embodiments, the drug is delivered to the target coronary artery tissue (e.g., passively or actively) via the vasculature. In some embodiments, the present invention provides treatment or suppression of a cardiac disorder or condition selected from the following list: aortic dissection, cardiac arrhythmia (e.g., atrial arrhythmia (e.g., atrial premature beats, atrial ectopic pacemaker, multifocal atrial tachycardia, atrial flutter, atrial fibrillation, etc.), junctional arrhythmia (e.g., supraventricular tachycardia, atrioventricular nodal reentrant tachycardia, paroxysmal supraventricular tachycardia, junctional rhythm, junctional tachycardia, premature junctional complexes, etc.), atrioventricular arrhythmia, ventricular arrhythmia (e.g., ventricular premature beats, enhanced automatic ventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, ventricular fibrillation, etc.), congenital heart disease, myocardial infarction, dilated cardiomyopathy, hypertrophic cardiomyopathy, aortic valve regurgitation, aortic valve stenosis, mitral valve regurgitation, mitral valve stenosis, Ellis - van Creveld syndrome, familial hypertrophic cardiomyopathy, Holt - Oram syndrome, Marfan syndrome, Ward - Romano syndrome and / or similar diseases and conditions. In some embodiments, the cardiac disorder may be any one or more of cardiac arrhythmia, congestive heart failure, and coronary artery disease. For example, the cardiac disorder may be a cardiac arrhythmia, e.g., an atrial arrhythmia or a ventricular arrhythmia. The arrhythmia may be tachycardia or bradycardia. Exemplary arrhythmias include, for example, atrial fibrillation, atrial flutter, supraventricular tachycardia, Wolff - Parkinson - White syndrome, ventricular tachycardia, ventricular fibrillation, long QT syndrome, sick sinus syndrome, conduction block, etc.

[0028] The agent can be administered by any suitable route. The route of administration depends on the location of the intended delivery of the agent within the subject. For example, the agent can be administered by a catheter-based delivery method, a needle-based delivery method, a non-needle-based delivery method, a laparoscopic delivery method, a surgical (e.g., by thoracotomy) delivery method, a systemic delivery method (e.g., enteral or parenteral administration), a local delivery method, or a delivery method by an injection device. Exemplary devices are described in U.S. Patent Application Publication Nos. 20110245756 and 2011013728, each of which is incorporated herein by reference in its entirety. In some embodiments, the agent is administered to the coronary vascular system region using a catheter-based injection method.

[0029] In a detailed embodiment, the mode of administration is selected to avoid thoracotomy. For example, the agent may be delivered using a catheter inserted through a body location remote from the heart. The catheter can be inserted into any suitable body part and guided to the coronary vascular system region prior to administration of the agent to the region of the subject's coronary vascular system. For example, the catheter can be inserted into a vein or artery in a body part such as the leg, groin, underarm, etc., to enable delivery of the agent to a desired location within the heart without the need for thoracotomy.

[0030] The agent may be administered endocardially or epicardially. For example, the agent can be injected into the atrium or ventricle by endocardial injection or epicardial injection. In some embodiments, the agent is administered by intracoronary injection. Intracoronary injection involves injecting the agent into any suitable cardiac region (e.g., artery, vein, sinus venosus) without the need for direct application or injection into the atrium or ventricle.

[0031] Thus, in some embodiments, the coronary vasculature region may be the coronary veins entering the coronary sinus (e.g., the great cardiac vein, middle cardiac vein, small cardiac vein, posterior vein of the left ventricle, Marshall vein, etc.), the coronary veins directly entering the right atrium (e.g., the anterior cardiac vein, smallest cardiac vein (Thebesian vein), etc.), the aorta, aortic root, coronary arteries (e.g., right coronary artery, left main coronary artery, circumflex artery, left anterior descending artery), left marginal branch, right marginal branch, posterior descending artery, coronary sinus, great veins (e.g., superior vena cava, inferior vena cava), pulmonary veins (e.g., right pulmonary vein, left pulmonary vein), pulmonary arteries (left pulmonary artery, right pulmonary artery), brachiocephalic artery, carotid artery, subclavian artery, pericardial cavity, or a combination thereof. In some embodiments, the coronary vasculature region is selected to enable non-invasive delivery of a drug to the subject (e.g., an injection that does not require open-heart surgery, e.g., a catheter-based technique). In some embodiments, the coronary vasculature region is selected from the aortic root, coronary arteries, coronary sinus, and combinations thereof.

