Patent Application
20210260158
Several embodiments of the present application relate generally to methods and compositions for the generation of pacemaker cells (e.g., cardiac cells that have regular, rhythmic electrical activity). In particular, some embodiments of the invention relate to gene and cell therapy methods (and associated compositions) to generate pacemaker cells using transcription factors.
During cardiogenesis, cardiomyocytes become specialized to exhibit either ventricular, atrial, or pacemaker properties. The sinoatrial node (SAN), the primary pacemaker region of the heart, is a highly-specialized structure containing fewer than 10,000 pacemaker cells, which function to initiate contractions in the SAN. These SAN contractions then propagate to the rest of the excitable heart tissue and result in a heartbeat. Irregularities of excitable cardiac tissue and/or irregularities of pacemaker cells can lead to abnormalities in heart rhythm. Many cardiac abnormalities typically involve irregular heartbeat, tachycardia (where the heart rate is too high), or bradycardia (where the heart rate is too slow). These abnormalities are collectively known as arrhythmias.
Current therapies for cardiac arrhythmias typically rely on drug therapy, ablation, electronic pacemaker devices or combinations thereof. However, the usefulness of each of these therapies has met with limited and varying success. While antiarrhythmic drugs are widely prescribed and used, they may result in adverse systemic side effects in certain patient populations. Further, many drugs have a propensity to provoke new arrhythmic events, which can lead to an increase in morbidity. Radiofrequency ablation is used in some treatments of arrhythmias. Ablation involves permanent removal of the tissue identified as the source of, or critical to, the maintenance of the arrhythmias. While this method has found some success in the treatment of atrioventricular node reentry tachycardia, accessory pathway tachycardia, and atrial flutter, it has found limited success in the treatment of other arrhythmias. For instance, catheter ablation is less successful in treating more complex cases, such as atrial fibrillation (AF) or ventricular tachycardia (VT). Moreover, catheter ablation is not useful in the treatment of bradycardia. Electronic pacemaker devices can sustain heart rate, or deliver shocks to terminate tachycardias. However, the high cost of devices, and complications such as pulmonary collapse, hemorrhage, bacterial infection, and lead/generator failure or other types of malfunction represent limitations of the technology.
In light of the limitations associated with traditional therapies for dysfunctions of cardiac pacing and arrhythmias, there is a need for alternative methods and compositions that can be used to modulate cardiac pacing and rhythm to treat cardiac arrhythmias (both simple and complex varieties), to treat heart failure (as in cardiac resynchronization applications of electronic pacemakers), and/or supplement or obviate the need for electrically-powered pacemakers.
Currently, biological pacemakers typically elicit their effects through the gene delivery of nucleotides that transcribe mutant ion-channel proteins. In contrast, several embodiments of the present invention operate through delivery to cells of polynucleotides that encode one or more transcription factors, which allows direct somatic-to-somatic cell reprogramming (e.g., a non-pacemaker cell is reprogrammed to a pacemaker cell or a malfunctioning pacemaker cell is reprogrammed to a functional pacemaker cell). Thus, in several embodiments, pacemaker cells are generated without the induced alteration expression of ion-channel proteins. Additional embodiments involve the conversion of stem cells, or in some embodiments cardiomyocytes or combinations of stem cells and cardiomyocytes, to biological pacemaker cells in vitro (e.g., by administration of one or more transcription factors disclosed herein), followed by subsequent implantation of the converted cells. Thus, delivery of the transcription factors, or cells converted by those transcription factors, treats a variety of cardiac rhythmic abnormalities and/or lessens or obviates the needs for traditional therapies.
Thus, there is provided in several embodiments, methods for generating a biological pacemaker using transcription factors in order to modify the electrical activity of the cardiac tissue of a subject, the method comprising identifying a subject having cardiac tissue exhibiting abnormal electrical activity, wherein the subject has cardiac tissue comprising quiescent cells, wherein the quiescent cells comprise one or more of cardiomyocytes and stem cells, wherein the quiescent cells do not exhibit spontaneous, repetitive electrical activity; and administering one or more transcription factors to the quiescent cells to generate treated cells, wherein the treated cells exhibit spontaneous, repetitive electrical activity, thereby modifying electrical activity of the cardiac tissue the subject.
In several embodiments, the abnormal cardiac electrical activity is due to a cardiac arrhythmia. However, other abnormalities in cardiac electrical activity, signaling or function can be addressed with the methods disclosed herein. For example, in several embodiments, the subject is afflicted with a condition selected from the group consisting of sick sinus syndrome, sinus bradycardia, tachycardia-bradycardia syndrome, atrial fibrillation, atrioventricular block, chronotropic incompetence, prolonged QT syndrome, and heart failure.
In several embodiments, the administration occurs in vitro, and the method further comprises administering the treated cells to the subject. Advantageously, however, the methods disclosed herein allow the administration to occur in vivo.
There are also provided methods of converting a population of stem cells through the use of transcription factors into cells suitable for generation of a biological pacemaker, comprising obtaining a population of stem cells, culturing the stem cells in vitro, wherein the cultured stem cells comprise quiescent cells that do not exhibit spontaneous, repetitive electrical activity, delivering one or more transcription factors to the quiescent cells to generate converted cells, wherein the converted cells exhibit spontaneous, repetitive electrical activity, thereby converting the stem cells into cells capable of generating a biological pacemaker.
There is additionally provided a method of treating a cardiac arrhythmia using transcription factors comprising identifying a subject suffering from cardiac arrhythmia, wherein the subject has cardiac tissue comprising quiescent cells, wherein the quiescent cells comprise one or more of cardiomyocytes and stem cells, wherein the quiescent cells do not exhibit spontaneous, repetitive electrical activity; and administering one or more of Tbx18, Shox-2, Tbx3, Tbx5, functional fragments thereof, or combinations thereof, to the quiescent cells to generate treated cells, wherein the treated cells exhibit spontaneous, repetitive electrical activity; thereby treating the cardiac arrhythmia.
There is also provided a method of treating a cardiac arrhythmia by generating a biological pacemaker using transcription factors, comprising identifying a subject suffering from cardiac arrhythmia, obtaining a population of converted stem cells, wherein, prior to the conversion, the stem cells comprised quiescent cells that did not exhibit spontaneous, repetitive electrical activity, wherein one or more of Tbx18, Shox-2, Tbx3, Tbx5, functional fragments thereof, or combinations thereof were administered to the quiescent cells to generate converted cells that exhibit spontaneous, repetitive electrical activity; and administering the converted cells to the subject, wherein the administered converted cells engraft into the cardiac tissue of the subject and continue to exhibit spontaneous, repetitive electrical activity, thereby generating a biological pacemaker and treating the cardiac arrhythmia.
In several embodiments, the one or more transcription factors are regulators of embryonic sinoatrial node development and/or promote de novo cellular differentiation into sinoatrial nodal cells. In several embodiments, the one or more transcription factors define the sinus venosus during development. In several embodiments, the one or more transcription factors are negative regulators of Nkx2.5 in the sinus venosus.
In several embodiments, the one or more transcription factors is selected from the group consisting of Tbx18, Shox-2, Tbx3, Tbx5, functional fragments thereof, and combinations thereof. In several embodiments, the one or more transcription factors comprises Tbx-18. In several embodiments, Tbx18 (or other Tbox factors) are used to cause myocytes to generate spontaneous electrical activity. In several embodiments, the one or more transcription factors comprises Shox-2. In some embodiments, Shox-2 is used to convert stem cells to cells that generate spontaneous electrical activity. However, in additional embodiments, Shox-2 may optionally be used to convert myocytes to pacemaker cells, and/or Tbx18 is used to convert stem cells to pacemaker cells.
In several embodiments, the administration of the transcription factors is in vivo, and is to a site selected from the group consisting of the apex of the heart, right branch of the Bundle of His, the left branch of the Bundle of His, the Purkinje fibers, the inter-ventricular septum, the right ventricular free wall, the left ventricular free wall, the SA node, the AV node. In several embodiments the administration site is accessed via the right ventricle, via the right atrium, or by accessing the heart directly. In several embodiments, direct access is achieved by a map guided catheter injection system, by fluoroscopy guidance, by X-ray guidance, by echocardiography guidance, by guidance via magnetic resonance imaging, or combinations thereof. Each particular subject may present with symptoms or other relevant medical issues that determine an optimal rout of administration.
In several embodiments, the administration of the transcription factors is achieved by delivering to the target tissue a DNA delivery system comprising a polynucleotide encoding the one or more transcription factors. In several embodiments, the DNA delivery system comprises a viral vector. Various viral vectors are used, depending on the embodiment, such as, for example, adenovirus, adeno-associated virus, lentivirus, retrovirus, HJV, HIV, and/or HSV. In embodiments wherein more that one transcription factor is administered, the transcription factors can optionally be packed in different viral vectors. Alternatively, in some embodiments, multiple transcription factors can be packaged in a single viral vector.
In several embodiments, however, the DNA delivery system comprises a non-viral vector. Various non-viral vectors can be used, depending on the embodiment, such as for example, liposomal vectors, a cationic polymers, and/or DNA binding polymers. In several embodiments, the DNA delivery system comprises naked DNA. In several embodiments, a patient that is immuno-suppressed or otherwise has deficient immune function may benefit from a non-viral delivery system.
In several embodiments, in addition to functioning like pacemaker cells, the treated cells exhibit characteristics that are similar to those of natural pacemaker cells. However, certain of these characteristics need not be present in order to have the treated cells function as pacemaker cells. In several embodiments, the treated cells exhibit a length-to-width morphology substantially similar to a length-to-width morphology of native SAN cells. In several embodiments, this length-to-width ratio is at least about 10. In several embodiments, the treated cells exhibit an increase in spontaneous intracellular Ca++ oscillations. In several embodiments, the spontaneous, repetitive electrical activity increases in response to β-adrenergic stimulation. In several embodiments, the converted cells do not express atrial natriuretic peptide (ANP) or skeletal α-actin (αSkA).
In several embodiments, the subject has an electronic pacemaker to modify the electrical activity of the cardiac tissue, which in some patients may be implanted in the subject. In several embodiments, the generation of the biological pacemaker supplements the function of the electronic pacemaker (e.g., the workload of the electronic pacemaker is decreased due to the generation of the biological pacemaker). In several embodiments, the generation of the pacemaker cells serves as a temporary bridge until a replacement or alternative electronic pacemaker can be implanted in a subject. In several embodiments, however, the generation of the biological pacemaker functionally replaces the electronic pacemaker. In several embodiments, this functional replacement allows short-term, medium-term, long-term, or even permanent shut-off (or explanting) of the electronic pacemaker.
