WO2017029206A1 - Use of k2p potassium channel for altering the electrophysiology of the heart - Google Patents

Abstract

The invention relates to an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium (K_2P) channel or a variant thereof, for use in altering the cardiac electrophysiology in a patient and/or for use in preventing or treating cardiac arrhythmia, atrial dilation, and/or atrial fibrosis, as well as pharmaceutical composition comprising the expression-system, method of altering the cardiac electrophysiology of the heart, and methods of preventing or treating cardiac arrhythmia.

Description

Use of K2P Potassium Channel for Altering the Electrophysiology of the Heart

The invention relates to an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium

channel or a variant thereof, for use in altering the cardiac electrophysiology in a patient and/or for use in preventing or treating cardiac arrhythmia, atrial dilation, and/or atrial fibrosis, as well as pharmaceutical composition comprising the expression-system, method of altering the cardiac electrophysiology of the heart, and methods of preventing or treating cardiac arrhythmia.

Background

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in clinical practice. AF is characterized by atrial repolarization and conduction disturbances that are based on electrical and structural remodeling of the atria. These processes lead to imbalanced electrical properties of atrial cardiomyocytes, resulting in focal ectopic activity and electrical re-entry that contribute to the initiation and perpetuation of AF. The ability of an antiarrhythmic intervention to prevent AF largely depends on its capacity to suppress the underlying mechanisms.

Despite its epidemiological and clinical relevance, effective and safe management of AF still constitutes a major clinical challenge. Successful and safe pharmacologic treatment of AF is a primary yet unmet need in cardiovascular medicine. Patients with AF exhibit largely variable disease characteristics and continue to be at high risk for hospitalizations, heart failure and stroke due to limited effectiveness of unspecific pharmacological or interventional treatment. Antiarrhythmic drug therapy is not tolerated in a significant subset of patients, limited by a relatively high recurrence rate, and may induce life-threatening ventricular arrhythmia owing to ventricular off-target effects. Furthermore, some drugs cause non-cardiac side effects such as hyperthyroidism and fibrosis of the lung. Ablative therapy is effective in suppressing paroxysmal AF in patients without structural heart disease by 50-80%, but successful ablation is more difficult to achieve in chronic AF patients and in cases with concomitant cardiac disease where the effectiveness equals 20-45%. In addition, catheter ablation is associated with the risk of pericardial effusion, stroke, vascular access complications, atrio- esophageal fistula, and phrenic nerve palsy. Patient-tailored therapy is required to improve outcome of AF patients. However, mechanism-based approaches are currently limited by an insufficient understanding of precise molecular remodeling associated with AF. The development of alternatives to current approaches is of high interest in order to establish more effective therapies that increase quality of life and reduce symptoms and hospitalizations.

K⁺ (K₂p) channels facilitate AP repolarization, and regulation of K_2P currents dynamically determines cellular excitability. Cardiac K₂p3.1 (TASK-1, tandem of P domains in a weak inward rectifying K⁺ channel (TWIK) -related acid-sensitive K⁺ channel-1) currents are implicated in AP regulation. TWIK (tandem of P domains in a weak inwardly rectifying K⁺ channel)-related potassium channel 1 (TREK-1) ¾p channels mediate background potassium currents that stabilize the resting membrane potential and contribute to repolarization of action potentials. TREK-1 channels are expressed in human and porcine heart, and atrial TREK-1 downregulation has previously been observed in a pig model of AF.

In search of a novel therapeutics which overcome above described disadvantages, the present inventors sought to suppress AF by specific reversal of K⁺ channel remodeling via targeted K2P channel gene therapy. An important advantage of the provided K2P channel gene therapy is the ability to specifically correct electrical dysfunction at ion channel level within the cardiac area that is responsible for the development of cardiac arrhythmia. Specific targeting of a mechanism leading to arrhythmia by K2P channel gene therapy provides effective control of the heart rhythm while significantly reducing the risk of cardiac and non-cardiac off -target effects that are observed with the use of antiarrhythmic drugs. Finally, the provided K2P channel gene therapy exerts beneficial structural effects on left atrial size and cardiac fibrosis, respectively, which has not been demonstrated with antiarrhythmic drugs or catheter ablation.

Summary of the Invention

In a first aspect, the present invention relates to an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium

channel or a variant thereof, for use in altering the cardiac electrophysiology in a patient.

In a second aspect, the present invention relates to an expression system comprising one or more polynucleotide(s) encoding one or more two-pore -domain-potassium

channel or a variant thereof, for use in preventing or treating cardiac arrhythmia, atrial dilation, and/or atrial fibrosis.

In a third aspect, the present invention relates to a pharmaceutical composition comprising the expression-system comprising one or more polynucleotide(s) encoding one or more two-pore -domain- potassium (K2p) channel or a variant thereof, and a pharmaceutical acceptable carrier and/or excipient.

In a fourth aspect, the present invention relates to a method of altering the cardiac electrophysiology, in particular the electrophysiology of the atrium, sinoatrial and/or atrioventricular node of the heart, comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore -domain-potassium

channel, or a variant thereof.

In a fifth aspect, the present invention relates to an method of preventing or treating cardiac arrhythmia, comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium

channel, or a variant thereof.

List of Figures

Fig. 1: Downregulation of TREK-1 expression in AF patients and in a porcine AF model. (A)

Remodeling of TREK-1 channel mRNA in patients with sinus rhythm (SR; n = 9), paroxysmal atrial fibrillation (pAF; n = 9) and chronic AF (cAF; n = 8). (B) Suppression of TREK-1 transcript levels in sham-treated AF pigs after 14 days of atrial burst pacing (AF sham; n = 5) compared to SR controls (SR sham; n = 5). Data are expressed relative to controls as mean (± SEM) normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). LA, left atrium; RA, right atrium; RAA, right atrial appendage; *P<0.05 versus respective SR subjects

Fig. 2: In vitro efficacy of Ad-TREK-1 gene transfer in HL-1 atrial myocytes. (A) Map of the gene transfer shuttle vector p06-A5-CMV-KCNK2 encoding TREK-1 protein. (B) Channel protein expression was assessed in cells treated with either Ad-TREK-1 (MOI 200) or Ad- control and compared with untreated control cells. Optical density was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (n=3 assays). (C) Typical whole cell TREK-1 currents recorded using the depicted voltage protocol are shown with mean current densities obtained from untreated cells (n = 11) and after Ad-control (Ad-Ctrl., n = 14) or Ad- TREK-1 treatment (n = 15), respectively. *P < 0.05; ***P < 0.001 versus untreated cells.

Fig. 3: Action potential shortening after Ad-TREK-1 gene transfer in vitro in neonatal mouse cardiac myocytes. (A) Representative action potentials (AP) recorded at 1 Hz from neonatal mouse cardiac myocytes under baseline conditions and after application of Ad-TREK-1 for 24h. (B) Corresponding mean AP durations at 50% (APD50) and 90% repolarization (APD90) for controls (n=18) and following TREK-1 gene transfer (n=22). *P<0.05, **P<0.01 versus control conditions. Data are provided as mean+SEM.

Fig. 4: Rhythm control and correction of TREK-1 remodeling following Ad-TREK-1 gene therapy. (A) Prevalence of sinus rhythm (SR) in atrial fibrillation (AF) pigs receiving Ad- TREK-1 therapy (n = 5) compared to AF sham (n = 5) animals. Results from daily rhythm analyses (left) and cumulative SR prevalence during 14-day follow-up (right) are presented. In sham-treated AF animals, the prevalence of SR progressively declined over time, whereas SR was maintained in the majority of pigs receiving Ad-TREK-1. (B) Representative ECG recordings are provided for each animal group as indicated, showing sinus rhythm in SR sham, SR Ad-TREK-1, and AF Ad-TREK-1 animals, respectively. AF is apparent in AF sham animals that did not receive Ad-TREK-1 gene transfer. Note the artifacts induced by atrial burst pacing in AF sham and AF Ad-TREK-1 pigs. (C) Remodeling of TREK-1 protein expression in AF and reconstitution by Ad-TREK-1 treatment. TREK-1 protein levels in right atrial tissue were assessed by Western blot analysis, followed by quantification of optical density normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (n = 5 animals per group). Representative immunoblots of three animals from each study group (left panel) and mean values (right panel) are shown. BL, baseline measurement prior to pacemaker implantation and gene transfer or sham treatment; SR, sinus rhythm; ***P < 0.001 for cumulative follow-up determined by repeated-measures analysis of variance (panel A); *P < 0.05, **P < 0.01, ***P < 0.001 for indicated pair-wise comparisons (panel C).

Fig. 5: Mean heart rates of study pigs. Heart rates were assessed by daily ECG recordings. AF, atrial fibrillation; BL, baseline measurement prior to pacemaker implantation and gene transfer or sham treatment; bpm, beats per minute; SR, sinus rhythm; ***P < 0.001 for cumulative follow-up determined by repeated-measures analysis of variance. Fig. 6: Effects of TREK-1 gene therapy on cardiac electrophysiology in vivo. (A-C) Mean atrial refractory periods (AERP) were determined during electrophysiological study on the day of pacemaker implantation (baseline, BL) and after 14-day follow-up at 300 ms (A), 400 ms (B) and 500 ms (C) basic cycle length, respectively. (D) Mean Wenckebach cycle length assessed in indicated study groups. (E, F) Ventricular effective refractory periods (VERP) measured at baseline prior to study intervention and on the day of sacrifice at 400 ms (E) and 500 ms (F) basic cycle length. AF, atrial fibrillation; SR, sinus rhythm; *P < 0.05, **P < 0.01 versus respective baseline measurements (n = 5 each).

Fig. 7: Changes in left ventricular ejection fraction (LVEF) and left atrial (LA) diameter by rhythm status and study treatment. Echocardiography was performed during sinus rhythm at baseline (BL) and on day 14 in indicated animals to measure LVEF (A) and LA diameter (B). AF, atrial fibrillation; SR, sinus rhythm; *P < 0.05 and **P < 0.01 (n = 5 per group).

Fig. 8: Transcriptional analysis of atrial ion channel subunits in study animals. Changes in mean (±SEM) right atrial appendage mRNA expression levels were normalized to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) and are shown for indicated study groups in relation to sinus rhythm (SR) control pigs not receiving gene therapy. AF, atrial fibrillation; *P<0.05, **P<0.01, ***P<0.001, versus SR sham animals; #P<0.05 versus AF sham pigs. P values were calculated by ANOVA followed by Bonferroni post hoc testing (n=5 per group).

Fig. 9: Stretch- dependent reduction of TREK-1 mRNA expression in vitro. Mechanical stretch was applied to neonatal rat ventricular myocytes for indicated durations. Mean (±SEM)

TREK-1 mRNA expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and are provided in relation to control cells that were not exposed to stretch. *P<0.05, **P<0.01, versus control measurements (n=3 per group).

Fig. 10:Histological assessment of fibrosis and inflammation in study animals. (A) Representative microphotographs obtained from indicated study groups stained using Masson's trichrome or hematoxylin-eosin (HE) reflect fibrotic content and inflammation, respectively, in right atrium (RA), left atrium (LA), and left ventricle (LV). Scale bar, 400 μιη. (B) Quantification of cardiac fibrosis. (C) Quantitative analysis of inflammation according to the scoring system described in the text. AF, atrial fibrillation; SR, sinus rhythm; **P < 0.01, calculated by ANOVA followed by Bonferroni post hoc testing (n = 5 per group).

Fig. llrAtrial expression of type I collagen in study animals. Collagen I protein levels were analyzed by Western blot analysis in right atrial tissue, followed by quantification of optical density normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (n = 5 animals per group). Representative immunoblots of three animals from each study group (upper panel) and mean values (lower panel) are shown. Note the dual immunosignal at -140 kD and -130 kD, reflecting al and a2 chains of type I collagen. AF, atrial fibrillation; SR, sinus rhythm.

Fig. 12:Apoptosis in atrial tissue of study animals. (A) Fluorescence microphotographs obtained from indicated animals, corresponding to terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays in porcine right atrium. Green nuclear fluorescence is indicated by arrows and reflects endonucleolytic DNA degradation and apoptosis. Scale bar, 100 μιη. (B) Mean apoptosis rates. TUNEL-positive cells are expressed in relation to the total number of cells (n = 5 per group).

List of Sequences

SEQ ID NO: 1: Amino acid sequence of TREK- 1 (K₂p2.1);

SEQ ID NO: 2: Nucleotide sequence of KCNK2;

SEQ ID NO: 3: Amino acid sequence of TREK- 1 Variant 1 (K₂p2.1);

SEQ ID NO: 4: Nucleotide sequence of KCNK2 Variant 1 ;

SEQ ID NO: 5: Amino acid sequence of TREK-1 Variant 2 (K₂p2.1);

SEQ ID NO: 6: Nucleotide sequence of KCNK2 Variant 2;

SEQ ID NO: 7: Amino acid sequence of TWIK-1 (K2pl. l);

SEQ ID NO: 8: Nucleotide sequence of KCNK1 ;

SEQ ID NO: 9: Amino acid sequence of TASK- 1 (K2p3.1);

SEQ ID NO: 10: Nucleotide sequence of KCNK3;

SEQ ID NO: 11: Amino acid sequence of TRAAK (K2p4.1);

SEQ ID NO: 12: Nucleotide sequence of KCNK4;

SEQ ID NO: 13: Amino acid sequence of TASK-2 (K2p5.1);

SEQ ID NO: 14: Nucleotide sequence of KCNK5;

SEQ ID NO: 15: Amino acid sequence of TWIK-2 (K2p6.1);

SEQ ID NO: 16: Nucleotide sequence of KCNK6;

SEQ ID NO: 17: Amino acid sequence of TWIK-3 (K2p7.1);

SEQ ID NO: 18: Nucleotide sequence of KCNK7;

SEQ ID NO: 19: Amino acid sequence of TASK-3 (K2p9.1);

SEQ ID NO: 20: Nucleotide sequence of KCNK9;

SEQ ID NO: 21: Amino acid sequence of TREK-2 (K2pl0.1);

SEQ ID NO: 22: Nucleotide sequence of KCNK10;

SEQ ID NO: 23: Amino acid sequence of TREK-2 Variant 1 (K₂p2.1);

SEQ ID NO: 24: Nucleotide sequence of KCNK10 Variant 1 ;

SEQ ID NO: 25: Amino acid sequence of TREK-2 Variant 2 (K₂p2.1);

SEQ ID NO: 26: Nucleotide sequence of KCNK10 Variant 2;

SEQ ID NO: 27: Amino acid sequence of THIK-2 (K₂pl2.1);

SEQ ID NO: 28: Nucleotide sequence of KCNK12;

SEQ ID NO: 29: Amino acid sequence of THIK-1 (K2pl3.1);

SEQ ID NO: 30: Nucleotide sequence of KCNK13;

SEQ ID NO: 31: Amino acid sequence of TASK-5 (K2pl5.1);

SEQ ID NO: 32: Nucleotide sequence of KCNK15;

SEQ ID NO: 33: Amino acid sequence of TALK- 1 (K2pl6.1);

SEQ ID NO: 34: Nucleotide sequence of KCNK16; SEQ ID NO: 35 Amino acid sequence of TALK- 1 Variant 1 (K2pl6.1);

SEQ ID NO: 36 Nucleotide sequence of KCNK16 Variant 1 ;

SEQ ID NO: 37 Amino acid sequence of TALK-2 (also known as TASK-4) (K2pl7.1);

SEQ ID NO: 38 Nucleotide sequence of KCNK17;

SEQ ID NO: 39 Amino acid sequence of TRESK (also known as TRIK) (K2pl8.1);

SEQ ID NO: 40 Nucleotide sequence of KCNK18;

Detailed Description of the Invention

Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being "incorporated by reference ". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise. The term "about" when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value.

The term "expression system" as used herein refers to a system designed to produce one or more gene products of interest. Typically, such system is designed "artificially", i.e. by gene- technological means usable to produce the gene product of interest either in vitro in cell-free systems or in vivo in cell-based systems. It is understood that naturally occurring expression systems such as for instance native viruses are not encompassed by the expression system of the present invention. The term "expression system" further encompasses the expression of the gene product of interest comprising the transcription of the polynucleotides, RNA splicing, translation into a polypeptide, and post-translational modification of a polypeptide or protein.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein. In cell-free expression systems isolated polynucleotides are used as template for in vitro translation reactions. In cell-based expression systems polynucleotides are comprised on one or more vectors.

