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WO1999036538A1 - Renforcement du chronotropisme cardiaque - Google Patents

Renforcement du chronotropisme cardiaque Download PDF

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Publication number
WO1999036538A1
WO1999036538A1 PCT/US1999/000732 US9900732W WO9936538A1 WO 1999036538 A1 WO1999036538 A1 WO 1999036538A1 US 9900732 W US9900732 W US 9900732W WO 9936538 A1 WO9936538 A1 WO 9936538A1
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cardiac
gene
pacemaker
expression
heart
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Jay M. Edelberg
Robert D. Rosenberg
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Beth Israel Deaconess Medical Center Inc
Massachusetts Institute of Technology
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Beth Israel Deaconess Medical Center Inc
Massachusetts Institute of Technology
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Priority to AU21147/99A priority Critical patent/AU2114799A/en
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Priority to US09/614,326 priority patent/US6776987B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the natural pacemaker of the mammalian heart is the sinoatrial node (SA node) which is located in the high right atrium, and which comprises specialized pacemaker cells that generate electrical impulses characterized by an intrinsic rhythm.
  • the electrical impulse, or pacemaker potential results from the spontaneous depolarization (a bioelectrical process involving the influx and egress of ions which reduces a membrane potential to a less negative value) of the cardiomyocytes within the SA node. This depolarization spreads from the sinus node through the surrounding atrial tissue and then into the atrial-ventricular node (AV node) before proceeding into the ventricular conduction system.
  • a murine cDNA clironotropic test system was developed to evaluate the effects of expressing the human ⁇ 2 -adrenergic receptor ( ⁇ 2 .AR) in mice under in vitro, ex vivo and finally in vivo conditions.
  • ⁇ 2 AR human ⁇ 2 -adrenergic receptor
  • the ability of ⁇ 2 AR gene therapy to restore the normal function of endogenous cardiac tissue was further evaluated in a direct porcine cardiac gene therapy system.
  • the present invention describes a gene therapy strategy which utilizes localized expression of biological pacemakers to restore the function of the ⁇ -adrenergic signaling cascade. The strategy results in improved cardiac performance and is a useful modality to restore the chronotropic and inotropic responsiveness of dysfunctional or senescent mammalian cardiac tissue.
  • the biological cardiac pacemaker is a molecularly- mediated pacemaker.
  • the molecularly-mediated pacemaker is an expression construct comprising at least one gene encoding a cellular protein which either upregulates heart rate, alters cardiac rhythm, or encodes a receptor protein or signal transduction molecule which is essential to normal physiologic cardiac conductance.
  • the gene, or genes are operably linked to expression control sequences.
  • the expression construct comprising the molecularly-mediated pacemaker can mediate either transient or stable expression.
  • the molecularly-mediated pacemakers can be transiently expressed, and can comprise at least one gene selected from the group consisting of a ⁇ 2 .AR gene, a ⁇ j AR gene, and a G ⁇ s gene.
  • the gene can encode either the endogenous protein or a heterologous protein which is sufficiently homologous to the endogenous protein to possess biological activity in the recipient host cell.
  • the molecularly-mediated pacemaker can comprise at least one gene selected from the above listed group operably linked to expression control sequences suitable for transient expression under the control of a cardiac tissue specific promoter, which can be either constitutive or inducible.
  • the cardiac tissue promoter can be specific for atrial tissue.
  • the invention also encompasses methods of regulating in vivo cardiac pacemaking (chronotropic) activity in a mammal by introducing one of the biologic cardiac pacemakers described herein into the SA node region of an endogenous mammalian heart.
  • the mammal for example be a human.
  • the biological pacemaker is introduced into the heart of the mammal, for example, into the right atrium at a site which is localized to a region surrounding the sinoatrial node.
  • the chronotropic method can employ a molecularly-mediated cardiac biological pacemaker comprising at least one gene that upregulates heart rate or alters cardiac rhythm under the control of expression control elements which mediate either transient expression or stable expression, which is either constitutive or inducible.
