Perinuclear targeting by gene therapies for cardiopathology

Abstract

The present disclosure provides a method of treating or preventing a heart disease, by administering to a patient at risk of such heart disease, a pharmaceutically effective amount of a recombinant AAV-based or fusion protein-based composition that is localized to the outer nuclear membrane, binds to P-AR or suppresses perinuclear cAMP production, or a composition that expresses a protein that binds or a composition that binds to binds to P-AR, or suppresses perinuclear cAMP production. The composition may be in the form of a peptide that specifically inhibits P-AR near the nucleus, or in the form of a viral-based gene therapy vector encoding a fusion protein that inhibits perinuclear P-AR, or suppresses perinuclear cAMP production.

Description

Attorney Docket No. 65274.12WO01Customer No. 27683PERINUCLEAR TARGETING BY GENE THERAPIES FOR CARDIOPATHOLOGYCROSS-REFERENCE TO RELATED APPLICATION

[0000] This application claims priority and the benefit of the filing date of U.S. Provisional Application No. 63 / 699,794 filed September 26, 2024, the entire disclosure of which is hereby incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under R01HL166547 and R01HL153835 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on September 26, 2024, having the file name “24-0991-US-PRO_Sequence_Listing_ST26.xml,” and being 41809 bytes in size.BACKGROUND

[0003] The heart is capable of undergoing hypertrophic growth in response to a variety of stimuli. Hypertrophic growth may occur as the result of physical training such as running or swimming. However, it also occurs as the result of injury or in many forms of heart disease, including genetically acquired dilated and hypertrophic cardiomyopathies. When the growth is not accompanied by an increase in chamber size and primarily an increase in ventricular wall thickness, this is called concentric hypertrophy. Concentric hypertrophy is the primary mechanism by which the heart reduces stress on the ventricular wall in many acquired cardiovascular diseases but can be associated with cardiac diastolic dysfunction. When hypertrophy is associated with ventricular dilatation, this is called eccentric hypertrophy and in disease is often associated with systolic cardiac dysfunction. Hypertrophy occurs as the result of an increase in protein synthesis and in the size and organization of sarcomeres4906-5655-6138 v.l 1Attorney Docket No. 65274.12WO01Customer No. 27683 within individual myocytes. Due to its association with cardiac dysfunction, cardiac hypertrophy is a major risk factor for the development of heart failure. Traditional routes of treating heart failure include afterload reduction, blockage of beta-adrenergic receptors (|3- ARs) and use of mechanical support devices in afflicted patients. However, there is a need of additional mechanisms of preventing or treating cardiac hypertrophy.

[0004] Ventricular myocyte hypertrophy is the primary compensatory mechanism whereby the myocardium reduces ventricular wall tension when submitted to stress because of myocardial infarction, hypertension, and congenital heart disease or neurohumoral activation. It is associated with a nonmitotic growth of cardiomyocytes, increased myofibrillar organization, and upregulation of specific subsets of “fetal” genes that are normally expressed during embryonic life. The concomitant aberrant cardiac contractility, Ca2+handling, and myocardial energetics are associated with maladaptive changes that include interstitial fibrosis and cardiomyocyte death and increase the risk of developing heart failure and malignant arrhythmia. Increased in prevalence by risk factors such as smoking and obesity, heart failure is a syndrome that affects about six million Americans and has an annual incidence of 1% of senior citizens. Since the five-year survival rate after diagnosis of heart failure is still very poor (-50% depending upon the underlying disease), many efforts have been made during the last years to define the molecular mechanisms involved in this pathological process.

[0005] Cardiac hypertrophy can be induced by a variety of neuro-humoral, paracrine, and autocrine stimuli, which activate several receptor families including G protein-coupled receptors, cytokine receptors, and growth factor tyrosine kinase receptors. In this context, it is clear that A-kinase anchoring proteins (AKAPs) are scaffold proteins that can assemble multiprotein complexes that integrate hypertrophic pathways emanating from these receptors. In particular, recent studies have now identified anchoring proteins including mAKAP (AKAP6) and AKAP-Lbc and D-AKAP1 that play a central role in organizing and modulating hypertrophic pathways activated by stress signals.

[0006] In cardiomyocytes, mAKAPP is localized to the nuclear envelope through an interaction with nesprin- 1 a. mAKAP assembles a large signaling complex that integrates hypertrophic signals initiated by al-adrenergic receptors (al-ARs) and P-ARs, endothelin-1 receptors, and gpl30 / leukemia inhibitor factor receptors. The molecular mechanisms, as well as the signaling pathways whereby mAKAPP mediates cardiomyocyte hypertrophy, are being4906-5655-6138 v.l 2Attorney Docket No. 65274.12WO01Customer No. 27683 investigated and may serve as a source of therapeutic targets to combat cardiomyocyte hypertrophy and its associated problems, e.g., heart failure, which is important as current therapy for pathologic hypertrophy is generally limited to the broad downregulation of signaling pathways through the inhibition of upstream cell membrane receptors and ion channels. Novel drug targets may be revealed through the identification of signaling enzymes that regulate distinct pathways within the hypertrophic signaling network because of isoform specificity or association with unique multimolecular signaling complexes.SUMMARY

[0007] The following brief summary is not intended to include all features and aspects of the present disclosure, nor does it imply that any claim must include all features and aspects discussed in this summary.

[0008] In one aspect, the disclosure provides compositions that include a viral-based gene therapy vector encoding certain fusion proteins. In some embodiments of composition, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 98-1114 of SEQ ID NO:3. In some embodiments, the composition inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2-mediated Ca2+release in a perinuclear compartment organized by mAKAPp and nesprin-la.

[0009] In some embodiments, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 193-1209 of SEQ ID NO:5. In some embodiments, the composition inhibits adenylyl cyclase activity, binds to -AR, or suppresses perinuclear cAMP production in a perinuclear compartment organized by mAKAPp and nesprin-la.

[0010] In some embodiments, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80% identical to amino acids 3-15 of SEQ ID NO: 1 , and present in one or more copies; and (2) a nesprin-la amino4906-5655-6138 v.l 3Attorney Docket No. 65274.12WO01Customer No. 27683 acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 125- 1141 of SEQ ID NO: 1. In some embodiments, the composition inhibits CaMKII activity or binds to CaMKII in a perinuclear compartment organized by mAKAPp or nesprin- 1 a.

[0011] In some embodiments, the viral vector is adeno-associated virus (AAV), while in some embodiments, the AAV includes AAV2 and / or AAV9.

[0012] In another aspect, the disclosure provides compositions that include certain fusion protein. In some embodiments, the composition includes fusion protein that binds to mAKAPp and inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2- mediated Ca2+release. In some embodiments, the fusion protein includes (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80% identical to amino acids 3- 27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin-l sequence is at least 80% identical to amino acids 98-1114 of SEQ ID NO:3.

[0013] In some embodiments, the composition includes fusion protein that binds to mAKAPp and inhibits adenylyl cyclase activity, binds to P-AR , or suppresses perinuclear cAMP production. In some embodiments, the fusion protein includes (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 193-1209 of SEQ ID NO:5.

[0014] In some embodiments, the composition includes fusion protein that binds to mAKAPp and inhibits calmodulin-dependent protein kinase II (CaMKII) activity or binds to CaMKII. In some embodiments, the fusion protein includes (1) an Autocam tide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80% identical to amino acids 3-54 of SEQ ID NO:1, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 125-1141 of SEQ ID NO:1.

[0015] In some embodiments of this aspect, the fusion protein is expressed using a viral vector, which, in some embodiments, includes adeno-associated virus (AAV), e.g., AAV2 and / or AAV9.

[0016] In some embodiments of the various aspects of the disclosure, the fusion protein is formulated as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically4906-5655-6138 v.l 4Attorney Docket No. 65274.12WO01Customer No. 27683 acceptable salts include: (1) an acid selected from the group consisting of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and mandelic acid; (2) an inorganic base selected from the group consisting of sodium, potassium, ammonium, calcium, and ferric hydroxide; or (3) an organic base selected from the group consisting of isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine. In some embodiments, the fragment is modified with a cell membrane-penetrating sequence, e.g., transactivator of transcription (TAT) polypeptide, polyarginine peptide, or a penetratin peptide. In some embodiments, the fragment is modified with a lipid-derived group, e.g., a stearate.

[0017] In another aspect, the disclosure provides methods of treating or preventing a heart disease by administering one or more compositions related to activity in and around mAKAP|l In some embodiments, the method includes administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that binds to mAKAPp and inhibits RyR2 activity, binds to RyR2, or reduces RyR2-mediated Ca2+release. In some embodiments, the method of treating or preventing heart disease includes administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that binds to mAKAPp and inhibits adenylyl cyclase activity, binds to P-AR, or suppresses perinuclear cAMP production. In some embodiments, the method of treating or preventing heart disease includes administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that inhibits CaMKII activity, or binds to CaMKII.

[0018] In another aspect, the disclosure provides methods for treating or preventing pathological cardiac remodeling by administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that binds to mAKAPp and inhibits RyR2 activity, binds to RyR2, or reduces RyR2-mediated Ca2+release. In some embodiments, the method of treating or preventing pathological cardiac remodeling includes administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that binds to mAKAPp and inhibits adenylyl cyclase activity, binds to P-AR, or suppresses perinuclear cAMP production. In some embodiments, the method of treating or preventing pathological cardiac remodeling includes administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that inhibits CaMKII activity or binds to CaMKII.

[0019] In another aspect, the disclosure provides methods of treating or preventing a disease or condition associated with an increase in perinuclear Ca2+release, including4906-5655-6138 v.l 5Attorney Docket No. 65274.12WO01Customer No. 27683 delivering to a patient in need thereof, an amount of one or more compositions as described elsewhere herein sufficient to inhibit the perinuclear Ca2+release in the patient. In some embodiments, the disease or condition associated with an increase in perinuclear Ca2+release includes cardiac hypertrophy, e.g., ventricular myocyte hypertrophy or ventricular hypertrophy, or heart failure. In some embodiments, the disease or condition is caused by, e.g., hypertension, coronary artery disease, myocardial infarction, valvular disease, primary cardiomyopathy, congenital heart disease, arrhythmia, pulmonary disease, diabetes, anemia, or hyperthyroidism.

[0020] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a viral-based gene therapy vector encoding a fusion protein that includes (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 98-1114 of SEQ ID NO:3.

[0021] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a viral-based gene therapy vector encoding a fusion protein that includes (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 193-1209 of SEQ ID NO:5.

[0022] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a viral-based gene therapy vector encoding a fusion protein that includes (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-54 of SEQ ID NO:1, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 125-1141 of SEQ ID NO: 1.4906-5655-6138 v.l 6Attorney Docket No. 65274.12WO01Customer No. 27683

[0023] In some embodiments, the viral vector is adeno-associated vims (AAV), while in some embodiments, the AAV includes AAV2 and / or AAV9.

[0024] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a fusion protein that comprises (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin- la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 98-1114 of SEQ ID NO:3.

[0025] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a fusion protein that comprises (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 193-1209 of SEQ ID NO:5.

[0026] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a fusion protein that comprises (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-54 of SEQ ID NO:1, and (2) a nesprin- la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 125-1141 of SEQ ID NO:1.

[0027] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0028] These and other aspects of the present invention are described in more detail below.4906-5655-6138 v.l 7Attorney Docket No. 65274.12WO01Customer No. 27683BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0030] FIG. 1 shows a model of mAKAP signalosome with intracellular PAR, shown on Golgi apparatus. Norepinephrine shuttled in by Oct3, activates intracellular PAR which stimulates production of a perinuclear pool of cAMP which activates mAKAP-bound PKA and inevitably perinuclear calcineurin (CaN). Activated perinuclear calcineurin dephosphorylates the transcription factor NF AT, allowing for nuclear accumulation of NFAT. Taken together these receptors are required for hypertrophic gene upregulation by CaN stimulated NFAT.

[0031] FIG. 2 shows peptides utilized herein. FRET -based biosensors targeted to the mAKAP complex via nesprin-la were utilized to measure localized PKA activity, and calcineurin activity. The untargeted parent sensors measure activity in the cytosol. Nesprin- 1 a targeted, mCherry-tagged vectors were developed to manipulate perinuclear PKA. mCherry-TCL-nesprin contains TCL3-9, a pepducin targeting the central region of the third intracellular loop of P2-ARS, which promotes cAMP synthesis. mCherry-NB80-nesprin contains a conformation- specific single-domain camelid antibody (Nb80), which can be used to block Gsmediated signaling.

[0032] FIG. 3A to FIG. 3D show results using FRET biosensors. FIG. 3A shows neonatal rat ventricular myocytes (RNV) infected with adenovirus expressing cytosolic AKAR4 (left), or perinuclear AKAR4-nesprin and imaged using confocal microscopy. Bar - 10pm. FIG. 3B shows representative tracings of PKA activity in RNV treated with either AKAR4 or AKAR4-nesprin in response to lOnM norepinephrine (NE). FIG. 3C shows RNV infected with adenovirus expressing cytosolic CaNAR2 (left), or perinuclear CaNAR2- nesprin and imaged using confocal microscopy. FIG. 3D shows representative tracings of Calcineurin activity in RNV treated with either CaNAR2 or CaNAR2-nesprin in response to lOnM norepinephrine (NE). FIG. 3B and FIG. 3D show FRET ratio (R normalized to baseline Ro).4906-5655-6138 v.l 8Attorney Docket No. 65274.12WO01Customer No. 27683

[0033] FIG. 4A to FIG. 4H show perinuclear P-AR activation. FIG. 4A to FIG. 4DA show fluorescent images of RNV expressing mCherry-nesprin (left) stained for atrial natriuretic factor (ANF, right middle). DAPI (left middle) and Composite (right) image included. FIG. 4E shows average baseline PKA activity of each trace from RNV expressing either mCherry-nesprin or mCherry-ICL-nesprin and AKAR4 or AKAR4-nesprin as indicated. FIG. 4F shows average baseline CaN activity of each trace from RNV expressing either mCherry-nesprin or mCherry-ICL-nesprin and CaNAR2 or CaNAR2-nesprin as indicated. For FIG. 4E and FIG. 4F, Ro=net FRET - donor. FIG. 4G shows RNV expressing the CaN target NFATcl-GFP and either mCherry-nesprin or mCherry-ICL- nesprin that were given either no drug (Ctrl) or lOnM NE (NE) for 48 hours then measured for NFATcl-GFP nuclear translocation. Data is shown as fluorescence intensity in the nucleus versus the cytosol. FIG. 4H shows RNV expressing either mCherry-nesprin or mCherry-ICL-nesprin that were given either no drug (Ctrl) or lOnM NE (NE) for 48 hours then measured for expression of ANF. For FIG. 4GA and FIG. 4H, at least 50 cells from 3 biological replicates and 3 technical replicates were analyzed for each condition.

[0034] FIG. 5A to FIG. 5E show perinuclear -AR inhibition. FIG. 5A shows average peak PKA activity of each trace from RNV infected with either mCherry-nesprin or mCherry-NB80-nesprin and AKAR4 or AKAR4-nesprin as indicated. FIG. 5B shows average baseline CaN activity of each trace from RNV expressing either mCherry-nesprin or mCherry-NB80-nesprin and CaNAR2 or CaNAR2-nesprin as indicated. For FIG. 5A, FIG. 5B, and FIG. 5E, the FRET ratio is calculated as total normalized to baseline Ro, Ro=net FRET - donor. FIG. 5C shows RNV expressing NFATcl-GFP and either mCherry-nesprin or mCherry-NB80-nesprin that were given either no drug (Ctrl) or lOnM NE (NE) for 48 hours then measured for NFATcl-GFP nuclear translocation. Data is shown as fluorescence intensity in the nucleus versus the cytosol. FIG. 5D shows RNV expressing either mCherry- nesprin or mCherry-NB80-nesprin that were given either no drug (Ctrl) or lOnM NE (NE) for 48 hours then measured for expression of ANF. For FIG. 5C and FIG. 5D, at least 50 cells from 3 biological replicates and 3 technical replicates were analyzed for each condition. FIG. 5E shows Average peak PKA activity of each trace from RNV expressing mCherry-NB80 localized to subcellular compartments as indicated.

[0035] FIG. 6A to FIG. 6F show hypertrophy assays in adult rat cardiomyocytes (ARVM). FIG. 6A to FIG. 6D show fluorescent images of ARVM expressing mCherry-4906-5655-6138 v.l 9Attorney Docket No. 65274.12WO01Customer No. 27683 nesprin (left) stained for a-actinin (right middle). DAPI (left middle), with a composite (right) image included. FIG. 6E and FIG. 6F shows ARVM infected with either mCherry- nesprin or mCherry-NB80-nesprin or mCherry-ICL-nesprin as indicated that were treated for 48 hours with either no drug (Ctrl) or lOnM NE (NE) and then measured by length and width. At least 50 cells from at least 3 separate biological replicates with at least 2 technical replicates were analyzed for each condition.

[0036] FIG. 7 shows mAKAP domain structure. Panel A shows mAKAPp is the alternatively spliced isoform of mAKAP (AKAP6) expressed in striated myocytes. Numbering by rat mAKAPa. Well-defined partner binding sites are shown. AC5, adenylyl cyclase 5; - PACT domains of pericentrin or AKAP9, SR - spectrin repeats. Panel B shows mAKAP (AKAP6) mRNA was increased in the LV myocytes of a human PLN R14del DCM patient, as assayed by snRNA-seq. NPPA and NPPB were also up-regulated in this patient.

[0037] FIG. 8A to FIG. 8D show an mAKAP -dependent perinuclear Ca2+ compartment. FIG. 8A illustrates cameleon-nesprin, a ratiometric FRET Ca2+biosensor localized to the nuclear envelope. SR - spectrin repeats. FIG. 8B shows neonatal rat ventricular myocytes infected with adenovirus expressing Cameleon (left) or Cameleon- nesprin (right). FIG. 8C and FIG. 8D, show myocytes that were co-infected with adenovirus for Cameleon-nesprin (FIG. 8C), Cameleon (FIG. 8D), and either mAKAP or control shRNA. FRET imaging following 1 pM Iso stimulation. Representative tracings and peak amplitudes for FRET ratio (R normalized to baseline Ro) are shown. Note that imaging with Cameleon and most other genetically encoded sensors is too slow to detect action potentials associated with individual myocyte contractions but rather is an assay for average Ca2+concentration. Each data point in peak amplitude plots represents separate tracings (n 2= 6) obtained using cells from 3 independent myocyte preparations.

[0038] FIG. 9 shows RyR2 is responsible for the release of Ca2+to the mAKAP0- nesprin-la compartment. Panel A shows co-immunoprecipitation of nesprin- la, mAKAPp, and RyR2 from myocytes, n = 3. Panel B shows neonatal myocytes expressing Cameleon- nesprin and treated with 1 pm Iso + 5 pM ryanodine (Ry). Ctrl - control.

[0039] FIG. 10 shows that phosphorylation of perinuclear RYR2 by PKA is regulated by mAKAP . Neonatal myocytes infected with adenovirus expressing mAKAP or control shRNA before stimulation with 1 pM Iso as indicated, hr - hours. Immunoprecipitated protein4906-5655-6138 v.l 10Attorney Docket No. 65274.12WO01Customer No. 27683 complexes and whole cell extracts were assayed for RyR2 Ser-2808 (Panel A) and Ser- 2030 (Panel B) phosphorylation. n=3.

[0040] FIG. 11 shows perinuclear RyR2 regulates perinuclear, but not cytosolic Ca2+. Panel A illustrates tools to modulate perinuclear RyR2. GGGGSB is a flexible linker; myc - epitope tag; mCherry tag to allow detection of fusion protein expression during imaging. Neonatal myocytes were co-infected with adenovirus for perinuclear GCaMP6s-nesprin (Panel B, Panel C) or cytosolic parental GCaMP6s (Panel D, Panel E) intensiometric Ca2+sensor and either control, Bcl2BH4-, or B laCT-mCherry-nesprin. Average fluorescent signals (F) are provided normalized to control samples for mCherry-nesprin (Fo). Myocytes were treated with the adrenergic agonist norepinephrine (NE, 10 nM) or ryanodine (5 pM - inhibitory dose) as indicated. Datapoints represent multiple traces from individual preparations.

[0041] FIG. 12 shows inhibition of CaN-NFAT signaling and hypertrophy by perinuclear Bcl2BH4. Neonatal myocytes expressing nesprin-tagged NFATc3 and either mCherry- nesprin (Control) or Bcl2BH4-mCherry-nesprin (Bcl2BH4) were stimulated with 10 pM NE or 5 nM ryanodine (RyR2 activating dose) before assay for NF AT localization (Panel A) and ANF expression (Panel B). n = 3.

[0042] FIG. 13 shows activation of perinuclear RyR2 induces a DCM-like phenotype in mice. Panel A shows 7-8 week old male C57BL / 6N mice that were injected IV with 5 x10” vg AAV9 expressing under the control of the cardiac myocyte- specific cTnT promoter BlaCT- or control myc-tagged nesprin-l or no AAV at all. ITR - AAV inverted terminal repeats. Panel B shows serial 4-D echocardiography. **** p<0.0001 for BlaCT-nesprin vs. No AAV and Control Nesprin. Panel C shows indexed heart weight 8 weeks after AAV administration. **** p<0.0001. n = 6-9 mice per cohort.

[0043] FIG. 14A and FIG. 14B show the treatment of TM54 DCM mice by gene therapies that inhibits perinuclear RyR2 Ca2+ release (Bcl2CH4 AAV) or PAR (Nb80 AAV). FIG. 14A, Panel A shows 6-8 week-old TM54 or wildtype littermate mice of both sexes were injected IV with 5 xlO11vg AAV9 expressing Bcl2BH4-, Nb80 or control nesprin-la under the control of the cardiac myocyte-specific cTnT promoter. FIG. 14A, Panel B and Panel C show results of Vevo 3100 serial 4D echocardiography. All TM54 cohorts were significantly different from all NTG cohorts for both parameters (p<0.0001). * or fp<0.05;4906-5655-6138 v.l 11Attorney Docket No. 65274.12WO01Customer No. 27683** or ffpO.Ol; *** or ff p<0.001; **** or fftf p<0.0001. * compared to TM54 Nesprin cohort, t compared to non- injected mouse TM54 cohort, red - Nb80 AAV; blue - Bcl2BH4 AAV. FIG. 14B, Panel D shows indexed heart weight at 10-week endpoint.

