Promoter-enhancer sequence of human troponin t gene for selective expression in myocardial cells

Abstract

The present invention describes a novel gene regulatory sequence comprising the promoter and enhancer sequences of the human cardiac troponin T gene (TNNT2) that guide selective expression in cardiac muscle cells. The novel TNNT2 promoter / enhancer composition can be used for guiding adeno-associated virus gene expression, or used for constructing a cell type specific expression vector, or used for heart specific transgenosis. Uses of the novel promoter / enhancer compositions are demonstrated by expression of mAKAP shRNA, as well as anchor disrupting peptides derived from mAKAP, useful for the treatment of heart failure.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 484,543, filed February 13, 2023, which is hereby incorporated by reference in its entirety for all purposes. This application also incorporates by reference in its entirety for all purposes the following patents: U.S. Patent Nos. 9,132,174, 9,937,228, 10,617,737, 11,229,679, and U.S. Patent Application Serial No. 17 / 580,692, filed January 21, 2022, and U.S. Patent No. 10,907,153 and U.S. Patent Application Serial No. 16 / 818,771, filed March 13, 2020.

[0002] References to sequence listings

[0003] This application contains a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. This XML copy was created on February 12, 2024, is named 65274_4WO01_SL.xml, and is 292,801 bytes in size.

[0004] Government Funding Statement

[0005] This invention was made with government support under Contract Nos. R44 HL158318 and HL147631 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the Invention

[0006] In response to chronic stress, the heart's primary compensatory mechanism is cardiomyocyte hypertrophy—a non-mitotic increase in the volume of contractile cells (Hill and Olson 2008). Adult mammalian cardiomyocytes are roughly cylindrical in shape, and their growth can manifest as either widening or elongation. Because cardiomyocytes comprise the vast majority of cardiac mass (Jugdutt 2003), concentric and eccentric hypertrophy lead to thickening and dilation of the cardiac chamber walls, respectively. Theoretically, concentric cardiomyocyte widening, characterized by the parallel assembly of sarcomeres, reduces ventricular wall stress (Law of Laplace), while eccentric cardiomyocyte longitudinal elongation, characterized by the serial assembly of sarcomeres, allows for greater ventricular volume without stretching individual sarcomeres beyond their optimal contractile length (length-tension relationship) (Grossman, Jones, and McLaurin 1975). Although relatively symmetrical left ventricular hypertrophy can occur during physiological stresses such as pregnancy or exercise training, concentric ventricular hypertrophy is the primary initial response to the increased systolic wall stress associated with pressure-overload conditions such as hypertension or aortic stenosis. Eccentric ventricular hypertrophy predominates in states of volume overload, such as after myocardial infarction and during the transition from concentric hypertrophy to the dilated heart of heart failure with reduced ejection fraction (HFrEF) in some cardiovascular conditions characterized by predominantly pressure-overload conditions. Concentric and eccentric hypertrophy also occur in hereditary hypertrophic cardiomyopathy and dilated cardiomyopathy, respectively.

[0007] At the cellular level, cardiomyocyte hypertrophy is due to increased protein synthesis within individual cardiomyocytes, as well as an increase in the volume and structural reorganization of the sarcomeres. For a more detailed review of cardiac remodeling and hypertrophy, see (Nakamura and Sadoshima 2018) and (Burchfield, Xie and Hill 2013), which are incorporated herein by reference in their entirety. It is generally believed that myocardial hypertrophy plays a major role in the development and progression of heart failure. Traditional approaches to the treatment of heart failure include reducing afterload, blocking β-adrenergic receptors (β-ARs), and using mechanical support devices in affected patients. However, there is still a need in the art for additional mechanisms to prevent or treat pathological myocardial hypertrophy.

[0008] Studies have shown that mechanisms that induce “compensatory” concentric hypertrophy early in pressure-overload-related heart disease predispose the heart to systolic dysfunction and ultimately failure later in life (Schiattarella and Hill 2015). In this regard, targeting the RSK3-mAKAPβ complex has been shown to mitigate cardiac remodeling induced by pressure overload and prevent 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, when the heart is at a stage in the disease process characterized by eccentric growth and ventricular dilation leading to HFrEF, either in the late stages of pressure-overload-related disease or throughout the progression of volume-overload-related disease, measures to maintain signaling that promotes concentric hypertrophy and inhibits eccentric hypertrophy may also protect cardiac volumes and systolic function. In this regard, targeting the PP2A-mAKAPβ complex has been shown to attenuate cardiac remodeling after myocardial infarction (Martinez et al., 2022; Li et al., 2020). Therefore, in familial dilated cardiomyopathy, enhancing concentric hypertrophy and / or inhibiting eccentric hypertrophy may be beneficial.

[0009] AKAPs and cardiac remodeling

[0010] When the myocardium is stressed by myocardial infarction, hypertension, congenital heart disease, or neurohumoral activation, ventricular myocyte hypertrophy is the primary compensatory mechanism for the myocardium to reduce ventricular wall tension. This process is accompanied by non-mitotic growth of cardiomyocytes, enhancement of myofibril structure, and upregulation of a specific subset of "fetal" genes normally expressed during the embryonic period (Nakamura and Sadoshima 2018). At the same time, abnormal cardiac contractile function, Ca 2+ The pathological remodeling of cardiomyocytes is regulated by a complex intracellular signaling network that includes mitogen-activated protein kinases (MAPKs), cyclic nucleotides, Ca 2+, and Ca 3+. 2+ , hypoxia, and phosphatidylinositol-dependent signaling pathways (Heineke and Molkentin 2006; Nakamura and Sadoshima 2018).

[0011] Heart failure affects 6.7 million adults in the United States, with approximately 1 million new cases each year, driven by risk factors such as smoking and obesity (Tsao et al., 2023). The prevalence and incidence of heart failure continue to rise, primarily due to increased life expectancy but also due to an increase in the 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 treatment for heart failure includes β-adrenergic, angiotensin II, and mineralocorticoid receptor antagonists, angiotensin-converting enzyme and neprilysin metalloproteinase inhibitors, and sodium-glucose cotransporter 2 inhibitors (Heidenreich et al., 2022). Subsequent or replacement therapies include loop diuretics, thiazide diuretics, vasodilators, and I f Current blocking agents, as well as device-based therapies, have been used. Despite this, the 5-year mortality rate for symptomatic heart failure remains approximately 50%, including >40% after myocardial infarction (Heidenreich et al., 2013; Gerber et al., 2016).

[0012] Cardiac hypertrophy can be induced by a variety of 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 is becoming increasingly clear that A-kinase anchoring proteins (AKAPs) can assemble multiprotein complexes that integrate hypertrophic signaling pathways originating from these receptors. In particular, recent studies have identified anchoring proteins such as mAKAP, AKAP-Lbc, and D-AKAP1, which serve as scaffold proteins and play a central role in organizing and regulating hypertrophic pathways activated by stress signals (Kritzer et al., 2012).

[0013] As organizers of "nodes" in intracellular signaling networks, scaffold proteins have attracted considerable attention as potential therapeutic targets (Negro, Dodge-Kafka, and Kapiloff 2008). Within cells, scaffold proteins organize multimolecular complexes known as "signalosomes," a crucial mechanism for ensuring the specificity and efficiency of intracellular signal transduction (Scott and Pawson 2009). First, many signaling enzymes possess broad substrate specificity. Scaffold proteins can colocalize these pleiotropic enzymes with individual substrates, selectively enhancing substrate catalysis and providing a degree of specificity not inherent in the enzyme's active site itself (Scott and Pawson 2009). Second, some signaling enzymes are extremely low in abundance. Scaffold proteins can colocalize rare enzymes with their substrates, facilitating kinetics of signaling. Third, because many scaffold proteins possess multivalent binding capabilities, scaffold binding can coordinate the coordinated regulation of multiple enzymes on a single substrate effector. Muscle A-kinase anchoring protein (mAKAP, also known as AKAP6) is a large scaffold protein expressed in cardiomyocytes, skeletal muscle cells and neurons, which can bind to signal transduction enzymes with broad substrate specificity, such as protein kinase A (PKA) and Ca 2+ / calmodulin-dependent phosphatase calcineurin (CaN) and very low abundance signaling enzymes such as p90 ribosomal S6 kinase 3 (RSK3) binding ( 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 cardiomyocytes, where it localizes to the outer nuclear membrane by binding to the integral membrane protein nesprin-1α (Pare, Easlick et al., 2005).

[0014] Given the role of mAKAPβ as a scaffolding protein for stress-related signaling molecules in cardiomyocytes, deletion of mAKAPβ in rat neonatal ventricular myocytes in vitro inhibits hypertrophy induced by α-adrenergic, β-adrenergic, endothelin-1, angiotensin II, and leucine inhibitory factor / gp130 receptor signaling (Zhang et al., 2011; Pare, Bauman et al., 2005; Dodge-Kafka et al., 2005; Guo et al., 2015). In vivo, in addition to attenuating hypertrophy induced by short-term pressure overload and chronic β-adrenergic stimulation, mAKAP gene targeting in mice inhibited the development of heart failure after long-term pressure overload and conferred a survival benefit (Kritzer et al., 2014). Specifically, deletion of mAKAPβ in rat neonatal ventricular myocytes in vitro inhibited hypertrophy induced by α-adrenergic, β-adrenergic, endothelin-1, angiotensin II, and leucine inhibitory factor / gp130 receptor signaling (Zhang et al., 2011; Pare, Bauman et al., 2005; Dodge-Kafka et al., 2005; Guo et al., 2015). fl / flIn Tg(Myh6-cre / Esr1*) tamoxifen-induced conditional knockout mice, deletion of the mAKAP gene reduced left ventricular hypertrophy and significantly inhibited cardiomyocyte apoptosis, interstitial fibrosis, left atrial hypertrophy, and pulmonary edema (wet lung weight) induced by 16-week transverse aortic constriction (Kritzer et al., 2014).

[0015] Genetic targeting of mAKAPs has also been shown to be beneficial after myocardial infarction (Martinez et al., 2022). Permanent ligation of the left anterior descending coronary artery (LAD) in mice leads to myocardial infarction, characterized by extensive cardiomyocyte death, scarring, and subsequent left ventricular (LV) remodeling. Four weeks after LAD ligation, left ventricular size and function were preserved in mAKAP conditional knockout mice compared to a control cohort that had already undergone infarction. Left ventricular ejection fraction and corrected atrial mass were preserved in mAKAP conditional knockout mice compared to controls, while infarct size was significantly reduced.

[0016] Introduction to mAKAP and Cardiac Remodeling

[0017] mAKAPs were originally identified as novel cAMP-dependent protein kinase (PKA) regulatory subunit (R-subunit)-binding proteins, also known as A-kinase anchoring proteins or AKAPs, during cDNA library screening (Mccartney et al., 1995). Due to the size of the protein encoded by the original cDNA fragment, mAKAPs were initially designated "AKAP100" (Mccartney et al., 1995). Subsequently, the full-length mRNA sequence of mAKAPα, an alternatively spliced ​​isoform of mAKAP expressed in neurons, was determined. Studies have shown that wild-type mAKAPα is a 255 kDa scaffold protein (Kapiloff et al., 1999). Subsequently, the sequence of mAKAPβ, a 230 kDa alternatively spliced ​​isoform of mAKAP expressed in striated muscle cells, was obtained. Studies have shown that when mAKAPs are expressed in cardiac or skeletal muscle, they are translated from an internal initiation site corresponding to the methionine residue 245 of mAKAPα (Michel et al., 2005).

[0018] mAKAPs are localized to the nuclear envelope in neurons, striated cardiomyocytes, skeletal muscle cells, and osteoclasts, all cell types that specifically express mAKAPs (Kapiloff et al., 1999; Pare, Easlick et al., 2005; Michel et al., 2005; Becker et al., 2021; Vergarajauregui et al., 2020). mAKAPs are not transmembrane domain proteins but rather contain three spectrin-like repeat regions (residues 772–1187) that confer their localization capabilities (Kapiloff et al., 1999). Binding of the outer nuclear membrane protein nesprin-1α to the third spectrin repeat region (residues 1074–1187) of mAKAPs is both necessary and sufficient for mAKAP localization to the outer nuclear envelope, at least in myocytes and when expressed in heterologous cells (Pare, Easlick et al., 2005; Becker et al., 2021; Holt et al., 2019). Interestingly, mutations in laminA / C, emerin, and nesprin-1α have been associated with Emery-Dreyfuss muscular dystrophy and other cardiomyopathies (Bonne et al., 1999; Fatkin et al., 1999; Muchir et al., 2000; Bione et al., 1994; Zhang et al., 2007). However, no pathogenic mutations have been identified in human mAKAP genes to date, and knockout of mAKAPβ in mice during early cardiac development does not induce cardiomyopathy (Kritzer et al., 2014). In addition to binding to nesprin-1α, mAKAPβ also binds to phospholipase Cε (PLCε) via the first spectrin repeat region of mAKAP, which may enhance its association with the nuclear envelope (Zhang et al., 2011). Earlier reports indicated that mABAPβ was present in the sarcoplasmic reticulum (Mccartney et al., 1995; Marx et al., 2000; Yang et al., 1998), but these findings were likely due to technical issues with antibody specificity (Kapiloff, Jackson and Airhart 2001; Kapiloff et al., 1999).