  • Cardiac pacemaking activity can also be controlled by a method employing a cellular-based cardiac biological pacemaker comprising at least one myocyte transfected or transduced with at least one gene that upregulates heart rate or alters cardiac rhythm introduced (transplanted or grafted) into the SA node region of the right atria of the recipient host mammal.
  • a cellular-based cardiac biological pacemaker comprising at least one myocyte transfected or transduced with at least one gene that upregulates heart rate or alters cardiac rhythm introduced (transplanted or grafted) into the SA node region of the right atria of the recipient host mammal.
  • the invention also encompasses methods of enhancing the basal heart rate of a mammal by delivery into the mammal of a biological pacemaker comprising exogenous genes which upregulate heart rate or alters cardiac chronotropic or inotropic responsiveness.
  • the invention further encompasses methods of enhancing (upregulating) inotropic responsiveness (cardiac function) of cardiac tissue by utilizing one of the biological cardiac pacemakers described herein to upregulate heart rate or cardiac rhythm.
  • the chronotropic regulatory methods may further employ the in vivo administration of a receptor agonist having a specific cellular affinity for the molecule mediating the chronotropic or inotropic effect.
  • a receptor agonist having a specific cellular affinity for the molecule mediating the chronotropic or inotropic effect.
  • the method could further comprise the systemic or local administration of a cardioselective ⁇ - adrenergic agent such as isoproterenol.
  • Future treatments for chronotropic incompetence may obviate the need for mechanical pacemakers, by employing gene therapy strategies to develop therapeutics (biological pacemakers) which can specifically enhance the pacemaker potential of endogenous cardiac tissue. Therefore, it is useful to provide novel compositions and alternative methods are available to alleviate chronotropic incompetence without the necessity of surgical intervention, and the associated risk of mechanical or electronic failure.
  • Figures 1A and IB are graphs depicting the results of an experiment showing in vitro cardiac myocyte chronotropic recruitment.
  • A. The percentage of cardiac myocytes contracting, in the ⁇ 2 AR transfected cells (black boxes) and control cells (white circles), in the presence of increasing concentrations of isoproterenol (0 - 10 ⁇ 3 M).
  • B. The percentage of cardiac myocytes with a chronotropic rate greater than 60 bpm, in the ⁇ 2 AR transfected (black boxes) and control cells (white circles), in the presence of increasing concentrations of isoproterenol (0 - 10 "3 M).
  • Figure 2 is a graph showing in vitro murine cardiomyocyte chronotropic rates.
  • Figure 3B is a graph displaying the average heart rate of the transplanted hearts pre- and two days postinjection with the ⁇ 2 AR constract (white bars) or the control construct (black bars). * p ⁇ 0.001 ** p ⁇ 0.05.
  • Figure 4B is a graph displaying the average heart rate of murine hearts pre- and two days post injection with the ⁇ 2 AR construct (black bars) or the control construct (white bars). Bar 70 ⁇ m.
  • Figure 5 A are representative surface ECGs recorded 48 hrs after the intracardiac injection of either a control construct (encoding GFP) or a construct encoding B 2 AR.
  • Figure 5 B is a graph summarizing the average cycle lengths pre- and two days post-injection with the control construct (white bars) or the B 2 AR construct (black bars).
  • the physiologic depolarization of the heart originates in the sinus node located in the high right atrium. This depolarization spreads from the sinus node through the surrounding atrial tissue and then into the atrial-ventricular node before proceeding into the ventricular conduction system.
  • the rate of sinus node depolarization results from the spontaneous depolarization of myocytes within the node (DiFrancesco, D., Nature, 324(6096) .470-473 (1986)). These spontaneous cellular depolarizations are automatic, and are, in turn, subject to both sympathetic and parasympathetic regulation. Myocytes from other areas of the heart also depolarize spontaneously, but at physiologic frequencies significantly lower than those of sinus nodal myocytes.
