Treatment of arrhythmogenic cardiomyopathy

Abstract

The present invention relates to treating or preventing arrhythmogenic cardiomyopathy (ACM), Brugada syndrome (BrS) and hypertrophic cardiomyopathy (HCM), and agents for use in such methods. The agent is an inhibitor of the JAK / STAT pathway. The invention also provides methods of diagnosing a patient with ACM, BrS or HCM. The invention further provides methods of monitoring or evaluating the effect of treating ACM, BrS or HCM.

Description

[0001] TREATMENT OF ARRHYTHMOGENIC CARDIOMYOPATHY Field of the invention

[0002] The invention relates to methods of preventing and / or treating diseases which predispose patients to sudden cardiac death (SCD). Specifically, the present invention provides methods of treating or preventing arrhythmogenic cardiomyopathy (ACM), Brugada syndrome (BrS) and hypertrophic cardiomyopathy (HCM), and agents for use in such methods. The invention also provides methods of diagnosing a patient with ACM, BrS or HCM. The invention further provides methods of monitoring or evaluating the effect of treating ACM, BrS or HCM.

[0003] Background to the Invention

[0004] Arrhythmogenic cardiomyopathy (ACM) is a familial non-ischemic heart disease, affecting 1:2000 to 1:5000 individuals in the general population.1It is one of the leading causes of sudden cardiac death (SCD) in young individuals due to malignant ventricular arrhythmias.1,2Other causes of SCD are Brugada syndrome (BrS) and hypertrophic cardiomyopathy (HCM). Individuals with ACM may also progress to end-stage heart failure (HF). Currently, treatments such as antiarrhythmics, standard medical therapies for HF, and implantable cardioverter defibrillators (ICDs) only temporize symptoms, and heart transplantation represents the only curative therapy.3,4

[0005] ACM is primarily caused by mutations in genes encoding for cardiac desmosomal proteins.5Most ACM cases stem from mutations in the desmosomal genes plakophilin-2 (PKP2), desmoplakin (DSP) and desmoglein-2 (DSG2).5How these mutations result in ACM is not clearly understood but has been thought to involve abnormal NFKB, Wnt / 0-catenin, GSK30 and Hippo signalling pathways.6'9Recent studies have shown that ACM is associated with a striking cardiac and systemic inflammatory response.10It has previously been observed that using both a pharmacological and genetic approach to block NFKB signalling prevents myocardial loss and fibrosis, preserve contractile function and suppress arrhythmias in DSg2mut / mutmice.12NFKB signalling in cardiac myocytes leads to a 5 -fold increase in monocytes and macrophages expressing C-C motif chemokine receptor-2 (CCR2),9a potent chemotactic molecular that has been implicated in adverse cardiac remodelling and fibrosis.13'15Suppression of CCR2+monocyte and macrophage recruitment to the heart was sufficient to halt progression of ACM pathology in D g2mut / mutmice.9

[0006] However, effective mechanism-based drug therapy of ACM to reduce the risk of SCD would require long-term administration. Long-term inhibition of either GSK30 or NFKB may be expected to have unacceptable adverse consequences, including impaired immune response, increased susceptibility to infections, tissue damage and potential disruption of normal cellular functions.16,24Significant gaps were present in the understanding of how inflammatory monocyte and macrophage populations contribute to heart failure, myocardial inflammation, fibrosis and arrhythmogenesis. Such knowledge is critical to identify novel, effective yet safe, therapeutic targets for this devastating disease.

[0007] Summary of the Invention

[0008] Herein, the inventors provide, for the first time, evidence that inhibition of the JAK / STAT pathway can be used to treat ACM. Janus kinases (JAKs) are intracellular, non-receptor tyrosine kinases. JAK activation leads to phosphorylation of signal transducers and activators of transcription (STATs). STATs then enter the nucleus and initiate transcription of genes regulating immunity and inflammation.25

[0009] The inventors have surprisingly found that the clinically used JAK / STAT inhibitors tofacitinib and baricitinib showed a remarkable ability to rescue the ACM phenotype in a robust mouse model in vivo. The inventors have also provided evidence of JAK / STAT involvement in the pathogenesis of BrS and HCM, which are also associated with SCD. In contrast, there was no evidence of aberrant JAK / STAT activation in a range of other cardiac diseases. Taken together, these observations provide new insights into mechanisms of pathogenesis of SCD-causing entities and identify new therapeutic strategies to limit myocardial damage and reduce the risk of serious arrhythmias in patients with ACM, BrS or HCM. The inventors have thus demonstrated for the first time that inhibition of the JAK / STAT pathway may be therapeutically beneficial in ACM, BrS and HCM.

[0010] Accordingly, the invention provides a method of treating and / or preventing arrhythmogenic cardiomyopathy (ACM), Brugada syndrome (BrS) or hypertrophic cardiomyopathy (HCM), comprising administering a therapeutically effective amount ofan agent to a subject, wherein the agent is an inhibitor of one or more Janus Kinases (JAKs).

[0011] The invention also provides an agent for use in a method of treating and / or preventing ACM or BrS in a subject, wherein the method comprises administering a therapeutically effective amount of the agent to the subject, wherein the agent is an inhibitor of one or more JAKs.

[0012] The invention also provides a method of diagnosing ACM, BrS or HCM in a subject, comprising detecting and / or quantifying the activation of the JAK / Signal Transducer and Activator of Transcription (STAT) pathway in a biological sample taken from the subject.

[0013] The invention also provides a of monitoring and / or evaluating the effect of treatment of ACM, BrS or HCM, wherein the method comprises detecting and / or quantifying the activation of the JAK / STAT pathway according to the method of diagnosis provided by the invention.

[0014] Brief Description of the Figures

[0015] Figure 1: Relative quantification of proteins from the JAK / STAT pathway showing a positive score in cellular material from an ACM patient compared to a control.

[0016] Pathway enrichment analysis identified translocation of 9 JAK / STAT proteins (GRB2, HRAS, BCL2L1, EGFR, STAT3, STAT1, STAT6, STAM, PTPN11 and IFNGR1) to nuclear fraction in buccal cell material obtained from an ACM patient bearing a PKP2 mutation compared to an aged-matched control.

[0017] Figure 2: Distribution ofIFNGRI in patient myocardial samples

[0018] Representative IFNGR1 immunoperoxidase staining in myocardial samples from controls and patients with different forms of heart disease. IFNGR1 shows strong nuclear localization in samples from patients with ACM, BrS or HCM.

[0019] Figure 3: Distribution ofIFNGRI in patient buccal smear samples Representative confocal immunofluorescence images showing distribution of IFNGR1 in buccal smear samples from a control, a patient diagnosed with ACM (ACM5), a patient diagnosed with BrS (BrS9) and a silent carrier of an ACM-causing mutation (ACM 10). IFNGR1 showed strong nuclear localization only in the patients manifesting clinical evidence of disease.Figure 4: JAK / STAT activation in hiPSCs-cardiac myocytes from a patient diagnosed with HCM

[0020] Representative confocal immunofluorescence images from cultures of cardiac myocytes (CMs) derived from a control hiPSC cell line and a line from a patient with HCM. Control hiPSC-CMs showed normal cytoplasmic distribution of IFNGR1.

[0021] Marked nuclear accumulation of IFNGR1 was seen in all the hiPSC-CMs derived from the HCM patient.

[0022] Figure 5: JAK / STAT inhibition prevents ACM development in vivo

[0023] Group data for percent ejection fraction (A) and fractional shortening (B) in vehicle- and tofacitinib-treated Dsg2"1L'l / "lulmice. Quantitative data are shown as mean ±SEM; n=8 for vehicle-treated Dsg2"1L'l / "lulmice and n=8 for tofacitinib-treated Dsg2mut / mutmice. P<0.001 for tofacitinib-treated mice vs. vehicle-treated animals.

[0024] Figure 6: JAK / STAT inhibition decreases fibrosis in ACM mouse hearts

[0025] (A) Representative trichrome-stained hearts from mice at 16 weeks of age. Scale bars: 1mm. (B) Percent (%) myocardial fibrosis in tofacitinib-treated versus vehicle-treated animals.

[0026] Figure 7: Inflammatory / fibrotic mediators are at least partially normalised following tofacitinib treatment

[0027] Selected list of pro -inflammatory cytokines / chemokines and fibrokines / adipokines significantly down-regulated following JAK / STAT blockage in Dsg2mut / mut mice. Welch’s t-test was used for comparisons between cohorts.

[0028] Figure 8: Protein-protein interactions

[0029] Protein-protein interaction analysis was performed using the STRING database for the 37 candidate proteins shown to be significantly down-regulated by tofacitinib in the cytokine array. Nodes represent individual proteins, and edges indicate known or predicted functional associations based on experimental evidence, curated databases, co-expression, and text mining. JAK1 / JAK3, the main targets of tofacitinib, are shown as central hubs, with the majority of the array-identified proteins connected directly or indirectly to them. The network highlights extensive connectivity among the identified proteins and supports their convergence on JAK / STAT pathway-related signalling. Figure 9: Baricitinib decreases fibrosis in ACM mouse hearts(A) Representative trichrome-stained hearts from mice at 10 and 16 weeks of age. (B) Percent (%) myocardial fibrosis in baricitinib-treated versus vehicle-treated animals (p=0.0442 for 10-week animals and p=0.0051 for 16-week animals).

[0030] Figure 10: Baricitnib abolishes nuclear localisation ofIFNGRI

[0031] Representative immunoperoxidase images showing the distribution of IFNGR1 in myocardial samples from Dsg2mut / mutanimals treated with baricitinib versus vehicle.

[0032] Figure 11: Baricitnib abolishes nuclear localisation ofRelA

[0033] Representative confocal immunofluorescence images showing the distribution of RelA in myocardial samples from Dsg2mut / mutanimals treated with baricitinib versus vehicle.

[0034] Figure 12: Baricitinib treatment restored plakoglobin signal at the intercalated disks to levels comparable to those observed in wildtype animals

[0035] Representative confocal immunofluorescence images showing the distribution of plakoglobin in myocardial samples from Dsg2mut / mutanimals treated with baricitinib versus vehicle.