[0019] In addition to PKA, PLCε, and nesprin-1α, mABAPβ also binds to several proteins that are critical for cardiomyocyte stress response: adenylate cyclase type 5 (AC5), cAMP-activated exchange protein 1 (Epac1), cAMP-specific phosphodiesterase 4D3 type (PDE4D3), MEK5, and ERK5. MAP kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK1), p90 ribosomal S6 kinase 3 (RSK3), protein kinase Cε (PKCε), protein kinase D (PKD1, also known as PKCμ), the protein phosphatases calcineurin (CaN) Aβ and PP2A, ryanodine receptor type 2 (RyR2), the sodium / calcium exchanger NCX1, ubiquitin E3 ligases involved in HIF1α regulation, and myopodin (Pare, Bauman et al., 2005; Pare, Easlick et al., 2005; Dodge-Kafka et al., 2005; Marx et al., 2000; Kapiloff, Jackson, and Airhart et al., 2003). 2001; Michel et al., 2005; Li 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, after binding to mABAPβ, co-regulate the transcription factors hypoxia-inducible factor 1α (HIF1α), myocyte enhancer factor-2 (MEF2), serum response factor (SRF), and nuclear factor of activated T cells (NFATc), as well as type II histone deacetylases ( 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). Some of these molecules bind directly, some indirectly, some constitutively, and some regulated. Therefore, the composition of the mAKAPβ signalosome likely depends on the intrinsic state of the cardiomyocyte. As mAKAPβ is studied, its list of binding partners continues to expand, further supporting its role as a key orchestrator of the signaling pathways required for remodeling. Much of our understanding of mAKAPβ is based on studies in cultured neonatal rat ventricular myocytes, where mAKAPβ was early shown to be required for hypertrophy induction by multiple upstream receptors, including α- and β-adrenergic and cytokine receptors (Pare, Bauman et al., 2005; Dodge-Kafka et al., 2005). However, recently published phenotypic studies of cardiomyocyte-specific conditional mAKAPβ knockout mice have confirmed the central role of mAKAPβ in the remodeling process (Kritzer et al., 2014; Martinez et al., 2022). Within the mAKAPβ signalosome, multiple upstream inputs, downstream effectors (outputs), and integrative circuits exist that collectively influence pathological cardiac remodeling.

[0020] mAKAPβ—a typical A-kinase anchoring protein

[0021] Like most AKAPs, mAKAPs contain an amphipathic α-helix (residues 2055-2072) that is responsible for binding to PKA (Kapiloff et al., 1999; Kritzer et al., 2012). PKA is a heterotetramer composed of two R-subunits and two catalytic subunits (C-subunits) in a CRRC configuration. In the holoenzyme, the N-terminal anchoring and dimerization domains of the R-subunits of PKA form an X-shaped, antiparallel four-helix bundle (Newlon et al., 1999). This helical bundle contains a hydrophobic groove that accommodates the hydrophobic face of the AKAP amphipathic helix. mAKAPβ binds to PKA with high affinity (K D=119 nM) selectively binds to type II PKA (including the RII subunit) (Zakhary et al., 2000). Notably, upon RIIα autophosphorylation, PKA binding to mAKAPβ increases 16-fold (Zakhary et al., 2000), potentially impacting PKA-mAKAPβ binding under conditions of altered β-adrenergic signaling. In addition to mAKAPβ, over a dozen other AKAPs are expressed in cardiomyocytes, each with distinct localization and binding partners (Kritzer et al., 2014). Notably, mAKAPβ is one of the rarest AKAPs in cardiomyocytes, and loss of mAKAP does not even affect the perinuclear localization of PKA (Kapiloff, unpublished observations). Despite extremely low expression levels of this scaffold protein, replacement of endogenous mAKAPβ with a full-length mAKAPβ mutant that is unable to bind PKA is sufficient to inhibit the induction of cardiomyocyte hypertrophy (Pare, Bauman et al., 2005). Therefore, the mAKAPβ signalosome not only serves as an example of how PKA signaling can be finely compartmentalized at the level of individual organelles, but also illustrates that the expression level of a protein or its complex does not necessarily reflect the functional importance of the protein.

[0022] mAKAPβ is remarkable because 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). cAMP is synthesized from ATP by adenylate cyclase (AC), while its metabolism to 5'-AMP is catalyzed by phosphodiesterase (PDE). Differential binding of different types of ACs and PDEs to AKAPs contributes to the compartmentalization of cAMP within the cell, allowing for local activation of cAMP effectors and regulating local cAMP levels through distinct 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 relevant binding partner for mAKAPβ (Kapiloff et al., 2009). The N-terminal, C1, and C2 domains of AC5 directly bind to a unique N-terminal site (residues 275–340) on mAKAPβ. AC5 activity is inhibited by feedback phosphorylation by PKA, a process that is promoted intracellularly by the formation of the mAKAPβ complex (Kapiloff et al., 2009). This negative feedback appears to be physiologically relevant for maintaining basal cAMP signaling. When a competitive peptide containing the mAKAP AC5 binding domain inhibits AC5 binding to mAKAPβ, cardiomyocyte cAMP levels and cell size increase even in the absence of hypertrophic stimuli (Kapiloff et al., 2009).

[0023] mAKAP was the first AKAP demonstrated to bind to PDEs (Dodge et al., 2001). The mAKAP region 1286-1831 contains a site that binds to the unique N-terminal domain of PDE4D3. Phosphorylation of serine residues 13 and 54 of PDE4D3 enhances its binding to the scaffold and increases its PDE catalytic activity, respectively (Dodge et al., 2001; Sette and Conti 1996; Carlisle Michel et al., 2004). Because increased PDE4D3 activity accelerates cAMP degradation, PKA and PDE4D3 form a negative feedback loop that regulates local cAMP levels and PKA activity (Dodge et al., 2001). When bound to mAKAPs, PDE4D3 not only functions as a PDE but also acts as an adaptor protein, recruiting members of the MAPK family, MEK5 and ERK5, as well as the cAMP-dependent Rap1 guanine nucleotide exchange factor, Epac1, to the scaffold (Dodge-Kafka et al., 2005). Upon activation of MEK5 and ERK5 by upstream signaling, ERK5 phosphorylates Ser-579 of PDE4D3, inhibiting PDE activity and promoting cAMP accumulation and PKA activation (Dodge-Kafka et al., 2005; Hoffmann et al., 1999; Mackenzie et al., 2008). Epac1 is less sensitive to cAMP than PKA, leading to additional activation of mAKAP-associated Epac1 only when cAMP levels are extremely high. Through Rap1, Epac1 inhibits ERK5 activity, thereby preventing the inhibition of PDE4D3 by MAPK signaling. This is presumably the result of maximal PDE4D3 activity upon PKA phosphorylation (Dodge-Kafka et al., 2005). Thus, Epac1, ERK5, and PDE4D3 form a third negative feedback loop that attenuates cAMP levels in the vicinity of the mAKAP complex, preventing cAMP from reaching critically high levels.

[0024] An additional layer of complexity arises from the binding of the serine-threonine phosphatase PP2A to the C-terminus of mAKAPs (residues 2083-2319) (Dodge-Kafka et al., 2010). PP2A catalyzes the dephosphorylation of Ser-54 of PDE4D3, thereby inhibiting this phosphodiesterase (PDE) in the absence of upstream stimuli. PP2A bound to the mAKAP complex contains the B56δB subunit, a substrate for PKA. PKA phosphorylation enhances PP2A's catalytic activity (Ahn et al., 2007). Therefore, phosphorylation of B56δ by mAKAP-bound PKA enhances PDE4D3 dephosphorylation, leading to PDE inhibition. This may elevate cAMP levels, constituting a positive feedforward loop that initiates cAMP signaling. Combined with the negative feedback loop formed by AC5 phosphorylation and the regulation of PDE4D3 by PKA and ERK5, it is speculated that cAMP levels at the mAKAPβ signalosome are tightly regulated by upstream β-adrenergic and MAPK signaling. Signaling upstream of AC5 and ERK5 promotes cAMP signaling, which is initially enhanced by feedforward signals from PP2A. Simultaneously, PKA and Epac1 activate PDE4D3 and inhibit AC5 through negative feedback, thereby limiting signaling. Interestingly, Rababa'h et al. demonstrated that mAKAP proteins carrying nonsynonymous polymorphisms differentially bind to PKA and PDE4D3 (Rababa'h et al., 2013). cAMP signaling may be differentially regulated by cross-talk with upstream signaling pathways or by human polymorphisms, highlighting the need for in-depth investigation of the interplay of this complex signaling network in cardiomyocytes. In addition to PDE4D3, recent studies have shown that mAKAPβ-bound PP2A can also catalyze the dephosphorylation of SRF and promote longitudinal growth of cardiomyocytes (Li et al., 2020). Targeting mAKAPβ-bound PP2A can improve cardiac function in mice after myocardial infarction.

[0025] mAKAPβ and MAP kinase-RSK3 signaling

[0026] The recruitment of ERK5 to the mAKAPβ complex by PDE4D3 was initially linked to the regulation of local cAMP via the aforementioned feedback loop (Dodge-Kafka et al., 2005). However, ERK5 has also been identified as an important factor in the induction of cardiomyocyte hypertrophy. In cultured cardiomyocytes, ERK5 preferentially induces longitudinal cell growth (eccentric hypertrophy) and, in vivo, plays a role in concentric hypertrophy induced by pressure overload (transverse aortic constriction in mice) (Nicol et al., 2001; Kimura et al., 2010). Notably, in cultured cardiomyocytes, inhibition of mAKAPβ expression using RNA interference (RNAi) blocked eccentric growth induced by the interleukin-6-like cytokine leukemia inhibitory factor (LIF) (Dodge-Kafka et al., 2005). A potential effector of mAKAPβ-bound ERK5 is the MEF2 transcription factor, discussed below. However, in both the heart and brain, mAKAPs bind PDK1, which, together with ERK (ERK1, 2, or 5), activates the MAPK effector p90RSK, which also binds to mAKAPs (Ranganathan et al., 2006; Michel et al., 2005). Importantly, PDK1 binding to mAKAPs eliminates the requirement for membrane localization for RSK activation (Michel et al., 2005). Taken together, these data suggest that mAKAPβ coordinates RSK activation in cardiomyocytes through upstream MAPK signaling.

[0027] p90RSK is a pleiotropic ERK effector that regulates multiple cellular processes, including cell proliferation, survival, migration, and invasion. Most hypertrophic stimuli increase RSK activity in cardiomyocytes (Anjum and Blenis 2008; Sadoshima et al., 1995). Furthermore, elevated RSK activity has been found in human myocardial tissue from 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 activation requires sequential phosphorylation of the C-terminal and N-terminal kinase activation loops by ERK and PDK1, respectively. Therefore, phosphorylation of Ser-218 of the N-terminal domain by PDK1 is a hallmark of full enzyme activation. There are four ubiquitously expressed RSK family members in mammals, but only RSK3 binds to mABAPβ (Li, Kritzer et al., 2013). A unique N-terminal domain (1-30) of RSK3 directly binds to residues 1694–1833 of mAKAPβ, termed the RSK3 binding domain (RBD), explaining the selective binding of this isoform to this scaffold protein (Li, Kritzer et al., 2013). Although RSK3 is expressed at lower levels in cardiomyocytes than other RSK family members, studies have shown that blocking RSK3 binding to mAKAPs via RSK3 RNAi, inactivation of the RSK3 N-terminal kinase domain, or the use of anchor-disrupting peptides can all attenuate hypertrophic responses in neonatal rat cardiomyocytes (Li, Kritzer et al., 2013). Importantly, in vivo RSK3 expression is required for pressure overload- and catecholamine-induced cardiac hypertrophy, as well as for the development of heart failure in a mouse model of familial hypertrophic cardiomyopathy (α-myosin Glu180Gly) (Li, Kritzer et al., 2013; Passariello et al., 2013). Furthermore, consistent with the reported role of ERK1 / 2 MAP kinases in selectively inducing centripetal hypertrophy (Kehat et al., 2011), RSK3 gene deletion inhibited Raf1 in a mouse model of Noonan syndrome. L613V Mutations in this gene induce centripetal hypertrophy (Passariello et al., 2016). Given the essential nature of this specific RSK isoform in cardiac remodeling, it represents a promising therapeutic target.