  • the cardiac ⁇ -adrenoreceptor signaling pathway is made up of the ⁇ ,- and ⁇ 2 -adrenoreceptor, which are coexpressed in the myocardium.
  • ⁇ -adrenergic receptors ⁇ -AR
  • ⁇ -AR ⁇ -adrenergic receptors
  • ⁇ -adrenergic receptor ( ⁇ .AR) system plays a major role in cardiac contraction. These signaling pathways involve both G ⁇ s -direct and cAMP-mediated interactions with ion channels involved in myocyte depolarization. Agonist-mediated stimulation of the ⁇ AR activates adenylyl cyclase and triggers the production of cyclic adenosine-3' 5' monophosphate. Stimulation of ⁇ AR increases heart rate as well as cardiac inotropic force.
  • blockade of ⁇ AR decreases heart rate and cardiac contractility.
  • the ⁇ AR-regulated response is also seen in cultured cardiac myocytes which exhibit an increased spontaneous depolarization rate as well as an augmented contractile force.
  • the period of automatic depolarization of the heart is shortened by stimulation of ⁇ .AR in part through an increase in the flux of diastolic depolarization current (I f ) in cardiac myocytes (Guth, B. D., and Dietze, T., Basic Res Cardiol. 90(3): 192-202 (1995)).
  • I f diastolic depolarization current
  • the sinus node has a higher density of ⁇ AR compared with the surrounding atrium (Beau, S. L. et al, Circ Res.
  • the Examples provided herein provide a combination of in vitro, ex vivo, and in vivo gene transfer techniques useful for the to identification and characterization of genes that could be employed to selectively upregulate heart rate and alter cardiac rhythm in an intact heart.
  • the data presented herein demonstrate that the local delivery of expression constructs and/or molecularly engineered cells can increase cardiac pacemaking activity for varying periods of time.
  • Atrial targeting of a transgene may be achieved with previously described atrial specific promoters (Field, L. J., Science, 239(4843): 1029-33 (1988)), and has been employed to overexpress the human beta-1 adrenergic receptor (Bertin, B. et al, Cardiovasc Res., 27(9): 1606-12 (1993)).
  • the atrial specific atrial natriuretic factor promoter was used in the first reported murine transgenic overexpression model of human ⁇ ,AR (Bertin, B. et al, Cardiovasc Res., 27(9):1606-12 (1993)).
  • mice unlike the data from the ⁇ 2 AR transgenic mice, indicated no enhanced chronotropy. This effect was potentially due to pronounced down-regulation of the constitutively overexpressed receptor. Atria derived from these mice were subsequently found to possess enhanced basal function and reduced heart rate variability (Mansier, P. et al, Am. J. Physiol 270: 1465-1472 (1996).
  • inducible elements in concert with the atrial specific promoter may decrease such down-regulation, and may be valuable in the final evaluation of candidate genes, particularly if their expression could specifically be targeted to the sinus node or other critical conduction tissue.
  • Ye et al have recently reported the regulated (rapamycin-inducible) delivery of a therapeutic recombinant protein after in vivo somatic cell gene transfer (Ye, X. et al, Science 283: 88-91 (1999)). Delivery strategies such as these facilitate the stable transduction of cells and allow for the selective induction of the transgene by pharmacologic means.
  • the term "pacemaker” connotes an object or substance that influences the rate at which a particular phenomenon occurs.
  • the relevant phenomenon is the depolarization of the sinoatrial node.
  • heart rate refers to the number of heart beats per minute
  • heart rhythm or “rhythm” refers to the regularity of the heart beat.
  • chronotropy refers to the speed of impulse (electrical signal resulting from sinoatrial node depolarization) formation.
  • the terai “inotropy” refers to the force of cardiac contraction.
  • the term “Cahronotropism” describes the act or process of affecting the regularity of the heart beat (or heart rate).
  • the molecularly-mediated pacemaker can comprise at least one gene selected from the above-identified group operably linked to expression control sequences suitable for transient expression under the control of a cardiac tissue specific promoter, which can be either constitutive or inducible.
