Therapeutic connexin 45 inhibitors

Abstract

To provide compositions for treating, preventing, inhibiting the progression of or reducing the likelihood of occurrence of a myocardial infarction-related complication, a cardiac disorder characterized by abnormal conduction and other cardiac conditions.SOLUTION: The present disclosure relates to compositions comprising one or more inhibitors of Connexin 45 (Cx45). Preferably, the inhibitor of Cx45 is a small molecule, protein or nucleic acid. More preferably, the inhibitor of Cx45 is a nucleic acid molecule comprising the DNA sequence encoding the RNA molecule. The present invention also discloses the use of the inhibitor of Cx45 in producing pharmaceuticals for treating, preventing, inhibiting the progression of or reducing the likelihood of occurrence of a myocardial infarction-related complication in a subject.SELECTED DRAWING: Figure 14

Description

[Technology Field]

[0001] Related applications This application claims priority under Australian Provisional Application No. 2015903282, filed on 14 August 2015, the entirety of which is incorporated herein by reference.

[0002] This disclosure generally relates to methods for treating, preventing, inhibiting the progression of, or reducing the likelihood of the occurrence of myocardial infarction-related complications, cardiac dysfunction characterized by abnormal conduction, and other cardiac conditions. This disclosure also generally relates to one or more inhibitors of connexin 45. [Background technology]

[0003] Cardiovascular disease remains the sole most common cause of natural death in developed countries. Sudden cardiac death (commonly also called “out-of-hospital death,” “death upon arrival,” or “sudden death”) is estimated to account for approximately 50% of all deaths from cardiovascular causes. Common causes of sudden cardiac death include, for example, ventricular tachycardia, where the resting heart rate is faster than normal; ventricular fibrillation, where there is uncoordinated contraction of the ventricular myocardium, causing the muscle to tremble without proper contraction; or ventricular arrhythmias, which include arrhythmias in which both ventricular tachycardia and ventricular fibrillation are present. Ventricular arrhythmias are often triggered by acute cardiac events, occurring in people with or without known heart disease, or with structural cardiac abnormalities.

[0004] Several molecular mechanisms underlying heart disease have been elucidated by studying the role of gap junctions. Gap junctions are channels that enable intercellular communication. In mammalian tissues, these channels are expressed eccentrically and serve a variety of biological functions (Saez, 2003). Within the heart, gap junctions are essential for the propagation of electrical activity necessary for cardiac function. In addition, gap junction remodeling occurs in heart disease and may contribute to the pathophysiology of cardiac arrhythmias.

[0005] Functional gap junctions consist of two hemichannels, each supplied by two adjacent cells (Saez, 2003). Each hemichannel consists of six connexin (Cx) proteins. Protein assembly can result in homomeric or heteromeric channels. Of the 20 connexins in vertebrates, the most abundant in the heart are Cx37, Cx40, Cx43, and Cx45. Cx37, Cx40, and Cx43 are involved in the propagation of electrical impulses in the heart and in cardiac development. Cx45 is essential for embryonic cardiac development and may not be necessary at all for normal cardiac function after birth. In this regard, Bao et al. (2011) recently reported that activation and conduction were unchanged in hearts overexpressing Cx45, and no difference in conduction velocity or pattern was observed in Cx45-deficient hearts.

[0006] Modern clinical management of ventricular arrhythmias consists of electrotherapy based on pharmacologically active drugs and devices. Drugs are moderately effective and, in most patients, prevent the need for prophylactic implantable cardioverter-defibrillators (ICDs). In addition, many drugs used to prevent ventricular tachycardia and / or ventricular fibrillation have serious side effects, including arrhythmia induction. While ICDs are effective in detecting ventricular tachycardia and / or ventricular fibrillation by either anti-tachycardia pacing or defibrillation, using an ICD has many disadvantages, including the pain of receiving a direct electric shock while conscious, the anticipatory anxiety and cost of the incoming shock, as well as the risk of death associated with implantation and the maintenance of the hardware. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there remains an unmet need for methods and compositions for the treatment of myocardial infarction-related complications, cardiac dysfunction characterized by abnormal conduction, arrhythmias, and other cardiac conditions. [Means for solving the problem]

[0008] The inventors have for the first time demonstrated that inhibition of connexin 45 (Cx45) significantly reduces the incidence of cardiac dysfunction characterized by abnormal conduction, including myocardial infarction-related complications. Therefore, this disclosure provides a novel therapeutic indication for Cx45 inhibitors in treating cardiac dysfunction and myocardial infarction-related complications.

[0009] Accordingly, in one embodiment, the present disclosure provides a method for treating, preventing, inhibiting the progression of, or reducing the likelihood of myocardial infarction-related complications in a subject, the method comprising the step of administering a Cx45 inhibitor to the subject.

[0010] Myocardial infarction-related complications may be selected from a group consisting of arrhythmias, abnormal conduction, sudden cardiac death, left ventricular dysfunction, and ventricular dyssynchrony. In a specific case, the myocardial infarction-related complication was an arrhythmia.

[0011] The disclosure also provides a method for treating, preventing, inhibiting the progression of, or reducing the likelihood of the occurrence of cardiac dysfunction characterized by abnormal conduction in a subject, the method comprising the step of administering a Cx45 inhibitor to the subject.

[0012] Cardiac dysfunction may be selected from the group consisting of arrhythmias, left bundle branch block, right bundle branch block, bundle branch block, atrioventricular block, nonspecific intraventricular conduction delay, nonspecific interventricular conduction delay, left ventricular dysfunction, and ventricular dyssynchrony.

[0013] In any of the methods described herein, abnormal conduction may be a slowing of conduction velocity in cardiac cells. Abnormal conduction may cause arrhythmias such as one or more of the following: ventricular arrhythmia, ventricular fibrillation, or ventricular tachycardia.

[0014] In any of the methods described herein, the Cx45 inhibitor may be administered in an amount effective to reduce or inhibit the prolongation of the QRS interval and / or PR interval in the subject.

[0015] Any of the methods described herein may further include the step of selecting subjects for treatment by identifying the presence or likelihood of developing cardiac dysfunction or myocardial infarction-related complications characterized by abnormal conduction in the subjects before administering a Cx45 inhibitor to the subjects. For example, any of the methods described herein may include the step of selecting subjects who have previously suffered a myocardial infarction and administering a Cx45 inhibitor described herein to those subjects.

[0016] Cx45 inhibitors may be small molecules, proteins, or nucleic acids.

[0017] In one example, the Cx45 inhibitor is an RNA molecule. The RNA molecule may include a first sequence and a second sequence, the first sequence being at least 12 nucleotides long and having at least 70% sequence identity to the target region of the mRNA transcript shown in SEQ ID NO: 1, and the second sequence having at least 70% sequence identity to the reverse complement of the first sequence. The first sequence may have 100% sequence identity to the target region. The second sequence may have 95% sequence identity to the reverse complement of the first sequence. In a particular example, the target region may be shown in any one of SEQ ID NOs: 3, 15, or 18.

[0018] This disclosure also provides the use of Cx45 inhibitors in the manufacture of pharmaceuticals for treating, preventing, inhibiting the progression of, or reducing the likelihood of myocardial infarction-related complications in subjects.

[0019] This disclosure also provides the use of Cx45 inhibitors in the manufacture of pharmaceuticals for treating, preventing, inhibiting the progression of, or reducing the likelihood of the occurrence of cardiac dysfunction characterized by abnormal conduction in subjects.

[0020] This disclosure also provides inhibitors of Cx45 for use in treating, preventing, inhibiting the progression of, or reducing the likelihood of myocardial infarction-related complications in subjects.

[0021] The present disclosure also provides an inhibitor of Cx45 for use in treating, preventing, inhibiting the progression of, or reducing the likelihood of occurrence of, a cardiac dysfunction characterized by abnormal conduction in a subject.

[0022] The present disclosure also provides an RNA molecule comprising a first sequence and a second sequence, wherein the first sequence is at least 12 nucleotides in length and has at least 70% sequence identity to a target region of the mRNA transcript shown in SEQ ID NO: 1, and the second sequence has at least 70% sequence identity to the reverse complement of the first sequence. The first sequence may have 100% sequence identity to the target region. The second sequence may have 95% sequence identity to the reverse complement of the first sequence. In a specific example, the target region may be shown in any one of SEQ ID NO: 3, 15, or 18. The first sequence may hybridize to the second sequence.

[0023] The present disclosure also provides a nucleic acid encoding the RNA molecule of the present disclosure.

[0024] The present disclosure also provides a vector comprising the RNA molecule of the present disclosure, the nucleic acid of the present disclosure, or the vector of the present disclosure. The vector may be a viral vector such as a recombinant adeno-associated virus vector. In one example, the recombinant adeno-associated virus vector is an adeno-associated virus serotype 2 / 9 virus vector.

[0025] The present disclosure also provides a cell comprising the RNA molecule of the present disclosure, the nucleic acid of the present disclosure, or the vector of the present disclosure.

[0026] The present disclosure also provides a pharmaceutical composition comprising the RNA molecule of the present disclosure, the nucleic acid of the present disclosure, or the vector of the present disclosure, and a pharmaceutically acceptable carrier or diluent.

[0027] Key to the nucleotide sequence listing SEQ ID NO: 1 - Nucleotide sequence of the mRNA transcript of Homo sapiens connexin 45 (Cx45) (derived from the cDNA sequence of Cx45 identified by NCBI accession number U03493) Nucleotide sequence of the mRNA transcript of connexin 45 (Cx45) from Rattus norvegicus, derived from the cDNA sequence of Cx45 identified by accession number NM_001085381 (SEQ ID NO: 2) SEQ ID NO: 3 - Cx45-shRNA#1 target sequence SEQ ID NO: 4 - Cx45-shRNA#1 forward oligonucleotide sequence SEQ ID NO: 5 - Cx45-shRNA#1 reverse oligonucleotide sequence SEQ ID NO: 6 - Cx45-shRNA#2 target sequence SEQ ID NO: 7 - Cx45-shRNA#2 forward oligonucleotide sequence SEQ ID NO: 8 - Cx45-shRNA#2 reverse oligonucleotide sequence SEQ ID NO: 9 - Cx45-shRNA#3 target sequence SEQ ID NO: 10 - Cx45-shRNA#3 forward oligonucleotide sequence SEQ ID NO: 11 - Cx45-shRNA#3 reverse oligonucleotide sequence SEQ ID NO: 12 - Cx45-shRNA#4 target sequence SEQ ID NO: 13 - Cx45-shRNA#4 forward oligonucleotide sequence SEQ ID NO: 14 - Cx45-shRNA#4 reverse oligonucleotide sequence SEQ ID NO: 15 - Cx45-shRNA#5 target sequence SEQ ID NO: 16 - Cx45-shRNA#5 forward oligonucleotide sequence SEQ ID NO: 17 - Cx45-shRNA#5 reverse oligonucleotide sequence SEQ ID NO: 18 - Cx45-shRNA#6 target sequence SEQ ID NO: 19 - Cx45-shRNA#6 forward oligonucleotide sequence SEQ ID NO: 20 - Cx45-shRNA#6 reverse oligonucleotide sequence SEQ ID NO: 21 - Cx45-shRNA#7 target sequence Sequence ID 22 - Cx45-shRNA#7 forward oligonucleotide sequence Sequence ID 23 - Cx45-shRNA#7 reverse oligonucleotide sequence Sequence ID 24 - Cx45-shRNA#8 target sequence Sequence ID 25 - Cx45-shRNA#8 forward oligonucleotide sequence Sequence ID 26 - Cx45-shRNA#8 reverse oligonucleotide sequence Sequence ID 27 - Nucleotide sequence of the loop region. [Brief explanation of the drawing]

[0028] [Figure 1] This is a fluorescence microscope image showing the cellular localization of Cx45 in ventricular cardiomyocytes of neonatal rats after transduction using a vector that overexpresses Cx45. [Figure 2] This is a representative blot showing the expression level of Cx45 in ventricular cardiomyocytes of neonatal rats after transduction using a vector that overexpresses Cx45. [Figure 3] This is a confocal microscope image showing the co-localization of Cx43 and Cx45 in a ventricular section of rat myocardium. [Figure 4] A representative blot showing the co-immunoprecipitation of Cx43 and Cx45 is shown. Lysates from ventricular tissue were immunoprecipitated with either a Cx43 antibody or a Cx45 antibody, and immunoblotting was performed using antibodies against each other's proteins to detect interactions. Cx45 was detected in the Cx43 immunoprecipitate (Figure 4A), and Cx43 was detected in the Cx45 immunoprecipitate (Figure 4B). Specific protein bands representing Cx45 and Cx43 are indicated by arrows on the right side of the blot. [Figure 5] This table shows ECG parameters after transduction using a vector overexpressing Cx43 or Cx45. [Figure 6] This graph shows the trend of ventricular arrhythmias in rats after transduction using a vector overexpressing Cx43 or Cx45. [Figure 7] This graph shows the inflammation score in rats after transduction using a vector overexpressing Cx43 or Cx45. [Figure 8] This graph shows the percentage of intracardiac fibrosis in rats after transduction using a vector overexpressing Cx43 or Cx45. [Figure 9] This is a representative blot showing the expression levels of Cx45 in a pseudo-rat model (pseudo) or a rat model of myocardial infarction (MI). [Figure 10] This is a fluorescence microscope image showing Cx45 expression in rats co-expressing the Cx45-GFP fusion protein and Cx45-targeting shRNAs (Cx45-shRNA#1, Cx45-shRNA#2, Cx45-shRNA#3, Cx45-shRNA#4) or a control scramble sequence (shNS). [Figure 11] These are fluorescence microscopy images showing Cx45 expression in rats that overexpress Cx45 and are treated with shRNA targeting Cx45 (Cx45-shRNA#1) or a control scrambled sequence (shNS). [Figure 12] This is a representative blot showing the expression levels of Cx43 and Cx45 in rats that overexpress Cx45 and are treated with shRNA targeting Cx45 (Cx45-shRNA#1) or a control scrambled sequence (shNS). [Figure 13] This is a representative blot showing the levels of Cx43 and Cx45 in rats treated with shRNA targeting Cx45 (Cx45-shRNA#1) or a control scrambled sequence (shNS). [Figure 14] This graph shows the trend of ventricular arrhythmias in rats that developed infarction after transduction with a Cx45-targeting shRNA (Cx45-shRNA#1) or a control scramble sequence (shNS). [Modes for carrying out the invention]

[0029] Description of the Embodiment general Unless otherwise specified, all technical and scientific terms used herein shall be understood to have the same meaning as those generally understood by those skilled in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0030] Unless otherwise indicated, the recombinant protein, cell culture, virology, microbiology, and immunology techniques used in this invention are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991), DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JE Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), DNA Cloning: A Practical Approach, Volumes I and II (D. Glover ed.), Oligonucleotide Synthesis (N. Gait ed., 1984), Nucleic Acid Hybridization (B. Hames and S. Higgins eds., 1985), Transcription and Translation (B. This is described and explained throughout the literature of sources such as Hames and S. Higgins (eds., 1984), and Animal Cell Culture (ed. R. Freshney, 1986).