[0036] Figure 13: Baricitinib treatment restored Cx43 signal at the intercalated disks to levels comparable to those observed in wildtype animals

[0037] Representative confocal immunofluorescence images showing the distribution of Cx43 in myocardial samples from Dsg2mut / mutanimals treated with baricitinib versus vehicle.

[0038] Figure 14: Nuclear localisation of STAT3 was observed in ACM samples from boxer dog myocardial tissue

[0039] Representative immunoperoxidase staining for STAT3 in myocardial tissue from control Boxer dogs and Boxer dogs diagnosed with ACM. Prominent nuclear localisation of STAT3 is observed in ACM samples, whereas control specimens show no detectable nuclear signal.

[0040] Figure 15: Nuclear localisation of STAT3 was observed in ACM samples from boxer dog buccal smears

[0041] Representative confocal immunofluorescence images illustrating STAT3 distribution in buccal smear samples obtained from one control Boxer dog and two Boxer dogs diagnosed with ACM. Strong nuclear localisation of STAT3 is observed insamples from ACM-affected dogs, whereas no nuclear signal is detected in the control specimen.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0044] In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an amino acid sequence” includes two or more such sequences, and the like.

[0045] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise. In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0046] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0047] Reference to “significant” in the context of a difference, for example, a “significant increase”, “significant decrease” or “significant difference”, refers to a statistically significant difference, as determined using an appropriate statistical test. Appropriate statistical tests will be known to those skilled in the art.

[0048] Agent for use in method of treating or preventing ACM, BrS, or HCM

[0049] The agent used in the treatment or prevention of ACM, BrS or HCM according to the invention is an inhibitor of one or more Janus Kinases (JAKs). Therefore, the agentreduces the activity of the one or more JAKs. In other words, the activity of the one or more JAKs is lower in the presence of the agent than in the absence of the agent.

[0050] A “therapeutically effective amount” of the agent for use as described herein will be a dose that will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of an agent for use as described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically effective in 50% of a population). Efficacy can be determined in terms of therapeutic effects or in terms of inhibition of the JAK / STAT pathway, as described below. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. An agent for use that exhibits large therapeutic indices is preferred. In one embodiment, the agent for use according to the invention, exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject.

[0051] Janus kinases (JAKs) are intracellular, non-receptor tyrosine kinases. 4 members of the JAK family are known: Janus Kinase 1 (JAK1), JAK2, JAK3 and TY2K.

[0052] In some embodiments, the agent inhibits all of JAK1, JAK2, JAK3 and TYK2. In other words, the agent may be a pan- JAK inhibitor.

[0053] In some embodiments, the agent selectively or specifically inhibits one particular JAK, or a subset of JAKs. In the context of the present invention, by “selectively” inhibiting a particular JAK or a subset of JAKs, the agent inhibits that JAK or subset of JAKs to a significantly greater degree than other JAKs. The degree of inhibition of each JAK can be quantified by measuring inhibition of the JAK / STAT pathway as described below, before and after administration of the agent, and the inhibition of each JAK by the agent compared using an appropriate statistical test. In such embodiments, it may be that the selective JAK inhibitor has a greater binding affinity for that particular JAK orthe subset of JAKs than other types of JAK. Binding refers to the formation of a stable complex between multiple molecules. In some embodiments, a complex may be considered stable if it has a dissociation constant (KD) of 500 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM or less than 0.5 nM, as measured in a surface plasmon resonance assay.

[0054] Accordingly, an agent which selectively inhibits a JAK may have a greater tendency to form stable complexes with that specific JAK than with other JAKs.

[0055] However, an agent which selectively inhibits one JAK may still bind or inhibit other types of JAK which are not the JAK which it selectively inhibits. For example, an agent which is a selective inhibitor of JAK1 inhibits JAK1 to a greater extent than it inhibits JAK2, JAK3 and TYK2, though in some embodiments a selective inhibitor of JAK1 may nonetheless bind and / or inhibit JAK2, JAK3 and / or TYK2, albeit to a lesser extent than JAK1.

[0056] In some embodiments, the agent selectively inhibits JAK1. In some embodiments, the agent selectively inhibits JAK2. In some embodiments, the agent selectively inhibits JAK3. In some embodiments, the agent selectively inhibits TYK2.

[0057] In some embodiments, the agent selectively inhibits JAK1 and JAK2. In some embodiments, the agent selectively inhibits JAK1 and JAK3. In some embodiments, the agent selectively inhibits JAK1 and TYK2.

[0058] In some embodiments, the agent selectively inhibits JAK2 and JAK3. In some embodiments, the agent selectively inhibits JAK2 and TYK2. In some embodiments, the agent selectively inhibits JAK3 and TYK2.

[0059] In preferred embodiments, the agent selectively inhibits JAK1, JAK2 and JAK3. In some embodiments, the agent selectively inhibits JAK1 and JAK3, and inhibits JAK2 to a lesser extent than it inhibits JAK1 and JAK3, while not significantly inhibiting TYK2.

[0060] In preferred embodiments, the agent selectively inhibits JAK1 and JAK2.

[0061] In some embodiments, the agent selectively inhibits JAK1, JAK2 and JAK3. In other words, the agent inhibits TYK2 to a lesser extent than it inhibits JAK1 , JAK2 andJAK3. In some embodiments, the agent selectively inhibits TYK2, JAK2 and JAK3. In other words, the agent inhibits JAK1 to a lesser extent than it inhibits TYK2, JAK2 and JAK3. In some embodiments, the agent selectively inhibits TYK2, JAK1 and JAK3. In other words, the agent inhibits JAK2 to a lesser extent than it inhibits TYK2, JAK2 and JAK3. In some embodiments, the agent selectively inhibits JAK1, JAK2 and TYK2. In other words, the agent inhibits JAK3 to a lesser extent than it inhibits JAK1 , JAK2 and JAK3.

[0062] In some embodiments, the agent specifically inhibits one particular JAK. In the context of the invention, by “specifically” inhibiting a particular JAK, the agent does not significantly inhibit other types of JAK. The degree of inhibition of each JAK can be quantified by measuring inhibition of the JAK / STAT pathway as described below, before and after administration of the agent, and the inhibition of each JAK by the agent compared using an appropriate statistical test. In such embodiments, it may be that the specific JAK inhibit does not form stable complexes with JAKs other than the JAK which it specifically inhibits. An agent does not significantly inhibit a JAK if the activity of JAK in the presence of the agent is not significantly different from the activity of the JAK in the absence of the agent.

[0063] In some embodiments, the agent specifically inhibits JAK1. In some embodiments, the agent specifically inhibits JAK2. In some embodiments, the agent specifically inhibits JAK3. In some embodiments, the agent specifically inhibits TYK2.

[0064] JAK activation leads to phosphorylation of signal transducers and activators of transcription (STATs). STATs then enter the nucleus and initiate transcription of genes regulating immunity and inflammation.25Accordingly, the inhibitor reduces the ability of the one or more JAKs to phosphorylate STATs.

[0065] Therefore, inhibition of the one or more JAKs may be confirmed by quantifying the phosphorylation of at least one appropriate STAT in the presence and in the absence of the agent, and comparing the two measurements. For example, if phosphorylation of the STAT is significantly lower in the presence of the agent compared to in the absence of the agent, this may be used to indicate that the agent is inhibiting the one or more JAKs.

[0066] Alternatively, inhibition of the one or more JAKs may be confirmed by quantifying the nuclear translocation of at least one appropriate STAT, or interferongamma receptor 1 (IFNGR1), in the presence of the agent and in the absence of the agent, and comparing the two measurements. Nuclear translocation of a STAT or IFNGR1 can be quantified by carrying out subcellular protein fractionation, then quantifying the amount of the STAT or IFNGR1 in the nuclear fraction relative to nonnuclear fractions, as described in the Examples. For example, if the nuclear translocation of the STAT or IFNGR1 is significantly greater in the presence of the agent than in the absence of the agent, this may be used to indicate that the agent is inhibiting the one or more JAKs.

[0067] Alternatively, nuclear translocation of a STAT or IFNGR1 may be determined by immunofluorescence performed on buccal smears taken from a subject or a group of subjects. Buccal smears (obtained as described herein) may be obtained from a subject who has not undergone treatment with the agent, and from the same subject who is undergoing treatment with the agent. Antibodies which are specific for the STAT or IFNGR1 may be used to stain the buccal smears. Immunofluorescence may then used to determine the intracellular distribution (i.e. detecting and / or quantifying the signal from the STAT or IFNGR1 in the nucleus, optionally relative to non-nuclear signal from the STAT or IFNGR) in the subject when they have not been treated with the agent, and in the same subject who is undergoing treatment with the agent.

[0068] Typically, the agent directly inhibits one or more JAKs, i.e. through binding of the agent to the one or more JAKs, or direct interaction of the agent with the one or more JAKs. However, in some instances the agent may also indirectly inhibit the one or more JAKs. In this scenario, the agent may bind to / interact with another target, but a downstream effect is the inhibition of the one or more JAKs.

[0069] In some embodiments, the agent is a reversible inhibitor of the one or more JAKs. In some embodiments, the agent is an irreversible inhibitor of the one or more JAKs. In some embodiments, the agent is a competitive inhibitor of the one or more JAKs, i.e. it binds the JAK’s active site. In some embodiments, the agent is an allosteric inhibitor of the one or more JAKs i.e. it binds to a region on the JAK outside the JAK’s active site.

[0070] In some embodiments, the agent prevents the activation of the one or more JAKs by cytokines and / or interferons. JAKs are often present downstream of a receptor for one or more cytokines and / or interferons, which activate the one or more JAKs upon activation of the receptor by its ligand by causing the JAK to dimerise andautophosphorylate. Accordingly, in some embodiments, the agent may uncouple ligand binding to the receptor from the activation of JAK. In some embodiments, the agent prevents the dimerization of the JAK. In some embodiments, the agent prevents the JAK from autophosphorylating.

[0071] In some embodiments, the agent prevents the activation of the one or more JAKs by cytokines and / or interferons by binding a receptor for a cytokine or interferon(s). Accordingly, in some embodiments, the agent is an antagonist of a receptor for one or more cytokines or interferons. In such embodiments, the agent binds to the receptor but does not activate signalling through the receptor, therefore the binding of cytokines or interferons fail to activate one or more JAKs through the receptor, thus the agent inhibits one or more JAKs. In some embodiments, the agent is an antagonist antibody of a receptor for one or more cytokines or interferons.