  • a cardiac tissue specific promoter can be either constitutive or inducible.
  • the cardiac tissue promoter can be specific for atrial tissue.
  • Transfection or transduction of the cells with an expression construct as described above for the molecularly-mediated embodiments of the invention can be accomplished by a variety of techniques which are well .known to one of skill in the art.
  • the invention further encompasses methods of regulating in vivo cardiac pacemaking (chronotropic) activity in an animal by introducing one of the biologic cardiac pacemakers described herein into the S A node region of an endogenous mammalian heart.
  • the mammal can be for example a human.
  • the biological pacemaker is introduced into the heart, for example, into the right atrium localized to a region surrounding the sinoatrial node.
  • the biological pacemaker composition is preferably delivered in a pharmaceutical composition comprising, for example, the molecularly-mediated expression vector in a volume of phosphate buffered saline with 5% sucrose.
  • a therapeutically effective amount of the biological pacemaker is delivered to a site-specific location (e.g. an area in the upper portion of the right atria).
  • a therapeutically effect amount is that amount which corrects or improves the chronotropic or inotropic defect which characterizes the recipient tissue.
  • the therapeutically effective amount can be delivered preferably in a single administration, although multiple dose are also contemplated.
  • the chronotropic method can employ a molecularly-mediated cardiac biological pacemaker comprising at least one gene that upregulates heart rate or alters cardiac rhythm under the control of expression control elements which mediate transient expression.
  • the biological pacemaker e.g., cDNA, an expression vector or genetically-manipulated cells
  • cDNA an expression vector or genetically-manipulated cells
  • the biological pacemaker can be directly injected into the myocardium in the generalized region of the sinoatrial node via a transthoracic or mini-thoracotomy procedure, or may be delivered by using a electrophysiology recording catheter modified for endocardial transfection of the cardiomyocytes located in the vicinity of the sinoatrial node.
  • the invention also pertains to a cellular-based biological cardiac pacemaker utilizing genetically modified cells (transplanted or grafted) into the S A node region of the right atria of the recipient host mammal.
  • a cellular-based cardiac pacemaker can comprise at least one cell transfected or transduced with at least one gene that upregulates heart rate or alters cardiac rhythm, for example a ⁇ 2 AR gene a ⁇ ,.AR gene or a G ⁇ s gene.
  • Transfection or transduction of the cells with an expression construct as described above for the molecularly-mediated embodiments of the invention can be accomplished by a variety of techniques which are well k . nown to one of skill in the art.
  • the cardiac chronotropy compositions and methods described herein can be used for an individual suffering from cardiac conductive tissue incompetence (arrhythmias) indicative of a underlying disorder of cardiac impulse generation, or to treat an older patient experiencing age-related defects in cardiac performance.
  • the method may be useful in clinical conditions characterized by an abnormal sinus rhythm including but not limited to individuals having sick sinus syndrome, sinus bradycardia, or heartblock.
  • the method may also be useful in treating cardiac conductive disturbances responsible for atrial fibrillation, to the extent that the technique can establish a dominant alternative foci of automatic activity capable of reproducing the normal function of the sinoatrial node. Atrial fibrillation results from disorganized electrical activity in the atria.
  • the disclosed chronotropic methods may also find utility in an individual experiencing a heart attack or transient depression of heart rate.
  • the chronotropic compositions and methods of the present invention can also be used for permanently regulating cardiac pacemaking activity in an animal by introducing a stable cellular-based cardiac pacemaker comprising at least one myocyte transfected or transduced with at least one gene that upregulates heart rate or alters cardiac rhythm, or by introducing a molecularly-mediated cardiac pacemaker which is transcriptionally regulated under the control of an inducible promoter.
  • a stable cellular-based cardiac pacemaker comprising at least one myocyte transfected or transduced with at least one gene that upregulates heart rate or alters cardiac rhythm
  • a molecularly-mediated cardiac pacemaker which is transcriptionally regulated under the control of an inducible promoter.