[0032] In some embodiments, one or more regions may be occluded during administration of the drug to the coronary vasculature region, e.g., one or more regions in the heart or coronary vasculature may be occluded to prevent inflow of the drug to unintended tissue. Exemplary occlusion methods include, for example, balloon occlusion. Along with administration of the drug, the occlusion procedure can be performed without requiring open-heart surgery. Any suitable region can be occluded as it is necessary to prevent inflow of the drug to unintended tissue, including one or more arteries or veins within the heart. As an example, the drug may be injected into the coronary artery and the coronary sinus may be occluded (e.g., by balloon occlusion). As another example, the drug may be injected into the aortic root and the proximal aorta may be occluded. In some embodiments, the occlusion prevents the drug from reaching unintended sites within the coronary vasculature and / or contacting unintended tissue. In some embodiments, the occlusion allows the drug to move passively by diffusion rather than being carried by the blood flow within the coronary vasculature.

[0033] In some embodiments, the target coronary tissue (e.g., the tissue in which local distribution of a drug is intended to occur) is one or more of the left atrium, right atrium, left ventricle, and right ventricle. For example, for a method of treating atrial arrhythmia, the target coronary tissue may be the left atrium and / or the right atrium. As another example, for a method of treating ventricular arrhythmia, the target coronary tissue may be the left ventricle and / or the right ventricle.

[0034] The methods described herein further include electroporating or sonoporating the target coronary tissue. Electroporation or sonoporation can be performed before, simultaneously with, and / or after drug delivery to an area of the subject's coronary vasculature. For example, electroporation or sonoporation can be performed less than 1 hour before drug delivery. For example, electroporation or sonoporation can be performed less than 1 hour, less than 55 minutes, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 45 seconds, less than 30 seconds, less than 15 seconds, less than 10 seconds, less than 5 seconds, or less than 1 second before drug delivery. Alternatively, or in combination therewith, electroporation or sonoporation can be performed simultaneously with drug delivery. Alternatively, or in combination therewith, electroporation or sonoporation can be performed after drug delivery. For example, electroporation or sonoporation can be performed less than 1 second, less than 5 seconds, less than 10 seconds, less than 15 seconds, less than 30 seconds, less than 45 seconds, less than 1 minute, less than 2 minutes, less than 3 minutes, less than 5 minutes, less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 20 minutes, less than 25 minutes, less than 30 minutes, less than 35 minutes, less than 40 minutes, less than 45 minutes, less than 50 minutes, less than 55 minutes, or less than 1 hour after drug delivery.

[0035] Electroporation or sonoporation can be performed any suitable number of times and at any suitable intervals to achieve the desired effect. For example, electroporation or sonoporation can be performed one or more times. Electroporation can be performed endocardially or epicardially. For example, electroporation can be performed by epicardial electroporation. Alternatively, or in combination therewith, electroporation can be performed by endocardial electroporation. Any suitable device can be used for electroporation or sonoporation. For example, electroporation can be performed by proximity-sensing bipolar electrodes. Alternatively, electroporation can be performed by either a bipolar catheter or a multipolar catheter. An example of a multipolar catheter that can assist in facilitating endocardial electroporation is a commercially available basket catheter. Such a catheter typically covers substantially the entire surface area of one atrium. Electroporation from such a catheter can thus be performed in such a manner that the entire atrium can be subjected to electroporation during the process of intracoronary gene injection. This enables selective gene transfer to occur throughout the atrial region where electroporation is performed. Alternatively, electroporation of the entire atrium can be performed by placing a catheter (e.g., a multipolar catheter) at a first position in the atrium and electroporating the tissue, moving the catheter to a second position and electroporating the tissue, moving the catheter to a third position and electroporating the tissue, etc., until the agent has been delivered throughout the atrium. Using this method, any catheter, including a small catheter, can adequately deliver the agent throughout the atrium.

[0036] In some embodiments, a separate device can be used for drug delivery to regions of the coronary vasculature and for electroporation or sonoporation of target coronary tissue. In other embodiments, the same device can be used for delivery and for electroporation or sonoporation. Suitable devices are described in U.S. Patent Application Publication Nos. 20110245756 and 2011013728, which are hereby incorporated by reference in their entirety.