"Nucleic acid" molecules are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention referred to nucleic acid molecules include but are not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein. The nucleic acids, can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). Aptamers are nucleic acids which bind with high affinity to a polypeptide, here mirl46-a. Aptamers can be isolated by selection methods such as SELEmirl46-a (see e.g. Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981) from a large pool of different single-stranded RNA molecules. Aptamers can also be synthesized and selected in their mirror-image form, for example as the L-ribonucleotide (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). Forms which have been isolated in this way enjoy the advantage that they are not degraded by naturally occurring ribonucleases and, therefore, possess greater stability. Nucleic acids may be degraded by endonucleases or exonucleases, in particular by DNases and RNases which can be found in the cell. It is, therefore, advantageous to modify the nucleic acids in order to stabilize them against degradation, thereby ensuring that a high concentration of the nucleic acid is maintained in the cell over a long period of time (Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94; WO 95/11910; WO 98/37240; WO 97/29116). Typically, such a stabilization can be obtained by introducing one or more internucleotide phosphorus groups or by introducing one or more non-phosphorus internucleotides. Suitable modified internucleotides are compiled in Uhlmann and Peyman (1990), supra (see also Beigelman et al. (1995) Nucleic Acids Res. 23:3989-94; WO 95/11910; WO 98/37240; WO 97/29116). Modified internucleotide phosphate radicals and/or non-phosphorus bridges in a nucleic acid which can be employed in one of the uses according to the invention contain, for example, methyl phosphonate, phosphorothioate, phosphor amidate, phosphorodithioate and/or phosphate esters, whereas non-phosphorus internucleotide analogues contain, for example, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamidate bridges and/or thioether bridges. It is also the intention that this modification should improve the durability of a pharmaceutical composition which can be employed in one of the uses according to the invention. Nucleic acids may be selected from the group consisting of a polynucleotide probe, a primer(s) (e.g. a primer pair), preferably a primer(s) for polymerase chain reaction (PCR), reverse transcription (RT) reaction, or DNA sequencing, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), a microRNA (miRNA), and a small interfering RNA (siRNA).

The term "open reading frame" (ORF) refers to a sequence of nucleotides, that can be translated into amino acids. Typically, such an ORF contains a start codon, a subsequent region usually having a length which is a multiple of 3 nucleotides, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) in the given reading frame. Typically, ORFs occur naturally or are constructed artificially, i.e. by gene -technological means. An ORF codes for a protein where the amino acids into which it can be translated form a peptide-linked chain.

The term "expression level" (of a gene, here e.g. of a K2P channel) refers to the amount of gene product present in the body or a sample at a certain point of time. The expression level can e.g. be measured/quantified/detected by means of the protein or mRNA expressed from the gene. The expression level can for example be quantified by normalizing the amount of gene product of interest (e.g. K2P channel mRNA or protein) present in a sample with the total amount of gene product of the same category (total protein or mRNA) in the same sample or a reference sample (e.g. a sample taken at the same time from the same individual or a part of identical size (weight, volume) of the same sample) or by identifying the amount of gene product of interest per defined sample size (weight, volume, etc.). The expression level can be measured or detected by means of any method as known in the art, e.g. methods for the direct detection and quantification of the gene product of interest (such as mass spectrometry) or methods for the indirect detection and measurement of the gene product of interest that usually work via binding of the gene product of interest with one or more different molecules or detection means (e.g. primer(s), probes, antibodies, protein scaffolds) specific for the gene product of interest, here for a K2P channel. The determination of the level of gene copies of a K2P channel comprising also the determination of the absence or presence of one or more fragments (e.g. via nucleic acid probes or primers, e.g. quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR) is also within the knowledge of the skilled artisan.

The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modification. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes and protein domains) can be further modified by chemical modification. This means such a chemically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to the variants usable in the present invention include without limitation: PEGylation, glycosylation of non-glycosylated parent polypeptides, or the modification of the glycosylation pattern present in the parent polypeptide.

In the context of the different aspects of present invention, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins, but is commonly shorter in length. The shortest peptide is a dipeptide, consisting of two amino acids joined by a single peptide bond. There can also be a tripeptide, tetrapeptide, pentapeptide, etc. Preferably, the peptide has a length of up to 8, 10, 12, 15, 18 or 20 amino acids. A peptide has an amino end and a carboxyl end, unless it is a cyclic peptide.

In the context of the different aspects of present invention, the term "polypeptide" refers to a single linear chain of amino acids bonded together by peptide bonds and preferably comprises at least about 21 amino acids. A polypeptide can be one chain of a protein that is composed of more than one chain or it can be the protein itself if the protein is composed of one chain.

In the context of the different aspects of present invention, the term "protein" refers to a molecule comprising one or more polypeptides that resume a secondary and tertiary structure and additionally refers to a protein that is made up of several polypeptides, i.e. several subunits, forming quaternary structures. The protein has sometimes non-peptide groups attached, which can be called prosthetic groups or cof actors.

As used herein, the term "polyprotein" refers to an amino acid chain that comprises, or essentially consists of or consists of two amino acid chains that are not naturally connected to each other. The polyprotein may comprise one or more further amino acid chains. Each amino acid chain is preferably a complete protein, i.e. spanning an entire ORF, or a fragment, domain or epitope thereof. The individual parts of a polyprotein may either be permanently or temporarily connected to each other. Parts of a polyprotein that are permanently connected are translated from a single ORF and are not later separated co- or post-translationally. Parts of polyproteins that are connected temporarily may also derive from a single ORF but are divided co-translationally due to separation during the translation process or post-translationally due to cleavage of the peptide chain, e.g. by an endopeptidase. Additionally or alternatively, parts of a polyprotein may also be derived from two different ORF and are connected post-translationally, for instance through covalent bonds. Proteins or polyproteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes and protein domains) can be further modified by chemical modification. This means such a chemically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to the variants usable in the present invention include without limitation: PEGylation, glycosylation of non-glycosylated parent polypeptides, or the modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications applicable to the variants usable in the present invention may occur co- or post-translational.

"Tandem-pore-domain potassium channels" form a family of 15 members which are known as "leak channels". These channels are regulated by several mechanisms including oxygen tension, pH, mechanical stretch, and G-proteins. Their name is derived from the fact that the a subunits consist of four transmembrane segments forming two pore loops. As such, they structurally correspond to two inward-rectifier a subunits, and functional channels are thus formed by a subunit dimers in the membrane. The two-pore-domain potassium channel family includes TREK-1 (K2p2.1; NCBI accession numbers: EF165335, KF182338, and KF182339), TWIK-1 (K_2P1.1; NCBI accession number: NM_002245), TASK-1 (K_2P3.1; NCBI accession number: NM_002246), TRAAK (K_2P4.1; NCBI accession number: NMJ 3310), TASK-2 (K_2P5.1; NCBI accession number: NMJX 740), TWIK-2 (K_2P6.1; NCBI accession number: NM_004823), TWIK-3 (K_2P7.1; NCBI accession number: NMJ 3347), TASK-3 (K_2P9.1, NCBI accession number: NM_001282534), TREK-2 (K_2P10.1; NCBI accession numbers: EU978938, EU978939, EU978940), THIK-2 (K_2P12.1; NCBI accession number: NM_022055), THIK-1 (K_2P13.1; NCBI accession number: NM_022054), TASK-5 (K_2P15.1; NCBI accession number: NM_022358), TALK-1 (K_2P16.1; NCBI accession numbers: EU978943, NM_032115), TALK-2 (also known as TASK-4) (K_2P17.1; NCBI accession number: NMJ 1460), and TRESK (also known as TRIK) (K_2P18.1; NCBI accession numbers: NM_181840), as well as its respective variant(s). These proteins are encoded by the genes KCNK2, KCNK1, KCNK3, KCNK4, KCNK5, KCNK6, KCNK7/KCNK8 (both of which encode K_2P7.1), KCNK9, KCNK10, KCNK12, KCNK13, KCNK 15/KCNK 11 /KCNK 14 (all of which encode K_2P15.1, KCNK16, KCNK17, and KCNK18, respectively, as well as its respective variant(s).

Potassium channels form potassium-selective pores that span cell membranes and function to conduct potassium ions down their electrochemical gradient, doing so both rapidly (up to the diffusion rate of K+ ions in bulk water) and selectively (excluding, most notably, sodium despite the sub- angstrom difference in ionic radius). In particular, tandem-pore-domain potassium channel contribute to the resting membrane potential and repolarise action potentials.

As used herein, the term "variant" is to be understood as a polynucleotide or protein which differs in comparison to the polynucleotide or protein from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a protein or nucleic acid variant is derived is also known as the parent polypeptide or polynucleotide. The term "variant" comprises "fragments" or "derivatives" of the parent molecule. Typically, "fragments" are smaller in length or size than the parent molecule, whilst "derivatives" exhibit one or more differences in their sequence in comparison to the parent molecule. Also encompassed modified molecules such as but not limited to post-translationally modified proteins (e.g. glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. Also mixtures of different molecules such as but not limited to RNA-DNA hybrids, are encompassed by the term "variant". Typically, a variant is constructed artificially, preferably by gene-technological means whilst the parent polypeptide or polynucleotide is a wild-type protein or polynucleotide. However, also naturally occurring variants are to be understood to be encompassed by the term "variant" as used herein. Further, the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent molecule, i.e. is functionally active.

As used herein, the term protein or segment "variant" is to be understood as a polypeptide (or segment) which differs in comparison to the polypeptide (or segment, epitope, or domain) from which it is derived by one or more changes in the amino acid sequence. The polypeptide from which a protein variant is derived is also known as the parent polypeptide. Likewise, the segment from which a segment variant is derived from is known as the parent segment. Typically, a variant is constructed artificially, preferably by gene -technological means. Typically, the parent polypeptide is a wild-type protein or wild-type protein domain. In the context of the present invention it is further preferred that a parent polypeptide (or parent segment) is the consensus sequence of two or more wild-type polypeptides (or wild-type segments). Further, the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent polypeptide or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent polypeptide. The changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal truncations, or C-terminal truncations, or any combination of these changes, which may occur at one or several sites. In preferred embodiments, a variant usable in the present invention exhibits a total number of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) changes in the amino acid sequence (i.e. exchanges, insertions, deletions, N-terminal truncations, and/or C-terminal truncations). The amino acid exchanges may be conservative and/or non- conservative. In preferred embodiments, a variant usable in the present invention differs from the protein or domain from which it is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid exchanges, preferably conservative amino acid changes. Alternatively or additionally, a "variant" as used herein, can be characterized by a certain degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 80% sequence identity to its parent polypeptide. A polynucleotide variant in the context of the present invention exhibits at least 80% sequence identity to its parent polynucleotide. Preferably, the sequence identity of protein variants is over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, the sequence identity of polynucleotide variants is over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides.

The term "at least 80% sequence identity" is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide. Preferably, the polypeptide in question and the reference polypeptide exhibit the indicated sequence identity over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids or over the entire length of the reference polypeptide. Preferably, the polynucleotide in question and the reference polynucleotide exhibit the indicated sequence identity over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides or over the entire length of the reference polypeptide.

Variants may additionally or alternatively comprise deletions of amino acids, which may be

N-terminal truncations, C-terminal truncations or internal deletions or any combination of these. Such variants comprising N-terminal truncations, C-terminal truncations and/or internal deletions are referred to as "deletion variant" or "fragments" in the context of the present application. The terms "deletion variant" and "fragment" are used interchangeably herein. A fragment may be naturally occurring (e.g. splice variants) or it may be constructed artificially, preferably by gene -technological means. Preferably, a fragment (or deletion variant) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids at its N-terminus and/or at its C-terminus and/or internally as compared to the parent polypeptide, preferably at its N- terminus, at its N- and C-terminus, or at its C-terminus. In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID, if not specifically indicated otherwise. For example, a peptide sequence consisting of 50 amino acids compared to the amino acid sequence of protein F according to SEQ ID NO: 1 may exhibit a maximum sequence identity percentage of 10.04% (50/498) while a sequence with a length of 249 amino acids may exhibit a maximum sequence identity percentage of 50.00% (249/498). The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on http://www.ebi.ac.uk/Tools/clustalw/ or on http://www.ebi.ac.uk/Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl ?page=/NPSA/npsa_clustalw.html. Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score = 100, word length = 12, to obtain polynucleotide sequences that are homologous to those nucleic acids which encode F, N, or M2-1. BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to the F polypeptide, N polypeptide, or M2-1 polypeptide. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

"Hybridization" can also be used as a measure of sequence identity or homology between two nucleic acid sequences. A nucleic acid sequence encoding F, N, or M2-1, or a portion of any of these can be used as a hybridization probe according to standard hybridization techniques. The hybridization of an F, N, or M2-1 probe to DNA or RNA from a test source is an indication of the presence of the F DNA or RNA, N DNA or RNA, or M2-1 DNA or RNA, respectively, in the test source. Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1-6.3.6, 1991. "Moderate hybridization conditions" are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in IX SSC, 0.1% SDS at 50°C. "Highly stringent conditions" are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45 °C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.

Additionally or alternatively a deletion variant may occur not due to structural deletions of the respective amino acids as described above, but due to these amino acids being inhibited or otherwise not able to fulfill their biological function. Typically, such functional deletion occurs due to the insertions to or exchanges in the amino acid sequence that changes the functional properties of the resultant protein, such as but not limited to alterations in the chemical properties of the resultant protein (i.e. exchange of hydrophobic amino acids to hydrophilic amino acids), alterations in the post- translational modifications of the resultant protein (e.g. post-translational cleavage or glycosylation pattern), or alterations in the secondary or tertiary protein structure. Additionally or alternatively, a functional deletion may also occur due to transcriptional or post-transcriptional gene silencing (e.g. via siRNA) or the presence or absence of inhibitory molecules such as but not limited to protein inhibitors or inhibitory antibodies.

In the context of the present invention it is preferred that a protein (or a segment or a domain or an epitope) being "functionally deleted" refers to the fact that the amino acids or nucleotides of the corresponding sequence are either deleted or present but not fulfilling their biological function.

As used herein, the term "consensus" refers to an amino acid or nucleotide sequence that represents the results of a multiple sequence alignment, wherein related sequences were compared to each other. Such consensus sequence is composed of the amino acids or nucleotides most commonly observed at each position. In the context of the present invention it is preferred that the sequences used in the sequence alignment to obtain the consensus sequence are sequences of different viral subtypes strains isolated in various different disease outbreaks worldwide. Each individual sequence used in the sequence alignment is referred to as the sequence of a particular virus "isolate". A more detailed description of the mathematical methods to obtain such consensus is provided in the Example section. In case that for a given position no "consensus nucleotide" or "consensus amino acid" can be determined, e.g. because only two isolates were compared, than it is preferred that the amino acid of each one of the isolates is used. The resulting protein is assessed for its respective B cell and/or T cell inducing ability.

Semi-conservative and especially conservative amino acid substitutions, wherein an amino acid is substituted with a chemically related amino acid are preferred. Typical substitutions are among the aliphatic amino acids, among the amino acids having aliphatic hydroxyl side chain, among the amino acids having acidic residues, among the amide derivatives, among the amino acids with basic residues, or the amino acids having aromatic residues. Typical semi-conservative and conservative substitutions are:

Amino acid Conservative substitution Semi-conservative substitution

A G; S; T N; V; C

C A; V; L M; I; F; G

D E; N; Q A; S; T; K; R; H

E D; Q; N A; S; T; K; R; H

F W; Y; L; M; H I; V; A

G A S; N; T; D; E; N; Q

H Y; F; K; R L; M; A

I V; L; M; A F; Y; W; G

K R; H D; E; N; Q; S; T; A

L M; I; V; A F; Y; W; H; C

M L; I; V; A F; Y; W; C;

N Q D; E; S; T; A; G; K; R

P V; I L; A; M; W; Y; S; T; C; F

Q N D; E; A; S; T; L; M; K; R

R K; H N; Q; S; T; D; E; A

S A; T; G; N D; E; R; K

T A; S; G; N; V D; E; R; K; I

V A; L; I M; T; C; N

w F; Y; H L; M; I; V; C

Y F; W; H L; M; I; V; C Changing from A, F, H, I, L, M, P, V, W or Y to C is semi-conservative if the new cysteine remains as a free thiol. Furthermore, the skilled person will appreciate that glycines at sterically demanding positions should not be substituted and that P should not be introduced into parts of the protein which have an alpha-helical or a beta-sheet structure.

A "peptide linker" (or short: "linker") in the context of the present invention refers to an amino acid sequence of between 1 and 100 amino acids. In preferred embodiments, a peptide linker according to the present invention has a minimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In further preferred embodiments, a peptide linker according to the present invention has a maximum length of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less. It is preferred that peptide linkers provide flexibility among the two amino acid proteins, fragments, segments, epitopes and/or domains that are linked together. Such flexibility is generally increased if the amino acids are small. Thus, preferably the peptide linker of the present invention has an increased content of small amino acids, in particular of glycins, alanines, serines, threonines, leucines and isoleucines. Preferably, more than 20%, 30%, 40%, 50%, 60% or more of the amino acids of the peptide linker are small amino acids. In a preferred embodiment the amino acids of the linker are selected from glycines and serines. In especially preferred embodiments, the above -indicated preferred minimum and maximum lengths of the peptide linker according to the present invention may be combined, if such a combination makes mathematically sense. In further preferred embodiments, the peptide linker of the present invention is non-immunogenic; in particularly preferred embodiments, the peptide linker is non-immunogenic to humans.