  • the invention also encompasses methods of enhancing the basal heart rate of a mammal by delivery into the mammal of a biological pacemaker comprising exogenous genes which upregulate heart rate or alters cardiac chronotropic or inotropic responsiveness.
  • the invention further encompasses methods of enhancing (upregulating) inotropic responsiveness of cardiac tissue by utilizing one of the biological cardiac pacemakers described herein to upregulate heart rate or cardiac rhythm.
  • the chronotropic regulatory methods may further employ the in vivo administration of a receptor agonist having a specific cellular affinity for the molecule mediating the chronotropic or inotropic effect.
  • the term "agonist” means a drug that has an affinity for, and whose binding to, a cell surface receptor, triggers a biochemical response which mediates a physiologic activity.
  • the method can further comprise the systemic or local administration of a cardioselective agent, such as a ⁇ -adrenergic agonist, for example isoproterenol.
  • Example 2 transient transfection of cultured myocytes with expression vectors and lipofectamine was employed as the initial screen for assessing candidate genes that upregulate heart rate or alter cardiac rhythm.
  • Example 2 a similar approach was utilized to locally deliver exogenous genes to the intact contracting murine heart transplanted into the mouse ear which permits a rapid appraisal of the action of the candidate gene at the whole organ level that can be used for rapid evaluation of multiple constructs.
  • the exogenous gene was injected into the right atrium of the intact murine heart to determine its effect on heart rate and cardiac rhythm under conditions approaching the situation under which it will be ultimately utilized.
  • Example 4 a porcine cardiac gene transfer system was established to evaluate the use of biological pacemakers in the endogenous heart of a large animal.
  • the Yorkshire pig was specifically chosen for these experiments for its anatomic and physiologic similarity to the human cardiovascular system. Moreover, the porcine model has been successfully been employed in gene therapy studies involving cardiac vasculature. This system also provided an opportunity to develop a transvenous catheter delivery approach that could potentially be employed in human clinical trials.
  • the results obtained with cultured cardiac myocytes transfected with the ⁇ 2 AR suggested that expression of the receptor in cardiac tissue should result in an increased heart rate.
  • the above hypothesis was initially tested in an ex vivo model, described in Example 2.
  • the transplanted neonatal hearts served as an intermediate test in the progression from in vitro to in vivo models of ⁇ 2 AR gene transfer.
  • the subdermally transplanted heart as compared to the native heart, possesses the advantage of being easily accessible which permits injection of constructs under direct observation without the need for complex surgical procedures.
  • ECGs of the transplanted hearts can be recorded from leads attached to the host ear, and are electrically isolated from the host heart which can be utilized as a control.
  • Example 3 ⁇ 2 .AR constructs were injected into the right atrium of native murine hearts and were observed to generate a marked increase in cardiac rate as compared to control plasmids for several days presumably during the expression of the ⁇ 2 AR construct. Similar to observations made with the ex vivo model, minimal changes were noted in the electrocardiograms of ⁇ 2 .AR transfected hearts except for the increased basal rate. The expression of the encoded human ⁇ 2 .AR is confined to the right atrium of the injected hearts, as demonstrated by immunohistochemical analyses which suggests that ⁇ 2 .AR-enhanced stimulation is initiated in the right atrium and then proceeds through the normal conduction system of the heart.
  • Example 4 constructs encoding either the human b 2 adrenergic receptor ( ⁇ 2 AR) or green fluorescent protein (GFP) were injected into the right atrium of native Yorkshire pig hearts.
  • the ⁇ 2 AR construct significantly enhanced chronotropy, as compared to control injections.
  • the average cycle length of the pig heart rate was 567 +/- 100 ms prior to injection.
  • Two days after injection with plasmid encoding the ⁇ 2 AR the cycle length decreased to 327 +/- 60 ms, as compared to the control cycle length 488 +/- 130 ms (p ⁇ .03).