[0072] Typically, the agent is a small molecule. In some embodiments, the agent is abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, or upadacitinib. In preferred embodiments, the agent is tofacitinib, baricitinib or ruxolitinib. In particularly preferred embodiments, the agent is baricitinib.

[0073] An appropriate therapeutically effective amount can be determined by the person skilled in the art.

[0074] In some embodiments, the agent is tofacitinib, and the therapeutically effective amount is a 5mg or lOmg dose administered orally twice daily. In other words, the method of the invention comprises administering 5mg or lOmg tofacitinib orally twice daily to the subject.

[0075] In some embodiments, the agent is baricitinib, and the therapeutically effective amount is a 2mg oral dose administered twice daily. In other words, the method of the invention comprises administering 2mg baricitinib orally twice daily to the subject.

[0076] In some embodiments, the agent is ruxolitinib, and the therapeutically effective amount is a 5-20mg oral dose administered twice daily. Blood tests may be performed on the patient regularly during treatment to determine whether ruxolitinib is affecting platelet numbers, and this may be used to inform an appropriate dose of the ruxolitinib. In other words, the method of the invention comprises administering 5-20mg ruxolitiniborally twice daily to the subject. The method may further comprise performing blood tests on the subject regularly during treatment to inform dosage of ruxolitinib.

[0077] Therapeutic effects

[0078] As described above, the invention provides a method of treating and / or preventing arrhythmogenic cardiomyopathy (ACM) or Brugada syndrome (BrS) or hypertrophic cardiomyopathy (HCM), comprising administering a therapeutically effective amount of an agent to a subject, wherein the agent is an inhibitor of one or more JAKs, and agents for use in such methods. Treatment may result in reduced activity of the JAK / STAT pathway, which may be measured as described above.

[0079] Treatment or prevention may result in one or more therapeutic effects. In some embodiments, the treatment or prevention reduces the risk of sudden cardiac death (SCD). Sudden cardiac death (SCD) is a sudden and unexpected death due to a cardiovascular cause. In other words, a population of subjects with ACM, BrS or HCM who are treated with the agent may have an incidence of SCD which is significantly lower than a population of subjects with ACM, BrS or HCM who are not treated with the agent. Overall survival may also be significantly higher in the group who are treated with the agent than the group who are not treated with the agent.

[0080] In some embodiments, the treatment or prevention prevents or reduces myocardial necrosis. Myocardial necrosis refers to the death of cardiomyocytes.

[0081] Myocardial necrosis may be detected in a subject using serological markers of myocardial necrosis such as cardiac troponins e.g. cardiac troponin I and cardiac troponin T. Accordingly, a subject to whom the agent is administered may have significantly reduced levels of cardiac troponins in serum compared to the same subject before administration of the agent and when compared to subjects to whom the agent has not been administered. In other words, treatment with the agent may significantly reduce the levels of cardiac troponins in serum in a subject compared to the same subject before administration of the agent. Moreover, a subject who has been treated with the agent may have significantly lower levels of cardiac troponins in their serum when compared to equivalent subjects who have not been treated. Alternatively,myocardial necrosis may result in abnormalities in an ECG. Abnormalities in an ECG are described elsewhere herein.

[0082] In some embodiments, the treatment or prevention prevents or reduces myocardial fibrosis. Typically, the treatment or prevention prevents further myocardial fibrosis from occurring, or reduces the extent of further myocardial fibrosis which may occur. Myocardial fibrosis may be determined, for example, by Cardiovascular Magnetic Resonance (CMR) imaging. In some embodiments, the subject to whom the agent is administered may have significantly reduced myocardial fibrosis as detected by CMR imaging, compared to the same subject before administration of the agent and / or a subject to whom the agent has not been administered. In some embodiments, reduced myocardial fibrosis may be determined by a reduced fibrotic area as detected by CMR imaging. In other words, treatment with the agent may significantly reduce the fibrotic area in the myocardium in a subject compared to the same subject before treatment with the agent. Moreover, a subject who has been treated may have a significantly smaller fibrotic area in the myocardium when compared to equivalent subjects who have not been treated with the agent.

[0083] In some embodiments, the treatment or prevention prevents or reduces myocardial inflammation. CMR imaging, for example, may also be used to detect myocardial inflammation, by detecting associated tissue changes such as oedema, hyperaemia, monocyte injury and fibrosis. In some embodiments, the subject to whom the agent is administered may have significantly reduced myocardial inflammation (as detected by observation of associated tissue changes such as oedema, hyperaemia, monocyte injury and fibrosis by CMR imaging) compared to a subject to whom the agent has not been administered, or compared with the same subject before administration of the agent. In other words, treatment with the agent may significantly reduce myocardial inflammation (as detected by observation of associated tissue changes such as oedema, hyperaemia, monocyte injury and fibrosis by CMR imaging) compared to the same subject before treatment with the agent. Moreover, a subject who has been treated may have significantly lower myocardial inflammation (as detected by observation of associated tissue changes such as oedema, hyperaemia, monocyte injury and fibrosis by CMR imaging) compared to equivalent subjects who have not been treated with the agent.ACM, BrS and HCM are associated with a progressive deterioration of ventricular contractile function resulting from the development of extensive myocardial necrosis, fibrosis and inflammation. In some embodiments, the treatment or prevention slows or prevents the deterioration of ventricular contractile function. Ventricular contractile function can be measured, for example, by the ventricular ejection fraction, preferably the left ventricular ejection fraction. The ejection fraction is the proportion of blood in the ventricle which is ejected with each ventricular contraction. The ejection fraction may be measured using a suitable imaging technique, such as echocardiogram, magnetic resonance imaging, computerised tomography or nuclear medicine scan. Accordingly, a change or deterioration in ventricular contractile function of a subject may be measured by measuring the ventricular ejection fraction periodically, such as every 3 months or every year, and the change between the periodic measurements determined. A reduction in the ventricular ejection fraction measured over a series of periodic measurements may indicate a deterioration in ventricular contractile function. Measurements of ventricular ejection fraction may also be taken periodically before the agent is administered to the subject. In some embodiments, the treatment or prevention results in the decrease in ventricular ejection fraction measured over a series of periodic measurements in the subject who has been treated with the agent being of a significantly smaller magnitude than the reduction in ventricular ejection fraction in the same subject before the agent has been administered to the subject, and / or compared to the reduction in ventricular ejection fraction in a different subject to whom the agent has not been administered.

[0084] In some embodiments, the treatment or prevention prevents deterioration of ventricular contractile function. In such embodiments, the treatment or prevention means that the ventricular ejection fraction of the subject who is treated does not significantly decrease during the period in which periodic measurements are taken. In some embodiments, the treatment or prevention may improve ventricular contractile function. In such embodiments, the treatment or prevention means that the ventricular ejection fraction significantly increases during the period in which periodic measurements are taken.

[0085] In some embodiments, the treatment or prevention reduces arrhythmia in the subject. Arrythmia can be detected, for example, by monitoring the electrical activity ofthe heart using an electrocardiogram (ECG) and optionally comparing the trace to a healthy (non-arrhythmic) control, noting and optionally quantifying the frequency or proportion of abnormal heartbeats or noting and optionally quantifying the frequency of abnormalities in the heartbeat. An abnormal heartbeat is a heartbeat as identified by ECG which contains an abnormality, such as those described below. In some embodiments, the ECG may be taken using a Holter monitor, which allows for recording over a period of 24 hours or more.

[0086] Arrythmia also includes an abnormal heart rate, such as tachycardia or bradycardia, which may also be detected using an ECG or a heart rate monitor, and optionally comparing to a healthy control who does not have an abnormality in the heart rate. Arrythmia may also include the presence of ectopic heartbeats, detectible in an ECG trace by the presence of small beats of lower than normal amplitude between normal heartbeats.

[0087] A baseline measurement for a subject may be taken by performing an ECG to detect arrythmia before the agent is administered. In some embodiments, the treatment or prevention means that the subject to whom the agent has been administered has a significantly lower frequency or proportion of abnormal heartbeats as determined by ECG compared to the same subject before the agent has been administered to the subject, and / or compared to a different subject to whom the agent has not been administered.

[0088] In some embodiments, the treatment or prevention means that the subject to whom the agent has been administered has a significantly lower frequency of abnormalities in a heartbeat shown in an ECG trace compared to the same subject before the agent has been administered to the subject, and / or compared to a different subject to whom the agent has not been administered. Abnormalities in a heartbeat may include T-wave inversions, QRS prolongation or epsilon waves.

[0089] In some embodiments, the treatment or prevention abolishes arrythmia. In such embodiments, the treatment or prevention means that when an ECG is performed, no abnormalities in the heartbeat or abnormal heartbeats are detected, or the frequency of abnormalities in the heartbeat or abnormal heartbeats does not significantly differ from the number in a comparable subject who does not have arrythmia, or the subject has aheart rate which does not significantly differ from a healthy control who has a normal heart rate.

[0090] The methods of the invention may also improve or eliminate signs and / or symptoms of ACM, including fatigue, palpitations, light-headedness, fainting, and shortness of breath.

[0091] The methods of the invention may also improve or eliminate signs and / or symptoms of BrS, including fainting, palpitations, shortness of breath and seizures.

[0092] The methods of the invention may also improve or eliminate signs and / or symptoms of HCM, including chest pain, shortness of breath, fatigue, palpitations, fainting, and swelling of the legs, feet, ankles and / or abdomen.

[0093] Method of diagnosing ACM, BrS or HCM

[0094] In another aspect, the invention also provides a method of diagnosing ACM, BrS or HCM in a subject, comprising detecting and / or quantifying the activation of the JAK / Signal Transducer and Activator of Transcription (STAT) pathway in a biological sample taken from the subject. ACM, BrS or HCM as selected when practicing embodiments of this aspect of the invention may be referred to as the “condition to be diagnosed”. In some embodiments, if a positive diagnosis is made, the method further comprises treating the subject with the agent for use according to the invention, as described herein.