Preferably, peptide linkers have a length between 5 and 40 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34 35, 36, 37, 38, 39, 40 amino acids), more preferably between 5 and 20 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids), most preferably 8 to 15 amino acids (i.e. 8,

9, 10, 11, 12, 13, 14, 15 amino acids). A suitable length allowing for the sterical seperation of fused modules from the HD2 domain or from further connected modules can be selected by the skilled person using routine methodology well-known in the art.

Particularly preferred are flexible peptide linkers. Flexible linkers are composed of amino acids without bulky side chains that impede rotation or bending of the amino acid chain. Flexible linkers preferably comprise G, S, T, and A residues. Preferably at least 50% of the amino acids of the flexible linker peptide consists of amino acids selected from the group consisting of G, S, T, and A. More preferably at least 60%, 70%, 80%, 90%, 95% or 100% of the amino acids of the linker consists of amino acids selected from the group consisting of G, S, T, and A.

The term "cleavage site" as used herein refers to an amino acid sequence or nucleotide sequence where this sequence directs the division, e.g. because it is recognized by a cleaving enzyme, and/or can be divided. Typically, a polypeptide chain is cleaved by hydrolysis of one or more peptide bonds that link the amino acids and a polynucleotide chain is cleaved by hydrolysis of one or more of the phosphodiester bond between the nucleotides. Cleavage of peptide- or phosphodiester-bonds may originate from chemical or enzymatic cleavage. Enzymatic cleavage refers to such cleavage being attained by proteolytic enzymes including but not limited to restriction endonuclease (e.g. type I, type II, type II, type IV or artificial restriction enzymes) and endo- or exo-peptidases or -proteases (e.g. serine-proteases, cysteine-proteases, metallo-proteases, threonine proteases, aspartate proteases, glutamic acid proteases). Typically, enzymatic cleavage occurs due to self-cleavage or is effected by an independent proteolytic enzyme. Enzymatic cleavage of a protein or polypeptide can happen either co- or post-translational. Accordingly, the term "endopeptidase cleavage site" used herein, refers to a cleavage cite within the amino acid or nucleotide sequence where this sequence is cleaved or is cleavable by an endopeptidase (e.g. trypsin, pepsin, elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8, cathepsins). Alternatively, the term "cleavage site" refers to an amino acid sequence or nucleotide sequence that prevents the formation of peptide- or phosphodiester-bonds between amino acids or nucleotides, respectively. For instance, the bond formation may be prevented due to co-translational self -processing of the polypeptide or polyprotein resulting in two discontinuous translation products being derived from a single translation event of a single open reading frame. Typically, such self-processing is effected by a "ribosomal skip" caused by a pseudo stop-codon sequence that induces the translation complex to move from one codon to the next without forming a peptide bond. Examples of sequences inducing a ribosomal skip include but are not limited to viral 2A peptides or 2A-like peptide (herein both are collectively referred to as "2A peptide" or interchangeably as "2A site" or "2A cleavage site") which are used by several families of viruses, including Picornavirus, insect viruses, Aphtoviridae, Rotaviruses and Trypanosoma. Best known are 2A sites of rhinovirus and foot-and-mouth disease virus of the Picornaviridae family which are typically used for producing multiple polypeptides from a single ORF.

Accordingly, the term "self-cleavage site" as used herein refers to a cleavage site within the amino acid or nucleotide sequence where this sequence is cleaved or is cleavable without such cleavage involving any additional molecule or where the peptide- or phosphodiester-bond formation in this sequence is prevented in the first place (e.g. through co-translational self-processing as described above).

It is understood that cleavage sites typically comprise several amino acids or are encoded by several codons (e.g. in those cases, wherein the "cleavage site" is not translated into protein but leads to an interruption of translation). Thus, the cleavage site may also serve the purpose of a peptide linker, i.e. sterically separates two peptides. Thus, in some embodiments a "cleavage site" is both a peptide linker and provides above described cleavage function. In this embodiment the cleavage site may encompass additional N- and/or C-terminal amino acids.

As used herein, the term "vector" refers to a protein or a polynucleotide or a mixture thereof which is capable of being introduced or of introducing the proteins and/or nucleic acid comprised therein into a cell. In the context of the present invention it is preferred that the genes of interest encoded by the introduced polynucleotide are expressed within the cell upon introduction of the vector or vectors. Examples of suitable vectors include but are not limited to viruses, plasmids, cosmids, phages, bacterial spores, or artificial chromosomes.

The term "tissue" as used herein, refers to an ensemble of cells of the same origin which fulfil a specific function concertedly. Examples of a tissue include but are not limited to bone, cartilage, connective tissue, muscle tissue, nervous tissue, and epithelial tissue. Multiple tissues together form an "organ" to carry out a specific function. Examples of an organ include but are not limited to heart, brain, blood, liver, kidney, stomach, joint, skeleton, muscle, and skin.

For example, the "heart" refers to a muscular organ in humans and other animals, which pumps blood through the blood vessels of the circulatory system. In humans, other mammals and birds the heart is divided into four chambers: upper left and right atria; and lower left and right ventricles. Commonly the right atrium and ventricle are referred together as the "right heart" and their left counterparts as the "left heart". Fish in contrast have two chambers, an atrium and a ventricle, while reptiles have three chambers. In a healthy heart blood flows one way through the heart due to heart valves, which prevent backflow. The heart is enclosed in a protective sac, the pericardium, which also contains a small amount of fluid. The wall of the heart is made up of three layers: epicardium, myocardium, and endocardium. Two of the great veins, the venae cavae, and the great arteries, as well as the aorta and the pulmonary artery, are attached to the upper part of the heart, called the base. The four chambers of mammal's and bird's heart comprise the two upper atria, which function as the receiving chambers, and the two lower ventricles, which function as discharging chambers. The atria are connected to the ventricles by the atrioventricular valves and separated from the ventricles by the coronary sulcus. The heart comprises four valves which lie along the same plane. The valves ensure unidirectional blood flow through the heart and prevent backflow. Between the right atrium and the right ventricle is the "tricuspid valve". This consists of three cusps (flaps or leaflets), made of endocardium reinforced with additional connective tissue. Each of the three valve -cusps is attached to several strands of connective tissue, the chordae tendineae (tendinous cords), sometimes referred to as the heart strings. Between the left atrium and left ventricle is the "mitral valve", also known as the bicuspid valve due to its having two cusps, an anterior and a posterior medial cusp. The tricuspid and the mitral valves are the atrioventricular valves. During the relaxation phase of the cardiac cycle, the papillary muscles are also relaxed and the tension on the chordae tendineae is slight. The "semilunar pulmonary valve" is located at the base of the pulmonary artery. This has three cusps, which are not attached to any papillary muscles. When the ventricle relaxes blood flows back into the ventricle from the artery and this flow of blood fills the pocket-like valve, pressing against the cusps which close to seal the valve. The "semilunar aortic valve" is at the base of the aorta and also is not attached to papillary muscles. This too has three cusps which close with the pressure of the blood flowing back from the aorta. The normal rhythmical heart beat, called sinus rhythm, is established by the "sinoatrial node (SA node)", the heart's pacemaker. Here an electrical signal is created that travels through the heart, causing the heart muscle to contract. The sinoatrial node is found in the high right atrium. The electrical signal generated by the sinoatrial node travels through the right atrium in a radial way. It travels to the left atrium via "Bachmann's bundle", such that both left and right atrium contract together. The signal then travels to the "atrioventricular node (AV node)". This is found at the bottom of the right atrium in the interatrial septum-the boundary between the right atrium and the left atrium. The signal then travels along the "Bundle of His" to left and right bundle branches through to the ventricles of the heart. In the ventricles the signal is carried by specialized tissue called the "Purkinje fibers" which then transmit the electric charge to the cardiac muscle.

The terms "tissue status", "status of a tissue", "tissue state" and "state of a tissue" are used interchangeably herein referring to the condition of a tissue. The state of a tissue may be characterised by a specific morphology of such tissue or may be characterised by the expression of one or more specific molecules such as but not limited to peptides, proteins, and nucleic acids, or combinations thereof. The status of a tissue may be regarded as "healthy" or "normal" in case it resembles the condition of such tissue when being free from illness or injury and efficiently fulfilling its specific function. The status of a tissue may be regarded as "degenerative", "diseased" or "abnormal" in case such tissue fails to fulfil its function due to an illness or injury. Additionally or alternatively, the status of a tissue may be regarded as "degenerative", "diseased" or "abnormal" in case the morphology of the tissue or its molecule expression pattern is "altered" or "changed" in comparison to normal tissue. Accordingly, the morphology of a tissue or the expression pattern of specific molecules in a tissue may be an indicator for the state of a tissue. Examples of a tissues status include but are not limited to tissue degradation such as cartilage degradation, bone degradation, and degradation of the synovium, tissue inflammation such as cartilage inflammation, or inflammation of the synovium, tissue remodelling such as bone remodelling or cartilage remodelling, sclerosis, liquid accumulation, or proliferative tissue such as proliferation in wound healing processes, cyst formations, or in cancer.

The term "disease" and "disorder" are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a tissue, an organ or an individual is not able to efficiently fulfil its function anymore. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a tissue, an organ or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease. A progression of a disease is typically characterised by an increase or decrease of such symptoms or signs which may indicate a "worsening" or "bettering" of the disease. The "worsening" of a disease is characterised by a decreasing ability of a tissue, organ or organism to fulfil its function efficiently, whereas the "bettering" of a disease is typically characterised by an increase in the ability of a tissue, an organ or an individual to fulfil its function efficiently. A tissue, an organ or an individual being at "risk of developing" a disease is in a healthy state but shows potential of a disease emerging. Typically, the risk of developing a disease is associated with early or weak signs or symptoms of such disease. In such case, the onset of the disease may still be prevented by treatment. Examples of a disease include but are not limited to traumatic diseases, inflammatory diseases, infectious diseases, cutaneous conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, and various types of cancer. Diseases of the heart include but are not limited to cardiac arrhythmia (e.g. atrial arrhythmia, junctional arrhythmia, ventricular arrhythmia), atrial dilation, atrial fibrosis, atrial fibrillation (AF; such as first detected AF, paroxysmal AF, and chronic AF (cAF) which includes but is not limited to persistent, long-standing persistent, or permanent AF), sinus bradycardia, sinus tachycardia, premature atrial contractions (PACs, wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, supraventricular tachycardia (SVT, atrial flutter, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions (PVCs, accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation.

"Symptoms" of a disease are implication of the disease noticeable by the tissue, organ or organism having such disease and include but are not limited to pain, weakness, tenderness, strain, stiffness, and spasm of the tissue, an organ or an individual. "Signs" or "signals" of a disease include but are not limited to the change or alteration such as the presence, absence, increase or elevation, decrease or decline, of specific indicators such as biomarkers or molecular markers, or the development, presence, or worsening of symptoms.

The term "cardiac electrophysiology" as used herein refers to electrical properties of the heart that underlie cardiac impulse conduction, excitation-contraction coupling, automaticity, and arrhythmogenesis. These include the resting membrane potential and the cardiac action potential and are determined by ion channels, by cardiac structure, and by hemodynamics of the heart.

In particular, the term "transmembrane potassium current" refers to potassium ion movement across the cell membrane and includes but is not limited to background potassium current, calcium- activated potassium current, and voltage-gated potassium current. Transmembrane potassium current may occur through constantly open "leak channels" such as e.g. two-pore -domain potassium channel. Alternatively, transmembrane potassium current may occur in a calcium-dependent manner through calcium-activated potassium channel, or in a voltage dependent manner through voltage-gated potassium channel which open and close in response to calcium or voltage changes, respectively.

The term "transmembrane background potassium current" refers in particular to those potassium ion movement across the cell membrane which occur through constituently open potassium channel such as e.g. two-pore -domain potassium channel. Transmembrane background potassium current are active during the resting membrane potential to stabilize the negative resting membrane potential and counterbalance depolarization. Transmembrane background potassium current also occurs as transmembrane potassium ion movement during action potentials.

The term "cardiac arrhythmia" refers to heart rhythm disorders which include but are not limited to atrial fibrillation (AF; such as first detected AF, paroxysmal AF, and chronic AF (cAF) which includes but is not limited to persistent, long-standing persistent, or permanent AF), sinus bradycardia, sinus tachycardia, premature atrial contractions (PACs, wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, supraventricular tachycardia (SVT, atrial flutter, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions (PVCs, accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation.

In particular, the term "atrial arrhythmia" refers to heart rhythm disorders that originate from or involve the cardiac atrium which includes but is not limited to right atrium, left atrium, right atrial appendage, left atrial appendage, pulmonary veins, coronary sinus, venae cavae, sinoatrial node, atrioventricular node, and the bundle of HIS.

In particular, the term "junctional arrhythmia" refers to heart rhythm disorders that originate from or involve the atrioventricular junction which includes but is not limited to right atrium, left atrium, coronary sinus, bundle of HIS, interatrial septum, and interventricular septum.

In particular, the term "ventricular arrhythmia" refers to heart rhythm disorders that originate from or involve the ventricle which includes but is not limited to right ventricle, right ventricular outflow tract, left ventricle, left ventricular outflow tract, pulmonary valve, pulmonary artery, aortic valve, aorta, coronary sinus, papillary muscle, and Punkinje fibers.

The term "atrial dilation" refers to enlargement of the atrium of the heart, which includes left atrium, right atrium, left atrial appendage, right atrial appendage. Enlargement refers to but is not limited to increase in diameter, volume, tissue volume, number of cells which include but are not limited to cardiac myocytes, cardiac progenitor cells, fibroblasts, myofibroblasts, endothelial cells and adipocytes.

The term "atrial fibrosis" refers to increases in fibrous connective tissue which includes but is not limited to connective tissue deposition, increasing size and number of fibroblasts, increasing size and number of myofibroblasts, and deposition of extracellular matrix proteins.

The term "atrial fibrillation (AF)" refers to a heart rhythm disorder in which the electrical impulses in the atria degenerate from their usual organized rhythm into a rapid chaotic pattern.

In particular, the term "first detected AF" refers to the first episode of AF detected in a given patient.

In particular, the term "paroxysmal AF" refers to patients with spontaneous termination of AF within 7 days of its onset.

In particular, the term "chronic AF (cAF)" refers to persistent, long-standing persistent, or permanent AF. The term "persistent AF" refers to patients with sustained AF beyond 7 days. The term "long-standing persistent AF" refers to patients with uninterrupted AF for more than 1 year. The term "permanent AF" refers to patients in which efforts to restore normal sinus rhythm have either failed or been forgone. These categories are not mutually exclusive.

The term "bradycardia" refers to slow heartbeat usually of below 60 beats per minute.

The term "tachycardia" refers to rapid heartbeat usually of over 100 beats per minute.

The term "sinus bradycardia" refers to sinus rhythm with heart rates below 60 beats per minute.

The term "sinus tachycardia" refers to sinus rhythm with heart rates above 100 beats per minute. The term "premature atrial contractions (PACs)" refers to premature heartbeats originating from the atrium, which includes but is not limited to right atrium, left atrium, pulmonary veins, coronary sinus, venae cavae, sinoatrial node, atrioventricular node, and the bundle of HIS.

The term "wandering atrial pacemaker" refers to cardiac arrhythmia resulting from shifting of the pacemaker site between different regions of the atrium which includes but is not limited to right atrium, left atrium, pulmonary veins, coronary sinus, venae cavae, sinoatrial node, atrioventricular node, and the bundle of HIS.

The term "atrial tachycardia" refers to tachycardia originating from the atrium which includes but is not limited to right atrium, left atrium, pulmonary veins, coronary sinus, venae cavae, sinoatrial node, atrioventricular node, and the bundle of HIS. Atrial tachycardia does not require the atrioventricular (AV) junction, accessory pathways, or ventricular tissue for its initiation and maintenance.

The term "multifocal atrial tachycardia" refers to tachycardia originating from the atrium which includes but is not limited to right atrium, left atrium, pulmonary veins, coronary sinus, venae cavae, sinoatrial node, atrioventricular node, and the bundle of HIS. Multifocal atrial tachycardia is characterized but not limited to two or more different mechanisms which include but are not limited to sites of origin and electrical reentry pathways. Multifocal atrial tachycardia does not require the atrioventricular (AV) junction, accessory pathways, or ventricular tissue for its initiation and maintenance.