[0037] Electroporation or electropermeabilization refers to a significant increase in the electrical conductivity and permeability of cell membranes that can be caused by an externally applied electric field. Any suitable level of current can be delivered to the target coronary tissue within a subject. In some embodiments, the level of current applied to the tissue is selected based on the subject (e.g., species, size, age, etc.), the treatment site (e.g., epicardium, endocardium, etc.), and other considerations known to those of ordinary skill in the art. In some embodiments, the current is delivered continuously. The current can be delivered continuously over any suitable time period. For example, the current can be delivered for a period ranging from 1 microsecond to 1 hour. For example, the current can be applied for 1 microsecond, 10 microseconds, 50 microseconds, 100 microseconds, 150 microseconds, 200 microseconds, 250 microseconds, 300 microseconds, 350 microseconds, 400 microseconds, 450 microseconds, 500 microseconds, 550 microseconds, 600 microseconds, 650 microseconds, 700 microseconds, 750 microseconds, 800 microseconds, 850 microseconds, 900 microseconds, 950 microseconds, 1000 microseconds, 10 milliseconds, 20 milliseconds, 30 milliseconds, 40 milliseconds, 50 milliseconds, 60 milliseconds, 70 milliseconds, 80 milliseconds, 90 milliseconds, 100 milliseconds, 150 milliseconds, 200 milliseconds, 250 milliseconds, 300 milliseconds, 350 milliseconds, 400 milliseconds, 450 milliseconds, 500 milliseconds, 550 milliseconds, 600 milliseconds, 650 milliseconds, 700 milliseconds, 750 milliseconds, 800 milliseconds, 850 milliseconds, 900 milliseconds, 950 milliseconds, 1 second, 2 seconds, 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, or more). In some embodiments, the current is pulse-driven. The length of the pulse, the current applied, and the interval between pulses can be selected based on appropriate criteria determined by those of ordinary skill in the art or a clinician. In some embodiments, the pulse is 1 microsecond to 10 seconds in length.For example, the current can be delivered in pulses of 1 microsecond, 10 microseconds, 50 microseconds, 100 microseconds, 150 microseconds, 200 microseconds, 250 microseconds, 300 microseconds, 350 microseconds, 400 microseconds, 450 microseconds, 500 microseconds, 550 microseconds, 600 microseconds, 650 microseconds, 700 microseconds, 750 microseconds, 800 microseconds, 850 microseconds, 900 microseconds, 950 microseconds, 1000 microseconds, 10 milliseconds, 20 milliseconds, 30 milliseconds, 40 milliseconds, 50 milliseconds, 60 milliseconds, 70 milliseconds, 80 milliseconds, 90 milliseconds, 100 milliseconds, 150 milliseconds, 200 milliseconds, 250 milliseconds, 300 milliseconds, 350 milliseconds, 400 milliseconds, 450 milliseconds, 500 milliseconds, 550 milliseconds, 600 milliseconds, 650 milliseconds, 700 milliseconds, 750 milliseconds, 800 milliseconds, 850 milliseconds, 900 milliseconds, 950 milliseconds, 1 second, 2 seconds, 5 seconds, or 10 seconds. The pulses can be spaced at any suitable time intervals (e.g., from microseconds to 10 seconds). For example, the pulses can be spaced 1 microsecond, 10 microseconds, 50 microseconds, 100 microseconds, 150 microseconds, 200 microseconds, 250 microseconds, 300 microseconds, 350 microseconds, 400 microseconds, 450 microseconds, 500 microseconds, 550 microseconds, 600 microseconds, 650 microseconds, 700 microseconds, 750 microseconds, 800 microseconds, 850 microseconds, 900 microseconds, 950 microseconds, 1000 microseconds, 10 milliseconds, 20 milliseconds, 30 milliseconds, 40 milliseconds, 50 milliseconds, 60 milliseconds, 70 milliseconds, 80 milliseconds, 90 milliseconds, 100 milliseconds, 150 milliseconds, 200 milliseconds, 250 milliseconds, 300 milliseconds, 350 milliseconds, 400 milliseconds, 450 milliseconds, 500 milliseconds, 550 milliseconds, 600 milliseconds, 650 milliseconds, 700 milliseconds, 750 milliseconds, 800 milliseconds, 850 milliseconds, 900 milliseconds, 950 milliseconds, 1 second, 2 seconds, 5 seconds, or 10 seconds apart. Any suitable number of pulses can be delivered to the tissue within the desired time frame.In some embodiments, the pulses can be delivered over a total of 1 second to 1 hour, and the length of each pulse and each interval between pulses is counted. For example, the pulses can be delivered over a total of 1 second, 2 seconds, 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 45 minutes, or 1 hour).