[0044] FIG. 15 illustrates peptide tools utilized to assess what is the source of Ca2+in the mAKAP|B signalosome. The untargeted parent sensors measure activity in the cytosol.Nesprin- 1 a targeted, mCherry-tagged vectors were developed to manipulate perinuclear Ryanodine receptor activity. The BH4 domain “Bcl2BH4” of the anti-apoptotic protein Bcl-2 that binds all 3 RyR subtypes, inhibiting Ca2+release; 2) “DAc” comprising residues 671-690 of the dihydropyridine receptor (DHPR) al S subunit, shown to inhibit Ca2+-induced Ca2+release from cardiac SR vesicles without affecting resting leak; and 3) “BlaCT” comprising the C-terminal 35 residues of the DHPR pia subunit that will bind and increase RyR pore opening. Expression of Bcl2BH4-, DAc-, and BlaCT mCherry-nesprin fusion proteins will specifically modulate RyR2 in the mAKAPp compartment.

[0045] FIG. 16A and FIG. 16B show the effects of RyR inhibitors and activators on Ca2+levels in the cytosol (FIG. 16A, Panel B; FIG. 16B, Panel D) versus perinuclear signalosome region (FIG. 16A, Panel A; FIG. 16B, Panel C). Average fluorescent signals (F) are provided normalized to control samples for mCherry-nesprin (Fo). Myocytes were treated with the adrenergic agonist norepinephrine (NE, 10 nM) with or without RyR inhibitor (5 pM; Bcl2BH4-mCherry-nesprin, “Inhibitor 1”; or DAc-mCherry-nesprin, “Inhibitor 2”) or RyR activator (5 pM; blaCT-mCherry- nesprin, “Inhibitor”) as indicated. Datapoints represent multiple traces from individual preparations.

[0046] FIG. 17 shows mAKAPP-associated RyR induces NFAT nuclear localization. Rat neonatal myocytes expressing GFP-tagged NFATc3 and mCherry-nesprin and either Bcl2BH4-mCherry-nesprin (Panel A), or DAc-mCherry-nesprin (Panel B), or BlaCT- mcherry-nesprin (Panel C), were stimulated for 48 hours with NE, before assay of NFAT localization. Ratio of nuclear to cytoplasmic GFP fluorescent is shown.

[0047] FIG. 18 shows mAKAPP-associated RyR induces ANF expression. Rat neonatal myocytes expressing mCherry-nesprin and either Bcl2BH4-mCherry-nesprin (Panel A), DAc- mCherry -nesprin (Panel B), or B laCCT-mCherry-nesprin (Panel C), were stimulated for 48 hours with NE before staining for the hypertrophy marker ANF. Ratio of ANF positive cells is shown.4906-5655-6138 v.l 12Attorney Docket No. 65274.12WO01Customer No. 27683

[0048] FIG. 19A and FIG. 19B show mAKAPP-associated perinuclear Ca2+is PKA dependent. FRET imaging of neonatal rat ventricular myocytes expressing Cameleon-nesprin (FIG. 19A) or CaNAR2-nesprin (FIG. 19B) were stimulated with isoproterenol (1 pmol / L) and inhibited with H89 PKA inhibitor (1 pmol / L) as indicated.

[0049] FIG. 20A to FIG. 20D show experiments measuring mAKAP -associated PKA activity. (FIG. 20A) AKAR4 consists of a forkhead-associated domain (FHA1) and PKA substrate (LRRATLVD) flanked by Cerulean and cp Venus; PKA substrate phosphorylation induces FHA1 binding, increasing FRET signal. mCherry-PDE-nesprin contains a mutated PDE4D catalytic domain (T349A, K455 / 456A, S579A, FQF597-599AAA). (FIG. 20B) Neonatal rat ventricular myocytes were infected with adenovirus for AKAR4 or AKAR4- nesprin and imaged by confocal microscopy. Bar - 10 pm. (FIG. 20C) FRET imaging of neonatal rat ventricular myocytes expressing CaNAR2-nesprin were stimulated with isoproterenol (1 pmol / L) and inhibited with H89 PKA inhibitor (1 pmol / L) as indicated. (FIG. 20D) FRET imaging of neonatal myocytes expressing CaNAR2-nesprin and either mCherry-PDE-nesprin or mCherry-nesprin control and stimulated with norepinephrine (NE, 10 nmol / L).FIG. 21A to FIG. 21C show mAKAPP-associated PKA activity is dependent on internal PAR. FRET imaging of rat neonatal myocytes expressing either AKAR4-nesprin (FIG. 21A) or AKAR4 (FIG. 21B). Rat neonatal myocytes were treated with either cell-impermeable P- blocker sotalol cell-permeable P-blocker propranolol, or organic cation transporter 3 (Oct3) blocker corticosterone (FIG. 21C) as indicated.

[0050] FIG. 22 shows perinuclear-associated CaN activity is dependent on internal PAR. FRET imaging of norepinephrine (NE) -treated neonatal cardiomyocytes infected with either CaNAR-nesprin and treated with either cell-impermeable P-blocker sotalol or the cell- permeable P-blocker propranolol as indicated, measuring cytosolic (Panel A) and perinuclear (Panel B) CaN activity.

[0051] FIG. 23A and FIG. 23B show that internal PAR are required for myocyte hypertrophy. Rat neonatal myocytes expressing GFP-tagged NFATc3 were treated with either no drug (Control), NE, cell-impermeable P-blocker sotalol or cell-permeable P-blocker propranolol for 48 hours before assay of NFAT localization. Ratio of nuclear to cytoplasmic GFP fluorescent is shown (FIG. 23A, Panel A). Rat neonatal myocytes were treated with4906-5655-6138 v.l 13Attorney Docket No. 65274.12WO01Customer No. 27683 either no drug (Control), NE, cell-impermeable P-blocker sotalol or cell-permeable P-blocker propranolol for 48 hours before staining with atrial natriuretic factor antibody (ANF). Fraction of cells with prominent ANF staining was determined (FIG. 23A, Panel B). Adult rat cardiomyocytes (ARVM) were treated with either no drug (Control), NE, cell- impermeable P-blocker sotalol, or cell-permeable P-blocker propranolol for 48 hours before staining with a-actinin and fluorescent imaging. Myocyte length (FIG. 23B, Panel C) and width (FIG. 23B, Panel D) were measured.

[0052] FIG. 24A and FIG. 23B show that internal PAR are required for myocyte hypertrophy. Rat neonatal myocytes expressing GFP-tagged NFATc3 were treated with either no drug (Control), NE, or organic cation transporter 3 (Oct3) blocker corticosterone for 48 hours before assay of NF AT localization. Ratio of nuclear to cytoplasmic GFP fluorescent is shown (FIG. 24A, Panel A). Rat neonatal myocytes were treated with either no drug (Control), NE, or organic cation transporter 3 (Oct3) blocker corticosterone for 48 hours before staining with atrial natriuretic factor antibody (ANF). Fraction of cells with prominent ANF staining was determined (FIG. 24A, Panel B). Adult rat cardiomyocytes (ARVM) were treated with either no drug (Control), NE, or organic cation transporter 3 (Oct3) blocker corticosterone for 48 hours before staining with a-actinin and fluorescent imaging. Myocyte length (FIG. 24B, Panel C) and width (FIG. 24B, Panel D) were measured.

[0053] FIG. 25A to FIG. 25D show that pAR-induced apoptosis is dependent on expression of mAKAPp. FIG. 25A shows myocyte specific cell line H9C2 cells that were infected with adenovirus expressing mAKAP (shmAKAP; third row) or control shRNA (shControl; top row) and cultured in minimal medium for two days with and without 10 pmol / L with the beta-adrenergic agonist Denopamine (second and fourth rows) that is specific for the type 1 receptor before TUNEL staining. TUNEL - green (center column); Dapi nuclear stain - blue (left column); bar - 100 pm. Arrowheads indicate nuclei with detectable TUNEL labeling (second row, middle and right columns). FIG. 25B shows quantification of TUNEL-positive cells. FIG. 25C shows adult rat myocytes that were infected with adenovirus expressing mAKAP (shmAKAP) or control shRNA (shControl) and cultured in minimal medium for two days with and without 10 pmol / L with the beta- adrenergic agonist Denopamine that is specific for the type 1 receptor before active Caspase staining (row layout the same as FIG. 25A). Active caspase - red (center column); Hoescht4906-5655-6138 v.l 14Attorney Docket No. 65274.12WO01Customer No. 27683 nuclear stain - blue (left column); bar - 100 |im. Arrowheads indicate nuclei with detectable TUNEL labeling. FIG. 25D shows quantification of Caspase-positive cells.

[0054] FIG. 26 shows that SRF phosphorylation at serine 103 is PKA sensitive. Panel A shows 1 pg of purified SRF protein was incubated in kinase buffer (50 mM Tris-HCL pH 7.5, 5mM MgCh) containing PKA catalytic subunit (833 units / pl) plus and minus 100 nM PKI for 30 minutes before running on SDS-Page and subjection to Western Blot. Antibodies were used at 1 : 10,000. n=5. Panel B shows the proportion of phosphorylated SRF positive H9C2 cells treated with denopamine in the presence or absence of H89, n=5. * p <0.05; ** p<0.01. Panel C shows the proportion of phosphorylated SRF positive H9C2 cells infected with adenovirus for control shRNA or mAKAPp shRNA and treated with denopamine, n=2.Panel D shows representative images of phosphorylated SRF stained H9C2 cells treated with denopamine in the presence or absence of H89. Bar -100 pm.

[0055] FIG. 27A and FIG. 27B show that SRF phosphorylation at serine 102 is required for AR-induced apoptosis. ARVMs were infected with adenovirus expressing a phosphomimetic S103D mutant, a phosphoablative S103A mutant, or wild type (WT) SRF. The expression of these adenoviruses requires co-infection with a TET-ON adenovirus (TET). After infection and treatment with denopamine, the cells were subjected to TUNEL staining. FIG. 27A shows the proportion of TUNEL positive ARVMs averaged over all images. FIG. 27B shows TUNEL positive nuclei in left ventricular tissue slices from mice infected with adenovirus expressing the S103D mutant, SI 03 A mutant, WT SRF, or GFP, n=5. ** p <0.01, *** p <0.001.

[0056] FIG. 28 shows a model of the mAKAPp Signalosome in a myoblast cell line: (1) A release of perinuclear Ca2+from mAKAPp bound RyRl results in a perinuclear Ca2+pool that sustains the activation of mAKAPp bound CaN. (2) Dephosphorylation of transcription factor NFAT by CaN and (3) subsequent nuclear translocation of the transcription factor drives (4) myogenic differentiation.

[0057] FIG. 29 shows peptides utilized herein. Nesprin- 1 a contains 5 spectrin repeats and a transmembrane KASH domain that localizes the protein to the nuclear envelope. mCherry-Parvalbumin-nesprin contains a calcium binding protein that removes the perinuclear pool of calcium around mAKAPP; mCherry-Bcl2BH4-nesprin is derived from the anti-apoptotic protein Bcl-2 and inhibits the mAKAPp associated RyRs; mCherry-D Ac-4906-5655-6138 v.l 15Attorney Docket No. 65274.12WO01Customer No. 27683 nesprin is derived from the DHPR alS subunit and is inhibits RyR; mCherry-BlaCT-nesprin contains the terminal peptide from the DHPR pia subunit to activate mAKAPP-associated Ry Rs; CaNAR2 is a Forster Resonance Energy Transfer (FRET) sensor that allows for detection of CaN activity in live cells at nesprin and globally throughout the cell.

[0058] FIG. 30A to FIG. 30F show calcineurin activity in an immortalized myoblast cell line: C2C12 cells expressing Bcl2BH4 (RyR inhibitor 1) + / - 5pM Ryanodine (FIG. 30A, FIG. 30B), DAc (RyR inhibitor 2) + / - 5pM Ryanodine (FIG. 30C, FIG. 30D), and BlaCT (RyR activator) + / - IpM / L Cyclosporin A (FIG. 30E, FIG. 30F) and parent or mAKAPP localized CaNAR2 sensor + / - differentiation overnight. Each data point is the average normalized baseline CaNAR2 level of 6 cells imaged on the same day. Data were compared using a two-way ANOVA with Tukey Post-Hoc testing. * p $ 0.05; ** p =£ 0.01; *** p =£ 0.001.

[0059] FIG. 31 shows mAK PP RyRl colocalization in immortalized myoblast cells: Images of C2C12 cells stained for mAKAPP (Panel A), RyRl (Panel B), and a composite image (Panel C). Cells were stimulated to differentiate overnight.

[0060] FIG. 32A to FIG. 32E show NFAT nuclear localization assays in immortalized myoblast cells: FIG. 32A shows images of C2C12 cells expressing both the nesprin targeted peptide and NFATcl GFP and stained with DAPI. C2C12 cells expressing Parvalbumin + / - 5 pM Ryanodine (FIG. 32B), Bcl2BH4 (RyR inhibitor 1) + / - 5pM Ryanodine (FIG. 32C), DAc (RyR inhibitor 2). + / - 5pM Ryanodine (FIG. 32D), and BlaCT (RyR activator) + / - 5nM Ryanodine (FIG. 32E) with NFATc3 GFP + / - differentiation overnight (n=3). Data were compared using a two-way ANOVA with Tukey Post-Hoc testing. * p =4 0.05; ** p =5 0.01.

[0061] FIG. 33A to FIG. 33C show NFAT diffusion in live immortalized myoblast cells: FIG. 33A shows a Representative Fluorescence Redistribution after Photobleaching (FRAP) fluorescence recovery graph of C2C12 cells expressing NFATcl GFP + / - differentiation stimulation overnight. Diffusion Coefficient (pm2 / s) (FIG. 33B) and mobile fraction (FIG. 33C) of C2C12 cells expressing NFATcl GFP + / - differentiation stimulation overnight. Each data point is the average value of 6 cells imaged on the same day. Analysis performed using Virtual Cell software. Data were compared using an unpaired, two-tailed t-test. * p $ 0.05; ** p sj 0.01 ;0.001.4906-5655-6138 v.l 16Attorney Docket No. 65274.12WO01Customer No. 27683

[0062] FIG. 34A to FIG. 34E show myogenic differentiation in an immortalized myoblast cell line: (FIG. 34A) Images of C2C12 cells expressing the nesprin targeted peptide and stained with DAPI and Myosin Heavy Chain (MHC) antibody. C2C12 cells expressing Parvalbumin + / - 5pM Ryanodine (FIG. 34B), Bcl2BH4 (RyR inhibitor 1) + / - 5pMRyanodine (FIG. 34C), DAc (RyR inhibitor 2) + / - 5pM Ryanodine (FIG. 34D), and B laCT (RyR activator) + / - 5nM Ryanodine (FIG. 34E) and stained for MHC + / - differentiation overnight. Data were compared using a two-way ANOVA with Tukey Post-Hoc testing. * p 0.05; 0.01.

[0063] FIG. 35 shows regulation of ANF expression by perinuclear CaMKII. AIP4 - 4 tandem copies of the CaMKII autocamptide inhibitor peptide and AKAP185c - AKAP185 aa 238-266 CaMKII activating peptide are shown (Panel A). Cells transfected with plasmids for mCherry-nesprin and AlP4-mCherry-nesprin (Panel B, and results in Panel C, left graph) or AKAP188c-mCherry-nesprin (Panel C, right graph) were treated for 24 hours with 10 nM NE before staining for ANF. n = 3 separate myocyte preparations for AIP4, 2 for AKAP185c.

[0064] FIG. 36 shows perinuclear CaMKII activity is required for Pi AR- stimulated myocyte apoptosis. Neonatal myocytes expressing either AIP4-nesprin (Panel A) or mCherry-Parv-nesprin (Panel B) were stimulated with 10 pM denopamine for 48 hours. Cell Death was measured by TUNEL staining, n = 4 separate myocyte preparations.

[0065] FIG. 37A to FIG. 37D show an intracellular pool of -adrenergic receptors activates AKAP6P-bound PKA. FIG. 37A: model for experimental design of FIG. 37 A to FIG 37D, wherein differentially permeable P-blockers and an organic cation transporter 3 (Oct3) blocker were utilized to test if intracellular P-adrenergic receptors support AKAP6P- bound PKA activity. AKAR4 biosensor design features a forkhead-associated domain (FHA1) and PKA substrate (LRRATLVD; SEQ ID NO:8) with fluorescent proteins Cerulean and cpVenus on either side. Phosphorylation of PKA substrate results in FHA1 binding, leading to increased FRET signal. ONM- AKAR4 was localized to AKAP6P via nesprin 1 a which contains spectrin-like repeat domains and a C-terminal transmembrane Klarsicht, ANC-1, Syne Homology (KASH) domain conferring nuclear envelope localization. FIG.37B shows iamges of neonatal rat ventricular myocytes (RNV) that were infected with either AKAR4 or 0NM-AKAR4 adenovirus as indicated and imaged with a confocal microscope. Bar - 10 pm. Cyan image is shown in grayscale. FIG. 37C and FIG. 37D show FRET4906-5655-6138 v.l 17Attorney Docket No. 65274.12WO01Customer No. 27683 imaging of norepinephrine (NE)-treated neonatal cardiomyocytes infected with either AKAR4 (FIG. 37C, top and bottom panels) or 0NM-AKAR4 (FIG. 37D, top and bottom panels) and treated with either cell-impermeable P-blocker sotalol, cell-permeable P-blocker propranolol, or organic cation transporter 3 (Oct3) blocker corticosterone as indicated. Representative tracings and peak amplitudes for FRET ratio (R normalized to baseline Ro) are shown.

[0066] FIG. 38A to FIG. 38F show that perinuclear P-adrenergic receptors are necessary and sufficient for activation of AKAP6P-bound PKA. FIG. 38A shows a model for the experimental design of FIG. 38A to FIG. 38F, where novel peptides localized to AKAP6P were utilized to test whether perinuclear PKA activity relies on P-adrenergic receptors within the AKAP6P signaling zone. ONM-Nb80 consists of camelid single chain nanobody Nb80 linked to nesprin la with an mCherry tag, which binds to active Pi and P2 halting downstream signaling. ONM-ICL3-9 contains pepducin ICL3-9 derived from the third intracellular loop of P2 and screened for agonist independent activation of P-adrenergic receptors, linked to nesprin la with an mCherry tag. ONM-control peptide only contains nesprin la with an mCherry tag. FIG. 38B shows images of RNV infected with either AKAR4 or ONM- AKAR4 as indicated and either ONM-Nb80 or ONM-ICL3-9, as indicated, followed by imaging by confocal microscopy. Bar - 10 pm. Cyan image is shown in grayscale. FIG. 38C and FIG. 38D show the results of FRET imaging of NE-treated RNV infected with either AKAR4 or 0NM-AKAR4 and either ONM-Control or ONM-Nb80, as indicated.Representative tracings and peak amplitudes for FRET ratio (R normalized to baseline Ro) are shown. FIG. 38E and FIG. 38F show graphical results of FRET imaging of baseline PKA activity (Ro) in RNV infected with either AKAR4 or 0NM-AKAR4 and either ONM- Control or ONM-ICL3-9 as indicated.

[0067] FIG. 39A to FIG. 391 show Golgi-localized pARs activate PKA in AKAP6P signalosomes. FIG. 39A shows a model for experimental design of FIG. 39A-I where novel peptides were localized to the plasma membrane (PM), endosome (Endo), or Golgi apparatus (Golgi) in order to discern the location of AKAP6P-associated P-adrenergic receptors. PM- Nb80 and PM-ICL3-9 are flanked by an mCherry tag and directed to the plasma membrane by fusion to the N-terminal domain of AKAP7a (aa 1-25). Endo-Nb80 and Endo-ICL3-9 are flanked by an mCherry tag and directed to the endosomes via fusion to tandem FYVE domains from hepatocyte growth factor 1 - regulated tyrosine kinase substrate (Hrs aa 147-4906-5655-6138 v.l 18Attorney Docket No. 65274.12WO01Customer No. 27683223). Golgi-Nb80 and Golgi-ICL3-9 are mCherry tageged and directed to the Golgi via beta- 1 ,4-galactosyltransferase 1 targeting sequence (GalT aa 2-79). SR-l-mCherry consists of the first spectrin repeat of AKAP6P with an mCherry tag to disrupt AKAP6P and AKAP9 binding. FIG. 39B shows images of RNV that were infected with AKAR4 (panel not shown) and transfected with either PM-Nb80, Endo-Nb80, or Golgi-Nb80 as indicated and imaged by confocal microscopy. Bar - 10 pm. Cyan image is shown in grayscale. FIG. 39C to FIG. 39F show graphical results from FRET imaging of RNV infected with either AKAR4 or ONM- AKAR4 and transfected with either PM-Nb80, Endo-Nb80, Golgi-Nb80, PM-ICL3-9, Endo- ICL3-9, or Golgi-ICL3-9 as indicated. RNV in FIG. 39C and FIG. 39D were treated with NE. Peak amplitudes for FRET ratio (R normalized to baseline Ro) or baseline PKA activity (Ro) are shown. In FIG. 39E and FIG. 39F, data shown for ONM-Control with either FRET sensor is repeated from previous figure for ease of comparison. FIG. 39G shows images of RNV transfected with either SR-l-mCherry or mCherry that were stained for the Golgi apparatus and imaged by fluorescence microscopy. Bar - 10 pm. FIG. 39H shows graphical results for FRET imaging of RNV infected with either AKAR4 or 0NM-AKAR4 and transfected with either mCherry or SRI -mCherry and treated with NE as indicated. Peak amplitudes for FRET ratio (R normalized to baseline Ro). FIG. 391 shows graphical results of FRET imaging of RNV infected with 0NM-AKAR4 and ONM-ICL3-9 and transfected with either mCherry or SRI -mCherry as indicated. Cells were treated with PKA inhibitor H89 as indicated. Baseline PKA activity is shown (Ro).