In several embodiments, there is also provided a method of generating a biological pacemaker using transcription factors to treat a cardiac arrhythmia comprising identifying a subject suffering from cardiac arrhythmia, wherein the subject has cardiac tissue comprising quiescent cells, wherein the quiescent cells comprise one or more of cardiomyocytes and stem cells, wherein the quiescent cells do not exhibit spontaneous, repetitive electrical activity; and administering one or more transcription factors to the quiescent cells to generate treated cells, wherein the treated cells exhibit spontaneous, repetitive electrical activity; thereby treating the cardiac arrhythmia.
In several embodiments, the subject is mammalian, and in several embodiments is a human. In several embodiments, the stem cells that are converted into pacemaker cells are embryonic stem cells, while in additional embodiments, they are adult stem cells, induced stem cells, or resident stem cells.
There is also provided herein a method of treating a dysfunction in cardiac electrical activity comprising obtaining cells converted into pacemaker cells according to the methods disclosed herein and administering the converted cells to a subject suffering from a dysfunction in cardiac electrical activity, wherein the converted cells exhibit spontaneous, repetitive electrical activity, thereby treating the dysfunction in cardiac electrical activity.
In several embodiments, the dysfunction in cardiac electrical activity comprises a cardiac arrhythmia. In several embodiments, the method further comprises isolating the converted cells prior to the administration.
In several embodiments the spontaneous, repetitive electrical activity is within about 65% to about 100% of the normal activity of pacemaker cells. In several embodiments, the administration of transcription factors causes a change in the rhythm of the heart of the subject. In several embodiments, the changed rhythm of the heart corresponds to a new heart rate within about 25% to about 35% of a normal heart rate.
Additionally provided are compositions for the generation of a biological pacemaker comprising: a DNA delivery system, the system comprising, a viral vector encoding Tbx18, Shox-2, Tbx3, Tbx5, functional fragments thereof, or combinations thereof, and a eukaryotic promoter.
In several embodiments, the viral vector is an adenoviral vector. In several embodiments, the adenoviral vector further comprises the following operably linked components in sequence, a first inverted terminal repeat sequence (ITR), a first lox P site, a packaging site (W, psi), a cytomeglovirus promoter, a sequence encoding Tbx18, Shox2, or combinations thereof, an internal ribosome entry site (IRES), a polyadenylation signal (An), a second lox P site, a sequence encoding the adenovirus early region 2 and early region 4 genes; and a second inverted repeat sequence (ITR).
Several embodiments disclosed herein relate to a population of cells for the generation of a biological pacemaker comprising a plurality of stem cells, wherein the stem cells have been contacted with one or more transcription factors selected from the group consisting of Tbx18, Shox-2, Tbx3, Tbx5, functional fragments thereof, and combinations thereof, wherein the one or more transcription factors induce an increase in the spontaneous, repetitive electrical activity of the cells, wherein the increase in the spontaneous, repetitive electrical activity of the cells is capable of generating an ectopic contraction of the cells, and wherein the stem cells are suitable for administration to a subject in need of biological pacemaker function.
In several embodiments, the stem cells are selected from the group consisting of embryonic stem cells, non-embryonic stem cells, bone marrow-derived stem cells, adipose-derived stem cells, induced pluripotent stem cells, and cardiac stem cells.
There is also provided for herein a use of one or more transcription factors selected from the group consisting of Tbx18, Shox-2, Tbx3, Tbx5, functional fragments thereof, and combinations thereof to convert quiescent cells that do not exhibit spontaneous, repetitive electrical activity into pacemaker cells that exhibit spontaneous, repetitive electrical activity.
In several embodiments, there are provided methods for generating a biological pacemaker using transcription factors comprising identifying a subject having cardiac tissue comprising quiescent cells that do not exhibit spontaneous, repetitive electrical activity and administering one or more transcription factors to the quiescent cells to generate treated cells, wherein the treated cells exhibit spontaneous, repetitive electrical activity, thereby treating said cardiac arrhythmia. In several embodiments, the subject was previously suffering from cardiac arrhythmia.
In several embodiments, there is also provided a method for treating cardiac arrhythmia comprising identifying a subject having cardiac tissue comprising quiescent cells, the subject suffering from cardiac arrhythmia, wherein the quiescent cells do not exhibit spontaneous repetitive electrical activity, and administering one or more of Tbx18 and/or Shox2 (or fragments thereof) to the quiescent cells to generate treated cells, wherein the treated cells exhibit spontaneous repetitive electrical activity, thereby treating the cardiac arrhythmia
In several embodiments, the quiescent cells comprise cardiomyocytes. In other embodiments, the quiescent cells comprise stem cells. In still additional embodiments, the quiescent cells comprise malfunctioning sinoatrial node cells (e.g. sinoatrial node cells that signal at levels that are too fast or too slow relative to normal functioning pacemaker cells). In other embodiments, the quiescent cells comprise other cells found in the heart and/or other somatic cells not found in the heart.
In several embodiments, there is also provided a method of converting a population of stem cells into cells suitable for generation of a biological pacemaker, the method comprising obtaining a population of stem cells, culturing the stem cells in vitro, wherein the cultured stem cells comprise quiescent cells that do not exhibit spontaneous repetitive electrical activity, and administering one or more transcription factors to the quiescent cells to generate converted cells. In several embodiments, the converted cells exhibit spontaneous repetitive electrical activity, and thus are capable of generating a biological pacemaker in vivo. In several embodiments, the stem cells comprise cardiac stem cells. In several embodiments, in vitro conversion can be performed on cardiomyocytes, other cells found in the heart, and/or other somatic cells not found in the heart.
In several embodiments, there are also provided methods of treating a cardiac arrhythmia by generating a biological pacemaker using transcription factors, the method comprising identifying a subject suffering from cardiac arrhythmia, obtaining a population of converted stem cells, wherein, prior to the conversion, the stem cells comprised quiescent cells that did not exhibit spontaneous repetitive electrical activity, wherein one or more of Tbx18 and Shox2 (or fragments thereof) were administered to the quiescent cells to generate converted cells, the converted cells exhibiting spontaneous repetitive electrical activity, and administering the converted cells to the subject, wherein the converted cells engraft into the cardiac tissue of the subject and continue to exhibit spontaneous repetitive electrical activity, thereby generating a biological pacemaker and treating the cardiac arrhythmia. In several embodiments, the converted cells are isolated, purified, selected for, or otherwise concentrated prior to administration to the subject. As discussed above, in several embodiments, instead of, or in conjunction with stem cells, other quiescent cells types are converted to pacemaker cells. In several embodiments, cells converted in vitro to cells that exhibit spontaneous repetitive electrical activity include cardiomyocytes.
In several embodiments, the methods provided herein utilize mammalian stem cells to treat mammalian subjects. In several embodiments, the subject is a human.
In several embodiments, the one or more transcription factors are regulators of embryonic sinoatrial node development. In several embodiments, the one or more transcription factors promote de novo cellular differentiation into sinoatrial nodal cells. In still additional embodiments, the one or more transcription factors not only regulate embryonic sinoatrial node development but also promote de novo cellular differentiation of precursor cells into sinoatrial nodal cells. In further embodiments, the one or more transcription factors provide a positive or negative regulatory input on such development or differentiation pathways. In several embodiments, the one or more transcription factors define the sinus venosus during cardiac development. In some embodiments, the one or more transcription factors are negative regulators of Nkx2.5 in the sinus venosus (or other regions of the developing heart). For example, the one or more transcription factors may negatively regulate a pathway in which Nkx2.5 signaling is involved. Further, the one more transcription factors may negatively regulate the expression (either at the RNA or protein level) of Nkx2.5. Moreover, the one or more transcription factors may indirectly regulate Nkx2.5, or a signaling pathway related thereto (e.g., the one or more transcription factors regulate a pathway that thereafter negatively regulates Nkx2.5).
In several embodiments, the one or more transcription factors is selected from the group consisting of Tbx18, Shox2, Tbx3, Tbx5 or combinations thereof. In several embodiments, variants of Tbx18, Shox2, Tbx3, Tbx5 are used alone or in combination. In some embodiments, functional fragments of Tbx18, Shox2, Tbx3, Tbx5 are used either alone or in combination. In additional embodiments, related family members of these transcription factors may also be used. In some embodiments, human transcription factors are used, while in other embodiments homologs from different species are used (either in place of or in conjunction with the human transcription factor(s)). In further embodiments, additional transcription factors, proteins, biologic molecules, or pharmacological agents are administered prior to, concurrently with, or subsequently to the one or more transcription factors in order to enhance the effect of the one or more transcription factors in generation of a biological pacemaker or conversion of stem cells. In some embodiments, for example, a traditional pharmacological agent that is used to treat cardiac arrhythmia is administered concurrently with the one or more transcription factors in order to provide a short-term bridge to allow the generated biological pacemaker to take full functional effect in a subject. In several embodiments, the one or more transcription factors comprises Tbx18 while in some embodiments, the one or more transcription factors comprises Shox-2. In still additional embodiments, Tbx18 and Shox2 are both used. However, in some embodiments, these two transcription factors are administered at discrete time frames (that optionally overlap in some embodiments). Additionally, Tbx18 and Shox2, are administered separately in some embodiments (e.g., different delivery systems are used). In several embodiments, a functional fragment of one transcription factor is used in conjunction with one (or more) full length transcription factors. In still additional embodiments, combinations of functional fragments of transcription factors are used.
In some embodiments, administration of the one or more transcription factors comprises administration of a DNA delivery system comprising a polynucleotide encoding the one or more transcription factors. In several embodiments, the DNA delivery system comprises a viral vector. A variety of different viral vectors are used, depending on the embodiment, based on the transcription factor(s) to be delivered, the size of the polynucleotide(s), the route of administration, and the general health status of the patient, among other variables. In some embodiments, the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, retrovirus, HJV, HIV, and HSV. As discussed above, more than a single transcription factor is administered in certain embodiments. In some embodiments, each of the transcription factors delivered is delivered in the same type of virus. In some embodiments, however, a first transcription factor is delivered in a first type of virus, while a second transcription factor is delivered in a second type of virus. In this manner, a desired expression profile of each of the transcription factors can be generated based on the characteristics of the virus chosen (e.g., the time from infection with the virus to expression of the transgene carried by the virus). For example, certain viruses provide longer-term expression of the polynucleotides that they carry as compared to other viruses. In still other embodiments, more than one viral vector can be used to deliver a single transcription factor.