[0095] In some embodiments, the method further comprises comparing the activation of the JAK / STAT pathway in the biological sample obtained from the subject to the activation of the JAK / STAT pathway in a biological sample obtained from a different subject who does not have the condition to be diagnosed i.e. ACM, BrS or HCM or a subject who does not have any diagnosed inflammatory condition (referred to as a negative control sample). An inflammatory condition is a disease characterised by pathological long-term inflammation. Inflammatory conditions include cystic fibrosis, chronic inflammatory intestinal diseases like, for example, ulcerative colitis or Crohn's disease, vasculitis, in particular Kawasaki disease, chronic bronchitis, inflammatory arthritis diseases like, for example, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, and systemic onset juvenile rheumatoid arthritis (SOJRA, Still's disease), graft-versus-host disease, asthma, psoriasis and systemic lupus erythematosus. The negative control may be determined from a group of subjects. In some embodiments, the subject is diagnosed as having the condition to be diagnosed if the activation of the JAK / STAT pathway in the biological sample taken from the subject is significantly higher than the activation of the JAK / STAT pathway in the negative control sample.

[0096] In some embodiments, the method further comprises comparing the activation of the JAK / STAT pathway in the biological sample obtained from the subject to the activation of the JAK / STAT pathway in a biological sample obtained from a different subject who has the condition to be diagnosed i.e. ACM, BrS or HCM (referred to as a positive control sample). Once again, the positive control may be based on a group of subjects with the condition. In some embodiments, the subject is diagnosed as having the condition to be diagnosed if the activation of the JAK / STAT pathway in the biological sample taken from the subject is not significantly lower than the activation of the JAK / STAT pathway in the positive control sample.

[0097] In some embodiments, the method comprises comparing the activation of the JAK / STAT pathway in the biological sample taken from the subject to both a positive and negative control sample. In some embodiments, the subject is diagnosed with the condition if the activation of the JAK / STAT pathway in the biological sample taken from the subject is significantly higher than the activation of the JAK / STAT pathway in the negative control sample, and the activation of the JAK / STAT pathway in the biological sample taken from the subject is not significantly lower than the activation of the JAK / STAT pathway in the positive control sample.

[0098] Proteins which may be monitored to determine activation JAK / STAT signalling pathway include Growth factor receptor-bound protein2 (GRB2), H-Ras protein (HRAS), Bcl-2-like protein 1 (BCL2L1), epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 1 (STAT1), signal transducer and activator of transcription 6 (STAT6), signal transducing adapter molecular 1 (STAM), tyrosine -protein phosphatase non-receptor type 11 (PTPN11) and interferon gamma receptor 1 (IFNGR1)].

[0099] In some embodiments, the detecting and / or quantifying the activation of the JAK / Signal Transducer and Activator of Transcription (STAT) pathway in a biologicalsample taken from the subject comprises detecting and / or quantifying nuclear localisation of one or more STATs or interferon gamma receptor 1 (IFNGR1). Nuclear translocation can be quantified by carrying out subcellular protein fractionation of cells in the biological sample taken from the subject, then quantifying the amount of the STAT or IFNGR1 in the nuclear fraction relative to non-nuclear fractions, as described in the Examples.

[0100] In preferred embodiments, the biological sample obtained from the subject is a buccal sample. A buccal sample may be obtained by collecting material from the inside of the cheek by using scraping motions as described in the Examples. In other embodiments, the sample is a myocardial sample. A myocardial sample may be obtained by a solid tissue biopsy.

[0101] Subject

[0102] In some embodiments of any of the aspects of the invention, the subject is an individual who exhibits signs and / or symptoms of ACM, BrS, or HCM. In embodiments of the diagnostic methods of the invention, the condition of which the subject is exhibiting signs and / or symptoms may be the condition to be diagnosed. Signs and symptoms of ACM include: arrythmia, fatigue, ankle swelling, palpitations, fainting, shortness of breath, chest pain. Signs and symptoms of BrS include: fainting, palpitations, shortness of breath, seizures, arrythmia. Signs and symptoms of HCM include: chest pain, shortness of breath, fatigue, palpitations, fainting, swelling of the legs, feet, ankles and abdomen.

[0103] In some embodiments of any of the aspects of the invention, the subject is identified as having one or more mutations associated with ACM, BrS or HCM. In some embodiments, the method of treating or preventing ACM, BrS or HCM may further comprise a first step of genetically testing the subject for one or more mutations associated with ACM, BrS and / or HCM. The one or more mutations may comprise one or more heterozygous, nonsense, point, frameshift or splice site mutations. The one or more mutations may comprise one or more dominant or recessive mutations.

[0104] Mutations in desmosomal genes may be associated with ACM. Around 60% of patients identified as having ACM have mutations in desmosomal genes. In some embodiments, the one or more mutations may include one or more mutations in one ormore desmosomal genes, such as any combination of plakophilin-2 (PKP2), desmoglein-2 (DSG2), desmoplakin (DSP), and rarely, desmocollin-2 (DSC) and plakoglobin (JUP). In some embodiments, the one or more desmosomal genes comprises or consists of DSG2. In some embodiments, the one or more desmosomal genes comprises or consists of PKP2. The subject may, for example, have heterozygous nonsense, point, frameshift or splice site mutations in PKP2.

[0105] Extra- desmosomal genes may also be associated with ACM. Around 20% of patients identified as having ACM have mutations in extra-desmosomal genes. In some embodiments, the one or more mutations include mutations in one or more extra-desmosomal genes. In some embodiments, the one or more mutations comprises mutations in any combination of the following extra- desmosomal genes: filamin C, titin, desmin and Transmembrane Protein 43 (TMEM43).

[0106] Mutations in genes encoding sarcomere proteins, such as Myosin Heavy Chain 7 (MYH7), Troponin T2, Cardiac Type (TNNT2), Troponin 13, Cardiac Type (TNNI3), Actin Alpha Cardiac Muscle 1 (ACTC1) and Myosin Binding Protein C3 (MYBPC3) are associated with HCM. In some embodiments, the one or more mutations comprise or consist of mutations in one or more genes encoding sarcomere proteins. In some embodiments, the one or more genes comprises mutations in any combination of the following genes: MYBPC3, MYH7, TNNT2, TNNI3 and ACTC1.

[0107] In preferred embodiments, subjects to be treated according to the invention are subjects identified as having one or more mutations associated with ACM, BrS or HCM, but who do not yet exhibit any clinical signs or symptoms of ACM, BrS or HCM. Signs and symptoms of ACM, BrS and HCM are described above.

[0108] Method of monitoring and / or evaluating

[0109] In another aspect, the invention also provides a method monitoring and / or evaluating the effect of treatment of ACM, BrS or HCM in a subject, wherein the method comprises detecting and / or quantifying the activation of the JAK / STAT pathway according to the method of diagnosis provided by the invention. In some embodiments, the treatment is treatment with an agent for use according to the invention.In some embodiments, the method comprising detecting and / or quantifying the activation of the JAK / STAT pathway before and after treatment. In some embodiments, the method may comprise detecting and / or quantifying the activation of the JAK / STAT pathway before, after and during treatment. In some embodiments, the method comprises comparing the activation of the JAK / STAT pathway after and / or during treatment with the activation of the JAK / STAT pathway before treatment.

[0110] In some embodiments, the subject is considered as having a positive response to treatment if the activation of the JAK / STAT pathway after and / or during treatment is significantly lower than the activation of the JAK / STAT pathway before treatment. In some embodiments, the subject is considered as having a positive response to treatment if during or after treatment with the agent, the activation of the JAK / STAT pathway is not significantly higher than in a negative control sample, wherein the negative control sample is taken from a subject who does not have the condition being treated. In some embodiments, the subject is considered as having a positive response to treatment if during or after treatment with the agent, the activation of the JAK / STAT pathway is significantly lower than in a positive control sample, wherein the positive control sample is taken from a subject who has the condition being treated.

[0111] In some embodiments, the subject is considered as having a negative response to treatment if the activation of the JAK / STAT pathway after and / or during treatment is not significantly lower than the activation of the JAK / STAT pathway before treatment. In some embodiments, the subject is considered as having a negative response to treatment if during or after treatment with the agent, the activation of the JAK / STAT pathway is significantly higher than in a negative control sample. In some embodiments, the subject is considered as having a negative response to treatment if during or after treatment with the agent, the activation of the JAK / STAT pathway is not significantly lower than in a positive control sample.EXAMPLES

[0112] METHODS

[0113] Ethical approval: This study was approved by the UK National Health Service Research Ethics Committee (hearts: 17 / LO / 0747, buccal smears / pellets: 17 / LO / 0840). Informed consent was provided by next-of-kin at the time of autopsy and by study participants whose buccal cells were sampled.

[0114] Buccal mucosa sampling for mass spectrometry: Sterile, individually-wrapped toothbrushes were used for collection of buccal cells from an ACM patient bearing a frameshift mutation in PKP2 and an age-matched control with no history of heart disease. Material was collected from the inside of the cheek by using scraping motions for about 30 seconds on each side. Immediately after collection, the buccal cell material was washed in PBS, centrifuged (1800rpm, 5 mins) and the pellets were processed for subcellular protein fractionation.

[0115] Subcellular protein fractionation: The Thermo Scientific™ Subcellular Protein Fractionation Kit (#87790) was used for the stepwise separation and preparation of cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein extracts from the buccal cells pellets as per the manufacturer’s instructions.