[0068] FIG. 40A to FIG. 40F show experiments defining the dimensions of the AKAP6P cAMP compartment. FIG. 40A shows a model for experimental design of panel I where we test whether PDE4D3 activity and / or buffering by RII limits the diffusion of cAMP in the AKAPP signaling compartment. 4D3 -mCherry is an mCherry tagged PDE4D3 -derived peptide which disrupts the N-terminus of PDE4D3 which binds AKAP6. AKAP-ZS-mCherry is an mCherry-tagged peptide which competitively binds the RII binding domain on AKAP6. FIG. 40B to FIG. 40D, and FIG. 40F show graphical results of FRET imaging of RNV treated with no drug or NE as indicated after co-infection with either Epac2-camps, ONM- Epac2-camps, or ONM-Epac2-camps-50 and either ONM-mCherry or ONM-ICL3-9 followed by transfection with mCherry, 4D3 -mCherry, or SuperAKAP-ZS’-mCherry (FIG. 40F only). Peak amplitudes for FRET ratio (1 divided by R normalized to baseline Ro) or baseline cAMP release (1 divided by Ro) are shown. FIG. 40E shows model (left, center, and4906-5655-6138 v.l 19Attorney Docket No. 65274.12WO01Customer No. 27683 right panels) for experimental design that measures the dimensions of the AKAP60 compartment using a nesprin-targeted FRET sensor Epac2-camps flanked by molecular linkers of varying size in conjunction with our perinuclear 0-adrenergic receptor activator peptide ONM-ICL3-9. The structure of Epac2-camps is previously described. 0NM-Epac2- camps is localized to AKAP60 via nesprin la addition, while ONM-Epac2-camps-50 also contains an intervening rigid spacer containing 50 copies of the pentapeptide EAAAK, which increases distance from the ONM by -10-26 nm.

[0069] FIG. 41A to FIG. 41N and FIG. 42A to FIG. 421 show regulation of Camdependent calcineurin signaling by AKAP60-associated 0ARs. FIG. 41A and FIG 42A show models for experimental design of FIG. 41B-N, and FIG. 42B-J, respectively, wherein differentially permeable 0-blockers and Oct3 blocker were utilized, and novel ONM-Nb80 and ONM-ICL3-9 peptides to test Ca2+and calcineurin signaling and activation of hypertrophic gene transcription factor and calcineurin target NFAT. CaNAR2 contains fluorescent proteins Cerulean 3 and YPet on either side of the N-terminal regulatory domain of NFATc2, which remains highly phosphorylated in the cytosol under control conditions until calcineurin dephosphorylation induces the conformational change leading to increased FRET. ONM-CaNAR2 retains these components but is restricted to AKAP6 via a nesprin 1 addition. GCaMP6s is an intensiometric Ca2+sensor with calmodulin-binding M13 peptide, circularly permuted green fluorescent protein (cpGFP), and a variant mCam, which upon M13-mCam binding results in increased cpGFP fluorescence. ONM-GCaMP6s retains these components but is restricted to AKAP6 via a nesprin 1 addition. The NFAT nuclear translocation assay requires infection of adenoviral NFATcl which has a GFP tag. FIG. 41B, RNV were co-infected with either CaNAR2, ONM-CaNAR2, GCaMP6s, or ONM-GCaMP6s and ONM-mCherry followed by confocal imaging as shown. Bar - 10 pm. Sensor images are shown in grayscale. FIG. 41C-F, Confocal imaging of RNV treated with no drug, NE, or H89 overnight as indicated after co-infection of either GCaMP6s or ONM-GCaMP6s and either ONM-Control, ONM-Nb80 or ONM-ICL3-9. ONM-Control with no drug was treated as baseline for normalization purposes. Average change in fluorescence from each biological replicate is shown. FIG 41G-J, FRET imaging of RNV treated with either CaNAR2 or ONM-CaNAR2 as indicated. Baseline calcineurin activity is shown (Ro) (FIG. 42D-E). FIG 41G-H, RNV were treated with either cell-impermeable 0-blocker sotalol, cell-permeable 0- blocker propranolol, or organic cation transporter 3 (Oct3) blocker corticosterone as4906-5655-6138 v.l 20Attorney Docket No. 65274.12WO01Customer No. 27683 indicated. (FIG. 42B, FIG. 42C, FIG. 42F, FIG. 42G) FIG 41I-J RNV were treated with either ONM-Control, ONM-Nb80, or ONM-ICL3-9 and stimulated with NE as indicated. FIG. 41K, RNV infected with adenovirus expressing GFP-tagged NFATcl were treated with either no drug or NE for 48 hours and then imaged by fluorescence microscopy. Bar - 10 pm. L, RNV infected with adenovirus expressing GFP-tagged NFATcl were treated with and either no drug (Control), NE, cell-impermeable P-blocker sotalol, cell-permeable P-blocker propranolol, or organic cation transporter 3 (Oct3) blocker corticosterone as indicated (FIG. 42G-J). FIG. 41M-N, RNV infected with adenovirus expressing GFP-tagged NFATcland either ONM-Control, ONM-Nb80, or ONM-ICL3-9 were treated with no drug (control) or NE for 48 hours. FIG. 41L-N, After treatment, RNV were fixed and assayed for change in NFAT localization by fluorescence microscopy. Fraction of cells with majority NFAT nuclear localization was determined. All data were analyzed by matched 2-way ANOVA and Tukey post-hoc testing. * p < 0.05; ** p < 0.01; *** p < 0.001 ; **** p < 0.0001.

[0070]

[0071] FIG. 43A to FIG. 430 show regulation of myocyte hypertrophy by perinuclear P- adrenergic receptors. Adult rat cardiomyocytes (ARVM) were treated with either no drug (Control), NE, cell-impermeable P-blocker sotalol, cell-permeable P-blocker propranolol, or organic cation transporter 3 (Oct3) blocker corticosterone for 48 hours before staining with a- actinin and fluorescent imaging. Myocyte length and width were measured (FIG. 43A to FIG. 43D). ARVM expressing either ONM-Control, ONM-Nb80, or ONM-ICL3-9 were treated with either no drug (Control) or NE for 48 hours before staining with a-actinin and fluorescent imaging. Myocyte length and width were measured (FIG. 43E to FIG. 43H). Each data point represents the mean value of > 50 cells that were measured for each condition for each individual biological replicate; n = 3-6 independent myocyte preparations. All data were analyzed by matched 2- way ANOVA and Tukey post-hoc testing. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0072] FIG. 44A to FIG. 44Q show perinuclear P-adrenergic receptors regulate pathological cardiac remodeling in vivo. FIG. 44A shows an illustration of a cardiomyocyte- selective AAV9 vectors containing a cTnT promoter were used to express versions of ONM- Control, ONM-ICL3-9, and ONM-Nb80 lacking the mCherry tag. FIG. 44B to FIG. 44D show graphical results of C57BL6 / NJ mice treated at 6-8 weeks of age with 5xlOnvg i.p AAV9 or no virus (NI) that were followed by serial “4D’' echocardiography for 8 weeks, n =4906-5655-6138 v.l 21Attorney Docket No. 65274.12WO01Customer No. 276836-9. * vs. ONM-Control,fvs. NI controls. FIG. 44E shows indexed heart weight determined gravimetrically at 8 week endpoint, and FIG. 44F and FIG. 44G show myocyte cross- sectional area at endpoint determined by wheat-germ agglutinin staining and fluorescent microscopy. FIG. 44H and FIG. 441 show interstitial myocardial fibrosis at endpoint determined by Picrosirius Red staining and polarized light microscopy. FIG. 44J to FIG. 44L show FVB / N TM54 and non-transgenic (NTG) littermate mice were mice treated with AAV as above and followed by serial “4D” echocardiography for 8 weeks, n = 8-15. * vs. ONM-Control,fvs. NI controls. All corresponding TM54 and NTG cohorts were significantly different at all time points (p < 0.01). (G,H, and unlabelled graph on your version) See Videos S4-S9. FIG. 44M shows indexed heart weight determined gravimetrically at 10 week endpoint. FIG. 44N and FIG. 440 show myocyte cross-sectional area at endpoint determined by wheat-germ agglutinin staining and fluorescent microscopy. FIG. 44P and FIG. 44Q show interstitial myocardial fibrosis at endpoint determined by Picrosirius Red staining and polarized light microscopy.DETAILED DESCRIPTION

[0073] AKAP-based signaling complexes play a central role in regulating physiological and pathological cardiac events. As such, the present inventors have examined inhibiting the signaling properties of individual signaling complexes involving mAKAPP (AKAP6) using interventions that inhibit the activity of specific components of mAKAPP signalosomes as an approach for limiting cardiac pathological processes. Such a therapeutic strategy offers an advantage over classical therapeutic approaches since it allows the selective inhibition of defined cellular responses by the localized inhibition of signaling proteins only in the intracellular compartment relevant to the cellular process of interest.

[0074] Anchoring proteins like mAKAP, factors (e.g., proteins) associated with or bound to the anchoring proteins, as well as secondary factors (e.g., proteins) that interact with the factors associated with or bound to mAKAP, are therapeutic targets for the treatment of, e.g., cardiac hypertrophy and heart failure. In particular, the present inventors have found that disrupting AKAP-mediated protein-protein interactions or inhibiting the activity of proteins associated with the AKAP scaffold can be used to inhibit the ability of mAKAP to coordinate the activation of enzymes that play a central role in activating key transcription factors that initiate the remodeling process leading to cardiac hypertrophy.4906-5655-6138 v.l 22Attorney Docket No. 65274.12WO01Customer No. 27683

[0075] In particular, the inventors have found that peptide disruptors (e.g., an antibody, antibody fragment, fusion protein, small peptide, pepducin, modified peptide, peptidic foldamer, structural mimetic, mechanistic mimetic, and the like) can be used to target specific protein functions within AKAP-based complexes. Peptide disruptors can be identified by combining rational design and screening approaches. Such compounds can be designed to target- specific binding surfaces on AKAPs, AKAP-associated proteins, or proteins that interact with AKAPs and / or AKAP-associated proteins, to affect the interaction between them or their intrinsic activity, resulting in stimulation or inhibition of wild-type function. For instance, mAKAPP is localized to the nuclear envelope in both neonatal and adult cardiomyocytes via binding to nesprin-la. Functional PARs have also been detected on the nuclear envelope and on adjacent golgi apparatus and transverse tubules. It is shown herein that such ARs are responsible for mAKAPP signalosome activation (through adenylyl cyclase 5) and subsequent development of cardiac hypertrophy, and disruption of the activity of PARs associated physically with mAKAPP complexes (signalosomes) inhibited the development of cardiac hypertrophy.

[0076] Such peptide inhibitors can be delivered to the cell, e.g., through the use of a cellpenetrating sequence, or delivered by intracellular expression via viral-based gene therapy vectors.

[0077] As will be described in greater detail elsewhere herein, the present disclosure generally relates to methods of treating a heart disease (cardiac dysfunction) by administering to a patient at risk of a heart disease a pharmaceutically effective amount of a composition that, e.g., localized to the outer nuclear membrane binds to adjacent PAR, or suppresses perinuclear cAMP production, as well as peptide and gene therapy compositions related thereto. Some methods and related compositions described herein include binding to mAKAPP or protein associated with or bound to mAKAPP and inhibiting adenylyl cyclase activity, binding to a P-AR, and / or suppressing perinuclear cAMP production.

[0078] A number of terms are introduced below, which are used to describe the invention of the present disclosure. In instances where a technical or scientific term is not specifically defined herein, they will have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly4906-5655-6138 v.l 23Attorney Docket No. 65274.12WO01Customer No. 27683 used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0079] The term “muscle” is well understood by the skilled person. Preferably, the muscle is a heart muscle, and the muscle is a muscle of a vertebrate, of a mammal, or a human subject. Preferably, the muscle is a striated muscle.

[0080] The term “muscle cell” or “myocyte” or “myoblast” relate to cells which are found in muscles (muscle tissue), or which are derived from muscle tissue or differentiate into muscle tissue. Muscle cells can be primary cells or a cell line (such as C2C12 or H2K cells (myoblast cell line) or, e.g., neonatal (NRVM) or adult rat ventricular myocytes (ARVM) , or H9C2 cells (cardiac cell line)). The muscle cells can be in vivo (e.g. in muscle tissue) or in vitro (e.g. in cell culture). Myocytes as found in muscle tissue are typically long, tubular cells that develop from myoblasts to form muscles in a process known as myogenesis. The term muscle cells or myocytes as used herein includes myocytes from cardiac muscle (cardiomyocytes).

[0081] As used herein, the term “heart cell” refers to a cell which can be: (a) part of a heart present in a subject, (b) part of a heart which is maintained in vitro, (c) part of a heart tissue, or (d) a cell which is isolated from the heart of a subject. For example, the cell can be a muscle cell, such as a cardiac myocyte (cardiomyocyte) or smooth muscle cell. Heart cells of the invention can also include endothelial cells within the heart, for example, cells of a capillary, artery, or other vessel. A heart cell includes pacemaker cells and the like.

[0082] As used herein, the term “heart” refers to a heart present in a subject or to a heart which is maintained, ex vivo outside a subject.

[0083] As used herein, the term “heart tissue” refers to tissue which is derived from the heart of a subject.

[0084] As used herein, the term “heart disorder” refers to a structural or functional abnormality of the heart that impairs its normal functioning. For example, the heart disorder can be heart failure, ischemia, myocardial infarction, congestive heart failure (CHF), arrhythmia, cardiomyopathy, defect in cardiac contractility, transplant rejection and the like. The term includes disorders characterized by abnormalities of contraction, abnormalities in Ca2+metabolism, and disorders characterized by arrhythmia.4906-5655-6138 v.l 24Attorney Docket No. 65274.12WO01Customer No. 27683

[0085] The terms “heart disease” and “heart disorder” refer to structural or functional abnormalities of the heart that impair normal functioning, which can manifest acutely or chronically. A heart disease / disorder is often associated with a decrease in cardiac contractile function and / or ventricular relaxation, may be associated with an observable decrease in blood flow to the myocardium (e.g., as a result of coronary artery disease) and can be, e.g., heart failure, cardiomyopathies, ischemia, myocardial infarction, congestive heart failure (CHF), arrhythmia, cardiomyopathy, defect in cardiac contractility, transplant rejection and the like. The term includes disorders characterized by abnormalities of contraction, abnormalities in Ca2+metabolism, and disorders characterized by arrhythmia.

[0086] The term “cardiomyopathies” refers to a group of diseases giving rise to congestive heart failure. Cardiomyopathies are a heterogeneous group of diseases of the myocardium associated with mechanical and / or electrical dysfunction that usually exhibit inappropriate ventricular hypertrophy or dilatation, e.g., left ventricular remodeling, restrictive cardiomyopathy, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM; ischemic and non-ischemic), arrhythmogenic right ventricular cardiomyopathy (ARVC), and idiopathic dilated cardiomyopathy (IDCM). Remodeling that manifests a disease state can be referred to as “pathological cardiac remodeling”.

[0087] As used herein, the term “cardiomyopathy” refers to a deterioration of function of the myocardium (i.e., heart muscle). Cardiomyopathy can be extrinsic (e.g., wherein the primary pathology resides outside of the myocardium itself, for example, caused by ischemia or hypertension) or intrinsic (e.g., wherein the weakness in the heart muscle is not due to an identifiable external cause).

[0088] The term “congestive heart failure” or “CHF” is also interchangeably with “heart failure” or “chronic heart failure” is a manifestation of pathological conditions affecting the heart and refers to the inability of the heart to pump blood at an adequate rate to meet the metabolic demands of the body. The term “heart failure” is clinically defined as a condition in which the heart does not provide adequate blood flow to the body to meet metabolic demands. Symptoms include breathlessness, fatigue, weakness, leg swelling, and exercise intolerance. On physical examination, patients with heart failure tend to have elevations in heart and respiratory rates, rales (an indication of fluid in the lungs), edema, jugular venous distension, and, in many cases, enlarged hearts. Patients with heart failure suffer a high mortality; typically, 50% of the patients die within five years of developing the condition. In4906-5655-6138 v.l 25Attorney Docket No. 65274.12WO01Customer No. 27683 some cases, heart failure is associated with severe coronary artery disease (“CAD”), typically resulting in myocardial infarction and either progressive chronic heart failure or an acute low output state, as described herein and in the art. In other cases, heart failure is associated with dilated cardiomyopathy without associated severe coronary artery disease. Stated differently, the term “heart failure” refers to any of a number of disorders in which the heart has a defect in its ability to pump adequately to meet the body’s needs. In many cases, heart failure is the result of one or more abnormalities at the cellular level in the various steps of excitationcontraction coupling of the cardiac cells.

[0089] Heart failure can be divided into two groups: (a) reduced ejection fraction (HFrEF) and (b) preserved ejection fraction (HFpEF) (ejection fraction >45%); the two forms confer a similar prognosis and have similar prevalence. HFrEF patients have a prominent defect in myocardial contraction, which can occur for many reasons, the most common of which include ischemic damage to the myocardium, excessive mechanical resistance to the outflow of blood from the heart, overloading of the cardiac chambers due to defective valve function, infection or inflammation of the myocardium, or congenitally poor myocardial contractile function. While HFrEF includes prominent systolic cardiac dysfunction, HFpEF is more closely associated with diastolic dysfunction. HFpEF typically presents with multiple comorbidities including hypertension, atrial fibrillation, diabetes, anemia, and chronic kidney disease. Most importantly, population studies have repeatedly found that about 2 / 3 of those with HFpEF are women. Although there are established regimens for the treatment of HFrEF, including “Guideline Directed Medical Therapy,” there are currently no therapies for HFpEF that decrease mortality. In addition, HFrEF caused by dilated cardiomyopathy also is associated with high mortality and is the leading cause of cardiac transplantation.

[0090] The term “myocardial ischemia” or “MI” is a condition in which the heart muscle does not receive adequate levels of oxygen and nutrients, which is typically due to inadequate blood supply to the myocardium (e.g., as a result of coronary artery disease).

[0091] The terms “coronary artery disease” and “acute coronary syndrome” as used interchangeably herein, and refer to myocardial infarction refer to a cardiovascular condition, disease or disorder, include all disorders characterized by insufficient, undesired or abnormal cardiac function, e.g. ischemic heart disease, hypertensive heart disease and pulmonary hypertensive heart disease, valvular disease, congenital heart disease and any condition which leads to congestive heart failure in a subject, particularly a human subject. Insufficient or4906-5655-6138 v.l 26Attorney Docket No. 65274.12WO01Customer No. 27683 abnormal cardiac function can be the result of disease, injury and / or aging. By way of background, a response to myocardial injury follows a well-defined path in which some cells die while others enter a state of hibernation where they are not yet dead but are dysfunctional. This is followed by infiltration of inflammatory cells, deposition of collagen as part of scarring, all of which happen in parallel with in-growth of new blood vessels and a degree of continued cell death.

[0092] As used herein, the term “ischemia” refers to any localized tissue ischemia due to reduction of the inflow of blood. The term “myocardial ischemia” refers to circulatory disturbances caused by coronary atherosclerosis and / or inadequate oxygen supply to the myocardium. For example, an acute myocardial infarction represents an irreversible ischemic insult to myocardial tissue. This insult results in an occlusive (e.g., thrombotic or embolic) event in the coronary circulation and produces an environment in which the myocardial metabolic demands exceed the supply of oxygen to the myocardial tissue.

[0093] As used herein, the term “contractility” (as in myocardial contractility) refers to the performance of cardiac muscle. It is often defined as: the intrinsic ability of a cardiac muscle fiber to contract at a given fiber length.

[0094] As used herein, the term “restricting blood flow” refers to substantially blocking the flow of blood through a vessel, e.g., flow of blood into the distal aorta and its branches. For example, at least 50% of the blood flowing out of the heart is restricted, preferably 75% and more preferably 80, 90, or 100% of the blood is restricted from flowing out of the heart. The blood flow can be restricted by obstructing the aorta and the pulmonary artery, e.g., with clamps.

[0095] The term “nucleic acid” as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose4906-5655-6138 v.l 27Attorney Docket No. 65274.12WO01Customer No. 27683 and / or 2-deoxyribose common in naturally occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term “nucleic acid” further preferably encompasses DNA, RNA and DNA RNA hybrid molecules, specifically including hnRNA, shRNA, siRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA or DNA RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partly or entirely, chemically or biochemically synthesized. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0096] The term “peptide”, “polypeptide”, “protein”, and the like, as used herein, refers to a molecule that is formed using naturally occurring L-amino acids or analogs thereof, like D- amino acids, or N-alkylated amino acids, or the like. Preferred amino acids are selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai. Additionally, modifications such as fluorescence dyes or biotin are also contemplated.

[0097] “Functional derivatives” of proteins are also contemplated, in which a functional derivative refers to a “chemical derivative,” “fragment,” “polymorph” or “variant” of the polypeptide or nucleic acid of the invention. A functional derivative retains at least a portion of the function of the protein, which permits its utility in accordance with the invention. It is well known in the art that, due to the degeneracy of the genetic code, numerous different nucleic acid sequence can code for the same amino acid sequence. It is also well known in the art that conservative changes in amino acid can be made to arrive at a protein or polypeptide that retains the functionality of the original. In both cases, all permutations are intended to be covered by this disclosure.4906-5655-6138 v.l 28Attorney Docket No. 65274.12WO01Customer No. 27683

[0098] Another functional derivative intended to be within the scope of the present invention is a “variant” polypeptide, which either lacks one or more amino acids or contains additional or substituted amino acids relative to the native polypeptide. Such variants having added, substituted and / or additional amino acids retain the functional portion of the original polypeptide. A functional derivative of a protein with deleted, inserted and / or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art (e.g., site-directed mutagenesis). Alternatively, proteins with amino acid deletions, insertions and / or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.