The term "supraventricular tachycardia (SVT)" refers to tachycardia that begins in the atrium which includes but is not limited to right atrium, left atrium, right atrial appendage, left atrial appendage, pulmonary veins, coronary sinus, venae cavae, sinoatrial node, atrioventricular node, and the bundle of HIS. SVT includes but is not limited to sinus tachycardia, atrial fibrillation, atrial flutter, atrial tachycardia, multifocal atrial tachycardia, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia and junctional tachycardia.

The term "atrial flutter" refers to supraventricular tachycardia that results from a rapid electrical circuit in the atrium which includes but is not limited to right atrium, left atrium, right atrial appendage, left atrial appendage, pulmonary veins, coronary sinus, venae cavae, sinoatrial node, atrioventricular node, and the bundle of HIS.

The term "AV nodal reentrant tachycardia" refers to supraventricular tachycardia that involves but is not limited to reentrant electrical activation that requires the presence of two ore more AV nodal pathways.

The term "atrioventricular reciprocating tachycardia" refers to supraventricular tachycardia that involves but is not limited to reentrant electrical activation that requires an extra electrical pathway linking the atria and the ventricles of the heart. Atrioventricular reciprocating tachycardia occurs in but is not limited to Wolff-Parkinson-White syndrome.

The term "junctional rhythm" refers to abnormal heart rhythm resulting from impulses coming from an area that involves but is not limited to the right atrium, left atrium, coronary sinus, bundle of HIS, interatrial septum, and interventricular septum. The term "junctional tachycardia" refers to supraventricular tachycardia resulting from impulses coming from the atrioventricular junction that involves but is not limited to the right atrium, left atrium, coronary sinus, bundle of HIS, interatrial septum, and interventricular septum.

The term "premature junctional contraction" refers to premature heartbeats originating from the atrioventricular junction that involves but is not limited to the right atrium, left atrium, coronary sinus, bundle of HIS, interatrial septum, and interventricular septum.

The term "premature ventricular contractions (PVCs)" refers to premature heartbeats originating from the ventricle which includes but is not limited to right ventricle, right ventricular outflow tract, left ventricle, left ventricular outflow tract, pulmonary valve, pulmonary artery, aortic valve, aorta, coronary sinus, papillary muscle, and Punkinje fibers.

The term "accelerated idioventricular rhythm" refers to abnormally high or accelerated heart rhythm resulting from impulses coming from the ventricle which includes but is not limited to right ventricle, right ventricular outflow tract, left ventricle, left ventricular outflow tract, pulmonary valve, pulmonary artery, aortic valve, aorta, coronary sinus, papillary muscle, and Punkinje fibers.

The term "monomorphic ventricular tachycardia" refers to tachycardia that begins in the ventricle which includes but is not limited to right ventricle, right ventricular outflow tract, left ventricle, left ventricular outflow tract, pulmonary valve, pulmonary artery, aortic valve, aorta, coronary sinus, papillary muscle, and Punkinje fibers. In monomorphic ventricular tachycardia, ECG characteristics of ventricular activations are largely similar between heartbeats.

The term "polymorphic ventricular tachycardia" refers to tachycardia that begins in the ventricle which includes but is not limited to right ventricle, right ventricular outflow tract, left ventricle, left ventricular outflow tract, pulmonary valve, pulmonary artery, aortic valve, aorta, coronary sinus, papillary muscle, and Punkinje fibers. In monomorphic ventricular tachycardia, ECG characteristics of ventricular activations differ between heartbeats.

The term "ventricular fibrillation" refers to heart rhythm disorder characterized by rapid, erratic electrical impulses of the ventricle which includes but is not limited to right ventricle, right ventricular outflow tract, left ventricle, left ventricular outflow tract, pulmonary valve, pulmonary artery, aortic valve, aorta, coronary sinus, papillary muscle, and Punkinje fibers, resulting rapid, uncoordinated quivering of the heart.

As used herein, a "patient" means any mammal, reptile or bird that may benefit from the present invention. As used herein, a "healthy subject" means any mammal, reptile or bird which is not afflicted with a disease or disorder, in particular with a disease or disorder which is treated or prevented by the present invention. In particular, the "healthy subject" or the "patient" is selected from the group consisting of laboratory animals (e.g. mouse, rat or rabbit), domestic animals (including e.g. guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, duck, camel, cat, dog, turtle, tortoise, snake, or lizard), or primates including chimpanzees, bonobos, gorillas and human beings. It is particularly preferred that the "healthy subject" or the "patient" is a human being.

The term "sample" or "sample of interest" are used interchangeably herein, referring to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual. Upon analysis a sample provides information about the tissue status or the health or diseased status of an organ or a subject or patient. Examples of samples include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as tissue extracts, cartilage, bone, synovium, perichondrium, capsule, and connective tissue. Further examples of samples are cell cultures or tissue cultures. Analysis of a sample may be accomplished on a visual or chemical basis. Visual analysis includes but is not limited to microscopic imaging or radiographic scanning of a tissue, organ or individual allowing for morphological evaluation of a sample. Chemical analysis includes but is not limited to the detection of the presence or absence of specific indicators or alterations in their amount or level.

The term "control" as used herein refers to a measure allowing for the evaluation of the result obtained when analysing the sample of interest. The results obtained when analysing the sample may be compared to the control in order to assess whether the sample of interest differs from the healthy status of a healthy tissue, organ or subject, or if the sample of interest is identical to or similar to the diseased status of a diseased tissue, organ or patient. The term "control" includes reference samples or reference values.

The term "reference sample" as used herein, refers to a sample which is analysed in a substantially identical manner as the sample of interest and whose information is compared to that of the sample of interest. A reference sample thereby provides a standard allowing for the evaluation of the information obtained from the sample of interest. A reference sample may be derived from a healthy or normal tissue, organ or individual, thereby providing a standard of a healthy status of a tissue, organ or individual. Differences between the status of the normal reference sample and the status of the sample of interest may be indicative of the risk of disease development or the presence or further progression of such disease or disorder. A reference sample may be derived from an abnormal or diseased tissue, organ or individual thereby providing a standard of a diseased status of a tissue, organ or individual. Differences between the status of the abnormal reference sample and the status of the sample of interest may be indicative of a lowered risk of disease development or the absence or bettering of such disease or disorder. A reference sample may also be derived from the same tissue, organ, or individual as the sample of interest but has been taken at an earlier time point. Differences between the status of the earlier taken reference sample and the status of the sample of interest may be indicative of the progression of the disease, i.e. a bettering or worsening of the disease over time. A reference sample was taken at an earlier or later time point in case a period of time has lapsed between taking of the reference sample and taking of the sample of interest. Such period of time may represent years (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 years), months (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months), weeks (e.g. 1, 2, 3, 4, 5, 6, 7, 8 weeks), days (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 days), hours (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours), minutes (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 minutes), or seconds (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 seconds). The terms "lowered" or "decreased" level of an indicator, e.g. a K2P channel, refer to the level of such indicator in the sample being reduced in comparison to the reference or reference sample. The terms "elevated" or "increased" level of an indicator, e.g. a K2p-channel, refer to the level of such indicator in the sample being higher in comparison to the reference or reference sample. E.g. a K2P channel that is detectable in lower amounts in a patient suffering from AF than in a subject not suffering from AF, has a decreased level. For K2P channels, a lower level in a sample may indicate the presence of AF or increased susceptibility or increased probability to develop AF.

As used herein, "treat", "treating" or "treatment" of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in an individual that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in individuals that were previously symptomatic for the disorder(s).

As used herein, "prevent", "preventing", "prevention", or "prophylaxis" of a disease or disorder means preventing that such disease or disorder occurs in patient.

The terms "pharmaceutical", "medicament" and "drug" are used interchangeably herein referring to a substance and/or a combination of substances being used for the identification, prevention or treatment of a tissue status or disease.

"Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "active ingredient" refers to the substance in a pharmaceutical composition or formulation that is biologically active, i.e. that provides pharmaceutical value. A pharmaceutical composition may comprise one or more active ingredients which may act in conjunction with or independently of each other. The active ingredient can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as but not limited to those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The terms "preparation" and "composition" are intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.

The term "carrier", as used herein, refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, or vehicle with which the therapeutically active ingredient is administered. Such pharmaceutical carriers can be liquid or solid. Liquid carrier include but are not limited to sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The term "adjuvant" refers to agents that augment, stimulate, activate, potentiate, or modulate the immune response to the active ingredient of the composition at either the cellular or humoral level, e.g. immunologic adjuvants stimulate the response of the immune system to the actual antigen, but have no immunological effect themselves. Examples of such adjuvants include but are not limited to inorganic adjuvants (e.g. inorganic metal salts such as aluminium phosphate or aluminium hydroxide), organic adjuvants (e.g. saponins or squalene), oil-based adjuvants (e.g. Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g. IL-Ι β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF- γ) particulate adjuvants (e.g. immuno-stimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g. monophosphoryl lipid A, or muramyl peptides), synthetic adjuvants (e.g. non-ionic block copolymers, muramyl peptide analogues, or synthetic lipid A), or synthetic polynucleotides adjuvants (e.g polyarginine or polylysine).

An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art. Embodiments

In a first aspect, the present inventions provides an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium (K2p) channel or a variant thereof, for use in altering the cardiac electrophysiology in a patient. In particular embodiments, the patient has a reduced expression level of said one or more K2P channel in comparison to a control. In particular, the patient has a reduced expression level of said one or more K2P channel in comparison to the expression level in a healthy subject, in particular in a subject not afflicted with a reduced expression level of said one or more K2P channel, in particular in a subject not afflicted with any heart disease.

In particular embodiments, the patient has a reduced expression level of said one or more K2P channel in comparison to a value representing the average expression level in a healthy subject, in particular in a subject not afflicted with a reduced expression level of said one or more K2P channel, in particular in a subject not afflicted with any heart disease.

In further embodiments, the cardiac electrophysiology is altered by modulating the transmembrane potassium current, preferably the transmembrane background potassium current. In particular, the transmembrane potassium current is increased. In particular, the transmembrane background potassium current is altered, in particular increased.

In a second aspect, the present inventions provides an expression system comprising one or more polynucleotide(s) encoding one or more two-pore -domain-potassium (K₂p) channel or a variant thereof, for use in preventing or treating cardiac arrhythmia, atrial dilation, and/or atrial fibrosis.

In embodiments of the second aspect, the cardiac arrhythmia is selected from the group consisting of atrial, junctional and ventricular arrhythmia. In particular, the cardiac arrhythmia is selected from the group consisting of atrial fibrillation (AF), sinus bradycardia, sinus tachycardia, premature atrial contractions (PACs), wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, supraventricular tachycardia (SVT), atrial flutter, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions (PVCs), accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation.

In particular embodiments, the AF is selected from the group consisting of first detected, paroxysmal, or chronic AF (cAF). In particular embodiments, chronic AF is persistent, long-standing persistent, or permanent AF.

In further embodiments, the atrial fibrosis is selected from the group of fibrosis of the atria, fibrosis of the sinus node (SA node), and fibrosis of the atrioventricular node (AV node).

In particular embodiments of the second aspect, the expression system does not comprise a polynucleotide encoding the K_2P channel K₂p3.1 in case the cardiac arrhythmia is a chronic atrial fibrillation and the patient shows normal cardiac function. In embodiments of the first or second aspect of the present invention, the one or more two- pore-domain-potassium (K₂p) channel(s) are selected from the group consisting of TREK-1 (K₂p2.1), TWIK-1 (K₂pl.l), TASK-1 (K_2P3.1), TRAAK (K_2P4.1), TASK-2 (K_2P5.1), TWIK-2 (K_2P6.1), TWIK-3 (K_2P7.1), TASK-3 (K_2P9.1), TREK-2 (K_2P10.1), THIK-2 (K_2P12.1), THIK-1 (K_2P13.1), TASK-5 (K_2P15.1), TALK-1 (K_2P16.1), TALK-2 (also known as TASK-4) (K_2P17.1), and TRESK (also known as TRIK) (K₂pl 8.1), and variants thereof. In particular embodiments, the variant is a functional variant of the respective K₂p-channel, i.e. is able to fulfil the same function as the respective K₂p-channel.

In particular embodiments of the first or second aspect of the present invention, the one or more K₂p channel(s) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39. In particular embodiments, the K_2P channel(s) comprises or consists of an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In particular embodiments, the variant exhibits an amino acid sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference sequence.

In particular embodiments of the first or second aspect, the polynucleotide comprises one or more of gene sequences selected from the group consisting of KCNK2, KCNK1 , KCNK3, KCNK4, KCNK5, KCNK6, KCNK7, KCNK8, KCNK9, KCNK10, KCNK11 , KCNK12, KCNK13, KCNK14, KCNK15, KCNK16, KCNK17, and KCNK18, and a variant thereof.

In particular embodiments of the first or second aspect of the present invention, the polynucleotide comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40. In particular embodiments, the polynucleotide comprises or consists of a nucleotide sequence according to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In particular embodiments, the variant exhibits a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference sequence.

In embodiments of the first or second aspect of the present invention, wherein the expression system comprises more than one, i.e. two or more, polynucleotide(s) encoding two-pore -domain- potassium (K₂p) channel, the two or more polynucleotides encode identical or different K₂p-channel. In particular embodiments, the two or more polynucleotides encode two or more different K₂p-channels.

In embodiments of the first or second aspect of the present invention, wherein the expression system comprises more than one, i.e. two or more, polynucleotide(s) encoding two or more two-pore- domain-potassium

channel, the two or more polynucleotides may be present in the expression system in two or more separate open-reading frames or in a single open-reading frame. In embodiments, wherein the two or more polynucleotides are present in a single open-reading frame, the two or more polynucleotides are connected via a nucleotide sequence encoding a linker, in particular, a nucleotide sequence encoding a peptide linker which sterically separate the different K_2P channels.

In particular embodiments, the peptide linkers have a length between 5 and 40 amino acids

(i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 30, 31 , 32, 33, 34 35, 36, 37, 38, 39, 40 amino acids), more preferably between 5 and 20 amino acids (i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids), most preferably 8 to 15 amino acids (i.e. 8, 9, 10, 11 , 12, 13, 14, 15 amino acids). In particular embodiments, the linker is a flexible peptide linkers. Flexible linkers are composed of amino acids without bulky side chains that impede rotation or bending of the amino acid chain. Flexible linkers preferably comprise G, S, T, and A residues. Preferably at least 50% of the amino acids of the flexible linker peptide consists of amino acids selected from the group consisting of G, S, T, and A. More preferably at least 60%, 70%, 80%, 90%, 95% or 100% of the amino acids of the linker consists of amino acids selected from the group consisting of G, S, T, and A.

In further embodiments, the linker comprises one or more cleavage sites, i.e. one or more sequence areas wherein the linker sequence may be chemically or enzymatically cleaved by division of one or more peptide -bonds. Enzymatic cleavage may be attained by proteolytic enzymes including but not limited to restriction endonuclease (e.g. type I, type II, type II, type IV or artificial restriction enzymes) and endo- or exo-peptidases or -proteases (e.g. serine -proteases, cysteine -proteases, metallo- proteases, threonine proteases, aspartate proteases, glutamic acid proteases). In particular embodiments, the one or more cleavage sites comprise one or more endopeptidase cleavage sites, i.e. wherein the sequence is cleaved or is cleavable by an endopeptidase such as but not limited to trypsin, pepsin, elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8, and/or cathepsins. In further embodiments, the one or more cleavage sites comprise one or more self-cleavage sites.

In particular embodiments of the first or second aspect of the present invention, the expression system further comprises regulatory elements. In particular embodiments, the regulatory elements are selected from the group consisting of promoter, enhance, silencer, and Rheo-Switch.

In particular embodiments, the promoter is selected from the group consisting of a CMV promoter, a ANF promoter, a ALC-1 promoter, a MLC-2v promoter, and a v-MHC promoter.

In particular embodiments of the first or second aspect of the present invention, the expression system is a viral vectors, plasmid vectors, cosmid vectors, phage vectors, or bacterial spores.

In particular embodiments, the expression system is a viral vectors selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpes virus vectors, measles virus vectors, pox virus vectors, vesicular stomatitis virus vectors, retrovirus vectors, lentivirus vectors, and viral like particles.

In particular embodiments, the adenovirus vector is an Ad5 adenovirus vector. In particular embodiments, the AVV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, and AAV 12.

In a third aspect, the present invention provides a pharmaceutical composition comprising the expression-system comprising one or more polynucleotide(s) encoding one or more two-pore-domain- potassium (K2p) channel or a variant thereof, and a pharmaceutical acceptable carrier and/or excipient.

In particular embodiments, the pharmaceutical of the third aspect comprises a further ingredient. In particular, the additional ingredient is selected from the group consisting of an adjuvant and an active ingredient.