[0031] The term "and / or," for example, "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and clear supporting evidence shall be provided for both meanings or either meaning.

[0032] Unless otherwise clearly indicated by the context, the singular forms of words like "a," "an," and "the" also include their plural forms.

[0033] Unless otherwise stated, the term "approximately" refers to ±20%, more preferably ±10%, of the specified value. To avoid ambiguity, the term "approximately" following a specified value should be interpreted as including the specified value itself (for example, "approximately 10" includes exactly 10).

[0034] The terms “hybridize” and “anneal,” and their grammatical synonyms, are used interchangeably herein and refer to nucleotide sequences that can form Watson-Crick base pairs due to their complementarity. Those skilled in the art will expect to understand that non-Watson-Crick base pairing is also possible, particularly in relation to RNA sequences. For example, so-called “fluctuation pairs” can be formed between guanosine and uracil residues in RNA.

[0035] Throughout this specification, the term “comprise,” or variations such as “comprises” or “comprising,” are understood to include the element, integer, or step, or group of elements, integers, or steps described, but not to exclude any other element, integer, or step, or group of elements, integers, or steps.

[0036] Connexin 45The terms “connexin 45” or “Cx45” are understood to refer to the specific connexin protein contained in gap junctions. Cx45 may have structural and / or biochemical functions associated with gap junctions and electromechanical junctions. Cx45 can originate from any mammal. Representative mammals include, but are not limited to, humans, primates, domestic animals (e.g., sheep, cattle, horses, donkeys, pigs), or companion animals (e.g., dogs, cats). In one example, Cx45 is mammalian Cx45. In a particular example, Cx45 is human Cx45. The amino acid and cDNA sequences of Cx45 have been determined and are publicly available through many online databases, such as UniProt, Swiss-Prot, TrEMBL, and NCBI. Those skilled in the art will understand how to obtain mRNA transcript sequences from DNA sequences. For example, nucleotide sequencing software that converts DNA sequences to mRNA transcripts is publicly available. For example, a human Cx45 mRNA transcript may be derived from the Cx45 cDNA sequence found in NCBI deposit number U03493. A human Cx45 mRNA transcript may contain or consist of the nucleotide sequence shown in SEQ ID NO: 1. Alternatively, a human Cx45 mRNA transcript may contain or consist of a nucleotide sequence that, over its entire length, has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 1. A human Cx45 mRNA transcript may incorporate such sequence mutations (which may be naturally occurring mutations), provided that the transcript codes for a functional Cx45 protein. A rat Cx45 mRNA transcript may be derived from the Cx45 cDNA sequence found in NCBI deposit number NM_001085381. The rat Cx45 mRNA transcript may contain or consist of the nucleotide sequence shown in SEQ ID NO: 2. Alternatively, the rat Cx45 mRNA transcript may contain the nucleotide sequence shown in SEQ ID NO: 1 throughout its entire length. In contrast, it may contain or consist of nucleotide sequences having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity. A human Cx45 mRNA transcript may incorporate such sequence mutations (which may be native mutations), provided that the transcript encodes a functional Cx45 protein.

[0037] Cx45 inhibitors The inhibitors described herein may be any compound capable of inhibiting the expression or function of Cx45. As used herein, the terms “inhibit” or “to inhibit” refer to the ability of a compound to block, partially block, interfere with, reduce or diminish the expression or function of Cx45. Thus, those skilled in the art will know that the term “inhibit” includes the complete and / or partial loss of Cx45 expression and / or activity. For example, an inhibitor may inhibit Cx45 expression and / or activity by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% or 100% compared to Cx45 expression and / or activity in the absence of the inhibitor. The ability of a compound to inhibit Cx45 expression may be tested by preferred methods known in the art. Such methods include, for example, techniques such as Western blotting using antibodies specific to Cx45. Anti-Cx45 antibodies are well-known in the art and are available from many commercial sources. Alternatively, anti-Cx45 antibodies may be prepared by routine methods known in the art. Other methods for determining the ability of compounds to inhibit Cx45 expression and / or activity may include RT-PCR utilizing primers specific to Cx45 mRNA, or immunofluorescence techniques on transformed cells in culture.

[0038] The inhibitors described herein may be small molecules, proteins, or nucleic acid molecules. Proteins may be gene-editing proteins. For example, gene-editing proteins include zinc finger nucleases, TALENs, (International Publication Nos. WO2013 / 176772, WO2014 / 018423, WO2014 / 022702, WO2014 / 065596, WO2014 / 089290, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635) The inhibitor may be a clustered and regularly arranged short palindromic sequence repeat (CRISPR)-related protein, nuclease, meganuclease, or nickas (as defined in one or more of the following publications: WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, or any repetition of the technologies described in these publications). The inhibitor may also contain a CRISPR-Cas chimeric RNA (chiRNA). The chiRNA may contain a guide sequence that can hybridize to the target sequence described herein (i.e., the Cx45 target sequence), tracer mate sequence, and / or tracer sequence. The inhibitor may also contain a polynucleotide encoding the chiRNA. The inhibitor may further contain a polynucleotide encoding a CRISPR enzyme containing at least one nuclear localization sequence. The polynucleotide may be contained within any preferred vector known to be suitable for resulting in the regulation of CRISPR-Cas-mediated gene expression, either as described herein or otherwise. When expressed in the target cells, the tracer-mate sequence may hybridize to the tracer sequence, and the guide sequence may direct the CRISPR complex to sequence-specific binding to the target sequence. The CRISPR complex may include a CRISPR enzyme complexed with a guide sequence hybridized to the target sequence and / or a tracer-mate sequence hybridized to the tracer sequence. Any preferred CRISPR enzyme may be used. The enzyme is preferably a Cas enzyme. In a particular example, the enzyme is a Cas9 enzyme.

[0039] The nucleic acid molecule may originate from any of the ribonucleic acids, such as messenger RNA (mRNA), transfer RNAs (tRNA), mitochondrial RNA (mtRNA), ribosomal RNA (rRNA), and nuclear RNA (nRNA).

[0040] The inhibitors described herein may be RNA molecules. In one example, an RNA molecule may inhibit the transcription and / or translation of Cx45. Suitable RNA molecules include clustered inhibitory RNAs, dicer substrates, DNA-dependent RNAi (ddRNAi), single-stranded RNAi (ssRNAi), microRNA (miRNA) antagonists, microRNA mimes, microRNA agonists, blockmirs, microRNA addbacks, and supermiRs, including small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), reverse repeats, small hairpin RNAs (shRNAs), small temporally regulated RNAs (stRNAs), radial clustered inhibitory RNAs, asymmetric clustered inhibitory RNAs, linear clustered inhibitory RNAs, and complexed or compounded clustered inhibitory RNAs. Preferably, the RNA molecule is shRNA. It is understood that shRNA can be processed in a target to form siRNA. Therefore, in another specific example, the RNA molecule is siRNA.

[0041] An RNA molecule may contain a first sequence. The first sequence may be of any length. For example, the first sequence may be of a length of at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides. Thus, an RNA molecule may contain or be composed of 12 to 50 nucleotides, for example, 12 to 25 nucleotides or 12 to 21 nucleotides. In another example, an RNA molecule may consist of or contain 19 to 21 molecules. An RNA molecule may contain a second sequence. The second sequence may be of any length. The length of the second sequence may be the same as the length of the first sequence. Alternatively, the length of the second sequence may be different from the length of the first sequence (i.e., longer or shorter). For example, the second sequence may consist of or contain at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length.

[0042] The first and second sequences may form a single, continuous RNA molecule. For example, the RNA molecule may include a loop region located between the first and second sequences. The loop region may be of any length. The length of the loop region may be sufficient to allow the RNA molecule to fold so that the first and second sequences hybridize with each other. For example, the loop region may consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

[0043] The first sequence and the second sequence may form two distinct molecules. The two distinct sequences may hybridize with each other to form a single RNA molecule.

[0044] The first sequence may have at least 70% sequence identity with respect to the target region of the Cx45 mRNA transcript. For example, the first sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to the target region of Cx45. The target region may be selected using methods known in the art. For example, the target region may be selected to allow the maximum level of inhibition achieved through the use of the RNA molecule described herein. Experimental methods described herein (e.g., those described in the Examples section herein) may be used to select a suitable target region (and therefore a suitable RNA molecule or other nucleotide inhibitor).

[0045] The second sequence may have at least 70% sequence identity with respect to the reverse complement of the first sequence. For example, the second sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to the reverse complement of the first sequence.

[0046] RNA molecules may contain overhangs at their 3' and / or 5' ends. For example, the overhangs may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long. In one example, the overhang may be 2 nucleotides long. Thus, RNA molecules may have (or be processed to have) 2-nucleotide overhangs at their 3' and 5' ends. The 2-nucleotide overhang(s) may contain the sequence UU.

[0047] RNA molecules may contain one or more native nucleotides. RNA may contain one or more modified nucleotides. Any suitable modified nucleotide may be used. For example, the modified nucleotide may be selected to increase the stability of the RNA molecule. Typical modified nucleotides include, but are not limited to, 2'-O-methyl modified nucleotides, nucleotides having a 5'-phosphorothioate group, and terminal nucleotides linked to cholesteryl derivatives. Alternatively, the modified nucleotide may be selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramic acids, and non-native bases including nucleotides. Other modified nucleotides are expected to be known to those skilled in the art.

[0048] The term “sequence identity” can be used interchangeably with the terms “homology,” “homology percentage,” “identity percentage,” or “similarity percentage.” Sequence identity is used to refer to the similarity or relationship between two sequences. Those skilled in the art will readily understand how to determine sequence identity. For example, sequence identity can be calculated manually or by using computer-based sequence comparison and identification tools such as BLAST (Basic Local Alignment Search Tool; Altschul et al., 1993); see also www.ncbi.nih.gov / BLAST / , and appropriate parameters can be selected for each particular sequence comparison, as will be understood by those skilled in the art.

[0049] The term "target region" can refer to any sequence of nucleotides in a Cx45 mRNA transcript. The target region can be located in any region of the Cx45 mRNA transcript. The target region can be selected using methods known in the art. For example, guidelines for identifying the target region are described by Lagana et al., 2014.

[0050] The target region may be located near 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more nucleotides downstream of the AUG translation start codon in the Cx45 mRNA transcript. The term “near” generally means within 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide of the stated position. The term “downstream” means toward the 3' end of the Cx45 mRNA transcript. The target region may be located near nucleotides 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more upstream of the UAA, UAG, or UGA translation stop codon of the Cx45 mRNA transcript. The term “upstream” means toward the 5’ end of the Cx45 mRNA transcript.

[0051] The target region may be located near positions 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 850, 900, 950, 1000, 1050, 1100, and 1150 of SEQ ID NO: 1. For example, the target region may contain or consist of the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 15, or SEQ ID NO: 18.

[0052] Treatment method The inventors have shown that reducing the overexpression of Cx45 reduces the incidence of arrhythmias. Therefore, this disclosure provides methods for treating, preventing, inhibiting the progression of, or reducing the likelihood of myocardial infarction-related complications, cardiac dysfunction characterized by abnormal conduction, and other cardiac conditions by administering Cx45 inhibitors as described herein.

[0053] The terms “to treat,” “to treat,” and “treatment” refer to the eradication or remission of a disease, disorder, or condition, or one or more symptoms associated with such a disease, disorder, or condition. For example, the term may refer to minimizing the transmission or exacerbation of such disease, disorder, or condition, as brought about by the administration of one or more Cx45 inhibitors to a subject having the disease, disorder, or condition. The term “remission” refers to any reduction, whether permanent or temporary, sustained or transient, that is attributable to or may be associated with the administration of a Cx45 inhibitor. Methods for determining the degree of treatment or remission will be known to those skilled in the art. For example, methods used herein (such as those disclosed in the Examples section) for determining the level of CX45 expression, or PR intervals, or QRS intervals may be used. Alternative methods particularly adapted for determining the degree of treatment or remission in human subjects may also be used.