Alternatively, in several embodiments, the DNA delivery system comprises a non-viral vector. Non-viral vectors, in some embodiments, are selected from liposomal vectors, cationic polymers, and/or DNA binding polymers. In additional embodiments naked DNA encoding the one or more transcription factors is delivered. In still additional embodiments wherein more than one transcription factor is administered, a combination of viral and non-viral delivery systems may be used.
In several embodiments, the methods disclosed herein result in the generation of cells that exhibit spontaneous, repetitive, electrical activity. In some embodiments that spontaneous, repetitive, electrical activity is between about 50% to about 100% of the normal activity of healthy pacemaker cells. In some embodiments, a biological pacemaker can be generated even when the generated cells exhibit spontaneous, repetitive, electrical activity that is between about 5% to about 50% of the normal activity of healthy pacemaker cells. Thus, the methods disclosed herein are suitable for generating a biological pacemaker when the generated cells exhibit spontaneous, repetitive, electrical activity between about 5% to about 15%, about 15% to about 25%, about 25% to about 35%, about 35% to about 45%, about 45% to about 55%, about 55% to about 65%, about 65% to about 75%, about 75% to about 85%, about 85% to about 95%, or about 95% to about 100% of the normal activity of healthy pacemaker cells. In several embodiments, the generated cells exhibit spontaneous, repetitive, electrical activity of greater than 100% of the normal activity of healthy pacemaker cells.
In addition to changing the electrical activity of the target cells, in several embodiments administration of one or more transcription factors (or cells converted into biological pacemaker cells) causes a subsequent change in the rhythm of the heart. In several embodiments, the resultant heart rhythm is within about 5% to about 15%, about 15% to about 25%, about 25% to about 35%, about 35% to about 45%, about 45% to about 55%, about 55% to about 65%, about 65% to about 75%, about 75% to about 85%, about 85% to about 95%, or about 95% to about 100% of normal heart rhythm (taking into account the age and health history of a particular patient). In several embodiments, increases in heart rhythm of greater than 100% are achieved.
In several embodiments, the administration of one or more transcription factors (or cells converted into biological pacemaker cells) is to a subject having an implanted electronic pacemaker. In some embodiments, the generation of a biological pacemaker due to set administration reduces the dependence of the subject on the implanted electronic pacemaker. In some embodiments, generation of the biological pacemaker is for the purpose of providing a bridge therapy during which time all or a portion of the electronic pacemaker can be replaced (e.g., if a portion of the electronic pacemaker has become infected). In still additional embodiments, the generation of a biological pacemaker obviates the need for an implanted electronic pacemaker, and as such, the implanted electronic pacemaker can be permanently removed from the subject. In further embodiments, an electronic pacemaker may be implanted in conjunction with administration of one or more transcription factors (or converted biological pacemaker cells) such that reliance on the electronic pacemaker is reduced.
Multiple routes of administration may optionally be used, depending on the embodiment. Various factors determine what target site will be used including, but not limited to, where healthy tissue is available to generate a biological pacemaker, if the methods disclosed herein are to be used to re-functionalize a malfunctioning pacemaker cell, if one or more transcription factors are to be administered or if converted cells are to be administered. In some embodiments, administration is into the apex of the heart. In some embodiments, administration is to the inter-ventricular septum. In some embodiments, administration is to the right ventricular free wall. In some embodiments, administration is to the left ventricular free wall. In some embodiments, administration is via the right ventricle. In some embodiments, administration is via the right atrium. In several such embodiments, a right-sided approach advantageously reduces the risk of embolism and/or stroke. In some embodiments, administration is made via more than one of the above routes, either concurrently or at different times.
As discussed above, in some embodiments, the methods disclosed herein are used to restore (either partially or fully) the function of an existing pacemaker cell, or a cell within the cardiac conduction system. In some embodiments, administration is to the right or left branch of the Bundle of His. In some embodiments, administration is to the Purkinje fibers. In still additional embodiments, administration is to the SA node and/or to the AV node.
In certain embodiments wherein converted cells are delivered, intravenous administration is used. In some embodiments, intra-coronary administration is used.
Depending on the circumstance, in some embodiments administration (of one or more transcription factors or of converted biological pacemaker cells) is made by accessing the heart directly (e.g., injection during removal of an electronic pacemaker). In some embodiments, a catheter is used for administration. In some embodiments, the catheter comprises a map guided catheter injection system. In some embodiments, fluoroscopy guidance is used to guide a catheter (or other administration system) to the target tissue within the heart. In some embodiments, x-ray guidance is used. Echocardiography guidance is used in additional embodiments as well as magnetic resonance imaging, in certain embodiments. In some embodiments, more than one of the above modes of guidance vehicles are used concurrently or at different times during administration.
In addition to the methods disclosed above, there is also provided herein a composition for the generation of a biological pacemaker comprising a DNA delivery system, the system comprising a viral vector encoding Tbx18, Shox2, or combinations thereof, and a eukaryotic promoter. In one embodiment, the viral vector is an adenoviral vector however, as discussed above a variety of different viral vectors may also be used, depending on the embodiment. In several embodiments, the adenoviral vector further comprises the following operably linked components in sequence: a first inverted terminal repeat sequence (ITR), a first lox P site, a packaging site (V, psi), a cytomeglovirus promoter, a sequence encoding Tbx18, Shox2, or combinations thereof, an internal ribosome entry site (IRES), a polyadenylation signal (An), a second lox P site, a sequence encoding the adenovirus early region 2 and early region 4 genes, and a second inverted repeat sequence (ITR).
In several embodiments, the dose of a viral construct to be administered is based on plaque-forming units, which is a well-established unit of measurement in the viral arts. In some embodiments, a dose of between about 1×108 and 1×1010 pfu (in volumes ranging from 50 to 200 microliters) are used. With respect to delivery of cells converted to be biological pacemaker cells, doses of cells range between 1×105 and 1×108 cells. Higher or lower doses may be used, depending (among other factors) on the severity of cardiac arrhythmia in the subject, the presence or absence of an electronic pacemaker, and/or the size of the heart of the subject.
Also provided for herein is a population of cells for the generation of a biological pacemaker comprising a plurality of stem cells, wherein the stem cells have been contacted with one or more transcription factors selected from the group consisting of Tbx18, Shox2, and combinations thereof, wherein the one or more transcription factors induce an increase in the spontaneous, repetitive electrical activity of the cells, wherein the increase in the spontaneous, repetitive electrical activity of the cells generates spontaneous, repetitive ectopic contractions, and wherein the stem cells are suitable for administration to a subject in need of biological pacemaker function.
In several embodiments, the stem cells are selected from the group consisting of embryonic stem cells, non-embryonic stem cells, bone marrow-derived stem cells, adipose-derived stem cells, induced pluripotent stem cells, and cardiac stem cells. In one embodiment, the population of cells comprises harvested adult cardiac stem cells that have been converted to cells with the biological pacemaker phenotype.
Also provided for herein is the use of one or more transcription factors selected from the group consisting of Tbx18 and Shox2 to convert quiescent cells that do not exhibit spontaneous, repetitive electrical activity into pacemaker cells that exhibit spontaneous, repetitive electrical activity.
Cardiac arrhythmias belong to a heterogeneous group of conditions in which there is abnormal electrical activity in the heart. As the result of an arrhythmia, heart rate may be too fast, too slow and/or may be regular or irregular. Normal electrical activity in the heart results from an electrical impulse that originates from the right atrium of the heart, in particular the sinus node (also referred to as the sino-atrial node, the SA node and/or SAN). This impulse induces contraction of both atria. The impulse then passes through the atrioventricular (or AV) node and through both ventricles via the Bundle of His and the Purkinje fibers. The result is a synchronized contraction of the heart muscle, and thus, blood flow. Normal adult heart rates range from 60 to 80 beats per minute, while the resting heart rate in children is typically much faster.
Bradycardias (HR of <60 bpm) have multiple possible etiologies, namely slowed signals from the sinus node (sinus bradycardia), pauses in the normal activity of the sinus node (sinus arrest), or blockages of the electrical impulse from the atria to the ventricles (AV block). Tachycardias (resting HR of >100 bpm) may cause mere palpitations (a subject becomes abnormally aware their heart beat) and may simply be the result of sympathetic nervous system stimulation of the sinus node (known as sinus tachycardia), for example during exercise or physical stress. Tachycardia that is not sinus tachycardia may result from abnormal impulses in addition to the normal cardiac cycle. Abnormal impulses can begin by one of three mechanisms: automaticity, reentry or triggered activity.
Certain cardiac tissues are capable of initiating an electrical impulse on their own, which is known as automaticity. Normally, such automatic cells are located in the conduction system of the heart (the SA node, AV node, Bundle of His and Purkinje fibers). The sinoatrial node is a single specialized location in the atrium which has a higher automaticity (a faster pacemaker) than the rest of the heart, and therefore is usually responsible for setting the heart rate, and initiating each heartbeat.
Re-entry arrhythmias occur when an electrical impulse recurrently circulates through a small region of the heart, rather than propagating from the atria to the ventricles. If conduction is abnormally slow in some areas of the heart, for example due to damaged or diseased cardiac tissue, impulse propagation times will vary, and certain impulses may potentially be treated as an entirely new impulse. Such disjointed impulse propagation can produce sustained abnormal circuit rhythms, which are responsible for atrial flutter, most paroxysmal supraventricular tachycardia, and dangerous ventricular tachycardia.
When an entire chamber of the heart has multiple reentry circuits, the chamber is considered to be in fibrillation, and quivers due to the chaotic electrical stimulation, rather than smoothly contracting and delivering blood. The lack of smooth and sustained blood and chaotic contraction can result in cardiogenic shock, cessation of effective blood circulation, and sudden cardiac death. Fibrillation can affect the atrium (atrial fibrillation) or the ventricle (ventricular fibrillation); ventricular fibrillation is imminently life-threatening.