[0116] Mass spectrometry: Subcellular fractions were concentrated, and the buffer was exchanged to 100 mM triethylammonium bicarbonate with the Amicon Ultra Centrifugal Filter, 3 kDa MWCO (4 x 400 pL). Proteins were reduced and alkylated with 5 mM TCEP (Thermo, Bond-breaker) and 10 mM iodoacetamide (IAA) respectively, followed by overnight digestion with trypsin at 50 ng / pL. Peptides were SpeedVac dried, reconstituted in 200 pL of 0.1% TFA and 10 pL were injected for LC-MS Data Independent Acquisition (DIA) analysis. DIA analysis was performed on an UltiMate 3000 system coupled with the Orbitrap Ascend Mass Spectrometer (Thermo) using a 25 cm capillary column (Waters, nanoE MZ PST BEH130 C18, 1.7 pm, 75 pm x 250 mm) over a 100 min gradient 5%-27% mobile phase B composed of 80% acetonitrile, 0.1% formic acid. MSI spectra were collected with mass resolution of 60K in the m / z range of 380-985, with Maximum Injection Time 100 ms and AGC 4x105. DIA MS2 spectra were collected with HCD fragmentation CE 32%, orbitrap resolution 15K with isolation window 10 m / z and 1 m / z overlap, Maximum Injection Time 40 ms and AGC 1 x 105. Raw data were processed in the DIA-NN software version 1.8.1 forprotein identification and quantification in the library-free mode using a fasta file containing reviewed UniProt human proteins. Carbamidomethylation of C and oxidation of M were defined as fixed and variable modifications respectively. RT-dependent cross-run normalization was selected with MBR enabled and proteins were filtered at 1% FDR. Data were additionally normalized so that the total protein intensity is equal between the same subcellular fraction of the two individuals (group normalization). The normalized abundances were scaled so that row mean is 100, and ARVC quantitation was corrected for the respective Control sample values using linear regression per protein (x=Control, y=ARVC). Pathway analysis was performed on the residuals of the linear regression in the Perseus platform.20

[0117] Myocardial samples: Myocardial samples were analysed from 123 patients with different forms of heart disease. Diagnosis and tissue source for all patient samples are included in Table 1. Myocardial samples from aged-matched subjects who had died of non-cardiac causes and had no family history of heart disease acted as negative controls (n=19).

[0118] Immunohistochemistry of myocardial samples: Formalin-fixed, paraffin-embedded ventricular myocardium from patients and age-matched controls was analysed by immunoperoxidase staining using a primary antibody against IFNGR1 (Abeam #Ab 134070). Sections (5 pm thick) were deparaffinized, dehydrated, rehydrated and exposed to 3% hydrogen peroxide solution for 10 minutes to block endogenous peroxidase activity. Sections were first incubated with 5% donkey serum (Stratech Scientific) and 5% BSA (Merck Life Science) in lx TBS blocking solution for Ihr then overnight at 4°C with a rabbit anti-IFNGRl polyclonal antibody (LSBiosciences, LS-C375948). The following day, sections were incubated with horseradish peroxidase donkey anti -rabbit secondary antibody (ThermoFisher Scientific) for Ihr at room temperature. Peroxidase-conjugated antibodies were detected by the 3,3'-diaminobenzidine (DAB) substrate kit (AbCam). Slides were then counterstained with Mayer’s hematoxylin (Cellavision) and bright field images were taken with a Nikon Eclipse 80i microscope and recorded with Nikon DS-Fil camera.

[0119] Masson’s Trichrome Staining: Hearts were formalin-fixed, paraffin-embedded (FFPE) and blocks cut at 5pm (2-3 cuts per slide) and stained with either Masson’s Trichrome (Sigma; Cat. No. HT15-1KT) or Hematoxylin and Eosin (H&E Kit; Abeam,Cat. No. ab245880) following the manufacturer’s protocol. Myocardial sections were traced using ImageJ to achieve total area. Then blue fibrotic sections were traced to record total fibrosis. Percent myocardial fibrosis was determined by the sum of all fibrotic areas (within one slice) divided by total myocardial area using ImageJ version 1.53e software. Each slice was then averaged to obtain percent myocardial fibrosis for one mouse.

[0120] Patient selection for buccal smears: Buccal mucosa smears were obtained from 23 BrS patients, 7 patients diagnosed with ACM and 4 silent carriers of ACM-causing mutations. Clinical diagnostic and genetic information is shown in Table 2. Buccal smears from 20 age-matched individuals without clinical signs or family history of heart disease were used as negative controls.

[0121] Buccal smear preparation: Clean, individually- wrapped cytology brushes were used for collection of buccal cells. Material was collected from the inside of the cheek by using slight rolling and scraping motions for about 30 seconds on each side.

[0122] Immediately after collection, the buccal mucosa material was smeared on positively-charged microscopy slides, which were sprayed with a fixative (M-FIX, Merck) from a distance of ~30 cm. The slides were allowed to air-dry and stored at room temperature before being immunostained.

[0123] Immunocytochemistry of buccal smears: Buccal smears were first incubated with blocking solution for 1 hr and then with a rabbit monoclonal anti-IFNGRl antibody (abl34070, AbCam) overnight at 4°C. The following day, the smears were incubated with anti-rabbit IgG Cy3 -labeled secondary antibody, counterstained with 4',6-diamidino-2-phenylindole (DAPI) and mounted with ProLong Gold. Images from 5 random fields / section were recorded using a Nikon AIR confocal microscope.

[0124] Animals: All animal studies were in full compliance with policies of Beth Israel Deaconess Medical Centre and conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH publication no. 85-23, revised 1996). Mice were housed in a 12-hour-light / dark cycle, climate-controlled facility with ad libitum access to water and standard rodent chow. Studies were performed in wild type (WT) mice and mice with homozygous knock-in of a variant in DSG2, the gene encoding the desmosomal cadherin desmoglein-2 (pSg2mut / mutmice) aspreviously described.8,9,12This variant entails loss of exons 4 and 5 which causes a frameshift and premature termination of translation.

[0125] JAK / STAT inhibition and characterization of disease phenotypes in DSg2mut / mutmice: The effects of a JAK / STAT inhibitor were studied in age-matched WT (n=8) and DSg2mut / mutmice (n=8). 10-week-old animals were randomized to receive tofacitinib (30 mg / kg / day through intraperitoneal injections) or vehicle (DMSO) for 6 weeks. Echocardiograms were obtained in both groups at 16 weeks of age as previously described.8,9,12Briefly, measurements were made according to American Society of Echocardiography guidelines in non-sedated mice with a Vevo 2100 Visualsonic imaging system.21Parasternal long-axis views of the left ventricle, at the level of the papillary muscles, were acquired at a sweep speed of 200 mm / s. Three to five measurements were acquired from each mouse and averaged to assess left ventricular function. Thereafter, animals were euthanized and hearts were collected for additional studies.

[0126] Cytokine measurements: Myocardial cytokines were measured using Proteome Profiler Mouse XL Cytokine Array Kits (R&D Systems, Cat. No. ARY028) (6). Frozen heart samples were lysed in RIPA buffer containing 1:100 protease and phosphatase inhibitor cocktails. Protein content in lysates were quantified via BCA assay and 40pg / pl of protein lysate were probed on cytokine array blots. Following the manufacturer’s protocol, blots were incubated with ECL substrate, imaged on an Azure Biosystems 400 imager and analysed using Quick Spots image analysis software (Version 25.5.1.2, Ideal Eyes Systems).

[0127] Statistical analysis: Ejection fraction (EF) and fractional shortening (FS) data are presented as mean±SEM. A value of P<0.05 was considered statistically significant. Differences in measured variables were assessed with 1-way ANOVA with Tukey post hoc analysis to correct for multiple comparisons. In view of sample sizes, gaussian distributions were assumed. Fibrosis staining data are presented as mean ± standard deviation (SD). Statistical analyses were performed using Prism 8 software (GraphPad Software, Inc., USA). Comparisons between two groups were conducted using the Mann- Whitney test.

[0128] Patient-derived induced pluripotent stem cell (iPSC) generation: Whole blood was collected by regular venepuncture in BD Vacutainer© CPT™ tubes (BDBiosciences, #362761). Peripheral blood mononuclear cells (PMBCs) were isolated from whole blood using Ficoll™ Hypaque™ Solution, according to the Institute of Molecular Medicine (iMM) biobank, Lisbon, Standard Operating Procedure (SOP) protocol, and cryopreserved in 20 % DMSO (Merck, #D8418) in foetal bovine serum (FBS) (Life Technologies, #10500). For reprogramming, PBMCs were thawed and cultured in StemPro™-34 medium (Gibco #10639011), with the appropriate cytokines (IL-3, IL-6, FLT-3 and SCF -Prepotech / Tebu-bio, #200.03, 200.06, 300-19, 300-07, respectively) for 4 days. Cells were then plated at a density of 5x105 cells / ml and transduced using CytoTune™-iPS 2.0 Sendai (SeV) Reprogramming Kit (Thermo Fisher Scientific, #A16517), according to the manufacturers’ instructions. At day 3 post transduction, cells were reseeded into 9.5 cm2culture dishes coated with Matrigel™ (Corning, #354230) and cultured in mTeSR™ Plus (mTeSR + ) Medium (STEMCELL Technologies, #100-0276). Based on stem cell morphology, colonies were individually picked and transferred to new Matrigel coated-plates in the first 5 passages. Cells were then passaged when the colonies covered ~ 80 % of the surface area of the culture dish approximately every 2-3 days, using 0.5 mM EDTA dissociation buffer (ThermoFisher Scientific, #15575020). Medium was changed every two days. Cells were continuously passaged and from passage 10 were constantly monitored for SeV clearance by qRT-PCR (see below). All the experiments documented here were performed on SeV-free iPSCs, in an incubator at 37 °C and 5 % CO2 in normoxia conditions. iPSCs were confirmed to be mycoplasma free using qPCR Mycoplasma Test (Mycoplasmacheck, Euro fins Genomics) in accordance with the manufacturer’s instructions.22

[0129] Cell lines generated: The control cell line was reprogrammed from PBMCs obtained from an individual with no clinical evidence or family history of heart disease. The HCM cell line was reprogrammed from PBMCs obtained from an individual diagnosed with HCM. The patient, who shows maximal wall thickness of 29mm, has a defibrillator and is currently awaiting cardiac transplantation. She is bearing a deep intronic variant in the gene encoding for cardiac myosin binding protein 3 (MYBPC3,' c.1224-52 G>A), predicted to be pathogenic on spliceAI software by disruption of splicing resulting in haploinsufficiency.

[0130] iPSC cardiac myocyte (CM) differentiation: iPSCs were differentiated to CMs using StemMACS™ CardioDiff Kit XF, human (Miltenyi Biotec, #130-125-289)according to the manufacturer’s instructions. Briefly, iPSCs were maintained in StemFlex™ Medium (Gibco, A3349401) on culture dishes coated with Matrigel™ (Corning, #354230). When iPSCs covered ~80% of the surface area of the culture dish, cells were dissociated using Accutase® (Merck, #A6964) and plated on 1.9cm2culture dishes coated with Matrigel™ at a density of 250,000 cells / cm2in Mesoderm Induction Medium. 24 hours later, medium was replaced with Cardiac Cultivation Medium. A further 24 hours later, medium was changed to Cardiac Induction Medium. Medium was then exchanged with Cardiac Cultivation Medium every 24 hours for the following 8 days until dissociation. iPSC-CMs were observed to be spontaneously beating 8-10 days after cardiac induction.

