Promoter-enhancer sequences of the human troponin T gene for selective expression in cardiomyocytes

Abstract

This invention describes a novel gene regulatory sequence containing the promoter and enhancer sequences of the human cardiac troponin T gene (TNNT2) that selectively induces expression in cardiomyocytes. This novel TNNT2 promoter / enhancer composition can be used to induce adeno-associated virus gene expression, construct cell-type-specific expression vectors, or perform cardiac-specific transgenesis. The use of this novel promoter / enhancer composition is demonstrated by the expression of mAKAP shRNA and a mAKAP-derived anchor-disrupting peptide useful for the treatment of heart failure.

Description

[Background technology]

[0001] In response to chronic stress, the heart's primary compensatory mechanism is myocyte hypertrophy, a nonmitotic expansion of contractile cell volume (Hill and Olson 2008). Adult mammalian myocytes are roughly cylindrical and grow in both width and length. Because cardiomyocytes account for the majority of the cardiac myocardial mass (Jugdutt 2003), concentric and eccentric myocyte hypertrophy result in ventricular wall thickening and ventricular dilation, respectively. Theoretically, "concentric" myocyte width expansion, involving parallel sarcomere assembly, reduces ventricular wall stress (Laplace's law), whereas "eccentric" myocyte length expansion, involving serial sarcomere assembly, can accommodate larger ventricular volumes without stretching individual sarcomeres beyond their optimal length for contraction (length-tension relationship) (Grossman, Jones, and McLaurin 1975). Although the left ventricle hypertrophies relatively symmetrically in response to physiological stressors such as pregnancy or exercise training, concentric ventricular hypertrophy predominates as an initial response to increased systolic wall stress, as seen in pressure-overload disorders such as hypertension or aortic stenosis. Eccentric ventricular hypertrophy predominates during states of volume overload, such as after myocardial infarction, and during the transition from concentric hypertrophy to dilated heart in heart failure with reduced ejection fraction (HFrEF) in several cardiovascular disorders, including those characterized primarily by pressure overload. Concentric and eccentric hypertrophy are also seen in hereditary hypertrophic and dilated cardiomyopathy, respectively.

[0002] At the cellular level, cardiomyocyte hypertrophy occurs as a result of increased protein synthesis and increased sarcomere size and organization within individual cardiomyocytes. For more detailed reviews of cardiac remodeling and hypertrophy, see Nakamura and Sadoshima 2018 and Burchfield, Xie, and Hill 2013, which are incorporated herein by reference in their entireties. The general consensus is that cardiac hypertrophy plays a major role in the development of heart failure. Conventional heart failure treatments include afterload reduction, beta-adrenergic receptor (β-AR) blockade, and the use of mechanical assist devices. However, the art is lacking novel mechanisms to prevent or treat pathological cardiac hypertrophy.

[0003] Research suggests that mechanisms that induce "compensatory" concentric hypertrophy early in pressure-overload-related heart disease later lead to systolic dysfunction and eventual failure (Schiattarella and Hill 2015). In this regard, targeting the RSK3-mAKAPβ complex has been shown to prevent pressure-overload-induced cardiac remodeling and heart failure (Kritzer et al. 2014; Li, Kritzer, et al. 2013; Li et al. 2020). Therefore, inhibiting signaling pathways that induce remodeling, including concentric hypertrophy, may be desirable early in pressure-overload disease. Conversely, efforts to maintain signals that promote concentric hypertrophy and counteract eccentric hypertrophy, initiated when the heart is in the stage of the disease process characterized by eccentric hypertrophy and ventricular dilation leading to HFrEF, may preserve cardiac volume and contractility, whether in the later stages of pressure-overload-related disease or during the progression of volume-overload-related disease. In this regard, targeting the PP2A-mAKAPβ complex has been shown to suppress myocardial remodeling after myocardial infarction (Martinez et al. 2022; Li et al. 2020). Therefore, promoting concentric myocyte hypertrophy and / or suppressing eccentric myocyte hypertrophy may be beneficial in familial dilated cardiomyopathy.

[0004] [AKAP and cardiac remodeling] Ventricular myocyte hypertrophy is a major compensatory mechanism by which the myocardium reduces ventricular wall tension when exposed to stresses such as myocardial infarction, hypertension, congenital heart disease, and neurohumoral activation. This is associated with increased cardiomyocyte impermeability, increased myofibrillar organization, and upregulation of a specific subset of "fetal" genes normally expressed during the embryonic stage (Nakamura and Sadoshima 2018). Cardiac contractility, Ca 2+ Abnormalities in myocardial energy handling are associated with maladaptive changes, including interstitial fibrosis and cardiomyocyte death, increasing the risk of developing heart failure and malignant arrhythmias. These adaptations combine to cause systolic and diastolic dysfunction, the extent of which varies depending on the underlying disease (Sharma and Kass 2014). Pathological remodeling of myocytes involves the activation of mitogen-activated protein kinases (MAPKs), cyclic nucleotides, and Ca. 2+ It is regulated by a complex intracellular signaling network including hypoxia- and phosphoinositide-dependent signaling pathways (Heineke and Molkentin 2006; Nakamura and Sadoshima 2018).

[0005] Heart failure, with its prevalence increasing due to risk factors such as smoking and obesity, affects 67 million adults in the United States each year, with 1 million new cases diagnosed (Tsao et al. 2023). The prevalence and incidence of heart failure are increasing primarily due to increased lifespan, but also due to increased prevalence of risk factors (hypertension, diabetes, dyslipidemia, and obesity) and improved survival from other types of cardiovascular disease (myocardial infarction [MI] and arrhythmias) (Heidenreich et al. 2013). First-line medications for heart failure include beta-adrenergic agonists, angiotensin II, mineralocorticoid receptor antagonists, angiotensin-converting enzyme inhibitors, neprilysin metalloproteinase inhibitors, and sodium-glucose cotransporter 2 inhibitors (Heidenreich et al. 2022). Subsequent treatments or alternative therapies include loop diuretics, thiazide diuretics, vasodilators, If current blockers, and device therapy. Nevertheless, the 5-year mortality rate for symptomatic heart failure remains approximately 50%, including mortality rates of over 40% after MI (Heidenreich et al. 2013; Gerber et al. 2016).

[0006] Cardiac hypertrophy is induced by various neurohumoral, paracrine, and autocrine stimuli that activate several receptor families, including G protein-coupled receptors, cytokine receptors, and growth factor tyrosine kinase receptors (Nakamura and Sadoshima 2018). In this context, it has become increasingly clear that muscle A-kinase anchoring proteins (AKAPs) can assemble multiprotein complexes that integrate the hypertrophic pathways emanating from these receptors. In particular, recent studies have identified anchoring proteins such as mAKAP, AKAP-Lbc, and D-AKAP1, which function as scaffolding proteins and play a central role in orchestrating and regulating hypertrophic pathways activated by stress signals (Kritzer et al.).

[0007] As organizers of the "nodes" of intracellular signaling networks, scaffolding proteins have emerged as potential therapeutic targets (Negro, Dodge-Kafka, and Kapiloff 2008). Within cells, scaffolding proteins organize multimolecular complexes called "signalosomes," which constitute a key mechanism responsible for the specificity and efficacy of intracellular signaling (Scott and Pawson 2009). First, many signaling enzymes have broad substrate specificity. Scaffolding proteins can colocalize these multipotent enzymes with individual substrates, selectively enhancing substrate catalysis and providing specificity not present in the enzyme's active site (Scott and Pawson 2009). Second, some signaling enzymes are low in abundance. Scaffolding proteins can colocalize rare enzymes with their substrates, providing kinetic advantages for signaling. Third, many scaffolds are multivalent, allowing scaffold binding to coordinate the cooperative regulation of individual substrate effectors by multiple enzymes. Muscle A-kinase anchoring protein (mAKAP, also known as AKAP6) is a large scaffold expressed in cardiac myocytes, skeletal muscle cells, and neurons, and mediates the activation of protein kinase A (PKA) and Ca 2+ It binds to both broad-substrate-specific signaling enzymes, such as the calmodulin-dependent kinase calcineurin (CaN), and significantly less abundant signaling enzymes, such as p90 ribosomal S6 kinase 3 (RSK3) (Figure 16) (Wang et al. 2015; Pare, Easlick, et al. 2005; Michel et al. 2005; Kapiloff et al. 1999). mAKAPβ is an alternatively spliced ​​isoform expressed in muscle cells, where it localizes to the nuclear envelope by binding to the integral membrane protein nesprin-1α (Pare, Easlick, et al. 2005).

[0008] Consistent with the role of mAKAPβ as a scaffolding protein for stress-related signaling molecules in cardiomyocytes, depletion of mAKAPβ in neonatal rat ventricular myocytes in vitro inhibited hypertrophy induced by α-adrenergic, β-adrenergic, endothelin-1, angiotensin II, and leucine inhibitor / gp130 receptor signaling (Zhang et al. 2011; Pare, Bauman, et al. 2005; Dodge-Kafka et al. 2005; Guo et al. 2015). In vivo, mAKAP gene targeting in mice suppressed the development of heart failure after chronic pressure overload and conferred a survival benefit (Kritzer et al. 2014). Specifically, mAKAP fl / fl Deletion of the mAKAP gene in Tg(Myh6-cre / Esr1*), a tamoxifen-inducible conditional knockout mouse, suppressed left ventricular hypertrophy and, at the same time, significantly reduced myocyte apoptosis, interstitial fibrosis, left atrial hypertrophy, and pulmonary edema (wet lung weight) induced by aortic arch coarctation over a 16-week period (Kritzer et al. 2014).

[0009] mAKAP gene targeting is also effective after myocardial infarction (Martinez et al. 2022). Permanent ligation of the left anterior descending coronary artery (LAD) in mice results in myocardial infarction, including extensive cardiomyocyte death, scar formation, and subsequent left ventricular (LV) remodeling. Four weeks after LAD ligation, mAKAP conditional knockout mice exhibited preserved LV dimensions and function compared with infarcted control cohorts. Compared to control mice, mAKAP conditional knockout mice exhibited preserved LV ejection fraction and atrial mass, but significantly reduced infarct size.

[0010] [Overview of mAKAP and cardiac remodeling] mAKAP was originally identified in a cDNA library screen for novel cAMP-dependent protein kinase (PKA) regulatory subunit (R-subunit)-binding proteins, i.e., muscle A-kinase anchor proteins or AKAPs (Mccartney et al. 1995). mAKAP was initially named "AKAP100" after the size of the protein encoded by the original cDNA fragment (Mccartney et al. 1995). The full-length mRNA sequence of mAKAPα, an alternatively spliced ​​isoform of mAKAP expressed in neuronal cells, was subsequently determined, revealing that wild-type mAKAPα is a 255 kDa scaffold (Kapiloff et al. 1999). The sequence of mAKAPβ, a 230 kDa alternatively spliced ​​isoform of mAKAP expressed in striated muscle cells, was subsequently obtained, and it was shown that when expressed in cardiac or skeletal muscle, mAKAP is translated from an internal initiation site corresponding to residue Met-245 of mAKAPα.

[0011] mAKAP is localized to the nuclear envelope in all cell types where it is specifically expressed: neurons, striated cardiomyocytes, skeletal muscle cells, and osteoclasts (Kapiloff et al. 1999; Pare, Easlick, et al. 2005; Michel et al. 2005; Becker et al. 2021; Vergarajauregui et al. 2020). mAKAP is not a transmembrane domain protein; instead, it contains three spectrin-like repeats (residues 772-1187) that confer its localization (Kapiloff et al. 1999). Binding of the third spectrin repeat of mAKAP (residues 1074–1187) to the nuclear envelope protein nesprin-1α is necessary and sufficient for the outer nuclear membrane localization of mAKAP, at least in muscle cells and when expressed in heterologous cells (Pare, Easlick, et al. 2005; Becker et al. 2021; Holt et al. 2019). Interestingly, mutations in lamin A / C, emerin, and nesprin-1α are associated with Emery-Dreifuss muscular dystrophy and other forms of cardiomyopathy (Bonne et al. 1999; Fatkin et al. 1999; Muchir et al. 2000; Bione et al. 1994; Zhang et al. 2007). However, no disease-causing mutations have yet been identified in the human mAKAP gene, and knockout of mAKAPβ during early cardiac development in mice does not induce cardiomyopathy (Kritzer et al. 2014). In addition to binding to nesprin-1α, mAKAPβ also binds to phospholipase Cε (PLCε) through the first spectrin repeat of mAKAP, potentially enhancing its association with the nuclear membrane (Zhang et al. 2011). Early reports of the presence of mAKAPβ in the sarcoplasmic reticulum (Mccartney et al. 1995; Marx et al. 2000; Yang et al. 1998) likely stem from technical issues related to antibody specificity (Kapiloff, Jackson, and Airhart 2001; Kapiloff et al. 1999).

[0012] In addition to PKA, PLCε, and nesprin-1α, mAKAPβ binds to various proteins important for the stress response of muscle cells: adenylyl cyclase type 5 (AC5), exchange protein activated by cAMP-1 (Epac1), cAMP-specific phosphodiesterase type 4D3 (PDE4D3), MEK5 and ERK5 MAP kinases, 3-phosphoinositide-dependent protein kinase-1 (PDK1), p90 ribosomal S6 kinase 3 (RSK3), protein kinase Cε (PKCε), protein kinase D (PKD1, PKCμ), protein kinases calcineurin (CaN), Aβ, and PP2A, ryanodine receptor type 2 (RyR2), sodium / calcium exchanger NCX1, ubiquitin E3 ligases involved in HIF1α regulation, and myopodin (Pare, Bauman, et al. 2005; Pare, Bauman, et al. 2005). Easlick, et al. 2005;Dodge-Kafka et al. 2005;Marx et al. 2000;Kapiloff, Jackson, and Airhart 2001;Michel et al. 2005;Li et al.;Wong et al. 2008;Zhang et al. 2011;Dodge-Kafka and Kapiloff 2006;Vargas et al. 2012;Faul et al. 2007;Schulze et al. 2003;Kapiloff et al. 2009;Zhang et al. 2013). These signaling molecules bind to mAKAPβ and co-regulate the transcription factors hypoxia-inducible factor 1α (HIF1α), myocyte enhancer factor 2 (MEF2), serum response factor (SRF), nuclear factor of activated T cells (NFATc), and type II histone deacetylase (Figure 7) (Kritzer et al. 2014; Li, Vargas, et al. 2013; Li et al. 2010; Wong et al. 2008; Li et al. 2019; Dodge-Kafka et al. 2018; Li et al. 2020).These molecules include direct and indirect binding, as well as constitutive and regulated binding. Therefore, the composition of the mAKAPβ signalosome appears to depend on the basal state of the myocyte. As research on mAKAPβ continues, the list of its binding partners expands, supporting the hypothesis that it plays a key role as a regulator of signaling pathways required for remodeling. Most of what is known about mAKAPβ is based on studies using cultured neonatal rat ventricular myocytes, where it was early recognized that mAKAPβ is required for hypertrophy induction by various upstream receptors, including α-adrenergic receptors, β-adrenergic receptors, and cytokine receptors (Pare, Bauman, et al. 2005; Dodge-Kafka et al. 2005). However, the phenotype of conditional cardiomyocyte-specific mAKAPβ knockout mice has recently been published, confirming the importance of mAKAPβ in remodeling (Kritzer et al. 2014; Martinez et al. 2022). The mAKAPβ signalosome, which influences pathological cardiac remodeling, has various upstream inputs, downstream effectors (outputs), and integrated circuits.