[0054] The terms “prevent,” “prevention,” and “prevention” refer to the prevention of the onset, recurrence, or transmission of a disease, disorder, or condition, or one or more symptoms associated with a disease, disorder, or condition. For example, these terms refer to treatment or administration of Cx45 inhibitors to subjects at risk of developing, in particular, the diseases, disorders, or conditions described herein, prior to the onset of symptoms. These terms encompass the inhibition or reduction of symptoms of specific diseases, disorders, or conditions. Subjects with a family history of a disease, disorder, or condition are potential candidates for a preventive regimen. In addition, subjects with a history of symptom recurrence are also potential candidates for prevention. In this regard, the term “prevention” may be used interchangeably with the term “preventive treatment.”

[0055] The term "inhibiting progression" refers to the ability of a Cx45 inhibitor to block, partially block, interfere with, reduce, or lessen the progression of a disease, disorder, or condition, or the symptoms of a disease, disorder, or condition.

[0056] The term “reducing the likelihood of occurrence” refers to reducing the risk of developing a condition associated with a disease, disorder, or state provided herein. The degree of reduction is not limited. For example, the reduction may be any detectable reduction. Alternatively, a threshold level of “reduction” that achieves a particular therapeutically beneficial outcome may be selected according to methods known in the art as a preferred indicator of significant “reduction.”

[0057] The term "myocardial infarction-related complications" refers to any disease, disorder, or condition associated with, related to, or resulting from myocardial infarction. Such diseases, disorders, or conditions may be selected from the group consisting of arrhythmias, cardiac dysfunction characterized by abnormal conduction, sudden cardiac death, left ventricular dysfunction, and ventricular dyssynchrony.

[0058] The term “arrhythmia” refers to any cardiac abnormality, including any disruption of the initialization and / or propagation of impulses in the heart. Arrhythmias can result in an irregular rhythm, decreased or increased heart rate, or desynchronization of myocardial contraction that can reduce the mechanical function of the heart. Arrhythmias can be atrial, atrioventricular, or ventricular. For example, an arrhythmia can be a ventricular tachyarrhythmia. In one example, a ventricular tachyarrhythmia may be ventricular fibrillation. In one example, a ventricular tachyarrhythmia may be ventricular tachycardia. In one example, a ventricular tachyarrhythmia may be ventricular fibrillation and ventricular tachycardia. Methods for identifying arrhythmias are well known to those skilled in the art. For example, arrhythmias can be determined based on echocardiography, coronary angiography, electrophysiological examination, or by any other suitable method known in the art.

[0059] The term "echocardiography" refers to a group of tests that use ultrasound to examine the heart and record information in the form of reflected ultrasound waves. Magnetic resonance imaging may also be used as a non-invasive means to determine intracardiac pressure and the anatomical structure of the heart.

[0060] The term "coronary angiography" refers to a procedure that involves X-ray examination of the blood vessels of the heart.

[0061] "Electrophysiological testing of the heart" refers to a method in which a probe with registration electrodes is inserted or introduced into the esophageal lumen. Certain portions of the esophagus are closely adjacent to the posterior walls of the left atrium and left ventricle, and therefore, intraesophageal ECG signals selectively register the activity of these cardiac compartments. Intraesophageal electrocardiography may be used to diagnose ventricular arrhythmias.

[0062] The term “cardiac dysfunction characterized by abnormal conduction” also refers to conditions associated with, or resulting from, abnormal conduction or proarrhythmic regions in any part of the cardiac conduction system of the subject, or abnormal conduction between cardiomyocytes. A proarrhythmic region is defined as a region of cardiac tissue involved in the initiation or perpetuation of an arrhythmia. Cardiac dysfunction may include abnormal conduction from the sinoatrial node to the right atrium, or from the right atrium to the left atrium, or at any location within the right atrium, resulting in a delayed conduction of the normal excitation front of depolarization. For example, cardiac dysfunction may be slow electrical conduction or slow conduction velocity of the heart in cardiomyocytes. Those skilled in the art understand that delayed electrical conduction or reduced conduction velocity of the heart in cardiomyocytes can manifest as QRS interval prolongation and / or PR interval prolongation. QRS interval or PR interval prolongation refers to an electrocardiographic change of QRS interval or PR interval that is prolonged beyond the normal interval based on the subject's age, sex, medical history, and other clinical findings. Normal QRS intervals and PR intervals are obvious to those skilled in the art. For example, in a 20-year-old male, a normal QRS interval can have a duration between 80 and 120 ms (two to three small squares) and / or a normal PR interval can have a duration between 120 and 200 ms (three to five small squares). Cardiac dysfunction characterized by abnormal conduction also includes abnormal conduction through or between the following structures: ventricles, AV node, bundle of His, right and left bundle branches, Purkinje network, or ventricular cardiomyocytes. Abnormal conduction may be present continuously or intermittently in the cardiac conduction system of interest. Examples of abnormal conduction disorders include sinoatrial block, intraatrial conduction delay, atrioventricular nodal block, right bundle branch block, left bundle branch block, and intraventricular conduction delay. Cardiac dysfunction can cause arrhythmias. Methods for identifying cardiac dysfunction characterized by abnormal conduction are well known to those skilled in the art. Cardiac dysfunction may be determined based on abnormal heart rate, electrocardiogram, electrophysiological examination, or by any other suitable mechanism known to those skilled in the art. The functional effects of conduction abnormalities can also be evaluated using electrocardiography, echocardiography, cardiac catheterization, cardiac magnetic resonance imaging, cardiac nuclear medicine imaging, or any other suitable method.

[0063] The term "sudden cardiac death" refers to any condition characterized by a sudden loss of cardiac function. In this regard, the term "sudden cardiac death" may be used interchangeably with the terms "cardiac arrest," "sudden cessation," "sudden death," and "sudden cardiac arrest." Death occurs shortly after the onset of cardiovascular symptoms. For example, sudden cardiac death may occur within one hour, forty-five minutes, thirty minutes, fifteen minutes, ten minutes, or five minutes of symptom onset.

[0064] The term "left ventricular dysfunction" refers to any condition characterized by a left ventricular ejection fraction of less than 40%, as defined by echocardiography.

[0065] The term "ventricular dyssynchrony" refers to any condition characterized by a delay in the timing of contraction in various myocardial segments within the ventricles.

[0066] The therapeutic methods described herein may include a step of administering a therapeutically effective dose of a Cx45 inhibitor to a target. The terms “therapeutically effective dose” or “effective dose” of a Cx45 inhibitor mean an amount of the Cx45 inhibitor sufficient to provide a therapeutic effect in the treatment or management of any disease, disorder, or condition described herein, or to alleviate, delay, or minimize one or more symptoms associated with any disease, disorder, or condition described herein, either alone or in combination with one or more other agents. For example, an amount of the Cx45 inhibitor may be sufficient to reduce or inhibit the prolongation of the QRS interval and / or PR interval. Those skilled in the art can detect any electrocardiographic change in the QRS interval or PR interval. The term “effective dose” may encompass an amount that improves the overall treatment and reduces or avoids the symptoms of any disease, disorder, or condition described herein.

[0067] The dose range for administering the Cx45 inhibitors described herein is sufficiently large to produce the desired therapeutic effect. For example, the dose may be sufficient to reduce or inhibit the prolongation of the QRS interval and / or PR interval. The dose suitable for use in the subjects described herein is preferably one that avoids or minimizes excessive adverse effects. Adverse effects may include, but are not limited to, nausea, vomiting, toxicity, irritation, allergic reactions, and death. An adverse effect may be considered "excessive" if the risks outweigh the benefits provided by the Cx45 inhibitor described herein.

[0068] In general, the dose may vary depending on age, weight, sex, condition, the severity of the disease, disorder or condition described herein, and / or the intended purpose. The dose may be adjusted in the event of any contraindications, resistance, or similar conditions. Those skilled in the art can readily assess such factors and, based on this information, determine the specific effective concentration of the Cx45 inhibitor to be used for the intended purpose.

[0069] Cx45 inhibitors may be administered by any appropriate method. Methods of administration include, but are not limited to, intravenous injection, bolus injection via catheter during percutaneous transcatheter coronary angioplasty, direct injection into coated or impregnated implants such as stents, or into target tissue. For example, the target tissue may be in or around the infarcted area of ​​the heart. Examples of implantable devices and stents include, but are not limited to, those described in U.S. Patent Nos. 6,709,379, 6,273,913, 5,843,172, 4,355,426, 4,101,984, 3,855,638, 5,571,187, 5,163,958 and 5,370,684; U.S. Patent Application Publications US2002 / 0098278 and US2004 / 0073284; International Publication WO2004 / 043292; and European Patent Application Publication 0875218.

[0070] The term "subject" includes any human or non-human animal. Preferably, the subject is human. The term "non-human animal" includes, but is not limited to, non-human primates, rodents such as sheep, dogs, cats, rabbits, ferrets, mice, rats, and guinea pigs, birds such as chickens, amphibians, and vertebrates such as reptiles. The terms "subject," "patient," and "individual" may be used interchangeably herein. The subject may have a cardiovascular injury or be at risk of one in the future. The term "cardiovascular injury" refers to any form of injury caused by any means. For example, a cardiovascular injury may be myocardial infarction, arrhythmia, cardiac dysfunction characterized by abnormal conduction, sudden cardiac death, left ventricular dysfunction, ventricular dyssynchrony, or any other condition associated with cardiovascular dysfunction. For example, the subject may have (or have had) a myocardial infarction or be at risk of one in the future. Myocardial infarction may result from one or more of the following causes: hypertension, ischemic heart disease, exposure to cardiotoxic compounds, myocarditis, thyroid disease, viral infection, gingivitis, drug poisoning, alcoholism, pericarditis, arteriosclerosis, vascular disease, hypertrophic cardiomyopathy, acute myocardial infarction, left ventricular systolic failure, coronary artery bypass surgery, starvation, eating disorders, genetic abnormalities, injury, or other causes. Subjects may have (or have had) or be at risk of having cardiac dysfunction characterized by abnormal conduction.

[0071] Any method known in the art may be used to determine whether a subject has or may have a future risk of cardiovascular injury. For example, a risk factor score may be used to predict the future risk of cardiovascular injury based on the measurement of any known risk factor. Examples of risk factors include, but are not limited to, age, sex, systolic blood pressure, smoking, impaired glucose tolerance, left ventricular hypertrophy, total cholesterol, low-density lipoprotein levels, and high-density lipoprotein levels. Methods for measuring these risk factors will be known to those skilled in the art. In one example, a risk factor score may be determined using the Framingham risk factor score. Methods described herein may be used to monitor a subject's risk of suffering cardiovascular injury. For example, measuring the levels of Cx45 expression and / or activity at a first and second time point may provide an indication that the subject is at risk of cardiovascular injury. For example, an increased level of Cx45 expression and / or activity at a second time point compared to a first time point may indicate that the subject is at risk of cardiovascular injury. For example, measuring the duration of the QRS interval and / or PR interval at a first and second time point can provide an indicator of the subject's risk of cardiovascular injury. For instance, a prolonged QRS interval and PR interval at the second time point compared to the first time point may indicate that the subject is at risk of cardiovascular injury.

[0072] vector The Cx45 inhibitors described herein may be incorporated into any suitable vector to enable their expression in the target. Suitable expression vectors are known in the art. Generally, expression vectors are designed to encode exogenous or heterologous nucleic acid sequences. Manipulated nonviral vectors are known in the art, for example, described in Ramamoorth and Narvekar, 2011. A variety of viral vector systems are also known in the art. Suitable viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus (AVV) vectors, herpesvirus vectors, retrovirus vectors, poxvirus vectors, baculovirus vectors, rabies virus vectors, bovine papillomavirus vectors, lentivirus vectors, vaccinia virus vectors, polyomavirus vectors, or Sendai virus vectors. In one example, the vector may be an adenovirus vector. In one example, the vector may be an adeno-associated virus (AVV) vector. For example, the AAV vector may be an AAV6, AAV2, rAAV2 / 1, rAAV2 / 2, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, rAAV2 / 9, or rAAV2 / 10 vector. In one example, the vector may be a herpesvirus vector. For example, the herpesvirus vector may be a herpes simplex virus type 2 vector, a human cytomegalovirus vector, an Epstein-Barr virus vector, a varicerazoster virus vector, or a Kaposi's sarcoma-associated herpesvirus vector. In one example, the vector may be a retrovirus vector. The retrovirus vector may be a Moloney's mouse leukemia virus vector or a lentivirus vector. For example, the lentivirus vector may be an HIV virus vector, a feline immunodeficiency virus vector, a bovine immunodeficiency virus vector, or a Simian immunodeficiency virus vector. In one example, the vector may be a baculovirus vector.

[0073] The vectors described herein may be used to deliver the Cx45 inhibitors described herein into target cells. Thus, the vectors may be used to transform the target cells. Preferred methods for transforming (or "transfecting") the target cells are known to those skilled in the art. Examples include, but are not limited to, direct delivery of DNA such as perfusion, naked DNA transfection, liposome-mediated transfection, encapsulation, and receptor-mediated endocytosis. Non-viral methods may also be employed.

[0074] Further details of specific vectors that may be used are described, for example, in U.S. Patent Application No. 2013 / 287739. Therefore, preferred vectors include the following:

[0075] Adeno-associated virus (AVV) Adeno-associated viruses (AVVs) have shown promise in human clinical trials for gene delivery in gene therapy. As the only viral vector system based on a non-pathogenic replication-deficient virus, recombinant AAV viruses have been successfully used to establish efficient and sustained gene delivery in both proliferating and terminally differentiated cells in diverse tissues.