In particular embodiments of the third aspect, the pharmaceutical is for use in preventing or treating cardiac arrhythmia, atrial dilation, and/or atrial fibrosis. In embodiments of the third aspect, the cardiac arrhythmia is selected from the group consisting of atrial, junctional and ventricular arrhythmia. In particular, the cardiac arrhythmia is selected from the group consisting of atrial fibrillation (AF), sinus bradycardia, sinus tachycardia, premature atrial contractions (PACs), wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, supraventricular tachycardia (SVT), atrial flutter, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions (PVCs), accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation. In particular embodiments, the AF is selected from the group consisting of first detected, paroxysmal, or chronic AF (cAF). In particular embodiments, chronic AF is persistent, long-standing persistent, or permanent AF. In further embodiments, the atrial fibrosis is selected from the group of fibrosis of the atria, fibrosis of the sinus node (SA node), and fibrosis of the atrioventricular node (AV node).

In particular embodiments of the third aspect, the expression system does not comprise a polynucleotide encoding the K2P channel K2p3.1 in case the cardiac arrhythmia is a chronic atrial fibrillation and the patient shows normal cardiac function.

In embodiments of the third aspect of the present invention, the one or more two-pore -domain- potassium (K₂p) channel(s) are selected from the group consisting of TREK-1 (K₂p2.1), TWIK-1 (K₂pl.l), TASK-1 (K_2P3.1), TRAAK (K_2P4.1), TASK-2 (K_2P5.1), TWIK-2 (K_2P6.1), TWIK-3 (K_2P7.1), TASK-3 (K_2P9.1), TREK-2 (K_2P10.1), THIK-2 (K_2P12.1), THIK-1 (K_2P13.1), TASK-5 (K_2P15.1), TALK-1 (K_2P16.1), TALK-2 (also known as TASK-4) (K_2P17.1), and TRESK (also known as TRIK) (K₂pl8.1), and variants thereof. In particular embodiments, the variant is a functional variant of the respective K₂p channel, i.e. is able to fulfil the same function as the respective K₂p channel.

In particular embodiments of the third aspect of the present invention, the one or more K₂p channel(s) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39. In particular embodiments, the K₂p channel(s) comprises or consists of an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In particular embodiments, the variant exhibits an amino acid sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference sequence.

In particular embodiments of the third aspect, the polynucleotide comprises one or more of gene sequences selected from the group consisting of KCNK2, KCNK1, KCNK3, KCNK4, KCNK5, KCNK6, KCNK7, KCNK8, KCNK9, KCNK10, KCNK11, KCNK12, KCNK13, KCNK14, KCNK15, KCNK16, KCNK17, and KCNK18, and a variant thereof.

In particular embodiments of the third aspect of the present invention, the polynucleotide comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40. In particular embodiments, the polynucleotide comprises or consists of a nucleotide sequence according to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In particular embodiments, the variant exhibits a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference sequence.

In embodiments of the third aspect of the present invention, wherein the expression system comprises more than one, i.e. two or more, polynucleotide(s) encoding two-pore -domain-potassium (K2p) channel, the two or more polynucleotides encode identical or different K2P channel. In particular embodiments, the two or more polynucleotides encode two or more different K2P channels.

In embodiments of the third aspect of the present invention, wherein the expression system comprises more than one, i.e. two or more, polynucleotide(s) encoding two or more two-pore -domain- potassium (K2p) channel, the two or more polynucleotides may be present in the expression system in two or more separate open-reading frames or in a single open-reading frame. In embodiments, wherein the two or more polynucleotides are present in a single open-reading frame, the two or more polynucleotides are connected via a nucleotide sequence encoding a linker, in particular, a nucleotide sequence encoding a peptide linker which sterically separate the different K2P channels.

In particular embodiments, the peptide linkers have a length between 5 and 40 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34 35, 36, 37, 38, 39, 40 amino acids), more preferably between 5 and 20 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids), most preferably 8 to 15 amino acids (i.e. 8, 9, 10, 11, 12, 13, 14, 15 amino acids). In particular embodiments, the linker is a flexible peptide linkers. Flexible linkers are composed of amino acids without bulky side chains that impede rotation or bending of the amino acid chain. Flexible linkers preferably comprise G, S, T, and A residues. Preferably at least 50% of the amino acids of the flexible linker peptide consists of amino acids selected from the group consisting of G, S, T, and A. More preferably at least 60%, 70%, 80%, 90%, 95% or 100% of the amino acids of the linker consists of amino acids selected from the group consisting of G, S, T, and A.

In further embodiments, the linker comprises one or more cleavage sites, i.e. one or more sequence areas wherein the linker sequence may be chemically or enzymatically cleaved by division of one or more peptide -bonds. Enzymatic cleavage may be attained by proteolytic enzymes including but not limited to restriction endonuclease (e.g. type I, type II, type II, type IV or artificial restriction enzymes) and endo- or exo-peptidases or -proteases (e.g. serine -proteases, cysteine -proteases, metallo- proteases, threonine proteases, aspartate proteases, glutamic acid proteases). In particular embodiments, the one or more cleavage sites comprise one or more endopeptidase cleavage sites, i.e. wherein the sequence is cleaved or is cleavable by an endopeptidase such as but not limited to trypsin, pepsin, elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8, and/or cathepsins. In further embodiments, the one or more cleavage sites comprise one or more self-cleavage sites.

In particular embodiments of the third aspect of the present invention, the expression system further comprises regulatory elements. In particular embodiments, the regulatory elements are selected from the group consisting of promoter, enhance, silencer, and Rheo-Switch. In particular embodiments, the promoter is selected from the group consisting of a CMV promoter, a ANF promoter, a ALC-1 promoter, a MLC-2v promoter, and a v-MHC promoter.

In particular embodiments of the third aspect of the present invention, the expression system is a viral vectors, plasmid vectors, cosmid vectors, phage vectors, or bacterial spores. In particular embodiments, the expression system is a viral vectors selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpes virus vectors, measles virus vectors, pox virus vectors, vesicular stomatitis virus vectors, retrovirus vectors, lentivirus vectors, and viral like particles. In particular embodiments, the adenovirus vector is an Ad5 adenovirus vector. In particular embodiments, the AVV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV 12.

In a fourth aspect, the present invention provides a method of altering the cardiac electrophysiology comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium

channel, or a variant thereof.

In particular embodiments of the fourth aspect, the electrophysiology of the atrium, sinoatrial and/or atrioventricular node of the heart is altered.

In further embodiments, the cardiac electrophysiology is altered by modulating the transmembrane potassium current, in particular the transmembrane background potassium current. In particular, the transmembrane potassium current is increased. In particular, the transmembrane background potassium current is altered, in particular increased.

In particular embodiments, the cardiac electrophysiology is altered in a patient, in particular in a patient with a reduced expression level of said one or more K2P channel in comparison to a control. In particular, the patient has a reduced expression level of said one or more K2P channel in comparison to the expression level in a healthy subject, in particular in a subject not afflicted with a reduced expression level of said one or more K2P channel, in particular in a subject not afflicted with any heart disease.

In particular embodiments, the patient has a reduced expression level of said one or more K2P channel in comparison to a value representing the average expression level in a healthy subject, in particular in a subject not afflicted with a reduced expression level of said one or more K2P channel, in particular in a subject not afflicted with any heart disease.

In particular embodiments, the expression system is administered locally or systemically. In embodiments, the expression system is administered locally by direct injection or via an endocardial catheter.

In further embodiments, the expression system is administered systemically through the oral, intravenous, intramucosal, intraarterial, intramusculuar or intracoronal route.

In embodiments, wherein the expression system is administered systemically, the expression system comprises a heart-specific promotor. In particular embodiments, the heart-specific promotor is an atrial or ventricular-specific promotor. In particular embodiments the heart-specific promotor is selected from the group consisting of atrial natriuretic factor (ANF), atrial myosin light chain 1 (ALC- 1), myosin light chain 2v (MLC-2v), and ventricular myosin heavy chain (v-MHC). In a fifth aspect, the present invention provides a method of preventing or treating cardiac arrhythmia, comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium

channel, or a variant thereof.

In embodiments of the fifth aspect, the cardiac arrhythmia is selected from the group consisting of atrial, junctional and ventricular arrhythmia. In particular, the cardiac arrhythmia is selected from the group consisting of atrial fibrillation (AF), sinus bradycardia, sinus tachycardia, premature atrial contractions (PACs), wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, supraventricular tachycardia (SVT), atrial flutter, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions (PVCs), accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation.

In particular embodiments, the AF is selected from the group consisting of first detected, paroxysmal, or chronic AF (cAF). In particular embodiments, chronic AF is persistent, long-standing persistent, or permanent AF.

In further embodiments, the atrial fibrosis is selected from the group of fibrosis of the atria, fibrosis of the sinus node (SA node), and fibrosis of the atrioventricular node (AV node).

In particular embodiments of the fifth aspect, the expression system does not comprise a polynucleotide encoding the K_2P channel K₂p3.1 in case the cardiac arrhythmia is a chronic atrial fibrillation and the patient shows normal cardiac function.

In particular embodiments, the expression system is administered locally or systemically. In embodiments, the expression system is administered locally by direct injection or via an endocardial catheter.

In further embodiments, the expression system is administered systemically through the oral, intravenous, intramucosal, intraarterial, intramusculuar or intracoronal route.

In embodiments, wherein the expression system is administered systemically, the expression system comprises a heart-specific promotor. In particular embodiments, the heart-specific promotor is an atrial or ventricular-specific promotor. In particular embodiments the heart-specific promotor is selected from the group consisting of atrial natriuretic factor (ANF), atrial myosin light chain 1 (ALC- 1), myosin light chain 2v (MLC-2v), and ventricular myosin heavy chain (v-MHC).

In embodiments of the fourth or fifth aspect of the present invention, the one or more two- pore -domain-potassium (K₂p) channel(s) are selected from the group consisting of TREK-1 (K_2P 2.1), TWIK-1 (K₂pl.l), TASK-1 (K_2P3.1), TRAAK (K_2P4.1), TASK-2 (K_2P5.1), TWIK-2 (K_2P6.1), TWIK-3 (K_2P7.1), TASK-3 (K_2P9.1), TREK-2 (K_2P10.1), THIK-2 (K_2P12.1), THIK-1 (K_2P13.1), TASK-5 (Κ_2Ρ15.1), TALK-1 (Κ_2Ρ16.1), TALK-2 (also known as TASK-4) (K_2P17.1), and TRESK (also known as TRIK) (K₂pl 8.1), and variants thereof. In particular embodiments, the variant is a functional variant of the respective K₂p channel, i.e. is able to fulfil the same function as the respective K₂p channel.

In particular embodiments of the fourth or fifth aspect of the present invention, the one or more K₂p channel(s) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39. In particular embodiments, the K_2P channel(s) comprises or consists of an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In particular embodiments, the variant exhibits an amino acid sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference sequence.

In particular embodiments of the fourth or fifth aspect, the polynucleotide comprises one or more of gene sequences selected from the group consisting of KCNK2, KCNK1, KCNK3, KCNK4, KCNK5, KCNK6, KCNK7, KCNK8, KCNK9, KCNK10, KCNK11, KCNK12, KCNK13, KCNK14, KCNK15, KCNK16, KCNK17, and KCNK18, and a variant thereof.

In particular embodiments of the fourth or fifth aspect of the present invention, the polynucleotide comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40. In particular embodiments, the polynucleotide comprises or consists of a nucleotide sequence according to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In particular embodiments, the variant exhibits a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference sequence.

In embodiments of the fourth or fifth aspect of the present invention, wherein the expression system comprises more than one, i.e. two or more, polynucleotide(s) encoding two or more two-pore- domain-potassium (K₂p) channel, the two or more polynucleotides may be present in the expression system in two or more separate open-reading frames or in a single open-reading frame. In embodiments, wherein the two or more polynucleotides are present in a single open-reading frame, the two or more polynucleotides are connected via a nucleotide sequence encoding a linker, in particular, a nucleotide sequence encoding a peptide linker which sterically separate the different K₂p channels.

In particular embodiments, the peptide linkers have a length between 5 and 40 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34 35, 36, 37, 38, 39, 40 amino acids), more preferably between 5 and 20 amino acids (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids), most preferably 8 to 15 amino acids (i.e. 8, 9, 10, 11, 12, 13, 14, 15 amino acids). In particular embodiments, the linker is a flexible peptide linkers. Flexible linkers are composed of amino acids without bulky side chains that impede rotation or bending of the amino acid chain. Flexible linkers preferably comprise G, S, T, and A residues. Preferably at least 50% of the amino acids of the flexible linker peptide consists of amino acids selected from the group consisting of G, S, T, and A. More preferably at least 60%, 70%, 80%, 90%, 95% or 100% of the amino acids of the linker consists of amino acids selected from the group consisting of G, S, T, and A.

In further embodiments, the linker comprises one or more cleavage sites, i.e. one or more sequence areas wherein the linker sequence may be chemically or enzymatically cleaved by division of one or more peptide -bonds. Enzymatic cleavage may be attained by proteolytic enzymes including but not limited to restriction endonuclease (e.g. type I, type II, type II, type IV or artificial restriction enzymes) and endo- or exo-peptidases or -proteases (e.g. serine -proteases, cysteine -proteases, metallo- proteases, threonine proteases, aspartate proteases, glutamic acid proteases). In particular embodiments, the one or more cleavage sites comprise one or more endopeptidase cleavage sites, i.e. wherein the sequence is cleaved or is cleavable by an endopeptidase such as but not limited to trypsin, pepsin, elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8, and/or cathepsins. In further embodiments, the one or more cleavage sites comprise one or more self-cleavage sites.

In particular embodiments of the fourth or fifth aspect of the present invention, the expression system further comprises regulatory elements. In particular embodiments, the regulatory elements are selected from the group consisting of promoter, enhance, silencer, and Rheo-Switch.

In particular embodiments, the promoter is selected from the group consisting of a CMV promoter, a ANF promoter, a ALC-1 promoter, a MLC-2v promoter, and a v-MHC promoter.

In particular embodiments of the fourth or fifth aspect of the present invention, the expression system is a viral vectors, plasmid vectors, cosmid vectors, phage vectors, or bacterial spores.

In particular embodiments, the expression system is a viral vectors selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpes virus vectors, measles virus vectors, pox virus vectors, vesicular stomatitis virus vectors, retrovirus vectors, lentivirus vectors, and viral like particles.

In particular embodiments, the adenovirus vector is an Ad5 adenovirus vector. In particular embodiments, the AVV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, and AAV 12.

Further aspects of the present invention are the following:

1. An expression system comprising one or more polynucleotide(s) encoding one or more two- pore -domain-potassium (K2p) channel or a variant thereof, for use in altering the cardiac electrophysiology in a patient. The expression system of aspect 1 , wherein the patient has a reduced expression level of said one or more K_2P channel in comparison to a control, in particular in comparison to the expression level in a healthy subject or in comparison to a value representing the average expression level in a healthy subject.

The expression system of aspect 1 or 2, wherein the cardiac electrophysiology is altered by modulating, preferably increasing, the transmembrane potassium current, preferably the transmembrane background potassium current.

An expression system comprising one or more polynucleotide(s) encoding one or more two- pore -domain-potassium (K2p) channel or a variant thereof, for use in preventing or treating cardiac arrhythmia, atrial dilation, and/or atrial fibrosis.

The expression system of aspect 4, wherein the cardiac arrhythmia is selected from the group consisting of atrial, junctional and ventricular arrhythmia.

The expression system of aspect 4 or 5, wherein the cardiac arrhythmia is selected from the group consisting of atrial fibrillation (AF) (in particular first detected, paroxysmal, or chronic AF (cAF; i.e. persistent, long-standing persistent, or permanent AF), sinus bradycardia, sinus tachycardia, premature atrial contractions (PACs), wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, supraventricular tachycardia (SVT), atrial flutter, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions (PVCs), accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation.

The expression system of aspect 4 or 5, wherein the expression system does not comprise a polynucleotide encoding the K_2P channel K₂p3.1 in case the cardiac arrhythmia is a chronic atrial fibrillation and the patient shows normal cardiac function.

The expression system of any of aspects 4 to 7, wherein the atrial fibrosis is selected from the group of fibrosis of the atria, fibrosis of the sinus node (SA node), and fibrosis of the atrioventricular node (AV node).

The expression system of any of aspects 1 to 8, wherein the one or more two-pore-domain- potassium (K_2P) channel(s) are selected from the group consisting of TREK-1 (K₂p2.1), TWIK-1 (K₂pl.l), TASK-1 (K_2P3.1), TRAAK (K_2P4.1), TASK-2 (K_2P5.1), TWIK-2 (K_2P6.1), TWIK-3 (K_2P7.1), TASK-3 (K_2P9.1), TREK-2 (K_2P10.1), THIK-2 (K_2P12.1), THIK-1 (K_2P13.1), TASK-5 (K_2P15.1), TALK-1 (K_2P16.1), TALK-2 (also known as TASK-4) (K_2P17.1), and TRESK (also known as TRIK) (K_2P18.1), and variants thereof. 10. The expression system of any of aspects 1 to 9, wherein the one or more two-pore-domain- potassium (K_2P) channel(s) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID

NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39.