[0013] [mAKAPβ - Prototype muscle A-kinase anchor protein] Like most AKAPs, mAKAP possesses an amphipathic helix (residues 2055-2072) that is responsible for binding to PKA (Kapiloff et al. 1999; Kritzer et al. 2012). PKA is a heterotetramer consisting of two R-subunits and two catalytic C-subunits in a CRRC configuration. Within the holoenzyme, the N-terminal docking and dimerization domains of the PKA R subunits form an X-shaped antiparallel four-helix bundle (Newlon et al.). This bundle contains a hydrophobic groove that accommodates the hydrophobic face of the AKAP amphipathic helix. mAKAPβ binds type II PKA (containing the RII subunit) with high affinity (K D= 119 nM) (Zakhary et al. 2000). Interestingly, PKA-mAKAPβ binding increases 16-fold after autophosphorylation of RIIα (Zakhary et al. 2000), potentially affecting PKA-mAKAPβ binding under conditions of altered β-adrenergic signaling. In addition to mAKAPβ, there are more than 10 AKAPs expressed in muscle cells, each with its own unique localization and binding partners (Kritzer et al. 2014). Surprisingly, mAKAP is one of the rarest AKAPs in muscle cells, and its deletion does not even affect the nuclear PKA localization (Kapiloff, unpublished observations). Despite low expression levels of the scaffold, replacing endogenous mAKAPβ with a full-length mAKAPβ mutant that cannot bind PKA in muscle cells is sufficient to inhibit hypertrophy induction in muscle cells (Pare, Bauman, et al. 2005). Thus, the mAKAPβ signalosome serves as an example both of how PKA signaling is finely compartmentalized, even on individual organelles, and of how the expression level of a protein or protein complex does not necessarily indicate the functional importance of that protein.

[0014] mAKAPβ is notable in that it binds not only to effectors of cAMP signaling but also to enzymes responsible for cAMP synthesis and degradation (Kapiloff et al. 2009; Dodge et al. 2001). The synthesis of cAMP from ATP is catalyzed by adenylyl cyclase (AC), and the metabolism of cAMP to 5'AMP is catalyzed by phosphodiesterases (PDEs). The differential association of ACs and PDEs with AKAPs contributes to intracellular cAMP compartmentalization, providing both local activation of cAMP effectors and control of local cAMP levels through unique regulatory feedback and feedforward loops (Scott, Dessauer, and Tasken 2013). mAKAPs can bind to both AC2 and AC5, but in the heart, AC5 appears to be the binding partner for mAKAPβ (Kapiloff et al. 2009). The N-terminus, C1, and C2 domains of AC5 directly bind to a unique N-terminal site (residues 275–340) on mAKAPβ. AC5 activity is inhibited intracellularly by PKA feedback phosphorylation, which is promoted by mAKAPβ complex formation (Kapiloff et al. 2009). This negative feedback is thought to be physiologically relevant for maintaining basal cAMP signaling. When AC5 binding to mAKAPβ was inhibited by a competitor peptide containing the mAKAP AC5-binding domain, both the cAMP content and size of myocytes increased in the absence of hypertrophic stimuli (Kapiloff et al. 2009).

[0015] mAKAP was the first AKAP shown to bind to PDEs (Dodge et al. 2001). A site within mAKAP1286-1831 binds to the unique N-terminal domain of PDE4D3. Phosphorylation of serine residues 13 and 54 of PDE4D3 increases scaffold binding and PDE catalytic activity, respectively (Dodge et al. 2001; Sette and Conti 1996; Carlisle Michel et al. 2004). Because increased PDE4D3 activity promotes cAMP degradation, PKA and PDE4D3 form a negative feedback loop that can regulate local cAMP levels and PKA activity (Dodge et al. 2001). PDE4D3 bound to mAKAP functions not only as a PDE but also as an adaptor protein, recruiting the MAPKs MEK5 and ERK5 and the cAMP-dependent Rap1-guanine nucleotide exchange factor Epac1 to the scaffold (Dodge-Kafka et al. 2005). Activation of MEK5 and ERK5 by upstream signals phosphorylates PDE4D3 at Ser-579, inhibiting PDEs and promoting cAMP accumulation and PKA activation (Dodge-Kafka et al. 2005; Hoffmann et al. 1999; Mackenzie et al. 2008). Because Epac1 is less sensitive to cAMP than PKA, very high cAMP levels further activate Epac1 in association with mAKAP. It is speculated that Epac1, via Rap1, can inhibit ERK5 activity, thereby preventing MAPK signaling from inhibiting PDE4D3, and consequently, the concomitant PKA phosphorylation maximizes PDE4D3 activity (Dodge-Kafka et al. 2005). As a result, Epac1, ERK5, and PDE4D3 form a third negative feedback loop that suppresses cAMP levels near the mAKAP complex, preventing excessively high levels of cAMP.

[0016] Further complexity is provided by the binding of the serine-threonine phosphatase PP2A to the C-terminus of mAKAP (residues 2083–2319) (Dodge-Kafka et al. 2010). PP2A can catalyze the dephosphorylation of PDE4D3 Ser-54, inhibiting PDE in the absence of upstream stimuli. PP2A associated with the mAKAP complex contains the B56δ B subunit, a substrate for PKA. PKA phosphorylation enhances the catalytic activity of PP2A (Ahn et al. 2007), and phosphorylation of B56δ by mAKAP-bound PKA increases the dephosphorylation of PDE4D3, inhibiting PDE. This likely increases cAMP levels, forming a positive feed-forward loop for the initiation of cAMP signaling. Combined with the negative feedback loop based on AC5 phosphorylation and PDE4D3 regulation by PKA and ERK5, cAMP levels in the mAKAPβ signalosome are predicted to be tightly controlled by upstream β-adrenergic and MAPK signaling. AC5 and ERK5 upstream signaling promotes cAMP signaling, initially driven by PP2A feedforward signaling, while PKA and Epac1 negative feedback activates PDE4D3 and inhibits AC5 signaling. Interestingly, Rababa'h et al. demonstrated how mAKAP proteins with nonsynonymous polymorphisms differentially bind to PKA and PDE4D3 (Rababa'h et al. 2013). Further studies using muscle cells are needed to demonstrate the relevance of this complex signaling network, as crosstalk between upstream signaling pathways or human polymorphisms may differentially regulate cAMP signaling. Besides PDE4D3, PP2A bound to mAKAPβ was recently shown to catalyze the dephosphorylation of SRF and promote myocyte length growth (Li et al. 2020). Targeting mAKAPβ-bound PP2A improved cardiac function after myocardial infarction in mice.

[0017] [mAKAPβ and MAP kinase-RSK3 signaling] It was first shown that PDE4D3-mediated recruitment of ERK5 to the mAKAPβ complex is involved in the local regulation of cAMP through the aforementioned feedback loop (Dodge-Kafka et al. 2005). However, it has been recognized that ERK5 is also a key inducer of myocyte hypertrophy, preferentially inducing increased body length (eccentric hypertrophy) in cultured myocytes, while also being important in vivo for concentric hypertrophy induced by pressure overload (aortic arch coarctation in mice) (Nicol et al. 2001; Kimura et al. 2010). Notably, RNA interference (RNAi) inhibition of mAKAPβ expression in cultured myocytes inhibited eccentric growth induced by the interleukin-6-type cytokine leukemia inhibitory factor (LIF) (Dodge-Kafka et al. 2005). A potential effector of mAKAPβ-bound ERK5 is the MEF2 transcription factor, as discussed below. However, in both the heart and brain, mAKAP associates with PDK1, a kinase that can activate the MAPK effector p90RSK, together with ERK (ERK1, 2, or 5), and p90RSK also associates with mAKAP (Ranganathan et al. 2006; Michel et al. 2005). Importantly, binding of PDK1 to mAKAP abolished the requirement for membrane association for RSK activation (Michel et al. 2005). Together, these data suggest that mAKAPβ may regulate RSK activation in muscle cells in response to upstream MAPK signals.

[0018] p90RSK is a pleiotropic ERK effector that regulates many cellular processes, including cell proliferation, survival, migration, and invasion. RSK activity is elevated in myocytes by most hypertrophic stimuli (Anjum and Blenis 2008; Sadoshima et al. 1995). Furthermore, elevated RSK activity has been found in cardiac tissue from human end-stage dilated cardiomyopathy (Takeishi et al. 2002). RSK family members contain two catalytic domains: an N-terminal kinase domain and a C-terminal kinase domain (Anjum and Blenis 2008). The N-terminal kinase domain phosphorylates RSK substrates and is activated by sequential phosphorylation of the activation loops of the C- and N-terminal kinase domains by ERK and PDK1, respectively. Phosphorylation of Ser-218 in the N-terminal domain of PDK1 indicates full activation of the enzyme. The ubiquitously expressed mammalian RSK family contains four members, but only RSK3 binds to mAKAPβ (Li, Kritzer, et al. 2013). The unique N-terminal domain (1-30) of RSK3 directly binds to mAKAPβ residues 1694-1833, termed the RSK3-binding domain ("RBD"), explaining the selective binding of this isoform to scaffolds (Li, Kritzer, et al. 2013). Despite RSK3 being less expressed in muscle cells than other RSK family members, hypertrophy of neonatal muscle cells was found to be suppressed by RSK3 RNAi, inactivation of the RSK3 N-terminal kinase domain, and disruption of RSK3 binding to mAKAPβ using an anchor-disrupting peptide (Li, Kritzer, et al. 2013). Importantly, in vivo RSK3 expression was required for the induction of cardiac hypertrophy by both pressure overload and catecholamine infusion, and for heart failure associated with a familial hypertrophic cardiomyopathy mouse model (α-tropomyosin Glu180Gly) (Li, Kritzer, et al. 2013; Passariello et al. 2013).Furthermore, consistent with the reported role of ERK1 / 2 MAP-kinases in selectively inducing concentric hypertrophy (Kehat et al. 2011), deletion of the RSK3 gene suppressed concentric hypertrophy induced by the Raf1L613V mutation in a mouse model of Noonan syndrome (Passariello et al. 2016). The recognition that this specific RSK isoform is required for cardiac remodeling makes it a promising therapeutic target.

[0019] Recently, it has been shown that RSK3 in AKAPβ phosphorylates the SRF transcription factor and promotes cardiomyocyte growth (Li et al. 2020). Replacing RSK3 from mAKAPβ by expression of an RBD peptide in mice also suppressed the development of hypertrophy in response to pressure overload and the subsequent development of heart failure.

[0020] [mAKAPβ and phosphatidylinositide signaling] The cAMP effector Epac1 activates Rap1 in the mAKAPβ complex, affecting ERK5 signaling (Dodge-Kafka et al. 2005). Furthermore, Epac1-Rap1 activates PLCε, a phospholipase whose Ras-associated domain directly binds to the first spectrin repeat-like domain of mAKAPβ (Zhang et al.). Similar to mAKAPβ, PLCε was required for neonatal myocyte hypertrophy when inhibited by RNAi or by displacing mAKAPβ with a competitive binding peptide. An elegant paper from the Smrcka lab showed that mAKAPβ-bound PLCε regulates the activation of PKCε and PKD through a novel phosphatidylinositol-4-phosphate (PI4P) pathway in which PLCε selectively converts perinuclear PI4P to diacylglycerol and inositol-1,4-bisphosphate (Zhang et al. 2013). PKD1 phosphorylates type II histone deacetylases (HDACs 4 / 5 / 7 / 9), inducing their nuclear export and derepressing hypertrophic gene expression (Monovich et al. 2010; Xie and Hill 2013). Smrcka et al. found that PLCε is required for pressure overload-induced PKD activation, type II HDAC phosphorylation, and hypertrophy in vivo (Zhang et al.). Subsequently, mAKAPβ was found to be required for PKD activation and HDAC4 phosphorylation in response to pressure overload in vivo (Kritzer et al. 2014). Surprisingly, mAKAPβ can form a ternary complex with PKD and HDAC4. These results demonstrate how local cAMP signaling can influence cardiac gene expression regulation.

[0021] Recently, it was reported that mAKAPβ scaffolds HDAC5 in cardiomyocytes, forming a signalosome containing HDAC5, PKD, and PKA (Dodge-Kafka et al.). Inhibition of mAKAPβ expression suppressed PKD- and PKA-mediated HDAC5 phosphorylation in response to α- and β-adrenergic receptor stimulation, respectively. Importantly, disruption of the mAKAPβ-HDAC5 anchoring prevented α-adrenergic receptor signaling and PKD phosphorylation-induced HDAC5 nuclear export. In addition, disruption of the mAKAPβ-PKA anchoring prevented β-adrenergic receptor-mediated inhibition of α-adrenergic-induced HDAC5 nuclear export. Taken together, these data demonstrate that the mAKAPβ signalosome plays a role in bidirectionally regulating the nucleocytoplasmic localization of class IIa HDACs. Thus, the mAKAPβ scaffold functions as a node in a muscle cell regulatory network that controls both the repression and activation of pathological gene expression in health and disease, respectively.