[0076] The AAV genome is a linear, single-stranded DNA molecule containing approximately 4681 nucleotides. Generally, the AAV genome contains an internal non-repeatable genome with terminal inversion sequences (ITRs) at each end. ITRs are approximately 145 base pairs (bp) long. ITRs have multiple functions, including serving as the starting point for DNA replication and as a packaging signal for the viral genome. The internal non-repeatable portion of the genome contains two large open reading frames known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes encode viral proteins that enable the virus to replicate and package into virions. In particular, families of at least four viral proteins are expressed from the AAV rep region, Rep78, Rep68, Rrep52, and Rep40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3.

[0077] AVVs are engineered to deliver target genes by removing internal non-repeatable regions of the AVV genome (i.e., rep and cap genes) and inserting heterologous genes between ITRs. These heterologous genes are typically functionally or operably linked to heterologous promoters (constitutive, cell-specific, or inducible) that can promote gene expression in the patient's target cells under appropriate conditions. Termination signals, such as polyadenylation sites, may also be included.

[0078] As used herein, the term “AAV vector” means, without limitation, vectors derived from adeno-associated viruses and their variants, including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. AAV vectors may retain functional facilitational ITR sequences, but may have one or more of the AAV wild-type genes, preferably rep and / or cap genes, removed in whole or in part. Despite high homology, different serotypes exhibit different tissue orientations. While the receptor for AAV1 is unknown, it is known that AAV1 transduces into skeletal and smooth muscle more efficiently than AAV2. While we do not wish to be bound by theory, it is clear that biological differences are more related to the capsid than the genome, as most studies have been conducted using pseudotype vectors in which vector DNA facilitation of an AAV2 ITR is packaged into a capsid of a different serotype. Recent evidence indicates that DNA expression cassettes packaged in AAV1 capsids are at least 1 log10 more efficient than those packaged in AAV2 capsids in transducing cardiomyocytes.

[0079] Functional ITR sequences are necessary for the rescue, replication, and packaging of AAV virions. Therefore, an AAV vector is defined herein as comprising at least a sequence (e.g., a functional ITR) that is required to be cis for viral replication and packaging. The ITR does not need to be a wild-type nucleotide sequence and may be modified, for example, by nucleotide insertion, deletion, or substitution, as long as the sequence provides functional rescue, replication, and packaging.

[0080] AAV vectors must have one copy of an AAV terminal inversion sequence (ITR) at each end of the genome in order to be replicated, packaged into AAV particles, and efficiently integrated into cell chromosomes. However, the nucleic acid facilitated by the ITR may be any desired sequence. For example, the nucleic acid may encode an RNA molecule described herein (e.g., one that, when expressed in the cells of interest, can inhibit the expression and / or activity of Cx45).

[0081] The ITR consists of nucleotides 1–145 at the left end of the AAV DNA genome and corresponding nucleotides 4681–4536 at the right end of the AAV DNA genome (i.e., the same sequence). Therefore, an AAV vector must have a terminal sequence of at least 300 nucleotides in total. Consequently, in order to package a large coding region into an AAV vector particle, it is crucial to develop the smallest possible regulatory sequences, such as transcription promoters and poly-A addition signals.

[0082] Therefore, as used herein, AAV refers to all serotypes of AAV (i.e., 1-9) and their variants. Thus, in this art, it is common practice to use ITR sequences from other serotypes of AAV, as it is expected that the ITRs of all AAV serotypes will have similar structures and functions with respect to replication, integration, excision, and transcription mechanisms.

[0083] AAV is also a helper-dependent virus. That is, AAV requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia) to form an AAV virion. Without co-infection with a helper virus, AAV establishes a latent state in which the viral genome is inserted into the host cell chromosome, but no infectious virion is produced. Subsequent infection with a helper virus "rescues" the integrated genome, allowing it to replicate and package that genome into an infectious AAV virion. While AAV can infect cells of different species, herpesviruses must be of the same species as the host cell. Therefore, for example, human AAV replicates in dog cells co-infected with canine adenovirus.

[0084] The term "AAV helper function" refers to an AVV-derived coding sequence that, when expressed, can provide a genomic product that functions trans for replication of proliferative AAV. Thus, AAV helper function includes both major AVV open reading frames (ORFs), rep and cap. Rep expression products have been shown to have many functions, including, among others, recognition, binding, and nicking of AAV start sites for DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. Cap expression products provide the necessary packaging function. AAV helper function is used herein to complement the trans-functioning AAV lost from an AAV vector.

[0085] Therefore, the term “AAV helper construct” generally refers to a nucleic acid molecule containing a nucleotide sequence that provides the AAV function removed from an AAV vector, used to construct a transduction vector for delivery of the desired nucleotide sequence. AAV helper constructs are generally used to provide transient expression of AAV rep and / or cap to compensate for the lost AAV function necessary for the replication of lytic AAV, but helper constructs lack AAV ITR and cannot replicate or package themselves. AAV helper constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Many AAV helper constructs and vectors encoding Rep and / or Cap expression products have been described.

[0086] Typically, recombinant AAV (rAAV) viruses are prepared by co-introducing a plasmid containing the molecule of interest (such as a nucleotide inhibitor as described herein) adjacent to two AAV terminal repeats, and / or an expression plasmid containing the wild-type AAV coding sequence without terminal repeats, e.g., pIM45. Cells are also infected and / or transfected with an adenovirus and / or plasmid carrying the adenovirus gene necessary for AAV helper function. The rAAV virus stock solution prepared in this manner is contaminated with adenovirus that must be physically separated from the rAAV particles (e.g., by cesium chloride density centrifugation or column chromatography). Alternatively, adenovirus vectors containing the AAV coding region and / or cell lines containing some or all of the AAV coding region and / or adenovirus helper genes may be used. Cell lines carrying rAAV DNA as an incorporated provirus may also be used.

[0087] As used herein, the term “accessory function” refers to a non-AAV-derived viral and / or cellular function on which AAV depends for its replication. Thus, the term encompasses the capture of proteins and RNA necessary for AAV replication, including regions involved in the transcription of AAV genes, the splicing of step-specific AAV mRNA, the replication of AAV DNA, the synthesis of Cap expression products, and the activation of AAV capsid assembly. The virus-based accessory function may be derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0088] Therefore, "accessory function vectors" generally refer to nucleic acid molecules containing nucleotide sequences that provide accessory function. Accessory function vectors are transfected into suitable host cells, which can then support the production of AAV virions in the host cells. Infectious viral particles, such as adenoviruses, herpesviruses, or vaccinia virus particles, are naturally occurring and are therefore clearly excluded from this term. Thus, accessory function vectors may take the form of plasmids, phages, transposons, or cosmids.

[0089] In particular, it has been demonstrated that a complete complement of the adenovirus gene is not required for accessory helper function. Specifically, adenovirus mutants that are unable to replicate DNA and synthesize late genes have been shown to be tolerant of AAV replication. Similarly, mutants in the E2B and E3 regions have been shown to support AAV replication, suggesting that the E2B and E3 regions are probably not involved in providing accessory function. However, adenoviruses with an incomplete E1 region or those with a removed E4 region cannot support AAV replication. Therefore, the E1A and E4 regions are probably required for AAV replication, either directly or indirectly. Other characterized Ad mutants include: E1B; E2A; E2B; E3; and E4. Studies of accessory function provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, but it has recently been reported that E1B55% is required for AAV virion production, while E1B19K is not.

[0090] Typical accessory functional vectors include the adenovirus VA RNA coding region, the adenovirus E4 ORF6 coding region, the adenovirus E2A 72kD coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the complete E1B 55k coding region.

[0091] The phrase "can support efficient rAAV virion production" means the ability of an accessory function vector or system to provide sufficient accessory function to complement rAAV virion production in a particular host cell at a level substantially equal to or higher than that obtainable with infection of host cells by an adenovirus helper virus. Thus, the ability of an accessory function vector or system to support efficient rAAV virion production can be determined by comparing the rAAV virion titer obtained using the accessory function vector or system with the titer obtained using infection with infectious adenovirus. More specifically, the accessory function vector or system supports efficient rAAV virion production substantially equal to or greater than that obtained using infectious adenovirus when the amount of virions obtained from the same number of host cells is 200 times less or less, more preferably about 100 times less or less, and most preferably equal to or greater than the amount obtained using adenovirus infection.

[0092] Therefore, "AAV virion" refers to a complete viral particle, such as a wild-type (wt) AAV virus particle (containing a linear single-stranded AAV nucleic acid genome with an AVV capsid protein coat). In this regard, in terms of complementarity, for example, a single-stranded AAV nucleic acid molecule, either a "sense" or "antisense" strand, can be packaged in either one AAV virion, and both strands are equally infectious. Clearly, the vector described herein may be an AAV virion.

[0093] Similarly, “recombinant AAV virion” or “rAAV virion” is defined herein as an infectious replication-deficient virus comprising an AVV protein shell that capsids a heterologous nucleotide sequence of interest (such as a nucleotide inhibitor described herein) flanked on both sides by AAV ITRs. rAAV virions are produced in an AAV vector, a suitable host cell having AAV helper and accessory functions into which the AAV vector has been introduced. In this way, the host cell can encode the AAV polypeptide necessary to package the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery. Thus, the vector may be an rAVV virion.

[0094] The vectors described herein may also include sequences encoding selection markers. The terms “selection marker” or “selection gene product” as used herein refer to the use of genes that confer resistance to the drug G418 in mammalian cells (also known as the neogene); the bacterial hygromycin G phosphotransferase (hyg) gene that confers resistance to the antibiotic hygromycin; and the bacterial xanthine-guanine phosphoribosyltransferase gene that confers the ability to grow in the presence of mycophenolic acid (also known as the gpt gene). In addition, vectors described herein may further include genes encoding visually detectable markers, such as green fluorescent protein (GFP) or any other standard of detectable markers in the art, which are recognized and available to those skilled in the art.

[0095] Those skilled in the art have circumvented some of the limitations of adenovirus-based vectors by using adenovirus "hybrid" viruses that incorporate desired characteristics from other types of viruses as well as adenoviruses, as a means of creating unique vectors with highly specialized properties. For example, viral vector chimeras have been created between adenovirus and adeno-associated virus (AAV). Such hybrid viruses or viral vector chimeras may also be used to transfect target cells with the Cx45 inhibitors described herein. Thus, this disclosure also provides hybrid viruses or viral vector chimeras containing Cx45 inhibitors.

[0096] An alternative method for the delivery of nucleotide Cx45 inhibitors described herein involves the use of an adenovirus expression vector. As used herein, the term “adenovirus expression vector” is intended to include a construct containing an adenovirus sequence sufficient to (a) assist in the packaging of constructs and / or (b) ultimately express tissue and / or cell-specific constructs cloned therein.

[0097] The expression vector may contain a genetically modified form of adenovirus. Knowledge of the adenovirus's genetic makeup, a 36kb linear double-stranded DNA virus, allows for the substitution of large portions of adenovirus DNA with foreign sequences of 7kb or less (Grunhaus and Horwitz, 1992). In contrast to retroviruses, adenovirus infection of host cells does not result in integration into chromosomes because adenovirus DNA can replicate episomal without producing potential genotoxicity. Furthermore, adenoviruses are structurally stable, and no genomic rearrangements have been detected after extensive amplification.

[0098] The propagation and handling of adenoviruses are known to those skilled in the art, and they exhibit a broad host range in vitro and in vivo. Viruses in this group exhibit high titers, e.g., 10 per mL. 9 ~1011 These can be obtained as plaque-forming units, which are highly infectious. The adenovirus life cycle does not require integration into the host cell genome. Because the exogenous polynucleotides delivered by adenovirus vectors are episomal, they have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and / or therapeutic potential as in vivo polynucleotide delivery vectors.

[0099] Adenovirus vectors have been used in eukaryotic gene expression and vaccine development. Recently, animal studies have suggested that recombinant adenoviruses may be used for gene therapy (see, e.g., Stratford-Perricaudet et al., 1991; Rich et al., 1993). Studies involving the administration of recombinant adenoviruses to various tissues include intramuscular injection, peripheral intravenous injection, and stereotactic inoculation into the brain. Both recombinant adenoviruses and adeno-associated viruses can infect and transduce non-dividing human primary cells.

[0100] The use of adenovirus vectors is conceivable, but such use in gene therapy trials is currently limited by short-lived transgene expression (Vassalli et al., 2003). This is due to cellular immunity to adenovirus antigens. Improved "non-viable" adenovirus vectors have reduced immunogenicity, but they are still ineffective when maximum transgene expression for more than six months is required or desirable for therapeutic efficacy. AAV vectors demonstrate long-term expression (>1 year) and are preferred vectors for therapeutic efficacy when long-term expression is necessary.

[0101] Retrovirus vectors Retroviruses can be selected as inhibitor delivery vectors because they integrate their polynucleotides into the host genome, introduce large amounts of foreign genetic material, and have the ability to infect a broad spectrum of species and cell types, as well as to be packaged to specific cell lines.

[0102] The retroviral genome contains three genes, gag, pol, and env, which encode the capsid protein, polymerase enzyme, and envelope component, respectively. The sequence found upstream of the gag gene contains the signal for packaging the genome into a virion. Two terminal repeat sequences (LTRs) are located at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration into the host cell genome.

[0103] To construct a retroviral vector, a nucleic acid encoding the desired polynucleotide is inserted into the viral genome in place of a specific viral sequence, resulting in a replication-deficient virus. To produce virions, a packaging cell line is constructed that contains the gag, pol, and / or env genes but lacks the LTR and / or packaging components. When a recombinant plasmid containing cDNA along with the retroviral LTR and packaging sequence is introduced into this cell line (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium. The culture medium containing the recombinant retrovirus is then collected, optionally enriched, and used for gene transfer. Retroviral vectors can infect a wide range of cell types. However, integration and stable expression require host cell division.