[0028] Recent studies have shown that mAKAPβ-bound RSK3 phosphorylates the SRF transcription factor, thereby promoting lateral growth of cardiomyocytes (Li et al., 2020). In mice, expressing an RBD peptide to displace RSK3 from mAKAPβ inhibits pressure overload-induced hypertrophy and the subsequent development of heart failure.

[0029] mAKAPβ and phosphatidylinositol signaling

[0030] The cAMP effector Epac1 activates Rap1 within the mAKAPβ complex, thereby influencing ERK5 signaling (Dodge-Kafka et al., 2005). Furthermore, Epac1-Rap1 activates PLCε, a phospholipase that directly binds to the first spectrin-like repeat domain of mAKAPβ via its Ras-association domain (Zhang et al., 2011). Like mAKAPβ, PLCε is required for neonatal cardiomyocyte hypertrophy, whether its expression is inhibited by RNAi or displaced from mAKAPβ by expression of a competitive binding peptide. In an elegant study, the Smrcka laboratory demonstrated that mAKAPβ-bound PLCε regulates the activation of PKCε and PKD through a novel phosphatidylinositol 4-phosphate (PI4P) pathway. In this pathway, PLCε selectively converts perinuclear PI4P into diacylglycerol and inositol 1,4-bisphosphate (Zhang et al., 2013). PKD1 phosphorylates type II histone deacetylases (HDAC4 / 5 / 7 / 9), inducing their nuclear export and thereby relieving the repression of hypertrophy-related gene expression (Monovich et al., 2010; Xie and Hill, 2013). Smrcka and colleagues found that pressure overload-induced PKD activation, type II HDAC phosphorylation, and cardiac hypertrophy in vivo are all dependent on PLCε (Zhang et al., 2013). Subsequent studies further confirmed that pressure overload-induced PKD activation and HDAC4 phosphorylation in vivo also require mAbApβ (Kritzer et al., 2014). Notably, mAbApβ can form a ternary complex with PKD and HDAC4. Taken together, these results demonstrate how local cAMP signaling influences the regulation of cardiac gene expression.

[0031] Recent studies have demonstrated that mAKAPβ serves as a scaffold for HDAC5 in cardiomyocytes, forming a signalosome comprising HDAC5, PKD, and PKA (Dodge-Kafka et al., 2018). Inhibition of mAKAPβ expression attenuates HDAC5 phosphorylation by PKD and PKA in response to α- and β-adrenergic receptor stimulation, respectively. Importantly, disruption of the mAKAPβ-HDAC5 anchorage prevents HDAC5 nuclear export induced by α-adrenergic receptor signaling and PKD phosphorylation. Furthermore, disruption of the mAKAPβ-PKA anchorage prevents β-adrenergic receptor stimulation from inhibiting α-adrenergic-induced HDAC5 nuclear export. Taken together, these data establish that the mAKAPβ signalosome bidirectionally regulates the nuclear-cytoplasmic localization of class IIa HDACs. Thus, the mAKAPβ scaffold serves as a node in the cardiomyocyte regulatory network, controlling the repression and activation of pathological gene expression in both healthy and diseased states.

[0032] mAKAPβ and calcium signaling

[0033] In addition to cAMP, phosphatidylinositol, and MAP kinase signaling, mAbApβ is also involved in coordinating Ca 2+ The second binding partner of mAKAPβ identified is the ryanodine receptor Ca 2+ release channel (RyR2), which is responsible for the passage of Ca from intracellular stores 2+ Induced Ca 2+ The most well-known role of RyR2 is its involvement in excitation-contraction coupling, during which a large amount of Ca 2+Release of RyR2 triggers sarcomere contraction. PKA phosphorylation enhances RyR2 currents (Valdivia et al., 1995; Dulhunty et al., 2007; Bers 2006), although the importance of PKA-catalyzed RyR2 phosphorylation in excitation-contraction coupling remains highly controversial (Houser 2014; Dobrev and Wehrens 2014). A small fraction of RyR2, presumably located in perinuclear dyads (Escobar et al., 2011), can be immunoprecipitated using mAbA-dependent neprin-1α antibodies (Pare, Easlick et al., 2005; Kapiloff, Jackson, and Airhart 2001). By binding to RyR2, mAbA-dependent neprin-1α appears to bring together components of the excitation-contraction coupling apparatus with signaling molecules that regulate nuclear events associated with pathological remodeling. β-adrenergic stimulation of primary cardiomyocytes increases PKA phosphorylation of RyR2 bound to mAKAPβ (Pare, Bauman et al., 2005). Notably, only the RyR2 located at the outer nuclear membrane and bound to nesprin-1α is dependent on mAKAPβ for PKA phosphorylation, while RyR2 in other parts of the sarcoplasmic reticulum is independent of mAKAPβ (Turcotte et al., 2022). Under conditions of increased sympathetic excitation, PKA-catalyzed RyR2 phosphorylation may promote local Ca2+ in the vicinity of the mAKAPβ signalosome. 2+ More importantly, β-adrenergic stimulation induces Ca release at nesprin-1α near the outer nuclear membrane. 2+ The increase in Ca levels is dependent on the expression of mABAPβ in cardiomyocytes, while the overall cytosolic Ca 2+ levels were not affected by it (Turcotte et al., 2022).

[0034] Although the small amount of RyR2 bound to mAKAPβ does not seem to significantly affect the overall contractile function of cardiomyocytes, the perinuclear Ca 2+ One target of elevation is Ca 2+Calcineurin (CaN), a calmodulin-dependent phosphatase, binds to scaffold proteins (Turcotte et al., 2022). The catalytic subunit of CaN has three isoforms (α, β, and γ), but only the CaNAβ-mAKAPβ complex has been detected in cardiomyocytes (Li et al., 2010). Notably, CaNAβ is the CaNA isoform that plays an important role in inducing cardiac hypertrophy and post-ischemic cardiomyocyte survival in vivo (Bueno et al., 2002; Bueno et al., 2004). CaNAβ directly binds to a unique site in mAKAPβ (residues 1286-1345) (Pare, Bauman et al., 2005; Li et al., 2010). Intracellularly, the binding of CaNAβ to mAKAPβ is enhanced by adrenergic stimulation and can be inhibited by Ca 2+ The CaNβ-mAKAPβ binding is directly enhanced by calcitonin / calmodulin (Li et al., 2010). Importantly, in vitro, CaNβ-mAKAPβ binding is required for α-adrenergic-induced neonatal cardiomyocyte hypertrophy (Li et al., 2010).

[0035] mAKAPβ and gene expression

[0036] Among its many substrates, CaN can activate NFATc and MEF2 transcription factors. The NFATc family of transcription factors comprises four CaN-dependent isoforms, all of which are expressed in cardiomyocytes and involved in the induction of cardiomyocyte hypertrophy (Wilkins et al., 2004). Generally, NFATc family members are retained in the cytoplasm when hyperphosphorylated on multiple serine-rich motifs within their N-terminal regulatory domains. Upon dephosphorylation of these motifs by CaN, NFATc translocates to the nucleus. Several NFATc family members bind to mAKAPβ, and this binding is required for the CaN-dependent dephosphorylation of NFATc3 in cardiomyocytes (Li et al., 2010). Consequently, mAKAPβ expression is also required for nuclear translocation of NFAT and its transcriptional activity in vitro (Li et al., 2010; Pare, Bauman et al., 2005). These results are consistent with in vivo observations that cardiomyocyte-specific knockout of mABAPβ attenuated NFAT-dependent gene expression following transverse aortic constriction (Kritzer et al., 2014).

[0037] 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 all contain a conserved DNA-binding domain that includes the MADS box and the MEF2 homology domain (Potthoff and Olson 2007). The DNA-binding domain of MEF2D directly binds to the N-terminal domain of mAbAp (Vargas et al., 2012; Kim et al., 2008). CaN and MEF2D are crucial 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 in the binding of MEF2 to mAKAPβ attenuates MEF2 transcriptional activity and expression of endogenous MEF2 target genes in C2C12 skeletal myoblasts (Vargas et al., 2012). Furthermore, disruption of the MEF2-mAKAP complex inhibits the differentiation of C2C12 myoblasts into myotubes, as demonstrated by reduced cell fusion and decreased expression of differentiation markers (Vargas et al., 2012). Notably, in cardiomyocytes, CaN binding to MEF2 is dependent on mAKAPβ (Li, Vargas et al., 2013). Thus, disruption of CaN-mAKAPβ binding inhibits MEF2 transcriptional activity and cardiomyocyte hypertrophy in C2C12 cells (Li, Vargas et al., 2013). Similar to NFATc2, pressure overload-induced dephosphorylation of MEF2D is attenuated by conditional knockout of mAKAPβ in vivo, which correlates with decreased expression of MEF2 target genes, including atrial natriuretic factor (Kritzer et al., 2014).

[0038] During in vivo pressure overload, mAKAPβ regulation of NFATc, MEF2, and HDAC4 demonstrates the importance of mAKAPβ in stress-regulated gene expression (Kritzer et al., 2014). Published studies have shown that at mAKAPβ, NFATc and MEF2 are regulated by 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, ERK5, which associates with mAKAPβ, activates MEF2 by phosphorylating this 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 proposed that phosphorylation of HDAC4 by PKA can inhibit MEF2 activity by generating a new HDAC4 proteolytic fragment (Backs et al., 2011). How the activities of numerous mAbAPβ binding partners are ultimately integrated to control gene expression is a question that can be further studied both in vitro and in vivo.

[0039] Other mAKAPβ binding partners

[0040] mAKAPβ also has other binding partners whose significance for anchoring to this scaffold is less well characterized, including phospholamban, myopodin, and NCX1 (Faul et al., 2007; Schulze et al., 2003; Hakem-Zadeh et al., 2019). Perinuclear binding of AKAP9 to mAKAPβ has been described in striated myocytes and osteoclasts, where it plays an important role in the structure of the microtubule organizing center at the nuclear envelope and the proximity of the nuclear envelope to the Golgi apparatus (Becker et al., 2021; Vergarajauregui et al., 2020).

[0041] HIF-1α is a transcription factor that regulates the systemic response to hypoxia and also binds to mAKAPβ (Wong et al., 2008). Under normoxic conditions, HIF-1α abundance in cells is maintained at low levels through ubiquitin-mediated proteasomal degradation. HIF-1α is hydroxylated by a class of oxygen-sensitive dioxygenases called prolyl hydroxylases (PHD1, PHD2, and PHD3) (Ohh et al., 2000). Hydroxylated HIF-1α is subsequently recognized by the von Hippel-Lindau protein (pVHL), which recruits the Elongin C ubiquitin ligase complex, ubiquitinating HIF-1α and promoting its proteasome-dependent degradation (Maxwell et al., 1999). Under hypoxic conditions, PHDs are inactivated, reducing HIF-1α degradation, leading to nuclear accumulation of HIF-1α, where it dimerizes with HIF-1β to promote transcription of target genes. In cultured neonatal cardiomyocytes, mAKAPβ assembles a signaling complex consisting of HIF-1α, PHD, pVHL, and the E3 ubiquitin 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, Siah2 activation induces proteasomal degradation of the bound PHD, thereby promoting HIF-1α accumulation (Wong et al., 2008). Knockdown of mAKAPβ may impair cardiomyocyte survival after ischemia-reperfusion.

[0042] mAKAPβ—the conductor of reshaping harmony

[0043] The above discussion demonstrates that multiple signaling pathways known to be critical for cardiac hypertrophy and pathological remodeling are regulated through the binding of key signaling intermediaries to the mAKAPβ scaffold. Cardiomyocyte-specific, conditional mAKAP knockout mice have been characterized, demonstrating the importance of the mAKAPβ signalosome in vivo (Kritzer et al., 2014; Martinez et al., 2022). mAKAPβ in cardiomyocytes is required for cardiac hypertrophy induced by transverse aortic constriction and isoproterenol infusion. Most strikingly, however, mAKAPβ deficiency under chronic pressure overload prevents pathological remodeling (including cardiomyocyte apoptosis and interstitial fibrosis) and preserves cardiac function, significantly prolonging mouse survival (Kritzer et al., 2014). Similar beneficial effects were observed in mice following myocardial infarction (Martinez et al., 2022). These results establish mAKAPβ as the first scaffold protein whose deletion confers a survival benefit in heart disease. Importantly, mAKAPβ appears to be dispensable for both the development and maintenance of normal adult cardiac function, as no overt phenotype was observed in 6-month-old mice using an Nkx2-5-driven Cre deletion line (Kritzer et al., 2014). Although mAKAPβ knockout does attenuate physiological hypertrophy induced by forced exercise (swimming), targeting the mAKAPβ complex remains relevant in disease states.