[0022] [mAKAPβ and calcium signaling] In addition to cAMP, phosphoinositide, and MAP kinase signaling, mAKAPβ mediates Ca 2+ A second binding partner of mAKAPβ has been identified, which is Ca(2+) transporter from intracellular stores. 2+ Inducible Ca 2+ Ryanodine receptors involved in Ca release 2+ The release channel (RyR2) was the most well-known (Kapiloff, Jackson, and Airhart 2001; Marx et al. 2000). RyR2 is best known for its role in excitation-contraction coupling, where bulk Ca 2+This releases PKA, inducing sarcomere contraction. PKA phosphorylation can enhance RyR2 currents (Valdivia et al. 1995; Dulhunty et al. 2007; Bers 2006), but whether PKA-catalyzed RyR2 phosphorylation is important for excitation-contraction coupling remains controversial (Houser 2014; Dobrev and Wehrens 2014). A small fraction of RyR2 is likely located in perinuclear dyads (Escobar et al. 2011) and can be immunoprecipitated with mAKAPβ and nesprin-1α antibodies (Pare, Easlick, et al. 2005; Kapiloff, Jackson, and Airhart 2001). By binding to RyR2, mAKAPβ and nesprin-1α likely connect elements of the excitation-contraction coupling system with signaling molecules that regulate nuclear events important for pathological remodeling. β-Adrenergic stimulation of primary muscle cell cultures increases PKA phosphorylation of RyR2 associated with mAKAPβ (Pare, Bauman, et al.). Notably, only PKA phosphorylation of RyR2 associated with nesprin-1α at the nuclear envelope is dependent on mAKAPβ, not on RyR2 elsewhere in the sarcoplasmic reticulum (Turcotte et al. 2022). PKA-catalyzed RyR2 phosphorylation is mediated by local Ca2+ accumulation near the mAKAPβ signalosome during sympathetic stimulation. 2 In particular, it enhances Ca release near nesprin-1α in the nuclear envelope upon β-adrenergic stimulation. 2+ The increased levels depend on the expression of mAKAPβ in cardiomyocytes, rather than in the general cytoplasm (Turcotte et al. 2022).

[0023] RyR2 bound to the few mAKAPβs did not significantly affect the contractile force of the whole muscle cell, but increased perinuclear Ca 2+ The target of Ca is a Ca molecule that can bind to the scaffold. 2+The CaNAβ-calmodulin-dependent kinase is calcineurin (CaN) (Turcotte et al. 2022). There are three isoforms of the catalytic subunit of CaN (α, β, and γ), but only the CaNAβ-mAKAPβ complex has been detected in myocytes (Li et al. 2010). Surprisingly, CaNAβ is the CaNA isoform that is important for the induction of cardiac hypertrophy in vivo and for the survival of cardiomyocytes after ischemia (Bueno et al. 2002; Bueno et al. 2004). CaNAβ directly binds to a unique site (residues 1286–1345) within mAKAPβ (Pare, Bauman, et al. 2005; Li et al. 2010). CaNAβ binding to mAKAPβ is induced intracellularly by adrenergic stimulation and directly by Ca 2+ / calmodulin enhances CaNAβ-mAKAPβ binding (Li et al. 2010). Notably, CaNAβ-mAKAPβ binding was required for α-adrenergic-induced neonatal myocyte hypertrophy in vitro (Li et al. 2010).

[0024] [mAKAPβ and gene expression] Among its many substrates, CaN is involved in the activation of NFATc and MEF2 transcription factors. The NFATc transcription factor family has four CaN-dependent isoforms, all of which are expressed in muscle cells and contribute to muscle cell hypertrophy induction (Wilkins et al. 2004). NFATc family members are generally retained in the cytoplasm upon robust phosphorylation of multiple serine-rich motifs within their N-terminal regulatory domains. Dephosphorylation of these motifs by CaN induces NFATc translocation into the nucleus. Multiple NFATc family members can bind mAKAPβ, and mAKAPβ binding was required for CaN-dependent dephosphorylation of NFATc3 in muscle cells (Li et al. 2010). Accordingly, mAKAPβ expression was also required for NFAT nuclear translocation and transcriptional activity in vitro (Li et al. 2010; Pare, Bauman, et al. 2005). These results correlate with the observation that NFAT-dependent gene expression in vivo is suppressed by cardiomyocyte-specific knockout of mAKAPβ after aortic arch coarctation surgery (Kritzer et al. 2014 ).

[0025] Like NFATc2 and NFATc3, MEF2D is a transcription factor required for cardiac hypertrophy in vivo (Kim et al. 2008; Wilkins et al. 2002; Bourajjaj et al. 2008). MEF2 family members contain a conserved DNA-binding domain containing both a MADS box and an MEF2 homology domain (Potthoff and Olson 2007). The DNA-binding domain of MEF2D directly binds to the N-terminal domain of mAKAP (Vargas et al. 2012; Kim et al. 2008). CaN and MEF2D are important not only in the heart but also in skeletal muscle (Naya et al. 1999; Naya and Olson 1999; Black and Olson 1998; Friday et al. 2003; Wu et al. 2001). Interference with MEF2-mAKAPβ binding blunted MEF2 transcriptional activity and expression of endogenous MEF2 target genes in C2C12 skeletal myoblasts (Vargas et al. 2012). In addition, disruption of the MEF2-mAKAP complex inhibited differentiation of C2C12 myoblasts into myotubes, as evidenced by the suppression of cell fusion and the expression of differentiation markers (Vargas et al. 2012). Surprisingly, in cardiomyocytes, CaN-MEF2 binding is mAKAPβ-dependent (Li, Vargas, et al. 2013). Accordingly, disruption of CaN-mAKAPβ binding inhibited both MEF2 transcriptional activity and cardiomyocyte hypertrophy in C2C12 cells (Li, Vargas, et al.). Similar to NFATc2, dephosphorylation of MEF2D in vivo in response to pressure overload was suppressed after mAKAPβ conditional knockout and correlated with reduced expression of MEF2 target genes, including atrial natriuretic factor expression (Kritzer et al. 2014 ).

[0026] Regulation of NFATc, MEF2, and HDAC4 by mAKAPβ during pressure overload in vivo demonstrates the importance of mAKAPβ in stress-regulated gene expression (Kritzer et al. 2014). It has been reported that mAKAPβ regulates NFATc and MEF2 through CaN, while HDAC4 and HDAC5 are regulated by PKD and PKA (Li, Vargas, et al. 2013; Zhang et al. 2013; Li et al. 2010; Dodge-Kafka et al. 2018). mAKAPβ appears to facilitate the regulation of these gene regulatory proteins by other signaling enzymes. For example, mAKAPβ-bound ERK5 can phosphorylate MEF2, activating the transcription factor (Kato et al. 2000). Furthermore, PKA can phosphorylate MEF2, affecting its DNA-binding affinity (Wang et al. 2005). On the other hand, Olson's group has proposed that PKA phosphorylation of HDAC4 can inhibit MEF2 activity through the generation of novel HDAC4 proteolytic fragments (Backs et al. 2011). How the activities of multiple mAKAPβ binding partners are ultimately integrated to regulate gene expression can be investigated both in vitro and in vivo.

[0027] [Other mAKAPβ binding partners] There are other binding partners of mAKAPβ, such as phospholamban, myopodin, and NCX1, whose docking to the scaffold has not yet been fully elucidated (Faul et al. 2007; Schulze et al. 2003; Hakem Zadeh et al. 2019). AKAP9 and its pericentric binding to mAKAPβ have been reported in striated muscle cells and osteoclasts and are important for the organization of the nuclear membrane microtubule-organizing center and the proximity of the nuclear membrane to the Golgi apparatus (Becker et al. 2021; Vergarajauregui et al. 2020).

[0028] HIF-1α, a transcription factor that regulates the systemic response to hypoxia, also binds to mAKAPβ (Wong et al. 2008). Under normoxic conditions, the abundance of HIF-1α in cells is maintained low by ubiquitin-mediated proteasomal degradation. HIF-1α is hydroxylated by a family of oxygen-sensitive dioxygenases called prolyl hydroxylases (PHD1, PHD2, and PHD3) (Ohh et al. 2000). The hydroxylated HIF-1α is then recognized by the von Hippel-Lindau protein (pVHL), which recruits the Elongin C ubiquitin ligase complex to ubiquitinate HIF-1α and promote its proteasome-dependent degradation (Maxwell et al. 1999). Under hypoxic conditions, PHD is inactivated, HIF-1α degradation is reduced, and HIF-1α accumulates in the nucleus, where it dimerizes with HIF-1β to promote target gene transcription. In cultured neonatal myocytes, mAKAPβ can assemble a signaling complex containing HIF-1α, PHD, pVHL, and the E3 ligase Siah2 (seven in absentia homolog 2) (Wong et al. 2008). Under normoxic conditions, mAKAPβ-anchored PHD and pVHL promote HIF-1α ubiquitination and degradation (Wong et al. 2008). However, under hypoxic conditions, activation of Siah2 induces proteasomal degradation of the bound PHD, promoting HIF-1α accumulation (Wong et al. 2008). mAKAPβ knockout can affect cardiomyocyte survival after ischemia-reperfusion.

[0029] [Conductor of the mAKAPβ-remodeling symphony] These findings demonstrate how multiple signaling pathways known to be important for cardiac hypertrophy and pathological remodeling are regulated by the binding of key signaling intermediates to the mAKAPβ scaffold. Cardiomyocyte-specific conditional mAKAP knockout mice have been characterized, demonstrating the relevance of the mAKAPβ signalosome in vivo (Kritzer et al. 2014; Martinez et al. 2022). mAKAPβ was required for the induction of cardiac hypertrophy in cardiomyocytes by aortic arch coarctation and isoproterenol infusion. However, most notably, it prevented pathological remodeling, including myocardial apoptosis and interstitial fibrosis, and preserved cardiac function in the face of prolonged pressure overload, resulting in a significant increase in mouse survival (Kritzer et al. 2014). Beneficial outcomes were also demonstrated in mice after myocardial infarction (Martinez et al. 2022). These results establish mAKAPβ as the first scaffold whose ablation can confer a survival benefit in cardiac disease. Importantly, mAKAPβ does not appear to be required for the development or maintenance of normal adult cardiac function, as no obvious phenotype was observed by 6 months of age using an Nkx2-5-directed cre deletion strain (Kritzer et al. 2014). Although mAKAPβ knockout suppressed physiological hypertrophy induced by forced exercise (swimming), targeting the mAKAPβ complex in disease remains relevant.

[0030] Various strategies for targeting the mAKAPβ complex in humans have been envisioned, including siRNA knockdown of the scaffold. However, a relatively detailed understanding of the structure and function of the mAKAPβ signalosome offers new approaches to targeting these pathways. For example, expression of peptides targeting key protein-protein interactions involving mAKAPβ, such as anchor-disrupting peptides targeting mAKAPβ-CaNAβ, mAKAPβ-MEF2D, mAKAPβ-PLCε, mAKAPβ-PP2A, and mAKAPβ-RSK3 binding, has already been shown to be effective in vitro (Li, Vargas, et al. 2013; Li, Kritzer, et al. 2013; Vargas et al. 2012; Zhang et al. 2011; Li et al. 2020). Heart failure, a leading cause of death, results in a 50% mortality rate within 5 years of diagnosis despite modern treatments and costs the United States alone more than $30 billion annually (Go et al. 2014). The efficacy of targeting the mAKAPβ signalosome by targeting the mAKAPβ-PP2A and mAKAPβ-RSK3 junctions using adeno-associated virus (AAV) gene therapy vectors was recently demonstrated in mice (Li et al. 2020). Many potential targets in cardiac disease are multidirectional, complicating the development of drugs with sufficient specificity in vivo. Specific targeting of the mAKAPβ signalosome in cardiomyocytes offers an opportunity to target relatively rare protein-protein interactions that appear to be specialized for pathological cardiac remodeling, and their ablation may be facilitated without significant side effects.

[0031] Adeno-associated virus (AAV) is a small, single-stranded DNA virus with a genome of ~4.7 kb that belongs to the Parvoviridae family and the Dependoparvovirus genus (Wang, 2019). AAV is non-pathogenic and can deliver essentially any DNA sequence to target tissues in mammalian species, making it a gene therapy vector of choice for both basic research and clinical applications. Since the approval of alipogene tiparvovec (Glybera) for lipoprotein lipase deficiency in Europe in 2012, the US approval of RPE365 has been expanded to include other therapeutics. - / - With the approval of voretigene neparvovec-rzyl (Luxturna) for Leber's congenital amaurosis and retinitis pigmentosa, and the approval of onasemnogene abeparvovec (Zolgensma) for spinal muscular atrophy in the United States in 2019, the number of clinical trials for AAV-based therapies has increased dramatically, with over 200 clinical trials registered to date (Kuzmin et al., 2021). Adeno-associated virus species are designated by the serotype of their capsid protein. Among commonly used serotypes, AAV serotype 9 (AAV9) has been frequently used for skeletal, cardiac, and neurological applications, including the AAV9 biologic onasemnogene abeparvovec. Regarding the heart, AAV9 has become the dominant serotype in preclinical studies (Kieserman et al., 2019).

[0032] A key issue in any drug development is biodistribution. Despite recognition that AAV9 administered via the vascular system can be delivered to other organs, few studies have documented AAV9 biodistribution in pigs, or its dependence on local or peripheral delivery sites. AAVs containing serotype 9 have a strong tropism for the liver, and robust extrahepatic delivery of AAV9 biologics has been observed when injected via the coronary arteries (Ishikawa et al., 2018). Preclinical testing of muscle-targeting therapeutics often involves transplantation from rodents to pigs to demonstrate potential clinical relevance. Demonstrating the flexibility of AAV gene therapy, AAV9 biologics have been studied in pigs for the expression of inhibitor-1c and S100A1 proteins in heart failure (Fish et al., 2013; Pleger et al., 2011), somatic gene editing in Duchenne muscular dystrophy (Moretti et al., 2020), and RNA interference of the Hippo signaling pathway in cardiac regeneration (Liu et al., 2021). Adeno-associated viruses have been introduced into the heart by direct intramyocardial injection (Liu et al., 2021) and intracoronary injection (Fish et al., 2013). Intracoronary injection via a percutaneous catheter approach has been performed in a variety of ways. Although retrograde procedures have been performed with or without coronary artery / venous occlusion and vector recirculation, retrograde delivery is perhaps the simplest intracoronary approach and can be easily applied to patients undergoing standard cardiac catheterization ( Ishikawa et al., 2018 ).

[0033] Further discussion of the design and use of AAV vectors can be found, for example, in U.S. Pat. Nos. 11,129,908 and 5,139,941 and WO 1998 / 046728, which are incorporated by reference herein in their entireties for all purposes. The use of AAV vectors in the treatment of cardiac disease is also disclosed in U.S. Patent Nos. 9,132,174, 9,937,228, 10,617,737, and 11,229,679, U.S. Application No. 17 / 580,692 (filed January 21, 2022), and U.S. Patent No. 10,907,153 and U.S. Application No. 16 / 818,771 (filed March 13, 2020), Li et al., 2022; Li et al., 2020, and Martinez, et al., 2023, each of which is incorporated by reference in its entirety for any purpose.

[0034] There is a clear need to develop new and effective therapies not only to treat patients with heart failure, but also to prevent the development of heart failure in the context of other cardiovascular diseases such as coronary artery disease, hypertension, and valvular disease. Summary of the Invention

[0035] The following brief summary is not intended to be inclusive of all features and aspects of the present invention, nor is it intended to imply that the present invention must include all features and aspects discussed in this summary.