[0131] Dissociation was performed using Collagenase. iPSC-CMs were washed with HBSS (Gibco, #14175-053), and incubated with Collagenase type II (Worthington Biochemical Corporation, #LS0004176) for 1.5hrs. Cells were then blocked in RPMI (Gibco, #21875) supplemented with DNase (Sigma, D8764-30KU), and replated on Matrigel™ coated dishes. iPSC-CMs were maintained in DMEM (Sigma-Aldrich, #D5546) with 10% FCS (Sigma- Aldrich, #F4135) and 1% insulin (Gibco, # 51500056). iPSC-CMs were cultured for 35 days total.

[0132] iPSC-CMs immunofluorescence staining: iPSC-CMs were fixed for immunostaining on day 35 of differentiation (following contractility and calcium analysis). Cells were fixed with 4% PFA for 10 minutes at RT, permeabilised with 0.1% Triton X-100, and blocked with 10% normal goat serum and 10% gold buffer (GB) (Cytiva, #RPN3006) in lx PBS (Sigma) for 30 minutes at RT. Cells were incubated in rabbit monoclonal anti-IFNGRl primary antibody (Abeam, #ab 134070) diluted in blocking solution overnight at 4°C. The following day, an anti-rabbit IgG Cy3 -labeled secondary antibody diluted in 10% GB in lx PBS was added for 1 hour at RT. After nuclear counterstaining with 4',6-diamidino-2-phenyl-indole (DAPI, 0.2 mg / ml; Sigma, #D9542), dishes were mounted Mowiol (Sigma). Fluorescent images were acquired using a Nikon AIR confocal microscope.

[0133] RESULTS:

[0134] Proteomics analysis uncovers a new player in the pathogenesis of ACM: To gain insights into the entire range of cell signalling pathways that are aberrantlyactivated in patients with ACM, buccal cell material was obtained from a patient bearing a PKP2 mutation and an aged-matched control. Buccal cells exhibit similar protein distribution and signalling abnormalities to those present in cardiac myocytes of ACM patients and have previously been used as a surrogate for the myocardium.

[0135] Cellular material was subjected to subcellular proteome fractionation and quantitative mass spectrometry. Gene ontology, KEGG pathways and Corum protein complexes were used for ID annotation enrichment analysis23and computation of enrichment scores, p-values and Benjamini-Hochberg FDR. Among the significant terms with Benjamini-Hochberd FDR< 0.05, the KEGG ‘’JAK / STAT signalling pathway” encompassing 9 proteins [Growth factor receptor-bound protein2 (GRB2), H-Ras protein (HRAS), Bcl-2-like protein 1 (BCL2LI), epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STATS'), signal transducer and activator of transcription 1 (STAT1), signal transducer and activator of transcription 6 (STAT6), signal transducing adapter molecular 1 (STAM), tyrosine-protein phosphatase non-receptor type 11 (PTPN1F) and interferon gamma receptor 1 (IFNGRT)] showed a positive score (significantly increased abundance) in the nuclei of the buccal cells obtained from the ACM patient compared to the control indicating aberrant activation of the pathway (Fig 1).

[0136] To confirm these insights, myocardial tissue samples from patients with ACM were immunostained (n=36) for IFNGR1. These included 29 samples from SCD victims with either an antemortem or a postmortem diagnosis of ACM, 6 samples from the explanted hearts of ACM patients who had undergone cardiac transplantation and 1 endomyocardial biopsy sample obtained from an ACM patient during ICD placement. Myocardial samples from individuals who had died of non-cardiac causes were subjected to the same protocol as negative controls (n=19). All 36 ACM samples but none of the 19 controls showed strong nuclear signal for IFNGR1 (Fig 2).

[0137] Myocardial sample analysis indicates aberrant JAK / STAT activation in ACM, BrS and HCM:

[0138] To establish the specificity of this finding, the myocardial samples from patients with different forms of heart disease were immunostained (n=87). Such included patients with a diagnosis of Brugada syndrome (BrS; n=13), catecholaminergicpolymorphic ventricular tachycardia (CPVT, n=12), Long QT syndrome 1 (LQT1, n=5), Long QT syndrome 2 (LQT2, n=6), hypertrophic cardiomyopathy (HCM, n=15), dilated cardiomyopathy (DCM, n=5), Ischemic cardiomyopathy (ICM, n=5), cardiac sarcoidosis (CS, n=9), giant cell myocarditis (GCM, n=8) and viral myocarditis (n=9). The source of all samples is included in Table 1 below.

[0139] IFNGR1 showed strong nuclear localization in myocardial samples from patients with BrS and HCM but not in samples from controls or patients with CPVT, LQT1, LQT2, DCM, ICM, CM, GCM or viral myocarditis (Fig 2

[0140] JAK / STAT is aberrantly activated in buccal cells of symptomatic ACM and BrS patients:

[0141] To investigate whether the insights obtained from the myocardial sample analysis are translated into the buccal epithelium, the distribution of IFNGR1 was analysed by immunocytochemistry in buccal smears from BrS patients (n=23), patients with a diagnosis of ACM (n=7) and silent carriers of ACM-causing mutations (n=4). Clinical and genetics data on all sampled participants are shown in Table 2. IFNGR1 showed strong nuclear localization in all BrS samples and all samples of ACM mutation-carriers manifesting evidence of disease (n=7). IFNGR1 signal showed a diffuse cytoplasmic distribution in all controls (n=20) and all ACM-mutation carriers with no clinical evidence of disease (n=4) (Fig 3). This indicates that aberrant activation of JAK / STAT does not occur until and unless there is clinical manifestation of disease.

[0142] Key to abbreviations used in Tables 1 and 2

[0143] Abbreviations: BRS: Brugada syndrome; M: male, F: female, FHx: family history, PPM: permanent pacemaker, AV: atrioventricular, SCD: sudden cardiac death, N / A: not available, ACM: arrhythmogenic cardiomyopathy, GDE: gadolinium enhancement, MRI: magnetic resonance imaging, ICD: implantable cardioverter defibrillator, AT: atrial tachycardia, LV: left ventricle, EF: ejection fraction, DSP: desmoplakin, RV: right ventricle, LA: left atrium, NSVT: non-sustained ventricular tachycardia, DSG2: desmoglein 2SCD: sudden cardiac death, ACM: arrhythmogenic cardiomyopathy; BrS: Brugada syndrome, CPVT: catecholaminergic polymorphic ventricular tachycardia, LQT1: Long QT 1, LQT2: Long QT 2, HCM: hypertrophic cardiomyopathy, DCM: dilated cardiomyopathy, ICM: ischemic cardiomyopathy, CS: cardiac sarcoidosis, GCM: giant cell myocarditis.Table 1: Source of patient myocardial samples;

[0144] No of samples Diagnosis Tissue source

[0145] 29 ACM SCD; autopsy

[0146] 6 ACM Explanted hearts at cardiac transplantation 1 ACM Endomyocardial biopsy during internal defibrillator placement 13 BrS SCD; autopsy

[0147] 12 CPVT SCD; autopsy

[0148] 5 LQT1 SCD; autopsy

[0149] 6 LQT2 SCD; autopsy

[0150] 10 HCM SCD; autopsy

[0151] 5 HCM Explanted hearts at cardiac transplantation 5 DCM Explanted hearts at cardiac transplantation 5 ICM Explanted hearts at cardiac transplantation 9 CS SCD; autopsy

[0152] 8 GCM SCD; autopsy

[0153] 9 Viral SCD; autopsy

[0154] myocarditis

[0155]

[0156] Table 2: Clinical, genetic and immunocytochemical data for buccal smear samples

[0157] Study No Age Sex Genetics Clinical information IFNGR1 distribution BRS1 37 M Negative Palpitations, FHx of SCD, positive EPS Nuclear study, ICD

[0158] BRS2 32 M Negative Spontaneous Type 1 ECG, Hx of VT, ICD Nuclear BRS3 43 F Negative Hx of palpitations, drug-induced Type 1 Nuclear ECG, FHx ofBrS

[0159] BRS4 41 M SCN5A Hx of syncope, FHx of SCD, spontaneous Nuclear type 1 ECG, ICD

[0160] BRS5 52 M Negative Hx of palpitations, drug-induced pattern, Nuclear FHx of SCD

[0161] BRS6 38 F Negative Palpitations, presyncope, FHx of SCD Nuclear BRS7 32 M N / A Spontaneous Type 1 ECG, FHx ofBrS Nuclear BRS8 50 M Negative Drug-induced pattern, low risk EPS, FHx of Nuclear SCD BRS9 28 F SCN5A Spontaneous Type 1 ECG, Hx of VT, ICD Nuclear BRS10 46 M Negative Drug-induced pattern, suspected arrhythmic Nuclear syncope, FHx ofBrS

[0162]

[0163] BRS11 35 M N / A Hx of palpitations, presyncope, spontaneous Nuclear Type 1 pattern

[0164] BRS12 44 F Negative Spontaneous Type 1 ECG, Hx of Nuclear polymorphic VT, previous ablation

[0165] BRS13 30 M Negative Drug-induced Type 1 pattern, FHx of SCD Nuclear BRS14 51 M Negative Palpitations, negative EPS, FHx of SCD Nuclear BRS15 71 F Negative Suspected arrhythmic syncope, previous Nuclear SVT, FHx of SCD

[0166] BRS16 75 F Negative Arrhythmic syncope, NSVT, FHx of SCD Nuclear BRS17 47 M N / A Asymptomatic, FHx of BrS Nuclear BRS18 75 M Negative Asymptomatic, low risk EPS Nuclear BRS19 57 F SCN5A Presyncope, syncope, palpitations, FHx of Nuclear BrS

[0167] BRS20 46 F Negative Asymptomatic, FHx of SCD Nuclear BRS21 84 M N / A Conduction disease, PPM in view of Nuclear nocturnal pause with AV dissociation

[0168] BRS22 46 M SCN5A Suspected arrhythmic syncope, progressive Nuclear conduction disease, VT, Hx of ablation

[0169] BRS23 37 M Negative Palpitations, negative EPS, FHx of SCD Nuclear

[0170]

[0171] ACM1 17 M Negative Out of hospital cardiac arrest, extensive Nuclear GDE on MRI, ICD, widespread

[0172] intraventricular conduction delay and flat T- waves

[0173] ACM2 18 F Negative Multifocal AT, narrow complex Nuclear tachycardia, outflow tract ectopy, intraventricular conduction delay,

[0174] widespread repolarization abnormalities

[0175] ACM3 20 M Negative Post exertional syncope, extensive GDE on Nuclear MRI, abnormal septal motion on echo

[0176] ACM4 18 M Negative Extensive GDE on MRI, LV dilatation, Nuclear reduced LVEF (46%), low voltages on

[0177] ECG, intraventricular conduction delay, widespread repolarization abnormalities

[0178] ACM5 14 F DSP Widespread repolarization abnormalities Nuclear Leu622Pro and inverted T-waves on ECG. EF: 20%.