[0099] The terms “identity”, “identical”, “similar”, “similarity”, “homology”, “homologous”, and the like, refer to the “likeness” or “sameness” of two or more sequences, e.g., between two nucleic acid sequences or two peptide sequences, often expressed as a percentage.Sequence identity is the number of characters which match exactly between two different sequences, where gaps are not counted, and the measurement is relational to the shorter of the two sequences. This has the effect that sequence identity is not transitive, i.e. if sequence A=B and B=C then A does not necessarily equal C (in terms of the identity distance measure): Consider exemplary sequences A: AAGGCTT, B: AAGGC, and C:AAGGCAT. Here identity / A, B)=100% (5 identical nucleotides / min(length(A),length(B))).Identity(B,C)=100%, but identity(A,C)=85% ((6 identical nucleotides 11)). So, 100% identity does not necessarily indicate two sequences are the same. Sequence similarity accounts for sequence identity and conservative substitutions with positive scores in substitution matrices. E.g., a leucine to isoleucine substitution would receive partial “similarity” credit while not for a sequence identity calculation. Methods for aligning sequences for comparison are well- known in the art, and a detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0100] The term “synthetic” in the present application means a nucleic acid or peptide molecule that does not occur in nature. Synthetic nucleic acids and amino acids of the disclosure are produced artificially, typically by recombinant technologies or de novo synthesis. Such synthetic nucleic acids may contain naturally occurring sequences (e.g. promoter, enhancer, intron, and other such regulatory sequences), but these are present in a non-naturally occurring context. For example, a synthetic gene (or portion of a gene) typically contains one or more nucleic acid sequences that are not contiguous in nature4906-5655-6138 v.l 29Attorney Docket No. 65274.12WO01Customer No. 27683(chimeric sequences), and / or may encompass substitutions, insertions, and deletions and combinations thereof.

[0101] “Transfection” in the present application refers broadly to any process of deliberately introducing nucleic acids into cells, and covers introduction of viral and non-viral vectors, and includes or is equivalent to transformation, transduction and like terms and processes. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al. (1986) Nature 319:791-3); lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; whiskers-mediated transformation; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

[0102] As used herein, the phrase “transgene” refers to an exogenous nucleic acid sequence. In one example, a transgene is a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable trait. In yet another example, the transgene encodes useful nucleic acid such as an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. The transgene preferably encodes a therapeutic product, e.g. a protein.

[0103] The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated.

[0104] As used herein, the terms “virus vector,” “vector” or “gene delivery vector” refer to a virus (e.g., AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or any other AAV now known or later discovered) particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA [vDNA])4906-5655-6138 v.l 30Attorney Docket No. 65274.12WO01Customer No. 27683 packaged within a virion. Alternatively, in some contexts, the term “vector” may be used to refer to the vector genome / vDNA alone.

[0105] An “rAAV vector genome” or “rAAV genome” is an (recombinant) AAV genome that comprises one or more heterologous nucleic acid sequences. rAAV vectors generally require only the inverted terminal repeat(s) (TR(s)) in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans. Typically, the rAAV vector genome will only retain the one or more TR sequence so as to maximize the size of the transgene that can be efficiently packaged by the vector. The structural and non- structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell). In embodiments of the invention the rAAV vector genome comprises at least one ITR sequence (e.g., AAV TR sequence), optionally two ITRs (e.g., two AAV TRs), which typically will be at the 5' and 3' ends of the vector genome and flank the heterologous nucleic acid, but need not be contiguous thereto. The TRs can be the same or different from each other. As used herein, “AAV” and “rAAV” are used synonymously unless an instance of “AAV” specifically states a wild type AAV is being described.

[0106] The term “terminal repeat” or “TR” includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., an ITR that mediates the desired functions such as replication, virus packaging, integration and / or provirus rescue, and the like). The TR can be an AAV TR or a non- AAV TR. For example, a non- AAV TR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable virus sequence (e.g., the SV40 hairpin that serves as the origin of SV40 replication) can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and / or addition. Further, the TR can be partially or completely synthetic, such as the “double - D sequence” as described in U.S. Pat. No. 5,478,745 to Samulski et al.

[0107] An “AAV terminal repeat” or “AAV TR,” including an “AAV inverted terminal repeat” or “AAV ITR” may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or any other AAV now known or later discovered. An AAV terminal repeat need not have the native terminal repeat sequence (e.g., a native AAV TR or AAV ITR sequence may be altered by insertion, deletion, truncation and / or missense4906-5655-6138 v.l 31Attorney Docket No. 65274.12WO01Customer No. 27683 mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and / or provirus rescue, and the like.

[0108] Further, the viral capsid or genomic elements can contain other modifications, including insertions, deletions and / or substitutions.

[0109] Included within the scope of the invention are functional equivalents of the herein- described isolated nucleic acid molecules. The degeneracy of the genetic code permits substitution of certain codons by other codons that specify the same amino acid and hence would give rise to the same protein. The nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon. The encoded amino acid sequence thereof would, however, be preserved.

[0110] In addition, the nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5 '-end and / or the 3 '-end, provided that its addition, deletion or substitution does not alter the amino acid sequence described herein, which is encoded by the nucleotide sequence. For example, the nucleic acid molecule of the present invention may have restriction endonuclease recognition sites added to its 5'-end and / or 3'-end.

[0111] Further, it is possible to delete codons or to substitute one or more codons with codons other than degenerate codons to produce a structurally modified polypeptide, but one which has substantially the same utility or activity as the polypeptide produced by the unmodified nucleic acid molecule. As recognized in the art, the two polypeptides are functionally equivalent, as are the two nucleic acid molecules that give rise to their production, even though the differences between the nucleic acid molecules are not related to the degeneracy of the genetic code.

[0112] The term “fragment” is used to indicate a polypeptide derived from another polypeptide having a length less than the full-length polypeptide from which it has been derived. Such a fragment may, for example, be produced by proteolytic cleavage of the full- length protein. Such a fragment may also be obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites of the C-terminus, N-terminus, and / or within the native sequence. Such fragments retain the functional portion of the native protein.4906-5655-6138 v.l 32Attorney Docket No. 65274.12WO01Customer No. 27683

[0113] Unless indicated otherwise, “efficient transduction” or “efficient tropism,” or similar terms, can be determined by reference to a suitable control (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more of the transduction or tropism, respectively, of the control). In particular embodiments, the virus vector efficiently transduces or has efficient tropism for neuronal cells and cardiomyocytes. Suitable controls will depend on a variety of factors including the desired tropism and / or transduction profile.

[0114] The terms “heterologous nucleotide sequence” and “heterologous nucleic acid molecule” are used interchangeably herein and refer to a nucleic acid sequence that is not naturally occurring in the virus. Generally, the heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame that encodes a polypeptide and / or nontranslated RNA of interest (e.g., for delivery to a cell and / or subject).

[0115] As used herein, the term “administering” refers to the actual physical introduction of a composition into or onto (as appropriate) an individual, subject, patient, or cell. Any and all methods of introducing the composition into the subject, host or cell are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art and also are exemplified herein. “Providing” means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing. The “administration” of a composition to a subject includes any route of introducing or delivering to a subject the agent to perform its intended function.Administration can be carried out by any suitable route, including orally, intranasally, intraocularly, ophthalmically, parenterally (intravascularly, intramuscularly, including intracardiac muscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another. Intravenous or intraarterial administration is of particular interest in the present invention.

[0116] The terms “individual”, “subject”, and “patient” are used interchangeably, and refer to any individual subject with a disease or condition in need of treatment. For the purposes of the present disclosure, the subject may be a primate, preferably a human, or another mammal, such as a dog, cat, horse, pig, goat, or bovine, and the like.4906-5655-6138 v.l 33Attorney Docket No. 65274.12WO01Customer No. 27683

[0117] As used herein, an individual, subject, or patient is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0118] As used herein, the term “pharmaceutically acceptable” refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a subject, preferably a human or a non-human subject. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0119] As used herein, the terms “treat,” “treating,” and “treatment” include inhibiting the pathological condition, disorder, or disease, e.g., arresting or reducing the development of the pathological condition, disorder, or disease or its clinical symptoms; or relieving the pathological condition, disorder, or disease, e.g., causing regression of the pathological condition, disorder, or disease or its clinical symptoms. Treatment means any way the symptoms of a pathological condition, disorder, or disease are ameliorated or otherwise beneficially altered. Preferably, a subject in need of such treatment is a mammal, and particularly a human, but is not so limited as defined below. Treatment also means providing an active compound to a patient in an amount sufficient to measurably reduce any disease symptom, slow disease progression or cause disease regression. These terms also encompass therapy and cure.

[0120] As used herein, the term "effective amount" or “therapeutically effective amount” refers to the amount of a therapy, which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, inhibit or prevent the advancement of a disorder, cause regression of a disorder, inhibit or prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy e.g., prophylactic or therapeutic agent). An effective dose can refer to, e.g, plasma concentration in a subject that provides the desired specific pharmacological effect, e.g. to express a therapeutic gene in cardiac tissue or the heart, and / or secretion into the plasma, or successfully deliver to the cell a peptide (fusion protein) that successfully enters the cell. A therapeutically effective amount can include a therapeutically significant reduction in a4906-5655-6138 v.l 34Attorney Docket No. 65274.12WO01Customer No. 27683 symptom, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject. Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for a disease or disorder. It will be understood that the total daily usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated.

[0121] An effective amount can require more than one dose. As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0122] Effective amounts may vary depending upon the biological effect desired in the individual, condition to be treated, and / or the specific characteristics of the composition according to the present invention and the individual. In this respect, any suitable dose of the composition can be administered to the patient (e.g. , human), according to the type of disease to be treated. Various general considerations taken into account in determining the “effective amount” are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman’s: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington’s Pharmaceutical Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.

[0123] When using an experimental animal model, efficacy of treatment is evidenced when a reduction in a symptom of the cardiovascular disease or disorder or improvement in a clinically relevant test, for example, a reduction in one or more symptom of dyspnea, chest pain, palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue and high blood pressure or an improvement in cardiac function as detected by echocardiography, catheterization, or post-mortem tissue gravimetric or histological analyses, which occurs earlier in treated, versus untreated animals. By “earlier” is meant that a decrease, for example in the size of the tumor occurs at least 5% earlier, but preferably more, e.g., one day earlier, two days earlier, 3 days earlier, or more.4906-5655-6138 v.l 35Attorney Docket No. 65274.12WO01Customer No. 27683

[0124] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein. As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0125] The terms "therapeutic" or "therapeutic agent" refer to a compound or molecule that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof. The present invention contemplates a broad range of therapeutic agents and their use in conjunction with the liposome compositions, as further described herein.

[0126] The term “pharmaceutical compositions” means compositions comprising at least one active agent, and at least one other substance, such as a carrier. Pharmaceutical compositions meet the U.S. FDA’s GMP (good manufacturing practice) standards for human or non-human drugs.

[0127] The term “carrier” refers to a diluent, excipient, or vehicle with which an active compound is administered. A “pharmaceutically acceptable carrier” means a substance, e.g., excipient, diluent, or vehicle, that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier” includes both one and more than one such carrier.

[0128] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated.Generally, unless otherwise expressly stated herein, "weight" or "amount" as used herein with respect to the percent amount of an ingredient refers to the amount of the raw material comprising the ingredient, wherein the raw material may be described herein to comprise less than and up to 100% activity of the ingredient. Therefore, weight percent of an active in a composition is represented as the amount of raw material containing the active that is used and may or may not reflect the final percentage of the active, wherein the final percentage of the active is dependent on the weight percent of active in the raw material.4906-5655-6138 v.l 36Attorney Docket No. 65274.12WO01Customer No. 27683

[0129] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art of this disclosure.

[0130] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

[0131] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited. For example, if a size range is stated as 1 nm to 100 nm (or concentrations, degrees, mass amounts, and the like), it is intended that values such as 2 nm to 90 nm, 10 nm to 70 nm, 30 nm to 95 nm, 75 nm to 100 nm, or 2 nm to 27 nm, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0132] Furthermore, when "about", "approximately" and / or "substantially" is / are utilized to describe a value, this is meant to encompass minor variations (up to + / - 10%) from the stated value. Where no stated value is provided, an element described as "substantially" means at least about 60%, 70%, 80%, 90%, 95%, 99%, or more of the element, as is logically coherent within in the context. Unless specifically stated to the contrary, for ranges specified using "about" language, the about applies to both ends of the recited range whether specified4906-5655-6138 v.l 37Attorney Docket No. 65274.12WO01Customer No. 27683 or not. For example, "between about 10 mM and 10 M" is equivalent to "between about 10 mM and about 10 pM".

[0133] As used herein, the terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.

[0134] When introducing elements of the present disclosure or the aspects and embodiment thereof, the articles "a," "an," and "the" are intended to mean that there are one or more of the elements. Similarly, the adjective "another," when used to introduce an element, is intended to mean one or more elements.

[0135] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0136] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."4906-5655-6138 v.l 38Attorney Docket No. 65274.12WO01Customer No. 27683

[0137] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0138] The phrase "one or more," as used herein, means at least one, and thus includes individual components as well as mixtures / combinations of the listed components in any combination.

[0139] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0140] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise. Furthermore, the terms first, second, etc., as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers.

[0141] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in4906-5655-6138 v.l 39Attorney Docket No. 65274.12WO01Customer No. 27683Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. LIV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0142] Moving to the specifics of the subject matter, disclosed herein are methods of treating a heart disease (cardiac dysfunction) by administering to a patient at risk of a heart disease a pharmaceutically effective amount of a peptide-based or gene therapy-based composition that acts on the scaffold protein mA K APf> (which organizes multimolecular signaling complexes on the nuclear membrane of differentiated myocytes (e.g., cardiac) also known as a signalosome), acts on a factor (e.g., protein) associated with or bound to mAKAPp, or acts on a secondary factor (e.g., protein) that interacts with a factor associated with or bound to mAKAPp. Such compositions and methods are described in more detail below.

[0143] COMPOSITIONS

[0144] In one aspect, the disclosure provides compositions that include a viral-based gene therapy vector encoding certain fusion proteins. In some embodiments of composition, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 98-1114 of SEQ ID NO:3. In some embodiments, the composition inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2-mediated Ca2+release. In some embodiments, the fragment of4906-5655-6138 v.l 40Attorney Docket No. 65274.12WO01Customer No. 27683 a Bcl-2 amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 3-27 of SEQ ID NO:3, and the nesprin-la amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 98-11 14 of SEQ ID NO:3, while in some embodiments, the fragment of a Bcl-2 amino acid sequence is amino acids 3-27 of SEQ ID NO:3, and the nesprin-la amino acid sequence is amino acids 98-1114 of SEQ ID NO:3.

[0145] In some embodiments of the compositions, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 193-1209 of SEQ ID NO:5. In some embodiments, the composition inhibits adenylyl cyclase activity, binds to P-AR, or suppresses perinuclear cAMP production. In certain embodiments, the Nb80 nanobody amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin- 1 a amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 193-1209 of SEQ ID NO:5, while in some embodiments, the Nb80 nanobody sequence is amino acids 3-122 of SEQ ID NO:5, and the nesprin-la amino acid sequence is amino acids 193-1209 of SEQ ID NO:5.

[0146] In some embodiments, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes (1) one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, and the like) Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequences, wherein the AIP amino acid sequence(s) is / (are) at least 80% identical to amino acids 3-15 of SEQ ID NO:1 ; and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 125-1 141 of SEQ ID NO:1. In some embodiments, the composition inhibits calmodulin-dependent protein kinase II (CaMKII) activity, or binds to CaMKII. In some embodiments, the AIP amino acid sequence is at least 90%, 95% , or 99% identical to amino acids 3-54 of SEQ ID NO: 1, and the nesprin-la amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 125-1 141 of SEQ ID NO:1 , while in some embodiments, the AIP amino acid sequence is amino acids 3-54 of SEQ ID NO:1, and the nesprin-la amino acid sequence is amino acids 125-1141 of SEQ ID NO:1.

[0147] In some embodiments of this aspect, the fusion protein is expressed using a viral vector, which, in some embodiments, includes adeno-associated virus (AAV), e.g., AAV2 and / or AAV9. Additional viral vector disclosure is provided elsewhere herein.4906-5655-6138 v.l 41Attorney Docket No. 65274.12WO01Customer No. 27683

[0148] In some embodiments, the fusion protein is delivered by intracellular expression via a viral-based gene therapy vector.

[0149] In some embodiments, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes one of (i) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-27 of SEQ ID NO:3, (ii) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-122 of SEQ ID NO:5, or (iii) one or more Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequences, wherein the AIP amino acid sequence(s) is / (are) at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-15 of SEQ ID NO:1; and (a) one or more of a fragment of a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

[0150] In some embodiments, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes a fragment of a RyR inhibitor including spinophilin, sorcin, calsequestrin, junctin, FKBP12 / 12.6 (calstabin), homer, natrin, triadin and calmodulin, wherein the amino acid sequence of each is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence; and (a) one or more of a fragment of a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.4906-5655-6138 v.l 42Attorney Docket No. 65274.12WO01Customer No. 27683

[0151] In some embodiments, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes (i) a 0-AR nanobody amino acid sequence according to Table 1 of US 9,453,065 (incorporated herein by reference in its entirety), wherein the P-AR nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acid sequences described in US 9,453,065, or (ii) a Nb80 peptide mimetic, such as those described in C. Martin, et al., Chem. Eur. J. 2017, 23, 9632 (incorporated by reference in its entirety); and (a) one or more of a fragment of a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916- 975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

[0152] In some embodiments, the viral-based gene therapy vector encodes a fusion protein, which fusion protein includes one or more fragments of a CaMKII inhibitor comprising one or more Autocamtide-3 inhibitor (AC3-I), CN27 (also known as CaMKIINtide), CN21, and CN19o amino acid sequences, wherein the AC3-I, CaMKIINtide, CN21, and CN19o amino acid sequences are at least 80%, 90%, 95%, 99% or 100% identical to their canonical sequences; and (a) one or more of a fragment of a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

[0153] In another aspect, the disclosure provides compositions that include one or more fusion proteins that interact with one or more components of the mAKAPp complex and / or molecules that interact with the complex. In some embodiments of the composition, the fusion protein binds to mAKAPp and inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2-mediated Ca2+release. In some embodiments of composition, the4906-5655-6138 v.l 43Attorney Docket No. 65274.12WO01Customer No. 27683 fusion protein includes (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 98- 1114 of SEQ ID NO:3. In some embodiments, the fragment of a Bcl-2 amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 3-27 of SEQ ID NO:3, and the nesprin- l amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 98-1114 of SEQ ID NO:3, while in some embodiments, the fragment of a Bcl-2 amino acid sequence is amino acids 3-27 of SEQ ID NO:3, and the nesprin-la amino acid sequence is amino acids 98-1114 of SEQ ID NO:3.

[0154] In some embodiments of the fusion protein-based compositions, the fusion protein inhibits adenylyl cyclase activity, binds to P-AR, or suppresses perinuclear cAMP production. In some embodiments, the fusion protein includes (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 193-1209 of SEQ ID NO:5. In certain embodiments, the Nb80 nanobody amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin-la amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 193-1209 of SEQ ID NO:5, while in some embodiments, the Nb80 nanobody sequence is amino acids 3-122 of SEQ ID NO:5, and the nesprin-la amino acid sequence is amino acids 193-1209 of SEQ ID NO:5.

[0155] In some embodiments of the fusion protein-based compositions, the fusion protein inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2-mediated Ca2+release. In some embodiments, the fusion protein includes (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80% identical to amino acids 3-54 of SEQ ID NO:1 ; and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 125-1141 of SEQ ID NO:1. In some embodiments, the composition inhibits calmodulin-dependent protein kinase II (CaMKII) activity, or binds to CaMKII. In some embodiments, the AIP amino acid sequence is at least 90%, 95% , or 99% identical to amino acids 3-54 of SEQ ID NO: 1 , and the nesprin- 1 a amino acid sequence is at least 90%, 95%, or 99% identical to amino acids 125-1141 of SEQ ID NO:1, while in some embodiments, the AIP amino acid4906-5655-6138 v.l 44Attorney Docket No. 65274.12WO01Customer No. 27683 sequence is amino acids 3-54 of SEQ ID NO: 1, and the nesprin-la amino acid sequence is amino acids 125-1141 of SEQ ID NO: 1.

[0156] In some embodiments of the fusion protein-based compositions, the fusion protein is administered directly or using a viral vector.

[0157] In some embodiments of the fusion protein-based compositions, the fusion protein includes one of (i) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-27 of SEQ ID NO:3, (ii) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-122 of SEQ ID NO:5, or (iii) one or more Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequences, wherein the AIP amino acid sequence(s) is / (are) at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-15 of SEQ ID NO: 1; and (a) one or more of a fragment of a nesprin-la amino acid sequence, wherein the nesprin- 1 a sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

[0158] In some embodiments of the fusion protein-based compositions, the fusion protein includes a fragment of a RyR inhibitor including spinophilin, sorcin, calsequestrin, junctin, FKBP12 / 12.6 (calstabin), homer, natrin, triadin and calmodulin, wherein the amino acid sequence of each is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.4906-5655-6138 v.l 45Attorney Docket No. 65274.12WO01Customer No. 27683

[0159] In some embodiments of the fusion protein-based compositions, the fusion protein includes (i) a P-AR nanobody amino acid sequence according to Table 1 of US 9,453,065 (incorporated herein by reference in its entirety), wherein the -AR nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acid sequences described in US 9,453,065, or (ii) a Nb80 peptide mimetic, such as those described in C. Martin, et al., Chem. Eur. J. 2017, 23, 9632 (incorporated by reference in its entirety); and (a) one or more of a fragment of a nesprin-1 a amino acid sequence, wherein the nesprin-1 a sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

[0160] In some embodiments of the fusion protein-based compositions, the fusion protein includes one or more fragments of a CaMKII inhibitor comprising one or more Autocamtide- 3 inhibitor (AC3-I), CN27 (also known as CaMKIINtide), CN21, and CN19o amino acid sequences, or combinations thereof, wherein the AC3-I, CaMKIINtide, CN21, and CN19o amino acid sequences are at least 80%, 90%, 95%, 99% or 100% identical to their canonical sequences; and (a) one or more of a fragment of a nesprin- la amino acid sequence, wherein the nesprin- la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1 , nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP), (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

[0161] In some embodiments of the various aspects of the disclosure, the fusion protein is formulated as a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable salt includes: (1 ) an acid selected from the group consisting of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and mandelic acid; (2) an inorganic base selected from the group consisting of sodium, potassium, ammonium, calcium, and ferric hydroxide; or (3) an organic base selected from the group consisting of isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.4906-5655-6138 v.l 46Attorney Docket No. 65274.12WO01Customer No. 27683

[0162] Because of the necessity for the inhibitor to reach the cytosol, a peptide in accordance with the invention may need to be modified in order to allow its transfer across cell membranes. Thus, in some embodiments, the fragment is modified with a cell membrane-penetrating sequence, e.g., transactivator of transcription (TAT) polypeptide, polyarginine peptide, a penetratin peptide, or the like, among . In some embodiments, the fragment is modified with a lipid-derived group, e.g., a stearate.