[0044] Various strategies are conceivable for targeting the mAKAPβ complex in humans, including siRNA knockdown of this scaffold protein. However, the relatively advanced understanding of the structure and function of the mAKAPβ signalosome has provided additional approaches for targeting these pathways. For example, studies have demonstrated in vitro the effectiveness of expressing peptides targeting key protein-protein interactions involved in mAKAPβ, including anchor-disrupting peptides targeting mAKAPβ-CaNAβ, mAKAPβ-MEF2D, mAKAPβ-PLCε, mAKAPβ-PP2A, and mAKAPβ-RSK3 binding (Li, Vargas et al., 2013; Li, Kritzer et al., 2013; Vargas et al., 2012; Zhang et al., 2011; Li et al., 2020). Heart failure is a leading cause of death, and despite modern medical treatments, the 5-year mortality rate remains as high as 50%, with associated costs exceeding $30 billion annually in the United States alone (Go et al., 2014). Recently, proof-of-concept for the efficacy of targeting the mAKAPβ signalosome has been demonstrated in mice by targeting the mAKAPβ-PP2A and mAKAPβ-RSK3 binding using an adeno-associated viral (AAV) gene therapy vector (Li et al., 2020). Many potential cardiac disease targets have pleiotropic effects, which complicates the development of drugs with sufficient in vivo specificity. Specific targeting of the mAKAPβ signalosome in cardiomyocytes offers an opportunity to target relatively rare protein-protein interactions that appear specialized for pathological cardiac remodeling and facilitate their ablation without significant side effects.

[0045] Adeno-associated virus (AAV)

[0046] Adeno-associated virus (AAV) is a small single-stranded DNA virus with a genome of approximately 4.7 kb, belonging to the genus Dependoparvovirus of the family Parvoviridae (Wang, 2019). Due to its non-pathogenicity and ability to deliver almost any DNA sequence to tissues of interest in mammalian species, AAV has become the preferred gene therapy vector for basic research and clinical applications. Since the approval of alipogene tiparvovec (Glybera) in Europe for the treatment of lipoprotein lipase deficiency in 2012 and the approval of voretigene neparvovec-rzyl (Luxturna) in the United States for the treatment of RPE365 in 2017, AAV has become a promising candidate for gene therapy in both basic research and clinical applications. - / -Since the approval of onasemnogene abeparvovec (Zolgensma) for the treatment of Leber congenital amaurosis and retinitis pigmentosa in the United States in 2019, and the 2019 approval of onasemnogene abeparvovec (Zolgensma) for the treatment of spinal muscular atrophy in the United States, the number of clinical trials for AAV-based therapeutics has increased dramatically, with over 200 clinical trials registered to date (Kuzmin et al., 2021). Adeno-associated viruses are designated by the serotype of their capsid protein. Among the commonly used serotypes, AAV serotype 9 (AAV9) has been frequently used for skeletal muscle, cardiac, and neurological applications, including the AAV9 biologic drug onasemnogene abeparvovec. For cardiac applications, AAV9 has become the dominant serotype in preclinical research (Kieserman et al., 2019).

[0047] A key issue in any drug development process is biodistribution. Although it is recognized that AAV9 delivered via the vasculature can be delivered to other organs, little has been reported on the biodistribution of AAV9 in pigs, or whether it depends on the local or peripheral site of introduction. AAV, including serotype 9, has a pronounced tropism for the liver, and AAV9 biologics can experience significant off-target delivery to the liver when administered via coronary arteries (Ishikawa et al., 2018). Preclinical testing of muscle-directed therapies often involves transitioning from rodents to pigs to reveal potential clinical relevance. The flexibility of AAV gene therapy has been demonstrated in pigs using AAV9 biologics for 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 targeting the Hippo signaling pathway in cardiac regeneration (Liu et al., 2021). Adeno-associated viruses have been delivered to the heart via direct intramyocardial injection (Liu et al., 2021) and intracoronary infusion (Fish et al., 2013). Percutaneous catheter-based intracoronary infusion has been performed using a variety of approaches. Retrograde techniques can be performed with or without coronary artery / venous occlusion or vector recirculation, while antegrade delivery is perhaps the simplest intracoronary approach and is easily applicable to patients undergoing standard cardiac catheterization (Ishikawa et al., 2018).

[0048] Further discussion of the design and use of AAV vectors can be found, for example, in U.S. Patent Nos. 11,129,908 and 5,139,941 and WO 1998 / 046728, which are hereby incorporated by reference in their entirety for all purposes. In addition, the use of AAV vectors to treat heart disease is also disclosed in U.S. Patent Nos. 9,132,174, 9,937,228, 10,617,737, 11,229,679, and U.S. Patent Application Serial No. 17 / 580,692 filed on January 21, 2022, as well as U.S. Patent No. 10,907,153 and U.S. Patent Application Serial No. 16 / 818,771 filed on March 13, 2020, Li et al., 2022; Li et al., 2020; and Martinez et al., 2023, all of which are hereby incorporated by reference in their entirety for all purposes.

[0049] Clearly, there is an urgent need to develop new and effective therapies to treat patients with heart failure and to prevent its development in the context of other cardiovascular diseases such as coronary artery disease, hypertension, and valvular heart disease. Summary of the Invention

[0050] The following brief summary is not intended to be inclusive of all features and aspects of the present invention, nor does it mean that the present invention must include all features and aspects discussed in this summary.

[0051] The present inventors have discovered improved compositions and methods for delivering transgenes to cardiomyocytes using AAV plasmids containing novel promoter-enhancer regulatory sequences comprising a novel combination of promoter, exon, and intron sequences from the human TNNT2 gene to direct expression of the transgene.

[0052] In certain aspects, these improved compositions and methods can be used to treat cardiac pathological processes by inhibiting the signaling properties of individual mABAP signaling complexes using drugs that target unique protein-protein interactions. This therapeutic strategy has advantages over classical treatment approaches because it allows for the selective inhibition of well-defined cellular responses.

[0053] In certain aspects, the compositions and methods of the present invention can disrupt mAKAP-mediated protein-protein interactions to inhibit the ability of mAKAP to coordinately activate enzymes that play a central role in activating key transcription factors that initiate cellular processes that lead to pathological cardiac remodeling. Inhibiting mAKAPβ binding activity can protect the heart from damage that leads to heart failure, for example, after myocardial infarction.

[0054] In one aspect, the 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.

[0055] In another aspect, the invention relates to compositions and methods for inhibiting the interaction of mAKAPβ with RSK3, for example, using AAV vectors comprising novel promoter-enhancer regulatory sequences and sequences targeting the RSK3 binding domain (RBD) of mAKAPβ.

[0056] In yet another aspect, the 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.

[0057] In yet another aspect, the present invention relates to a vector comprising cardiac troponin T promoter 2 (hTNNT) and a skeletal muscle enhancer with a splice consensus site.

[0058] In a further aspect, the present invention relates to codon-optimized vectors and methods of using the same.

[0059] The compositions and methods described above can be used to protect the heart from injury by administering to a patient at risk for such injury a pharmaceutically effective amount of a composition that inhibits the expression, binding and / or activity of mABAPβ.

[0060] The foregoing and other objects, features, and advantages of the present invention will become apparent from the following more particular description of the preferred embodiments of the present 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 drawn to scale, emphasis instead being placed upon illustrating the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] This patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0062] Figure 1 Shown is the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence (TNNT2 regulatory sequence marked in green in the figure), which contains a novel combination of promoter, exon and intron sequences of the human TNNT2 gene to direct the expression of human mAKAP shRNA.

[0063] Figures 2-1 to 2-4 Show Figure 1The nucleic acid sequence of the AAV plasmid (SEQ ID NO: 1).

[0064] Figure 3 Shown is the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence (TNNT2 regulatory sequence marked in green in the figure) that comprises a novel combination of promoter, exon, and intron sequences of the human TNNT2 gene to direct the expression of the human mAKAP RBD sequence.

[0065] Figures 4-1 to 4-3 Show Figure 3 The nucleic acid sequence of the AAV plasmid (SEQ ID NO: 2).

[0066] Figure 5 Shown is the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence (TNNT2 regulatory sequence marked in green in the figure) that comprises a novel combination of promoter, exon, and intron sequences of the human TNNT2 gene to direct the expression of the human mAKAP PBD sequence.

[0067] Figures 6-1 to 6-3 Show Figure 5 The nucleic acid sequence of the AAV plasmid (SEQ ID NO: 3).

[0068] Figure 7 Shown is the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence comprising a novel combination of the human calsegregant enhancer and promoter and exonic sequences of the human TNNT2 gene to direct the expression of human mAKAP shRNA.

[0069] Figures 8-1 to 8-3 Show Figure 7 The nucleic acid sequence of the AAV plasmid (SEQ ID NO: 4).

[0070] Figure 9 Shown is the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence comprising a novel combination of the human calsequestrin enhancer and promoter and exonic sequences of the human TNNT2 gene to direct the expression of the human mAKAP RBD sequence.

[0071] Figures 10-1 to 10-3 Shown Figure 9 The nucleic acid sequence of the AAV plasmid (SEQ ID NO: 5).

[0072] Figure 11Shown is the design of an AAV plasmid containing a novel promoter-enhancer regulatory sequence comprising a novel combination of the human calsequestrin enhancer and promoter and exonic sequences of the human TNNT2 gene to direct the expression of the human mAKAP PBD sequence.

[0073] Figures 12-1 to 12-3 Shown Figure 11 The nucleic acid sequence of the AAV plasmid (SEQ ID NO: 6).

[0074] Figure 13 Shown is a nucleotide alignment view of newly introduced silent single nucleotide mutations in the open reading frame of the human PBD (mAKAP 2132-2319) designed to reduce the number of CpG-containing immunostimulatory motifs.

[0075] Figure 14 Shown is a nucleotide alignment view of newly introduced silent single nucleotide mutations in the open reading frame of the human RBD (mAKAP 1696-1835) designed to reduce the number of CpG-containing immunostimulatory motifs.

[0076] Figures 15-1 to 15-3 Shown Figure 1 -6 The origin of the "hTNNT2 regulatory sequence" contained in the plasmid shown.

[0077] Figure 16 A model of mAKAPβ-regulated, SRF-dependent gene expression is shown. Anchored RSK3, a Gq protein-coupled receptor-ERK effector, phosphorylates SRF associated with the perinuclear mAKAPβ complex. mAKAPβ-anchored PP2A is activated by cAMP-dependent protein kinase A (PKA), antagonizing SRF phosphorylation. Phosphorylated SRF induces the expression of genes that promote centripetal hypertrophy.

[0078] Figures 17-1 to 17-4 The complete nucleotide sequence (SEQ ID NO: 9) and the deduced amino acid sequence (SEQ ID NO: 10) of human RSK3 (Homo sapiens ribosomal protein S6 kinase A2 (RPS6KA2), transcript variant 1, mRNA, NCBI Reference Sequence: NM_021135.6) are shown. The deduced RSK3 protein sequence is represented by the single-letter amino acid code, starting with the first methionine residue before the open reading frame at codon 733 and ending with an asterisk. The N-terminal region unique to RSK3 (with no homology to RSK1 or RSK2) is indicated.

[0079] Figures 18-1 to 18-5The nucleotide sequence of human mAKAPa is shown (SEQ ID NO: 11), and the translation of its open reading frame is given (SEQ ID NO: 12).

[0080] Figures 19-1 to 19-2 The amino acid sequence of human mAKAP is shown (SEQ ID NO: 12). mAKAPα starts at residue 1 and mAKAPβ starts at residue 243. The PBD is in bold.

[0081] Figure 20 The amino acid sequence of human PBD expressed in AAV is shown (SEQ ID NO: 14).

[0082] Figure 21 Shown is an alignment of RBDs (SEQ ID NOs: 16-42) from multiple species. Figure 3 The sequence expressed by the vector is shown in row 1 (SEQ ID NO: 15).

[0083] Figure 22 An alignment of PBDs from various species (SEQ ID NOs: 45-64) is shown. Figure 5 The sequence expressed by the vector is shown in row 1 (SEQ ID NO: 68).

[0084] Figure 23 A map of the human PBD AAV shuttle plasmid pscAAV-hmAKAP PBD is shown.

[0085] Figures 24-1 to 24-2 The nucleotide sequence of the pscAAV-hmAKAP PBD plasmid is shown (SEQ ID NO: 67).

[0086] Figures 25-1 to 25-4 The sequence of human mAKAP (AKAP6) mRNA (SEQ ID NO: 69) is shown, with reference sequence XM_017021808.1, and shRNA sequences (#1-3) are indicated. Numbers correspond to the nucleotide sequence, and the encoded amino acids are indicated above.

[0087] Figure 26 A map of the pscA-TnT-mAKAP shRNA (#3) plasmid is shown.