[0036] The present inventors have discovered an improved composition for inducing transgene expression and a method for delivering transgenes to cardiomyocytes using an AAV plasmid containing a novel promoter-enhancer regulatory sequence that includes a novel combination of promoter, exon, and intron sequences of the human TNNT2 gene.

[0037] In some embodiments, these improved compositions and methods can be used to treat cardiac pathological processes by using drugs that target specific protein-protein interactions to inhibit the signal transduction properties of individual mAKAP signal transduction complexes.This therapeutic strategy is more advantageous than traditional therapeutic approaches because it can selectively inhibit defined cellular responses.

[0038] In certain embodiments, the compositions and methods of the present invention can disrupt mAKAP-mediated protein-protein interactions, so as to inhibit the ability of mAKAP to regulate the activation of enzymes that play a central role in the activation of key transcription factors that initiate the cellular processes that lead to pathological cardiac remodeling.Inhibiting the binding activity of mAKAPβ can protect the heart from damage that leads to heart failure, for example, after myocardial infarction.

[0039] In one embodiment, the present invention relates to compositions and methods for reducing the expression level of mAKAPβ, for example, using AAV vectors containing novel promoter-enhancer regulatory sequences that provide shRNA molecules against mAKAPβ.

[0040] In another aspect, the present invention relates to compositions and methods for inhibiting the interaction of mAKAPβ with RSK3, for example, using an AAV vector comprising a novel promoter-enhancer regulatory sequence and a sequence that targets the RSK3-binding domain (RBD) of mAKAPβ.

[0041] In yet another aspect, the present invention relates to compositions and methods for inhibiting the interaction of mAKAPβ with PP2A, for example, using AAV vectors comprising novel promoter-enhancer regulatory sequences and sequences targeting the PP2A-binding domain (PBD) of mAKAPβ.

[0042] In yet another embodiment, the present invention relates to a vector comprising the cardiac troponin t promoter 2 (hTNNT) and a skeletal muscle enhancer with splicing consensus sites.

[0043] In a further aspect, the invention relates to codon-optimized vectors and methods of using same.

[0044] The above-described compositions and methods can be used to protect the heart from damage by administering to a patient at risk of such damage a pharmaceutically effective amount of a composition that inhibits the expression, binding, and / or activity of mAKAPβ.

[0045] The foregoing and other objects, features and advantages of the present invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0046] The patent or application file contains at least one color drawing. Copies of this patent or patent publication will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1 shows the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence (TNNT2 regulatory sequence in green), which contains a novel combination of promoter, exon, and intron sequences from the human TNNT2 gene to drive expression of human mAKAP shRNA. [Figure 2] 2-1 to 2-4 show the nucleic acid sequence (SEQ ID NO: 1) of the AAV plasmid of FIG. [Figure 3]Figure 3 shows the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence (TNNT2 regulatory sequence in green) that contains a novel combination of promoter, exon, and intron sequences from the human TNNT2 gene to drive expression of the human mAKAP RBD sequence. [Figure 4] 4-1 to 4-3 show the nucleic acid sequence (SEQ ID NO: 2) of the AAV plasmid of FIG. [Figure 5] Figure 5 shows the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence (TNNT2 regulatory sequence in green) that contains a novel combination of promoter, exon, and intron sequences from the human TNNT2 gene to drive expression of the human mAKAP PBD sequence. [Figure 6] 6-1 to 6-3 show the nucleic acid sequence (SEQ ID NO: 3) of the AAV plasmid of FIG. [Figure 7] Figure 7 shows the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence, including the human calsequestrin enhancer and a novel combination of promoter and exon sequences from the human TNNT2 gene, to drive expression of a human mAKAP shRNA. [Figure 8] 8-1 to 8-3 show the nucleic acid sequence (SEQ ID NO: 4) of the AAV plasmid of FIG. [Figure 9] FIG. 9 shows the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence, including the human calsequestrin enhancer and a novel combination of promoter and exon sequences from the human TNNT2 gene, to drive expression of the human mAKAP RBD. [Figure 10] 10-1 to 10-3 show the nucleic acid sequence (SEQ ID NO: 5) of the AAV plasmid of FIG. [Figure 11] FIG. 11 shows the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence, including the human calsequestrin enhancer and a novel combination of promoter and exon sequences from the human TNNT2 gene, to drive expression of the human mAKAP PBD sequence. [Figure 12] 12-1 to 12-3 show the nucleic acid sequence (SEQ ID NO: 6) of the AAV plasmid of FIG. [Figure 13] FIG. 13 shows a nucleotide alignment of silent single base mutations newly introduced into the open reading frame of human PBD (mAKAP 2132-2319) to reduce the amount of CpG containing the immunostimulatory motif. [Figure 14] FIG. 14 shows a nucleotide alignment of newly introduced silent single-nucleotide mutations in the open reading frame of human RBD (mAKAP 1696-1835) to reduce the amount of CpGs containing immunostimulatory motifs. [Figure 15] Figures 15-1 to 15-3 show the origin of the "hTNNT2 regulatory sequence" contained in the plasmids shown in Figures 1 to 6. [Figure 16] Figure 16 shows a model of mAKAPβ-regulated SRF-dependent gene expression. Anchored RSK3 is a Gq-protein-coupled receptor-ERK effector that phosphorylates SRF bound to the perinuclear mAKAPβ complex. mAKAPβ-anchored PP2A, which can be activated by cAMP-dependent protein kinase A (PKA), opposes the phosphorylation of SRF. Phosphorylated SRF induces gene expression that promotes concentric hypertrophy. [Figure 17] Figures 17-1 to 17-4 show the complete nucleotide (SEQ ID NO: 9) and deduced amino acid (SEQ ID NO: 10) sequence of human RSK3 (Homo sapiens ribosomal protein S6 kinase A2 (RPS6KA2). Transcript variant 1, mRNA, NCBI reference sequence: NM_021135.6). The deduced RSK3 protein sequence is shown in single-letter amino acid code, beginning with the first methionine residue preceding the 733-codon open reading frame and ending with an asterisk. The unique N-terminal region of RSK3 (which has no homology to RSK1 or RSK2) is shown. [Figure 18]Figures 18-1 to 18-5 show the nucleotide sequence of human mAKAPα (SEQ ID NO: 11) having a translated open reading frame (SEQ ID NO: 12). [Figure 19] Figures 19-1 and 19-2 show the amino acid sequences of human mAKAP (SEQ ID NO: 12). mAKAPα starts at residue 1, and mAKAPβ starts at residue 243. The PBD is in bold. [Figure 20] FIG. 20 shows the amino acid sequence of the human PBD (SEQ ID NO: 14) expressed in AAV. [Figure 21] Figure 21 shows an alignment of RBDs from various species, with SEQ ID NOS: 16 to 42 and the sequence (SEQ ID NO: 15) expressed by the vector in Figure 3 shown in the first row. [Figure 22] Figure 22 shows an alignment of PBDs from various species, with SEQ ID NOS: 45 to 64 and the sequence (SEQ ID NOS: 68) expressed by the vector of Figure 5 shown in the first row. [Figure 23] Figure 23 shows the map of the human PBD AAV shuttle plasmid, pscAAV-hmAKAP PBD. [Figure 24] Figures 24-1 and 24-2 show the nucleotide sequence of the pscAAV-hmAKAP PBD plasmid (SEQ ID NO: 67). [Figure 25] Figures 25-1 to 25-4 show the nucleotide sequence of human mAKAP (AKAP6) mRNA (SEQ ID NO: 69)-ref seq XM_017021808.1 along with the shRNA sequences (#1-3). Numbers refer to the nucleotide sequence. The encoded amino acids are listed above. [Figure 26] Figure 26 is a map of the pscA-TnT-mAKAP shrnA(#3) plasmid. [Figure 27] Figures 27-1 and 27-2 show the nucleotide sequence of the pscA-TnT-mAKAP shrnA(#3) plasmid (SEQ ID NO: 70), with key features and some restriction enzyme sites indicated. [Figure 28]Figure 28 shows the target of the scAAV-mAKAP shRNA biologic. The mAKAP mRNA sequences are human (SEQ ID NO: 71), swine (SEQ ID NO: 72), mouse (SEQ ID NO: 73), and rat (SEQ ID NO: 74). The boxed sequence is shRNA target #3. [Figure 29] Figure 29 shows the amino acid sequence of rat mAKAP PBD expressed in an AAV vector (SEQ ID NO: 75), including an N-terminal myc tag. [Figure 30] Figure 30 shows cardiac-selective delivery of different AAV compositions. Data are mRNA levels in the left ventricle (LV) of the heart relative to mRNA levels in the liver, brain, and skeletal muscle (skm). Data were obtained for tissues from three pigs collected 3 months after intravenous administration of 2E12 vg / kg AAV9 vector. RT-qPCR was performed after RNA extraction and reverse transcription (RT) of 2 µg of total RNA into cDNA. AAV viral genomes (vg) were quantified by qPCR using genomic DNA extracted from the same tissues. Data shown here were calculated for gene transfer to the same tissues using mRNA levels after normalization to housekeeping gene 18S mRNA levels. The graph shows the fold enrichment of transgene expression averaged across nine different cardiac LV regions (anterior base, lateral base, posterobasal, anterior central, lateral central, posterocentral, anterior apex, lateral apex, and posteroapex) compared to the liver, cerebral cortex (brain), and five different skeletal muscles (triceps brachii medial head, triceps brachii long head, triceps brachii gluteal, and deltoid). "WPRE" refers to AAVs generated using the plasmids in Figure 23 and similar plasmids encoding human RBD and mAKAP shRNA. "Calseq" refers to AAVs generated with the plasmids in Figures 7-12. "enh.int" refers to AAVs generated with the plasmids in Figures 1-6. [Figure 31]Figures 31-1 to 31-4 show the nucleotide sequence of the TNNT2 gene (5167 bp extracted from NCBI chromosome reference NC_000001.11 201378367..201373201; the gene is in the antisense orientation). Exon 1 is located at bp 688-745 of the TNNT2 gene (NC_000001.11 201377680..201377623), and exon 2 is located at bp 5100-5154 (NC_000001.11 201373268..201373214). [Figure 32] Figure 32 shows CaMKII. (a) Ca2+ / calmodulin (CaM) binding releases autoinhibition of the CaMKII pseudosubstrate domain. Post-translational modifications at the N-terminus of the regulatory domain (exons 11-12 of exons 11-19 (Duran, Nickel et al. 2021)) positively (+) and negatively (-) regulate CaMKII activity (SEQ ID NO: 80). (b) CaMKII dimeric holoenzyme (PDB IDs: 5VLO, 2VN9, 3SOA) color-coded as in panel a. (c) The three most highly expressed CaMKIIδ variants in the adult heart (SEQ ID NOs: 81-82). Figure reproduced from Reyes Gaido, et al. (Reyes Gaido, Nkashama et al. 2023). [Figure 33] Figure 33 shows the association of endogenous mAKAPβ with CaMKII in muscle cells. Neonatal muscle cells expressing myc-GFP or myc-RBD-GFP were used in immunoprecipitation assays using CaMKII antibody and control IgG antibody. n=3. [Figure 34] Figure 34 shows that mAKAPβ 1694-1833 binds to various CaMKII isoforms. Myc-tagged RBD was coexpressed with Flag-tagged and mCherry-tagged CaMKII isoforms by transfection into COS-7 cells. Protein complexes were immunoprecipitated using myc-tag antibodies. [Figure 35]Figure 35 shows that RSK3 and aMKII bind to overlapping sites on mAKAPβ. Myc-GFP-tagged mAKAP fragments were coexpressed with CaMKIIγ-mCherry-Flag or mCherry-HA-RSK3 in COS-7 cells by transfection. Protein complexes were immunoprecipitated using myc-tag antibodies. n=2 (RSK3), 3 (CaMKIIγ). [Figure 36] Figure 36 shows that expression of mAKAP 1694-1833 inhibits HDAC4 phosphorylation in neonatal myocytes treated with Ang II. HDAC4 was immunoprecipitated from adenovirus-infected myocytes treated with 100 nM Ang II for 1 hour. n=3 biological replicates. The lower band in the p-HDAC4 blot corresponds to HDAC4. DETAILED DESCRIPTION OF THE INVENTION

[0048] As mentioned above, AKAP-based signaling complexes play a central role in the regulation of physiological and pathological cardiac events. Therefore, we investigated the use of drugs that target specific protein-protein interactions to inhibit the signaling properties of individual AKAP signaling complexes as an approach to limit cardiac pathological processes. Such a therapeutic strategy has advantages over classical therapeutic approaches because it can selectively inhibit defined cellular responses.

[0049] Anchoring proteins, including mAKAPβ, are therapeutic targets for the treatment of pathological cardiac hypertrophy and heart failure. Specifically, we have found that disrupting mAKAPβ-mediated protein-protein interactions inhibits the ability of mAKAPβ to regulate the activation and function of enzymes that play a central role in the activation of key transcription factors and chromatin-modifying enzymes that initiate and / or promote the remodeling process leading to heart failure.

[0050] One aspect of the present invention is to inhibit the progression of heart disease to heart failure by inhibiting intracardiac signaling that promotes the expression of pathological cardiac genes, as evidenced by improved ventricular morphology. For example, changes in cardiomyocyte signaling can be prevented or treated by reducing the LV internal diameter in eccentric heart disease by inhibiting cardiomyocyte elongation, or by suppressing cardiomyocyte hypertrophy and increased LV wall thickness in concentric heart disease. Gene therapy vectors based on expression from muscle A-kinase anchoring protein (mAKAP, also known as AKAP6) have been demonstrated to prevent cardiac dysfunction: (1) mAKAP shRNA (see, e.g., U.S. Patent No. 10,907,153, subsequently published by Martinez et al. 2022, which are incorporated by reference in their entireties for all purposes); (2) RBD peptides (see, e.g., U.S. Patent Nos. 9,132,174, 9,937,228, 10,617,337, and 11,229,679, subsequently published by Li et al. 2020, which are incorporated by reference in their entireties for all purposes); and (3) PBD peptides (see, e.g., U.S. Application No. 16,818,771, which are incorporated by reference in their entireties for all purposes).

[0051] In particular, the inventors have discovered a novel vector containing the human cardiac troponin t2 (hTNNT2) promoter, which showed increased expression levels of this molecule in the left ventricle compared to other tissues.

[0052] In one embodiment, such a vector also contains a skeletal muscle enhancer with splicing consensus sites.

[0053] In some embodiments, the vectors also contain codons optimized for expression and / or to reduce immunogenicity.