[0163] The rAAV vectors and, for peptide-based compositions the fusion proteins, as disclosed herein (e.g., SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6) for use in the methods of administration as disclosed herein can be formulated in a pharmaceutical composition with a pharmaceutically acceptable excipient, i.e., one or more pharmaceutically acceptable carrier substances and / or additives, e.g., buffers, carriers, excipients, stabilizers, etc. The pharmaceutical composition may be provided in the form of a kit. Pharmaceutical compositions comprising the rAAV vectors as disclosed herein for use in the methods of administration as disclosed herein and uses thereof are known in the art.

[0164] Accordingly, a further aspect of the invention provides a pharmaceutical composition comprising a rAAV vector or fusion protein as disclosed herein for use in the methods of administration as disclosed herein. Relative amounts of the active ingredient (e.g. a rAAV vector or fusion protein as disclosed herein), a pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and / or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w / w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 5 and 50%, between 1-30%, between 5- 80%, at least 80% (w / w) active ingredient.

[0165] The pharmaceutical compositions can be formulated using one or more excipients or diluents to, e.g., increase stability; increase cell transfection or transduction; permit the sustained or delayed release of the payload; alter the biodistribution (e.g., target the payload to specific tissues or cell types); for rAAV increase the translation of encoded protein; alter the release profile of encoded protein (or directly administered fusion protein) and / or allow for regulatable expression of the rAAV payload. In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least4906-5655-6138 v.l 47Attorney Docket No. 65274.12WO01Customer No. 2768399%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia. Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[0166] The rAAV vectors and fusion proteins as disclosed herein for use in the methods of administration as disclosed herein may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present invention. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In some embodiments, the delivery of one treatment (e.g., gene therapy vectors) is still occurring when the delivery of the second (e.g., one or more therapeutic) begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.”

[0167] In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second4906-5655-6138 v.l 48Attorney Docket No. 65274.12WO01Customer No. 27683 treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The compositions described herein and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the gene therapy vectors described herein can be administered first, and the one or more therapeutic can be administered second, or the order of administration can be reversed. The gene therapy vectors and the one or more therapeutic can be administered during periods of active disorder, or during a period of remission or less active disease. The gene therapy vectors can be administered before another treatment, concurrently with the treatment, posttreatment, or during remission of the disorder.

[0168] When administered in combination, the rAAV vectors and fusion proteins as disclosed herein for use in the methods of administration as disclosed herein and the one or more therapeutic (e.g., second or third therapeutic), or all, can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of each used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of a rAAV vector or fusion protein as disclosed herein for use in the methods of administration as disclosed herein and the one or more therapeutic (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each used individually. In other embodiments, the amount or dosage of the rAAV vector or fusion proteins as disclosed herein for use in the methods of administration as disclosed herein and the one or more therapeutic (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of a cardiovascular disease or heart disease) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each individually required to achieve the same therapeutic effect.

[0169] In some embodiments, the methods of administration of a rAAV vector or fusion protein as disclosed herein can deliver a rAAV vector or fusion protein disclosed herein alone, or in combination with an additional agent. In some embodiments, the additional agent4906-5655-6138 v.l 49Attorney Docket No. 65274.12WO01Customer No. 27683 is a muscle enhancing protein or peptide, e.g., to improve blood flow and enhance muscle function in the treated muscle. Furthermore, if desired, in some embodiments, the additional agent a vasoactive agent which can be employed in conjunction with these methods and compositions, as described herein, in order to further enhance gene delivery at the target site. Exemplary vasoactive agents include but are not limited to histamine, a histamine agonist, a nitric oxide donor, or a VEGF protein, and can be used to increase the efficiency of gene transfer at a gene vector dose. In some embodiments, a vasoactive agent is useful the to limit the amount of vector required to be administered in order to achieve a given therapeutic effect.

[0170] Compounds which may be used in combination with the AAV particles or fusion proteins described herein include, but are not limited to, agents currently used for treatment of congestive heart failure including angiotensin converting enzyme (ACE) inhibitors, betablockers, compounds that induce inotropic effects (e.g., increase of force of contraction of the heart) and compounds that increase urine flow, diuretics, angiotensin II (Ang II) and mineralocorticoid receptor antagonists, neprilysin metalloprotease inhibitors, and sodiumglucose co- transporter 2 inhibitors. In some embodiments, the rAAV vector or fusion protein is administered according to the methods as disclosed herein, in combination with another active agent, such as a food-intake-reducing, or plasma glucose-lowering or plasma lipid- lowering agent, such as amylin, an amylin agonist, a CCK, or a leptin, or a cardiac treatment agent such as angiotensin converting enzyme (ACE) inhibitors.

[0171] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0172] In a further aspect, a rAAV vector or fusion protein as disclosed herein is prepared for use as medicament for use in the methods of administration as disclosed herein.

[0173] In a further aspect, a rAAV vector or fusion protein as disclosed herein is prepared for use as medicament for use in the methods of administration as disclosed herein for use in4906-5655-6138 v.l 50Attorney Docket No. 65274.12WO01Customer No. 27683 therapy, i.e. the prevention or treatment of a medical condition or disease, e.g., a cardiovascular disease or a heart disease or disorder as disclosed herein. Exemplary medical conditions or diseases relevant to the present aspect are elsewhere herein.

[0174] In a further aspect, there is provided a cell comprising a rAAV vector as disclosed herein, or a variant thereof. In some embodiments the cell is a mammalian cell, optionally a human cell. Suitably, the cell is a cardiac cell. Suitably the cell may be a cardiomyocyte, e.g., ventricular cardiomyocyte. Suitably the cell may be a human cardiomyocyte, e.g., human ventricular cardiomyocyte.

[0175] In some embodiments, the pharmaceutical composition comprises rAAV vector in a buffer (e.g., excipient) of about pH 7.0 to about pH 8.0. In some embodiments, the pH of the buffer is from about 7.0 to about 7.5. In preferred embodiment, the pH of the buffer is less than 7.5. In several embodiments, the buffer is phosphate buffer saline (PBS) or a phosphate buffer (e.g., 10 mM Phosphate pH 7.4, 350 mM NaCl, 2.7 mM KC1, 5% Sorbitol, 0.001% (w / v) poloxamer 188). In certain embodiments, the buffer or, excipient comprises ions selected from the group consisting of sodium, potassium, phosphate, chloride, calcium, magnesium, sulfate, citrate and any combination thereof. The pharmaceutical composition may further comprise polyol, sugar or, similar. In some embodiment, the pharmaceutical composition comprises glycerol or, propylene glycol, or, polyethylene glycol, or, sorbitol, or mannitol. In several embodiments, the sorbitol concentration ranges from about 1% (w / v) to about 10% (w / v). In some embodiments, the sorbitol concentration ranges from about 2%(w / v) to about 8%(w / v). In preferred embodiments, the sorbitol concentration ranges from about 3%(w / v) to about 6% (w / v). In certain embodiments, the sorbitol concentration is 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or, 10% (w / v). The pharmaceutical composition further comprises a non-ionic surfactant. In some embodiments, the non-ionic surfactant is selected from the group consisting of polyoxy ethylene-poly oxypropylene block copolymers, alkylglucosides, alkyl phenol ethoxylates, polysorbates, polyoxyethylene alkyl phenyl ethers, and any combinations thereof. In some embodiments, the non-ionic surfactant is poloxamer 188 or, Ecosurf SA- 15. In certain embodiments, poloxamer 188 or, Ecosurf SA- 15 concentration is 0.0005% (w / v), 0.0008% (w / v), 0.0009% (w / v), 0.001% (w / v), 0.002% (w / v), 0.0025% (w / v), 0.003% (w / v), 0.0035% (w / v), 0.004% (w / v), 0.0045% (w / v), 0.005% (w / v), 0.006% (w / v), 0.007% (w / v), 0.008% (w / v), 0.009% (w / v), or, 0.01% (w / v).4906-5655-6138 v.l 51Attorney Docket No. 65274.12WO01Customer No. 27683

[0176] rAAV pharmaceutical compositions can comprise at least IxlO9vg / ml recombinant AAV vector as disclosed in the present invention. In some embodiments the pharmaceutical composition comprises about IxlO9vg / ml to about IxlO14vg / ml recombinant AAV vector. In some embodiments, the pharmaceutical composition comprises about IxlO11vg / ml to about IxlO14vg / ml recombinant AAV vector.

[0177] Fusion protein-based compositions a suitable dose of the therapeutic agent for achievement of therapeutic benefit, may, for example, be in a range of about 1 microgram (pg) to about 100 milligrams (mg) per kilogram body weight of the recipient, or in a range of about 10 pg to about 50 mg per kilogram body weight, or in a range of about 10 pg to about 10 mg per kilogram body weight.

[0178] METHODS OF TREATMENT

[0179] In another aspect, the disclosure provides methods of treating or preventing a heart disease (cardiac dysfunction) by administering one or more compositions related to activity in and around mAKAPp. In some embodiments, the method includes administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that binds to mAKAP and inhibits RyR2 activity, binds to RyR2, or reduces RyR2-mediated Ca2+release. In some embodiments, the method of treating or preventing heart disease includes administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that binds to mAKAPp and inhibits adenylyl cyclase activity, binds to P-AR, or suppresses perinuclear cAMP production. In some embodiments, the method of treating or preventing heart disease includes administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that binds to mAKAPp and inhibits CaMKII activity, inhibits CaMKII activity, or binds to CaMKII.

[0180] In various method aspects and embodiments described herein, the heart disease (cardiac dysfunction) includes, e.g., cardiac hypertrophy, ventricular hypertrophy, ventricular myocyte hypertrophy, dilated cardiomyopathy, heart failure, and other cardiac dysfunctions as described elsewhere herein.

[0181] In another aspect, the disclosure provides methods for treating or preventing pathological cardiac remodeling by administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that binds to mAKAPp and inhibits RyR2 activity, binds to RyR2, or reduces RyR2-mediated Ca2+release. In some4906-5655-6138 v.l 52Attorney Docket No. 65274.12WO01Customer No. 27683 embodiments, the method of treating or preventing pathological cardiac remodeling includes administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that binds to mAKAPP and inhibits adenylyl cyclase activity, binds to -AR, or suppresses perinuclear cAMP production. In some embodiments, the method of treating or preventing pathological cardiac remodeling includes administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that binds to mAKAPP and inhibits CaMKII activity, inhibits CaMKII activity, or binds to CaMKII.

[0182] In some embodiments, pathological cardiac remodeling includes concentric or eccentric cardiac hypertrophy, e.g., ventricular myocyte hypertrophy, ventricular hypertrophy, or heart failure, and other cardiac dysfunctions as described elsewhere herein.

[0183] In another aspect, the disclosure provides methods of treating or preventing a disease or condition associated with an increase in perinuclear Ca2+release, including delivering to a patient in need thereof, an amount of one or more compositions as described elsewhere herein sufficient to inhibit the perinuclear Ca2+release in the patient. In some embodiments, the disease or condition affects the heart.

[0184] In some embodiments, the disease or condition associated with an increase in perinuclear Ca2+release includes cardiac hypertrophy, e.g., ventricular myocyte hypertrophy or ventricular hypertrophy, or heart failure. In some embodiments, the disease or condition is caused by, e.g., hypertension, coronary artery disease, myocardial infarction, valvular disease, primary cardiomyopathy, congenital heart disease, arrhythmia, pulmonary disease, diabetes, anemia, or hyperthyroidism.

[0185] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a viral-based gene therapy vector encoding a fusion protein that includes (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin-lot sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 98-1114 of SEQ ID NO:3.

[0186] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac4906-5655-6138 v.l 53Attorney Docket No. 65274.12WO01Customer No. 27683 remodeling includes a viral-based gene therapy vector encoding a fusion protein that includes (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin- la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 193-1209 of SEQ ID NO:5.

[0187] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a viral-based gene therapy vector encoding a fusion protein that includes (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-54 of SEQ ID NO:1, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 125-1141 of SEQ ID NO: 1.

[0188] In some embodiments, the viral vector is adeno-associated virus (AAV), while in some embodiments, the AAV includes AAV2 and / or AAV9.

[0189] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a fusion protein that comprises (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin- la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 98-1114 of SEQ ID NO:3.

[0190] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac remodeling includes a fusion protein that comprises (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 193-1209 of SEQ ID NO:5.

[0191] In some method embodiments of the various aspects described herein, the composition administered to treat or prevent a heart disease or pathological cardiac4906-5655-6138 v.l 54Attorney Docket No. 65274.12WO01Customer No. 27683 remodeling includes a fusion protein that comprises (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-54 of SEQ ID NO:1, and (2) a nesprin- I ot amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 125-1141 of SEQ ID NO:1.

[0012] In some embodiments, the fusion protein is administered directly.

[0193] Evaluation of Treatment

[0194] In some embodiments, the methods to treat a heart disease or pathological cardiac remodeling as disclosed herein result in improvement in at least one parameter from a baseline level in the patient, where the at least one parameter includes (i) ejection fraction (EF or, interchangeably used as Left ventricular ejection fraction or, LVEF), (ii) end systolic volume (ESV), (iii) cardiac contractility, selected from ejection fraction (EF) and fractional shortening (FS), (iv) cardiac volumes selected from any of: end diastolic volume (DV) and end systolic volume (ESV), or (v) a decrease in any of: mortality risk due to the heart disease, reduced hospitalization due to the heart disease symptoms, or therapeutic intervention for treatment of the heart disease.

[0195] In some embodiments, a clinically meaningful change in End Systolic Volume (ESV) is a 10% decrease, or greater than 10% decrease in ESV measured at least 1 month or, at least 3 months, or, at least 6-months, or at least 12-months after administration of the rAAV or fusion protein composition according to the methods as disclosed herein, as compared to the ESV prior to administration of the rAAV or fusion protein composition to the subject. In some embodiments, a clinically meaningful change in ESV is a 10% decrease, or about 11%, or about 12%, or about 13%, or about 14%, or about 15%, or greater than a 15% decrease in ESV measured at least 3 months, or at least 6-months, or at least 12-months after administration of the rAAV or fusion protein composition according to the methods as disclosed herein, as compared to the ESV prior to administration of the rAAV to the subject.

[0196] In some embodiments, a clinically meaningful change in ESV is a 20 ml decrease, or greater than 20 ml decrease in ESV measured at least 1 month, or, at least 3 months, or, at least 6-months, or at least 12-months after administration of the rAAV or fusion protein composition according to the methods as disclosed herein, as compared to the ESV prior to administration of the rAAV or the fusion protein composition to the subject. In some4906-5655-6138 v.l 55Attorney Docket No. 65274.12WO01Customer No. 27683 embodiments, a clinically meaningful change in ESV is a 20 ml decrease, or about 22 ml, or about 23 ml or about 24 ml, or about 25 ml, or about 26 ml, or about 27 ml, or about 28 ml, or about 29 ml, or about 30 ml, or greater than a 30 ml decrease in ESV measured at least 1 month, or, at least 3 months, at least 6-months, or at least 12-months after administration of the rAAV according to the methods as disclosed herein, as compared to the ESV prior to administration of the rAAV to the subject.

[0197] In some embodiments, the methods also include administration of a rAAV vector or fusion protein composition as disclosed herein, according to the methods as disclosed herein, to prevent, inhibit, slow the progression, or at least lessen deleterious or pathological cardiac remodeling, e.g., left ventricular remodeling. Cardiac remodeling may be measured by any method known in the art, including the methods, such as, e.g., echocardiography. As an example, left ventricle chamber size may be used as a measure for cardiac remodeling. In evaluating attenuation of cardiac remodeling, an attenuation of the increase in size of the left ventricle may be an attenuation of any amount as compared with the left ventricle size before administration of a rAAV vector or fusion protein as disclosed herein, according to the methods as disclosed herein.

[0198] In some embodiments, an attenuation of the increase in size of the left ventricle may be an attenuation of any amount as compared with the left ventricle size of a matched control subject receiving vehicle only. Left ventricle chamber size may be measured, for example, by assaying left ventricle end diastolic dimension (LVEDD) or left ventricle end systolic dimension (LVESD). In an example, the change in LVEDD at least 3- or at least 6- months after administration with a rAAV vector or fusion protein as disclosed herein, according to the methods as disclosed herein may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more than about 200% as compared to the levels measured at, or before administration of the rAAV or fusion protein. In another example, the change in LVESD at least 3- or at least 6-months after administration with a rAAV vector or fusion protein as disclosed herein, according to the methods as disclosed herein may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more than about 200% as compared to the LVESD level measured at, or before administration of the rAAV or fusion protein.

[0199] ADMINISTRATION4906-5655-6138 v.l 56Attorney Docket No. 65274.12WO01Customer No. 27683

[0200] One aspect of the technology described herein relates to a method to administer a rAAV vector, where the method is a single administration of a total dose of a rAAV to the subject, where the single administration comprises delivery of a total dose of rAAV that is divided into at least 2, or 3, or 4, or 5 or more sub-doses within the single administration. That is, in some embodiments, the method comprises administering a bolus of rAAV vector to the subject in a single administration, where the single administration of the bolus comprises the administration of rAAV from least 2, or 3, or 4, or 5 doses. Other aspects of the technology described herein relate to a method to administer a rAAV vector, where the method comprises at least one administration or, more than one administration of rAAV to the subject. In some aspects of the embodiment, the method to administer rAAV vector comprises two administrations, three administrations, four administrations, or, five administrations of rAAV to the subject, where each administration comprises delivery of a total dose of rAAV that is divided into at least 2, or, 3, or, 4, or, 5 or, more sub doses.

[0201] In some embodiments, the method to administer rAAV vectors is a single injection that comprises within the single injection, discrete pulses of delivery of the rAAV vector. That is, in a single injection administration, the rAAV delivery is divided into a number of temporally spaced sub-administrations. For example, a single administration can be a total amount (or total dose, also referred to as “TD”) of rAAV that is divided into at least 2, or at least 3, or at least 4, or at least 5 or more sub-doses (“SD”), where each sub-dose is administered in a sub-administration, where each sub-administration is temporally spaced by a pre-defined period of time, e.g., at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 or more than 10 minutes between each sub-administration of each sub-dose. For example, a single administration of a total dose of rAAV can include a series of pulses of sub-doses, and each sub-dose is injected in a sub-administration (i.e., pulses of a single administration).

[0202] Without being limited to theory, an exemplary method of administration comprises administration of a single administration of a total dose (TD) of rAAV vector between about 1013vg (vector genomes) to about 1015vg, which can be divided into at least 2, or at least 3, or at least 4, or at least 5 or more sub-doses (SD), wherein the sub-doses are administered to the subject spaced at least 5 seconds, or at least 10 seconds, or at least 20 seconds, or at least 30 seconds, or at least 40 seconds, or at least 50 seconds or at least 1 minute, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at4906-5655-6138 v.l 57Attorney Docket No. 65274.12WO01Customer No. 27683 least 8, or at least 9, or at least 10 or more than 10 minutes apart, wherein the administration of all the sub-doses for the total rAAV dose takes between about 10 minutes to about 30 minutes, or between about 10 minutes to about 20 minutes, or between about 15 minutes to about 25 minutes, or between about 15 minutes to about 30 minutes, or, between about 25 minutes to about 30 minutes, or, between about 20 minutes to about 40 minutes, or between about 40 minutes to about 60 minutes, or more than 60 minutes, and wherein each subdose is administered over a period of time of 1 minute, or about 2 minutes, or, about 3 minutes or, about 4 minutes, or about 5 minutes, or about 6 minutes, or about 7 minutes or, about 8 minutes or about 9 minutes, or about 10 minutes or longer. In some embodiments, wherein the total rAAV dose administration is performed for about 10 minutes, or, about 15 minutes, or about 20 minutes, or about 25 minutes or about 30 minutes or about 35 minutes or, about 40 minutes or, about 45 minutes, or about 50 minutes, or about 60 minutes or longer. In certain embodiments, the total rAAV dose administration is performed for about 20 minutes to about 30 minutes. In certain aspects of the embodiments, the rAAV is AAV2, AAV6, AAV8, or AAV9. In some aspects of the embodiments, the rAAV administration is performed for one to five minutes in each of total five subdoses, e.g. five syringes, wherein each subdose has 8 ml, or 9 ml, or 10 ml, or 12 ml, or 15 ml, or 20 ml, or 25 ml or more volume of diluent. In certain aspects of the embodiments, the total volume of rAAV administration is 20 ml, or 25 ml, or 30 ml, or 35 ml, or 40 ml, or 45 ml, or 50 ml, or 60 ml, or 70 ml, or 80 ml, or 90 ml, or 100 ml or, more. Without limiting to any theory, the diluent can be saline, or different ratios of saline-blood mixture.

[0203] In some embodiments, where a single administration of a total dose (TD) of rAAV vector is divided into at least 2, or at least 3, or at least 4, or at least 5 or more sub-doses (SD), each sub-dose can be administered or injected into the subject over a pre-defined time period, e.g., at least 1 , or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 or more than 10 minutes, and wherein there is an interval of at least 1 , or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 or more than 10 minutes between administration of each sub-dose. In some embodiments, each sub-dose is administered over a period of 1-5 minutes. In some embodiments, the time interval between the sub-doses can be consistent, e.g., same time in-between each sub-dose, or can vary. For example, in an exemplary administration method where the total dose (TD) of a rAAV vector is divided into4906-5655-6138 v.l 58Attorney Docket No. 65274.12WO01Customer No. 276835 sub-doses (sdl, sd2, sd3, sd4, sd5), the interval between administration of sdl and sd2 can be, e.g., at least 2 minutes, and the interval between administration of sd3 after sd2 can be, e.g., 5 minutes.