[0104] Herpes virus Herpes simplex virus (HSV) has garnered considerable interest in treating nervous system disorders due to its neurotropic nature. Furthermore, the ability of HSV to establish latent infection in non-dividing neurons without integration into host cell chromosomes or altering host cell metabolism, combined with the presence of a promoter that is active during the incubation period, makes HSV an attractive vector. While much attention has been focused on the neurotropic applications of HSV, this vector, given its broad host range, could also be utilized in other tissues.

[0105] Another factor that makes HSVs attractive vectors is their genome size and composition. Because HSVs are large, incorporating multiple nucleotides or expression cassettes is less problematic than with other smaller viral systems. In addition, the availability of various viral control sequences with varying capabilities (such as temporal and intensity) allows for greater control of expression than in other systems. The fact that this virus has a relatively small number of spliced ​​messages, further facilitating genetic manipulation, is also beneficial.

[0106] HSV is also relatively easy to handle and can be grown to high titers. Therefore, there are fewer delivery problems in terms of both the volume required to achieve sufficient infection efficiency (MOI) and the need for repeated administration. For a review of HSV as a gene therapy vector, see Glorioso et al. (1995). Non-pathogenic variants of HSV have been developed and are readily available for use in gene therapy settings (U.S. Patent No. 5,672,344).

[0107] lentiviral vectors Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. This high complexity allows the virus to regulate its life cycle during latent infection. Some examples of lentiviruses include human immunodeficiency virus (HTV1, REV2) and Simian immunodeficiency virus (SIV). Lentiviral vectors are created by attenuating HIV pathogenic genes multiple times, for example, by removing genes env, vif, vpr, vpu, and nef to make the vector biologically safe.

[0108] Lentiviral vectors are known in the art; see, for example, U.S. Patents 6,013,516 and 5,994,136. Generally, vectors are plasmid-based or virus-based and are configured to carry sequences essential for incorporating foreign nucleic acids, for selection, and for introducing the nucleic acids into host cells. The gag, pol, and env genes of the vector of interest are also known in the art. Therefore, a suitable polynucleotide is cloned into the selected vector and then used to transform the target cells of interest.

[0109] U.S. Patent No. 5,994,136 describes a recombinant lentivirus capable of infecting non-dividing cells, wherein a suitable host cell is transfected with two or more vectors carrying packaging functions, i.e., gag, pol, and env, as well as rev and tat. This describes a first vector capable of providing nucleic acids encoding the viral gag and pol genes, and another vector capable of providing nucleic acids encoding the viral env, for the production of packaging cells. Introducing a vector providing heterologous genes into these packaging cells produces a producing cell that releases infectious viral particles carrying the desired foreign gene. Preferably, env is an amphotropic envelope protein that enables transduction of human and other species cells.

[0110] Vaccinia virus vector Vaccinia virus vectors have been widely used due to their ease of construction, the relatively high expression levels they achieve, their broad host range, and their large capacity for carrying DNA. Vaccinia contains a linear double-stranded DNA genome of approximately 186 kb, exhibiting significant "AT" preference. Approximately 10.5 kb of terminal inversion sequences adjoins the genome. Most essential genes appear to be located within the central region, which is the most highly conserved among poxviruses. The putative open reading frames of vaccinia virus range from 150 to 200. Both strands encode, but extensive overlap of reading frames is uncommon.

[0111] At least 25 kb can be inserted into the vaccinia virus genome. The prototype vaccinia vector contains a trans gene inserted into the viral thymidine kinase gene by homologous recombination. The vector is selected based on the tk phenotype. Inclusion of the untranslated reader sequence of encephalomyocarditis virus results in higher expression levels than conventional vectors, due to the trans gene accumulating in more than 10% of the protein in infected cells within 24 hours.

[0112] Polyomavirus vectors Empty capsids of papovaviruses, such as mouse polyomavirus, have attracted attention as potential vectors for gene transfer. The use of empty polyomas was first described when polyoma DNA and purified empty capsids were incubated in a cell-free system. The DNA of the new particles was protected from the action of pancreatic DNase. The reconstituted particles were used to introduce transforming polyoma DNA fragments into rat FIII cells. Both the empty capsids and the reconstituted particles consist of all three polyoma capsid antigens: VP1, VP2, and Vp3. U.S. Patent No. 6,046,173 discloses the use of pseudocapsids formed from the major papovavirus capsid antigen, with non-major capsid antigens excluded, and incorporating exogenous material for gene transfer.

[0113] Other viral vectors Other viral vectors, such as those derived from viruses like Sindbisvirus or cytomegalovirus, may be used as expression constructs in this disclosure. These offer several attractive features for various mammalian cells (see, for example, Friedmann, 1989; Horwich et al., 1990).

[0114] Recognizing defective hepatitis B viruses has provided new insights into the structure-function relationships of various viral sequences. In vitro studies showed that despite deletions of up to 80% of its genome, the virus was able to retain its ability for helper-dependent packaging and reverse transcription (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with exogenous gene material. Chang et al. introduced the chloramphenicol acetyltransferase (CAT) gene into the duck hepatitis R virus genome instead of polymerase, surface, and / or pre-surface coding sequences. This was co-introduced into chicken hepatocellular carcinoma cells along with wild-type virus. A medium containing high-titer recombinant virus was used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991).

[0115] Modified virus Cx45 inhibitors may be delivered within infectious viruses engineered to express specific binding ligands. Thus, the viral particles specifically bind to the homologous receptors of target cells and deliver their contents to the cells. For example, a novel approach designed to enable specific targeting of retroviral vectors has been developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification may allow for specific infection of hepatocytes via sialycoprotein receptors. Alternative proteins may be used to enable the vectors described herein to specifically target cardiac cells.

[0116] Another approach for targeting recombinant retroviruses was designed, using biotinylated antibodies against retroviral envelope proteins or specific cell receptors. The antibodies were conjugated via the biotin component by using streptavidin. They demonstrated in vitro infection of diverse human cells possessing major histocompatibility complex class I and / or class II antigens with narrow-host viruses using antibodies against these surface antigens. Suitable antibodies can be used to specifically target cardiac cell markers and may be used to deliver the vectors described herein to cardiac cells.

[0117] Nonviral introduction The Cx45 inhibitors described herein may be delivered to cells. For example, nucleic acids may be introduced using nonviral methods.

[0118] Several nonviral methods for introducing expression constructs into cultured mammalian cells are considered in this disclosure. Preferred methods for nucleic acid delivery for use in this disclosure include the methods described herein or methods expected to be known to those skilled in the art. Such methods include, but are not limited to, direct delivery of "naked" DNA plasmids via the vascular system (U.S. Patent No. 6,867,196); liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); stirring with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents No. 5,302,523 and 5,464,765); cationic lipids; use of naked DNA; or microencapsulated DNA (U.S. Patent Application No. 2005 / 0037085). Despite the application of such techniques, target cells or tissues may be transformed stably or transiently.

[0119] Once a construct is delivered to a cell, the nucleic acid encoding the therapeutic gene can be positioned and expressed at various sites. For example, the nucleic acid encoding the therapeutic gene can be stably integrated into the cell's genome. This integration can be at a congenerally related position and orientation via homologous recombination (gene substitution), or it may be integrated at a random, nonspecific position (gene enhancement). For example, the nucleic acid can be stably maintained in the cell as a distinct episomal segment of DNA. Such a nucleic acid segment or "episome" encodes a sequence sufficient to allow maintenance and replication independent of or synchronized with the host cell cycle. How the expression construct is delivered to the cell and where the nucleic acid remains in the cell depends on the type of expression construct employed.

[0120] Vectors or expression constructs may be encapsulated in liposomes. Liposomes are vesicle structures characterized by a phospholipid bilayer and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. These form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, encapsulating the water-dissolved solute between the lipid bilayers. Addition of DNA to cationic liposomes induces a topological transition from the liposome to an optically birefringent liquid-crystal-enriched sphere. These DNA-lipid complexes are potential non-viral vectors for use in gene therapy.

[0121] Liposome-mediated nucleic acid delivery and in vitro expression of foreign DNA have been highly successful. Using the β-lactamase gene, researchers demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chicken embryos, HeLa, and hepatocellular carcinoma cells. Successful liposome-mediated gene transfer after intravenous injection in rats has also been achieved. A variety of commercial approaches, including "lipofection" technology, are also being explored.

[0122] Liposomes may be complexed with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote the entry of liposome-encapsulated DNA into the cell. For example, liposomes may be complexed with or used together with nuclear nonhistone chromosome protein (HMG-1). For example, liposomes may be complexed with or used together with both HVJ and HMG-1. These are applicable to the present disclosure in that such expression constructs have been successfully employed for the in vitro and in vivo introduction and expression of nucleic acids.

[0123] Another vector delivery system that can be employed to deliver nucleic acids encoding Cx45 inhibitors into cells is receptor-mediated delivery vehicles. These utilize the selective uptake of macromolecules via receptor-mediated endocytosis in almost all eukaryotic cells. Due to the cell-type specific distribution of various receptors, delivery is highly specific (Wu and Wu, 1993). When liposomes are employed, other proteins that bind to cell membrane surface proteins associated with endocytosis, such as capsid proteins or fragments thereof that are tropic to specific cell types, antibodies against proteins that undergo internal migration in circulation, and proteins that target intracellular localization and enhance intracellular half-life, may be used for targeting and / or to facilitate uptake.

[0124] Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA binder. Several ligands have been used for receptor-mediated polynucleotide delivery. The most widely characterized ligands are asialorosomucoid (ASOR) and transferrin (Wagner et al., 1990). Synthetic neoglycoproteins that recognize the same receptor as ASOR have been used as gene delivery vehicles. Epidermal growth factor (EGF) has also been used to deliver genes to squamous cell cancer cells.

[0125] For example, the vector or delivery vehicle may include a ligand and liposomes. For instance, researchers have observed increased uptake of the insulin gene by hepatocytes using lactosylceramide, galactose-terminal asialoganglioside, incorporated into liposomes. Thus, it is feasible that nucleic acids encoding Cx45 inhibitors can also be specifically delivered to cell types such as cardiac cells by any number of receptor-ligand systems, with or without liposomes.

[0126] The vector or expression construct may simply consist of naked recombinant DNA or plasmid. The introduction of the construct may also be carried out by either of the aforementioned methods of physical or chemical permeation across the cell membrane. This is particularly applicable to in vitro introduction, but can be similarly applied to in vivo use. It is expected that therapeutic DNA can also be introduced in vivo in a similar manner. Wolff et al. (U.S. Patent No. 6,867,196) teach that efficient gene delivery into cardiac tissue can be achieved by injection of plasmid DNA solution into the cardiac vein or artery. Wolff also teaches the administration of RNA, non-plasmid DNA, and viral vectors.

[0127] The vectors useful in this disclosure have varying transfection efficiencies. As a result, viral or nonviral vectors can transduce at least about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and 100% of cells in a targeted vascular region, or more or equal to that percentage. More than one vector (viral or nonviral, or a combination thereof) may be used simultaneously or sequentially. This may be used to introduce one or more polynucleotides and / or target one or more cell types. When multiple vectors or multiple drugs are used, more than one transfection / transfection efficiency may occur.

[0128] cell The Cx45 inhibitors described herein can be taken up by cells. These cells can be cultured in vitro and used for transplantation into the target myocardium. Cells can be obtained from fresh or frozen media. Suitable cells include muscle cells, (mesenchymal and hematopoietic) stem cells, fibroblasts, and cardiac cells. Cells may be endogenous cells (obtained from a host) or derived from suitable cultured cell lines. Cells may be collected from mammalian subjects and / or by biopsy (e.g., muscle biopsy) to enable autologous transplantation of recombinant cell lines into host myocardium.

[0129] Pharmaceutical composition The Cx45 inhibitors described herein may be incorporated into pharmaceutical compositions for human or animal use. These pharmaceutical compositions may contain one or more pharmaceutically acceptable diluents, carriers, or excipients. Carriers or diluents acceptable for therapeutic use are well-known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by AR Gennaro, 1985). The choice of pharmaceutical carrier, excipient, or diluent may be selected in relation to the intended route of administration and standard pharmacopoeia.

[0130] Depending on the various delivery systems employed, there may be a need for various compositions / formulations. For example, pharmaceutical compositions may be formulated for parenteral administration (e.g., intravenous, transdermal, subcutaneous, intramuscular, intraperitoneal, intrathecal, intracardiac, intraarticular, intrapleural, intracerebral, intracranial, intramyocardial, intraarterial, or intravenous), mucosal administration (e.g., oral, intrarectal, intranasal, buccal, vaginal, respiratory), intestinal administration (e.g., oral, such as by tablets, capsules, or drops), or transdermal administration (topical, e.g., transdermal, inhalation, intranasal, or ophthalmic).

[0131] Compositions / formulations suitable for parenteral administration may include aqueous or non-aqueous injectable solutions of Cx45 inhibitors. These compositions / formulations may also contain buffers and solutes to make the composition / formulation isotonic with the blood of the target subject. Aqueous or non-aqueous suspensions may also contain suspending agents and thickeners.

[0132] The composition / formulation may comprise an injectable, stable, sterile composition containing a Cx45 inhibitor or a salt thereof. The Cx45 inhibitor may be provided in the form of a lyophilized product that can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. Those skilled in the art will understand that the composition / formulation may comprise agents that contribute to the pharmaceutically acceptable nature of the composition / formulation. Examples of such agents include, but are not limited to, saline, phosphatidylcholine, or glucose.