[0088] Figures 27-1 to 27-2 The nucleotide sequence of the pscA-TnT-mAKAP shRNA (#3) plasmid (SEQ ID NO: 70) is shown, with important features and some restriction enzyme sites indicated.

[0089] Figure 28The targets of the scAAV-mAKAP shRNA biopharmaceuticals are shown. The mAKAP mRNA sequences for human (SEQ ID NO: 71), porcine (SEQ ID NO: 72), mouse (SEQ ID NO: 73), and rat (SEQ ID NO: 74) are listed. The sequence in the box is shRNA target #3.

[0090] Figure 29 The amino acid sequence of rat mAKAP PBD expressed in an AAV vector is shown (SEQ ID NO: 75), including an N-terminal myc tag.

[0091] Figure 30 The results of cardiac selective delivery of different AAV compositions are shown. The data are mRNA levels in the left ventricle (LV) of the heart relative to the liver, brain or skeletal muscle (skm). The data were obtained from tissues collected from 3 pigs 3 months after intravenous infusion of 2E12vg / kg AAV9 vector. RNA extraction and 2μg total RNA were reverse transcribed (RT) into cDNA and then RT-qPCR was performed. AAV viral genome (vg) was quantified by qPCR using genomic DNA extracted from the same tissue. The data shown here are calculated for the gene delivery amount of the same tissue after the mRNA level was standardized by the housekeeping gene 18S mRNA level. Shown in the figure is the average transgene expression enrichment fold in 9 different left ventricular regions (front base, side base, back base, front middle section, side middle section, back middle section, front top, side top, back top) compared with the liver, cerebral cortex (brain) and 5 different skeletal muscles (triceps medial head, triceps long head, triceps lateral head, deltoid muscle). “WPRE” means using Figure 23 AAVs generated with the indicated plasmids and similar plasmids encoding human RBD and mABAP shRNAs. Figure 7-1 2. "enh.int" refers to the AAV generated using the plasmid Figure 1 AAV generated with the plasmids shown in Figure ​-6.

[0092] Figures 31-1 to 31-4 The sequence of the TNNT2 gene is shown (5167 bp extracted from the NCBI chromosome reference sequence NC_000001.11201378367..201373201, the gene is in the antisense direction). In the TNNT2 gene, exon 1 is located at 688-745 bp (NC_000001.11 201377680..201377623), and exon 2 is located at 5100-5154 bp (NC_000001.11 201373268..201373214).

[0093] Figure 32 shows CaMKII. (a) Ca 2+ Binding of calmodulin (CaM) releases the autoinhibition of the CaMKII pseudosubstrate domain. Post-translational modifications at the N-terminus of the regulatory domain (exons 11-12 within exons 11-19 (Duran, Nickel et al. 2021)) positively (+) and negatively (-) regulate CaMKII activity (SEQ ID NO:80). (b) Dodecameric CaMKII holoenzyme, color-coded as in panel a (PDBIDs 5VLO, 2VN9, and 3SOA). (c) The three most highly expressed CaMKIIδ variants in the adult heart (SEQ ID NOs:81-82). This figure is reproduced from Reyes Gaido et al. (Reyes Gaido, Nkashama et al. 2023).

[0094] Figure 33 The interaction between endogenous mAbA-Pβ and CaMKII in myocytes is shown. Co-immunoprecipitation assays were performed using CaMKII antibodies and control IgG antibodies in neonatal myocytes expressing myc-GFP or myc-RBD-GFP. n = 3.

[0095] Figure 34 mABAPβ1694-1833 binds to various CaMKII isoforms. COS-7 cells were transfected to co-express Myc-tagged RBD and Flag- and mCherry-tagged CaMKII isoforms. The protein complexes were immunoprecipitated using a Myc-tag antibody.

[0096] Figure 35 shows overlapping binding sites between RSK3 and CaMKII on mAKAPβ. COS-7 cells were transfected with Myc-GFP-tagged mAKAP fragments and co-expressed with either CaMKIIγ-mCherry-Flag or mCherry-HA-RSK3. Protein complexes were immunoprecipitated using a Myc-tag antibody. n = 2 (RSK3), n = 3 (CaMKIIγ).

[0097] Figure 36 shows that expression of mAKAP 1694-1833 inhibits HDAC4 phosphorylation in Ang II-treated neonatal myocytes. Adenovirus-infected myocytes were treated with 100 nM Ang II for 1 hour, and HDAC4 was immunoprecipitated. n = 3 biological replicates. The lower band in the p-HDAC4 blot corresponds to HDAC4. Detailed Description of the Invention

[0098] As discussed above, AKAP-based signaling complexes play a central role in regulating both physiological and pathological cardiac events. Therefore, the present inventors investigated the use of drugs targeting distinct protein-protein interactions to inhibit the signaling properties of individual AKAP signaling complexes as a means of limiting cardiac pathological progression. This therapeutic strategy offers the advantage of selectively inhibiting well-defined cellular responses compared to conventional treatment approaches.

[0099] Anchoring proteins, including mABAPβ, are therapeutic targets for treating pathological cardiac hypertrophy and heart failure. In particular, the inventors have discovered that disrupting mABAPβ-mediated protein-protein interactions can be used to inhibit mABAPβ's ability to orchestrate the activation and function of enzymes that play a central role in activating key transcription factors and chromatin-modifying enzymes that initiate and / or promote remodeling processes that lead to heart failure.

[0100] One aspect of the present invention is to inhibit intracellular signaling in cardiomyocytes that promotes pathological cardiac gene expression, as may be manifested by improvements in ventricular configuration, which will inhibit the progression of heart disease to heart failure. For example, heart failure can be prevented or treated by altering cardiomyocyte signaling: in centrifugal heart disease, by inhibiting cardiomyocyte elongation, thereby reducing the left ventricular internal diameter; or conversely, in concentric heart disease, by preventing cardiomyocyte thickening and increasing left ventricular wall thickness. Evidence for preventing cardiac dysfunction has been obtained for gene therapy vectors based on the expression of sequences derived from muscle A kinase anchoring protein (mAKAP, also known as AKAP6): (1) mAKAP shRNA (e.g., see U.S. Patent No. 10,907,153, which is incorporated herein by reference in its entirety for all purposes, and subsequently published in (Martinez et al., 2022)); (2) RBD peptides (e.g., see U.S. Patent Nos. 9,132,174, 9,937,228, 10,617,337, 11,229,679, which are incorporated herein by reference in their entirety for all purposes, and subsequently published in (Li et al., 2020)); and (3) PBD peptides (e.g., see U.S. Application Serial No. 16 / 818,771, which is incorporated herein by reference in its entirety for all purposes, and subsequently published in (Li et al., 2020)).

[0101] In particular, the present inventors have discovered novel vectors comprising the human cardiac troponin t 2 (hTNNT2) promoter, demonstrating that these vectors increase the expression levels of this molecule in the left ventricle compared to expression levels in other tissues.

[0102] In one embodiment, these vectors further comprise a skeletal muscle enhancer with a splice consensus site.

[0103] In some embodiments, these vectors also contain codon optimization for expression and / or reduced immunogenicity.

[0104] In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are fully explained in the literature. See, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th ed., 2012); "Current Protocols in Molecular Biology" Vol. I-III [Ausubel, RM, ed. (1994)]; "CellBiology: A Laboratory Handbook" Vol. "Immunology" Volume I-III [Coligan, JE, ed. (2005)]; "Oligonucleotide Synthesis" (MJGait ed.1984); "Nucleic Acid Hybridization" [BDHames&S.J.Higgins eds.(1985)]; "TranscriptionAnd Translation" [BDHames&S.J.Higgins, eds.(1984)]; "Animal Cell Culture" [RI Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "APractical Guide To Molecular Cloning" (1984); C. Machida, "ViralVectors for Gene Therapy: Methods and Protocols" (2010); J. Reidhaar-Olson and C. Rondinone, “Therapeutic Applications of RNAi: Methods and Protocols” (2009).

[0105] The definitions and abbreviations used in this article are as follows:

[0106] AAV - adeno-associated virus.

[0107] AC5-type adenylate cyclase

[0108] ACE - angiotensin-converting enzyme

[0109] ANF ​​- atrial natriuretic factor

[0110] ARVM-adult rat ventricular myocytes

[0111] CaN-calcineurin

[0112] CarG Box-(CC9AT)6GG

[0113] CPT-cAMP-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate

[0114] CsA-cyclosporine A

[0115] CTKD - C-terminal kinase domain

[0116] ERK - extracellular signal-regulated kinase

[0117] FBS-fetal bovine serum

[0118] Fsk-Forskolin

[0119] GFP - green fluorescent protein

[0120] GPCR-G protein-coupled receptor, HDAC-histone deacetylase

[0121] Gs - stimulatory G protein

[0122] GST-glutathione-S-transferase, HIF1α-hypoxia-inducible factor 1α

[0123] HFrEF - heart failure with reduced ejection fraction

[0124] IBMX-3-isobutyl-1-methylxanthine

[0125] Iso-isoproterenol

[0126] LIF-Leukemia Inhibitory Factor

[0127] The MADS-(MCM1, agamous, deficiens, SRF) domain mediates DNA binding to the CArG box (CC9AT) 6GG serum response element (SRE); the MADS-box gene family was later named after the acronyms of these four basic members, omitting ARG80:

[0128] MCM1 - from the budding yeast, Saccharomyces cerevisiae,

[0129] AGAMOUS - from Arabidopsis thaliana,

[0130] DEFICIENS-from snapdragon (Antirrhinum majus),

[0131] SRF - from humans (Homo sapiens).

[0132] mAKAP - muscle A kinase anchoring protein

[0133] mA KAPα - alternatively spliced ​​isoform expressed in neurons; 255 kDa

[0134] mAKAPβ - alternatively spliced ​​isoform expressed in striated muscle cells; 230 kDa

[0135] MAPK - Mitogen-activated protein kinase

[0136] MEF2 - myocyte enhancer factor-2

[0137] MgAc-magnesium acetate

[0138] MI - myocardial infarction

[0139] NCX1 - sodium / calcium exchanger

[0140] NFATc - nuclear factor of activated T cells

[0141] NRVM-neonatal rat ventricular myocytes

[0142] NTKD - N-terminal kinase domain

[0143] OA-okadaic acid

[0144] PBD-PP2A anchor disruptor—reducing eccentric hypertrophy

[0145] PDE4D3 - cAMP-specific phosphodiesterase type 4D3

[0146] PDK1-3'-phosphatidylinositol-dependent kinase 1

[0147] PE-phenylephrine

[0148] PHD - Prolyl Hydroxylase

[0149] PI4P - phosphatidylinositol 4-phosphate

[0150] PKA - Protein Kinase A

[0151] PKD - protein kinase D

[0152] PKI - protein kinase inhibitor

[0153] PLCε-phospholipase Cε

[0154] PKA-cAMP-dependent protein kinase

[0155] PP2A-protein (serine-threonine) phosphatase—makes Ser 103 Dephosphorylation

[0156] PP2B - calcium / calmodulin-dependent protein phosphatase 2B

[0157] RBD - isoform-specific N-terminal RSK3 domain that binds to a discrete "RSK3 binding domain" (RBD) located at residues 1694-1833 in mAKAPβ

[0158] RSK-p90 ribosomal S6 kinase

[0159] RyR2 - ryanodine receptor type 2

[0160] scAAV-self-complementary AAV

[0161] siRNA - small interfering RNA oligonucleotides

[0162] shRNA - short hairpin RNA

[0163] SRE - serum response element

[0164] SRF-serum response factor-transcription factor (SRF Ser 103 Phosphorylation of β-catenin induces centripetal myocyte and cardiac hypertrophy; inhibition of β-catenin phosphorylation improves cardiac structure and function)

[0165] siRNA - small interfering RNA

[0166] TAC - transverse aortic coarctation

[0167] TCA-Trichloroacetic acid

[0168] VSV - Vesicular Stomatitis Virus

[0169] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to any method and material described herein can be used in the practice or testing of the present invention, preferred methods and materials are still as described herein. In general, the nomenclature and techniques of cell and molecular biology and chemistry related to the present invention are well-known and commonly used in the art. Certain undefined experimental techniques are generally performed according to conventional methods known in the art and are described in various general and more specific references cited and discussed throughout this specification. For clarity, the following terms are defined as follows.

[0170] The present invention recognizes that the interaction between RSK3 and / or PP2A and mABAPβ mediates multiple intracellular signals and pathways that lead to cardiomyocyte hypertrophy and / or dysfunction. Therefore, the present inventors have discovered multiple methods to inhibit this interaction to prevent and / or treat cardiomyocyte hypertrophy and / or dysfunction.

[0171] Thus, the present invention includes a method of protecting the heart from injury by administering to a patient at risk of such injury 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 will be understood that a "pharmaceutically effective amount" can be determined empirically based on the delivery method and will vary depending on the delivery method.