Traditional arrhythmia treatments include pharmacological therapy, electronic pacemakers, implantable cardioverter-defibrillators (ICDs), ablation, and combinations thereof. While these traditional treatments have been used in the past to treat various types of cardiac arrhythmias, these approaches have several shortcomings. Implanted pacemakers and ICDs may cause complications from device implantation, malfunction or hardware infection. Pharmacological therapies may not be tolerated well in some patients, and have the capacity to induce additional arrhythmias during treatment. Thus, there is a need for alternatives or supplements to pharmacological therapies and implantable devices to modulate cardiac contractility and/or conductance.
There exist various approaches to generation of biological pacemakers that are distinct from those disclosed herein, in that several such methods employ delivery of ion channels, or subunits of ion channels to cardiac cells. In particular, dominant negative ion channels (or subunits thereof) have been investigated. Generally, as a result of expression of a dominant negative ion channel in cardiac cells, the ionic current across the cell membrane is altered, thereby resulting in pacemaker-like firing in these cells. Such methods generally rely on the delivery of genes that effectively manipulate the function of the treated cells by altering the ability of the cell with respect to conduction of certain ions. In other words, such approaches typically take the existing cell, and augment or alter its existing structures (e.g., ion channels) to change its function.
In contrast, several embodiments of the invention result in quiescent cells that are reprogrammed to become biological pacemaker cells. Several embodiments of the invention are particularly advantageous because the reprogramming of quiescent cells converts cells to their natural state, rather than treating cells with genetic sequences that did not exist naturally in the cell make-up. For example, several embodiments are advantageous because the reprogramming of the quiescent cells with transcription factors (e.g., not with ion channels) reduces the risk of induced abnormalities in the reprogrammed cell. In some embodiments, the transcription factors are less bulky (e.g., smaller genetic sequences can be used) which reduces logistical complications and opens up additional delivery options. Some embodiments of the invention are particularly beneficial because, by converting a cell to a natural (or native) pacemaker state, that cell will have a higher likelihood of success in generating and maintaining an appropriate pacemaker rhythm with reduced possibilities of side effects that may occur with other approaches (e.g., induction of arrhythmias due to the treatment itself). Further, several embodiments of the invention are beneficial in that they reduce the need for “fine-tuning” of the desired effect (e.g., requiring increased dose or number of treatments to achieve a particular result) as the conversion of the cells to a natural pacemaker-state enables the cells to operate at naturally defined, and therefore balanced, frequencies. As a result, several embodiments of the methods require fewer administrations (or doses) of the compositions in order to achieve conversion of a sufficient number of cells to generate a new pacemaker in the heart. Thus, in several embodiments, the methods disclosed herein may be less invasive to a patient, requiring fewer administration procedures, thereby presenting fewer risks to the patient and lowering morbidity due to the therapy itself.
Moreover, several embodiments of the methods disclosed herein are advantageous in that they do not rely on modification (by “non-native” sequences) of existing complex functional units of cardiac cells (e.g., the ion channels), rather, the conversion of the cells to a pacemaker state results in the generation of a complete complement of functional endogenous pacemaker channels. The reduced requirement for modification of existing channels reduces the likelihood that adverse results occur (e.g., mis-formation of channels or formation of channels that produce a greater or lesser impact on function than anticipated).
Additionally, several embodiments advantageously reduce the risk of unwanted side effects due to unbalanced electrical activity in the cells, as the cells converted to pacemakers by the methods herein have a complete and functionally balanced complement of ionic currents (and hence electrical activity), rather than cells having had a single current that was been exogenously manipulated.
As disclosed herein, the delivery of transcription factors involved in the early natural development of pacemaker cells (whose expression is reduced after development is complete) can unexpectedly reprogram non-pacemaker cells into pacemaker cells. These approaches are unexpected because the general view of cardiac cells is that they are terminally differentiated (e.g., once a contractile cell, always a contractile cell). However, several embodiments of the present methods allow the reprogramming of these cells, without direct exogenous alteration of their ion channels. Thus, rather than manipulating the functional units (ion channels) of the non-pacemaker cells, several embodiments of the methods disclosed herein change the functional fate and functional identity of the cells into pacemaker cells. As such, several embodiments of the methods and compositions disclosed herein result in the generation of a reprogrammed biological pacemaker, which lessens or obviates the need for such traditional pharmacological therapies, ablation, or artificial pacemakers.
The use of transcription factor based biological pacemakers reduces and/or obviates the need for traditional arrhythmia therapies. In several embodiments, generation of a biological pacemaker is used to supplement traditional therapies for bradycardias or other arrhythmias. In several embodiments, generation of a biological pacemaker reduces dependence (e.g., patients are weaned from) on traditional therapies. In several embodiments, cardiac arrhythmias are treated by generating a biological pacemaker that drives the heart at a normal or substantially normal rhythm that was not possible when a subject was untreated or when treated with a non-biological therapy (e.g., pharmaceutical or electronic pacemaker therapy). In several embodiments, generation of a biological pacemaker is used as a bridge therapy (e.g., for patients with cardiac damage sufficient to necessitate a transplant).
In several embodiments, the generation of a biological pacemaker comprises inducing the conversion of quiescent cardiac cells into pacemaker cells by transfer of transcription factors to cells (transfer can occur, for example, through the use of gene delivery techniques used to deliver polynucleotides that encode one or more of the transcription factors disclosed herein). As used herein the term “quiescent cardiac cells” shall be given its ordinary meaning and shall also refer to cardiac cells that exhibit no, little, or an inappropriate firing rate and/or cardiac cells are not spontaneously active. It shall be appreciated that identification of quiescent cardiac cells depends, in some embodiments, on the cell type being targeted. For example, ventricular and/or atrial myocardium normally responds to electrical signals generated by pacemaker cells, and thus typically has lower spontaneous firing rates as compared to normal pacemaker cells. Thus, if targeting the ventricular and/or atrial myocardium quiescent cells having little firing rate comprise, in some embodiments, cells having less than about 20%, less than about 15%, or less than about 10% of the spontaneous firing as compared to normal pacemaker cells. Alternatively, certain embodiments disclosed herein target a malfunctioning region (or regions) of the conduction system of the heart. For example, in several embodiments a malfunctioning region of the sinoatrial node is target, in for example, sick sinus syndrome. In additional embodiments, the atrioventricular (AV) node is targeted, for example, in patients having AV block. In still additional embodiments, the secondary conduction pathways of the heart (e.g., the His-Purkinje system and/or Bundle of His) are targeted. In such tissues that normally exhibit spontaneous repetitive electrical activity, quiescent cells are those that fire at a reduced rate compared to a normal cell in that region of the heart, with said reduced rate being insufficient to maintain an appropriate cardiac firing rate and/or cardiac output. Thus, quiescent cells, in some embodiments, are recognized as those cells which, if responsible for driving the electrical activity of the heart, would do so at a level of electrical firing that is insufficient to maintain appropriate blood flow throughout the cardiovascular system (e.g., those cells firing at a hemodynamically non-sustainable rate).
In several embodiments, the generation of a biological pacemaker results from delivery of transcription factors to cardiac tissue in vivo, resulting in the conversion of quiescent cardiomyocytes, endogenous cardiac stem cells, or combinations thereof, to pacemaker cells. In several embodiments, the generation of a biological pacemaker is performed by delivery of transcription factors in vitro resulting in the conversion of cultured somatic cells, cardiomyocytes, stem cells (including embryonic, induced pluripotent, pluripotent, multipotent, unipotent and/or adult stem cells), or combinations thereof to pacemaker cells. In several embodiments, these generated pacemaker cells can subsequently be implanted in vivo to treat abnormalities of cardiac rhythm.
In several embodiments, the conversion of somatic cells, cardiomyocytes, and/or stem cells to pacemaker cells is achieved using transcription factors that are regulators of embryonic sinoatrial node (SAN) development. Potential transcription factors that are regulators of embryonic SAN development include, but are not limited to: Tbx18, Shox2, Tbx3, and/or Tbx5. Tbx18 is a transcription factor that is required for embryonic development of the SAN head area, but, as discussed in more detail below, typically becomes undetectable by birth and remains undetectable in adulthood (
As described above, several embodiments of biological pacemaker generation are based on in vivo therapy. In several embodiments Shox2, Tbx18, or combinations thereof are delivered in vivo to induce ectopic pacemaker activity in non-pacemaker somatic cells, cardiomyocytes, endogenous stem cells, or combinations of the three. In several embodiments other combinations of SAN regulating transcription factors (or transcription factors that are related to cardiac development, but not specifically SAN regulation) can be used with or without Shox2 and Tbx18, or Shox2 and Tbx18 individually to convert cells in vivo to pacemaker cells. Those other transcription factors include, but are not limited to Tbx3 and Tbx5. Sequences for Tbx3, Tbx5, Tbx18, Shox2, and variants thereof are shown in Appendix A, B, C, and D, respectively.
In several embodiments, Tbx18, Shox2, or a combination of Tbx-18 and Shox2 are delivered to cardiomyocytes or stem cells in vitro, which, as discussed below, yields cultured pacemaker cells. These cells can subsequently be administered to patients as a cell therapy for biological pacemaker generations. In several embodiments, administration comprises direct administration of the cells to the heart of a subject (e.g., injection). Other administration routes disclosed herein are used. For example, in some embodiments, catheter-based administration is employed. In additional embodiments, the generated pacemaker cells are incorporated into a matrix, graft, or other biomaterial that aids in cell retention at a target site within the heart. Cells used for in vitro generation of biological pacemakers, are, in some embodiments, harvested from healthy tissue of the patient that they are to be transplanted into (e.g., autologous transplant of induced pacemaker cells). Then these cells can be converted to pacemaker cells by transcription factors in vitro and can be reinserted into the same patient for the treatment of arrhythmias. In other embodiments, allogeneic transplant of induced pacemaker cells is performed (e.g., cells are harvested from a first subject, cultured in vitro, contacted with one or more of the transcription factors disclosed herein, and then transplanted to a second subject). In several embodiments other SAN regulating transcription factors or combinations of such transcription factors are used (e.g., transcription factors in addition to or in place of Tbx18, Shox2, and/or Tbx-18+Shox2) to convert cells in vitro to pacemaker cells. Those other transcription factors include, but are not limited to Tbx3 and Tbx5.