11. The expression system of any of aspects 1 to 9, wherein the one or more two-pore -domain- potassium (K₂p) channel(s) comprises or consists of the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5. 12. The expression system of any of aspects 1 to 9, wherein the polynucleotide comprises one or more of gene sequences selected from the group consisting of KCNK2, KCNK1, KCNK3, KCNK4, KCNK5, KCNK6, KCNK7, KCNK9, KCNK10, KCNK12, KCNK13, KCNK15, KCNK16, KCNK17, and KCNK18, and a variant thereof.

13. The expression system of any of aspects 1 to 9, wherein the polynucleotide comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ

ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40. 14. The expression system of any of aspects 1 to 9, wherein the polynucleotide comprises or consists of the nucleotide sequence according to SEQ ID NO: 2, SEQ ID NO: 4, and/or SEQ ID NO: 6.

15. The expression system of any of aspects 1 to 14, further comprising regulatory elements, preferably selected from the group consisting of promoter, enhance, silencer, and Rheo- Switch.

16. The expression system of aspect 15, wherein the promoter is selected from the group consisting of a CMV promoter, a ANF promoter, a ALC-1 promoter, a MLC-2v promoter, and a v-MHC promoter.

17. The expression system of any of aspects 1 to 16, wherein the expression system is a viral vectors, plasmid vectors, cosmid vectors, phage vectors, or bacterial spores.

18. The expression system of any of aspects 1 to 17, wherein the expression system is a viral vectors selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpes virus vectors, measles virus vectors, pox virus vectors, vesicular stomatitis virus vectors, retrovirus vectors, lentivirus vectors, and viral like particles.

19. The expression system of any of aspects 1 to 18, wherein the expression system is a Ad5 adenovirus vector or a AVV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, and AAV 12.

20. A pharmaceutical composition comprising the expression-system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium

channel or a variant thereof, and a pharmaceutical acceptable carrier and/or excipient.

21. The pharmaceutical composition of aspect 20, comprising a further active ingredient, preferably selected from the group consisting of an adjuvant and an active ingredient.

22. A method of altering the cardiac electrophysiology, in particular the electrophysiology of the atrium, sinoatrial and/or atrioventricular node of the heart, comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium

channel, or a variant thereof. 23. A method of preventing or treating cardiac arrhythmia, comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore -domain-potassium

channel, or a variant thereof.

24. The method of aspect 22 or 23, wherein the expression system is administered locally or systemically. 25. The method of aspect 24, wherein the expression system is administered locally by direct injection or via an endocardial catheter.

26. The method of aspect 24 or 25, wherein the expression system is administered systemically through the oral, intravenous, intramucosal, intraarterial, intramusculuar or intracoronal route.

27. The method of any of aspects 24 to 26, wherein the expression system administered systemically includes a heart-specific promotor, in particular an atrial or ventricular-specific promotor, in particular a promoter selected from the group consisting of atrial natriuretic factor (ANF), atrial myosin light chain 1 (ALC-1), myosin light chain 2v (MLC-2v), and ventricular myosin heavy chain (v-MHC).

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention. Examples

Example 1 :

Study patients were subjected to analysis of TREK-1 mRNA expression in the right atrium to substantiate the first hypothesis that AF is associated with reduced atrial TREK-1 levels.

The study involving human tissue samples was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of the University of Heidelberg (Germany; institutional approval number S-390/2011). Written informed consent was obtained from all patients. Animal experiments have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health (NIH publication No. 86-23, revised 1985) and with EU Directive 2010/63/EU, and the current version of the German Law on the Protection of Animals was followed. Experiments involving pigs have been approved by the local animal welfare authorities (institutional approval number G-165/12).

Quantitative real-time PCR (RT-qPCR) was performed using the StepOnePlus (Applied Biosystems, Foster City, CA, USA) system according to the manufacturer's protocol. All RT-qPCR reactions were performed in triplicate. Total RNA was isolated from indicated human and porcine cardiac regions using TRIzol-Reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. DNA synthesis was carried out by reverse transcription with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Waltham, MA, USA) using 3 μg of total RNA. Optical detection plates (96 wells; Applied Biosystems) were then loaded to a total volume of 10 μΐ per well, consisting of 0.5 μΐ cDNA, 5 μΐ TaqMan Fast Universal Master Mix (Applied Biosystems), and 6-carboxyfluorescein (FAM)-labeled TaqMan probes and primers (TaqMan Gene Expression Assays; Applied Biosystems) detecting porcine TREK-1 (AJ6RNHL; custom-designed; Life Technologies, Darmstadt, Germany) or human TREK-1 (Hs01005159_ml ; predesigned; Life Technologies). Pre -designed primers and probes detecting porcine (Ss 03374854_gl ; Life Technologies) or human (Hs02758991_gl ; Life Technologies) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used for normalization. The validity of GAPDH as housekeeping gene for RT-PCR analyses was previously indicated by a comparison between GAPDH, hypoxanthine phosphoribosyltransferase 1, and β-actin levels in pigs, confirming stable housekeeping gene expression throughout different cardiac regions and rhythm conditions.

A total of 30 patients (mean age, 51 ± 13 years; male/female, 20/10) with sinus rhythm (SR; n

= 10), paroxysmal (p)AF (n = 10), and chronic (c)AF (i.e., persistent, long-standing persistent or permanent AF; n = 10) undergoing heart transplantation were included (Table 1). Right and left atrial tissue samples were provided by the tissue bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany) in accordance with the regulations of the tissue bank and the approval of the Ethics Committee.

TREK-1 (K2p2.1) mRNA expression in the right atrium (RA) was reduced by 60.9% in pAF (n = 9; P = 0.24) and cAF patients (-75.6%; n = 8; P = 0.15) compared to individuals with SR (n = 9) (Fig. 1A, Table 1). In human left atrium (LA), pAF was not associated with TREK-1 remodeling (n = 8; P = 0.95), whereas in cAF patients left atrial TREK-1 transcripts were suppressed by 63.4% (n = 7) compared with SR subjects (n = 9; P = 0.07).

Table 1. Baseline characteristics of study patients.

ACE, angiotensin converting enzyme; AT, angiotensin receptor; AVD, aortic valve disease; CAD, coronary artery disease; cAF, chronic atrial fibrillation; LA, left atrial; LVEF, left ventricular ejection fraction; MVD, mitral valve disease; OAC, oral anticoagulation; pAF, paroxysmal atrial fibrillation; SR, sinus rhythm. ^*P < 0.05, ^**P < 0.01 versus SR/cAF; statistical comparisons were performed using ANOVA followed by Bonferroni correction for continuous variables and chi- square tests for categorical variables.

Similarly, TREK-1 downregulation was observed in an established porcine AF model. AF was induced by repetitive atrial burst pacing via an implanted pacemaker. At 14 day follow-up, TREK-1 transcript levels in right atrial appendage were reduced by 65.7% in AF animals (n = 5; P = 0.025) compared to control pigs carrying inactive pacemakers (n = 5) (Fig. IB). TREK-1 expression was not significantly changed in right atrial regions remote from the experimental AF "trigger" site (i.e., pacing site) or in left atrial tissue (Fig. IB) in this short-term follow-up animal cohort.

Atrial TREK-1 expression was reduced in AF patients with concomitant heart failure undergoing cardiac transplantation. Furthermore, the porcine AF model used in the present study characterized by a combined phenotype of AF with tachycardia-induced impairment of left ventricular function similarly displayed TREK-1 suppression. Downregulation of repolarizing TREK-1 potassium channels is consistent with prolonged atrial refractoriness and associated arrhythmogenesis. Prolongation of AERP or action potential duration is characteristic to AF complicated by reduced LVEF and heart failure in humans in animal models.

Example 2:

To assess transduction efficacy and functional effects of TREK-1 overexpression in vitro, mouse atrial cardiac myocytes (HL-1 cells) were subjected to gene transfer using serotype 5 adenovirus encoding human TREK-1 under a CMV promoter (Ad-TREK-1; Fig. 2 A) or adenovirus carrying the empty vector as control (Ad-control).

Cell culture and in vitro gene transfer

HL-1 cells, a cardiac muscle cell line derived from the AT-1 mouse atrial myocyte tumor lineage, were kindly provided by Dr. William Claycomb (Louisiana State University Health Science Center, New Orleans, LA, USA). Cells were cultured in supplemented Claycomb medium (JRH Biosciences, Lenexa, KS, USA) . Adenoviral gene transfer with a MOI of 200 was performed in expanded Claycomb medium (Sigma-Aldrich, St. Louis, MO, USA) when cells were 60-80% confluent. Cells were harvested 72 h after adenovirus application.

Adenovirus production and amplification

Gene transfer was performed using adenovirus owing to its ability to induce immediate transgene expression and high transduction efficiency in myocardial tissue. Adenovirus carrying human KCNK2 encoding TREK-1 (Ad-TREK-1) was custom-made (SIRION, Martinsried, Germany). Human and porcine TREK-1 channels share high sequence homology and display similar functional and regulatory properties, validating the pig as appropriate model for translational studies targeting TREK-1. The vector was generated from the 1.3 kb TREK-1 (KCNK2) coding region (GenBank accession number EF165335). The gel-purified fragment was cloned into the shuttle vector p06-A5- CMV, yielding the plasmid p06-A5-CMV-KCNK2. The CMV-KCNK2 portion of p06-A5-CMV- KCNK2 was then transferred via recombination in a BAC vector containing the genome of a replication deficient type 5 adenovirus-based vector with deleted E1/E3 genes. Following release of the recombinant viral DNA by restriction digest with Pad, HEK293 cells were transfected with the adenoviral DNA and intact infectious particles were generated. The amplified viral particles were chromatographically purified using the ViraBind Adenovirus Purification Kit (Cell Biolabs, San Diego, CA, USA). Infectious titers were determined via immunohistochemical detection of the adenoviral hexon protein in infected HEK293 cells.

The efficacy of TREK-1 gene transfer was evaluated in vitro by the commercial manufacturer in NIH-3T3 cells using RT-qPCR with a MOI of 200 and 36 h after transduction. Virus stock expansion and assessment of virus concentration were performed using an immunohistochemical approach with primary antibodies targeting adenovirus hexon protein and corresponding secondary antibodies conjugated to horseradish peroxidase (HRP). Signals were analyzed using a metal-enhanced diaminobenzidin (DAB) substrate (Adeno-X Rapid Titer Kit; Clontech, Mountain View, CA, USA). Cellular electrophysiology

Whole -cell patch-clamp recordings from HL-1 cells were carried out using a RK-400 amplifier (Bio-Logic SAS, Claix, France). Fire -polished capillary glass pipettes (World Precision Instruments, New Haven, CT, USA) were filled with the following solution: 100 niM K-aspartate, 20 niM KCl, 2 niM MgC12, 1 niM CaC12, 10 niM EGTA, 10 niM HEPES, 40 niM glucose, 5 niM K-ATP (pH adjusted to 7.2 with KOH). The external solution was composed of 140 niM NaCl, 5 niM KCl, 1 niM MgC12, 1.8 niM CaC12, 10 niM HEPES, 10 niM glucose (pH adjusted to 7.4 with NaOH). Recordings were carried out under constant perfusion at room temperature, and no leak subtraction was done during the experiments. From the holding potential (-80 mV), currents were evoked by 500 ms-voltage steps from -120 mV to +80 mV at 20 mV intervals, preceded by a constant voltage pulse to -50 mV (20 ms) to inactivate sodium channels. To analyze current densities, membrane capacitance was measured using the analogue compensation circuit of the patch clamp amplifier. Adenovirus carrying human KCNK2 encoding TREK-1 (Ad-TREK-1) was custom-made (SIRION, Martinsried, Germany).

The appropriate number of viral particles (multiplicity of infection, MOI) for optimal expression of TREK-1 was experimentally determined at 200 MOI, yielding a 2.7-fold increase of TREK-1 protein expression 72 h after Ad-TREK-1 treatment compared to untreated HL-1 cells (n = 3; P = 0.048; Fig. 2B). TREK-1 currents were enhanced by 1.7-fold following Ad-TREK-1 application (n = 15; P < 0.001) compared to control vector treatment (n = 11; Fig 2C). Example 3:

Neonatal cardiomyocytes were obtained from isolated hearts of 1-3 day old mice after euthanization by decapitation. Following thoracotomy, the hearts were excised, washed, and stored in ice cold ADS buffer (116.4 niM NaCl, 19.7 niM HEPES, 9.4 niM NaH2P04, 5.6 niM D-glucose, 5.4 niM KCl, 0.8 mM MgS04 (pH 7.4 adjusted with NaOH) prior to enzymatic cell isolation. Vascular and non-cardiac tissue (e.g. lung tissue) was removed. Tissue samples were mechanically dissected and enzymatically digested (0.03% porcine pancreatin, 0.025% collagenase; Sigma- Aldrich). The cell suspension was then filtered, and the number of fibroblasts was reduced by temporary plating in uncoated culture dishes. Freshly isolated cells were plated onto coverslips coated with collagen A (Merck, Darmstadt, Germany) and maintained in DMEM/F12 medium supplemented with GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA), 1% L-glutamine, 1% penicillin and streptomycin, 10% fetal bovine serum at 37°C and 5% C02 for at least 3 hours at a density of 5 x 104 viable cells per cm². The medium was then replaced by long-term medium (culture medium with 1% FBS instead of 10% FBS). Isolated myocytes were used for up to 10 days after isolation. Ad-TREK-1 gene transfer (MOI 200) was carried out in long-term medium. Infected cells were subjected to electrophysiological recordings 24 h after virus application.

Recordings from neonatal mouse cardiomyocytes were performed with an Axopatch 200B Amplifier (Axon Instruments) and Signal software (version 4.11 ; Cambridge Electronic Design, Cambridge, UK) in whole-cell mode. Data were acquired at 20 kHz and filtered at 2 kHz using a four- pole Bessel low-pass filter. Pipettes were pulled from borosilicate glass capillaries (GB 150-8P, Science Products, Hofheim am Taunus, Germany) using a DMZ Universal Puller (Zeitz Instruments, Martinsried, Germany) to achieve pipette resistances of 1.5 - 2.5 ΜΩ. Patch pipettes for murine cardiac action potential (AP) recordings were filled with 130 niM KC1, 1 inM MgC12, 5 inM EGTA, 5 niM MgATP, 10 inM HEPES, 10 inM NaCl (pH 7.2 adjusted with KOH). Extracellular buffer consisted of 137 niM NaCl, 5.4 niM KC1, 1.8 niM CaC12, 1 niM MgC12, 10 niM D-glucose, 10 niM HEPES, and 2 mM sodium pyruvate (pH 7.4 adjusted with NaOH). APs were elicited in current clamp mode from a membrane potential set at -90 mV with a holding command below 400 pA by injection of brief current pulses (3 ms, 0.3 nA) at 1 Hz stimulation rate. Recordings were carried out at room temperature. Cells were used for a recording period of maximum 3 h after which they were exchanged with a fresh coverslip. Data analysis was performed using custom written MATLAB routines (The MathWorks, Natick, MA, USA). For calculation of action potential durations (APD), the absolute amplitude of the AP was used as a reference point for determining the percent repolarization. Overexpression of repolarizing TREK-1 channels resulted in action potential duration (APD) shortening. APD at 90% repolarization (APD90) recorded from isolated native mouse cardiac myocytes was reduced by 36% from 50±6 ms (control cells; n=18) to 32±3 ms (Ad-TREK-1 ; n=22; p=0.009) 24h after infection (Fig. 3).

Example 4:

Antiarrhythmic efficacy of Ad-TREK-1 gene therapy was assessed in vivo in domestic pigs. Direct virus injection in an open chest procedure was performed for gene delivery. Adenovirus carrying human KCNK2 encoding TREK-1 (Ad-TREK-1) was custom-made (SIRION, Martinsried, Germany), as above.