[0179] LV, LA and RV dilatation, tricuspid / mitral valve regurgitation, on transplant list

[0180] ACM6 14 F DSP Extensive fibrosis on MRI, heavy ectopy on Nuclear Thr760Tyrfs*7 Holter monitoring

[0181]

[0182] ACM7 9 M DSP High ventricular ectopic burden, NSVT, Nuclear c.274G>A & ICD implanted. Low voltages, inverted T- c,1799T>C waves on ECG. Significant LV dilatation,

[0183] EF: 30%, impaired RV systolic function

[0184] ACM8 15 F PKP2 Normal cardiac investigations Cytoplasmic Thr50Serfs*61 Family history of ACM

[0185] ACM9 17 F PKP2 Normal cardiac investigations Cytoplasmic Thr50Serfs*61 Family history of ACM

[0186] ACM 10 13 F DSP Trpl342* Normal cardiac investigations Cytoplasmic Family history of ACM

[0187] ACM 11 16 M DSG2 Normal cardiac investigations Cytoplasmic Thr255Ala Family history of ACM

[0188]

[0189] hiPSCs-cardiac myocytes from a HCM patient show JAK / STAT activation:

[0190] To determine whether JAK / STAT activation occurs as a cell-autonomous mechanism in patients, the immunoreactive signal for IFNGR1 was characterised in hiPSCs obtained from a patient with documented HCM underlied by a deep intronic variant in MYBPC3 (c.1224-52 G>A). These cultures are composed of >90% pure cardiac myocytes, and they are devoid of lymphocytes and macrophages. As shown in Figure 4, when grown under basal conditions, these cells displayed marked nuclear accumulation of INFGR1, indicating activation of JAK / STAT signalling pathways. No nuclear signal for IFNGR1 was seen in control hiPSC-CMs. These observations provide independent evidence of activation of innate immune response in cardiomyocytes in HCM under the control of JAK / STAT signalling.

[0191] JAK / STAT signalling inhibition prevents the development of ACM disease features in vivo:

[0192] To determine whether inhibition of JAK / STAT signalling pathways mitigates the development of the ACM phenotype in vivo, the inventors treated ]JSG2mut / mutwith tofacitinib (through daily intraperitoneal injections) over a 6-week period beginning when the mice were 10 weeks old. As previously reported,8]jSG2mut / mutmice show minimal cardiac structural or functional derangements at 10 weeks of age, but during the ensuing 6 weeks, they develop a robust phenotype that recapitulates the most important clinical features seen in patients with ACM. This includes progressive deterioration of ventricular contractile function associated with the development of extensive myocardial necrosis, fibrosis and inflammation. As shown in Figure 5, treatment of ]JSG2mut / mutmice with tofacitinib substantially mitigated this phenotype. Contractile function, as depicted by echocardiography, was normalized. Specifically, the ejection fraction in tofacitinib-treated animals was >60% (versus <30% in untreated littermates) and the fractional shortening (FS) was >35% in the treated animals (versus <15% in vehicle-treated animals).

[0193] Tofacitinib reduces myocardial fibrosis in ACM mice:

[0194] It has been shown that 16-week old Dsg2"""z"'"' mice show considerable myocardial injury, indicated by the presence of extensive fibrosis, which replaces areasof degenerated cardiac myocytes. To investigate the effects of tofacitinib on myocardial injury, hearts were harvested from mouse cohorts and the extent of myocardial fibrosis was calculated. A significant decrease in fibrotic area was found in tofacitinib-treated □sg2'"'" / """ mice relative to vehicle-treated counterparts (Figure 6).

[0195] Tofacitinib reduces pro-inflammatory cytokines and chemokines in ACM mice:

[0196] Levels of proinflammatory cytokines and fibrokines are increased in hearts of 16-week-old Dsg2"'"' / "'"' mice. These mediators are also produced under basal conditions in vitro by cardiac myocytes derived from iPSCs from patients with ACM harbouring variants in PKP2 or DSG2. To investigate the effects of tofacitinib on myocardial cytokine / fibrokine production, harvested snap-frozen hearts were subjected to multiplex cytokine array analysis. Levels of major pro-inflammatory cytokines, including Interleukin-3 (IL-3), Interleukin-27 (IL-27), pentraxin 3, P-selectin and granulocyte colony stimulating factor (G-CSF) were significantly increased in hearts of 16-week-old Dsg2mut / mut mice compared with WT mice. Additionally, levels of major pro-fibrotic molecules, including osteopontin, Insulin-like Growth Factor Binding Protein 3 (IGFBP-3) and Cluster of Differentiation 93 (CD93) were also significantly upregulated in mutant mice compared to WT. Nearly all of these inflammatory / fibrotic mediators were at least partially normalized in hearts of tofacitinib-treated Dsg2"'"' / "'"' animals suggesting that cardiac myocyte JAK / STAT signalling trigger expression of inflammatory mediators in this model of ACM (Figures 7 and 8).

[0197] Baricitinib reduces myocardial fibrosis in ACM mice:

[0198] To determine whether the observed effects were specific to tofacitinib or instead reflected a broader class effect of JAK / STAT pathway inhibition, whether additional pharmacological inhibitors targeting this pathway could similarly influence disease progression in experimental models of ACM was investigated. In particular, the effects of baricitinib, a selective JAK1 / JAK2 inhibitor with a distinct inhibitory profile compared with tofacitinib were evaluated. Using the same DSg2mut / mutmouse model, the intervention protocol was repeated to assess whether baricitinib would likewise attenuate readily quantifiable ACM features.Two cohorts of mutant animals received baricitinib injections and were sacrificed at 10 and 16 weeks of age, respectively, after which their hearts were collected and processed for histological and molecular analyses. Masson’s trichrome staining demonstrated a significant reduction in myocardial fibrosis in baricitinib-treated □sg2'"'" / """ mice compared with vehicle-treated controls (Figure 9). In addition, immunohistochemical analysis revealed prominent nuclear localisation of IFNGR1 and RelA in cardiac tissue from vehicle-treated animals, whereas this nuclear signal was abolished in hearts from baricitinib-treated mice (Figures 10 and 11).

[0199] In parallel, analysis of intercalated disk components revealed a marked reduction in plakoglobin and connexin 43 (Cx43) signal intensity in vehicle-treated Dsg2"'"' / "'"' mice, consistent with disruption of cell-cell junctional integrity. Importantly, baricitinib treatment restored plakoglobin and Cx43 signal at the intercalated disks to levels comparable to those observed in wildtype animals, indicating recovery of intercellular coupling structures and preservation of myocardial tissue organisation (Figure 12 and 13).

[0200] JAK / STAT signalling is aberrantly activated in Boxer dogs with naturally occurring ACM:

[0201] Arrhythmogenic cardiomyopathy in Boxer dogs is a well-recognised, naturally occurring myocardial disease characterised predominantly by ventricular arrhythmias, syncope, and an increased risk of sudden cardiac death. The condition shares key pathological and clinical features with human ACM, including fibrofatty myocardial replacement, myocyte loss, and electrical instability, often in the absence of overt structural changes in early stages. Affected Boxers may present with frequent premature ventricular complexes, ventricular tachycardia, or sudden death despite preserved systolic function. Although a deletion in the striatin (STRN) gene has been associated with susceptibility in some lines, disease expression is variable and incompletely penetrant, and a proportion of affected dogs do not carry known pathogenic variants. Consequently, Boxer ACM is considered a complex, multifactorial disorder and represents one of the most extensively studied spontaneous large-animal models of arrhythmogenic cardiomyopathy.To establish the relevance of the above findings in a naturally occurring large-animal model of arrhythmogenic cardiomyopathy (ACM), myocardial tissue samples from Boxer dogs diagnosed with ACM (n=6) were subjected to immunohistochemical analysis for STAT3. All affected animals had experienced either sudden cardiac death (n=3) or death secondary to heart failure (n=3). Myocardial samples obtained from Boxer dogs that had died from non-cardiac causes were processed using an identical staining protocol and served as negative controls (n=3). Robust nuclear localisation of STAT3 was observed in all six ACM samples, whereas no nuclear signal was detected in any control specimens (Figure 14).

[0202] To assess whether this pathway activation was detectable in a peripheral epithelial surrogate tissue, STAT3 distribution was analysed by immunocytochemistry in buccal smears obtained from Boxer dogs with ACM (n=3). Buccal samples from Boxer dogs without a clinical diagnosis or family history of cardiac disease were processed in parallel as negative controls (n=2). Consistent with the myocardial findings, strong nuclear localisation of STAT3 was observed in all ACM samples but was absent in control samples (Figure 15).