Patients having abnormalities of excitable tissue that may be treated with the methods disclosed herein include mammals and in particular humans. Patients include those suffering from or diagnosed with one or more of the cardiac conditions disclosed herein or other conditions known in the art to affect cardiac activity, conductivity, rhythm, and the like.
Reprogramming cardiomyocytes is accomplished, in several embodiments disclosed herein by using gene delivery as a means of delivering exogenous genetic material to somatic cells, cardiomyocytes, stem cells, or combinations thereof. In several embodiments, polynucleotides are administered in a nucleic acid delivery system. In several embodiments, a nucleic acid delivery system comprises a non-viral vector linked to the polynucleotide. Examples of such non-viral vectors include the polynucleotide alone (e.g., naked DNA) or the polynucleotide in combination with a suitable protein, polysaccharide or lipid formulation.
In several embodiments, the nucleic acid delivery systems comprise one or more viral vectors, including but not limited to adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, lentivirus, retrovirus, or hemaglutinating virus of Japan-liposome (HVJ) complex. Various serotypes of adenovirus and/or AAV are also used in several embodiments. In several embodiments the viral vector comprises a eukaryotic promoter. In several embodiments cytomegalovirus (CMV) promoters are used. Other promoters, including tissue-specific promoters are well-established in the art and may be used in certain embodiments. Additional vectors include retroviral vectors such as moloney murine leukemia viruses and HIV-based viruses. In one embodiment, an HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors are used in some embodiments, which include, but are not limited to pox vectors such as orthopox or avipox vectors, herpes virus vectors such as a herpes simplex I virus (HSV) vector.
In several embodiments, polynucleotides (e.g. those encoding one or more transcription factors) are administered in vivo and/or in vitro to convert cells (stem, cardiomyocytes, and/or other somatic cells) into pacemaker cells. In some embodiments, polynucleotides that encode a functional fragment of the transcription factor are delivered in addition to, or in place, of the entire transcription factor. As used herein, the terms “fragment”, “functional fragment” or similar terms shall be given their ordinary meaning and shall refer to a portion of an amino acid sequence (or polynucleotide encoding that sequence) that has at least about 70%, preferably at least about 80%, more preferably at least about 90%, 95%, 96%, 97%, 98% or 99% of the function of the corresponding full-length amino acid sequence (or polynucleotide encoding that sequence). Methods of detecting and quantifying functionality of such fragments are established in the art.
In several embodiments, administration of the compositions (being transcription factors or cells) disclosed herein to modulate cardiac electrical activity is via direct cardiac injection (e.g., during electronic pacemaker implantation or explantation). In some embodiments, systemic injection is used. Intracoronary injection is used in some embodiments. In still additional embodiments, catheter-directed administration is used. In some embodiments, a map-guided catheter system (e.g., NOGA®) is used, in order to focally administer the compositions. Other mapping or guidance techniques are used in some embodiments. For example, in several embodiments fluoroscopy-based guidance is used. Electroanatomical guidance is also used in some embodiments. Mapping of specific structures (including but not limited to the His Bundle, the right or left portions of the bundle, the Purkinje fibers, etc) by intracardiac electrograms are also used in some embodiments. Moreover, X-rays or magnetic catheters are also used in some embodiments to guide delivery of a catheter, needle, or other delivery device(s) to a desired target location.
In several embodiments, a focal delivery approach advantageously reduces the time to generation of an active biological pacemaker. In some embodiments, the tissue-specific (or cell type-specific) delivery of several of the constructs disclosed herein is advantageous in that the construct is particularly suited for facilitating the generation of a biological pacemaker based on the expression profile of endogenous tissues. In some such embodiments, combinations of transcription factors are delivered to the same target site, while in other embodiments, individual constructs are delivered to distinct target sites, with the overall effect resulting in biological pacemaker generation.
In several embodiments, transduction is achieved by focal injection into the apex of the heart. In several embodiments, transduction is achieved by focal injection to the left ventricular apex. In several embodiments, a right-sided (e.g., right side of the heart, either atrium or ventricle) approach is used, in order to reduce the risk of stroke or other embolism. However, in several embodiments, left-sided approaches are used. In several embodiments, an injection catheter is introduced via the right atrium (rather than the right ventricle), in order to access the Bundle of His or AV node from above. In several embodiments, trans-septal catheter methods are used to introduce an injection catheter into the left atrium or left ventricle without the need for arterial access, thereby reducing stroke risk. In still additional embodiments, the introduction of an injection catheter is by way of the cardiac veins via the sinus of Valsalva for injection of a biologic as disclosed herein into various targets of the ventricles. Such an approach is similar to that used for the placement of pacer leads in cardiac resynchronization therapy.
Thus, in several embodiments, the compositions as disclosed herein can be used to deliver one or more transcription factors (or cells that have been previously contacted with the transcription factors) to either the right atrium, right ventricle, SA node, AV node, bundle of his, and/or left and right bundle branches. Moreover, through cannulation of the coronary sinus and its venous branches delivery to multiple left ventricular sites is achieved in several embodiments. Advantageously, in those patients with unfavorable coronary venous anatomy, access to the left side is achieved, in several embodiments, from the right side through a trans-septal puncture which allows direct access to left sided structures without the need of arterial access.
Supplemental methods are used in several embodiments and include administration of compounds to increase the microvascular permeability of the cardiac tissue. Suitable vascular permeability agents (administered prior to, during, or after administration of a gene transfer vector) include a solution having less than about 500 micromolar calcium: substance P, histamine, acetylcholine, an adenosine nucleotide, arachidonic acid, bradykinin, endothelin, endotoxin, interleukin-2, nitroglycerin, nitric oxide, nitroprusside, a leukotriene, an oxygen radical, phospholipase, platelet activating factor, protamine, serotonin, tumor necrosis factor, vascular endothelial growth factor, a venom, a vasoactive amine, or a nitric oxide synthase inhibitor, serotonin, vascular endothelial growth factor (VEGF), or a functional VEGF fragment.
In several embodiments, administration of transcription factors (or cells contacted with those transcription factors in vitro) disclosed herein induces or otherwise causes the spontaneous repetitive electrical signals to be generated in cells that previously responded to such signals, but did not generate them. For example, for treated myocardial cells that exhibited little (e.g., an index of automaticity between about 40% to about 30%, about 30% to about 20%, about 20% to about 10%, or about 10% to about 0%, or overlapping ranges thereof) or no firing rate, exhibit an increased frequency of firing rate or electrical signal output post-administration (e.g., an index of automaticity of between about 5% to about 15%, about 15% to about 25%, about 25% to about 35%, about 35% to about 45%, about 45% to about 55%, about 55% to about 65%, about 65% to about 75%, about 75% to about 85%, about 85% to about 95%, about 95% to about 100%, or more) as compared to the cells pre-administration.
The resultant changes in cardiac contraction and/or an electrical property of converted pacemaker cells, by the methods disclosed herein, modulate cardiac rhythm in several embodiments. In several embodiments, the methods and compositions disclosed herein achieve a heart rate within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, within about 2%, or within about 1% of a clinically desired heart rate. In several embodiments, the methods and compositions disclosed herein are used to treat subjects suffering from or susceptible to a disease or disorder such as a cardiac-related syncope (e.g., Stokes-Adam syncope), an abnormality of sinus node function such as persistent sinus bradycardia, sino-atrial (S-A) block manifested as S-A Wenckebach, complete S-A block or sinus arrest, and high-grade atrioventricular block; or bradycardia-tachycardia syndrome or other bradycardia related condition. In several embodiments, modulation is used to increase or slow down the function of an implanted pacemaker (e.g., to achieve a desired heart rate that the implanted pacemaker fails to provide on its own).
In several embodiments, the administration of Tbx18, Shox2, or a combination thereof produces several physiological changes to the contacted cells in addition to or separate from the generation of spontaneous repetitive electrical signals discussed above. In some embodiments, these physiological changes even if first seen in vitro, are also detectable in vivo, where they may serve as supplemental markers of the efficacy of biological pacemaker generation (e.g., they are recognized as characteristics or hallmarks of pacemaker cells). In some embodiments, the administration of one or more transcription factors results in an increased percentage of spontaneously beating monolayer cultures compared to control. In several embodiments, the presence, or amount, of spontaneous beating is use to screen cultures for functionality prior to transplant or to evaluate other combinations of transcription factors for their utility in generating pacemaker cells in vitro. In some embodiments, the administration of one or more transcription factors results in spontaneous intracellular Ca2+ oscillations of myocardial cells are administered. In some embodiments, the administration of one or more transcription factors results in a gradual phase-4 depolarization. In some embodiments, the delivery of one or more transcription factors disclosed herein increases or decreases the expression of HCN4 in cells. Because calcium flux is a primary component of cardiac electrical signaling and HCN4 expression is important in pacemaker cell function, changes in these endpoints correspond, in some embodiments, to more SAN-like behavior in induced biological pacemaker cells. In some embodiments, the administration of one or more transcription factors results in modulation of sub-sarcolemmal, spontaneous localized Ca2+ release events. In some embodiments, the administration of one or more transcription factors results in modulation of intracellular cAMP levels. Thus, as a result of administration of one or more of the above types of transcription factors, cardiac electrical activity can be modulated and abnormalities in excitable cardiac tissue can be treated.
In several embodiments, the administration of Tbx18, Shox2, or a combination thereof will result in cells new phenotypes of the contacted cells in addition to or separate from the generation of spontaneous repetitive electrical signals. In some embodiments, the administration of one or more transcription factors results in disorganized and markedly lower sarcomeric α-actin expression in transduced cells, which is indicative of pacemaker cells. In some embodiments, the administration of one or more transcription factors results in a change in cell size. In some embodiments, the administration of one or more transcription factors results in changes to the chromatin state. In some embodiments, the administration of one or more transcription factors results in chromatin modification and will cause lower or higher expression and or activity of one or more of the following genes: Cx43, Kir2.1, Actc2, and HCN4. These phenotypic changes, mirror those of natural SAN cells and as a result of the administration of one or more of these transcription factors, can be used, in several embodiments, as an additional means to evaluate the generation of biological pacemaker cells.