[0172] 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 of RSK3 and / or PP2A with mAKAPβ.

[0173] The present invention also relates to compositions that inhibit the interaction of RSK3 and / or PP2A with mABAPβ. In specific embodiments, these inhibitory compositions or "inhibitors" include peptide inhibitors, which can be administered by any known method, including delivery via gene therapy. In other embodiments, the inhibitors can be small molecule inhibitors.

[0174] Specifically, the present invention relates to methods and compositions for treating and / or protecting the heart from injury by administering to a patient at risk of such injury a pharmaceutically effective amount of a composition that (1) inhibits the interaction of RSK3 and / or PP2A with mAKAPβ; or (2) inhibits the activity of RSK3 and / or PP2A with mAKAPβ; or (3) inhibits the expression of RSK3, PP2A and / or mAKAPβ.

[0175] The present invention also relates to methods for treating or protecting against cardiac injury by administering to a patient at risk of such injury a pharmaceutically effective amount of a composition that inhibits cellular processes mediated by RSK3 and / or PP2A.

[0176] In one embodiment, the composition comprises a mAKAPβ peptide. In a preferred embodiment, the mAKAPβ peptide is obtained from the carboxyl terminus of the mAKAPβ amino acid sequence. In a particularly preferred embodiment, the mAKAPβ peptide is at least a fragment of amino acids 2083-2319 of the mAKAPβ amino acid sequence.

[0177] In a preferred embodiment, the mAKAPβ peptide is at least a fragment of amino acids 2133-2319 of the mAKAPβ amino acid sequence.

[0178] In one embodiment, the composition comprises a mAKAP[beta] peptide.

[0179] In a preferred embodiment, the mAKAPβ peptide comprises 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.

[0180] In another embodiment, the mAKAPβ peptide is obtained from the carboxyl terminus of the mAKAPβ amino acid sequence. In a particularly preferred embodiment, the mAKAPβ peptide is at least a fragment of amino acids 2083-2319 of the mAKAPβ amino acid sequence, or a nucleotide sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0181] 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.

[0182] In another embodiment, the composition comprises a small interfering RNA (siRNA) that inhibits the expression of any one of RSK3, PP2A and / or mAKAPβ. In a preferred embodiment, the siRNA that inhibits mAKAPβ expression is produced in vivo following administration of a short hairpin RNA expression vector or biological agent (shRNA).

[0183] The composition of the present invention can be administered directly, or can be administered using a viral vector. In a preferred embodiment, the vector is adeno-associated virus (AAV).

[0184] In another embodiment, the composition comprises a small molecule inhibitor. In a preferred embodiment, the small molecule is an inhibitor of RSK3, PP2A and / or mAKAPβ.

[0185] In another embodiment, the composition comprises a molecule that inhibits mAKAPβ binding, expression, or activity. In a preferred embodiment, the molecule is a mAKAPβ peptide. The molecule can be expressed using a viral vector (including adeno-associated virus AAV).

[0186] In yet another embodiment, the composition comprises a molecule that interferes with a cellular process mediated by mAKAPβ. In certain preferred embodiments, the molecule interferes with the binding of mAKAPβ to RSK3, or interferes with the anchoring of PP2A.

[0187] The present invention also relates to diagnostic assays for determining a predisposition to heart disease, wherein 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 to mAKAPβ. The present invention also provides test kits for use in such assays.

[0188] In other embodiments, the inhibitor includes any molecule that inhibits the expression of RSK3, PP2A and / or mABAPβ, including antisense RNA, ribozymes, and small interfering RNA (siRNA), including shRNA.

[0189] The present invention also includes an assay system for screening potential drugs that effectively inhibit the expression and / or binding of RSK3 and / or PP2A to mAKAPβ. In one example, a test drug can be administered to a cell sample containing RSK3 and / or PP2A and mAKAPβ, or to an extract containing RSK3 and / or PP2A and mAKAPβ, and the effect of the drug on the binding activity of RSK3 and / or PP2A to mAKAPβ can be determined by comparison with a control. The present invention also provides a test kit for such an assay.

[0190] When preparing the peptide compositions of the present invention, all or part of the amino acid sequence of RSK3 and / or PP2A or mAKAP 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 positions 1735-1833 or 2133-2319 of the mAKAP is used.

[0191] It should be recognized that various amino acid substitutions, deletions or insertions can also enhance the inhibitory ability of the inhibitory peptide to the interaction of RSK3 and / or PP2A with mAKAPβ. Such substitution mutations can change the amino acids in the resulting protein in a non-conservative manner (i.e., changing the amino acids belonging to an amino acid group with a specific size or property to amino acids belonging to another group) or in a conservative manner (i.e., changing the amino acids belonging to an amino acid group with a specific size or property to amino acids belonging to the same group). Such conservative changes generally result in fewer changes in the structure and function of the resulting protein. Non-conservative changes are more 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.

[0192] The following is an example of various amino acid groups:

[0193] Amino acids with nonpolar R groups : Alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine.

[0194] Amino acids with uncharged polar R groups : Glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine.

[0195] Amino acids with charged polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid.

[0196] basic amino acids (Positively charged at pH 6.0): lysine, arginine, histidine.

[0197] Another grouping could be the amino acids containing a benzene ring: phenylalanine, tryptophan, tyrosine.

[0198] Another grouping can be made based on molecular weight (i.e., R group size): 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), tryptophan (204).

[0199] Particularly preferred replacements are:

[0200] - Lysine (Lys) replaced by Arg or vice versa to maintain a positive charge;

[0201] - Glutamic acid (Glu) replaces aspartic acid (Asp) or vice versa to maintain the negative charge;

[0202] - Serine (Ser) replaces threonine (Thr) to maintain a free -OH;

[0203] - Glutamine (Gln) replaces asparagine (Asn) to maintain free NH2.

[0204] Amino acid substitutions can also be introduced to replace an original amino acid with an amino acid having particularly advantageous properties. For example, cysteine ​​(Cys) can be introduced to provide a potential site for disulfide bond formation with other Cys. Histidine (His) can be introduced as a special "catalytic" site (i.e., His can act as both an acid and a base and is the most common amino acid in biochemical catalysis). Proline (Pro) can be introduced because its unique planar structure can induce a beta-turn in the protein structure. Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, more preferably at least about 90% or 95%) in the two amino acid sequences are identical or comprise conservative substitutions.

[0205] Similarly, the nucleotide sequence used according to the present invention also can be replaced, deleted or inserted.For the degenerate codon of encoding specific amino acid, any codon of encoding this specific amino acid can be used.In addition, if need to replace another amino acid with a kind of amino acid, can modify the nucleotide sequence according to known genetic code.

[0206] Nucleotides and oligonucleotides can also be modified. Exemplary modifications are described in U.S. Patent No. 7,807,816 (which is hereby incorporated by reference in its entirety, particularly with respect to its description of modified nucleotides and oligonucleotides).

[0207] Two nucleotide sequences are "substantially homologous" or "substantially identical" when at least about 70% of the nucleotides in the two nucleotide sequences are identical (preferably at least about 80%, most preferably at least about 85%, 90%, 95% or 99%).

[0208] Two nucleotide sequences are "substantially complementary" when at least about 70% of the nucleotides in the two nucleotide sequences (preferably at least about 80%, most preferably at least about 85%, 90%, 95% or 99%) are capable of forming hydrogen bonds with a target sequence.

[0209] The term "standard hybridization conditions" refers to salt and temperature conditions that are essentially equivalent to 5×SSC and 65°C for hybridization and washing. However, those skilled in the art will understand that these "standard hybridization conditions" depend on specific conditions, including the concentrations of sodium and magnesium in the buffer, the length and concentration of the nucleotide sequence, the percentage of mismatches, the percentage of formamide, etc. In addition, important factors determining "standard hybridization conditions" include whether the two hybridizing sequences are RNA-RNA, DNA-DNA, or RNA-DNA. Such standard hybridization conditions can be easily determined by those skilled in the art according to well-known formulas, wherein the hybridization temperature is generally 10-20°C lower than the predicted or measured Tm, and if necessary, a higher stringency wash can be used.

[0210] The phrase "pharmaceutically acceptable" refers to a molecular entity or composition that is physiologically tolerable and does not generally produce allergic or similar adverse reactions (eg, stomach upset, dizziness, etc.) when administered to humans.

[0211] The phrase "therapeutically effective amount" is used herein to refer to an amount sufficient to prevent, and preferably reduce, clinically significant changes in cardiomyocyte characteristics by at least about 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0212] The preparation of therapeutic compositions containing polypeptides, analogs, or active fragments as active ingredients is well known in the art. Typically, such compositions are prepared as injectables in the form of liquid solutions or suspensions, however, they can also be prepared in solid forms suitable for dissolution or suspension in a liquid prior to injection. The formulations can also be emulsified. The active therapeutic ingredient is typically mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients include water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, which enhance the effect of the active ingredient.

[0213] Polypeptides, analogs or active fragments, and small molecule inhibitors can all be formulated into therapeutic compositions as neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptide or antibody molecules), which can be formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or with organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide), as well as organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0214] The therapeutic compositions of the present invention are typically administered intravenously, for example, by injection of a unit dose. The term "unit dose" when used to refer to the therapeutic compositions of the present invention refers to physically discrete units suitable as single dosages for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent (i.e., carrier or vehicle).

[0215] The composition is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount administered depends on the subject to be treated, the ability 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 to be administered depends on the physician's judgment and is specific to each individual. However, a suitable dosage range may be from about 0.1 to 20 mg per kg of individual body weight per day, preferably from about 0.5 to about 10 mg, more preferably from 1 to several mg of active ingredient, and depends on the route of administration. Suitable regimens for initial administration and booster injections also vary, but a typical approach is to perform an initial administration followed by repeated administration at intervals of one or more hours by subsequent injections or other modes of administration. Alternatively, a continuous intravenous infusion sufficient to maintain a concentration of 10 nanomolar to 10 micromolar in the blood can be used. In a preferred embodiment, the mAKAPβ peptide or shRNA is expressed by an AAV gene therapy vector. Suitable intravenous doses of AAV vectors range from 1×1012 viral genomes per kg body weight to 5×101 per kg body weight. 4 In particular, for AAV vectors containing serotype 9 capsids, the dosage is preferably 0.3-1×101 per kg body weight. 4 A viral genome.

[0216] Since the inhibitor must reach the cytoplasm of the cell, the peptide according to the present invention may need to be modified to allow it to be transported across the cell membrane, or may need to be expressed by a vector encoding the peptide inhibitor. Similarly, nucleic acid inhibitors (including siRNA, shRNA and antisense RNA) can also be expressed by a vector. According to the present invention, any vector that can enter the target cell can be used. In particular, viral vectors can "infect" cells and express the desired RNA or peptide. Any viral vector that can "infect" cells can be used. A particularly preferred viral vector is adeno-associated virus (AAV).

[0217] siRNA inhibits the translation of target mRNA through a mechanism called RNA interference. When siRNA is completely complementary to the target mRNA, siRNA works by promoting mRNA degradation. As a special form of siRNA, shRNA has certain advantages over siRNA prepared in the form of oligonucleotides. siRNA oligonucleotides are usually synthesized in the laboratory and delivered to cells using a delivery system to deliver siRNA to the cytoplasm. In contrast, shRNA is expressed as a small gene, which is delivered to the cell nucleus through a vector and, after transcription, is processed into mature siRNA species by enzymes in the cell (such as Drosha and Dicer). 99% of siRNA is usually degraded after 48 hours, while shRNA can be expressed for up to 3 years or even longer. In addition, shRNA can be delivered at a much lower copy number than siRNA (5 copies vs. low nM) and is less likely to produce off-target effects, immune activation, inflammation and toxicity. siRNA is suitable for acute diseases where high doses are tolerable, while shRNA is suitable for chronic, life-threatening diseases or conditions where low doses are required. (http: / / www.benitec.com / technology / sirna-vs-shrna).

[0218] Design guidelines for siRNA and shRNA can be found in the literature of Elbashir et al. (2001) and multiple websites, including 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, which are all incorporated herein by reference in their entirety. Preferably, the first nucleotide is A or G. siRNAs with a length of 25-29 nucleotides may be more effective than shorter sequences, but shRNAs with a double-strand length of 19-21 are as effective as longer sequences. The preferred length of siRNA and shRNA is 19-29 nucleotides. The loop sequence length of shRNA can be 3-9 nucleotides, with 5, 7 or 9 nucleotides being preferred.

[0219] Exemplary shRNA sequences of the invention include: GGTTGAAGCTTTGAAGAAA (SEQ ID NO: 77), GCTAAGAGATACAGAGCTT (SEQ ID NO: 78), or GGAGGAAATAGCAAGGTTA (SEQ ID NO: 79).