  • the difference in cycle length after control injection was not statistically significant (p > 0.3).
  • EXAMPLE 1 TRANSFECTION OF MURINE MYOCYTES PLASMID CONSTRUCTS: The human ⁇ 2 .AR cDNA was a gift from Dr. Robert J. Leftowitz (Duke).
  • the cells were then plated onto 48-well plates (Falcon Labware, Cockeysville, MD) precoated with 1% gelatin or onto 25 mm 2 square coverslips precoated with 1% gelatin and 20 ⁇ g/ml laminin at a density of 10 5 cell/ml in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal calf serum (FCS), and Streptomycin 100 ⁇ g/ml, and Penicillin (500 ⁇ g/mL).
  • DMEM Dulbecco's modified Eagle's media
  • FCS fetal calf serum
  • Streptomycin 100 ⁇ g/ml
  • Penicillin 500 ⁇ g/mL
  • the percentage of beating myocytes was determined for cells transfected with either the ⁇ 2 AR expression vector or the control construct.
  • the myocytes were identified by GFP as described above.
  • the total percentage of beating cells ( ⁇ 1 contraction/minute) was estimated visually from cotransfected GFP -positive myocytes (> 100 cells/point).
  • the percentage of myocytes that were beating faster than 60 beats per minute (bpm) was determined in identical fashion. Similar measurements of the percentages of both total and fast beating myocytes were conducted at various concentrations of isoproterenol (control, 10 "5 , 10 "4 , and 10 "3 M). Both the total percentage of beating myocytes and those with rates > 60 bpm were used as a measure of automaticity.
  • the average rate of contraction determined by motion detection was higher in the ⁇ 2 .AR transfected myocytes, as compared with control transfected cells under both baseline conditions and in the presence of 10 "3 M isoproterenol.
  • Figure 2 reveals that under control conditions the average rate of contraction of ⁇ 2 AR transfected myocytes was significantly higher as compared with control transfected cells (71 +/- 14 vs. 50 +/- 10 bpm;p ⁇ .001).
  • the average rate of contraction increased by a similar proportion in both populations with the addition of isoproterenol.
  • the transplanted hearts were assayed for visual pulsation and electrocardiographic activity. Visual pulsation of the transplanted tissue was observed in the anesthetized host mice under stereoscopic microscopy. Electrocardiograms (ECGs) of the transplanted hearts were also recorded. Host mice were anesthetized and electrocardiogram limb leads were clipped to the ear surrounding the transplanted heart. ECGs were recorded with a Silogic EC-60 monitor (Silogic Design Limited). Approximately 80% of the transplanted hearts were observed to have visual pulsations and electrocardiographic activity.
  • Transplanted hearts with both visual pulsations and electrocardiographic activity were then employed in DNA injection experiments.
  • expression vectors prepared as described in Example 1 were injected into the atrium of the transplanted hearts similarly as previously described in murine skeletal muscle injection (Wolff, J. A. et al, Science, 247 (4949 Pt 1): 1465-8 (1990)).
  • the ⁇ 2 AR expression vector or the control construct (5 ⁇ l of DNA (2 ⁇ g/ml) in 20% sucrose, 2% Evans Blue, in PBS) were injected into the transplanted hearts with a 33-gauge needle.
  • EXAMPLE 3 INTRACARDIAC DNA INJECTION OF ENDOGENOUS MURINE HEARTS
  • the right atria of 6-week-old adult B6D2F1 murine hearts were injected with expression vectors (which were prepared as described in Example 1).
  • Adult mice were anesthetized with avertin 2.5%, and a baseline ECG was recorded.
  • the heart was exposed as previously described (Selge, H. et al, Angiology, 11:398-407 (1960); Kitsis, R. N. et ⁇ /., Pr c Natl Acad Sci USA., ⁇ °S(10):4138-42 (1991)).
  • mice were then intubated and mechanically ventilated with a rodent ventilator (Model 683, Harvard Apparatus, Inc., South Natick, MA) with room air.