In several embodiments, the administration of the one or more transcription factors to the heart will result in frequent ectopic ventricular beats that originate from the site of gene injection resulting in targeted generation of pacemaker activity. In some embodiments, the administration of one or more transcription factors results in gene-related epigenetic changes in cells and de novo pacemaker activity. This pacemaker activity, in some embodiments, is a result of somatic reprogramming, and not due to dedifferentiation to a progenitor state. Somatic to somatic transdifferentiation will lower the threat of neoplasia from transduced cells (e.g., teratoma formation is reduced). In other embodiments, however, the administration of one or more transcription factors may result in dedifferentiation to a progenitor state. In certain such embodiments, differentiation into pacemaker cells is induced using one or more of the transcription factors disclosed herein. Using such methods, biological pacemaker cells can safely and effectively be made from a wide variety of cells.
Examples provided below are intended to be non-limiting embodiments of the invention.
The human Tbx18 gene with a C-terminal myc/FLAG tag was excised from pCMV6-Tbx18 (Origene, Rockville, Md.) by digestion with FseI and SalI. The Tbx18 gene was then sub cloned into a NotI- and XhoI digested lentiviral expression vector with the desired reporter gene, pLVX-IRES-ZsGreen1 (Clontech, Mountain View, Calif.), thus generating the pLV-Tbx18-IRES-ZsGreen1 (˜10.1 kb) vector. ZsGreen1 was used as the reporter protein for Tbx18-transduced cardiomyocytes, due to its similar spectral characteristics as the commonly used GFP. Thus, throughout the disclosure, the terms ZsGreen1 and GFP are used interchangeably. The recombinant target gene was then introduced to an adenovirus vector backbone by Gateway recombination cloning using Gateway-adapted vectors (Invitrogen, Carlsbad, Calif.). An LR recombination reaction was performed between the entry clone and the destination vector, pAd/CMV/V5-DEST (˜36.7 kb), to generate the desired adenoviral expression construct, pAd-CMV-TBX18-IRES-GFP (˜39.8 kb). Positive constructs were verified to have the correct target gene by DNA sequencing (Laragen, Los Angeles, Calif.).
Whole-cell electrophysiology recordings were performed as described below. Experiments were carried out using standard microelectrode whole-cell patch-clamp techniques with an Axopatch 200B amplifier (Axon instruments) with a sampling rate of 20 kHz and low-pass Bessel-filtered at 5 kHz. All experiments were performed at a room temperature. Cells were washed with a normal Tyrode's solution containing (mmol/L): NaCl 138, KCl 5, CaCl2), 2, glucose 10, MgCl2 0.5, and HEPES 10; pH 7.4. The micropipette electrode solution was composed of (mmol/L): K-glutamate 130, KCl 9, NaCl 8, MgCl2 0.5, HEPES 10, EGTA 2, and Mg-ATP 5; pH 7.2. Microelectrodes had tip resistances of 2 to 4 MΩ when filled with the internal recording solution. Voltage-clamp experiments were performed with an inter-episode interval of 2.5 seconds. Action potentials were either initiated by short depolarizing current pulses (2 to 3 ms, 500 to 800 pA) on GFP-NRVMs or recorded with I=0 mode on Tbx18-NRVMs. Data were corrected for the measured liquid junction potential (−mV). A xenon arc lamp was used to view GFP fluorescence at 488/530 nm (excitation/emission).
The expression constructs as discussed above were digested with PacI to expose inverted terminal repeats before transfecting into 293A cells to produce adenoviral stocks for use in subsequent expression of the transgene. Adenoviruses were plaque-purified, amplified, and affinity-column purified using Adenopure kit (Puresyn, Inc), and stored at −80° C.
NRVMs were isolated from 1-2 day old rat pups and cultured as a monolayer using established culture methods. Only the lower one third of the heart (from the apex to the midline) was excised in order to minimize contaminating atrioventricular nodal cells. In some embodiments, other portions of the heart are used, including the lower third, the middle third, and combinations of these portions, or whole hearts (or portions thereof) where endogenous pacemaker cells are selectively removed. In some embodiments, biopsies (e.g., guided biopsies) are used to obtain non-pacemaker tissue). A monolayer of NRVMs was transduced with either Ad-Tbx18-IRES-GFP or Ad-GFP (control vector; moi=1-10) one day after cell isolation, and cultured for 2-5 days. Tbx20, known to be critical for cardiac chamber differentiation, was employed in order to control for non-specific, embryonic transcription factor-related effects.
SA nodal myocytes were isolated from adult Sprague-Dawley rats. Animals were anesthetized with isoflurane. Hearts were quickly removed, the atria separated from the ventricles, and the sinoatrial node region dissected in Tyrode solution, which consisted of (in mM) 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 5 HEPES, 5.55 glucose, 1 MgCl2, 1.8 CaCl2); pH adjusted to 7.4 with NaOH. The rat sinoatrial node region was defined by the borders of the crista terminalis, the interatrial septum, and the superior and inferior vena cavae.
Mouse embryonic stem cells were transfected with an adenoviral vector expressing Shox2 or a control gene. Established embryoid body methods were used for differentiation. Pooled data are n≥3 with p<0.05 for all reported differences.
For measurements of intracellular Ca2+ oscillations, 2×106 NRVMs were plated in 35-mm glass bottom dishes (MatTek Cultureware) or 22 mm fibronectin-coated glass coverslips, transduced, and analyzed 4 days post-transduction. Cells were loaded with Rhod2-AM (2 μmol/L) (Molecular Probes) for 18 minutes, then washed once and subsequently placed in normal Tyrode's solution with 2 mmol/L calcium. Calcium transients were recorded at 37° C. from AdTbx18IS-IRES-GFP and Ad-GFP transduced NRVMs. Images were acquired on an inverted confocal laser-scanning microscope (Perkin Elmer/Nikon) or Leica SP5 confocal microscope. Offline analysis was performed using Ultraview (Perkin Elmer) and ImageJ. Whole-cell Ca2+ transients were obtained from confocal line scan images through single NRVMs by averaging the signal of an individual cell. Ca2+ transients are presented as background-subtracted, normalized fluorescence (F/F0). For 2-D confocal Ca2+ imaging calcium transients were obtained by averaging the signal through the entire cell.
Frozen sections of neonatal rat sinoatrial node and NRVMs 4 days post adenoviral transduction were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X 100 and then incubated with the appropriate primary antibody: sarcomeric α-actinin (Sigma-Aldrich; A5044; 1:800), ANP (AbCam; ab-14348; 1:1000), HCN4 (Abcam; ab85023; 1:500) and Alexa Fluor-conjugated secondary antibodies (Invitrogen).
Western blots were performed using specific antibodies against to Cx43 (Sigma-Aldrich, C6219; 1:1000), PLN (Alomone; A010-14: 1:5000), p-PLB (Alomone; A010-12: 1:5000) HCN4 (AbCam; ab85023; 1:500) Briefly, Ad-Tbx18, Ad-GFP transduced NRVMs, rat SAN and Left ventricle were homogenized in RIP A buffer containing a protease inhibitor cocktail (Sigma). Protein content was quantified by BCA assay and cell lysates (15 μg per lane) were run on a 12% SDS-PAGE gel and transferred onto a PVDF membrane. Then the transferred membrane was incubated with a primary antibody overnight at 4° C., followed by 1-hour incubation with a peroxidase-conjugated secondary antibody. Immunoreactivity was detected by chemiluminescence (ECL Western Blotting Analysis System, Amersham). Equal protein loading of the gels was assessed by re-probing the membrane with monoclonal anti-GAPDH antibody (Abeam; ab9482; 1:10000) or anti-β-actin (Sigma-Aldrich; A3848: 1:25000).
Adenoviruses were injected into the left ventricular apex of guinea pigs. Adult female guinea pigs (weight, 250 to 300 g; Charles River) were anesthetized with 4% isoflurane, intubated, and placed on a ventilator with a vaporizer supplying 1.5% to 2% isoflurane. After lateral thoracotomy, a 30-gauge needle was inserted at the free wall apex of the left ventricle. 100 μl of adenovirus containing 1×109 plaque-forming units of Ad-Tbx18-IRES-GFP or Ad-GFP (control group) was injected into the left ventricle apex. As discussed above, other delivery routes are used in some embodiments.
Methacholine (0.1-0.5 mg per kg of body weight in saline, Sigma-Aldrich, St. Louis, Mo.) was delivered via the jugular vein in order to slow the animals' sinus rhythm prior to ECG recordings under general anesthesia (2% isoflurane, 98% 02). ECGs were recorded using a 2-lead digital ECG system at 2 kHz (Lead I and Lead 3, BIOPAC Systems, Goleta, Calif.) and Lead 2 was offline calculated by Einthoven's triangle using Acqknowlege 3.7.3 software (BIOPAC Systems, Goleta, Calif.). In all animals, methacholine was administered until complete heart block was achieved. Heart block was accompanied by a reduction of the animals' sinus rhythm to <100 bpm. In most of the Tbx18-injected guinea pigs, ectopic ventricular rhythms were manifested well before the sinus rhythm reached 100 bpm (
For ex vivo, intact whole-heart ECG recordings, the heart was retrograde-perfused via the aorta at 60 mmHg with oxygenated Tyrode's solution at 36° C. The perfused heart was placed in a sylgard-coated plate filled with warm Tyrode's solution. ECG leads were stationed at appropriate sites to record leads I and II (
Rat sinoatrial node, left ventricle and Tbx18- and GFP-transduced NRVMs (4 days post transduction) were collected and mRNA was extracted (Qiagen mRNA Isolation Kit). The mRNA samples were converted to first strand cDNA, using the RT2 First Strand Kit (SA Biosciences). Then, the cDNA template was mixed with the RT2 qPCR Master Mix and the mixture was aliquoted into each well of the same plate containing pre-dispensed gene specific primer sets. PCR was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems/Life Technologies Corporation, Carlsbad, Calif.) and the relative expression of the genes was calculated.
cAMP Assay
The Cyclic AMP Elisa Assay Kit (catalog #STA-501; Cell Biolabs, INC) was used to determine cAMP levels in NRVMs transduced with Ad-Tbx18 or Ad-GFP. Briefly, 50 μl Tbx18- and GFP-NRVM cell lysates were added to the Goat Anti-Rabbit Antibody Coated Plate 96 well plate. 25 μl of diluted Peroxidase cAMP Tracer Conjugate was added to each tested well. Then, 50 μl of diluted Rabbit Anti-cAMP Polyclonal Antibody was added to each tested well and the plate was incubated at room temperature for 30 minutes with shaking. After washing 100 μl of Chemiluminescent Reagent was added to each well. After incubation at room temperature for 5 minutes on an orbital shaker, the plate was read for luminescence of each microwell on a plate luminometer. Measurement of light emission (RLU) allowed calculating the amount of cAMP in samples which were then normalized to β-actin for comparison of the samples.