AF was induced by rapid atrial burst pacing in domestic swine via an implanted cardiac pacemaker. Domestic swine were used and randomized to sham treatment and active atrial burst pacing (AF sham, n = 5) or inactive pacemaker programming (SR sham, n = 5) as reference groups, and to Ad-TREK-1 gene transfer in the absence (SR Ad-TREK-1, n = 5) or presence of AF (AF Ad- TREK-1, n = 5). In vivo gene transfer

Pigs were randomized following pacemaker implantation to either AF induction by activation of atrial burst pacing or to pacemaker deactivation (SR groups), in combination with Ad-TREK-1 gene transfer or application of sham solution without adenovirus, respectively. Gene transfer was performed in vivo employing a hybrid approach combining right atrial adenovirus injection and epicardial electroporation to increase plasmid DNA expression (see below). The animals underwent open chest surgery during general anaesthesia with median thoracotomy and incision of the pericardium to expose the heart under sterile conditions. A total of 1.5 ml solution containing Ad-TREK-1 (1.5x109 to 5x109 plaque forming units) was injected in aliquots of 0.1 ml into the high right atrial wall, carefully avoiding injections into the atrial cavity. Injection of adenoviruses was followed by electroporation using a paddle-style quadripolar rectangular array of 2x2 stainless steel electrodes (electrode length, 5 mm; gap size, 15 mm). Five square wave applications were carried out at the site targeted by gene therapy (20 V/100 ms; ECM 830, BTX Harvard Apparatus, Holliston, MA, USA). The electric field causes transient pores to form in the cells of the atrial tissue, improving adenovirus uptake into cells. Application of adenoviruses encoding for GFP using the hybrid approach previously yielded 50-70% gene transfer efficacy after two weeks. After gene transfer and approximation of the pericardium the thorax was closed.

Two acute periprocedural cases of death caused by uncontrollable bleeding prior to study treatment were observed. Those animals were excluded from the analysis and two additional pigs were randomized. All eligible animals were included in the analyses without defining or excluding outliers. The endpoint of the experimental study was the completion of 14 days experimental follow-up.

On day 14 all animals subjected to burst pacing and sham treatment displayed AF in 100% of ECG recordings (n = 5) (Fig. 4A). By contrast, AF animals receiving Ad-TREK-1 therapy showed SR in 40 ± 22% (n = 5; P = 0.015) of analyzed ECG segments. The cumulative prevalence of SR was calculated as percentage of non-pacing segments showing SR per animal on daily ECG recordings (Fig. 4, A and B). During 14-day follow up SR prevalence was low (35 ± 28%) in the AF sham group, while in the Ad-TREK-1 treatment group SR was preserved in the majority of ECG recordings (62 ± 15%; P < 0.001) (Fig. 4A). Control pigs receiving inactive pacemakers and either sham (n = 5) or Ad- TREK-1 treatment (n = 5) displayed SR during the entire 14-day observation period.

Following the initiation of atrial burst pacing, AF animals were characterized by rapid ventricular rate response with mean cumulative heart rates of 211 ± 15 beats per minute (bpm) in sham-treated pigs (n = 5) (Fig. 5). During SR, heart rates yielded 117 + 5 bpm during the 14 day follow-up period (n = 5; P < 0.001). Atrial application of Ad-TREK-1 did not significantly affect heart rates in the SR animal group (123 ± 6 bpm; n = 5) compared to SR controls (n = 5; P = 0.052). However, in AF pigs gene therapeutic TREK-1 overexpression resulted in reduced cumulative ventricular rates (182 ± 10 bpm; n = 5) in comparison with untreated AF animals (P < 0.001) owing to increased SR prevalence (Fig. 5). Western blot analysis

After data acquisition on day 14 anesthetized animals were killed by intravenous application of KC1 (1 M), and the hearts were removed and rinsed with phosphate buffered saline. The right atrial appendage of the heart was dissected, rapidly frozen in liquid nitrogen and stored at -80°C. Homogenized cardiac samples (Yellow line DI 18 basic homogenizer, IKA, Essex, UK) and HL-1 myocytes were subjected to cell lysis in radioimmunoprecipitation (RIP A) buffer containing 20 niM Tris-HCl, 0.5% NP-40, 0.5% sodium deoxycholate, 150 niM NaCl, 1 niM EDTA, 1 niM Na3V04, 1 niM NaF, and inhibitors of proteases (Complete) and phosphatases (PhosStop) (Roche Applied Science, Indianapolis, IN, USA). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were separated on 6-20% SDS polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and developed using primary antibodies directed against TREK-1 potassium channel (sc-11556; Santa Cruz Biotechnology, Heidelberg, Germany) or type I collagen (NB600-408, Novus Biologicals, Littleton, CO, USA) and appropriate horseradish peroxidase (HRP) -conjugated mouse anti-goat (sc- 2354; Santa Cruz Biotechnology) or donkey anti-rabbit (NA934V; GE Healthcare Life Sciences, Piscataway, NJ, USA) secondary antibodies. Signals were developed using the enhanced chemiluminescence assay (ECL Western Blotting Reagents, GE Healthcare, Buckinghamshire, UK) and quantified with ImageJ 1.41 software (National Institutes of Health, Bethesda, MD, USA). After removal of primary and secondary antibodies, the membranes were re-probed with anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (G8140-01; US Biological, Swampscott, MA, USA) and corresponding goat anti-mouse secondary antibodies (sc-2005; Santa Cruz Biotechnology). Protein content was normalized to GAPDH for quantification of optical density. Western blot analyses at the time of sacrifice revealed a 61% decrease of TREK-1 protein in AF animals (n = 5) compared to SR controls (n = 5; P = 0.039) (Fig. 4C). Effective gene transfer after Ad- TREK-1 treatment was demonstrated by 3.4-fold TREK-1 protein overexpression in right atrium (n = 5) compared to sham-treated AF pigs (P = 0.003). TREK-1 levels after Ad-TREK-1 treatment in AF pigs were not significantly different from expression levels found in sham-treated pigs during SR (P = 0.48) (Fig. 4C), indicating successful reconstitution of TREK-1 expression by gene therapy. Ad- TREK-1 application to SR animals (n = 5) resulted in 2.0-fold increase in protein expression compared to sham-treated SR pigs (n = 5; P = 0.0003).

Example 5:

The animal model employed in this study displays a combined phenotype of AF and reduced left ventricular ejection fraction (LVEF) due to rapid ventricular rate response. Prolongation of atrial effective refractory periods (AERP) is characteristic to AF complicated by ventricular dysfunction. AERP assessment by invasive electrophysiological study confirmed AERP prolongation in sham- treated AF pigs (n = 5) at different basic cycle lengths after 14 days compared to baseline conditions (AERP300, +107 ± 8.9 ms, P = 0.007; AERP400, +53 + 37 ms, P = 0.20; AERP500, +48 + 36 ms, P = 0.25) (Fig. 5, A to C). Prolongation of atrial refractoriness was attenuated in AF animals receiving Ad- TREK-1 treatment (n = 5) by 130 ± 61 ms at AERP300 (P = 0.11), by 18 ± 28 ms at AERP400 (P = 0.29), and by at AERP500 (-56 ± 31 ms; P = 0.05).

Electrophysiological study

Electrophysiological (EP) examination was performed in all study animals during SR before pacemaker implantation and on day 14. In animals exhibiting AF during follow-up, electrical cardioversion preceded EP studies by at least 30 min. Bipolar catheters were placed in the right atrium (RA) and right ventricle (RV) via the jugular vein. The EP Lab system (Bard Electrophysiology Division, Lowell, MA, USA) was used to record atrial effective refractory periods (AERP), Wenckebach cycle length (WCL), sinus node recovery times (SNRT), and ventricular effective refractory periods (VERP).

To determine AERP and VERP, repeated trains of 10 stimuli at a fixed cycle length (CL) of 500 ms, 400 ms or 300 ms (AERP only) were applied, followed by a single programmed premature stimulus. The coupling interval between the last basic stimulus and the premature stimulus was decreased in 10 ms-steps until no atrial or ventricular response was recorded. To study atrial and ventricular arrhythmia inducibility programmed stimulation was performed with up to two extrastimuli at three basic cycle lengths (500 ms, 400 ms, and 300 ms). Coupling intervals of extrastimuli were decreased in 10-ms intervals until coupling intervals of 140 ms were reached or refractoriness of all extrastimuli was achieved. Of note, ventricular S2S3 coupling intervals <200 ms were not tested to avoid nonspecific arrhythmia induction at very short coupling intervals. Atrial tachyarrhythmia was defined as any atrial arrhythmia of >5 s duration. Ventricular stimulation exclusively induced ventricular fibrillation requiring immediate defibrillation.

WCL was measured by decreasing basic atrial cycle length in 10 ms-steps until no ventricular response was recorded. To measure SNRT atrial simulation was applied at basic cycle lengths of 500 ms, 400 ms or 300 ms, respectively, for 30 seconds. SNRT was calculated by subtracting the intrinsic cycle length from the recorded time from last programmed stimulus to first intrinsic atrial activation.

Sinus node recovery times (SNRT) were measured in study animals to evaluate whether right atrial transfer of TREK-1 background K+ channels affected sinus node function. There was no inappropriate SNRT prolongation at different basic cycle lengths in any group (Table 1).

Table 1. Sinus node recovery times (SNRT) obtained at indicated basic cycle lengths.

Study group (n = 5 each) P value

Sinus rhythm SR (sham;

(SR) (sham; baseline) baseline)

SNRT500 (ms) 159 + 71 119 + 83 0.49

SNRT400 (ms) 140 ± 65 118 + 81 0.68

SNRT300 (ms) 100 ± 73 145 + 64 0.46

SR (Ad- SR (Ad- TREK-1; baseline) TREK-1; day 14)

SNRTsoo (ms) 79 + 49 93 + 12 0.60

SNRT400 (ms) 72 + 33 87 + 20 0.45

SNRT300 (ms) 83 + 13 125 + 61 0.35

Atrial AF (sham;

fibrillation (AF) day 14)

(sham; baseline)

SNRT500 (ms) 100 + 70 170 + 54 0.15

SNRT400 (ms) 100 + 50 296 + 286 0.21

SNRT300 (ms) 144 + 70 149 + 45 0.93

AF (Ad- AF (Ad- TREK-1; baseline) TREK-1; day 14)

SNRT500 (ms) 113 + 29 134 + 67 0.58

SNRT400 (ms) 112 + 30 125 + 51 0.67

SNRT300 (ms) 126 + 59 107 + 63 0.69

Atrioventricular (AV) conduction properties were studied by determining the Wenckebach cycle length (WCL) prior to atrial burst pacing and study treatment and on day 14. Significantly increased WCL was similarly observed in AF animals subjected to sham treatment (+56 + 26 ms; n = 5; P = 0.029) and to Ad-TREK-1 gene transfer (+52 + 15 ms; n = 5; P = 0.002) compared to baseline conditions (Fig. 6D). There were no effects of sham or Ad-TREK-1 treatment on WCL in SR pigs during follow-up.

Induction of atrial tachyarrhythmia by programmed atrial stimulation was successful in a total of 4 out of 20 animals at study start (SR Ad-TREK-1, n = 2; AF sham, n = 2) (Table 2). By contrast, no atrial tachyarrhythmias could be induced in any animal group following study treatment on day 14. In addition, ventricular effective refractory periods (VERP) were evaluated to detect potential electrophysiological off -target effects of Ad-TREK-1 gene transfer on ventricular myocardium. VERP was significantly prolonged during follow-up in both AF study groups (n = 5 each) irrespective of sham (VERP400, +68 + 8 ms, P = 0.003; VERP500, +94 + 16 ms, P < 0.001) or Ad-TREK-1 treatment (VERP400, +60 + 15 ms, P = 0.049; VERP500, +55 + 13 ms, P = 0.030) (Fig. 5, E and F). Finally, programmed ventricular stimulation resulted in unspecific induction of ventricular fibrillation (VF) under baseline conditions and after study treatment (Table 3). VF prevalence was not significantly different between Ad-TREK-1 application and sham treatment in both SR animals (P = 1) and AF pigs (P = 0.5), respectively. Table 2. Induction of atrial tachyarrhythmias in study animals.

Number of animals with inducible arrhythmia and total number of arrhythmias are provided. P values reflect statistical comparisons of animals displaying arrhythmia inducibility at baseline versus day 14, calculated using Fisher' s exact test.

Table 3. Induced ventricular tachyarrhythmias in study animals.

Number of animals with inducible arrhythmia and total number of arrhythmias are provided. P values reflect statistical comparisons of animals displaying arrhythmia inducibility at baseline versus day 14, calculated using Fisher's exact test.

In this proof-of -concept study, TREK-1 potassium channel gene therapy successfully reduced AF burden in pigs. Antiarrhythmic efficacy of the intervention was accompanied by attenuation of AERP prolongation. AERP reduction and antiarrhythmic efficacy following atrial TREK-1 gene transfer confirm a mechanistic role of TREK-1 downregulation in AF. Furthermore, ERP shortening through targeted overexpression of a repolarizing ¾p K⁺ channel represents a specific therapeutic mechanism of action that is effective in AF. Of note, adenoviral TREK-1 protein overexpression in SR control pigs significantly exceeded expression levels observed in SR controls but was well-tolerated and did not cause inappropriate AERP shortening or atrial arrhythmia inducibility in vivo. AF and reduced LVEF were accompanied by prolonged WCL and VERP in study animals irrespective of sham or Ad-TREK-1 treatment. By contrast, VERP and WCL were not affected in the SR rhythm group subjected to Ad-TREK-1 application, demonstrating that effects on AV conduction and ventricular refractoriness were induced by AF and/or associated LVEF impairment as opposed to the therapeutic intervention. Indeed, previous studies revealed that ventricular dysfunction induces downregulation of ventricular repolarizing IK_s and Ικι currents that accounts for VERP prolongation. AV conduction delays may be attributed to reduced atrial and AV nodal L-type calcium channels in structural heart disease. Example 6:

Effective rhythm control attenuated tachycardia-associated left ventricular ejection fraction (LVEF) reduction.

Echocardiography

Echocardiography was performed on the day of pacemaker implantation and gene transfer and prior to euthanization, respectively (Philips Healthcare Sonos 5500, Hamburg, Germany). Animals were sedated and anesthetized, and all examinations were done under equal conditions during SR. AF was electrically converted to SR prior to examination. Sizes of left atria and left ventricles were measured in M-mode. Left ventricular ejection fraction (LVEF) was calculated from M-mode measurements using the Teichholz formula (V = [7/(2.4 + LVId)]- [LVId]³).

Echocardiograms performed at the beginning of the study during sinus rhythm revealed equal LVEF in all animal groups (Fig. 6A). Fourteen days after initiation of atrial burst pacing we observed a significant reduction of LVEF in sham-treated pigs (81 ± 5% on day 0 versus 51 ± 5% on day 14; n = 5; P = 0.002) owing to AF and ventricular tachyarrhythmia. LVEF impairment was markedly attenuated in AF animals receiving Ad-TREK-1 after 14 days to 72 ± 5%; n = 5; P = 0.05 versus day 0) (Fig. 7A). No significant changes in LVEF were detected in SR pigs independent of Ad-TREK-1 application. In addition, there were no alterations in additional echocardiographic parameters of LV structure or function in animal groups during follow-up (Tables 4 to 7).

Table 4. Echocardiographic parameters obtained from sham-treated SR control animals.

Sinus rhythm (sham; Sinus rhythm (sham; P value baseline) (n = 5) day 14) (n = 5)

LVEF (%) 79 + 3 76 + 3 0.17

LVEDD (mm) 34 + 5 31 + 4 0.36

LVESD (mm) 19 + 3 20 + 4 0.68

IVSD (mm) 17 + 4 13 + 2 0.15

IVSS (mm) 19 + 4 14 + 4 0.16

LA diameter (mm) 33 + 3 35 + 2 0.31

LVPWs (mm) 17 + 1 21 + 2 0.007 LVPWd (mm) 15 + 4 17 + 4 0.40

LVEF, left ventricular ejection fraction; LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; IVSD, interventricular septal wall thickness at end diastole; IVSS, interventricular septal wall thickness at end systole; LA, left atrium; LVPWs, left ventricular posterior wall thickness at end systole; LVPWd, left ventricular posterior wall thickness at end diastole

Table 5. Echocardiographic parameters obtained from SR animals subjected to Ad-TREK-1 treatment.

LVEF, left ventricular ejection fraction; LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; IVSD, interventricular septal wall thickness at end diastole; IVSS, interventricular septal wall thickness at end systole; LA, left atrium; LVPWs, left ventricular posterior wall thickness at end systole; LVPWd, left ventricular posterior wall thickness at end diastole Table 6. Echocardiographic parameters obtained from sham-treated AF pigs.

LVEF, left ventricular ejection fraction; LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; IVSD, interventricular septal wall thickness at end diastole; IVSS, interventricular septal wall thickness at end systole; LA, left atrium; LVPWs, left ventricular posterior wall thickness at end systole; LVPWd, left ventricular posterior wall thickness at end diastole Table 7. Echocardiographic parameters obtained from AF animals receiving Ad-TREK-1 gene therapy.