[0038] In some embodiments, the level of the applied current is between 1 volt and 1000 volts. For example, the current can be 1 volt, 2 volts, 3 volts, 4 volts, 5 volts, 6 volts, 7 volts, 8 volts, 9 volts, 10 volts, 15 volts, 20 volts, 25 volts, 30 volts, 35 volts, 40 volts, 45 volts, 50 volts, 55 volts, 60 volts, 65 volts, 70 volts, 75 volts, 80 volts, 85 volts, 90 volts, 95 volts, 100 volts, 150 volts, 200 volts, 250 volts, 300 volts, 350 volts, 400 volts, 450 volts, 500 volts, 550 volts, 600 volts, 650 volts, 700 volts, 750 volts, 800 volts, 850 volts, 900 volts, 950 volts, or 1000 volts.

[0039] Sonoporation or cell sonication is the use of sound (e.g., ultrasonic frequency) to alter the permeability of cell membranes. In some embodiments, the device of the present invention directs sound energy (e.g., ultrasonic frequency) towards the treatment site to assist in the uptake of therapeutic agents (e.g., nucleic acids). In some embodiments, any suitable level of ultrasound can be delivered through the device of the present invention and applied to a site within the subject. In some embodiments, the level and / or frequency of the ultrasound applied to a site (e.g., treatment site, delivery site, etc.) is selected based on the subject (e.g., species, size, age, etc.), the treatment site (e.g., epicardium, endocardium, etc.), and other considerations known to those of skill in the art.

[0040] In some embodiments, the ultrasound is delivered continuously over a period of time (e.g., 1 second, 2 seconds, 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, or more). In some embodiments, the ultrasound is pulse-driven. In some embodiments, the length, level and / or frequency of the ultrasound, and the length of the pulse, are selected based on appropriate criteria determined by one of ordinary skill in the art or a clinician. In some embodiments, the frequency of the ultrasound applied by the device of the present invention is between 20 kHz and 200 MHz (e.g., 20 kHz, 50 kHz, 100 kHz, 200 kHz, 500 kHz, 1 MHz, 2 MHz, 5 MHz, 10 MHz, 20 MHz, 50 MHz, 100 MHz, 200 MHz). In some embodiments, the level of the ultrasound applied by the device of the present invention has a mechanical index (MI) between 0.01 and 5 (e.g., 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0). In some embodiments, the pulse is between 0.1 seconds and 10 seconds in length (e.g., 0.1 seconds, 0.2 seconds, 0.5 seconds, 1 second, 2 seconds, 5 seconds, 10 seconds), and is delivered over a period of 1 second to 1 hour (e.g., 1 second, 2 seconds, 5 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour).

[0041] An electroporation or sonoporation device can be provided in a target coronary tissue that does not require open heart surgery. For example, electroporation at a target coronary tissue site can be performed using a catheter-based electroporation device. For example, an electroporation device can be provided in a target coronary tissue using a non-invasive, catheter-based method. A catheter-based electroporation device can be inserted into any suitable site within the body away from the heart, such as in a vein or artery, and guided to the desired target coronary tissue. For example, a catheter-based electroporation device can be inserted via a vein or artery in the subject's leg, groin, arm, or any other suitable body region.

[0042] In some embodiments, the agent comprises a therapeutic agent (e.g., a biological agent) for the treatment of heart disorders in a subject. For example, the agent may be a nucleotide, oligonucleotide, protein, peptide, small molecule, or macromolecule. In some embodiments, the agent is a nucleotide (e.g., DNA (e.g., plasmid, minigene, etc.), RNA (e.g., siRNA, shRNA, etc.)). In some embodiments, the agent is a naked DNA plasmid. In other embodiments, the agent further comprises a carrier. For example, the carrier may be a vector. Any suitable vector, including viral vectors (e.g., adenovirus, adeno-associated virus, alphavirus, herpes virus, retrovirus, vaccinia virus, etc.) and non-viral vectors, can be used.