[0054] In accordance with the present invention, one skilled in the art may employ conventional molecular biology, microbiology, and recombinant DNA techniques, which are fully explained in the literature. For example, Sambrook et al, "Molecular Cloning: A Laboratory Manual" (4th Ed., 2012);"Current Protocols in Molecular Biology" Volumes I-III [Ausubel, RM, ed. (1994)];"Cell Biology: A Laboratory Handbook" Volumes I-III [JE Celis, 3rd ed. (2005))]; Volumes I-III [Coligan, JE, ed. (2005)];"Oligonucleotide Synthesis" (MJ Gait ed. 1984);"Nucleic Acid Hybridization" [BD Hames & SJ Higgins eds. (1985)];"Transcription And Translation" [BD Hames & SJ Higgins, eds. (1984)];"Animal Cell Culture" [RI Freshney, ed. (1986)];"Immobilized Cells And See "Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984); C. Machida, "Viral Vectors for Gene Therapy: Methods and Protocols" (2010); J. Reidhaar-Olson and C. Rondinone, "Therapeutic Applications of RNAi: Methods and Protocols" (2009).

[0055] The following definitions and abbreviations are used herein: AAV: adeno-associated virus AC5: adenylyl cyclase type 5 ACE: Angiotensin-converting enzyme inhibitor ANF: atrial natriuretic factor ARVM: adult rat ventricular myocytes CaN: Calcineurin CArG box: (CC9AT)6GG CPT-cAMP: 8-(4-chlorophenylthio) cyclic adenosine monophosphate CsA: Cyclosporin A CTKD: C-terminal kinase domain ERK: extracellular signal-regulated kinase FBS: fetal bovine serum Fsk: Forskolin GFP: green fluorescent protein GPCR: G protein-coupled receptor HDAC: histone deacetylase Gs: stimulatory G protein GST: glutathione S-transferase HIF1α: Hypoxia-inducible factor 1α HFrEF: heart failure with reduced ejection fraction IBMX: 3-isobutyl-1-methylxanthine Iso: Isoproterenol LIF: leukemia inhibitory factor MADS (MCM1, agamous, deficiens, SRF) domain: Mediates DNA binding to the CArG box (CC9AT)6GG of the serum response element (SRE). The MADS-box gene family is named after the initials of the four genes that were initially identified in this family, excluding ARG80: MCM1 from budding yeast (Saccharomyces cerevisiae), AGAMOUS from Arabidopsis thaliana, DEFICIENS derived from snapdragon (Antirrhinum majus) (Reference 10), SRF derived from humans (Homo sapiens). mAKAP: A kinase anchor protein mAKAPα: alternatively spliced ​​isoform, 255 kDa, expressed in neuronal cells mAKAPβ: alternatively spliced ​​isoform, 230 kDa, expressed in striated muscle cells MAPK: mitogen-activated protein kinase MEF2: Myocyte enhancer factor 2 MgAc: Magnesium acetate MI: Myocardial infarction NCX1: sodium / calcium exchanger NFATc: nuclear factor of activated T cells NRVM: neonatal rat ventricular myocytes NTKD: N-terminal kinase domain OA: Okadaic acid PBD: PP2A-anchor breaking factor-inhibition of eccentric hypertrophy PDE4D3: cAMP-specific phosphodiesterase type 4D3 PDK1: 3-phosphoinositide-dependent protein kinase-1 PE: Phenylephrine PHD: prolyl hydroxylase PI4P: phosphatidylinositol 4-phosphate PKA: protein kinase A PKD: Protein kinase D PKI: Protein kinase inhibitor PLCε: phospholipase Cε PKA: cAMP-dependent protein kinase PP2A: Protein (serine / threonine) phosphatase - SRF Ser 103 is phosphorylated. PP2B calcium / calmodulin-dependent protein phosphatase 2B RBD: isoform-specific N-terminal RSK3 domains bind to distinct "RSK3-binding domains" within mAKAPβ located at residues 1694-1833 (RBD) RSK: p90 ribosomal S6 kinase RyR2: type 2 ryanodine receptor scAAV: self-complementary AAV siRNA: small interfering RNA oligonucleotide shRNA: small hairpin RNA SRE: serum response element SRF: Serum response factor-transcription factor (SRF Ser 103 Phosphorylation induces afferent myocyte and cardiac hypertrophy; inhibition of phosphorylation improves cardiac structure and function. siRNA: small interfering RNA TAC: Aortic arch coarctation TCA: Trichloroacetic acid VSV: vesicular stomatitis virus

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although similar or equivalent methods and materials to those described herein can be used to practice or test the present invention, the nomenclature and techniques used in connection with cell biology and molecular biology and chemistry, which describe preferred methods and materials, are generally well known and commonly used in the art. Specific experimental techniques are not specifically defined, but are generally carried out according to conventional methods well known in the art, as described in various general and more specific references cited and discussed throughout this specification. For clarity, the following terms are defined:

[0057] The present invention recognizes that the interaction of RSK3 and / or PP2A with mAKAPβ mediates various intracellular signals and pathways that lead to cardiomyocyte hypertrophy and / or dysfunction. Therefore, the present inventors have discovered various methods for inhibiting this interaction to prevent and / or treat cardiomyocyte hypertrophy and / or dysfunction.

[0058] Therefore, the present invention includes a method for protecting the heart from damage by administering to a patient at risk of such damage a pharmaceutically effective amount of a composition that inhibits the interaction of RSK3 and / or PP2A with mAKAPβ or reduces the expression level of mAKAPβ. It should be understood that the "pharmaceutically effective amount" can be empirically determined based on the delivery method and varies depending on the delivery method.

[0059] The present invention also relates to a method for treating heart disease by administering to a patient a pharmaceutically effective amount of a composition that inhibits the interaction between RSK3 and / or PP2A and mAKAPβ.

[0060] The present invention also relates to compositions that inhibit the interaction of RSK3 and / or PP2A with mAKAPβ. In certain embodiments, these inhibitory compositions or "inhibitors" comprise peptide inhibitors, which can be administered by any known method, including by gene therapy delivery. In other embodiments, the inhibitors can be small molecule inhibitors.

[0061] Specifically, the present invention is directed to methods and compositions for treating or protecting the heart from damage by administering to a patient at risk of such damage a pharmaceutically effective amount of a composition that (1) inhibits the interaction between RSK3 and / or PP2A and mAKAPβ, (2) inhibits the activity of RSK3 and / or PP2A and mAKAPβ, or (3) inhibits the expression of RSK3, PP2A and / or mAKAPβ.

[0062] The present invention also relates to methods for treating or protecting the heart from damage by administering to a patient at risk of such damage a pharmaceutically effective amount of a composition that inhibits cellular processes mediated by RSK3 and / or PP2A.

[0063] In one embodiment, the composition comprises a mAKAPβ peptide. In a preferred embodiment, the mAKAPβ peptide is obtained from the carboxy terminus of the mAKAPβ amino acid sequence. In a particularly preferred embodiment, the mAKAPβ peptide is a fragment of at least amino acids 2083 to 2319 of the mAKAPβ amino acid sequence.

[0064] In a preferred embodiment, the mAKAPβ peptide is a fragment of at least amino acids 2133 to 2319 of the mAKAPβ amino acid sequence.

[0065] In one embodiment, the composition comprises a mAKAPβ peptide.

[0066] In preferred embodiments, the mAKAPβ peptide consists of nucleotides 1735-1833 of the mAKAP amino acid sequence, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0067] In other embodiments, the mAKAPβ peptide is derived from the carboxy terminus of the mAKAPβ amino acid sequence. In particularly preferred embodiments, the mAKAPβ peptide is a fragment of at least amino acids 2083 to 2319 of the mAKAPβ amino acid sequence, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0068] In a preferred embodiment, the mAKAPβ peptide is at least a fragment of amino acids 2133-2319 of the mAKAPβ amino acid sequence, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0069] In another embodiment, the composition comprises a small interfering RNA (siRNA) that inhibits the expression of any of RSK3, PP2A, and mAKAPβ. In a preferred embodiment, the siRNA that inhibits the expression of mAKAPβ is generated in vivo after administration of a small hairpin RNA expression vector or a biological agent (shRNA).

[0070] The compositions of the present invention can be administered directly or using a viral vector, hi a preferred embodiment, the vector is an adeno-associated virus (AAV).

[0071] In another embodiment, the composition comprises a small molecule inhibitor. In a preferred embodiment, the small molecule is an RSK3, PP2A and / or mAKAPβ inhibitor.

[0072] In another embodiment, the composition comprises a molecule that inhibits the binding, expression, or activity of mAKAPβ. In a preferred embodiment, the molecule is a mAKAPβ peptide. The molecule can be expressed using a viral vector, including an adeno-associated virus (AAV).

[0073] In yet another embodiment, the composition comprises a molecule that interferes with a cellular process mediated by mAKAPβ, hi some preferred embodiments, the molecule interferes with the binding of mAKAPβ to RSK3 or to the anchoring of PP2A.

[0074] The present invention also relates to diagnostic assays for determining a propensity for heart disease, in which the binding interaction of RSK3 and / or PP2A with mAKAPβ is measured directly or by measuring the downstream effects of the binding of RSK3 and / or PP2A with mAKAPβ. The present invention also provides test kits for such assays.

[0075] In yet other embodiments, the inhibitor comprises any molecule that inhibits the expression of RSK3, PP2A and / or mAKAPβ, including antisense RNA, ribozymes and small interfering RNA (siRNA).

[0076] The present invention also includes an assay system for screening potential agents effective in inhibiting the expression and / or binding of RSK3 and / or PP2A and mAKAPβ. In one example, a test agent can be administered to a cell sample having RSK3 and / or PP2A and mAKAPβ, or to an extract containing RSK3 and / or PP2A and mAKAPβ, to determine its effect on the binding activity of RSK3 and / or PP2A and mAKAPβ by comparison with a control. The present invention also provides a test kit for such an assay.

[0077] When preparing the peptide compositions of the present invention, all or part of the RSK3 and / or PP2A or mAKAP amino acid sequence can be used. Preferably, at least 10 amino acids of the mAKAP sequence are used. More preferably, at least 25 amino acids of the mAKAP sequence are used. Most preferably, a peptide segment from amino acids 1735-1833 or 2133-2319 of mAKAP is used.

[0078] It should be understood that various amino acid substitutions, deletions, or insertions can also enhance the ability of the inhibitory peptide to inhibit the interaction between RSK3 and / or PP2A and mAKAPβ. This type of substitution mutation can change the amino acids in the resulting protein non-conservatively (i.e., by changing an amino acid belonging to a group of amino acids with a particular size or property to an amino acid belonging to another group) or conservatively (i.e., by changing an amino acid belonging to a group of amino acids with a particular size or property to an amino acid belonging to the same group). Such conservative changes generally result in less change in the structure and function of the resulting protein. Non-conservative changes are likely to change the structure, activity, or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes that do not significantly change the activity or binding properties of the resulting protein.

[0079] Below are some examples of different groupings of amino acids: Amino acids with nonpolar R groups : Alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine. Amino acids with uncharged polar R groups: Glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine. Amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid. Basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine.

[0080] Furthermore, amino acids can be grouped into those with phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0081] Further, they can be grouped by molecular weight (i.e., size of the R group): glycine (75), alanine (89), serine (105), proline (115), valine (117), threonine (119), cysteine ​​(121), leucine (131), isoleucine (131), asparagine (132), aspartic acid (133), glutamine (146), lysine (146), glutamic acid (147), methionine (149), histidine (at pH 6.0) (155), phenylalanine (165), arginine (174), tyrosine (181), and tryptophan (204).

[0082] Particularly preferred substitutions are: -Lys to Arg and vice versa can maintain a positive charge; -Asp to Glu and vice versa can maintain a negative charge; -Thr can be changed to Ser to maintain a free -OH; and By changing -Asn to Gln, free NH2 can be maintained.

[0083] Amino acid substitutions can also be introduced to replace amino acids with particularly favorable properties. For example, Cys can be introduced as a potential site for disulfide bonding with another Cys. His may be introduced as a particularly "catalytic" site (i.e., His can function as an acid or a base and is the most common amino acid in biochemical catalysis). Pro may be introduced to induce a β-turn in the protein's structure, particularly a planar structure. Two amino acid sequences are "substantially homologous" if at least about 70% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the amino acid residues are identical or have conservative substitutions.

[0084] Similarly, the nucleotide sequences utilized in accordance with the present invention may be substituted, deleted, or inserted. If the codons encoding a particular amino acid are degenerate, any codon encoding that particular amino acid may be used. Furthermore, if it is desired to replace one amino acid with another, the nucleotide sequence may be modified in accordance with the known genetic code.

[0085] Nucleotides and oligonucleotides can also be modified. U.S. Patent No. 7,807,816, which is incorporated herein by reference in its entirety, describes exemplary modifications, particularly in its description of modified nucleotides and oligonucleotides.

[0086] Two nucleotide sequences are "substantially homologous" or "substantially identical" when at least about 70% (preferably at least about 80%, and most preferably at least about 85%, 90%, 95% or 99%) of the nucleotides are identical.

[0087] Two nucleotide sequences are "substantially complementary" when at least about 70% (preferably at least about 80%, and most preferably at least about 85%, 90%, 95% or 99%) of the nucleotides are capable of hydrogen bonding with the target sequence.

[0088] The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5xSSC and 65°C for both hybridization and washing. However, those skilled in the art will understand that such "standard hybridization conditions" depend on specific conditions, including the sodium and magnesium concentrations in the buffer, the length and concentration of the nucleotide sequence, the percentage of mismatches, the percentage of formamide, and the like. In determining "standard hybridization conditions," it is also important whether the two hybridizing sequences are RNA-RNA, DNA-DNA, or RNA-DNA. Such standard hybridization conditions are readily determined by those skilled in the art according to well-known formulas, in which hybridization typically occurs at a predicted or determined T m The reaction is carried out at a temperature 10-20°C lower than the standard temperature, and washed at higher stringency if necessary.

[0089] "Pharmaceutically acceptable" refers to a molecular entity or composition that is physiologically tolerable and does not produce allergic or similar unpleasant reactions, such as upset stomach, dizziness, etc., when administered to a human.

[0090] As used herein, a "therapeutically effective amount" means an amount sufficient to prevent, and preferably reduce by at least about 30%, 40%, 50%, 60%, 70%, 80% or 90%, clinically significant changes in cardiomyocyte characteristics.

[0091] The preparation of therapeutic compositions containing polypeptides, analogs, or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, although solid forms suitable for solution or suspension in liquid prior to injection can also be prepared. Preparations can also be emulsified. The therapeutically active ingredient is often mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Additionally, if desired, the composition can contain minor amounts of auxiliary substances, such as wetting agents, emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.

[0092] Polypeptides, analogs or active fragments, and small molecule inhibitors can be formulated in therapeutic compositions as neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), such as with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0093] The therapeutic composition of the present invention is conventionally administered intravenously, for example, by injection of a unit dose.When used in relation to the therapeutic composition of the present invention, the term "unit dose" refers to a physically separate unit suitable as a single dose for humans, each unit containing a predetermined amount of active substance calculated to produce a desired therapeutic effect in association with a required diluent; i.e., carrier or vehicle.