LVESD, left ventricular end systolic diameter; IVSD, interventricular septal wall thickness at end diastole; IVSS, interventricular septal wall thickness at end systole; LA, left atrium; LVPWs, left ventricular posterior wall thickness at end systole; LVPWd, left ventricular posterior wall thickness at end diastole

Left atrial (LA) dilation is a common finding in AF patients. Repetitive atrial burst pacing and AF resulted in pronounced dilation of the left atrium, resembling findings in humans. In the sham- treated AF group, LA diameter increased from 33 + 2 mm (day 0) to 41 + 4 mm (day 14; n = 5; P = 0.003) (Fig. 7B). Atrial dilation of pigs treated with Ad-TREK-1 was not statistically significant (day 0, 36 + 2 mm; day 14, 41 + 4 mm; n = 5; P = 0.08). SR control and SR Ad-TREK-1 animals showed no changes in LA dimensions.

Example 7:

To delineate the contribution of TREK-1 K+ channels to the regulation of atrial refractoriness, atrial APD is determined by multiple ion currents. In particular, AF and concomitant HF were associated with downregulation of TREK-1 two-pore-domain K+ channels and with prolongation of AERP. TREK-1 gene transfer effectively suppressed AF in pigs, highlighting a contribution of TREK- 1 channels to AF pathophysiology. To further assess the molecular basis of atrial electrical remodeling underlying inverse AERP regulation in the present model, study animals were screened for mRNA levels of 13 additional ion channel subunits relevant to atrial electrophysiology in a more comprehensive approach.

AF-associated remodeling was characterized by increased mRNA expression of KCNQ1 (IKs), KCNA5 (IKur), KCNJ3/KCNJ5 (IK,ACh), and CACNAIC (ICaL) (Fig. 8). Enhanced levels of voltage-gated or inwardly rectifying K+ channels and L-type calcium channels is expected to shorten rather than prolong atrial refractoriness, suggesting that reduction of TREK-1 may represent a mechanistic key factor in a subgroup of AF patients characterized by AERP prolongation. Following Ad-TREK-1 treatment, no significant expression changes among channels studied were detected compared with AF pigs undergoing sham procedures (Fig. 8). Apparent tendencies towards reduced mRNA levels of KCNJ3/KCNJ5 (IK,ACh), KCNH2 (IKr), or CACNA1C (ICaL) may reflect a compensatory biological mechanism that prevents inappropriate shortening of refractoriness and associated proarrhythmic potential after TREK-1 gene therapy. Of note, there was no significant interaction of Ad-TREK-1 therapy with expression levels of related TASK-1 (KCNK3, K2P3.1) channels that contribute to atrial electrophysiology and AF arrhythmogenesis as well.

Example 8:

The association of AF and HF with dilation of the left atrium (LA) and TREK-1 downregulation in humans and pigs suggests a triggering role for mechanical stretch in ionic remodeling. A neonatal rat ventricular myocyte model was subjected to defined stretch (15% elongation) for 2h, 6h, and 24h, respectively, to delineate potential direct mechanical effects on TREK-1 expression. In particular, freshly prepared neonatal rat ventricular cardiomyocytes were plated in DMEM medium containing 10% fetal calf serum (FCS) on collagen type I-coated Bioflex (Flexcell International Cooperation, Burlington, NC, USA) culture plates in a density of 1x106 cells per well. After 24 hours of incubation (37°C, 5% C02) the cells were washed once with phosphate buffered saline (PBS) and incubated for another 24 hours in serum-free DMEM. Then biaxial stretch was applied using the Flexcell FX -4000 tension system (Flexcell International Cooperation). Stretch settings were 15% elongation at 1 Hz. The experiment was started with the 24 h plates. After 18 and 22 hours, respectively, the 6 h and 2 h plates were added to the stretch device. Control cells plated on similar control plates were maintained at identical conditions (without stretch) for the same duration. RNA of all samples was prepared at the same time. RNA preparation was carried out using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. DNA-free RNA was subsequently subjected to further analyses.

Transcriptional analyses revealed downregulation of TREK-1 mRNA by 19% (2h; n=3; P=0.15), 47% (6h; n=3; P=0.020), and 49% (24h; n=3; P=0.007) compared with control conditions (n=3) (Fig. 9). Example 9:

AF is associated with fibroblast activation, increased amount of extracellular matrix (ECM) proteins, and fibrosis. In addition, targeted atrial gene therapy may induce local fibrosis. To assess the degree of interstitial fibrosis in right atrium (RA), left atrium (LA) and left ventricle (LV), the hearts of all study animals were subjected to histological analysis using Masson's trichrome stain (n = 5 pigs per group) (Fig. 10A).

Evaluation of fibrosis, inflammation, and apoptosis

Histological analyses of tissue samples from right atrium, left atrium, and left ventricle of study animals were carried out. Right atrium, left atrium, and left ventricle were dissected from the heart. Sections for microscopic analysis were fixed in 10% formalin, embedded in paraffin and cut to 7 μιη thickness. Slices were stained with Masson's trichrome to identify interstitial fibrosis or with hematoxylin-eosin to analyze inflammation. The extent of cardiac fibrosis was visualized using a Nikon Eclipse TE2000-E microscope (Nikon GmbH, Diisseldorf, Germany) and quantified with ImageJ 1.41 software (National Institutes of Health) in blinded fashion. Inflammation was graded using an arbitrary scale from 1 to 4, where 1 reflects normal cardiac tissue, 2 indicates inflltration in -20% of sections, 3 denotes less than 50% inflltration, and 4 was scored in cases of >50%> infiltration, respectively. Reported scores reflect average values of n = 3 blinded observers. Sections were examined at 64-fold magnification using the Olympus Provis AX 70 microscope (Olympus Life Science Europe, Hamburg, Germany).

Right atrial fibrosis was increased to 27 ± 6% in sham-treated pigs with AF compared with corresponding SR animals carrying inactive pacemakers (10 ± 2%; P = 0.008) (Fig. 10B). This finding is consistent with previous studies reporting increased atrial fibrosis in AF patients and animal models. Ad-TREK-1 gene therapy suppressed the formation of right atrial fibrosis in AF to 17 ± 4% that did not significantly differ from SR controls (P = 0.23) (Fig. 10B). Right atrial levels of type I collagen, a major extracellular matrix (ECM) protein, were increased in AF animals by 2.4-fold (n = 5) relative to SR control animals (n = 5) without achieving statistical significance (P = 0.13) (Fig. 11). Conversely, AF animals receiving Ad-TREK-1 treatment exhibited collagen I levels that were similar to SR groups. Cardiac fibrosis in LA or LV did not significantly differ between study groups (Fig. 10, A and B).

There was an apparent increase in right atrial inflammation in both gene transfer groups and in the AF sham group relative to sham-treated SR controls (Fig. 10, A and C). Inflammation levels were significantly increased by 1.33 units on an arbitrary scale ranging from 1 to 4 in SR Ad-TREK-1 animals compared to SR sham controls (P = 0.046). Furthermore, AF animals revealed mildly increased inflammation in the absence of gene transfer by 0.72 arbitrary units compared to SR control pigs (P = 0.062). No significant inflammatory dynamics were observed in LA and LV of all treatment groups.

Example 10:

Apoptosis has been implicated in AF-associated structural remodeling. In addition, adenoviral gene transfer may trigger apoptosis of atrial cells. To assess whether AF or TREK-1 gene were associated with programmed cell death, apoptotic activity was evaluated by TUNEL fluorescence of right atrial tissue sections (Fig. 12A). TUNEL staining

Apoptosis was detected by TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling). Sections of right atrial tissue were embedded in paraffin and cut to 10 μιη thickness. Embedded tissue was de -waxed and rehydrated. After washing with PBS, sections were post-fixed in ice-cold acetone, rinsed with PBS, and incubated in 200 ml 0.1 M citrate buffer (Merck, Darmstadt, Germany) and 0.1 % Triton X-100 (Merck) (pH 6.0). TUNEL reaction mixture (285 μΐ label solution + 15 μΐ enzyme solution; Roche Applied Science, Mannheim, Germany) was then added to the sections, and slides were incubated for 60 min at 37°C. After removal of the TUNEL reagent slides were rinsed with PBS and stained with blue nuclear stain (Hoechst 33258, Sigma-Aldrich) to visualize cell nuclei. TUNEL -positive cells were counted using a fluorescence microscope (Zeiss, Oberkochen, Germany), and ImageJ 1.41 software was used to calculate the percentage of TUNEL- positive cells in randomly selected fields.

We found that apoptosis rates were low and not significantly increased by either AF or gene transfer in groups of 5 animals each (Fig. 12B).

Example 11 :

To assess potential systemic effects of atrial Ad-TREK-1 application on cardiac, renal or liver function, serological parameters were analyzed on day 14 in all study groups. We did not detect significant differences in multiple markers of myocardial injury or renal, liver and pancreatic dysfunction during SR (Table 8) and AF (Table 9), respectively. Reduced alanine transaminase (ALT) levels in AF animals following Ad-TREK-1 gene therapy compared to sham-treated controls further indicate a lack of liver damage during short-term follow-up (Table 9). Table 8. Serological findings in sinus rhythm pigs following Ad-TREK-1 treatment compared to sham-treated control animals at 14-day follow-up.

Sinus rhythm (sham) Sinus rhythm (Ad- P value

(ii = 5) TREK-1) (n = 5)

Creatinine (mg/dl) 1.2 ± 0.1 1.5 + 0.2 0.29

Urea (mg/dl) 31 + 3 31 + 2 0.88

Creatine kinase (total,

1124 + 160 1224 + 267 0.78

U/l)

Creatine kinase (MB

334 + 38 293 + 47 0.56

isoenzyme, U/l)

High sensitive

214 + 81 55 + 23 0.13

troponin T (pg ml)

Aspartic acid

47 + 5 41 + 6 0.46

transaminase (U/l)

Alanine transaminase

41 + 2 37 + 5 0.54

(U/l)

Alkaline phosphatase

73 + 8 86 + 8 0.33

(U/l)

Gamma-

39 + 7 34 + 5 0.62

glutamyltransferase (U/l)

Table 9. Serological findings in pigs subjected to atrial tachypacing and Ad-TREK-1 treatment compared to sham-treated animals at 14-day follow-up.

Safety of atrial TREK-1 gene transfer

Serological analyses at the end of the follow-up period provided no indication of cardiac, renal or liver damage following Ad-TREK-1 application in both SR and AF animals, respectively. Adenoviral-mediated gene transfer is known to induce pro-inflammatory signaling and local inflammation. We observed interstitial inflammation in the right atrium (i.e. in the area of gene transfer and arrhythmogenic burst pacing) in sham-treated AF animals and following Ad-TREK-1 application in both AF and SR pigs. This finding suggests that inflammation was triggered by a dual mechanism comprising adenovirus injection and AF-associated migration of activated lymphocytes into atrial myocardium (32). Of note, atrial apoptosis rates were low at baseline and not significantly increased by gene therapy.

In addition, right atrial TREK-1 overexpression did not markedly prolong SNRT, reflecting negligible effects on sinoatrial node function. Localized overexpression of ion channels may induce gene therapy-related proarrhythmia. In the present study, no atrial arrhythmias were triggered by programmed atrial stimulation upon completion of follow-up in any study group in spite of atrial arrhythmia induction in 4/20 animals on the day of pacemaker implantation. Furthermore, localized atrial transgene application prevented undesired treatment effects on ventricular repolarization (VERP), highlighting a potential advantage of gene therapy compared to systemic drug application. Finally, the prevalence of VF upon programmed ventricular stimulation was not significantly affected by atrial Ad-TREK-1 treatment compared with sham procedures in SR or AF study groups, respectively.

Statistics

Continuous patient data are provided as mean ± standard deviation (SD), and categorical variables are given as frequency and percentage. Experimental animal data are expressed as mean ± SEM. Statistical analyses were performed with Graph-Pad Prism 6.0 software (GraphPad Software, La JoUa, CA, USA). Statistical differences of continuous variables were determined using paired and unpaired Student's t tests (two-sided tests) where appropriate. Categorical data were analyzed using the chi-square or Fisher's exact test (two-tailed test). P < 0.05 was considered statistically significant. Multiple comparisons were performed using one-way ANOVA. If the hypothesis of equal means could be rejected at the 0.05-level, pair wise comparisons of groups were made and the probability values were adjusted for multiple comparisons using the Bonferroni correction. Mean heart rates and cumulative prevalence of SR of study animals were compared using two-factor analysis of variance (ANOVA) with treatment and time as factors and repeated measures on 1 factor (time). In summary, the data provide novel mechanistic insights into AF arrhythmogenesis in humans and pigs with reduced cardiac function. In a porcine model of AF and reduced LVEF it was demonstrated that targeted atrial expression of K2p2.1 (TREK-1) K⁺ channels provides rhythm control and preserves cardiac function. Antiarrhythmic TREK-1 gene therapy could expand and personalize the current polymodal treatment strategy to eliminate the most debilitating of arrhythmias.

Claims

Claims

An expression system comprising one or more polynucleotide(s) encoding one or more two- pore-domain-potassium (K2p) channel or a variant thereof, for use in altering the cardiac electrophysiology in a patient.

The expression system of claim 1 , wherein the patient has a reduced expression level of said one or more K2P channel in comparison to a control, in particular in comparison to the expression level in a healthy subject or in comparison to a value representing the average expression level in a healthy subject.

The expression system of claim 1 or 2, wherein the cardiac electrophysiology is altered by modulating, in particular increasing, the transmembrane potassium current, preferably the transmembrane background potassium current.

An expression system comprising one or more polynucleotide(s) encoding one or more two- pore-domain-potassium (K₂p) channel or a variant thereof, for use in preventing or treating cardiac arrhythmia, atrial dilation, and/or atrial fibrosis.

The expression system of claim 4, wherein the cardiac arrhythmia is selected from the group consisting of atrial, junctional and ventricular arrhythmia.

The expression system of claim 4 or 5, wherein the cardiac arrhythmia is selected from the group consisting of atrial fibrillation (AF) (in particular first detected, paroxysmal, or chronic AF (cAF; i.e. persistent, long-standing persistent, or permanent AF), sinus bradycardia, sinus tachycardia, premature atrial contractions (PACs), wandering atrial pacemaker, atrial tachycardia, multifocal atrial tachycardia, supraventricular tachycardia (SVT), atrial flutter, AV nodal reentrant tachycardia, atrioventricular reciprocating tachycardia, junctional rhythm, junctional tachycardia, premature junctional contraction, premature ventricular contractions (PVCs), accelerated idioventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation.

The expression system of any of claims 1 to 6, wherein the one or more two-pore -domain- potassium (K₂p) channel(s) are selected from the group consisting of TREK-1 (K₂p2.1), TWIK-1 (K₂pl. l), TASK-1 (K_2P3.1), TRAAK (K_2P4.1), TASK-2 (K_2P5.1), TWIK-2 (K_2P6.1), TWIK-3 (K_2P7.1), TASK-3 (K_2P9.1), TREK-2 (K_2P10.1), THIK-2 (K_2P12.1), THIK-1 (K_2P13.1), TASK-5 (K_2P15.1), TALK-1 (K_2P16.1), TALK-2 (also known as TASK-4) (K_2P17.1), and TRESK (also known as TRIK) (K_2P18.1), and variants thereof.

8. The expression system of any of claims 1 to 7, wherein the expression system is a viral vectors, plasmid vectors, cosmid vectors, phage vectors, or bacterial spores, preferably a viral vectors selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpes virus vectors, measles virus vectors, pox virus vectors, vesicular stomatitis virus vectors, retrovirus vectors, lentivirus vectors, and viral like particles.

9. The expression system of any of claims 1 to 8, wherein the expression system is a Ad5 adenovirus vector or a AVV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, and AAV 12.

10. A pharmaceutical composition comprising the expression-system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium (K₂p) channel or a variant thereof, and a pharmaceutical acceptable carrier and/or excipient.

11. A method of altering the cardiac electrophysiology, in particular the electrophysiology of the atrium, sinoatrial and/or atrioventricular node of the heart, comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore-domain-potassium (K₂p) channel, or a variant thereof.

12. A method of preventing or treating cardiac arrhythmia, comprising administration of an effective amount of an expression system comprising one or more polynucleotide(s) encoding one or more two-pore -domain-potassium (K₂p) channel, or a variant thereof.

13. The method of claim 11 or 12, wherein the expression system is administered locally or systemically.

14. The method of claim 13, wherein the expression system is administered locally by direct injection or via an endocardial catheter, and/or wherein the expression system is administered systemically through the oral, intravenous, intramucosal, intraarterial, intramusculuar or intracoronal route.

15. The method of any of claims 12-14, wherein the expression system administered systemically includes a heart-specific promotor, in particular an atrial or ventricular-specific promotor, in particular a promoter selected from the group consisting of atrial natriuretic factor (ANF), atrial myosin light chain 1 (ALC-1), myosin light chain 2v (MLC-2v), and ventricular myosin heavy chain (v-MHC).