  • a rodent ventilator Model 683, Harvard Apparatus, Inc., South Natick, MA
  • a right anterolateral thoracotomy was then performed and the heart visualized.
  • the ⁇ 2 AR expression vector or the control construct was then introduced into the right atrial wall with a 30-gauge needle, as described above.
  • the lungs were reexpanded and the chest closed in three layers with 4-0 silk sutures.
  • the mice were then allowed to recover spontaneous respiration. Electrocardiographic activity was recorded daily for up to 7 days following the injections. The statistical significance of increased heart rate was determined by a Student's t Test analysis.
  • EXAMPLE 4 MOLECULAR ENHANCEMENT OF PORCINE CARDIAC CHRONTROPY
  • the experiment outlined in this example was directed at developing an in vivo gene transfer technique to identify and study genes that can be employed to selectively upregulate heart rate and alter cardiac rhythm in the intact heart in a large animal model.
  • the Yorkshire pig was chosen for its anatomic and physiologic similarity to the human cardiovascular system, and because porcine models have been successfully employed in other gene therapy studies involving caridac vascularure. Constructs encoding either the human b 2 adrenergic receptor ( ⁇ 2 .AR) or green fluorescent protein were injected into the right atrium of native Yorkshire pig hearts.
  • ⁇ 2 .AR human b 2 adrenergic receptor
  • the average atrial electrocardiogram to surface P wave interval at the injection site was similar in pigs injected with the ⁇ 2 AR and control constructs (14 +/- 10 vs. 12 +/- 10 ms).
  • the average PR interval and P wave axis were similar in the ⁇ 2 .AR- and control-animals, both at baseline and 48 hr post injection.
  • Injection of the ⁇ 2 AR construct significantly enhanced chronotropy, as compared to control injections.
  • the average cycle length of the pig heart rate was 567 +/- 100 ms prior to injection.
  • PLASMID CONSTRUCTS cDNA encoding the human ⁇ 2 AR was the kind gift of Dr. Robert J. Lefkowitz (Duke University Medical Center, Durham NC).
  • a 2.25 kb Sal 1-BamH 1 fragment, the human ⁇ 2 .AR SV40 cDNA was ligated into a Sal 1-BamH 1 site 3' to the ( ⁇ actin promoter) ( ⁇ AP) in a pBR322 vector to generate pBR322- ⁇ AP- ⁇ 2 AR-SV40.
  • the plasmid construct encoding the humanized green fluorescent protein (GFP) with a CMV promoter element was purchased from Clontech, and served as a control vector.
  • the injection vehicle was PBS with 20% sucrose and 2% Evans blue.
  • Electrophysiology recording catheters were custom designed and manufactured by Medtronic, Inc.
  • the polyurethane-coated catheter was 7F in size and was supported with an 8F sheath.
  • the distal end of the catheter was terminated with a 3 l ⁇ turn 33 gauge corkscrew shaped needle allowing it to impale securely onto tissues to record local intracardiac electrograms.
  • the proximal end of the catheter was terminated with a lure lock injection port allowing it to accept standard sized syringes.
  • the total unit had 70 ⁇ L of dead space).
  • the A-P interval (ms), cycle length (ms), PR interval (ms), and P wave axis (°) were measured.
  • the injection needle was rotated 270° into the atrial myocardium.
  • the catheter was then disengaged and removed from the animal. The animal was observed from an additional 10 min and monitored for complications.
  • the vascular sheath was then removed, the vein sutured, and the incision site closed. .Anesthesia was then discontinued. After regaining spontaneous respirations that animals were placed in individual pens. The animals were monitored on an hourly basis for the next three hours, and then daily until the termination of the experiments, 96 hr post injection.