Tyrode solution containing (mm): NaCl 140, KCl 5.4, CaCl2) 1.5, MgCl2 1.5, glucose 10 and Hepes 5; pH adjusted to 7.38 with NaOH. Ryanodine and Protein kinase I (PKI) were purchased from Tocris biosciences and caffeine was purchased from Sigma. Rhod-2/AM and was purchased from Invitrogen.
NRVMs transduced with either Tbx18 or GFP were fixed two- to four-days after viral vector transduction with 10% formaldehyde for 8 min at room temperature. Cells were sheared using a sonicator with ten pulses of 20 seconds each, with a 30-second rest on ice between each pulse. Chromatin immunoprecipitation was performed using ChIP-IT® Express Chromatin Immunoprecipitation kit (Active Motif, Carlsbad, Calif.) following the manufacturer's protocol. Primary antibodies for the H3K4me3 and H3K27me3 were purchased from Active Motif.
qPCR after Chromatin Immunoprecipitation
Gene specific (Cx43, Kir2.1, α-SA, HCN4) primers, already validated for qPCR in rat, were purchased from SA Biosciences. For each gene, three sets of primers were employed corresponding to −2 kb (upstream), −lkb, and +lkb (downstream) of the transcription start site. The ΔCt values from the three tiles were averaged from each experiment for data analyses.
Single myocytes were collected in PBS with a wide-opening patch pipette, placed on dry ice and then stored at −80° C. Tbx18 mRNA levels in individual myocytes were examined by quantitative real-time PCR with an Ambion® Single Cell-to-CT™ Kit (Life Technologies) according to manufacturer's instructions. Briefly, single cells were treated with cell lysis solution and DNase I for 5 minutes at room temperature. Reverse transcription was performed at 25° C. for 10 minutes, at 42° C. for 60 minutes and at 85° C. for 5 minutes after addition of SupersScript RT and VILO RT mix. Preamplification was performed with 14 cycles of 95° C. for 15 seconds and 60° C. for 4 minutes with addition of PreAmp mix and 0.2× TaqMan Gene Expression Assays, containing primers for human Tbx18 (assay ID: Hs01385457_ml), guinea pig GAPDH (assay ID: Cp03755742_g1), and guinea pig TnnT2 (assay ID: Cp04182357_g1). The custom primers were synthesized by Applied Biosystems (Carlsbad, Calif.). Preamplification products (1:20 dilution) were used for real-time PCR with TaqMan Gene Expression Assays using an Applied Biosystems 7900HT Fast Real-Time PCR System. Standard curves for each of the 3 primer sets were constructed with serial dilutions of input DNA templates (
Data were analyzed for mean, standard deviation and standard error of the mean (SEM). The quantitative figures in this work represent the mean±SEM. Data sets were statistically evaluated using an unpaired t test. p<0.05 was considered significant unless indicated otherwise.
As discussed above, present therapies for cardiac arrhythmias caused by abnormalities of excitable tissue rely primarily on pharmacotherapy, radiofrequency ablation, implantable devices, and other such related approaches. These methods, while useful for the treatment of some forms of arrhythmias, have limitations (as discussed above). Biological pacemakers offer advantages over the use of traditional pacemakers and can be used instead of, or in conjugation with traditional pacemakers. The present study evaluated the use of transcription factor inducing agents for the generation of biological pacemakers.
To demonstrate that transduction of transcription factors to cardiac cells produces biological pacemakers, singular, heterologous transduction of selected transcription factors in bicistronic adenoviral vectors was performed in freshly-isolated NRVMs. As an initial screen, the number of spontaneously-beating cultures 36-48 hrs post-transduction was analyzed. Tbx18-transfected NRVMs (Tbx18-NRVMs) exhibited an increased percentage of spontaneously-beating monolayer cultures compared to control and other transcription factors screened (minimum of five different cell isolations per group,
Although NRVMs exhibit spontaneous, syncytial contractions when cultured as monolayers, such a phenomenon is driven by a relatively small number of autonomously-beating cells.
The maximum diastolic potential (MDP) of −47±10 mV (n=6) in Tbx18-NRVMs was depolarized relative to the resting membrane potential (RMP) of −73±6 mV in GFP-NRVMs (n=5,
The change in diastolic potential, automaticity, and Ca2+ oscillation was complemented by a 78% reduction in IK1 density in Tbx18-NRVMs (
HCN4 is the molecular correlate of the hyperpolarization-activated current, If, which contributes to pacemaker activity in the SAN. Tbx18 transduction led to a 3.8-fold increase in the number of cells expressing HCN4 (
While membrane-delimited electrophysiological pathways contribute to pacemaking, SAN cells are triggered to fire rhythmically by finely-orchestrated, distinctive intracellular Ca2+ cycling events. Sub-sarcolemmal, spontaneous localized Ca2+ release events (LCRs) are a hallmark of automaticity in sinoatrial node cells. During late diastole, LCRs activate Na+-Ca2+ exchanger currents (INCX), which then contribute to the exponential phase of phase-4 depolarization. LCR measurements were performed on Tbx18-NRVMs to characterize their Ca2+ cycling profiles.
During late diastole, LCRs activate Na+-Ca2+ exchanger currents (INCX), which then contribute to the exponential phase of phase-4 depolarization. Line-scan confocal imaging of Tbx18-NRVMs resolved LCRs preceding each whole-cell Ca2+ transient (
The LCRs in Tbx18-NRVMs occurred at an average period of 343±8 ms, which was 72±1% of the whole-cell Ca2+ transient cycle length (474±7 ms,
Western blot experiments demonstrated a decrease in the total PLB levels and an increase in phosphorylated PLN (se16) akin to the adult rat SAN (
Phospholamban (PLN), in its unphosphorylated state, inhibits sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), thereby suppressing the reuptake of Ca2+ by internal stores. Such inhibition is relieved upon phosphorylation of the protein (P-PLB). The relative p-PLN (Ser16) level was 65-fold higher in Tbx-NRVMs in comparison to GFP-NRVMs (
In addition to the electrophysiological changes, cellular reprogramming replicates key features of cell structure in pacemaker tissue. Native sinoatrial node pacemaker cells are distinctive in their morphology: they are smaller and exhibit less-organized myofibrils than working cardiomyocytes. To test whether Tbx18-NRVMs had phenotypic changes, the morphology of the cells was investigated.
Sections of neonatal rat heart demonstrate that cardiac sarcomeric α-actinin (α-SA) expression is markedly lower and disorganized in the sinoatrial node compared to the adjacent right atrium (RA,
Whether the shifts to sinoatrial node-like phenotype are associated with a sinoatrial node-like chromatin state in Tbx18-NRVMs was then investigated. Trimethylation of lysine 27 in histone 3 (H3K27me3) is a heterochromatin mark which promotes the recruitment of Polycomb group proteins for gene silencing. Conversely, trimethylation of lysine 4 (H3K4me3) marks genes transcriptionally active. Histone modification profiles in the promoter regions of four genes, Cx43, Kir2.1, Actc2, and HCN4, were then investigated. Tbx18 is a transcription factor that is required for embryonic development of the sinoatrial node head area. Shox2 is a negative regulator of Nkx2.5 in the sinus venosus (as discussed above). Tbx3 is a potent regulator of sinoatrial node specialization, with developmental errors resulting from either deficiency or ectopic expression. Tbx5 is a positive regulator of Shox2 and Tbx3. These genes exhibit relevant molecular and functional changes in Tbx18-NRVMs. Tri-methylation level on H3K27 indicates that Tbx18 increased inactivity of Cx43, Kir2.1, and α-SA promoters while relieving its repressive epigenetic pressure on HCN4 promoter normalized to control. These results were measured by chromatin immunoprecipitation followed by qPCR. Meanwhile, H3K4me3 (
In
In several embodiments, at least one of Cx43, Kir2.1, and Actc2 may become epigenetically inactive in iSAN cells as compared to the quiescent cells. In several embodiments, expression of sarcomeric α-actinin in iSAN cells may be weak as compared to the quiescent cells. Furthermore, in several embodiments, the iSAN cells may exhibit myofibrillar disorganization. Only one of such changes may occur, or combinations thereof may occur, depending on the embodiment; however, each of the above are consistent with a shift of a cell towards a sinoatrial node-like phenotype. Moreover, in several embodiments pacemaker cells are generated without the induced alteration expression of ion-channel proteins, the genetic introduction of which may be contrary to the data above (e.g., the reduction in Kir2.1 that contributes, at least in part, to iSAN formation, would potentially be offset by genetic introduction of Kir2.1 channels).
The prior example established the ability of Tbx18 to induce changes in gene/protein expression and function in vitro. Thus, the possibility of reprogramming adult ventricular myocytes into pacemaker cells in vivo was also investigated. An adenoviral vector encoding Tbx18 was directly and focally injected into the apex of guinea pig hearts. In some embodiments, the injection will be to other areas of the heart and sometimes in several areas at once. It shall be appreciated that the guinea pig is an accepted model for use in cardiovascular studies, and data can be readily extrapolated to other mammals, including the human. In some embodiments, the administration of the Tbx18 adenoviral vector will be performed on various mammalian hearts, including that of the human heart.
Two to four days after injection into the guinea pig hearts, the hearts were checked for pacemaker-like signals originating from the site of injection. Upon slowing of the sinus rhythm, none of the seven control (GFP-injected) animals exhibited wide-complex escape rhythms, which would indicate pacemaker function (
The fidelity of reprogramming was tested by expressing Tbx18 in adult guinea pig ventricles and comparing the properties of Tbx18-transduced ventricular myocytes (Tbx18-VMs) with those of GFP-transduced ventricular myocytes (as controls; control-VMs) and native SAN cells. Adenoviruses co-expressing Tbx18 and GFP, or GFP alone, were directly injected in the apex of the guinea pig heart (4×107 cfu/heart). Five days after injection, the heart was harvested and cardiomyocytes were isolated from the site of gene injection.