[0094] The composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount administered depends on the subject being treated, the capacity of the subject's immune system to utilize the active ingredient, and the desired degree of inhibition of RSK3 and / or PP2A-mAKAPβ binding. The precise amount of active ingredient required for administration depends on the judgment of the skilled artisan and is unique to each individual. However, suitable dosages range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably 1 to several milligrams of active ingredient per kilogram of body weight per day, depending on the route of administration. Suitable regimes for initial administration and booster injections vary, but typically include an initial administration followed by subsequent injections or other administrations at intervals of one hour or more. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations between 10 nanomolar and 10 micromolar is contemplated. In a preferred embodiment, the mAKAPβ peptide or shRNA is expressed by an AAV gene therapy vector. An appropriate intravenous dose of AAV vector is 1×10 12 5 x 10 viral genomes / kilogram body weight 14 In particular, for AAV vectors containing serotype 9 capsids, the dosage is preferably 0.3 to 1 × 10 14 viral genomes / kilogram body weight.

[0095] Because the inhibitor needs to reach the cytoplasm, the peptide according to the present invention must be modified to allow translocation across the cell membrane or be expressed by a vector encoding the peptide inhibitor. Similarly, nucleic acid inhibitors (including siRNA, shRNA, and antisense RNA) can also be expressed by vectors. Any vector capable of entering the target cell can be used according to the present invention. In particular, viral vectors can "infect" cells and express the desired RNA or peptide. Any viral vector capable of "infecting" cells can be used. A particularly preferred viral vector is adeno-associated virus (AAV).

[0096] siRNAs inhibit the translation of target mRNAs through a process called RNA interference. When siRNAs are perfectly complementary to a target mRNA, they act by promoting mRNA degradation. shRNAs, as a special type of siRNA, offer certain advantages over siRNAs produced as oligonucleotides. siRNA oligonucleotides are typically synthesized in the laboratory and delivered to cells using a delivery system that delivers siRNA to the cytoplasm. In contrast, shRNAs are expressed as minigenes delivered to the cell nucleus via vectors, where, after transcription, they are processed into mature siRNA species by cellular enzymes such as Drosha and Dicer. While siRNAs are typically 99% degraded after 48 hours, shRNAs can be expressed for 3 years or longer. Furthermore, shRNAs can be delivered at much lower copy numbers than siRNAs (5 copies vs. low nM), making them much less likely to cause off-target effects, immune activation, inflammation, or toxicity. While siRNAs are suitable for acute diseases where high doses are tolerated, shRNAs are suitable for chronic or life-threatening diseases where lower doses are desired. (http: / / www.benitec.com / technology / sirna-vs-shrna)

[0097] Guidelines for siRNA and shRNA design can be found on various websites, including Elbashir et al. 2001, https: / / www.thermofisher.com / us / en / home / references / ambion-tech-support / rnai-sirna / general-articles / -sirna-design-guidelines.html, and http: / / www.invivogen.com / review-sirna-shrna-design, all of which are incorporated herein by reference in their entirety. Preferably, the first nucleotide is A or G. 25-29 nucleotide siRNAs may be more effective than shorter ones, while shRNAs with duplex lengths of 19-21 nucleotides appear to be as effective as longer ones. siRNAs and shRNAs are preferably 19-29 nucleotides long. The length of the shRNA loop sequence may be 3-9 nucleotides, with 5, 7, or 9 nucleotides being preferred.

[0098] Exemplary shRNA sequences of the invention include GGTTGAAGCTTTGAAGAAA (SEQ ID NO: 77), GCTAAGAGATACAGAGCTT (SEQ ID NO: 78) or GGAGGAAATAGCAAGGTTA (SEQ ID NO: 79).

[0099] Regarding small molecule inhibitors, any small molecule that inhibits the interaction of RSK3 and / or PP2A with mAKAPβ can be used. Furthermore, any small molecule that inhibits the activity of RSK3 and / or PP2A and / or mAKAPβ can be used.

[0100] Small molecule compounds with similar structures and functionality can also be determined by rational screening approaches.

[0101] Similarly, any small molecule that inhibits the expression of RSK3, PP2A and / or mAKAPβ can be used.

[0102] More particularly, the present invention is described by the following items, which represent preferred embodiments thereof:

[0103] 1. A composition comprising a regulatory nucleotide sequence for the expression of a second nucleotide sequence in cardiomyocytes, wherein the regulatory nucleotide sequence comprises an intron sequence comprising a splicing consensus site, and the intron sequence is derived from the human cardiac troponin T gene (hTNNT).

[0104] 2. The composition of paragraph 1, further comprising a TNNT2 promoter sequence.

[0105] 3. The composition of claim 1, wherein the regulatory nucleotide sequence is in a vector.

[0106] 4. The composition of paragraph 3, further comprising a transgene.

[0107] 5. The composition of paragraph 4, wherein the transgene is a muscle A kinase anchor protein beta (mAKAPβ) sequence.

[0108] 6. The composition described in paragraph 5, wherein the mAKAPβ sequence is an shRNA.

[0109] 7. The composition described in paragraph 6, wherein the shRNA comprises GGTTGAAGCTTTGAAGAAA (SEQ ID NO: 77), GCTAAGAGATACAGAGCTT (SEQ ID NO: 78) or GGAGGAAATAGCAAGGTTA (SEQ ID NO: 79).

[0110] 8. The composition of paragraph 3, wherein the vector encodes an amino acid sequence having at least 80% sequence identity to a fragment of mAKAPβ.

[0111] 9. The composition of paragraph 8, wherein the vector encodes an amino acid sequence having at least 90% sequence identity to a fragment of mAKAPβ.

[0112] 10. The composition of paragraph 9, wherein the amino acid sequence encodes a fragment of mAKAPβ.

[0113] 11. The composition of paragraph 8, wherein the amino acid sequence binds to a kinase.

[0114] 12. The composition of paragraph 11, wherein the kinase is p90 ribosomal S6 kinase 3 (RSK3).

[0115] 13. The composition of paragraph 12, wherein the amino acid sequence inhibits binding of mAKAPβ to RSK3.

[0116] 14. The composition of paragraph 10, wherein the amino acid sequence has at least 80% sequence identity to amino acids 1694-1757, 1735-1833, or 1694-1833 of mAKAP.

[0117] 15. The composition of claim 14, wherein the amino acid sequence has at least 90% sequence identity to amino acids 1735 to 1833 of mAKAP.

[0118] 16. The composition described in paragraph 12, wherein the amino acid sequence comprises the RSK3 binding domain (RBD) of mAKAPβ.

[0119] 17. The composition of claim 15, wherein the RBD comprises amino acids 1735 to 1833 of SEQ ID NO: 12.

[0120] 18. The composition of claim 11, wherein the amino acid sequence binds to protein phosphatase 2A (PP2A).

[0121] 19. The composition according to paragraph 18, wherein the amino acid sequence inhibits anchoring of PP2A to mAKAPβ.

[0122] 20. The composition of paragraph 19, wherein the amino acid sequence has at least 80% sequence identity to amino acids 2132 to 2319 of mAKAP.

[0123] 22. The composition of paragraph 20, wherein the amino acid sequence has at least 90% sequence identity to amino acids 2132 to 2319 of mAKAP.

[0124] 23. The composition described in paragraph 20, wherein the amino acid sequence comprises the PP2A binding domain (PBD) of mAKAPβ.

[0125] 24. The composition of paragraph 23, wherein the PBD comprises amino acids 2132-2319 of SEQ ID NO: 12.

[0126] 25. The kinase is Ca 2+ / The composition of any one of paragraphs 11, 14 or 15, wherein the protein is calmodulin-dependent protein kinase II (CaMKII).

[0127] 26. The composition of any one of paragraphs 3 to 25, wherein the vector is an adeno-associated virus (AAV).

[0128] 27. The composition of any one of paragraphs 3 to 26, wherein the vector further comprises an SV40 polyadenylation sequence.

[0129] 28. The composition of paragraph 5, wherein human mAKAP amino acids 2132 to 2319 (sequence number 11) are modified at one or more of the following positions: TCG to TCA at amino acid 2144; AGC to AGT at amino acid 2183; TCC to TCA at amino acid 2256; GCC to GCA at amino acid 2291; or CGA to AGA at amino acid 2313.

[0130] 29. The composition of paragraph 5, wherein human mAKAP amino acids 1696 to 1835 (SEQ ID NO: 11), encoding the RBD, are modified at one or more of the following positions: CCG to CCA at amino acid 1712; TCG to TCT at amino acid 1714; TCG to TCT at amino acid 1717; CGT to AGA at amino acid 1721; CGT to AGA at amino acid 1724; AGC to AGT at amino acid 1730; AGC to AGT at amino acid 1753; or GAC to GAT at amino acid 1775.

[0131] 30. A method for treating or preventing heart disease, comprising administering the vector described in any one of items 1 to 29 to cardiac cells of a patient.

[0132] 31. A method for treating or preventing cardiac disease, comprising administering to cardiac cells of a patient a vector according to paragraph 6 or 7, wherein said method inhibits expression of mAKAP.

[0133] Several recent clinical trials have reported varying degrees of immunotoxicity following recombinant adeno-associated virus (rAAV) administration, limiting the durability and success of gene therapy in humans (Wright 2020; Hamilton and Wright 2021). Molecules such as unmethylated CpG dinucleotide motifs (CpGs) signal AAV virus infection (Akira, Uematsu, and Takeuchi 2006; Kanneganti, Lamkanfi, and Nunez 2007) and promote the host innate immune response through activation of the Toll-like receptor (TLR9)-MyD88 signaling pathway, which leads to the recruitment of cytotoxic T lymphocytes (CTLs) to infected cells (Hartmann, Weiner, and Krieg 1999; Ohto et al. 2018; Zhu, Huang, and Yang 2009; Shirley et al. 2020; Xiang et al. 2020). Furthermore, vaccine research using oligonucleotides as adjuvants has introduced CpG-containing immunostimulatory motifs (e.g., ACGT, TCGT, CCGT) and inhibitory motifs (e.g., GCGG, CCGC, GCGC) to the scientific community (Wright 2020; Ohto et al. 2015; Bode et al. 2011; Pohar et al. 2017).

[0134] Due to differences in TLR / innate receptor sensitivity between humans and most other preclinical animal models (Tahtinen et al. 2022; Hawash et al. 2021), the immunostimulatory function of AAV gene therapy in human patients may not be manifested during preclinical development and cannot be expected in rodent or porcine studies. However, to prevent potential immunological reactions and loss of transgene expression in patients, new versions of mAKAPβ-targeting AAV biologics have been designed. The newly proposed viral genome (vg) configuration features a novel expression cassette designed to enhance expression in the heart and reduce immunogenicity in humans. The sequence has been optimized by removing CpG and CpG-containing immunostimulatory motifs and retaining CpG-containing immunosuppressive motifs.

[0135] Off-target effects can also be suppressed by using tissue-specific promoters that restrict expression of the gene of interest to the relevant cell type. The TNNT2 gene promoter has been used as a means of cardiomyocyte-selective expression of recombinant proteins and shRNA (Prasad et al. 2011; Martinez et al. 2022; Li et al. 2020) (U.S. Patent No. 11,129,908). The construct of the present invention contains a novel configuration of the human TNNT2 promoter, incorporating additional promoter sequences, sequences from exons 1 and 2, and the first intron, which confers greater cardiomyocyte specificity in expression and is useful for in vivo applications. This novel TNNT2 promoter composition is useful not only for AAV gene therapy, but also for gene transfer and transient expression in cardiomyocyte-specific applications based on both plasmid and viral vectors.

[0136] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. [Example]

[0137] The compositions and processes of the present invention will be better understood in connection with the following examples, which are intended as illustrations only and are not intended to limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications, including but not limited to those relating to the processes, formulations and / or methods of the invention, can be made without departing from the spirit of the invention and the scope of the appended claims.

[0138] [Example 1] (New AAV configuration) For each of the three AAV9 self-complementary biologics shown here, two alternative layouts are shown, representing alternative configurations for cardiomyocyte-specific gene therapy:

[0139] 1. Promoter, exon sequences, and skeletal muscle enhancer and splicing consensus sites (hTNNT+enh+int)+ Transgene + Human cardiac troponin T2 regulatory sequence including intron sequence encompassing SV40 polyadenylation (SV40polyA). The complete plasmid sequence and corresponding vector map are shown in Figures 1-6. Composition #1: pAAVsc.hTNNT+enh.int. shmAKAP .SV40polyA, Composition #2: pAAVsc.hTNNT+enh.int. hRBD .SV40polyA, Composition #3: pAAVsc.hTNNT+enh.int. hPBD .SV40polyA.

[0140] 2. Human calsequestrin enhancer (calseq) + human cardiac troponin T2 promoter (hTNNT) + SV40 16S synthetic intron (SV40int.) + Transgene +SV40 polyadenylation. We present the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence, including the human calsequestrin enhancer and a novel combination of promoter and exon sequences from the human TNNT2 gene, to drive expression of the human mAKAP PBD sequence. The complete plasmid sequence and corresponding vector map are shown in Figures 7-12. Composition #4: pAAVsc.calseq.hTNNT.SV40int. shmAKAP .SV40polyA, Composition #5: pAAVsc.calseq.hTNNT.SV40int. hRBD .SV40polyA, Composition #6: pAAVsc.calseq.hTNNT.SV40int. hPBD .SV40polyA.

[0141] We optimized the CpG content of all elements by selecting human genomic sequences that exhibited the fewest CpG dinucleotides, as well as CpG-containing tetranucleotides known to stimulate immune responses. We also performed codon optimization wherever possible to remove inflammatory motifs from open reading frame (ORF) sequences. Sequence alignments are shown in Figures 8 and 9. A detailed list of modifications, summarized per ORF, is provided here.

[0142] [Table 1]

[0143] (composition 1-3) In the first presented construct, cardiac-specific expression is achieved with a novel combination of regulatory elements. The novel TNNT2 transcriptional regulatory sequences include: 1) the TNNT2 promoter, exon 1, and adjacent intron 1 sequences from -673 to +79 (transcript_id: NM_000364.4; NCBI chromosome reference: NC_000001.11 201,378,353..201,377,602, corresponding to bp 1-752 of the new construct "New regulatory"); 2) the intronic enhancer (NC_000001.11 201,376,706..201,376,196, corresponding to bp 753-1263 of the new construct); and 3) the 3' sequence of intron 1 and partial sequence of exon 2 (NC_000001.11 201,373,323..201,373,261, corresponding to bp 1264-1326 of the new construct).