[0133] Any suitable pharmaceutically effective drug used for the treatment of myocardial infarction, arrhythmia, cardiac disease characterized by abnormal conduction, sudden cardiac death, left ventricular dysfunction, or ventricular dyssynchrony may be administered concurrently with the Cx45 inhibitors described herein.

[0134] The present invention is further described with reference to the following unlimited embodiments. [Examples]

[0135] [Example 1] Materials and methods plasmid Recombinant adeno-associated virus serotype 2 / 9 (rAAV) vectors were obtained from Dr. Jim Wilson at the University of Pennsylvania. The vectors contained Woodchuck hepatitis virus post-translational (WPRE) regulatory elements. Full-length cDNAs of rat Cx43 and Cx45 were PCR-amplified from a rat cardiac cDNA library and cloned into the multi-cloning site of the rAAV vector. The vectors were sequenced and named rAAV.Cx43 and rAAV.Cx45, respectively. For control experiments, GFP was subcloned into the rAAV vector. The control vectors were sequenced and named rAAV.GFP.

[0136] Small hairpin RNAs targeting Cx45 (Cx45-shRNAs) were designed using web-based prediction software. To ensure that the sequences were specific to rat Cx45, homology analysis was performed for each oligonucleotide using a rat genome database. A forward oligonucleotide containing a sense sequence (i.e., the first sequence) and an antisense sequence (i.e., the second sequence) connected by a loop region was designed. A reverse oligonucleotide complementary to the forward sequence was also constructed. The forward and reverse oligonucleotides were annealed to create a double-stranded DNA construct, which was then cloned into a lentiviral shRNA expression vector as described previously (Cingolani et al., 2007). The following Cx45-shRNAs were constructed using this approach. The target region of Cx45-shRNA #1 has the nucleotide sequence shown in SEQ ID NO: 3. The forward and reverse oligonucleotide sequences for Cx45-shRNA#1 (including not only the first sequence, loop region sequence, and second sequence, but also additional 5' and 3' sequences) are shown in SEQ ID NOs: 4 and 5, respectively. The target region of Cx45-shRNA#2 has the nucleotide sequence shown in SEQ ID NOs: 6. The forward and reverse oligonucleotide sequences for Cx45-shRNA#2 are shown in SEQ ID NOs: 7 and 8, respectively. The target region of Cx45-shRNA#3 has the nucleotide sequence shown in SEQ ID NOs: 9. The forward and reverse oligonucleotide sequences for Cx45-shRNA#3 are shown in SEQ ID NOs: 10 and 11, respectively. The target region of Cx45-shRNA#4 has the nucleotide sequence shown in SEQ ID NOs: 12. The forward and reverse oligonucleotide sequences for Cx45-shRNA#4 are shown in SEQ ID NOs: 13 and 14, respectively. The target region of Cx45-shRNA#5 has the nucleotide sequence shown in SEQ ID NOs: 15. The forward and reverse oligonucleotide sequences for Cx45-shRNA#5 are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively. The target region of Cx45-shRNA#6 has the nucleotide sequence shown in SEQ ID NO: 18.The forward and reverse oligonucleotide sequences for Cx45-shRNA#6 are shown in SEQ ID NOs. 19 and 20, respectively. The target region of Cx45-shRNA#7 has the nucleotide sequence shown in SEQ ID NO. 21. The forward and reverse oligonucleotide sequences for Cx45-shRNA#7 are shown in SEQ ID NOs. 22 and 23, respectively. The target region of Cx45-shRNA#8 has the nucleotide sequence shown in SEQ ID NO. 24. The forward and reverse oligonucleotide sequences for Cx45-shRNA#8 are shown in SEQ ID NOs. 25 and 26, respectively. For control experiments, scrambled control sequences (shNS) that are not complementary to any known mammalian gene sequences were also created.

[0137] To determine the efficacy of the designed shRNAs in inhibiting Cx45 expression, full-length rat Cx45 cDNA was cloned into a commercially available vector (Clontech) such that the Cx45 cDNA was fused with GFP cDNA within the frame to produce a Cx45 protein with a GFP tag for fluorescence quantification of Cx45 expression. The ability of individual shRNAs to inhibit Cx45 expression was tested in the HEK-293 cell line. Vectors expressing Cx45 and vectors expressing shRNAs targeting either Cx45 or a scrambled control sequence were co-introduced into HEK-293 cells. Cx45 expression was examined 48 hours after co-introduction. GFP expression levels were used as an indicator of Cx45 expression. Subsequently, for stable shRNA delivery, the selected shRNAs were cloned into the KpnI site downstream of the RNA polymerase III promoter in an rAAV vector. For control experiments, a scrambled control sequence was cloned into an rAAV vector.

[0138] animal All animal experiments were approved by the Western Sydney Area Health Service Animals Ethics Committee. Female Sprague-Dawley rats weighing approximately 200 grams were used in all experiments and were provided by the Australian Resource Centre (ARC, Canning Vale, West Australia). The rats were housed under conditions of constant ambient temperature (22°C), humidity, and a 12-hour light-dark cycle. The rats were fed commercially available pellets (Allied Foods, Sydney, Australia) and provided with access to feed and water. The rats were housed in wire cages (550 × 370 × 170 mm) with open tops and containing wood shavings for bedding. A maximum of three rats were placed per cage. After the protocol was completed, all animals were euthanized using a CO2 chamber.

[0139] Anesthesia was initiated in a custom-made Perspex chamber with a mixture of oxygen and 5% isoflurane (Aerrane; Baxter Healthcare) supplied via a flexible PVC tube from a small animal anesthesia machine (The Stinger; Advanced Anaesthesia Specialists). The flow rate into the chamber was set to a tidal volume of 4 mL. Once unconscious, the rat was placed dorsally on a custom-made intubation board to facilitate neck extension. Under direct pharyngoscopy, the tongue was pulled out with straight, serrated forceps (Fine Science Tools), and a rigid, non-bending hippocampal tool (Fine Science Tools) was inserted into the pharynx to support the anterior cervical tissue. Once the vocal cords were visible, an 18-gauge intravenous cannula (Becton, Dickinson and Company) was inserted under direct visualization using the Seldinger technique. The rat was then moved dorsally onto a bread cutting stand (Ikea). A flexible PVC tube was attached to a cannula and connected to a small animal ventilator (Harvard Apparatus) configured to deliver a tidal volume of 2.5 mL at a rate of 70 breaths per minute. The ventilator received a mixture of oxygen and 2% isoflurane from the small animal anesthesia machine.

[0140] Tail vein injection At the very tip, a 25-gauge needle was used to insert the cannula into the tail vein. Once blood flow was clearly visible through the syringe, the tourniquet was removed and the viral vector, i.e., rAAV.Cx43, rAAV.Cx45, rAAV.GFP, Cx45-shRNA#1, Cx45-shRNA#2, Cx45-shRNA#3, Cx45-shRNA#4, Cx45-shRNA#5, Cx45-shRNA#6, Cx45-shRNA#7, Cx45-shRNA#8 or rAAV.shNS(1×10⁻¹⁰)⁻¹⁰ viral vector was inserted. 12 The individual vector genomes (diluted in 500 μL of 0.9% saline) were slowly injected. Following the injection of the vector, the rats were rinsed with 500 μL of 0.9% saline. The rats were then removed from the nose cones and allowed to recover.

[0141] Isolation of neonatal rat ventricular cardiomyocytes Two littermates (1-3 days old Sprague-Dawley rats) were used for each culture of neonatal rat ventricular cardiomyocytes. The littermates were sprayed with 70% ethanol, decapitated with large, straight surgical scissors (Roboz Surgical Instrument Co), and thoracotomy was performed with curved, delicate scissors (Roboz Surgical Instrument Co). The ventricles were resected and finely chopped in ice-cold Hanks equilibrium salt solution (HBSS) (Sigma-Aldrich) that did not contain calcium and magnesium. After rinsing twice with ice-cold HBSS, the ventricular tissue was transferred to a glass jar containing 40 mL of trypsin / HBSS solution and stirred on an orbital shaker at 75 rpm at 4°C for 16 hours. The medium was then aspirated and discarded. Warm 10% serum medium was added and gently stirred in a 37°C water bath for 4 minutes. The medium was aspirated and discarded again. Fresh collagenase / HBSS (8 mL) was added to the tissue and gently stirred for 1 minute in a 37°C water bath. The collagenase / HBSS solution was aspirated and discarded. 8 mL of fresh collagenase / HBSS solution was added to the tissue and gently stirred for 1 minute in a 37°C water bath. To aid in the separation of muscle cells, the tissue and medium were passed up and down once using a 10 mL blood pipette. The medium was aspirated and added to ice-cold HBSS and kept on ice. Another 8 mL of collagenase / HBSS was added to the tissue, and this process was repeated three times. Four aliquots of the HBSS solution containing the separated cells were allowed to precipitate at 50 × g for 8 minutes at 4°C. The supernatant was aspirated, and the pellet was resuspended in 8 mL of ice-cold HBSS. The cell suspension was filtered through a 40 μm cell strainer (BD Biosciences). The filtrate was centrifuged at 50 × g for 6 minutes at 4°C, and the supernatant was aspirated and discarded. The pellet was resuspended in warm 10% serum medium and heated at 150 cm². 2 The cells were pre-seed in tissue culture flasks and incubated at 37°C and 5% CO2 for 2 hours. This pre-plating step allowed for the removal of fibroblasts.

[0142] Next, the culture medium containing non-adherent cardiomyocytes was aspirated and placed into a 50 mL polypropylene tube. The tissue culture flask was carefully rinsed with 10% serum medium, and the rinse solution was also added to the 50 mL polypropylene tube. The medium was gently mixed once by pipetting up and down. Further 10% serum medium was added to the cell suspension, and 7 × 10⁶ cells were added. 5 A final density of 100 cardiomyocytes / mL was achieved. A confluent monolayer was achieved 24 hours after plating at this concentration.

[0143] Twenty-four hours after plating the cardiomyocytes, the culture medium was aspirated, and the myocytes were washed once with pre-warmed PBS at 37°C. Fresh 10% serum medium, also pre-warmed at 37°C, was added to the myocytes, and the cells were returned to a humidified incubator. To prevent fibroblast proliferation, the culture medium was replaced with fresh 2% serum medium, also pre-warmed at 37°C, at 48 and 90 hours after plating.

[0144] Cells were cultured in 24-well plates. Newborn RVMs were cultured at a rate of 1 × 10⁶ per well. 11 Transduced into rAAV.Cx45 with vector genomes of the order of magnitude. After 48 hours, they were fixed and immunostained.

[0145] Preparation of ventricular tissue The rats were euthanized with CO2 and weighed. Their hearts were removed, rinsed with PBS, and then weighed.

[0146] For protein and mRNA studies, the heart was placed in a centrifuge tube, frozen with liquid nitrogen, and then stored at -80°C. Subsequently, the cardiac tissue was cut into 6 μm sections and placed on glass slides for immunohistochemical staining and studies of inflammation and fibrosis.

[0147] For histological and immunohistochemical studies, rats transduced with rAAV.Cx43 or rAAV.Cx45 were immediately embedded in Tissue-Tek OCT compound (Sakura). For rats transduced with rAAV.GFP, the heart was fixed overnight at 4°C in 4% PFA before embedding in Tissue-Tek OCT. All embedded tissues were frozen in liquid nitrogen and transferred to storage at -80°C.

[0148] Protein extraction Frozen ventricular tissue was placed in ice-cold round-bottom microcentrifuge tubes. The tissue used for protein extraction was flash-frozen with nitrogen at the time of collection. Using a surgical scalpel on a metal plate on the surface of dry ice, the tissue was divided into 50 mg sections, which were then placed in new microcentrifuge tubes and held on dry ice. Ice-cold steel balls (Qiagen) were inserted into the tubes with 500 μL of ice-cold dissolution (RIPA buffer (Sigma-Aldrich), 1% (v / v) phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and 4% (v / v) protease inhibitor cocktail (Sigma-Aldrich)). The tissue was subjected to 3 minutes of disruption using a bead mill with a TissueLyser (Qiagen) set to 30 Hz. This was repeated twice to ensure thorough homogenization of the tissue. The suspension was then centrifuged at 10,000 rpm for 5 minutes at 4°C. The supernatant was decanted into a second microcentrifuge tube and kept on ice, ready for protein evaluation.

[0149] Protein evaluation was performed using the Bradford method. Standards and samples were assayed three times in a row. Bovine serum albumin (1 mg / mL) was added to different wells of a 96-well plate in 1 μL increments from 1 μL to 8 μL and used to construct a standard curve. The extracted protein sample was diluted 5-fold with MQ H2O and 1 μL was added to each well. Protein assay dye reagent concentrate (Bio-Rad) was diluted 4-fold and 200 μL was added to each well. The absorbance used for measurement was 595 nm using a Victor2 Multilabel Counter (PerkinElmer Life Sciences). The amount of protein was calculated by comparing its absorbance to the standard curve. This was then corrected for dilution and averaged to determine the original protein concentration of each lysis supernatant.