[0204] In some embodiments, the single administration of the rAAV vector is coadministered with an additional agent, or therapeutic agent. In some embodiments, the additional agent is an immune modulator as disclosed herein. In some embodiments, the additional agent is administered before, or after, or both (before and after) the single injection of the complete rAAV dose. In some embodiments, the additional agent (e.g., immune modulator) is administered to the subject in the intervals between sub-doses of the rAAV, that is - for example, in an exemplary administration method where the total dose (TD) of a rAAV vector is divided into 5 sub-doses (sdl, sd2, sd3, sd4, sd5), the additional agent, e.g., immune modulator can be administered between any one or more of: between sdl and sd2, between sd2 and sd3, between sd3 and sd4, between sd4 and sd5. In some embodiments, the additional agent, e.g., immune modulator is present in the sub-doses of rAAV.

[0205] In some embodiments, the total-dose of the rAAV is selected from any of: about 1011vg, about 3xl0nvg, about 5xl0nvg, about 1012vg, about 3xl012vg, about 5xl012vg, about 1013vg, about 3xl013vg, about 1014vg, 3xl014vg, about 1015vg, or 3xl015vg, or more than about 1016vg. In some embodiments, total-dose of the rAAV is between about 1013vg to about 1016vg. In some embodiments, at least one, or at least two or at least three or more total doses of rAAV is administered. In some embodiments, each subdose of rAAV is between about 1011vg to about 1016vg. In certain embodiments, each subdose of rAAV is between about 1013vg to about 1016vg. In one embodiment, each subdose of the rAAV is selected from any of about 1011vg, about 3xl0nvg, about 5x 1011vg, about 1012vg, about 3xl012vg, about 5xl012vg, about 1013vg, about 3xl013vg, about 1014vg, 3xl014vg, or, about 1015vg, or more than about 1016vg.

[0206] In some embodiments, administration of rAAV vector or virion comprising the synthetic cardiac-specific promoter or expression cassette according to this invention is intravascular. Suitably, the rAAV vector or virion comprising the synthetic cardiac-specific promoter (e.g., cTnT) or expression cassette according to the disclosure may be administered in the veins of the dorsal hand or the veins of the anterior forearm. Suitable veins in the anterior forearm are the cephalic, median or basilic veins. This is because this administration route is generally safe for the patient.4906-5655-6138 v.l 59Attorney Docket No. 65274.12WO01Customer No. 27683

[0207] In some embodiments, the rAAV vector is directly injected into heart tissue. Direct injection or application of a viral vector into the myocardium can restrict expression of the transferred genes to the heart.

[0208] In some embodiments, the rAAV vector is introduced into the lumen of one or more coronary arteries. Passage of blood out of the coronary arteries can be restricted. The preparation comprising rAAV vectors can be delivered antegrade and allowed to reside in the arteries for between one to five minutes, e.g., between one to three minutes. Non- viral vehicles may be delivered by similar methods.

[0209] In some embodiments, the rAAV vector can be administered to a subject by standard methods. For example, the agent can be administered by any of a number of different routes including intravenous (systemic), intradermal, subcutaneous, oral (e.g., inhalation or ingestion), transdermal (topical), transmucosal or by catheter or, by syringes, or, by a combination of catheter and syringe. In one embodiment, the agent is administered by injection, e.g., intra-arterially, intramuscularly, or intravenously. In still another embodiment, the rAAV vector as disclosed herein is directly injected into a muscle of the heart.

[0210] In some embodiments, flow of blood through coronary vessels of the heart of the subject is restricted, and the rAAV vector as disclosed herein is introduced into the lumen of a coronary artery in the subject. In yet another embodiment, the heart is pumping while coronary vein outflow is restricted. In yet another embodiment, flow of blood through the coronary vessels is completely restricted. The restricted coronary vessels may comprise, without limitation: the left anterior descending artery (LAD), the distal circumflex artery (LCX), the great coronary vein (GCV), the middle cardiac vein (MCV), or the anterior interventricular vein (AIV). In yet another embodiment, the introduction of the rAAV vector as disclosed herein occurs after ischemic preconditioning of the coronary vessels. In still another embodiment, the rAAV vector as disclosed herein is injected into the heart of the subject while aortic flow of blood out of the heart is restricted, thereby allowing the nucleic acid molecule to flow into the heart.

[0211] In some embodiments, a rAAV vector as disclosed herein is injected into the heart by a method comprising the steps of: restricting aortic flow of blood out of the heart, such that blood flow is re-directed to coronary arteries; injecting the vector into lumen of the heart, aorta or coronary ostia such that the vector flows into the coronary arteries; permitting the4906-5655-6138 v.l 60Attorney Docket No. 65274.12WO01Customer No. 27683 heart to pump while the aortic flow of blood out of the heart is restricted; and reestablishing the aortic flow of blood. In a more specific embodiment, a rAAV vector as disclosed herein is injected into the heart with a catheter, and in an even more specific embodiment, a rAAV vector as disclosed herein is directly injected into a muscle of the heart.

[0212] The present disclosure also provides a method of gene therapy of a subject, preferably a human, in need thereof, the method comprising: administering to the subject (suitably introducing into the heart of the subject) a synthetic cardiac-specific expression cassette, vector, virion or pharmaceutical composition of the present invention, which comprises a gene encoding a fusion protein that includes nesprin- 1 a and at least a second domain such as a fragment of Bcl-2, Nb80, or AIP.

[0213] The method suitably comprises expressing a therapeutic amount of a fusion protein of nesprin- la and at least a second domain such as a fragment of Bcl-2, Nb80, or AIP, in the heart tissue of said subject.

[0214] Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo, are well-known in the art and will not be discussed in detail here. Briefly, they include intravenous or intraarterial administration (e.g., intra-carotid artery, intra-hepatic artery, intra-hepatic vein), intracerebroventricular, intracranial administration, intramuscular injection, interstitial injection, instillation in airways, application to endothelium and intra- hepatic parenchyma, of plasmid DNA vectors (naked or in liposomes) or viral vectors. Various devices have been developed for enhancing the availability of DNA to the target cell. While a simple approach is to contact the target cell physically with catheters or implantable materials containing the relevant vector, more complex approaches can use jet injection devices and the like. Gene transfer into mammalian heart cells can been performed using both ex vivo and in vivo procedures. The ex vivo approach typically requires harvesting of heart cells (e.g., cardiomyocytes), in vitro transduction with suitable expression vectors, followed by reintroduction of the transduced cardiomyocytes into the heart. This approach is generally less preferred due to the difficulty and danger of harvesting and reintroducing cardiomyocytes in the heart. However, ex vivo approaches may be useful for the pretreatment of hearts intended for cardiac transplantation to improve long term outcome for transplant patients. In vivo gene transfer has been achieved by injecting DNA or viral vectors directly into the heart, by intracranial injection, or by intravenous or intraarterial injection of viral vectors.4906-5655-6138 v.l 61Attorney Docket No. 65274.12WO01Customer No. 27683

[0215] In one embodiment, the gene therapy vector may be administered to a subject (e.g., to the heart of a subject) in a therapeutically effective amount to reduce the symptoms of a heart disease of a subject (e.g., determined using a known evaluation method).

[0216] As described above for rAAV compositions, the desired dose of fusion protein composition may be presented as one dose or two or more sub-doses administered at appropriate intervals throughout the dosing period (e.g., one week, two weeks, etc.). These sub-doses can be administered in unit dosage forms, for example, containing from about 10 pg to about 500 mg, or from about 50 pg to about 200 mg, or from about 50 pg to about 100 mg of active ingredient per unit dosage form. Alternatively, if the condition of the recipient so requires, the doses may be administered as a continuous infusion.

[0217] Viral vectors

[0218] AAV vectors have been extensively discussed in the art. AAV vectors are of particular interest as AAV vectors do not typically integrate into the genome and do not elicit immune response. AAV serotypes 1, 2, 4, 5, 8, 9, rhlO, DJ8 and 2g9 (AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, AAVrhlO, AAVDJ8 and AAV2g9) have been noted to achieve efficient transduction in the heart. Therefore, AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, AAVrhlO, AAVVDJ8, AAV2g9, AAV2i8 and derivatives thereof are preferred AAV serotypes. In some embodiments, AAV2 or AAV9 are preferred AAV vectors. In other embodiments, AAV9 is a particularly preferred AAV. Suitably an AAV vector comprises a viral genome which comprises a nucleic acid sequence of the present invention positioned between two inverted terminal repeats (ITRs). AAV vectors for use herein comprise a virus that has been reduced to the minimum components necessary for transduction of a nucleic acid payload or cargo of interest, i.e., rAAVs. In this manner, rAAV vectors are engineered as vehicles for specific delivery while lacking the deleterious replication and / or integration features found in wild-type viruses.

[0219] Methods of making AAV vectors are well known in the art and are described in e.g., U.S. Pat. Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948; or International Publication Nos. WO1996039530, W01998010088, WO 1999014354, WO 1999 / 015685, WO 1999 / 047691, W02000 / 055342,4906-5655-6138 v.l 62Attorney Docket No. 65274.12WO01Customer No. 27683W02000 / 075353 and W02001 / 023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O’Reilly et al, Baculovirus Expression Vectors, A LaboratoryManual, Oxford Univ. Press (1994); Samulski et al., J Fir.63:3822-8 (1989); Kajigaya et al, Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al, Vir., 219:37-44 (1996); Zhao et al, Vir.272: 382-93 (2000); the contents of each of which are herein incorporated by reference.

[0220] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0221] EXAMPLES

[0222] Example 1

[0223] Bcl2BH4

[0224] In disease, the heart undergoes pathological cardiac remodeling that includes the hypertrophy, impaired contractility, altered metabolism, and death of cardiomyocytes, as well as myocardial inflammation and fibrosis. This remodeling is controlled by a network of cardiomyocyte signaling pathways that together determine individual myocyte and overall cardiac phenotype. Accordingly, current guideline-directed medical therapy for DCM and other forms of heart failure with reduced ejection fraction is based upon the inhibition of proremodeling signaling pathways, including P-adrenergic (PAR), angiotensin II (Ang II), and mineralocorticoid receptors and more recently sodium-glucose co-transporter 2. However, the often poor outcome associated with DCM compels the development of new therapies. The identification of new drug targets has been complicated by the pleiotropy and ubiquitous expression of many relevant signaling enzymes. One approach to identifying therapeutically tractable targets is to focus on nodes within the myocyte signaling network where pathways converge to regulate remodeling. Nodes in signaling networks are often formed by multivalent scaffold proteins that bind signaling enzymes and ion channels in discrete intracellular compartments, providing the architectural underpinnings for the organization and co-regulation of intersecting signaling pathways. Scaffold proteins form “signalosomes” that confer specificity and efficacy by enhancing local signaling intermediate effective concentration, bringing together enzymes and relevant substrates, and facilitating pathway4906-5655-6138 v.l 63Attorney Docket No. 65274.12WO01Customer No. 27683 crosstalk. The approach to DCM taken herein involves targeting a downstream node in the cardiomyocyte pathological signaling network that is responsible, at least in part, for the development of the pathological phenotype characteristic of DCM.

[0225] The mAKAPp Perinuclear Signalosome

[0226] mAKAPp is a 230 kDa scaffold protein localized to the nuclear envelope by binding nesprin-l a in cardiac myocytes (FIG. 7, Panel A). There, mAKAPp organizes multimolecular signalosomes that integrate Ca2+, cAMP, phosphatidylinositol 4-phosphate, mitogen-activated protein kinase, and hypoxic upstream signaling regulating myocyte transcription factors and class Ila histone deacetylases. By orchestrating critical gene regulatory pathways, mAKAPp serves as a central node in the myocyte pathological remodeling signaling network. A key constituent of mAKAPp signalosomes is the Ca2+ / calmodulin-dependent phosphatase calcineurin (CaN) required for the induction of pathological hypertrophy. mAKAPp is necessary for the CaN-dependent nuclear translocation and activation of NFATc transcription factors, including NFATc2 and NFATc3 which are required for the induction of pathological cardiac hypertrophy. In addition, mAKAPP-bound CaN regulates MEF2D, another transcription factor required for pathological remodeling whose increased activity promotes a DCM phenotype. Consistent with its recruitment of CaN and other pro-remodeling signaling enzymes, it has been found that mAKAPp is required for the induction of pathological cardiac remodeling and heart failure by chronic pressure overload, catecholamine infusion, and myocardial infarction in mice. Thus, the mAKAPp signalosome is identified herein as a candidate target for the treatment of pathological cardiac remodeling in cardiovascular disease. Notably, mAKAP (AKAP6) expression is increased in left ventricular (LV) myocytes of a human PLN R14del DCM patient (FIG. 7, Panel B), supporting further the hypothesis elucidated herein that mAKAPp signalosomes play a key role in promoting the DCM pathological phenotype.

[0227] It has been discovered by the inventors that in cardiomyocytes, mAKAPp comprises a compartment in which Ca2+is regulated independently of cytosolic Ca2+mediating excitation-contraction coupling. To detect Ca2+transients in the mAKAPp compartment, the fluorescent Ca2+biosensor Cameleon was expressed in fusion to nesprin-la (FIG. 8A, FIG. 8B). By expression of mAKAP small hairpin RNA (shRNA), mAKAPp expression was found to be required for nesprin- 1 a-associated Ca2+transients induced by the PAR agonist isoproterenol (Iso, FIG. 8C, FIG. 8D). Inhibition of mAKAPp expression had4906-5655-6138 v.l 64Attorney Docket No. 65274.12WO01Customer No. 27683 no effect on PAR-induced Ca2+transients in the bulk cytosol, revealing the existence of a separately regulated Ca2+signaling compartment at mAKAP and nesprin-la. Accordingly, depletion of Ca2+in the mAKAPP compartment using a perinuclear localized Ca2+binding protein did not affect PAR-induced contractility of paced adult myocytes, while inhibiting PAR-induced myocyte hypertrophy.

[0228] Both IP3 receptor and RyR2 Ca2+release channels have been detected on or near the cardiomyocyte nuclear envelope. By co-immunoprecipitation, the association of RyR2 has been detected, but not IP3R with nesprin-la and mAKAPP (FIG. 9). Accordingly, perinuclear Ca2+transients in myocytes were inhibited by the RyR2 channel blocker ryanodine (Ry). RyR2 channel opening can be increased by PKA phosphorylation, albeit the relevance of PKA-dependent RyR2 phosphorylation to excitation-contraction (E-C) coupling is controversial. In response to PAR stimulation, the phosphorylation of nesprin-1- associated RyR2 on Ser-2808 and Ser-2030 was mAKAPP dependent, consistent with mAK PP recruitment of PKA to the perinuclear compartment (FIG. 10). Given that RyR2 is the major SR Ca2+release channel in the myocyte, the pool of RyR2 associated with nesprin-la and mAKAPP comprises a very small fraction of total myocyte RyR2. Consistent with mAKAPP perinuclear localization, mAKAPP was not required for PKA phosphorylation of the majority of RyR2 present in the myocyte.

[0229] To demonstrate the relevance of perinuclear RyR2 to Ca2+release into the mAKAPP compartment, nesprin-la fusion proteins have been deployed containing RyR2 binding peptides that limit the action of the modulatory peptides to near the outer nuclear membrane. A Bcl2BH4-mCherry-nesprin fusion protein, that contains a RyR2-inhibiting peptide, inhibited norepinephrine (NE) - induced perinuclear, but not cytosolic Ca2+transients (FIG. 11). Conversely, expression of the BlaCT-mCherry-nesprin, containing the C-terminal 35 residues of the DHPR pia subunit that will bind and increase RyR pore opening, selectively increased perinuclear, but not cytosolic Ca2+transients in the absence of adrenergic stimulation. Together these data support the hypothesis that the mAKAPP perinuclear compartment comprises a RyR2-dependent Ca2+signaling compartment independent of the SR dyad where RyR2 participates in E-C coupling.

[0230] CaN phosphatase is present in diverse intracellular compartments within the cardiomyocyte, where it serves both normal physiological functions and controls the induction of pathological hypertrophy. The involvement of CaN phosphatase in mAKAPP4906-5655-6138 v.l 65Attorney Docket No. 65274.12WO01Customer No. 27683 signalosomes has been characterized. In the heart mAKAPp binds the A isoform of CaN required for pathological hypertrophy. mAKAPp binds active CaNAp, and that binding is important for the dephosphorylation and nuclear translocation of NFAT transcription factors and the induction in vitro of myocyte hypertrophy. mAKAPp also brings together CaNAp and MEF2 transcription factor. mAKAPp / CaN-dependent MEF2D dephosphorylation promotes MED2D desumoylation and acetylation and a switch from HDAC5-repressed to p300 acetylase-activated MEF2D complexes that promote hypertrophy. Consistent with data showing that CaN activity within the mAKAPp compartment requires elevated local Ca2+levels activity. Bcl2BH4-mCherry-nesprin, which inhibited perinuclear RyR2 Ca2+release in cardiomyocytes (FIG. 11), inhibited NFAT nuclear translocation and the expression of atrial natriuretic factor (ANF), a MEF2-regulated marker for myocyte hypertrophy (FIG. 12). As a control, Bcl2BH4-mCherry-nesprin inhibition was prevented by stimulation of RyR2 with low dose (5 nM) ryanodine that locks the channel in active conformation. Conversely, expression of BlaCT-mCherry-nesprin, which activates perinuclear RyR2 in neonatal myocytes (FIG. 11), increased NFAT nuclear localization 2.9-fold and ANF expression 2.0- fold in the absence of NE stimulation.

[0231] The data suggest that perinuclear Ca2+within the mAKAPP-nesprin- 1 a-RyR2 compartment is a key mediator of pathological cardiac remodeling. To test this hypothesis by gain-of- function in vivo, the RyR2 activating perinuclear B 1 aCT fusion peptide was expressed under the control of the chicken cardiac troponin T (cTNT) promoter using AAV9 in adult wildtype C57BL / 6N mice. Note that the mCherry tag was removed due to AAV genome length limitations. Within two months of administration, mice injected with AAV9.BlaCT-nesprin exhibited prominent systolic dysfunction (decreased ejection fraction) and cardiac hypertrophy, while control mice injected with AAV9.nesprin or no AAV exhibited no apparent abnormal phenotype (FIG. 13). 8 weeks after injection, the BlaCT- nesprin cohort exhibited significant ventricular dilatation compared to nesprin control [end- systolic volume (pL): 40+1 vs. 27+1 ; p<0.001; end-diastolic volume (pL): 66+2 vs. 58+1, p = 0.01]. These results imply that activation of Ca2+signaling at mAKAPp is sufficient to induce a DCM-like phenotype and, in conjunction with results obtained by mAKAPp gene knock-out, define the perinuclear compartment as critical for the regulation of pathological cardiac remodeling.

[0232] Example 24906-5655-6138 v.l 66Attorney Docket No. 65274.12WO01Customer No. 27683

[0233] A perinuclear RyR2 Compartment for Ca2+Signaling regulating gene expression

[0234] The development of AAV9.Bcl2BH4-nesprin as a biologic drug is based upon the concept that mAKAPP organizes an independent Ca2+signaling compartment controlled by local RyR2 Ca2+release (FIG. 1). Like other second messengers, Ca2+is spatially restricted in the cardiomyocyte within discrete intracellular compartments resulting in differential regulation of downstream signaling pathways. In the cardiomyocyte, the sarcoplasmic reticulum (SR) and nuclear envelope form an interconnected high capacity Ca2+store, from where Ca2+can be differentially released into the cytosol or nucleoplasm depending upon the local activity of Ca2+release channels. For example, RyR2-mediated Ca2+release from junctional SR into SR-transverse tubule dyads initiates contraction during normal E-C coupling. The inventors have shown that on the cytosolic face of the outer nuclear membrane mAKAPP organizes an independent Ca2+compartment containing RyR2. This compartment exists even though the mAKAPP compartment is not separated from the bulk cytosol and myofibrils by a membrane barrier. mAKAPP does not directly regulate E-C coupling. Instead, local RyR2-dependent Ca2+release at mAKAPP signalosomes regulates mAKAPP- bound CaN, providing a mechanism for the compartmentalized regulation of gene expression and pathological hypertrophy. Inhibition of perinuclear RyR2-dependent Ca2+release constitutes a method for the inhibition of pathological cardiac remodeling which will not affect E-C coupling or induce arrythmia. It is shown in, e.g., Examples 1 and 3 that AAV9.Bcl2BH4-nesprin comprises a first-in-class biologic drug for the treatment of DCM representing this new approach to the inhibition of pathological cardiac remodeling.