[0043] The fundamental mechanisms in the formation of atrial fibrillation (AF) disease states, as well as several transgenes that selectively target these mechanisms in the atria, have been identified (References 1-3; incorporated herein by reference in their entirety). In some embodiments, the agent can be designed to target any one or more of these mechanisms that contribute to underlie a disease state (e.g., AF). In some embodiments, the devices and methods herein target either one or a combination of two fundamental mechanisms that contribute to electrical remodeling, oxidative stress and parasympathetic signaling in AF. In some embodiments, a nucleic acid (e.g., plasmid) expressing the following transgenes is used: NOX2 shRNA (this transgene inhibits NOX2, the major enzymatic source of oxidative stress) and / or C-terminal Gαi+Gαo hydrophobic peptides (these plasmids inhibit parasympathetic signaling in the atria). In some embodiments, the subject is administered a biological product comprising a combination of NOX2 shRNA + Gαi expression plasmid + Gαo expression plasmid. NOX2 shRNA completely blocks RAP-induced electrical remodeling (and AF).

[0044] NOX2 shRNA also inhibits atrial fibrillation in the HF model. Parasympathetic inhibition (by Gα i / o -ct) also significantly attenuates RAP-induced electrical remodeling and AF. NOX2 shRNA attenuates parasympathetic sprouting in dogs underlying RAP. This indicates a significant interaction between oxidative injury and parasympathetic signaling in the development of electrical remodeling in AF. Furthermore, NOX2 shRNA reverses electrical remodeling in RAP dogs with established AF, especially when combined with Gα i / o -ct.

[0045] In some embodiments, the agent may be a gene (e.g., DNA) with or without using a vector. Suitable targets for gene therapy include any target that contributes to the etiology of the heart disorder. For example, suitable targets for atrial or ventricular arrhythmia disorders may include targets that contribute to shortening of the action potential (e.g., ion channels, autonomic regulation), or conduction delays (e.g., gap junctions, structural remodeling) that may contribute to the development of the disorder. For example, the agent may be an ion channel modulator. For example, the agent may be a gene that prolongs the atrial action potential (e.g., variants of KCNH2, variants of the IKR subunit, etc.). As another example, the agent may target connexin biology thought to be associated with electrical conduction defects in the atria (e.g., connexin 40 and 43). The agent may target the development of local and systemic inflammation or fibrosis. For example, the agent may target enzymes known to be involved in inflammation and / or apoptosis (e.g., calpain, caspase-3, SOD1, etc.). As another example, the agent may target factors involved in other transcription factors known to affect the development of fibrosis (e.g., TGF-beta) or heart disorders (e.g., PITX2).

[0046] Both sympathetic and parasympathetic activities in the heart are mediated by the G-protein (GαGα3Gα) coupling pathway of heterotrimers initiated by G-protein coupled receptors (GPCRs). In some embodiments, the present invention provides a gene-based approach for selectively inhibiting the G-protein signaling pathway. In some embodiments, the present invention is used in an epicardial approach for administering to a tissue (e.g., atrium, ventricle) a minigene that expresses a G-protein inhibitory peptide to selectively inhibit the C-termini of Gαi and Gαs in this region. In some embodiments, the present invention provides electroporation and / or ultrasonic energy to enhance the effectiveness of gene therapy (e.g., for naked DNA and / or viral vectors). In some embodiments, the electroporation and / or ultrasonic energy source enhances intracellular gene transfer. In some embodiments, the present invention targets G-protein mediated autonomic signaling and / or other major signal transduction pathways (e.g., the TGF-beta pathway in the development of atrial fibrillation). In some embodiments, the present invention provides a targeted gene-based approach for weakening TGF-beta signaling in the left atrium to reduce the development of fibrosis in AF. In some embodiments, the present invention provides a method for blocking G-protein coupled receptor mediated signaling for targeting atrial fibrillation. (See U.S. Application No. 12 / 430,595, which is hereby incorporated by reference in its entirety.)

[0047] The methods described herein may be used in combination with other suitable therapies for the treatment of heart disorders in a subject. For example, the t method described herein may be used in combination with other suitable therapies for the treatment of cardiac arrhythmias (e.g., anticoagulants (e.g., warfarin, non-vitamin K antagonist oral anticoagulants), beta blockers, calcium channel blockers, cardiac glycosides (e.g., digoxigenin) antiarrhythmic drug therapy, defibrillation, catheter ablation, or other surgical procedures for restoring and maintaining normal sinus rhythm).