[0150] Immunoblotting Polyacrylamide 4–12% gradient Bis-Tris precast gels (NuPAGE; Invitrogen) were used. These were placed in a gel electrophoresis chamber (XCell SureLock Min-Cell; Invitrogen) containing electrophoresis buffer (MOPS, NuPAGE; Invitrogen). For each sample, 20 mg of protein was diluted in a microcentrifuge tube to a final volume of 20 μL containing 2 μL of reducing agent (NuPAGE; Invitrogen), 5 μL of LDS sample buffer (NuPAGE; Invitrogen), and MQH2O. The sample was then subjected to heat at 95°C for 10 minutes to denature the protein, and then added to a separate gel well. A pre-colored protein ladder (PageRuler Plus; Thermo Scientific) was used to assess protein separation and approximate protein size, and placed in a well adjacent to the test sample. A constant current gradient of 200 V was applied to the gel for 1 hour. Following separation by electrophoresis, proteins were transferred from the gel to the membrane using the IBLOT gel transfer system (Invitrogen). The gel was placed in the gel transfer stack (Invitrogen), and the IBLOT default 7-minute transfer protocol was used. The membrane was then blocked in TBST (0.05% Tween 20, Tris-buffered saline) with 5% skim milk solution for 2 hours, and then incubated with primary antibodies diluted in the blocking solution (mouse Cx45 monoclonal antibody, 1:200, Millipore; or rabbit Cx43 monoclonal antibody, 1:800, Millipore). These were placed on an orbital shaker for gentle agitation overnight at 4°C.

[0151] The following morning, the membranes were washed three times with TBST for 5 minutes each, and incubated for 1 hour with a secondary antibody (HRP-conjugated rabbit anti-mouse antibody, 1:10000, Sigma; or HRP-conjugated goat anti-rabbit antibody, 1:10000, Sigma) diluted in blocking solution. The membranes were again placed on an orbital shaker for gentle agitation. The membranes were washed again three times with TBST for 5 minutes each. SuperSignal Pico Chemiluminescent substrate (Thermo Fisher Scientific) blotting detection reagent was then added to the membranes over 5 minutes, mixed in a 1:1 ratio according to the manufacturer's instructions. To allow detection of weak signals, SuperSignal west Femto (Thermo Scientific) was mixed with the Pico substrate in a 1:10 ratio and then added to the membranes over 5 minutes. The membranes were transferred to an autoradiography cassette (Hypercassette; Amersham Biosciences). In a darkroom, X-ray film (Hyperfilm ECL; Amersham) was exposed to a nitrocellulose membrane in an autoradiography cassette. The exposure time depended on the requirements of the experiment. The film was then processed in an AGFA film processor according to the manufacturer's instructions.

[0152] co-immunoprecipitation Frozen ventricular tissue was pulverized, homogenized, and dissolved in a buffer solution containing 1.5% NP-40, 1% Triton X-100, 0.1% SDS, 0.1% BSA, and a protease inhibitor. For pull-down experiments, either anti-Cx43 monoclonal antibody or anti-Cx45 monoclonal antibody was incubated with Protein G Sepharose on a shaker at 4°C for 1 hour. The beads were then washed three times with lysis buffer at 4°C. Next, to immunoprecipitate the proteins bound to Cx43 and Cx45, respectively, lysates containing 1 mg equivalent of each protein were incubated with the antibody-bound beads in the presence of a protease inhibitor cocktail (Sigma catalog number P8340) at 4°C for 1 hour. Mouse IgG1 (Sigma catalog number 1538) was used as a negative control. The immunoprecipitation was allowed to settle by microcentrifugation at 16,000 × g for 15 minutes at 4°C, washed three times with lysis buffer containing BSA, suspended in a sample additive solution containing β-mercaptoethanol, and inactivated by heat at 95°C for 10 minutes before being subjected to the immunoblotting analysis described above. Rabbit Cx45 polyclonal antibody (donated by Dr. M. Koval) and rabbit Cx43 polyclonal antibody (Chemicon) were used for immunoblotting.

[0153] Preparation of ventricular tissue sections All frozen sectioning was performed using a Microm HM 505E microtome cryostat. Before beginning tissue handling and cutting, the temperature of the cooling chamber was set to -20°C. Frozen ventricular tissue embedded in OCT compound was placed on a cryostat chuck pre-cooled with dry ice and then placed on the cryostat arm. Using a microtome knife, the specimen was first trimmed, then 5 μm–7 μm sections of the ventricle were cut and transferred to glass slides (Superfrost Ultra Plus; Thermo Scientific), and kept in the chamber until moved to a -20°C freezer as needed for use.

[0154] Fixation, permeabilization, and immunohistochemical staining Ventricular tissue sections were fixed with 4% (w / v) paraformaldehyde and subjected to permeabilization with 0.1% (v / v) Triton X-100 (Sigma-Aldrich) in PBS for 15 minutes, followed by blocking with serum-free Dako Protein Block. The samples were then incubated overnight at 4°C with primary antibodies diluted in blocking solution (mouse Cx45 monoclonal antibody, 1:200, Millipore; and rabbit Cx43 monoclonal antibody, 1:800, Millipore). Secondary antibodies diluted in 0.1% (v / v) chilled fish skin gelatin in PBST (Alexa488-conjugated mouse antibody, 1:1000, Invitrogen; or Alexa594-conjugated mouse antibody, 1:2000, Invitrogen; or Alexa594-conjugated rabbit antibody, 1:2000, Invitrogen) were spread onto glass slides in the dark at room temperature for 1 hour. The sample was covered with a coverslip (Deckglaser; Menzel-Glaser) containing a Prolong Gold fading inhibitor with DAPI (Life Technologies), and fixed overnight in the dark at 4°C.

[0155] Co-immunostaining Ventricular tissue sections were fixed with 4% (v / v) paraformaldehyde at room temperature for 15 minutes. Fresh paraformaldehyde was prepared by dissolving 3.2 g in 80 mL of PBS containing calcium and magnesium at 56°C. The pH was adjusted to 7.4 with 5 M NaOH. The slides were washed three times with PBS for 5 minutes each. The samples were subjected to permeabilization with 0.1% (v / v) Triton X-100 (Sigma-Aldrich) in PBS for 15 minutes. This was followed by three gentle washes of 0.05% (v / v) Tween 20 (Sigma-Aldrich) (PBST) in PBS on an orbital shaker for 5 minutes each. Following washing and permeabilization, nonspecific areas were blocked for 1 hour with a blocking solution from Dako (Protein Block, serum-free). Next, the blocking solution was removed, and the sample was incubated overnight at 4°C with the primary antibody diluted in the blocking solution. Considering that this was for co-immunostaining, two primary antibodies were mixed and used (they had different host species to allow for the use of different secondary antibodies).

[0156] Inflammation-Hematoxylin and Eosin Staining Ventricular tissue sections were immersed in hematoxylin for a total of 4 minutes, Scott's blue-tinting solution for 1 minute, and 0.1% eosin for 1 minute. The samples were then dehydrated with anhydrous ethanol for 30 seconds and placed in xylene clearing solution. The coverslip was then mounted on a glass slide with a hydrophobic mounting agent containing colourfast (Fronine), Ultramount No. 4. The glass slide was fixed overnight. The degree of inflammation was then manually assessed and graded based on the scale by Igarrashi et al. (1993).

[0157] Fibrosis - Pico-Sirius Red staining Ventricular tissue sections were fixed with 4% paraformaldehyde for 15 minutes, then washed three times with PBS for 5 minutes each. The sections were then placed in a picrosilius red solution (0.1% Direct Red 80 (Sigma), 0.1% Fast Green FCF (Sigma), saturated picric acid (Sigma)) on an orbital shaker in the dark for 1 hour. The sections were then carefully washed twice with MQH20, followed by three washes with 100% ethanol for 1 minute each to aid in dehydration. They were then air-dried for 1 hour, and then immersed for 5 seconds in xylene clearing solution before mounting the coverslip with a hydrophobic mounting agent, Ultramount No. 4, containing Colorfast (Fronine). The slides were left to set overnight. The sections were then analyzed using custom-made software designed to objectively calculate the relative areas of red (collagen) and green (muscle cells).

[0158] Electrophysiological testing - Surface electrocardiogram Four unipolar needle electrodes (AD Instruments) were placed subcutaneously on both sides of the subclavian and iliac fossa regions. A single ground wire was also placed subcutaneously in the abdominal region. A two-lead electrocardiogram was created by combining the potentials from the left / right lower electrode pair and the right / left upper electrode pair. Biopotentials sampled at 2 kHz were amplified using an Ocal Bio Amp (AD Instruments) and digitized using a Powerlab 16 / 30 (AD Instruments) data acquisition system. Data was recorded via USB using a Windows-based version of Labchart Pro 7 (AD Instruments) running on a Dell laptop. No software filtering was used.

[0159] Electrophysiological testing - Transesophageal cardiac pacing A 5-French octa-electrode catheter (Biosense Webster) connected to an Octal Bio Amp was inserted through the oral cavity into the esophagus and positioned at the point where the amplitude of the ventricular electrocardiogram (VPO) recorded from the two intermediate electrodes was maximum. Recordings were made from only the two intermediate electrodes, and stimulation was delivered only through the two distal electrodes. The ventricles were captured using a constant 60-volt stimulation with a duration of 1 millisecond via an isolated pulse stimulator (Model 2100; AM Systems). Custom-designed software installed on a second Windows-based laptop (Hewlett-Packard) was used to drive the stimulator via its trigger input.

[0160] Programmed ventricular stimulation was used to induce ventricular tachycardia. The heart was paced with a drivetrain of 8 stimuli at 180-millisecond linked intervals, followed by up to four premature stimuli initiated at 150-millisecond intervals and continuously decreased by 10-millisecond intervals until ventricular refractory state. If the programmed ventricular stimulation failed to induce VT, a 30-second cycle with a 90-millisecond cycle length was repeated, and if necessary, a 60-millisecond cycle length was repeated after 3 minutes.

[0161] Stimulation was performed using programmed stimulation and burst pacing. Programmed stimulation included an 8-beat drivetrain with a 180-millisecond linking interval, followed by up to 4 premature beats. Burst pacing included electrical stimulation using a 90-millisecond linking interval for 30 seconds, followed by another 60-millisecond interval for 30 seconds.

[0162] myocardial infarction In short, rats were placed dorsally following anesthesia, ventilation, and baseline EP testing. A total of 10 μg of buprenorphine hydrochloride (Temgesic; Schering-Plough) and 1 mg of enrofloxacin (Baytril 50; Bayer Healthcare AG) were separately diluted in 1 mL of PBS and administered subcutaneously by injection. The hair on the anterior chest was moistened with 70% (v / v) ethanol (Fronine) and shaved off with an electric clipper (Remington; Spectrum Brands). The skin was disinfected with 10% w / v povidone-iodine solution (Povidine-Iodine) (Betadine; Sanofi-Aventis) or chlorhexidine, applied for at least 5 minutes prior to skin incision. 200 nL of 0.1% lignocaine was administered subcutaneously before incision, with the timing adjusted so that it would act before coronary artery ligation.

[0163] Using a surgical scalpel, a transverse incision was made in the skin from the ventral midline to the left anterior axillary line at the level of the fourth intercostal space. To move the surrounding skin, a blunt incision of the underlying superficial fascia was made approximately 1 cm on both sides of the incision using straight hemostatic forceps (Fine Science Tools). The muscles from the left chest wall surface to the ribs were also separated by a blunt incision using straight hemostatic forceps (Fine Science Tools). Using curved hemostatic forceps (Fine Science Tools), the thoracic cavity was punctured through the fourth intercostal space. The punctured intercostal muscle was bluntly incised with the same curved hemostatic forceps, and a Castrovio Joretractor (Roboz) was inserted to open and hold the incised intercostal space. The left atrium became visible, and just below it, a hole was made in the pericardium with curved forceps (Fine Science Tools). This was then carefully resected all the way to the distal ventricular apex. The LAD coronary artery was located at the level of the left atrial appendage, and a 6-0 75 cm suture was placed around the artery approximately 1–2 mm below the adnexa. Both ends of the suture were passed through polyethylene tubes to form a "snare"-like loop with a free end. The snare loop was then pulled for 10 seconds to assess the area of ​​cyanosis, and then released to demonstrate proper occlusion of the LAD by returning to normal color. When approximately 40–50% of the visible wall in the anterior portion was observed to be cyanotic, the LAD was permanently occluded again. If adjustment was necessary, the suture was removed and the final step was repeated to obtain an equal amount of cyanosis in all rats. Following LAD occlusion, the retractor was removed, and the intercostal space was closed with two stitches of 3.0 covered Vicryl suture (Ethicon). The skin incision was then closed using the horizontal mattress technique with 3.0 covered Vicryl suture (Ethicon). The rats were then placed in cages for recovery. The rats' drinking water was supplemented with 0.01% enrofloxacin (Baytril 25; Bayer Healthcare AG). The rats that underwent sham surgery received the exact same surgical procedure, except that their LAD (lead artery disease) was not ligated.

[0164] Data analysis and statistics NQuery Advisor (Statistical Solutions) was used to estimate the required sample size. Quantitative data were expressed as mean ± standard error of the mean (SEM). Continuous variables (such as QRS interval and PR interval) were analyzed using Student's t-test or Mann-Whitney U test. Statistical analysis of 2x2 tables was performed using Fisher's exact test (for cases with 5 or fewer samples in a cell) or using the two-sample Pearson chi-squared (X²) test (2x2 table) in SPSS (version 17) (e.g., for the induction of ventricular tachyarrhythmias). Significance was set at P ≤ 0.05.