[0144] The TNNT2 gene sequence (5167 bp excerpt) shown in Figure 31 is from NCBI chromosome reference NC_000001.11 201378367..201373201 (gene in antisense orientation). Exon 1 is located at bp 688-745 of the TNNT2 gene (NC_000001.11 201377680..201377623), and exon 2 is located at bp 5100-5154 of the TNNT2 gene (NC_000001.11 201373268..201373214). The hTNNT2 gene intron region from bp 1662 to 2172 in Figure 31 (NC_000001.11 201,376,706..201,376,196, bp 753-1263 in the new construct) contains an enhancer that has been reported to enhance expression in vitro in a differentiated mouse skeletal muscle cell line (C2C12) (Kwon et al. 2011). Sequences at the extreme 5' and 3' ends of intron 1 are included to facilitate mRNA splicing and promote transgene expression.

[0145] (composition 4-6) The second presented construct incorporates a previously described cardiomyocyte-specific transcriptional cis-regulatory motif (Chamberlain et al. 2018) in the first intron region of the human calsequestrin gene (NCBI chromosome reference NC_000001.11-115,768,786..115,768,977) and a -936 to +42 bp human TNNT2 promoter fragment (NCBI chromosome reference NC_000001.11-201,378,613..201,377,636) to promote cardiac-specific expression of therapeutic transgenes.

[0146] Human cardiac troponin T sequences with the following gene coordinates are listed in Table 1 of U.S. Pat. No. 11,129,908: −569 to +31 (TNNT2p-600 in Table 1 of U.S. Pat. No. 11,129,908); −469 to +31 (TNNT2p-500 in Table 1 of U.S. Pat. No. 11,129,908); −369 to +31 (TNNT2p-400 in Table 1 of U.S. Pat. No. 11,129,908); and −269 to +31 (TNNT2p-300 in Table 1 of U.S. Pat. No. 11,129,908). U.S. Pat. No. 11,312,943 discloses a human gene sequence with coordinates from −499 to +6, as well as a synthetic human troponin T promoter sequence featuring a unique 242-nucleotide sequence. Werfel et al. disclose the use of the hTNNT2 promoter, consisting of -499 to +45 (termed -502 to +42 by Werfel et al.) and a smaller fragment in AAV (Werfel et al. 2014).

[0147] Compositions 1-3 are the first to describe the use of the skeletal muscle enhancer of the human TNNT2 gene, either by itself or in combination with the human TNNT2 promoter, as part of the regulatory sequence directing cardiac expression. The promoter fragments in these compositions are larger than others previously described for expression vectors.

[0148] Other elements that characterize the rAAV expression cassette are the SV40 16S synthetic intron and polyadenylation element, features common to both plasmid and viral expression vectors, which are incorporated into the expression cassette to promote high-level and efficient gene expression. Finally, the AAV2 inverted terminal repeat (ITR) 3' to the "right" transgene was modified by partial deletion of the terminal resolution site, which allows for genomic hairpin formation, resulting in a self-complementary (sc) vector that maximizes vector efficacy while allowing for a lower systemic dose. The AAV2 ITR 5' to the "left" transgene is unmodified, as it is required in cis for both viral DNA replication and rAAV vector genome packaging.

[0149] [Example 2] After producing AAV serotype 9 virus using compositions 1–6, expression induced by the two new layouts (layouts 1 and 2 are abbreviated "enh.int" and "calseq.", respectively) was tested in 5–8 kg piglets by intravenous (i.v.) administration of ~2E12 vg / kg of each AAV. Gene expression and AAV biodistribution were assessed postmortem by quantitative polymerase chain reaction (qPCR) in liver, brain, and heart tissues 3 months after AAV injection. Unexpectedly, transgene expression (analyzed by reverse transcriptase-qPCR [qRT-PCR] and normalized to an 18S RNA control) of the enh.int compositions (shown in Figures 1–6) was ~800-fold higher in cardiac left ventricle than in skeletal muscle, and ~3-fold and ~20-fold higher in left ventricle than in liver and brain, respectively. Intravenously administered AAV9 in pigs preferentially transduces the liver, with similar transduction to the heart, skeletal muscle, and cerebral cortex (Li et al. 2022). Data presented in Figure 30 demonstrate that, when compared to the "WPRE" AAV composition similar to Figure 23, the new enh.int composition significantly (p<0.05) enhanced cardiac left ventricle selectivity of transgene expression (transgene expression normalized by delivery of the AAV9 genome to different tissues) by ~60-fold over skeletal muscle, while maintaining selectivity for the liver and brain. Additionally, expression in the left ventricle (normalized for delivery) was ~4-fold higher with the new composition than with the original "WPRE" composition.

[0150] [Example 3] (Ca 2+ / Calmodulin-dependent protein kinase II (CaMKII) - a key regulator in muscle cells In response to G protein-coupled receptor (GPCR) signals, CaMKII plays an important role in regulating cardiomyocyte ECC, gene transcription, inflammation, metabolism, and cell survival (Hegyi, Bers et al. 2019, Reyes Gaido, Nkashama et al. 2023). CaMKII is expressed by four alternatively spliced ​​genes, of which CaMKIIδ (CAMK2D) and γ (CAMK2G) are highly expressed in cardiac muscle (Duran, Nickel et al. 2021). Members of the CaMKII family share a common structure and function as Ca 2+ CaMKII isoforms undergo post-translational modifications that confer independence and activation (Figure 32). Functional differences between CaMKII isoforms have been observed, which may be due to differences in localization, given their similar intrinsic catalytic activities (Zhang, Kohlhaas et al. 2007). For example, CaMKIIδB has a nuclear localization signal and is cardioprotective, whereas CaMKIIδC and δ9 tend to be more abundant in the cytoplasm and are conversely associated with remodeling (Duran, Nickel et al. 2021). CaMKII is present in the plasma membrane, transverse tubules, sarcoplasmic reticulum (SR), mitochondria, nuclear membrane, and nucleus. SR ryanodine receptor 2 (RyR2) via muscle kinase anchoring protein 18δ (AKAP18δ) and SR Ca 2+ With the exception of CaMKII targeting to SR-ATPase type 2a (SERCA2a) and phospholamban (Pln) (Carlson, Aronsen et al. 2022), how CaMKII is localized within muscle cells is still poorly understood.

[0151] Elevated expression and activity of CaMKIIδ / γ are associated with cardiovascular disease and are thought to be drivers of arrhythmias and pathological remodeling (Beckendorf, van den Hoogenhof et al. 2018, Duran, Nickel et al. 2021, Reyes Gaido, Nkashama et al. 2023). CaMKII (γ, δC, but apparently not δB) physiologically regulate a variety of ion channels (Zhang, Kohlhaas et al. 2007, Kreusser, Lehmann et al. 2014) and fine-tune excitation-contraction coupling (Hegyi, Bers et al. 2019). Chronic elevated CaMKII activity may contribute to the regulation of excessive SR Ca. 2+ CaMKIIδ / γ gene deletion can induce leak and arrhythmias (Maier and Bers 2007, Beckendorf, van den Hoogenhof et al. 2018). Transgenic expression of key cardiac CaMKIIδ mutants induces hypertrophy in vivo and rapidly promotes heart failure, except for δB (Duran, Nickel et al. 2021). Conversely, CaMKIIδ deletion does not inhibit the early induction of hypertrophy by pressure overload, but inhibits eccentric hypertrophy and heart failure associated with prolonged pressure overload (Ling, Zhang et al. 2009). After myocardial infarction, CaMKIIδ / γ gene deletion prevents pathological remodeling (Weinreuter, Kreusser et al. 2014). Reactive oxygen species (ROS)-activated CaMKII δC and δ9 exacerbate muscle cell death through mitochondrial death, NF-κB death, inflammatory pathways, and impaired DNA repair (Feng and Anderson 2017, Yao, Li et al. 2022). CaMKII activation by oxidation and O-linked N-acetylglucosaminylation (O-GlcNAcylation), triggered by reactive oxygen species and hyperglycemia, is associated with diabetic cardiomyopathy (Hegyi, Bers et al. 2019, Veitch, Power et al. 2021).

[0152] Extensive data demonstrating that CaMKII signaling promotes heart failure and arrhythmias has spurred efforts to develop CaMKII-directed therapies, and it has generally been argued that loss of CaMKII activity in cardiomyocytes is well tolerated and beneficial (Nassal, Gratz et al. 2020, Lebek, Chemello et al. 2023). CaMKII family members have diverse physiological functions, including prominent roles for brain CaMKIIα and β in learning and memory (Beckendorf, van den Hoogenhof et al. 2018). Furthermore, CaMKII is required for physiological sympathetic responses, particularly in the sinoatrial node (Wu, Gao et al. 2009). Furthermore, through regulation of Pln and RyR2, maintenance of CaMKII activity is essential for early pressure-overload disease and contractile adaptation to exercise (Burgos, Yeves et al. 2017, Baier, Klatt et al. 2020). Furthermore, nuclear CaMKIIδB exerts cardioprotective effects through the phosphorylation of serine 133 of cAMP response element binding protein (CREB) (Wang, Xu et al. 2022).

[0153] [mAKAPβ, CaMKII, and HDAC4] In cardiomyocytes, the 230 kDa scaffolding protein A kinase anchoring protein β (mAKAPβ / AKAP6β) mediates Ca 2+The mAKAPβ signalosome orchestrates upstream signaling pathways, including cAMP, phosphatidylinositol 4-phosphate, mitogen-activated protein kinase, and hypoxia, regulating myocyte transcription factors and class IIa histone deacetylases (HDACs) (Dodge-Kafka, Gildart et al. 2019). Through post-translational modification of these gene regulators, the mAKAPβ signalosome influences both the extent of remodeling and the quality of cardiac hypertrophy (concentric or efferent) (Li, Tan et al. 2020). mAKAPβ is required for the induction of pathological cardiac remodeling and heart failure following chronic pressure overload, catecholamine infusion, and myocardial infarction (Kritzer, Li et al. 2014, Martinez, Li et al. 2023). mAKAPβ is not required for cardiac development, and cardiac-specific knockout of mAKAPβ has no apparent adverse effect on the response to swim training (Kritzer, Li et al. 2014).Recently, rat mAKAPβ 1694-1833, which binds to RSK3, was found to also bind to CaMKIIδ and CaMKIIγ.

[0154] Both pacing and pressure overload induce the accumulation of active threonine-287 autophosphorylated CaMKII at the myocyte nuclear membrane (Ljubojevic-Holzer, Herren et al. 2020). CaMKII phosphorylates HDAC4, inducing its 14-3-3 binding and nuclear export, and suppressing myocyte enhancer factor 2 (MEF2)-dependent gene expression, which promotes pathological remodeling (Backs, Song et al. 2006, Zhang, Kohlhaas et al. 2007, Li, Cai et al. 2011). Although CaMKII phosphorylates conserved sites among class IIa HDACs, CaMKII preferentially phosphorylates HDAC4 Ser-467 / 632, which is present only at the CaMKII docking site on HDAC4 (aa 585-608) (Backs, Song et al. 2006, Backs, Backs et al. 2009). During prolonged pressure overload, increased phosphorylation of HDAC4 was previously reported to be mAKAPβ-dependent (Kritzer, Li et al. 2014), consistent with CaMKII binding to mAKAPβ.

[0155] To identify novel RBD-binding partners, myc- and green fluorescent protein (GFP)-tagged mAKAP1694-1833 was expressed in adult rat ventricular myocytes by adenovirus infection. After dithiobis(succinimidyl propionate (DSP)) crosslinking, protein complexes were immunoprecipitated using myc-tag antibodies under stringent conditions and analyzed by mass spectrometry. Along with other candidate interactors, CaMKIIγ and δ were highly enriched in mAKAP immunoprecipitates (11 and 16 peptides, respectively, were identified by mass spectrometry). Binding of CaMKII to mAKAPβ was verified by co-immunoprecipitating endogenous proteins from neonatal rat ventricular myocytes infected with adenoviruses expressing either myc-GFP or myc-GFP-mAKAP1694-1833 (Figure 33). Immunoprecipitation of mAKAPβ with CaMKII antibodies was competed by expression of the 1694-1833 fusion peptide, verifying that the kinase bound to both the scaffold and the competing peptide. When coexpressed in COS-7 cells, mAKAP 1694-1833 bound to various CaMKII isoforms (Fig. 34), suggesting that mAKAPβ binds to a CaMKII domain common to all CaMKII family members.

[0156] A preliminary mapping study was performed to determine whether the binding sites for RSK3 and CaMKII differ within mAKAP aa 1694-1833 (Figure 35). Although the relative binding of RSK3 and CaMKIIγ to mAKAP aa 1694-1757 and 1735-1833 differs, both kinases appear to bind to a large overlapping region of aa 1694-1833.

[0157] HDAC4 phosphorylation at CaMKII sites during chronic pressure overload is dependent on mAKAPβ expression (Kritzer, Li et al. 2014). Our data show that both basal and angiotensin II-induced HDAC4 phosphorylation in neonatal myocytes is inhibited by expression of mAKAP 1694-1833, consistent with CaMKII inhibition (Figure 36).

[0158] The patent and scientific literature referred to herein demonstrates knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published documents, papers, manuscripts and scientific literature cited herein are incorporated herein by reference.

[0159] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0160] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 484,543, filed February 13, 2023, which is incorporated by reference herein in its entirety for all purposes. This application also incorporates by reference in its entirety U.S. Patent Nos. 9,132,174, 9,937,228, 10,617,737, 11,229,679, and U.S. Application No. 17 / 580,692, filed January 21, 2022, and U.S. Patent No. 10,907,153 and U.S. Application No. 16 / 818,771, filed March 13, 2020.

[0161] <Array list reference> This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on February 12, 2024, is named 65274_4WO01_SL.xml and is 292,801 bytes in size.