[0203] DISCUSSION:

[0204] ACM is a complex disease characterized by the early onset of arrhythmias followed by progressive myocardial damage that can lead to heart failure.1,2Exactly how mutations in desmosomal genes can cause such a complex clinical picture is incompletely understood, but evidence implicates abnormal activation of Wnt / 0-catenin7signalling via GSK308and NFKB.9, 12,19The fact that SB216763 (a smallmolecule inhibitor of GSK30)8and BAY11-7082 (a small-molecule inhibitor of NFKB),12can reverse both the myocardial injury and arrhythmia components of ACM shows that a common pathway drives the clinically important features of the disease. However, effective mechanism-based drug therapy of ACM to reduce the risk of SCD would require long-term administration. Long-term inhibition of either GSK30 or NFKB could have unacceptable adverse consequences.16,24

[0205] Herein, the inventors provide new evidence that the ACM disease pathway also involves aberrant JAK / STAT signalling. The clinically used JAK / STAT inhibitortofacitinib showed a remarkable ability to rescue the ACM phenotype in a robust mouse model in vivo. Evidence is also provided of JAK / STAT involvement in the pathogenesis of BrS and HCM. Taken together, these observations provide new insights into mechanisms of pathogenesis of SCD-causing entities and identify new therapeutic strategies to limit myocardial damage and reduce the risk of serious arrhythmias in patients with ACM, BrS or HCM.

[0206] The quantitative mass spectrometry approach indicated that 9 proteins involved in the JAK / STAT pathway are translocated to the nucleus in buccal cell material from an ACM patient bearing a PKP2 mutation. JAK / STAT has numerous activators including cytokines, interferons, growth factors, hormones and mechanical stretch.28

[0207] For the confirmatory studies, IFNGR1, one of the receptors of interferon gamma (IFN-y) was the focus. Specifically, IFN-y binds to IFNGR1 and the dimer translocates to the nucleus along with STAT1 as a single macromolecule.30The findings indicate both aberrant activation of JAK / STAT in ACM, BrS and HCM but also suggest that the pathway is triggered at least in part by IFNy. For ACM, it is speculated that higher numbers of CCR2+ cells secondary to NFKB activation9facilitate monocyte recruitment to sites of inflammation leading to increased IFNy production and JAK / STAT activation. Further experimental studies are required to establish the precise mechanisms through which JAK / STAT is activated in the three SCD entities.

[0208] Interestingly, IFNGR1 showed strong nuclear localization in buccal smears obtained from patients with an established ACM phenotype but not in silent carriers of ACM-causing mutations. This indicates that activation of JAK / STAT tracks with the phenotype and not the genotype raising the possibility that IFNGR1 translocation could be used as a biomarker of phenoconversion in ACM. Serial buccal smears from ACM-mutation carriers need to be analysed to correlate IFNGR1 nuclear translocation with clinical onset of disease.

[0209] Several studies indicate common pathological mechanisms underlying ACM and BrS adding evidence to the theory that the two disease entities may in fact be variants of the same spectrum.31However, no common pathogenetic mechanisms uniting ACM and HCM have been reported to date.

[0210] Currently, 11 JAK inhibitors have received approval for clinical use. They can be divided into selective and non-selective based on their inhibitory activity against JAKsor as reversible and irreversible based on their binding mode.25Specifically, tofacitinib was first approved by the FDA for the treatment of rheumatoid arthritis in 2012. It was also approved for the treatment of psoriatic arthritis (2017), ulcerative colitis (UC; 2018), juvenile idiopathic arthritis (2020) and active ankylosing spondylitis (2021).25Tofacitinib was selected for the purposes of this study on the basis of its nature to inhibit all JAKs. Tofacitinib is a small synthetic molecule administered orally, with a fast bioavailability and elimination rate, predictable pharmacokinetics and lack of immunogenicity, adding to both its efficacy and safety.32Its safety profile has been studied in patients with moderate to severe UC, in induction and maintenance therapy, with a follow-up in clinical trials of up to 6.1 years. The profile observed in these trials is acceptable and manageable. The most common adverse events reported were headache, nasopharyngitis, nausea and joint pain.32Similarly, few minor side effects were reported by alopecia areata patients treated with tofacitinib either orally or topically.33

[0211] Tofacitinib significantly reduced levels of major pro-inflammatory and pro-fibrotic cytokines / chemokines in the hearts of Dsg2"'"' """ animals.

[0212] Baricitinib treatment similarly resulted in a marked reduction in myocardial fibrosis in Dsg2"'"'z"'"' hearts and restored the normal cellular distribution of RelA and IFNGR1. In vehicle-treated animals, both proteins displayed prominent nuclear localisation, consistent with activation of pro-inflammatory signalling pathways, whereas baricitinib administration abolished this nuclear accumulation. These findings indicate that inhibition of JAK / STAT signalling can suppress downstream inflammatory and pro-fibrotic signalling cascades that contribute to myocardial injury in this model. Moreover, baricitinib treatment restored the expression levels of plakoglobin and Cx43 at the myocardial intercalated disks indicating recovery of intercellular coupling structures and preservation of myocardial tissue organisation.

[0213] Importantly, the observation that baricitinib — an inhibitor with a distinct JAK selectivity profile compared with tofacitinib — produces comparable protective effects supports the concept that the therapeutic benefit arises from inhibition of the JAK / STAT pathway itself, rather than from compound-specific properties. This result therefore strengthens the evidence that pharmacological blockade of JAK / STAT signalling represents a broadly applicable strategy to limit inflammatory signalling andfibrotic remodelling during ACM progression. More broadly, the demonstration that multiple structurally and pharmacologically distinct JAK inhibitors can attenuate key pathological features of ACM supports the concept that therapeutic targeting of the JAK / STAT pathway constitutes a generalizable intervention strategy for this disease.

[0214] The demonstration that JAK / STAT signalling is aberrantly activated in myocardial tissue and peripheral epithelial samples (cheek smears) from Boxer dogs with naturally occurring ACM, mirroring findings in human ACM, provides critical translational validation of this pathway as a disease-relevant therapeutic target.

[0215] Importantly, pathway activation is observed in a spontaneous large-animal disease model that does not uniformly depend on canonical desmosomal gene mutations, indicating that JAK / STAT dysregulation represents a convergent downstream mechanism rather than a mutation-specific epiphenomenon. This cross-species conservation of pathway activation supports the concept that JAK / STAT signalling contributes to core disease biology independent of primary genetic substrate, thereby strengthening the rationale for therapeutic intervention targeting this pathway across genetically heterogeneous forms of ACM.

[0216] To conclude, here is provided evidence of involvement of the JAK / STAT signalling pathway in the pathogenesis of three major sudden death entities. It has also been shown that FDA-approved JAK inhibitors ameliorate disease in robust models of ACM introducing the possibility of JAK inhibition as a mechanism-based treatment option for patients with these diseases.References

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Claims

1. CLAIMS1. A method of treating and / or preventing arrhythmogenic cardiomyopathy (ACM), Brugada syndrome (BrS) or hypertrophic cardiomyopathy (HCM), comprising administering a therapeutically effective amount of an agent to a subject, wherein the agent is an inhibitor of one or more Janus Kinases (JAKs).

2. The method of claim 1, wherein the agent is an inhibitor of Janus Kinase 1 (JAK1), Janus Kinase 2 (JAK2), Janus Kinase 3 (JAK3) and / or Tyrosine Kinase 2 (TYK2), or any combination thereof.

3. The method of claim 1 or 2, wherein the agent is a competitive inhibitor or allosteric inhibitor of one or more JAKs4. The method of any one of the preceding claims, wherein the agent is a small molecule inhibitor of one or more JAKs.

5. The method of any one of the preceding claims, wherein the agent selectively inhibits one JAK.

6. The method of any one of the preceding claims, wherein the agent specifically inhibits one JAK only.

7. The method of any one of any one of claims 1-5, wherein the agent inhibits more than one JAK.

8. The method of claim 7, wherein the agent inhibits JAK1, JAK2, JAK3 and TYK2.

9. The method of any one of claims 1-5 or 7, wherein the agent is a selective inhibitor of JAK1 and JAK2.

10. The method of any one of claims 1-5 or 7, wherein the agent is a selective inhibitor of JAK1 and JAK3.

11. The method of any one of the preceding claims, wherein the agent prevents the activation of the one or more JAKs by cytokines and / or interferons.

12. The method of any one of the preceding claims, wherein the agent does not reduce the tonic activation of the one or more JAKs.

13. The method of any one of the preceding claims, wherein the agent is abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, or upadacitinib.

14. The method of claim 13, wherein the agent is baricitinib, ruxolitinib or tofacitinib.

15. The method of any one of the preceding claims, wherein the treating and / or preventing prevents or reduces myocardial necrosis, myocardial fibrosis and / or myocardial inflammation.

16. The method of any one of the preceding claims, wherein the treating and / or preventing slows the deterioration of ventricular contractile function.

17. The method of any one of the preceding claims, wherein the treating and / or preventing reduces the risk of sudden cardiac death (SCD).

18. The method of any one of the preceding claims, wherein the treating and / or preventing reduces or prevents arrhythmia.

19. An agent for use in a method of treating and / or preventing ACM, BrS or HCM in a subject, wherein the method comprises administering atherapeutically effective amount of the agent to the subject, wherein the agent is an inhibitor of one or more JAKs.

20. The agent for use according to claim 19, wherein(a) the agent is as defined in any one of claims 2-14; and / or(b) the treating and / or prevention are as defined in any one of claims 15-18.

21. Use of an agent which is an inhibitor of one or more JAKs in the manufacture of a medicament for treating and / or preventing ACM or BrS.

22. The use according to claim 21, wherein(a) the agent is as defined in any one of claims 2-14; and / or(b) the treating and / or prevention are as defined in any one of claims 15-18.

23. A method of diagnosing ACM, BrS or HCM in a subject, comprising detecting and / or quantifying the activation of the JAK / Signal Transducer and Activator of Transcription (STAT) pathway in a biological sample taken from the patient.

24. The method of diagnosing ACM, BrS or HCM according to claim 23, wherein the detecting and / or quantifying the activation of the JAK / STAT pathway comprises detecting and / or quantifying nuclear translocation of STAT proteins.

25. The method of diagnosing ACM, BrS or HCM according to claim 23 or 24, wherein the detecting and / or quantifying the activation of the JAK / STAT pathway comprises detecting and / or quantifying nuclear translocation of interferon gamma receptor 1 (IFNGR1).

26. The method of diagnosing ACM, BrS or HCM according to claims 23-25, wherein the biological sample is a buccal cell sample.

27. A method of monitoring and / or evaluating the effect of treatment of ACM, BrS or HCM in a subject, wherein the method comprises detecting and / or quantifying the activation of the JAK / STAT pathway as defined in any one of claims 23-26.

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