[0165] [Example 2] Overexpression of connexin 45 significantly increases ventricular tachyarrhythmias. In vitro studies were conducted to investigate the role of Cx45 in cardiac physiology and arrhythmogenicity. Using the protocol described in Example 1, 1 × 10⁶ doses of rAAV.Cx45 were injected into ventricular cardiomyocytes of neonatal rats. 11 Transduction was performed using a vector genome. Broad expression of Cx45 was detected in transduced neonatal rat ventricular cardiomyocytes (Figure 1). In particular, localized Cx45 expression was observed in the junction membrane. Endogenous Cx45 expression was not detected in untransduced neonatal rat ventricular cardiomyocytes. Western blotting using an anti-Cx45 antibody confirmed Cx45 expression in transduced neonatal rat ventricular cardiomyocytes (Figure 2).

[0166] Cx43 (involved in cardiac electrical impulse propagation and cardiac development) is expressed in the junction membrane between adjacent cells. The expression of Cx45 was compared with that of Cx43. Using the protocol described in Example 1, Sprague-Dawley rats were expressed in 1 × 10¹⁶ units of either rAAV.Cx43 (n=15), rAAV.Cx45 (n=22), or rAAV.GFP (n=15). 12Rats received tail vein injection of vector genomes. On day 28 after injection, ventricular tissue was removed and the expression of Cx43 and Cx45 was investigated. As shown in Figure 3, rats transduced with either rAAV.Cx43 or rAAV.Cx45 yielded broadly transduced cardiomyocytes. Substantial co-localization of Cx43 and Cx45 was detected in intercalated plates. This finding is consistent with Cx43 and Cx45 being able to form connexons.

[0167] The identification of Cx45 as an interaction partner of Cx43 was confirmed by co-immunoprecipitation experiments on ventricular tissue from rats transduced with rAAV.Cx45. The immunoprecipitated proteins were analyzed by SDS-PAGE and subjected to immunoblotting using anti-Cx43 or anti-Cx45 antibodies as described in Example 1. As shown in Figure 4A, Cx45 was detected in the anti-Cx43 immunoprecipitate of ventricular tissue from rats transformed with rAAV.Cx45. In addition, Cx43 was detected in the anti-Cx45 immunoprecipitate of ventricular tissue from rats transformed with rAAV.Cx45 (Figure 4B). In the negative control, neither Cx43 nor Cx45 was observed. These data confirm that Cx43 and Cx45 interact with each other.

[0168] To investigate the electrophysiological effects of increased Cx45 expression, electrophysiological studies were performed in anesthetized rats on day 0 and day 28 after tail vein injection of rAAV.GFP, rAAV.Cx43, or rAAV.Cx45. The electrophysiological studies consisted of surface ECG and transesophageal cardiac pacing as described in Example 1. In short, 15 rats were injected with rAAV.GFP, 15 with rAAV.Cx43, and 22 with rAAV.Cx45. As shown in Figure 5, surface ECG in rats transduced with rAAV.Cx45 showed prolongation of the PR interval (45.8±6.0 vs. 59.0±12.0 ms) and QRS interval (14.8±1.9 vs. 16.2±1.8 ms) over 28 days. Neither rats transduced with rAAV.Cx43 nor rats transformed with rAAV.GFP showed any notable electrophysiological changes. Interestingly, atrioventricular block was observed in five rats transformed with rAAV.Cx45. These data suggest that cardiac overexpression of Cx45 delayed conduction, as evidenced by prolonged ECG parameters, namely prolonged PR and QRS intervals, and AV block.

[0169] In rats transduced with Cx45, an increased incidence of inductive ventricular tachyarrhythmias was also observed (Figure 6). On day 28, 13 rats transduced with rAAV.Cx45 had inductive ventricular tachyarrhythmias compared to 2 rats transduced with rAAV.Cx43. No inductive ventricular tachyarrhythmias were observed in any of the rats transduced with the control vector rAAV.GFP.

[0170] Histological analysis was performed to determine whether the increased tendency for inductive ventricular tachyarrhythmias was caused by inflammation or myocardial fibrosis. No significant differences in inflammation or myocardial fibrosis were observed among the groups (Figures 7 and 8). These data indicate that the increased tendency for inductive ventricular tachyarrhythmias observed in Cx45-transduced rats was unrelated to inflammation or myocardial fibrosis.

[0171] In summary, these results indicate that Cx45 overexpression is associated with delayed electrical propagation in the myocardium and an increased risk of proarrhythmia. Therefore, Cx45 plays a definite role in ventricular conduction.

[0172] [Example 3] Knockdown of shRNA-mediated connexin 45 reduces ventricular tachyarrhythmias in a rat model of myocardial infarction. To further understand the role of Cx45 in proarrhythmics, we investigated Cx45 expression levels in a rodent model of myocardial infarction. This model has been widely used in clinical trials applied to humans as a tool to evaluate cell therapy and gene therapy approaches for left ventricular dysfunction after myocardial infarction (Terrovitis et al., 2008; Terrovitis et al., 2009; del Monte et al., 2004).

[0173] Compared to rats subjected to sham surgery after myocardial infarction, Cx45 expression levels were significantly increased in ventricular tissue (Figure 9). To evaluate the effect of reducing Cx45 in this model, several shRNAs targeting Cx45 were designed as described in Example 1.

[0174] To determine the efficiency and specificity of candidate shRNAs, eight Cx45-shRNAs targeting various regions of the rat Cx45 mRNA transcript were constructed. These vectors were transfected into HEK-293 cells along with plasmids encoding rat Cx45 fused with GFP (Cx45-GFP). The ability of Cx45-shRNAs to inhibit Cx45 expression was examined by immunofluorescence. As shown in Figure 10, simultaneous introduction of Cx45-shRNA#1, Cx45-shRNA#2, Cx45-shRNA#3, and Cx45-shRNA#4 significantly reduced Cx45-GFP expression, as indicated by a decrease in the number of cells exhibiting green fluorescence, whereas the scrambled control sequence (shNS) did not.

[0175] To confirm that the candidate shRNA can inhibit Cx45 expression in vivo, rAAV.Cx45 and either candidate Cx45-shRNA or rAAV.shNS were co-introduced into rats. Adult Sprague-Dawley rats received an intravenous injection of 1×10 12 vector genomes of each vector using the protocol described in Example 1. On the 35th day after injection, Cx45 ventricular tissue was removed and examined for the expression of Cx43 and Cx45. As shown in Figure 11, Cx45-shRNA#1 was able to inhibit Cx45 expression 5 weeks after the first transduction. These data indicate the extended activity of the shRNA described herein. No change in Cx43 expression was observed (Figure 12). Since no detectable change in the expression level of Cx43 was observed, these data indicate that the observed inhibitory effect was specific to Cx45.

[0176] Since elevated levels of Cx45 were observed in ventricular tissue after myocardial infarction, the ability of candidate shRNAs to reduce myocardial infarction-related complications such as arrhythmia was investigated. After occlusion of the left anterior descending artery using the protocol described in Example 1, 22 rats were injected with Cx45-shRNA#1 and 17 rats were injected with rAAV.shNS. A significant reduction in Cx45 expression was observed 5 weeks after myocardial infarction and transduction (Figure 12). A significantly reduced incidence of inducible ventricular tachycardia arrhythmia was also observed in rats transduced with Cx45-shRNA#1 (Figure 13). Eight rats transduced with Cx45-shRNA#1 had inducible ventricular tachycardia arrhythmia compared to 12 rats transduced with rAAV.shNS. No evidence of spontaneous ventricular tachycardia arrhythmia was observed in either group.

[0177] These data clearly demonstrate that reducing Cx45 overexpression reduces the incidence of arrhythmias (such as ventricular tachyarrhythmias). Furthermore, these data demonstrate that reducing Cx45 expression has beneficial effects by mitigating the conduction deceleration associated with increased Cx45 expression. Therefore, the data provided herein validate the therapeutic use of Cx45 antagonists.

[0178] Those skilled in the art will understand that a great many variations and / or modifications can be made to the above embodiments without deviating from the broad general scope of this disclosure. Therefore, these embodiments should be considered illustrative and non-restrictive in all respects.

[0179] All references cited herein, including patents, patent applications, papers, textbooks, etc., and references cited therein unless already cited herein, are incorporated herein by reference as a whole.

[0180] Any discussion of documents, actions, materials, apparatus, articles, etc., contained herein is solely for the purpose of providing the environment for the present invention. None of these matters, in whole or in part, should be construed as forming part of the basis of the prior art, or as being common knowledge in the art related to the present invention because they existed before the priority date of each claim of this application.

[0181] References

Claims

1. Treating, preventing, inhibiting the progression of, or reducing the likelihood of occurrence of, complications associated with myocardial infarction in a subject for , Cx45 inhibitors A composition comprising .

2. Use of an inhibitor of Cx45 in the manufacture of a medicament for treating, preventing, inhibiting the progression of, or reducing the likelihood of occurrence of myocardial infarction-related complications in a subject.

3. 2. The method of claim 1, wherein the myocardial infarction-related complications are selected from the group consisting of arrhythmia, abnormal conduction, sudden cardiac death, left ventricular dysfunction, and ventricular dyssynchrony. The composition or use according to claim 2 .

4. The myocardial infarction-related complication is arrhythmia. 3 Described in Composition or Use .

5. Treating, preventing, inhibiting the progression of, or reducing the likelihood of the occurrence of cardiac dysfunction characterized by abnormal conduction in a subject for , an inhibitor of Cx45 A composition comprising .

6. Use of an inhibitor of Cx45 in the manufacture of a medicament for treating, preventing, inhibiting the progression of, or reducing the likelihood of occurrence of a cardiac dysfunction characterized by abnormal conduction in a subject.

7. the cardiac dysfunction is selected from the group consisting of arrhythmia, left bundle branch block, right bundle branch block, bundle branch block, atrioventricular block, nonspecific intraventricular conduction delay, nonspecific interventricular conduction delay, left ventricular dysfunction, and ventricular dyssynchrony; The composition according to claim 5 or the use according to claim 6 .

8. the abnormal conduction is a slowing of conduction velocity in cardiac cells; A composition according to claim 5 or 7 or a use according to claim 6 or 7. .

9. the abnormal conduction causes arrhythmia, A composition according to any one of claims 5, 7 and 8 or a use according to any one of claims 6 to 8 .

10. 2. The method of claim 1 , wherein the arrhythmia is a ventricular arrhythmia. 3, 4, 7 and 9 any one of the items Composition or Use .

11. The ventricular arrhythmia is ventricular fibrillation , ventricular tachycardia, or both A claim 10 Described in Composition or Use .

12. The Cx45 inhibitor is administered in an amount effective to reduce or inhibit prolongation of the QRS interval and / or PR interval in a subject.

12. A composition according to any one of claims 1, 3 to 5 and 7 to 11, or a use according to any one of claims 2 to 4 and 6 to 11, wherein .

13. The inhibitor of Cx45 is a small molecule, a protein, or a nucleic acid. A composition according to any one of claims 1, 3 to 5 and 7 to 12 or a use according to any one of claims 2 to 4 and 6 to 12 .

14. The Cx45 inhibitor is an RNA molecule a nucleic acid molecule comprising a DNA sequence encoding That is, A composition according to any one of claims 1, 3 to 5 and 7 to 13 or a use according to any one of claims 2 to 4 and 6 to 13 .

15. 15. The method of claim 14, wherein the RNA molecule comprises a first sequence and a second sequence, the first sequence being at least 12 nucleotides in length and having at least 70% sequence identity to a target region of an mRNA transcript set forth in SEQ ID NO: 1, and the second sequence having at least 70% sequence identity to the reverse complement of the first sequence. Composition or Use .

16. An RNA molecule comprising a first sequence and a second sequence. a nucleic acid molecule comprising a DNA sequence encoding wherein the first sequence is at least 12 nucleotides in length and has at least 70% sequence identity to the target region of the mRNA transcript set forth in SEQ ID NO:1, and the second sequence has at least 70% sequence identity to the reverse complement of the first sequence. nucleic acid molecule .

17. the first sequence has 100% sequence identity to the target region; 17. A composition or use according to claim 15 or a nucleic acid molecule according to claim 16. .

18. the second sequence has 95% sequence identity to the reverse complement of the first sequence; A composition or use according to claim 15 or 17, or a nucleic acid molecule according to claim 16 or 17. .

19. The target region is set forth in any one of SEQ ID NOs: 3, 15, or 18. A composition or use according to any one of claims 15, 17 and 18, or a nucleic acid molecule according to any one of claims 16 to 18. .

20. 20. A composition or use according to any one of claims 15 and 17 to 19, or a nucleic acid molecule according to any one of claims 16 to 19, wherein the first sequence comprises the sequence set forth in SEQ ID NO: 4 and the second sequence comprises the sequence set forth in SEQ ID NO:

5.

21. 21. The composition or use of any one of claims 14, 15 and 17 to 20, or the nucleic acid molecule of any one of claims 16 to 20, wherein the DNA sequence encoding the RNA molecule is operably linked to a promoter.

22. Claim Any one of items 16 to 21 The nucleic acid described in molecule A vector comprising:

23. 22. The composition or use of any one of claims 14, 15 and 17 to 21, wherein the nucleic acid molecule is contained within a vector.

24. Vector A viral vector. 22 The vector described in or the composition or use according to claim 23 .

25. The viral vector A recombinant adeno-associated virus vector. 24 The vector described in , composition or use .

26. 10. The recombinant adeno-associated virus vector of claim 9, wherein the recombinant adeno-associated virus vector is an adeno-associated virus serotype 2 / 9 virus vector. 25 The vector described in , composition or use .

27. Claim Any one of items 16 to 21 The nucleic acid described in molecule or claims 22 and any one of 24 to 26 A pharmaceutical composition comprising the vector according to any one of the preceding claims and a pharmaceutically acceptable carrier or diluent.

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