[0162] <Government support statement> This invention was made with Government support under Contracts R44 HL158318 and HL147631 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0163] <References> Ahn et al., 2007. Proc Natl Acad Sci USA, 104: 2979-84. Akira et al., 2006. Cell, 124: 783-801. Anjum et al., 2008. Nat Rev Mol Cell Biol, 9: 747-58. Backs et al. 2009. Proc Natl Acad Sci USA, 106(7): 2342-2347. Backs et al. 2006. J Clin Invest, 116(7): 1853-1864. Backs et al. 2011. J Cell Biol, 195: 403-15. Baier et al. 2020. J Mol Cell Cardiol 138: 212-221. Beckendorf et al. 2018. Basic Res Cardiol, 113(4): 29. Becker et al. 2021. Elife, 10. Bers et al. 2006. Biochem J, 396: e1-3. Bione et al. 1994. Nat Genet, 8: 323-7. Black and Olson. 1998. Annu Rev Cell Dev Biol, 14: 167-96. Bode et al. 2011. Expert Rev Vaccines, 10: 499-511. Bonne et al. 1999. Nat Genet, 21: 285-8. Bourajjaj et al. 2008. J Biol Chem, 283: 22295-303. Bueno et al. 2004. Circ Res, 94: 91-9. Bueno et al. 2002. Proc Natl Acad Sci U S A, 99: 4586-91. Burchfield et al. 2013. Circulation, 128: 388-400. Burgos et al. 2017. J Mol Cell Cardiol, 112: 16-26. Carlisle et al. 2004. Biochem J, 381: 587-92. Carlson et al. 2022. Circ Res, 130(1):27-44. Chamberlain et al. 2018. Hum Gene Ther, 29: 927-37. Dobrev and Wehrens. 2014. Circ Res, 114: 1311-9; discussion 19. Dodge-Kafka et al. 2010. J Biol Chem, 285: 11078-86. Dodge-Kafka et al.. 2018. J Mol Cell Cardiol, 118: 13-25. Dodge-Kafka et al. 2019. Cell Signal, 63: 109357. Dodge-Kafka and Kapiloff. 2006. Eur J Cell Biol, 85: 593-602. Dodge-Kafka et al. 2005. Nature, 437: 574-8. Dodge et al. 2001. EMBO J, 20: 1921-30. Dulhunty et al. 2007Pharmacol Ther, 113: 247-63. Duran et al. 2021. Front Cell Dev Biol, 9: 644630. Elbashir et al. 2001. Nature, 411: 494-8. Escobar et al. 2011. J Mol Cell Cardiol, 50: 451-9. Fatkin et al. 1999. N Engl J Med, 341: 1715-24. Faul et al. 2007. Mol Cell Biol, 27: 8215-27. Feng et al. 2017. J Mol Cell Cardiol, 103: 102-109. Fish et al. 2013. Circulation Heart failure 6: 310-317. Friday et al. 2003. Differentiation, 71: 217-27. Gerber et al. 2016. Circ Heart Fail, 9: e002460. Go et al. 2014. Committee American Heart Association Statistics, and Subcommittee Stroke Statistics. Circulation, 129: e28-e292. Grossman et al. 1975. Clin Invest, 56: 56-64. Guo et al. 2015. Int J Mol Med 5:1159–68. Hakem Zadeh et al. 2019. Physiol Rep, 7: e14144. Hamilton et al. 2021. Front Immunol, 12: 675897. Hartmann et al. 1999. Proc Natl Acad Sci USA, 96: 9305-10. Hawash et al. 2021. Proc Natl Acad Sci USA, 118(13):e2015855118. Heidenreich et al. 2013. Circ Heart Fail, 6: 606-19. Heidenreich et al. 2022. Circulation, 145: e895-e1032. Heineke and Molkentin. 2006. Nat Rev Mol Cell Biol, 7:589–600. Hegyi et al. 2019. J Mol Cell Cardiol, 127:246–25. Hill and Olson. 2008. N Engl J Med, 358: 1370-80. Hoffmann et al. 1999. EMBO J, 18: 893-903. Holt et a. 2019. Sci Rep, 9: 14202. Houser. 2014. Circ Res, 114: 1320-7; discussion 27. Ishikawa et al. 2018. Circ Res 123:601–613. Jugdutt 2003. Curr Drug Targets Cardiovasc Haematol Disord, 3: 1-30. KannegantiT. et al. 2007. Immunity, 27: 549-59. Kapiloff et al. 2001. J Cell Sci, 114: 3167-76. Kapiloff et al. 2009. J Biol Chem, 284: 23540-6. Kapiloff et al. 1999. J Cell Sci, 112 ( Pt 16): 2725-36. Kato et al. 2000. J Biol Chem, 275: 18534-40. Kehat et al. 2011. Circ Res, 108: 176-83. Kieserman et al. 2019. J Am Heart Assoc 8: e012239. Kim et al. 2008. J Clin Invest, 118: 124-32. Kimura et al. 2010. Circ Res, 106: 961-70. Kreusser et al. 2014. Circulation, 130(15):1262-1273. Kritzer et al. 2012. J Mol Cell Cardiol, 52: 351-8. Kritzer et al. 2014. Circ Heart Fail, 7: 663-72. Kuzmin et al. 2021. Nat Rev Drug Discov 20: 173-174. Kwon et al. 2011. PLoS Comput Biol, 7: e1002256. Lebek et al. 2023. Science, 379(6628): 179-185. Li et al. 2019. J Biol Chem, 294: 2543-54. Li et al. 2011. Biochem Biophys Res Commun, 409(1): 125-130. Li et al. 2022. Physiol. Genomics 54(7):261-272. Li et al. 2013. Circ Res, 112: 128-39. Li et al. 2010. J Mol Cell Cardiol, 48: 387-94. Li et al. 2020. Circulation, 142: 2138-54. Li et al. 2013. Exp Cell Res, 319: 447-54. Ling et al. 2009. J Clin Invest, 119(5):1230-1240. Liu et al. 2021. Science translational medicine 13: 2021. Ljubojevic-Holzer et al. 2020. Circ Res, 127(9): 1159-1178. Mackenzie et al. 2008. Biochem J, 411: 361-9. Maier et al. Cardiovasc, 2007. Res 73(4):631-640. Martinez et al. 2023. Gene Ther, 30(7-8): 543-551. Marx et al. 2000. Cell, 101: 365-76. Maxwell et al. 1999. Nature, 399: 271-5. McCarty et al. 2001. Gene Ther. 8(16):1248-54. McCartney et al. 1995. Journal of Biological Chemistry, 270: 9327-33. Michel et al. 2005. Mol Cell, 20: 661-72. Monovich et al. 2010. FEBS Lett, 584: 631-7. Moretti et al. 2020. Nat Med 26: 207-214. Muchir et al. 2000. Hum Mol Genet, 9: 1453-9. Nakamura and Sadoshima. 2018. Nat Rev Cardiol, 15: 387-407. Nassal et al. 2020. Front Pharmacol, 11: 35. Naya and Olson. 1999. 'Curr Opin Cell Biol, 11: 683-8. Naya et al. 1999. Development, 126: 2045-52. Black et al. 2008. Prog Pediatr Cardiol, 25: 51-56. Newlon et al. 1999. Nat Struct Biol, 6: 222-7. Nicol et al. 2001. EMBO J, 20: 2757-67. Ohh et al. 2000. Nat Cell Biol, 2: 423-7. Ohto et al. 2018. Immunity, 48: 649-58 e4. Ohto et al. 2015. Nature, 520: 702-5. Pare et al. 2005. J Cell Sci, 118: 5637-46. Pare et al. 2005. Exp Cell Res, 303: 388-99. Passariello et al. 2013. Am J Physiol Heart Circ Physiol, 305: H1010-9. Passariello et al. 2016. J Mol Cell Cardiol, 93: 98-105. Pleger et al. 2011. Science translational medicine 3: 92ra64. Pohar et al. 2017. J Immunol, 198: 2093-104. Potthoff and Olson. 2007. Development, 134: 4131-40. Prasad et al. 2011. Gene Ther, 18: 43-52. Rababa'h et al. 2013. J Mol Biol, 425: 3277-88. Ranganathan et al. 2006. Arch Biochem Biophys, 449: 8-16. Reyes Gaido et al. 2023. Annu Rev Pharmacol Toxicol, 63: 249-272. Sadoshima et al. 1995. Circ Res, 76: 1-15. Schiattarella and Hill. 2015. Circulation, 131: 1435-47. Schulze et al. 2003. J Biol Chem, 278: 28849-55. Scott et al. 2013. Annu Rev Pharmacol Toxicol, 53: 187-210. Scott and Pawson. 2009. Science, 326: 1220-4. Seven and Counts. 1996J Biol Chem, 271: 16526-34. Sharma and Kass. 2014. Circ Res, 115: 79-96. Shirley et al. 2020. Mol Ther, 28: 758-70. Tahtinen et al. 2022. Nat Immunol, 23: 532-42. Takeishi et al. 2002. Cardiovasc Res, 53: 131-7. ThermoFisher Scientific, retrieved June 16, 2017 <https: / / http: / / www.thermofisher.com / us / en / home / references / ambion-tech-support / rnai-sirna / general-articles / -sirna-design-guidelines.html>. Tsao et al. 2023. 'Heart Disease and Stroke Statistics-2023 Update: A Report From the American Heart Association', Circulation. Turcotte, M et al. 2022. J Mol Cell Cardiol, 172: 26-40. Valdivia et al. 1995. Science, 267: 1997-2000. Vargas et al. 2012. Cell Signal, 24: 1496-503. Veitch et al. 2021. Front Pharmacol, 12:695401. Vergarajauregui et al. 2020. Elife, 9. Wang et al. 2019. Nat Rev Drug Discov 18: 358-378, 2019. Wang et al. 2005. J Biol Chem, 280: 16705-13. Wang et al. 2015. EBioMedicine, 2: 1880-7. Wang et al. 2022. Circulation, 145(14): 1067-1083. Weinreuter et al. EMBO Mol Med, 6(10): 1231-1245. Werfel et al. 2014. Cardiovasc Res, 104: 15-23. Wilkins et al. 2004. Circ Res, 94: 110-8. Wilkins et al. 2002. Mol Cell Biol, 22: 7603-13. Wong et al. 2008. Sci Signal, 1: ra18. Wright 2020. Mol Ther, 28: 1756-58. Wu et al. 2009. Proc Natl Acad Sci U S A, 106(14): 5972-5977. Wu et al. 2001. EMBO J, 20: 6414-23. Xiang et al. 2020. Mol Ther, 28: 771-83. Xie et al. 2013. Circulation, 128: 1021-30. Xie and Hill. 2013. Trends Cardiovasc Med, 23: 229-35. Yang et al. 1998. J Cell Biol, 142: 511-22. Yao et al. 2022. Circ Res, 130(6): 887-903. Zakhary et al. 2000. J Biol Chem, 275: 41389-95. Zhang et al. 2007. J. Biol Chem, 282(48): 35078-35087. Zhang et al. 2011. J Biol Chem, 286: 23012-21. Zhang et al. 2013. Cell, 153: 216-27. Zhang et al. 2007. Hum Mol Genet, 16: 2816-33. Zhao et al. 1995. Mol Cell Biol, 15: 4353-63. Zhu et al. 2009. J Clin Invest, 119: 2388-98.

Claims

1. A composition comprising a regulatory nucleotide sequence for expression of a second nucleotide sequence in cardiomyocytes, wherein the regulatory nucleotide sequence comprises an intron sequence comprising a splicing consensus site, and the intron sequence is derived from the human cardiac troponin T gene (hTNNT).

2. The composition of claim 1 , further comprising a TNNT2 promoter sequence.

3. The composition of claim 1 , wherein the regulatory nucleotide sequence is in a vector.

4. The composition of claim 3 further comprising a transgene.

5. The composition of claim 4, wherein the transgene is a muscle A-kinase anchor protein beta (mAKAPβ) sequence.

6. The composition of claim 5 , wherein the mAKAPβ sequence is an shRNA.

7. The composition of claim 6, wherein the shRNA comprises GGTTGAAGCTTTGAAGAAA (SEQ ID NO: 77), GCTAAGAGATACAGAGCTT (SEQ ID NO: 78), or GGAGGAAATAGCAAGGTTA (SEQ ID NO: 79).

8. The composition of claim 3 , wherein the vector encodes an amino acid sequence having at least 80% sequence identity to a fragment of mAKAPβ.

9. The composition of claim 8 , wherein the vector encodes an amino acid sequence having at least 90% sequence identity to a fragment of mAKAPβ.

10. The composition of claim 9 , wherein the amino acid sequence encodes a fragment of mAKAPβ.

11. The composition of claim 8 , wherein the amino acid sequence binds to a kinase.

12. The composition of claim 11, wherein the kinase is p90 ribosomal S6 kinase 3 (RSK3).

13. The composition of claim 12 , wherein the amino acid sequence inhibits binding of mAKAPβ to RSK3.

14. 11. The composition of claim 10, wherein the amino acid sequence has at least 80% sequence identity to amino acids 1694-1757, 1735-1833, or 1694-1833 of mAKAP.

15. 15. The composition of claim 14, wherein the amino acid sequence has at least 90% sequence identity to amino acids 1735-1833 of mAKAP.

16. The composition of claim 12 , wherein the amino acid sequence comprises the RSK3 binding domain (RBD) of mAKAPβ.

17. 16. The composition of claim 15, wherein the RBD comprises amino acids 1735-1833 of SEQ ID NO:

12.

18. The composition of claim 11 , wherein the amino acid sequence binds to protein phosphatase 2A (PP2A).

19. The composition of claim 18, wherein the amino acid sequence inhibits anchoring of PP2A to mAKAPβ.

20. 20. The composition of claim 19, wherein the amino acid sequence has at least 80% sequence identity to amino acids 2132-2319 of mAKAP.

22. 21. The composition of claim 20, wherein the amino acid sequence has at least 90% sequence identity to amino acids 2132-2319 of mAKAP.

23. The composition of claim 20 , wherein the amino acid sequence comprises the PP2A binding domain (PBD) of mAKAPβ.

24. 24. The composition of claim 23, wherein the PBD comprises amino acids 2132-2319 of SEQ ID NO:

12.

25. The kinase ２＋ 16. The composition of any one of claims 11, 14 or 15, wherein the kinase is calmodulin-dependent protein kinase II (CaMKII).

26. The composition of any one of claims 3 to 25, wherein the vector is an adeno-associated virus (AAV).

27. The composition of any one of claims 3 to 26, wherein the vector further comprises an SV40 polyadenylation sequence.

28. The composition of claim 5, wherein human mAKAP amino acids 2132-2319 (SEQ ID NO: 12) are modified at one or more of the following positions: TCG to TCA at amino acid 2144; AGC to AGT at amino acid 2183; TCC to TCA at amino acid 2256; GCC to GCA at amino acid 2291; or CGA to AGA at amino acid 2313.

29. The composition of claim 5, wherein the human mAKAP amino acids 1696 to 1835 (SEQ ID NO: 12) encoding the RBD are modified at one or more of the following positions: CCG to CCA at amino acid 1712; TCG to TCT at amino acid 1714; TCG to TCT at amino acid 1717; CGT to AGA at amino acid 1721; CGT to AGA at amino acid 1724; AGC to AGT at amino acid 1730; AGC to AGT at amino acid 1753; and GAC to GAT at amino acid 1775.

30. A method for treating or preventing heart disease, comprising administering the vector according to any one of claims 3 to 29 to cardiac cells of a patient.

31. 10. A method for treating or preventing heart disease, comprising administering the vector of claim 6 or claim 7 to cardiac cells of a patient, wherein the method inhibits expression of mAKAP.

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