[0048] The methods described herein may further include monitoring a patient's response to a drug. For example, the method may further include monitoring the response to drug delivery after the drug has been delivered to the coronary vasculature region and / or after the target coronary tissue has been electroporated. Suitable methods for measuring a patient's response may include measuring the cardiac response to the drug. For example, the response may be measured by cardiac MRI imaging (which may be used in combination with ECG gating), electrocardiogram examination, photoplethysmography, echocardiogram, computed tomography, nuclear medicine scan, etc.

[0049] In some embodiments, delivery of the therapeutic agent and / or electroporation of the target coronary tissue may be continued until a favorable response in the subject is measured. For example, delivery and / or electroporation may be continued until the arrhythmia subsides in the subject (e.g., normal cardiovascular function is restored).

Examples

[0050] The atrial wall is very thin, and it can be very difficult to enable delivery of a sufficient volume / amount of gene into the atrial wall, not only safely (i.e., without making a hole). Therefore, described herein is a novel method for facilitating gene delivery into the heart (optionally into the atrium and / or ventricle) without the need to perform direct injection of the gene by a needle into the desired location within the heart.

[0051] The following experiments were conducted in a dog model. For the following experiments, 20 - 200 ml of Coomassie Blue at a concentration of 0.2 - 0.4 mg / 100 ml was injected. Electroporation was performed using 10 - 30 pulses of 75 - 200 volts over 10 msec, respectively. The pulses were at 1 - second intervals. Tissues were collected 10 minutes to 2 hours after injection.

[0052] Experiment 1: In this experiment, electroporation from a multipolar 'basket' catheter (64 electrodes) placed in the right atrium and simultaneous injection of a color dye (Coomassie Blue) at the aortic root (after clamping the proximal aorta with a Satinsky clamp) were performed. The results are shown in Figure 1.

[0053] Experiment 2: Electroporation from a multipolar 'basket' catheter (64 electrodes) placed in the right atrium and simultaneous injection of Coomassie Blue in the retrograde coronary sinus in a follow - up experiment. The results are shown in Figures 2 - 4. Figure 2 shows an angiogram of the retrograde coronary sinus injection of Coomassie Blue dye. The electroporation basket catheter is seen in the right atrium.

[0054] Figure 3 shows that the right atrium was stained with Coomassie Blue after electroporation. The left atrium also received Coomassie Blue dye by diffusion from the coronary sinus, but since the left atrium was not electroporated, no significant Coomassie Blue staining occurred.

[0055] Figure 4 shows that the left and right ventricles were also given Coomassie Blue dye via retrograde coronary sinus injection without using electroporation, but there was no significant Coomassie Blue staining.

[0056] Further experiments were conducted to examine whether the methods described herein are effective for gene delivery to the atria. In one animal, a cannula was inserted into the coronary sinus via the jugular vein approach. Using the femoral vein approach, a FirMap catheter (64 electrodes; Abbott - St. Jude) was advanced into the high right atrium. After balloon occlusion in the proximal coronary sinus, 1.5 mg of GFP - expressing plasmid (under the control of the CMV promoter) was diluted to 20 ml and then injected into the coronary sinus. During the injection, simultaneous electroporation was performed in the high right atrium (encodardially) via the FirmMap catheter (voltage 200 V; pulse duration 10 ms; number of pulses 20; pulse interval 1 second). A series of gene injections and electroporations were repeated three more times. Three days later, the animal was sacrificed and the heart was excised for further analysis.

[0057] The electroporated high right atrium and the non - electroporated posterior left atrium (control atrium) were tested for GFP expression using fluorescence microscopy. As shown in FIGS. 5A - B, GFP expression was observed in the electroporated atria. Furthermore, GFP expression was found to be transmural (i.e., from epicardium to endocardium). As shown in FIG. 5C, GFP expression was observed in the non - electroporated atria (e.g., left atrium). These results demonstrate that strong gene expression in the atria can be obtained via this unique new "needle - free" method.

[0058] Example 2 Coronary Sinus Gene Delivery and Simultaneous Targeted Atrial Electroporation - A Novel Transvenous Method for Obtaining Atrial Gene Delivery Coomassie blue injection: In two animals, the coronary sinus was cannulated via a jugular vein approach. In one animal, a FirMap catheter (64 electrodes; Abbott-St. Jude) was advanced into the high right atrium via a femoral vein approach. In the second animal, it was advanced into the left atrium via a transseptal puncture. In both animals, after balloon occlusion in the proximal coronary sinus, subsequent coronary sinus injection of Coomassie blue dye (80 mg of dye diluted to up to 20 ml) mixed with a contrast dye was performed. During the injection, simultaneous electroporation was performed via the FirMap catheter (voltage 200 V; pulse duration 10 ms; number of pulses 20; pulse interval 1 second) in the right atrium or left atrium. Figure 6 shows the FirMap catheter in the high right atrium; the figure also shows coronary sinus injection of the contrast dye. Each animal was sacrificed and the atria were tested for evidence of Coomassie blue uptake.