[0235] Example 3

[0236] Treatment of mouse model of human familial dilated cardiomyopathy (TM54 DCM mice) by gene therapies that inhibits perinuclear RyR2 Ca2+ release (Bcl2CH4 AAV) or PAR (Nb80 AAV) improved the cardiac structure and function of the TM54 mouse

[0237] To test whether perinuclear pARs and RyR2 are required for pathological cardiac remodeling, the inventors conducted a treatment study in a mouse model of human Familial Dilated Cardiomyopathy. The “TM54” FVB / N mouse contains a transgene that expresses under the control of the cardiac myocyte-specific a-myosin heavy chain promoter a mutant4906-5655-6138 v.l 67Attorney Docket No. 65274.12WO01Customer No. 27683 cDNA for the sarcomeric protein a-tropomyosin. The a-tropomyosin E54K mutation causes decreased myofilament Ca2+sensitivity and tension development, which manifests as prominent cardiac systolic dysfunction and early onset cardiomyopathy. 6-8-week-old TM54 mutant mice with obvious cardiac dysfunction (~20% decrease in ejection fraction) and wildtype littermate mice (NTG) with normal function were randomized for treatment with AAV to express AAV9.Nb80-nesprin, AAV9.Bcl2BH4-nesprin or AAV9.nesprin control or for no treatment at all. Following treatment, nesprin control or non-injected AAV-naive TM54 mice exhibited a progressive decline in cardiac systolic function, as evident by a significant decline in LV ejection fraction (11% and 9% decrease in LV ejection fraction, respectively, for non-injected and AAV9.nesprin cohorts, respectively, p<0.0001 vs. initial time point, FIG. 14A, Panel A). Remarkably, TM54 mice treated with AAV9.Nb80-nesprin and AAV9.Bcl2BH4-nesprin exhibited an improvement of 7.3 and 7.0% in ejection fraction (p<0.0001 vs. initial time point), resulting in a 17-20% improvement in ejection fraction at endpoint in comparison to the control cohorts (p < 0.0001). The improvement in systolic cardiac function was also evident by the significantly less LV end-systolic volume for treated compared to control mice (27-34% decreased ESV, p < 0.0001, FIG. 14A, Panel B). Notably, none of the AAV vectors had any significant effect on the cardiac function of wildtype littermates. The improvement in TM54 cardiac function conferred by AAV9.Nb80- nesprin and AAV9.Bcl2BH4-nesprin treatment was accompanied by decreased DCM- associated cardiac hypertrophy (FIG. 14A, Panel C). These results indicate that treatment with an AAV9.Nb80-nesprin or AAV9-Bcl2BH4-nesprin gene therapy vector improved the cardiac structure and function of the TM54 mouse, providing proof-of-concept for the use of these biologies in Dilated Cardiomyopathy.

[0238] Example 4

[0239] Perinuclear P-Adrenergic Receptors are Necessary and Sufficient for Cardiac Hypertrophy

[0240] Pathological cardiac hypertrophy development is controlled by networks of signaling pathways, integrated by scaffold proteins that localize signaling enzymes and facilitate crosstalk between these pathways, leading to upregulation of hypertrophic transcription factors. It is demonstrated below and herein that a protein scaffold at the nuclear envelope, muscle A-Kinase Anchoring Protein (mAKAPP), is required for the induction of cardiac hypertrophy. Among the various pathways affected, mAKAPP particularly tethers4906-5655-6138 v.l 68Attorney Docket No. 65274.12WO01Customer No. 27683 downstream targets of P-adrenergic receptors (PAR), and this localization provides the framework for stress-related gene transcription in cardiomyocytes. Although the traditional dogma of adrenergic receptor signaling posits that receptor activation begins at the plasma membrane, intracellular adrenergic receptor localization and activation is demonstrated herein, as with evidence of a perinuclear cAMP domain that is dependent on mAKAP expression. Novel peptides localized to mAKAP were used to stimulate or inhibit perinuclear PARs within proximity. Perinuclear pARs are shown to be necessary and sufficient for cardiac hypertrophy development in neonatal and adult rat cardiac myocytes, constituting a functionally independent cAMP domain.

[0241] Example 5

[0242] mAKAPp Associated Ryanodine Receptor Modulation of CalcineurinMediated Gene Transcription

[0243] Ca2+ / calmodulin dependent phosphatase calcineurin (CaN) promotes myocyte differentiation through dephosphorylation of the transcription factor NFAT. It is demonstrated herein that CaN associates with scaffold muscle-specific A-Kinase-Anchoring Protein (mAKAPp), located at the nuclear envelope via interaction with the nesprin-la protein in myocytes. Notably, this interaction requires a Ca2+release, ostensibly not obtained through cycled Ca2+during contraction. In cardiac myocytes the inventors have shown mAKAPp expression is required for dephosphorylation of NFAT; achieved through release of perinuclear Ca2+transients from perinuclear ryanodine receptors (RyRs). CaN activity in a myoblast cell line is shown to also be supported by perinuclear RyRs specifically within proximity of mAKAPp. Using novel, mAKAPP-targeted tools specific for RyR, the activity of perinuclear RyRs is shown to be necessary and sufficient for CaN-modulated NFAT activation and myogenic differentiation. Perinuclear RyR are shown to act independently from cytosolic RyR in a myoblast cell line using targeted fluorescent biosensors to measure CaN activity and Ca2+release, suggesting a novel, non-canonical function for RyR. Given the data shown below and herein, mAKAPP-associated RyR are promising candidates for therapeutic use.

[0244] Example 6

[0245] Nb80

[0246] An intracellular pool of f-adrenergic receptors activates AKAP6fi-bound PKA4906-5655-6138 v.l 69Attorney Docket No. 65274.12WO01Customer No. 27683

[0247] Compartmentation of cAMP-PKA signaling is conferred in part by association with AKAPs restricted to specific sites within the cell. AKAP6 is localized to the ONM by nesprin-la. In past studies of PKA activity at AKAP6 signalosomes in live cells, the ratiometric Fluorescence Resonance Energy Transfer (FRET) biosensor AKAR4-nesprin has been utilized, herein termed “0NM-AKAR4” (FIG. 37A,B), which is comprised of the PKA activity reporter AKAR4 in fusion to nesprin-la and has been shown to have a similar dynamic range as the parental AKAR4 sensor. Importantly, in neonatal and adult rat ventricular myocytes and hippocampal neurons, perinuclear PKA activity detected by ONM- AKAR4 has been shown to be dependent upon AKAP6 expression, while the diffusely localized cytosolic parent AKAR4 sensor is AKAP6 independent. The nuclear envelope is distant from the plasma membrane where PARs are canonically activated. The AKAR4 sensors are now used to test the hypothesis that an intracellular pool of PARs selectively activates AKAP6P-associated perinuclear cAMP-PKA signaling in the cardiac myocyte.

[0248] To investigate if AKAP6P-bound PKA is activated by intracellular pARs, a pharmacologic approach was initially used based upon the different membrane permeabilities of PAR antagonists. In primary neonatal rat ventricular myocytes, biosensor activity detected by AKAR4 and 0NM-AKAR4 in the cytosol and at the nuclear envelope, respectively, was similar in response to 10 nM norepinephrine (NE, FIG. 37C-D). Likewise, NE-stimulated PKA activity was inhibited by the membrane permeable P-blocker propranolol regardless of the location of the FRET biosensor. In contrast, the membrane impermeable P-blocker sotalol inhibited NE-stimulated cytosolic AKAR4, but not perinuclear 0NM-AKAR4 PKA signals. These results suggest that in contrast to PKA in the cytosol, the perinuclear AKAP6P compartment is not activated by PARs on the plasma membrane exposed to the outside of the cell.

[0249] While PARs on the plasma membrane can be activated by extracellular ligands, local activation of intracellular PARs presumably requires ligand entry into the cell. The endogenous catecholamines norepinephrine and epinephrine can enter cells through the non- selective organic cation transporter-3 (OCT3). Consistent with the inhibition of cytosolic AKAR4 signals by the membrane impermeant P-blocker sotalol, the OCT3 inhibitor corticosterone had no effect on NE-induced AKAR4 transients in neonatal myocytes (FIG. 37C). In contrast, corticosterone completely inhibited NE-induced 0NM-AKAR4 signals, which were resistant to sotalol inhibition (FIG. 37D). Together, these results indicated that4906-5655-6138 v.l 70Attorney Docket No. 65274.12WO01Customer No. 27683PKA confined to the perinuclear AKAP6 signalosome is activated by intracellular PARs binding ligand within the cell, while cytosolic PKA is stimulated by plasma membrane receptors binding ligand outside of the cell. Notably, despite the well-established differences in ultrastructure between cultured primary neonatal and adult rat ventricular myocytes, similar results were obtained for the -blockers and corticosterone in experiments employing adult myocytes (data not shown).

[0250] Perinuclear fl-adrenergic receptors are necessary and sufficient for activation of AKAP6 fl-bound PKA.

[0251] The camelid single chain nanobody Nb80 binds agonist-occupied Pi- and Pi- adrenergic receptors, inhibiting downstream signaling, including cAMP production. To target directly -adrenergic receptors near AKAP6 signalosomes, Nb80 was expressed in fusion to nesprin-la and the fluorescent tag mCherry (ONM-Nb80), restricting localization of the nanobody to the ONM (FIG. 38A-B). Over-expression of control mCherry-nesprin (ONM- control) had no effect on PKA activity in neonatal myocytes, either when detected with the cytosolic or perinuclear AKAR4 sensor (FIG. 38C-D). ONM-Nb80 expression had no effect on cytosolic PKA activity in myocytes (FIG. 38C). Remarkably, Nb80 tethered to the ONM by nesprin-la completely prevented NE- stimulation of PKA activity in the perinuclear compartment, demonstrating a requirement for active PAR at the ONM for activation of AKAP6P-bound PKA (FIG. 38D).

[0252] While Nb80 inhibits agonist-dependent PAR signaling, the pepducin ICL3-9, a peptide based upon the 2AR third intracellular loop, selectively activates AR-dependent Gas signaling in the absence of ligand binding and P-arrestin recruitment. In order to test whether PAR activation at or near the ONM is sufficient for activation of AKAP6 signalosomes, ICL3-9 was similarly expressed in fusion to nesprin-la and mCherry (ONM- ICL3-9, FIG. 38E-F). As expected, perinuclearly restricted expression of the pepducin had no effect on basal PKA activity in neonatal myocytes detected by the cytosolic AKAR4 parent sensor. Remarkably, expression of ONM-ICL3-9 increased 0NM-AKAR4 FRET ratio in the absence of any ligand stimulus when compared with ONM-control, implying direct activation of PARs near AKAP6 signalosomes on the ONM. Similar results for ONM-Nb80 and ONM-ICL3-9 were obtained using adult myocytes (data not shown). Thus, consistent with the results obtained using PAR and OCT3 inhibitors, expression of the Nb80 and ICL3-9 mcherry-nesprin- la fusion proteins demonstrated that activation of PAR at or near the ONM4906-5655-6138 v.l 71Attorney Docket No. 65274.12WO01Customer No. 27683 is necessary and sufficient for activation of PKA at AKAP6P signalosomes, thereby showing the functional relevance of intracellular PARs to compartment-specific signal transduction.

[0253] Golgi-localized i / \Rs activate PKA in AKAPAfi signalosomes.

[0254] Besides the canonical plasma membrane location of GPCRs, PARs have been detected on multiple internal membranes including endosomes, Golgi apparatus, and the nuclear envelope, both as part of receptor recycling and down-regulation, as well as mediating downstream signaling within the cell. It is, therefore, not obvious on which membranes are present the PARs activating AK P6P signalosomes. For example, in cardiac myocytes the Golgi is in close proximity to the ONM, such that the Golgi and ONM are bridged by nesprin-la - AKAP6P - AKAP9 - GM130 protein complexes (FIG. 39A). Deep segments of the transverse tubule system, which is an invagination of the plasma membrane, have also been found near the nuclear envelope, albeit the aforementioned pharmacological data do not support a role for PARs exposed to the outside of the myocyte in the activation of AKAP6P signalosomes. To determine which organelles might house PARs responsible for activation of PKA at AKAP6P and nesprin-la, in addition to nesprin-la fusion targeting to the ONM, Nb80 was targeted to the Golgi by fusion to a beta- 1 ,4-galactosyltransferase 1 targeting sequence (GalT aa 2-79, “Golgi-Nb80”), to endosomes by fusion to tandem FYVE domains from hepatocyte growth factor 1 - regulated tyrosine kinase substrate (Hrs aa 147- 223, “Endo-Nb80”), and to the plasma membrane by fusion to the N-terminal domain of AKAP7a (aa 1-25, “PM-Nb80”, FIG. 39B). In each case Nb80 was directed to the cytosolic face of these organelles. PARs have also been detected on the sarcoplasmic reticulum, which contains sections that are in close proximity to the ONM. Directing Nb80 solely to the sarcoplasmic reticulum proved elusive, as the ONM contains similar proteins as the sarcoplasmic reticulum, with which it is contiguous.

[0255] Consistent with the effects of propranolol, sotalol, and corticosterone suggesting that the cytosolic PKA compartment is regulated by PAR stimulated by extracellular agonists, expression of Nb80 on the plasma membrane of neonatal myocytes completely inhibited the NE-induction of PKA activity in the cytosol, while having no effect on NE-stimulated perinuclear PKA activity detected with 0NM-AKAR4 (FIG. 39C-D). Expression of Nb80 on endosomes did not significantly affect NE-stimulated cytosolic PKA activity, while having a modest but significant effect on perinuclear PKA activity. In contrast, localization of Nb80 to Golgi completely blocked the activation of perinuclear PKA as detected by 0NM-AKAR4,4906-5655-6138 v.l 72Attorney Docket No. 65274.12WO01Customer No. 27683 without impacting at all cytosolic PKA activity. Together with results obtained for ONM- Nb80, these results indicated that PAR within a compartment near or on both the Golgi and ONM was primarily responsible for activation of PKA at AKAP6 signalosomes.

[0256] To corroborate these results, the AR-activating pepducin ICL3-9 was similarly targeted to the plasma membrane, endosomes, and Golgi. Cytosolic PKA activity detected with the parent AKAR4 sensor was increased in unstimulated myocytes by PM-ICL3-9, while, surprisingly, resulting in a decrease in 0NM-AKAR4 FRET ratio (FIG. 39E-F). Targeting of the pepducin to endosomes had no effect on cytosolic PKA activity, while also suppressing perinuclear PKA activity. Notably, Golgi-ICL3-9 increased perinuclear PKA activity in unstimulated myocytes detected with 0NM-AKAR4, while having no effect on signals detected with the cytosolic parent sensor. Taken together, these results show that PAR on or near both the Golgi and ONM are necessary and sufficient for activation of PKA in the AKAP6P compartment.

[0257] The above results place the PAR activating AKAP6P-bound PKA in a perinuclear compartment. To distinguish between PARs that are on the ONM and the Golgi, that the Golgi can be separated from the nuclear envelope by disrupting the nesprin- 1 a - AKAP6 - AKAP9 - GM 130 protein bridge between the two organelles was utilized. While the third spectrin repeat domain (SR3) of AKAP6 binds nesprin- la, the first AKAP6 spectrin repeat domain (SRI) binds AKAP9, such that expression of an SRl-mCherry fusion protein will compete AKAP6-AKAP9 binding releasing the Golgi from its perinuclear location. As shown in FIG. 39G, Golgi, distinctly labelled by expression of a GalT-GFP fusion protein in control mCherry expressing cells, was dispersed in myocytes expressing SRl-mCherry. SRl- mCherry expression had no significant effect on AKAR4 signals induced by NE (FIG. 39H). Notably, SRl-mCherry expression completely suppressed NE-stimulated perinuclear PKA activity detected with ONM-AKAR4. In addition, SRl-mCherry-mediated Golgi dispersion completely blocked the activation of perinuclear PKA by ONM-ICL3-9 just like pharmacological PKA inhibition with H89 (FIG. 391, compared to FIG. 39F). These data indicate that PAR on the Golgi close enough to the ONM to be regulated by ICL3-9 and Nb80 localized to either membrane is responsible for cAMP signaling at AKAP6 signalosomes.

[0258] Defining the dimensions of the AKAP6 / 3 cAMP compartment4906-5655-6138 v.l 73Attorney Docket No. 65274.12WO01Customer No. 27683

[0259] That single polypeptide activators and inhibitors localized to the ONM or Golgi were able to regulate PKA activity detected with 0NM-AKAR4 without affecting cytosolic AKAR4 signals indicated that AKAP6P signalosomes are present within a discrete, independent signaling compartment. To estimate the size of the AKAP6P cAMP compartment, nesprin- 1 a fusion sensors containing the ratiometric cAMP FRET sensor Epac2-camps (FIG. 40A) were expressed in neonatal myocytes, one version with (ONM-50- Epac2-camps) and one without (ONM-Epac2-camps) an intervening rigid spacer comprising 50 copies of the pentapeptide EAAAK (SEQ ID NO:9), which would further distance the sensor from the ONM by -10-26 nm. Just as NE could activate PKA detected with both perinuclear and cytosolic AKAR4 (FIG. 37), NE similarly activated ONM-Epac2-camps, ONM-Epac2-50-camps and parental Epac2-camps sensors (FIG. 40B). In addition, cAMP levels at AKAP6P signalosomes detected with ONM-Epac2-camps were similar whether in response to NE or in myocytes expressing the PAR- activating ONM-ICL3-9 fusion protein (FIG. 40C). Remarkably, insertion of the (EAAAK)so spacer completely prevented the detection by the FRET biosensor of perinuclear cAMP induced by the pepducin fusion protein, while, as a control, the longer cAMP sensor was still equally responsive to NE stimulation (FIG. 40D). These results imply that cAMP levels at AKAP6P signalosomes drop precipitously from their local source of origin, within a distance at least 2 orders of magnitude smaller than the size of the overall nucleus (-10 pm in diameter).

[0260] Mechanisms limiting the size of cAMP compartments include physical membrane barriers, the activity of localized phosphodiesterases, and buffering by cAMP- binding proteins. The aforementioned results suggest that AKAP6P signalosomes are in a cleft between the Golgi and ONM, but the proximity of these membranes do not entirely explain the results with Epac-camps fusion sensors. AKAP6P binds the cAMP-specific, PKA activated phosphodiesterase PDE4D3, that in the absence of local stimulation can minimize cAMP levels and that in the presence of compartment activation can limit cAMP signaling via negative feedback regulation. AKAP6P, as well as potentially AKAP9 in the compartment, also binds PKA holoenzyme containing two Rll-subunits that bind cAMP. If PDE4D3 local cAMP hydrolysis and / or PKA RII buffering confer cAMP compartmentation, loss of PDE4D3 or PKA association with the signalosome should increase the distance at which cAMP can accumulate to levels capable of mediating signal transduction (FIG. 40E). To test this hypothesis, cytosolic cAMP was assayed in myocytes expressing the parental,4906-5655-6138 v.l 74Attorney Docket No. 65274.12WO01Customer No. 27683 cytosolic Epac2-camps sensor in the absence or presence of recombinant polypeptides that compete PDE4D3 or PKA binding to AKAP6P. 4D3(E)-mCherry contains a PDE4D3- derived peptide based upon the N-terminal domain of PDE4D3 that binds AKAP6, while SiiperAK AP- / .S'-mCherry contains a synthetic peptide that will compete PKA RII subunit binding to AKAPs. Consistent with results obtained for AKAR4, expression of perinuclearly localized pepducin (ONM-ICL3-9) had no effect on cAMP levels detected by the cytosolic Epac2-camps parent sensor (FIG. 40F). In addition, both PDE4D3 and PKA displacement from AKAP6P by expression of 4D3(E)-mCherry and SuperAK AP- / .S'-mCherry, respectively, had no effect on cytosolic cAMP levels in unstimulated cells expressing ONM-control. Remarkably, expression of either the PKA or PDE4D3 anchoring disruptor peptide in conjunction with activation of perinuclear pARs by ONM-ICL3-9 resulted in cytosolic accumulation of cAMP sufficient to activate Epac2-camps. To distinguish PKA effects on cAMP buffering from PDE4D3 feedback activation, ONM-ICL3-9 was also expressed in the presence of the PKA inhibitor H89, but in the absence of any anchoring disruptor peptide, resulting in no activation of cytosolic Epac2-cAMP (FIG. 391). As inhibition of PKA anchoring by SuperAKAP-ZS-mCherry, but not inhibition of PKA activity by H89, resulted in spread of cAMP into the cytosolic compartment, that cAMP buffering by PKA Rll-subunits, in conjunction with PDE4D3 local cAMP hydrolysis, restricts locally generated cAMP to the AKAP6P compartment, thereby conferring independence of this small compartment from cAMP signaling elsewhere in the cell.

[0261] Regulation of Co2 +-dependent calcineurin signaling by AKAP6f -associated fARs

[0262] The activation of cAMP signaling at AKAP6P signalosomes by local PARs would presumably entail the activation by downstream signaling known to be dependent upon perinuclear cAMP. Calcineurin is a Ca2+ / calmodulin-dependent phosphatase that activates NFAT and is required for pathological cardiac hypertrophy. In addition to orchestrating perinuclear cAMP signaling, AKAP6P organizes a perinuclear Ca2+signaling compartment that is independent of excitation-contraction Ca2+cycling and required for local activation of the calcineurin-NFAT pathway and induction of myocyte hypertrophy. In particular, perinuclear Ca2+- calcineurin signaling was dependent upon AKAP6P expression and elevated local cAMP levels. The effects of local cAMP were apparently through PKA- catalyzed phosphorylation of associated ryanodine receptors, which could release Ca2+from intracellular stores into the perinuclear compartment. Thus, in concert with the above results,4906-5655-6138 v.l 75Attorney Docket No. 65274.12WO01Customer No. 27683 it was hypothesized that Golgi PARs activating AKAP6 signalosomes serve to regulate local pro-hypertrophic Ca2+- calcineurin signaling.

[0263] To test whether perinuclear ARs regulate the perinuclear Ca2+signaling compartment, a nesprin- 1 a fusion protein containing the intensiometric Ca2+sensor GCaMP6s (ONM-GCaMP6s, FIG. 41 A) was first expressed in neonatal myocytes. Consistent with the inhibition of perinuclear PKA activity (FIG. 38D), inhibition of perinuclear PARs with ONM-Nb80 prevented the observed elevation in perinuclear [Ca2+] in response to PAR stimulation, while having no effect on cytosolic [Ca2+] assayed using the diffusely localized, parent GCaMP6s sensor (FIG. 41B-C). In addition, consistent with the activation of perinuclear PARs and PKA, ONM-ICL3-9 expression increased perinuclear [Ca2+] in unstimulated cells, while not affecting cytosolic [Ca2+] measured with the parent GCaMP6s sensor (FIG. 4 ID). Further, consistent with previous findings that Ca2+influx was dependent upon PKA activity, ONM-ICL3-9 activation of ONM-GCaMP6s was inhibited by H89. These results show that PARs associated with AKAP6P signalosomes not only regulate local PKA activity, but also Ca2+fluxes within that compartment.