  • SERI. ⁇ SURFACE ELECTROCARDIOGRAM RECORDINGS .AND ANALYSIS Serial surface electrocardiogram were recorded daily on all animals during the duration of the study. The pigs were anesthetized with ketamine as above. Simultaneous 6 lead surface electrocardiograms were recorded. The cycle length, PR interval, and P wave axis were measured. Statistical significance was determined by a Student's t-Test analysis.
  • b 2 .AR and control expression vector injected atria were sectioned to 10 mm sections and fixed with cold methanol for 10 min. The sections were then washed with PBS and blocked with 10% normal serum in PBS for 20 min. Samples were then incubated with rabbit-anti-human b 2 AR polyclonal antibody (Santa Cruz Biotechnologies) at 1.0 mg/mL for 1 lir. Samples were then incubated with the primary antibody at 1.0 mg/mL in PBS with 1% bovine serum albumin for one hr in a humid chamber at 25°C.
  • the animals were anesthetized, intubated, and venous access obtained as described above.
  • the injection catheter was advanced to the right lateral atrium under fluoroscopic guidance. Simultaneous surface and intracardiac electrocardiograms were recorded. The catheter was positioned at the site of the earliest atrial activity.
  • the atrial potential at the injection sight was similar in both the pigs injected with the control (14 +/- 10 ms) and the b 2 AR encoding constructs (12 +/- 10 ms).
  • both the average PR interval and P wave axis on the surface ECG was similar for both groups prior to injection, Table 1. All the animals tolerated the procedure well.
  • the increased heart rate was sustained for 1 to 2 days after which the heart rate trended to baseline levels. All animals survived until the termination of the experiment.
  • these studies demonstrate that the basal rate of the heart can be enhanced by local delivery of exogenous genes.
  • the present example demonstrates that local targeting of gene expression may be a feasible modality to regulate the cardiac pacemaking activity.
  • the porcine model system also provide an experimental basis for developing future human clinical gene transfer protocols designed to upregulate heart rate and alter cardiac rhythm.

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Abstract

La présente invention concerne des régulateurs cardiaques influencés par des récepteurs β-adrénergiques, à médiation moléculaire, et à fondement cellulaire. L'invention concerne également des procédés permettant d'utiliser ces compositions pour améliorer la réactivité cardiaque par une commande d'un débit cardiaque supérieur et une modification du rythme cardiaque.
PCT/US1999/000732 1998-01-13 1999-01-13 Renforcement du chronotropisme cardiaque Ceased WO1999036538A1 (fr)

Priority Applications (2)

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AU21147/99A AU2114799A (en) 1998-01-13 1999-01-13 Enhancement of cardiac chronotropy
US09/614,326 US6776987B1 (en) 1998-01-13 2000-07-12 Enhancement of cardiac chronotropy

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US7145698P 1998-01-13 1998-01-13
US60/071,456 1998-01-13

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WO2006127907A3 (fr) * 2005-05-25 2007-06-07 Massachusetts Inst Technology Administration localisee d'agents inotropes au niveau du coeur
US8562586B2 (en) 2008-08-26 2013-10-22 Massachusetts Institute Of Technology Devices and systems for local delivery of inotropic agents to the epicardium
US8859273B2 (en) 2003-12-24 2014-10-14 Medtronic, Inc. Methods of using HCN genes to treat cardiac arrhythmias

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8859273B2 (en) 2003-12-24 2014-10-14 Medtronic, Inc. Methods of using HCN genes to treat cardiac arrhythmias
US20150139966A1 (en) * 2003-12-24 2015-05-21 Medtronic, Inc. Methods of using hcn genes to treat cardiac arrhythmias
WO2006127907A3 (fr) * 2005-05-25 2007-06-07 Massachusetts Inst Technology Administration localisee d'agents inotropes au niveau du coeur
US8551961B2 (en) 2005-05-25 2013-10-08 Massachusetts Institute Of Technology Localized delivery of cardiac inotropic agents
US8562586B2 (en) 2008-08-26 2013-10-22 Massachusetts Institute Of Technology Devices and systems for local delivery of inotropic agents to the epicardium

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