[0059] As shown in Figure 7A, Coomassie blue was found only in the atrium where electroporation was performed (i.e., the right atrium), and the dye was not present in the atrium that was not electroporated (i.e., the left atrium). In the second animal, a transseptal puncture was performed and electroporation was performed in the posterior left atrium (during the coronary sinus injection of Coomassie blue). As shown in Figure 7B, Coomassie blue was found only in the left atrium (where electroporation was performed), and no dye was found in the right atrium.

[0060] Injection of GFP expression plasmid: In the third animal, 1.5 mg of green fluorescent protein (GFP) expression plasmid (under the control of the CMV promoter) was diluted to 20 ml and injected into the coronary sinus. Simultaneous electroporation was performed for Coomassie blue at the high right atrium (free wall of the right atrium) using the FirMap catheter as described above in the upper section. A series of gene injections and electroporations were repeated three times. Three days later, the animals were sacrificed and the hearts were excised for further analysis. The electroporated high right atrium (high and middle right atrium free walls) and the non-electroporated right atrium (low right atrium free wall, right atrial appendage, posterior right atrium) as well as the non-electroporated left atrium were tested for GFP expression using fluorescence microscopy and Western blot. As shown in Figures 8 and 9, GFP expression was observed only in the electroporated sites of the right atrium, namely the high and middle right atrium free walls, and no evidence of GFP was found in the non-electroporated right atrium or left atrium.

[0061] Furthermore, GFP expression was found to be trans-wall (i.e., from the epicardium to the endocardium). These results demonstrate that it is possible to obtain strong gene expression in the atrium via this unique novel "needleless" method.

[0062] References The following references are hereby incorporated by reference in their entirety herein: 1. Korantzopoulos P, Kolettis TM, Galaris D and Goudevenos JA. The role of oxidative stress in the pathogenesis and perpetuation of atrial fibrillation. Int J Cardiol. 2007;115:135 - 43. 2. Youn JY, Zhang J, Zhang Y, Chen H, Liu D, Ping P, Weiss JN and Cai H. Oxidative stress in atrial fibrillation: an emerging role of NADPH oxidase. J Mol Cell Cardiol. 2013;62:72-9. 3. Jeong EM, Liu M, Sturdy M, Gao G, Varghese ST, Sovari AA and Dudley SC, Jr. Metabolic stress, reactive oxygen species, and arrhythmia. J Mol Cell Cardiol. 2012;52:454-63.

Claims

1. A medicament for treating heart disorders in a subject, comprising a nucleotide, oligonucleotide, protein, peptide, small molecule, or macromolecule, wherein the medicament is delivered to an area of the subject's coronary vasculature, and the medicament is targeted to the subject's target coronary tissue by selective electroporation to the target coronary tissue, the target coronary tissue being selected from the subject's left atrium, right atrium, left ventricle, and right ventricle, the target coronary tissue being different from the area of the coronary vasculature, the medicament.

2. The medicament according to claim 1, wherein the heart disorder is cardiac arrhythmia.

3. The medicament according to claim 2, wherein the heart disorder is atrial fibrillation or ventricular tachycardia.

4. The medicament according to claim 1, wherein the area of the coronary vasculature is selected from the aorta, aortic root, coronary artery, coronary sinus, vena cava, pulmonary vein, pulmonary artery, brachiocephalic artery, carotid artery, subclavian artery, or pericardial cavity.

5. The medicament according to claim 4, wherein the area of the coronary vasculature is selected from the aortic root, coronary artery, and coronary sinus.

6. The medicament according to claim 1, wherein the electroporation is before delivery of the medicament to the area of the subject's coronary vasculature, simultaneous with delivery of the medicament, and / or after delivery of the medicament.

7. The medicament according to claim 1, wherein the electroporation comprises epicardial electroporation or endocardial electroporation.

8. The medicament according to claim 1, comprising DNA.

9. The medicament according to claim 1, wherein the subject is human.

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