Native sinoatrial node cells are smaller and leaner than nontransduced ventricular myocytes: length-to-width (LtW) ratios equal 14.7±1.5 (n=24) and 7.6±0.7 (n=9, p<0.05), respectively. Freshly-isolated control-ventricular myocytes maintained their native shape (with LtW=7.4±0.9, n=4); in contrast, Tbx18-ventricular myocytes were leaner (LtW=16.0±1.0, n=12, p<0.05) than control-ventricular myocytes and were often spindle-shaped, reproducing the morphological hallmark of SAN cells. Control-ventricular myocytes had stable resting potentials at −76 mV, with action potentials elicited only upon electrical stimulation. In contrast, Tbx18-VMs demonstrated diastolic depolarization (maximal diastolic potential=−59 mV) and fired spontaneous action potentials at 26 bpm. Thus the Tbx18 transduced cells function more like pacemaker cells. Whole-cell capacitance, a measure of cell size, was smaller in Tbx18-ventricular myocytes vs. control-ventricular myocytes (40.8±3.6 vs. 119±16 pF, respectively). The electrophysiological and morphological features of Tbx18-VMs generally resembled those of native SAN cells (
Tbx18 is a marker of multipotent progenitors in cardiac development, and has been associated with neoplasia. Pluripotent stem cells (embryonic or induced) are known to differentiate spontaneously into pacemaker cells. Thus, to reduce concern with neoplasia. testing was performed to ensure that Tbx18 transduction did not produce pacemaker cells via a pluripotent/neoplastic state, which could occur by accelerating the dedifferentiation known to occur in cultured cardiomyocytes. However, the data presented herein indicate that the SAN-like morphology of Tbx18-VMs is not indicative of nonspecific regression. For example, dedifferentiating adult VMs lose the longitudinal, ‘bricklike’ shape and become rather circular, similar to their neonatal counterparts, but do not become thinner. Further indicating that Tbx18 induces specific re-engineering rather than reversion to a fetal state are the following. First, dedifferentiation is accompanied by re-expression of genes characteristic of the fetal heart, including atrial natriuretic peptide (ANP) and skeletal α-actin (αSkA). Neither ANP nor αSkA was re-expressed in Tbx18-NRVMs (iSAN cells); in fact, ANP expression was strongly suppressed (
From these data it shall be appreciated that, in several embodiments, the quiescent cells will transdifferentiate to iSAN cells without first dedifferentiating to an embryonic/fetal state. Thus, in several embodiments the iSAN cells may exhibit similar morphology to that of native SAN cells consistent with conversion from quiescent cells to iSAN cells without dedifferentiation. Likewise, in several embodiments, expression of ANP and αSkA will be suppressed in the iSAN cells. Additionally, in several embodiments, iSAN cells may exhibit transcript levels of Nkx2.5 similar to that of mature native pacemaker cells. In several embodiments the iSAN cells will remain differentiated without any discernible increase in sternness factors. In several embodiments this transdifferentiation may occur in vitro, while in other embodiments this transdifferentiation may occur in vivo. In several embodiments, the quiescent cells will be mammalian, including several embodiments in which the quiescent cell is human.
Embryonic stem cells can spontaneously differentiate into heterogeneous aggregates of cardiomyocytes with atrial, ventricular, and pacemaker properties. Experiments were thus performed to test whether Embryonic stem cells could be biased from random cardiogenesis toward a dominant pacemaker phenotype. Shox2 is an embryonic transcription factor essential for the patterning of pacemaker cells in the sinoatrial node. Using Shox2 overexpression, the developmental program for the embryonic stem cells was tilted and dominated toward pacemaker myocytes. It will be appreciated that, in several embodiments, Tbx18 could be used instead of or in conjugation with Shox2. In several embodiments, one or more of the following transcription factors could be selected for use: Tbx18, Shox2, Tbx3, and Tbx5.
Mouse embryonic stem cells were cultured on a MEFs feeder layer. Embryoid body formation was cultivated using established hanging drop culture methods. On day 3, cultivation of embryoid bodies was performed. On day 6, plating of embryoid bodies on tissue culture plates was performed (
Endogenous levels of transcription factor RNA were measured at several time points. As shown in
Transient overexpression of Shox2 in embryonic stem cells increased the quantity of spontaneously-beating embryoid bodies by 8-fold (80±27%) compared to control (9±6%)(
Shox2 overexpression singularly biased embryonic stem cell differentiation toward more pacemaker cells, increased expression of NCX1, HCN4, Cx45, and down-regulated Cx43. All of these features are hallmarks of SA nodal cell biology. In several embodiments, one or more of the following transcription factors will be selected for use in differentiating embryonic stem cells: Tbx18, Shox 2, Tbx3, and Tbx5. Further, it will be appreciated that the use of multipotent and/or other pluripotent stem cells will also afford hallmarks of sinatrial nodal cell biology and/or pacemaker function when contact with one or more of the transcription factors disclosed herein. The data provide a novel and efficient platform to develop biological pacemakers from pluripotent cells.
SAN pacemaker cells respond to autonomic inputs with altered firing rates. To assess adrenergic and muscarinic responses in iSAN cells, Tbx18- and GFP-NRVMs were plated on multi-electrode arrays (MEAs) to record extracellular field potentials from spontaneously-beating cells (
A disease model of atrioventricular (AV) block, which is a common indication for the placement of an electronic pacemaker, was created to investigate autonomic regulation of induced biological pacemakers in the intact heart. Electrocardiographic recordings of the beating hearts, perfused ex vivo (
It shall be appreciated that, in several embodiments, Shox2 could be used instead of or in conjugation with Tbx18. In several embodiments, one or more of the following transcription factors could be selected for use: Tbx18, Shox2, Tbx3, and Tbx5. In several embodiments, the iSAN cells may generate ectopic ventricular beats. In several embodiments the Tbx18-NRVM cells (iSAN cells) may respond to autonomic regulation to alter the pacing ectopic beats in a way substantially similar to natural SAN cells. Thus, in several embodiments, iSAN cells will respond to autonomic regulation such as β-adrenergic stimulation and cholinergic suppression in vivo. In several other embodiments, iSAN cells converted from quiescent cells in vitro may be capable of responding to autonomic regulation. In several embodiments, the iSAN cells may generate ectopic beats at a rhythm controlled by autonomic regulation. It shall be appreciated that in several embodiments in which iSAN cells respond to autonomic regulation, the iSAN cells can function to treat a cardiac arrhythmia in a patient suffering from said arrhythmia. In several embodiments, iSAN cells responding to autonomic regulation can replace or supplement electronic pacemaker devices in a patient.
Reprogrammed cells remain altered without sustained transcription factor expression. In order to investigate the persistence of the SAN phenotype, Tbx18-VMs were isolated 6-8 weeks after the initial in vivo gene transfer, and the transcript levels of Tbx18 were quantified by single-cell, quantitative RT-PCR. Tbx18 transcripts exhibited a wide dynamic detection range in Tbx18-VMs isolated just 3 days after in vivo gene transfer (
The persistence of the induced pacemaker phenotype beyond the first few days of transduction was examined using the perfused intact heart AV block model. Three to four weeks after gene transfer, Tbx18-injected hearts demonstrated ectopic idioventricular rhythm at 165±14 bpm (n=3/3,
From this data it shall be appreciated that in several embodiments the iSAN cells will not continue to express Tbx18 after conversion from quiescent cells to pacemaker-like cells. Thus, in several embodiments, it shall be appreciated that a single administration of Tbx18 to the quiescent cells may be sufficient to convert the quiescent cells to iSAN cells and for the iSAN cells to maintain their pacemaker function for a period of at least 6-8 weeks in vivo even if Tbx18 expression wanes in the iSAN cells. In some embodiments, this period during which the converted iSAN cells maintain pacemaker function could be a period of less than 6 weeks. In some embodiments, this period could be a period of more than 8 weeks. In some embodiments, more than one administration of Tbx18 transcript may be administered to the quiescent cells. Furthermore, it shall be appreciated that, in several embodiments, Shox2 could be used instead of or in conjugation with Tbx18. In several embodiments, one or more of the following transcription factors could be selected for use: Tbx18, Shox2, Tbx3, and Tbx5 and/or functional fragments or combinations thereof. In some embodiments, the administration of the transcription factor or transcription factors could occur in vitro. It shall be appreciated that in several embodiments the ability of the iSAN cell to generate an ectopic rhythm after expression of Tbx18 in the iSAN cell has waned indicates that the iSAN cell may be suitable to replace or supplement an electronic pacemaker in a subject with a cardiac arrhythmia or act to treat said cardiac arrhythmia in said subject.
Although the embodiments of the inventions have been disclosed in the context of a certain preferred embodiments and examples, it will be understood by those skilled in the art that the present inventions extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the inventions and obvious modifications and equivalents thereof. In addition, while a number of variations of the inventions have been shown and described in detail, other modifications, which are within the scope of the inventions, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. For all of the embodiments described herein the steps of the methods need not be performed sequentially. Thus, it is intended that the scope of the inventions herein disclosed should not be limited by the particular disclosed embodiments described above.
Homo sapiens T-box 3 (TBX3),
Homo sapiens T-box 3 (TBX3),
Homo sapiens T-box 5 (TBX5),
Homo sapiens T-box 5 (TBX5),
Homo sapiens T-box 5 (TBX5),
Homo sapiens T-box 5 (TBX5),
Homo sapiens T-box 18 (TBX18), mRNA
Homo sapiens short stature homeobox 2
Homo sapiens short stature homeobox 2
Homo sapiens short stature homeobox 2
This application is a division of U.S. application Ser. No. 15/678,973 filed Aug. 16, 2017, which is a continuation of U.S. application Ser. No. 14/357,195, filed May 8, 2014, now U.S. Pat. No. 9,763,999, issued Sep. 19, 2017, which is a National Phase of International Application No. PCT/US2012/064204, filed Nov. 8, 2012, which designated the U.S. and that International Application was published under PCT Article 21(2) in English. This application also includes a claim of priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No. 61/557,812, filed Nov. 9, 2011.
| Number | Date | Country | |
|---|---|---|---|
| 61557812 | Nov 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15678973 | Aug 2017 | US |
| Child | 17194543 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14357195 | May 2014 | US |
| Child | 15678973 | US |