[0220] As for small molecule inhibitors, any small molecule that inhibits the interaction between RSK3 and / or PP2A and mAKAPβ can be used. In addition, any small molecule that inhibits the activity of RSK3 and / or PP2A and / or mAKAPβ can be used.

[0221] Small molecules with similar structures and functions can also be identified through rational design and screening strategies.

[0222] Likewise, any small molecule that inhibits the expression of RSK3, PP2A and / or mAKAPβ may be used.

[0223] The present invention is further described in detail by the following items, which represent preferred embodiments thereof:

[0224] 1. A composition comprising a regulatory nucleotide sequence for expressing a second nucleotide sequence in cardiomyocytes, wherein the regulatory nucleotide sequence comprises an intron sequence containing a splice consensus site, wherein the intron sequence is derived from the human cardiac troponin T gene (hTNNT).

[0225] 2. The composition according to item 1, further comprising a TNNT2 promoter sequence.

[0226] 3. The composition of item 1, wherein the regulatory nucleotide sequence is located in a vector.

[0227] 4. The composition according to item 3, further comprising a transgene.

[0228] 5. The composition of item 4, wherein the transgene is a muscle A kinase anchoring protein β (mAKAPβ) sequence.

[0229] 6. The composition of item 5, wherein the mAKAPβ sequence is shRNA.

[0230] 7. The composition of item 6, wherein the shRNA comprises GGTTGAAGCTTTGAAGAAA (SEQ ID NO: 77), GCTAAGAGATACAGAGCTT (SEQ ID NO: 78), or GGAGGAAATAGCAAGGTTA (SEQ ID NO: 79).

[0231] 8. The composition of item 3, wherein the vector encodes an amino acid sequence having at least 80% sequence homology to a fragment of mAKAPβ.

[0232] 9. The composition of item 8, wherein the vector encodes an amino acid sequence having at least 90% sequence identity with a fragment of mAKAPβ.

[0233] 10. The composition of item 9, wherein the amino acid sequence encodes a fragment of mAKAPβ.

[0234] 11. The composition of claim 8, wherein the amino acid sequence binds to a kinase.

[0235] 12. The composition of item 11, wherein the kinase is p90 ribosomal S6 kinase 3 (RSK3).

[0236] 13. The composition of item 12, wherein the amino acid sequence inhibits the binding of mAKAPβ to RSK3.

[0237] 14. The composition of item 10, wherein the amino acid sequence has at least 80% sequence homology with amino acids 1694-1757, 1735-1833, or 1694-1833 of mAKAP.

[0238] 15. The composition of item 14, wherein the amino acid sequence has at least 90% sequence identity with amino acids 1735-1833 of mAKAP.

[0239] 16. The composition of claim 12, wherein the amino acid sequence comprises the RSK3 binding domain (RBD) of mABAPβ.

[0240] 17. The composition of claim 15, wherein the RBD comprises amino acids 1735-1833 of SEQ ID NO: 12.

[0241] 18. The composition of claim 11, wherein the amino acid sequence binds to protein phosphatase 2A (PP2A).

[0242] 19. The composition of claim 18, wherein the amino acid sequence inhibits PP2A anchoring to mAKAPβ.

[0243] 20. The composition of item 19, wherein the amino acid sequence has at least 80% sequence homology with amino acids 2132-2319 of mAKAP.

[0244] 22. The composition of item 20, wherein the amino acid sequence has at least 90% sequence identity with amino acids 2132-2319 of mAKAP.

[0245] 23. The composition of claim 20, wherein the amino acid sequence comprises the PP2A binding domain (PBD) of mAKAPβ.

[0246] 24. The composition of claim 23, wherein the PBD comprises amino acids 2132-2319 of SEQ ID NO: 12.

[0247] 25. The composition of any one of items 11, 14 or 15, wherein the kinase is Ca 2+ / Calmodulin-dependent protein kinase II (CaMKII).

[0248] 26. The composition of any one of items 3-25, wherein the vector is adeno-associated virus (AAV).

[0249] 27. The composition of any one of items 3-26, wherein the vector further comprises an SV40 polyadenylation sequence.

[0250] 28. The composition of claim 5, wherein amino acids 2132-2319 of the human mAKAP (SEQ ID NO: 11) have been modified at one or more of the following positions: amino acid TCG at position 2144 is modified to TCA; amino acid AGC at position 2183 is modified to AGT; amino acid TCC at position 2256 is modified to TCA; amino acid GCC at position 2291 is modified to GCA; or amino acid CGA at position 2313 is modified to AGA.

[0251] 29. The composition of claim 5, wherein the RBD encoded by amino acids 1696-1835 of the human mAKAP (SEQ ID NO: 11) has been modified at one or more of the following positions: amino acid CCG at position 1712 is modified to CCA; amino acid TCG at position 1714 is modified to TCT; amino acid TCG at position 1717 is modified to TCT; amino acid CGT at position 1721 is modified to AGA; amino acid CGT at position 1724 is modified to AGA; amino acid AGC at position 1730 is modified to AGT; amino acid AGC at position 1753 is modified to AGT; and amino acid GAC at position 1775 is modified to GAT.

[0252] 30. A method for treating or preventing heart disease, comprising administering the vector of any one of items 1 to 29 to cardiac cells of a patient.

[0253] 31. A method for treating or preventing heart disease, comprising administering the vector of item 6 or item 7 to cardiac cells of a patient, wherein the method inhibits expression of mAKAP.

[0254] Several recent clinical trials have reported varying degrees of immunotoxicity following administration of recombinant adeno-associated virus (rAAV), limiting the durability and success of gene therapy in humans (Wright 2020; Hamilton and Wright 2021). For example, unmethylated CpG dinucleotide motifs (CpGs) are considered to signal AAV infection (Akira, Uematsu, and Takeuchi 2006; Kanneganti, Lamkanfi, and Nunez 2007), promoting host innate immune responses by activating the Toll-like receptor TLR9-MyD88 signaling pathway, thereby recruiting 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). In addition, experience with using oligonucleotides as adjuvants in vaccine research has revealed to the scientific community CpG-containing immunostimulatory motifs (e.g., ACGT, TCGT, CCGT) and immunosuppressive motifs (e.g., GCGG, CCGC, GCGC) (Wright 2020; Ohto et al., 2015; Bode et al., 2011; Pohar et al., 2017).

[0255] Due to differences in TLR / intrinsic receptor sensitivity between humans and most other preclinical animal models (Tahtinen et al., 2022; Hawash et al., 2021), the immunostimulatory properties of AAV gene therapy in human patients may not be apparent during preclinical development and may not be anticipated in rodent or pig studies. However, to prevent potential immune responses and loss of transgene expression in patients, new versions of AAV biologics targeting mAbApβ have been designed. The newly proposed viral genome (vg) configuration features a new expression cassette designed to achieve enhanced cardiac expression in humans with reduced immunogenicity. The sequence has been optimized by removing CpG and CpG-containing immunostimulatory motifs while retaining CpG-containing immunosuppressive motifs.

[0256] Off-target effects can also be suppressed by using tissue-specific promoters that limit the expression of the gene of interest to the relevant cell type. The TNNT2 gene promoter has been used as a means of selectively expressing recombinant proteins and shRNA in cardiomyocytes (Prasad et al., 2011; Martinez et al., 2022; Li et al., 2020) (U.S. Patent No. US11,129,908B2). This construct comprises a new human TNNT2 promoter configuration, which includes additional promoter sequences, sequences of exons 1 and 2, and the first intron, thereby conferring higher cardiomyocyte-specific expression and being suitable for in vivo applications. This new TNNT2 promoter composition can be used not only for AAV gene therapy, but also for cardiomyocyte-specific transgenic and transient expression applications based on plasmids and viral vectors.

[0257] The following examples are provided to aid the understanding of the present invention, the true scope of which is defined by the appended claims. It should be understood that modifications can be made in the procedures described without departing from the spirit of the invention. Example:

[0258] The compositions and methods of the present invention will be better understood with reference to the following examples, which are intended to be illustrative only and not 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 these changes and modifications, including but not limited to those related to the processes, formulations and / or methods of the present invention, may be made without departing from the spirit of the invention and the scope of the appended claims.

[0259] Example 1

[0260] Novel AAV compositions

[0261] Two alternative layouts are provided for each of the three AAV9 self-complementary biologics presented here, representing alternative compositions for cardiomyocyte-specific gene therapy:

[0262] 1- Human cardiac troponin T 2 regulatory sequence, which includes promoter, exon and intron sequences, and contains skeletal muscle enhancer and splicing consensus site (hTNNT+enh+int)+ genetically modified +SV40 polyadenylation (SV40polyA). The complete plasmid sequence and corresponding vector map are shown in Figure 1 -6.

[0263] Composition #1: pAAVsc.hTNNT+enh.int. shmAKAP .SV40polyA,

[0264] Composition #2: pAAVsc.hTNNT+enh.int. hRBD .SV40polyA,

[0265] Composition #3: pAAVsc.hTNNT+enh.int. hPBD .SV40polyA.

[0266] 2-human calponin enhancer (calseq) + human cardiac troponin T 2 promoter (hTNNT) + SV40 16S synthetic intron (SV40int.) + genetically modified +SV40 polyadenylation. The complete plasmid sequence and corresponding vector map are shown in Figure 7-1 2.

[0267] Composition #4: pAAVsc.calseq.hTNNT.SV40int. shmAKAP .SV40polyA,

[0268] Composition #5: pAAVsc.calseq.hTNNT.SV40int. hRBD .SV40polyA,

[0269] Composition #6: pAAVsc.calseq.hTNNT.SV40int. hPBD .SV40polyA.

[0270] All elements were optimized for CpG content by selecting human genomic sequences that exhibit CpG dinucleotides known to stimulate immune responses and the least number of CpG tetranucleotides. In addition, codon optimization was performed where possible to remove inflammatory motifs from open reading frame (ORF) sequences. Sequence alignments are shown in Figures 8 and Figure 9 This article provides a detailed list of modifications summarized for each ORF:

[0271] Human mAKAP amino acids 2132-2319 (PBD): Human mAKAP amino acids 1696-1835 (RBD):

[0272] Amino acid S2144_TCG>TCA Amino acid P1712_CCG>CCA

[0273] Amino acid S2183_AGC>AGT Amino acid S1714_TCG>TCT

[0274] Amino acid S2256_TCC>TCA Amino acid S1717_TCG>TCT

[0275] Amino acid A2291_GCC>GCA Amino acid R1721_CGT>AGA

[0276] Amino acid R2313_CGA>AGA Amino acid R1724_CGT>AGA

[0277] Amino acid S1730_AGC>AGT

[0278] Amino acid S1753_AGC>AGT

[0279] Amino acid D1775_GAC>GAT

[0280] Compositions 1-3

[0281] In the first proposed configuration, cardiac-specific expression is achieved through a novel combination of regulatory elements. The novel TNNT2 transcriptional regulatory sequence includes: 1) the TNNT2 promoter, exon 1, and adjacent intron 1 sequences from -673 to +79 (relative to transcript_id: NM_000364.4; NCBI chromosome reference: NC_000001.11201,378,353..201,377,602, novel construct "novel regulatory sequence" bp 1-752); 2) an intronic enhancer (NC_000001.11 201,376,706..201,376,196, novel construct bp 753-1263); and 3) intron 1 3' sequence and partial exon 2 sequence (NC_000001.11). 201,373,323..201,373,261, novel construct 1264-1326 bp).

[0282] The NCBI chromosome reference for the TNNT2 gene sequence (5167 bp fragment) shown in Figure 31 is NC_000001.11201378367..201373201 (the gene is in the antisense orientation). Exon 1 of the TNNT2 gene is located at bp 688-745 (NC_000001.11201377680..201377623), and exon 2 is located at bp 5100-5154 (NC_000001.11201373268..201373214). The hTNNT2 gene intron region 1662-2172 bp (NC_000001.11 201,376,706..201,376,196, bp 753-1263 in the novel construct) in Figure 31 contains an enhancer, which has been reported to enhance expression in the differentiated mouse skeletal muscle cell line C2C12 in vitro (Kwon et al., 2011). Sequences at the extreme 5' and 3' ends of intron 1 are included to facilitate mRNA splicing and enhance transgene expression.

[0283] Compositions 4-6

[0284] In the second proposed configuration, a cardiomyocyte-specific transcriptional cis-regulatory motif from the first intron region of the human calpain gene (NCBI chromosome reference NC_000001.11-115,768,786..115,768,977, described by Chamberlain et al. 2018) and a human TNNT2 promoter fragment from -936 to +42 bp (NCBI chromosome reference NC_000001.11-201,378,613..201,377,636) were introduced to promote cardiac-specific expression of the therapeutic transgene.

[0285] U.S. Patent No. 11,129,908 lists the following human cardiac troponin T sequences and their gene coordinates: -569 to +31 (TNNT2p-600 in Table 1 of U.S. Patent No. 11,129,908); -469 to +31 (TNNT2p-500 in Table 1 of U.S. Patent No. 11,129,908); -369 to +31 (TNNT2p-400 in Table 1 of U.S. Patent No. 11,129,908); -269 to +31 (TNNT2p-300 in Table 1 of U.S. Patent No. 11,312,943 discloses a synthetic human troponin T promoter sequence having a unique 242-nucleotide sequence in addition to the human gene sequence having coordinates -499 to +6. Werfel et al. disclosed the use of the hTNNT2 promoter encompassing -499 to +45 (denoted as -502 to +42 by Werfel et al.) and smaller fragments thereof in AAV (Werfel et al., 2014).

[0286] Compositions 1-3 describe for the first time the use of the skeletal muscle enhancer in the human TNNT2 gene as part of a regulatory sequence to direct cardiac expression, either alone or in combination with the human TNNT2 promoter. The promoter fragments in these compositions are longer than other previously described promoter fragments for expression vectors.

[0287] Other elements in the rAAV expression cassette include the SV40 16S synthetic intron and polyadenylation elements, which are common components of plasmid and viral expression vectors and are incorporated into the expression cassette to promote high-level, efficient gene expression. Finally, the "right" AAV2 inverted terminal repeat (ITR) located at the 3' end of the transgene is presented in a modified form with its terminal dissociation site partially deleted, allowing the genome to form a hairpin structure, resulting in a self-complementary (sc) vector, which will maximize vector potency while allowing for lower systemic doses. The "left" AAV2 ITR located at the 5' end of the transgene remains unmodified as it is required in cis for both viral DNA replication and packaging of the rAAV vector genome.

[0288] Example 2

[0289] After generating AAV serotype 9 viruses using Compositions 1-6, approximately 2E12 vg / kg of each AAV were administered intravenously (iv) to 5-8 kg piglets to test expression directed by two new layouts (Layouts 1 and 2, referred to as "enh.int" and "calseq.", respectively). Three months after AAV infusion, transgene expression and AAV biodistribution were assessed necropsy in liver, brain, and heart tissues by quantitative polymerase chain reaction (qPCR). Unexpectedly, for the enh.int type composition (shown in Figure 1 -6), analyzed by reverse transcriptase-qPCR (qRT-PCR) and normalized to an 18S RNA control, showed that transgene expression in the left ventricle of the heart was approximately 800-fold higher than in skeletal muscle, and transgene expression in the left ventricle was approximately 3-fold and 20-fold higher than in the liver and brain, respectively. In pigs, intravenously delivered AAV9 preferentially delivered to the liver, with similar delivery to the heart, skeletal muscle, and cerebral cortex (Li et al., 2022). Figure 30 The data shown show that Figure 23 Compared to a similar "WPRE" AAV composition, the new enh.int composition significantly (p < 0.05) increased transgene expression selectively in the left ventricle of the heart (normalized to transgene expression of AAV9 genome delivery in different tissues) by approximately 60-fold relative to skeletal muscle, while maintaining selectivity for the liver and brain. In addition, after normalization for delivery, expression of the new composition in the left ventricle was approximately 4-fold higher than that of the original "WPRE" composition.

[0290] Example 3

[0291] Ca 2+ / Calmodulin-dependent protein kinase II (CaMKII)—a key regulatory factor in cardiomyocytes

[0292] In response to G protein-coupled receptor (GPCR) signaling, CaMKII plays an important role in the regulation of 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 the myocardium (Duran, Nickel et al., 2021). Members of the CaMKII family share a common structure and undergo post-translational modifications, enabling them to acquire Ca. 2+ 32). Although the intrinsic catalytic activity of each CaMKII isoform is similar, their functions have been observed to differ due to differences in localization (Zhang, Kohlhaas et al., 2007). For example, CaMKIIδB contains a nuclear localization signal and has a cardioprotective effect, while CaMKIIδC and δ9 are mainly distributed in the cytoplasm and are associated with adverse remodeling (Duran, Nickel et al., 2021). CaMKII is present in the plasma membrane, transverse tubules, sarcoplasmic reticulum (SR), mitochondria, nuclear membrane and nucleus. In addition to A-kinase anchoring protein 18δ (AKAP18δ), which can target CaMKII to the type 2 ryanodine receptor (RyR2) of the SR, SRCa 2+ Apart from SERCA2a and phospholamban (Pln) (Carlson, Aronsen et al., 2022), the localization mechanism of CaMKII in cardiomyocytes remains poorly understood.

[0293] Increased expression and activity of CaMKIIδ / γ are associated with cardiovascular disease and are considered drivers of arrhythmias and pathological remodeling (Beckendorf, van den Hoogenhof, et al., 2018; Duran, Nickel, et al., 2021; Reyes Gaido, Nkashama, et al., 2023). Although CaMKII (γ, δC, but notably δB) regulates multiple ion channels under physiological conditions (Zhang, Kohlhaas, et al., 2007; Kreusser, Lehmann, et al., 2014) and fine-tunes excitation-contraction coupling (Hegyi, Bers, et al., 2019), chronically elevated CaMKII activity can induce SR Ca 2+Excessive leak and arrhythmias (Maier and Bers 2007; Beckendorf, van den Hoogenhof et al., 2018). Transgenic expression of the major cardiac CaMKIIδ variants induces cardiac hypertrophy in vivo and, with the exception of δB, rapidly promotes heart failure (Duran, Nickel et al., 2021). Conversely, although CaMKIIδ deficiency fails to suppress the initial induction of hypertrophy by pressure overload, it inhibits the eccentric hypertrophy and heart failure associated with chronic pressure overload (Ling, Zhang et al., 2009). After myocardial infarction, genetic deletion of CaMKIIδ / γ prevents pathological remodeling (Weinreuter, Kreusser et al., 2014). Reactive oxygen species (ROS)-activated CaMKIIδC and δ9 exacerbate cardiomyocyte death through mitochondrial and NF-κB death and inflammatory pathways, as well as impaired DNA repair (Feng and Anderson 2017; Yao, Li et al., 2022). Driven by ROS and hyperglycemia, CaMKII is activated through oxidation and O-linked-N-acetylglucosamine modification (O-GlcNAcylation), which is associated with diabetic cardiomyopathy (Hegyi, Bers et al., 2019; Veitch, Power et al., 2021).

[0294] Extensive data indicate that CaMKII signaling contributes to heart failure and arrhythmias, spurring efforts to develop therapeutic strategies targeting CaMKII. It is generally believed 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 the crucial role of 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, preserved CaMKII activity is essential in the early stages of disease after pressure overload and in contractile adaptation to exercise through regulation of Pln and RyR2 (Burgos, Yeves, et al., 2017; Baier, Klatt, et al., 2020). Furthermore, nuclear CaMKIIδB has a cardioprotective effect by phosphorylating cAMP response element binding protein (CREB) at serine 133 (Wang, Xu et al., 2022).

[0295] mAKAPβ, CaMKII and HDAC4

[0296] In cardiomyocytes, the 230 kDa scaffold protein muscle A kinase anchoring protein β (mAKAPβ / AKAP6β) organizes the signalosome, which integrates Ca 2+ , cAMP, phosphatidylinositol 4-phosphate, mitogen-activated protein kinases, and upstream signaling from hypoxia regulate cardiomyocyte transcription factors and class IIa histone deacetylases (HDACs) (Dodge-Kafka, Gildart, et al., 2019). Through post-translational modifications of these gene regulators, the mAKAPβ signalosome influences both the extent of remodeling and the nature of cardiac hypertrophy, i.e., whether it manifests as centripetal or eccentric hypertrophy (Li, Tan, et al., 2020). mAKAPβ is required for pathological cardiac remodeling and heart failure under conditions such as chronic pressure overload, catecholamine infusion, and myocardial infarction (MI) (Kritzer, Li, et al., 2014; Martinez, Li, et al., 2023). mAKAPβ is not essential for cardiac development, and cardiac-specific mAKAPβ ablation has no significant adverse effects on the response to swimming training (Kritzer, Li, et al., 2014). It was recently discovered that the amino acid region 1694-1833 of rat mAKAPβ can bind not only to RSK3 but also to CaMKIIδ and CaMKIIγ.

[0297] Both pacing and pressure overload induce the accumulation of CaMKII, an active form of CaMKII that is autophosphorylated at threonine 287, at the nuclear membrane of cardiomyocytes (Ljubojevic-Holzer, Herren et al., 2020). CaMKII phosphorylates HDAC4, inducing 14-3-3 binding and nuclear export, thereby relieving the inhibition of myocyte enhancer factor-2 (MEF2)-dependent gene expression and driving pathological remodeling (Backs, Song et al., 2006; Zhang, Kohlhaas et al., 2007; Li, Cai et al., 2011). Although CaMKII phosphorylation sites are conserved among class IIa HDACs, CaMKII preferentially phosphorylates serine 467 / 632 on HDAC4 due to a unique CaMKII docking site on HDAC4 (amino acids 585-608) (Backs, Song et al., 2006; Backs, Backs et al., 2009). It was previously reported that under long-term pressure overload, HDAC4 phosphorylation is elevated in a mAKAPβ-dependent manner (Kritzer, Li et al., 2014), which is consistent with the binding of CaMKII to mAKAPβ.

[0298] To identify novel RBD binding partners, mAKAP 1694-1833 tagged with myc and green fluorescent protein (GFP) was expressed in adult rat ventricular myocytes by adenovirus infection, followed by cross-linking with dithiobisuccinimidyl propionate (DSP). The protein complex was immunoprecipitated with a myc tag antibody under stringent conditions and analyzed by mass spectrometry. In addition to other candidate interacting proteins, CaMKIIγ and δ were highly enriched in mAKAP immunoprecipitates (11 and 16 peptides were identified by mass spectrometry, respectively). The binding of CaMKII to mAKAPβ was verified by co-immunoprecipitation of endogenous proteins from neonatal rat ventricular myocytes infected with adenovirus expressing myc-GFP or myc-GFP-mAKAP 1694-1833 ( Figure 33 Immunoprecipitation of mAKAPβ with CaMKII antibodies was competed by expression of the 1694-1833 fusion peptide, thus validating the binding of the kinase to the scaffold protein and the competing peptide. When co-expressed in COS-7 cells, mAKAP 1694-1833 bound to multiple CaMKII isoforms ( Figure 34 ), indicating that mABAPβ binds to the CaMKII domain shared by all CaMKII family members.

[0299] Initial mapping studies were performed to determine whether RSK3 and CaMKII bind to distinct sites within mAKAP amino acids 1694-1833 (Figure 35). Although RSK3 and CaMKIIγ differ in their relative binding to mAKAP amino acids 1694-1757 and 1735-1833, both kinases clearly bind to a large overlapping region at amino acids 1694-1833.

[0300] During chronic pressure overload, HDAC4 phosphorylation at the CaMKII site is dependent on mAKAPβ expression (Kritzer, Li et al., 2014). The present data demonstrate that expression of mAKAP 1694-1833 in neonatal myocytes inhibits basal and angiotensin II-induced HDAC4 phosphorylation, consistent with CaMKII inhibition (Figure 36).

[0301] The patents and scientific literature cited herein represent knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference.

[0302] While the 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.

[0303] 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 USA, 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., 2007 Pharmacol 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 al., 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. Kanneganti T. 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. Negro 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: 649-58e4. 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. Sette and Conti. 1996 J 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-2023Update: AReport 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 USA, 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 expressing a second nucleotide sequence in cardiomyocytes, wherein the regulatory nucleotide sequence comprises an intron sequence containing a splice consensus site, wherein the intron sequence is derived from the human cardiac troponin T gene (hTNNT). The composition according to claim 1 , further comprising a TNNT2 promoter sequence.

3. The composition of claim 1, wherein the regulatory nucleotide sequence is located 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 anchoring protein beta (mAKAPβ) sequence. The composition according to claim 5 , wherein the mAKAPβ sequence is 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 homology 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). The composition according to claim 12 , wherein the amino acid sequence inhibits the binding of mABAPβ to RSK3.

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

15. The composition of claim 14, wherein the amino acid sequence has at least 90% sequence identity with amino acids 1735-1833 of mAKAP. The composition of claim 12 , wherein the amino acid sequence comprises the RSK3 binding domain (RBD) of mA BAPβ.

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

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

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

22. The composition of claim 20, wherein the amino acid sequence comprises the PP2A binding domain (PBD) of mA BAPβ.

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

12.

24. according to the composition described in any one of claim 11, 14 or 15, wherein said kinase is Ca 2+ / Calmodulin-dependent protein kinase II (CaMKII).

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

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

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

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

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

30. A method of 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.

Images