[0264] Calcineurin is recruited during prolonged PAR stimulation to AKAP6P signalosomes, where elevated local [Ca2+] promotes calcineurin-NFAT signaling driving myocyte hypertrophy. To test whether elevated perinuclear PKA activity and [Ca2+] in response to chronic stimulation of perinuclear PARs was associated with activation of the calcineurin-NFAT pathway, Myocytes expressing a nesprin- 1 a-localized version of the calcineurin FRET biosensor CaNAR2 (ONM-CaNAR2, FIG. 42A-B) were imaged. Myocytes were studied as above using similar pharmacologic and molecular approaches. Like 0NM-AKAR4, propranolol, but not sotalol inhibited NE-induced perinuclear calcineurin activity, while, like AKAR4, both P-blockers inhibited NE-induced cytosolic calcineurin activity detected with the parental CaNAR2 sensor (FIG. 42C-D). Similarly, the OCT3 inhibitor corticosterone only inhibited pAR-induced calcineurin activity detected with the perinuclear ONM-CaNAR2 sensor, confirming a role for intracellular PARs in the regulation of perinuclear calcineurin signaling. Expression of ONM-Nb80, which inhibits perinuclear PARs, selectively inhibited NE-stimulated perinuclear calcineurin activity (FIG. 42E). Conversely, expression of ONM-ICL3-9, which activates perinuclear PARs, induced perinuclear calcineurin activity, but not calcineurin activity detected with the parent CaNAR24906-5655-6138 v.l 76Attorney Docket No. 65274.12WO01Customer No. 27683 sensor (FIG. 42F). Similar results were obtained for calcium signaling in adult myocytes (data not shown).

[0265] When dephosphorylated by calcineurin phosphatase, NFAT transcription factor translocates into the myocyte nucleus promoting hypertrophic gene expression. The nuclear accumulation of GFP-tagged NFATcl in neonatal myocytes chronically treated with NE was inhibited by the cell permeable P-blocker propranolol (FIG. 42G). NE-induced GFP-NFATcl nuclear translocation was not inhibited by membrane impermeant sotalol, while completely blocked by the OCT3 inhibitor corticosterone (FIG. 42H). Consistent with the regulation of perinuclear calcineurin, ONM-Nb80 inhibited NE-induced GFP-NFATcl nuclear translocation, while ONM-ICL3-9 induced GFP-NFATcl nuclear translocation in the absence of agonist (FIG. 421- J). Taken together, these results indicate that perinuclear PARs, which regulate cAMP-PKA signaling in the AKAP6P compartment, similarly regulate perinuclear [Ca2+] and the activity of the calcineurin - NFAT pathway.

[0266] Regulation of myocyte hypertrophy by perinuclear [i-adrenergic receptors

[0267] AKAP6P signalosomes, including AKAP6P-bound PKA and calcineurin, are required for myocyte hypertrophy (non-mitotic cell growth). The regulation of AKAP6P signalosomes by perinuclear PARs implies a unique role for these compartment-specific GPCRs in the regulation of myocyte hypertrophy. Culture of primary adult myocytes in the presence of NE for 48 hours results in an increase in length and width of the relatively columnar-shaped cells. To test directly whether perinuclear PARs regulate myocyte hypertrophy, adult myocytes chronically stimulated with NE were treated as above to alter perinuclear PAR activity. Treatment with propranolol and corticosterone, but not sotalol, prevented the NE-induced myocyte growth in length and width (FIG. 43A-D). In addition, expression of ONM-Nb80 inhibited NE-induced myocyte hypertrophy, while expression of ONM-1CL3-9 induced myocyte growth in length and width independently of GCPR stimulation (FIG. 43E-H). These results imply that activation of perinuclear PARs is necessary and sufficient for the induction of adult myocyte hypertrophy by AKAP6P signalosomes.

[0268] In contrast to other AKAP scaffolds, AKAP6P expression is not cardioprotective, while required for pathological cardiac remodeling in response to diverse pathophysiological insults. Symmetric growth in length and width of adult myocytes may represent physiological4906-5655-6138 v.l 77Attorney Docket No. 65274.12WO01Customer No. 27683 or pathological hypertrophy. However, elevated atrial natriuretic factor (ANF) expression is a marker for pathological myocyte hypertrophy readily detected in neonatal myocytes stimulated with GPCR agonists. Consistent with the above results, NE-induced ANF expression in neonatal myocytes was inhibited by propranolol and corticosterone, but not sotalol (FIG. 431- J). In addition, NE-induced ANF expression was suppressed by ONM- Nb80, that directly inhibits perinuclear PAR, while ANF expression was induced independently of NE stimulation by ONM-ICL3-9, that directly activates perinuclear PAR (FIG. 43K-L). Moreover, localization of Nb80 to the Golgi, but not the plasma membrane or endosomes, inhibited NE-induced ANF expression, while conversely, ICL3-9 pepducin localization to the Golgi, but not the plasma membrane or endosomes, resulted in agonistindependent ANF expression (FIG. 43M-N). Only Golgi-localized PAR induced an increase in pathological gene transcription, correlating to an increase in perinuclear-localized cAMP. cAMP increases in other sites of the cell were not sufficient to induce such an increase in pathological gene transcription. Finally, expression of SRl-mCherry, that disrupts AKAP6- AKAP9 binding, resulting in Golgi dispersion, also inhibited NE-induced ANF expression (FIG. 430). Taken together, these results suggest that Golgi-localized pARs regulate perinuclear AKAP6P signalosomes responsible for the coordinated activation of cAMP- and Ca2+-dependent signaling pathways inducing pathological myocyte hypertrophy.

[0269] Perinuclear fl-adrenergic receptors regulate pathological cardiac remodeling in vivo

[0270] The regulation of pathological myocyte hypertrophy by perinuclear PARs in vitro indicated that this compartmentalized GPCR pool could be similarly critical for the response of the heart in disease. To demonstrate that GPCRs in a privileged intracellular compartment is required for the determination of organ phenotype in vivo, ONM-Nb80 and ONM-ICL3-9 were expressed in mice using adeno-associated virus (AAV) gene therapy vectors. AAV serotype 9 vectors were generated that express under the control of the cardiac myocytespecific cardiac troponin T (cTnT) promoter the ONM-Nb80 and ONM-ICL3-9 constructs, in this case without the mCherry tag due to the limited length (~4.8 kb) of AAV genomes (FIG. 44A). When administered intravenously to adult mice, these vectors expressed ONM-Nb80 and ONM-control fusion proteins at lower levels in the heart than endogenous nesprin-la (FIG. 44K). ONM-ICL3-9 was expressed at even lower levels and was not consistently detected by western blot (data not shown).4906-5655-6138 v.l 78Attorney Docket No. 65274.12WO01Customer No. 27683

[0271] Expression of ONM-ICL3-9 in wildtype adult C57BL / 6NJ mice resulted in the development of prominent systolic cardiac dysfunction within 2 months of AAV administration, as detected by “4D” echocardiography (FIG. 44B-D). Left ventricular (LV) end-systolic volume was increased, while LV ejection fraction was decreased significantly by 6 weeks after AAV injection in comparison to ONM-nesprin control or non-treated naive mice, which had preserved cardiac function. Similar results were obtained by M-mode echocardiography. Although the hearts were not significantly dilated in diastole following 2 months of ONM-ICL3-9 expression, by gravimetric analysis the hearts exhibited significant ventricular hypertrophy (13% increased indexed heart weight, FIG. 44E). Likewise, cardiac myocyte cross-section area measured in wheat-germ agglutinin- stained LV tissue sections was increased in mice expressing ONM-ICL3-9, when compared to the two control cohorts (FIG. 44F). Taken together, these results demonstrated that, like in vitro, activation of perinuclear PAR is sufficient to induce cardiomyopathy in vivo.

[0272] To test whether perinuclear ARs are required for pathological cardiac remodeling, a treatment study in a mouse model of human Familial Dilated Cardiomyopathy was conducted. The “TM54” FVB / N mouse contains a transgene that expresses under the control of the cardiac myocyte-specific a-myosin heavy chain promoter a mutant cDNA for the sarcomeric protein a-tropomyosin. The a-tropomyosin E54K mutation causes decreased myofilament Ca2+sensitivity and tension development, which manifests as prominent cardiac systolic dysfunction and early onset cardiomyopathy. 6-8-week-old TM54 mutant mice with obvious cardiac dysfunction and wildtype littermate mice with normal function were randomized for treatment with AAV to express ONM-Nb80 or ONM -control or for no treatment at all. During the 10- weeks following treatment, ONM-control and non-injected AAV-naive TM54 mice exhibited a progressive decline in cardiac systolic function, as evident by a significant decline in LV ejection fraction (13% and 9% decrease in LV ejection fraction, respectively, for non-injected and ONM-control cohorts, respectively, p<0.0001 vs. initial time point, FIG. 441). Remarkably, TM54 mice treated with ONM-Nb80 exhibited an improvement over the 10-week study of 4.6% in ejection fraction (p - 0.03 vs. initial time point), resulting in a 16-17% improvement in ejection fraction at endpoint in comparison to the control cohorts (p < 0.0001). The improvement in function was also evident by the significantly less LV end-systolic volume for OMN-Nb80-treated mice than control mice (25% and 36% lower than ONM-Control and non-injected mice, respectively, p < 0.0001,4906-5655-6138 v.l 79Attorney Docket No. 65274.12WO01Customer No. 27683FIG. 44G). Notably, neither ONM-Nb80 nor ONM-Control had any significant effect on the cardiac function of wildtype littermates. The improvement in TM54 cardiac function conferred by ONM-Nb80 treatment was accompanied by decreased cardiac hypertrophy (FIG. 44H). Likewise, cardiac myocyte cross-section area measured in wheat-germ agglutinin-stained LV tissue sections was normalized in TM54 mice expressing ONM-Nb80, when compared to the two control TM54 cohorts and wildtype littermates (FIG. 441). In addition, the interstitial myocardial fibrosis associated with DCM was inhibited by ONM- Nb80 treatment (FIG. 44J). These results indicate that treatment with an ONM-Nb80 gene therapy vector improved the cardiac structure and function of the TM54 mouse. Taken together, results obtained by expression of OMN-ICL3-9 and OMN-Nb80 in vivo demonstrate the essential role of perinuclear PARs in pathological cardiac remodeling, while providing proof-of-concept for the targeting of this GPCR compartment in dilated cardiomyopathy.

[0273] Discussion

[0274] Example 6 provides molecular and physiological evidence of an autonomous, nanometer scale, GPCR compartment important for determination of cellular structure and function. AKAP6 organizes perinuclear multimolecular signalosomes, which in response to locally generated and restricted cAMP signals, regulate pathological cardiac myocyte hypertrophy, and retinal ganglion cell neuroprotection. Here, ARs located on Golgi membranes facing the ONM are defined as uniquely responsible for activation of these signalosomes, as studied in the context of pathological cardiac myocyte hypertrophy. Using an ONM localized activator and inhibitor of the PAR (ONM-ICL3-9 and ONM-Nb80), as well as a peptide that can disrupt AKAP6-AKAP9-mediated tethering of the Golgi to the ONM (SRl-mCherry), it is demonstrated that, in contrast to PAR on endosomes or the plasma membrane, Golgi-localized PAR directly stimulates perinuclear cAMP signaling, independently of cAMP signaling elsewhere in the cell. Perinuclear PARs thereby regulate perinuclear [Ca2+], activation of the calcineurin-NFAT pathway, myocyte hypertrophy, and ANF expression. The local regulation of AKAP6 signalosomes by perinuclear PARs permits the modulation of AKAP6-dependent signal transduction independently of other important PAR-dependent cellular processes in the myocyte, such as contractility, with biological relevance provided by perinuclear PAR gain- and loss-of-function in mouse models of cardiomyopathy. Taken together, this work demonstrates how compartmentalization provides4906-5655-6138 v.l 80Attorney Docket No. 65274.12WO01Customer No. 27683 specificity to GPCR signaling, illustrating how an understanding of the architecture of cellular compartmentation can be leveraged to alter selectivity cellular function, with potentially beneficial therapeutic effect.

[0275] The functional significance of GPCRs detected within the cell but away from the plasma membrane is of interest, and, in particular, the potential initiation of signal transduction at intracellular sites. In the cardiac myocyte, PARs have been detected on endosomes, Golgi apparatus, sarcoplasmic reticulum, and nuclear envelope, and the function of these internal receptors has been demonstrated using a pharmacological approach similar to that used in the initial experiments described herein. In contrast to endosomal GPCRs, Golgi-localized i AR apparently signal without prior internalization from the plasma membrane. Golgi-resident PiAR activated by OCT3-transported NE has been shown to induce neonatal myocyte hypertrophy via the Epac-mediated stimulation of phospholipase e (PLCe), that can hydrolyze phosphatidylinositol-4-phosphate (PI4P) to diacylglycerol and inositol bisphosphate (IPr). PLCe can bind AKAP6P, regulating histone deacetylase nuclear export. As a PLCs-derived AKAP6P-binding peptide inhibited PI4P hydrolysis, it may be that these Golgi-localized PiARs activate PLCe via AKAP6P-bound Epacl. However, Golgi PLCe appears insensitive to isoproterenol that stimulated cAMP production, while the OMN- AKAR4 sensor is robustly activated in myocytes by isoproterenol (at the same 1 M dose). As PIP4 was assayed using a biosensor (FAPP-PH-GFP) that labels the Golgi broadly, it is possible that different pools of Golgi- associated Pi AR and cAMP regulate AKAP6 signalosomes and Golgi PI4P hydrolysis. Like the plasma membrane that contains multiple discrete GPCR compartments, multiple separate GPCR compartments likely exist within the multilayered Golgi apparatus, regulating different signaling pathways, potentially under the control of different phosphodiesterases. Results obtained with ONM-Nb80 and 0NM-ICL3- 9, which can bridge the outer nuclear envelope and Golgi, indicate that ARs regulating the AKAP6P cAMP compartment are on a limited section of the Golgi directly facing the ONM.

[0276] To activate perinuclear AKAP6P signalosomes, NE must enter the myocyte via the OCT3 transporter and survive cellular monoamine oxidase to reach perinuclear Golgi receptors. While targeting of ICL3-9 and Nb80 to the Golgi and ONM regulated cAMP signaling at AKAP6P, targeting of ICL3-9 and Nb80 to the plasma membrane regulated signaling detected with the cytosolic parental AKAR4 sensor, thereby supporting the independence of the two compartments. Interestingly, exclusive activation of surface PAR4906-5655-6138 v.l 81Attorney Docket No. 65274.12WO01Customer No. 27683 with PM-ICL3-9 in neonatal myocytes robustly lowered baseline ONM-AKAR signals (FIG. 39F), an effect observed to a lesser degree with Golgi-ICL3-9.

[0277] A fundamental question in the field of signal transduction is how large are signaling compartments. It is known that in HEK293 cells glucagon-like peptide 1 (GLP-1) receptors control “receptor- associated independent cAMP nanodomains” (RAINs) with a radius of -60 nm from the plasma membrane receptor. Images obtained by electron microscopy suggest that Golgi membrane can be within 100 nm of the ONM. It is likely that at AKAP60 signalosomes, the Golgi and ONM are even closer, as the ONM-Nb80 and ONM-ICL3-9 polypeptides were able to bridge the ONM and Golgi PARs. In addition, the N-terminal domain of AKAP6 binds directly adenylyl cyclase 5. Ga proteins, including Gas, have long been recognized to reside on the Golgi. Due to the activation of adenylyl cyclase by G«s, the cyclase is likely located near PAR on the Golgi facing the nuclear envelope. As AKAP6 binds the conserved catalytic Cl and C2 domains of adenylyl cyclase 5, and Nb80 binds the cytoplasmic end of the PAR, both PAR and adenylyl cyclase 5 are presumably oriented with their cytoplasmic domains within the cleft between the Golgi and ONM. Thus, endogenous catecholamines would have to traverse both the plasma membrane and Golgi to bind AKAP6P signalosome-associated PARs within the Golgi lumen. NE presumably crosses both membranes via OCT3, which has been detected on both the plasma membrane and intracellular membranes. The close proximity of the ONM and Golgi at AKAP6P supports the inventors’ hypothesis that AKAP6P organizes a highly insulated, independent signaling nano-compartment located between the Golgi and ONM.

[0278] Within the Golgi-ONM cleft, a steep cAMP gradient was detected by comparing signals obtained with ONM-Epac2-camps and ONM-50-Epac2-camps, such that <26 nm away from the N-terminus of nesprin-la cAMP was decreased sufficiently to preclude sensor activation. This gradient is likely maintained primarily by two mechanisms involving AKAP6P binding partners. First, PKA regulatory subunits, which are in molar excess of catalytic subunits in most cells, can serve as a buffer for ambient cAMP. As shown in experiments herein with the SuperAKAP-ZS-mCherry fusion protein, the binding of PKA RII- subunits to AKAP6P limited cAMP to that compartment. Second, the binding of PKA- activated PDE4D3 by AKAP6P provides for the local degradation of cAMP, limiting its overall accumulation. Accordingly, displacement of PDE4D3 or PKA was sufficient to dissipate this gradient and to permit the detection of ONM-ICL3-dependent cAMP by soluble4906-5655-6138 v.l 82Attorney Docket No. 65274.12WO01Customer No. 27683Epac2-camps sensor. Additional mechanisms might also contribute to the small physical dimensions of the signaling compartment, e.g., Golgi membrane and ONM presumably provide a physical barrier to cAMP diffusion along that axis. The direct binding of adenylyl cyclase 5 to AKAP60 provides an explanation how, despite this steep gradient, local cAMP levels can be sufficient to activate AKAP6P-bound PKA. Interestingly, it has been suggested that in cells activated by endogenous ligands, cAMP levels are insufficient to promote the dissociation of PKA regulatory and catalytic subunits and, instead, induce PKA holoenzyme to undergo conformational shifts permitting the phosphorylation of adjacent substrates. The range at which PKA bound to an AKAP might phosphorylate a substrate has been predicted to be of the same scale (up to 25 nm) as the limit of the cAMP gradient detected herein. These results are intriguing because, like PDE4D3, AKAP60-bound adenylyl cyclase 5 itself is a target for phosphorylation by AK AP6f>-bound PKA. These results imply that relevant substrates for AKAP6P-bound PKA are similarly near the kinase.

[0279] In comparison to adult ventricular myocytes, cultured primary neonatal rat ventricular myocytes have a rudimentary transverse tubule and sarcoplasmic reticulum system and sparse myofibrils, with notable differences in Ca2+handling and excitationcontraction coupling. The differences in ultrastructure between neonatal and adult myocytes are in many ways similar to the differences between adult myocytes in the normal and failing heart and may explain why studies in neonatal myocytes have often been informative regarding the regulation of pathological cardiac remodeling. A remarkable feature of the work described herein is that results regarding signaling by PAR in the perinuclear compartment were similar whether obtained in neonatal or adult myocytes, while recapitulated in live mouse models of cardiovascular disease. This conservation of function likely reflects the close perinuclear location of the Golgi in both neonatal and adult myocytes and the highly restricted localization of AKAP6P in these cells. Despite early reports suggesting the presence of AKAP6P throughout the sarcoplasmic reticulum, AKAP6P (mAKAPP) is localized to the nuclear envelope in terminally differentiated myocytes by binding nesprin-la.

[0280] Consistent with its restricted intracellular localization, AKAP6P expression is required for the PKA-dependent phosphorylation of ryanodine receptors in the perinuclear compartment, but not ryanodine receptors throughout the sarcoplasmic reticulum. These perinuclear ryanodine receptors are likely responsible for releasing Ca2+into the Golgi-ONM4906-5655-6138 v.l 83Attorney Docket No. 65274.12WO01Customer No. 27683 cleft to activate the hypertrophic CaN-NFAT gene regulatory pathway though it is not clear on which membranes are located the ryanodine receptors that can be co-immunoprecipitated with nesprin-la and AKAP6P. Ryanodine receptors associated with AKAP60 signalosomes may be located on a section of sarcoplasmic reticulum near the ONM. Active CaN phosphatase is recruited to AKAP6P following NE stimulation, where maintenance of its active state by elevated perinuclear Ca2+levels is important for persistent activation of NFAT transcription factor. Consistent with the regulation of AKAP6P-bound PKA by nearby Golgi PARs, perinuclear Ca2+levels and local CaN activation were dependent upon the same pool of PARs. In addition, perinuclear PARs were found to be important for NFAT nuclear translocation and myocyte hypertrophy (e.g., Example 5, Example 6, above). These results support the inventor’s hypothesis that, through the modulation of relevant signaling pathways, the perinuclear AKAP6P compartment serves as an important switch promoting the induction of pathological cardiac hypertrophy in disease.

[0281] Elevated plasma norepinephrine levels (3-6 nM in heart failure compared to ~1 nM in normals) are a common finding among patients in heart failure and prior to the development of P-blocker therapies were highly predictive of mortality. Although PiAR signaling is overall downregulated in heart failure, recent studies have found that Pi AR signaling is differentially regulated in heart failure depending upon the myocyte compartment, including a shift from the plasma membrane to internal compartments. That the internal signaling compartment defined by PAR association with AKAP6P signalosomes is a critical target of P-blocker therapy is suggested herein. Complementing in vitro studies in primary myocytes, expression of the ONM-ICL3-9 activating protein was found in the mouse heart at relatively low levels rapidly induced systolic dysfunction and pathological remodeling in the absence of other primary disease. Based upon the requirement for activation of the AKAP6P compartment for myocyte hypertrophy, the inventors considered that, conversely, targeting of AKAP6P-associated perinuclear PARs would be efficacious for the treatment of heart failure, an approach avoiding the common side effects of P-blockers such as low heart rate (bradycardia) and blood pressure (hypotension). To test this hypothesis, a mouse model of familial, non-ischemic Dilated Cardiomyopathy was treated. Non-ischemic Dilated Cardiomyopathy, of which 30-50% cases are genetic, incurs a 3-year mortality of 12-20% due to heart failure and ventricular arrythmia despite modem therapy. Importantly, with a prevalence as high as 1 in 250 in the adult population, Dilated4906-5655-6138 v.l 84Attorney Docket No. 65274.12WO01Customer No. 27683Cardiomyopathy comprises a significant cause of cardiovascular mortality and is the most common indication for heart transplantation. Remarkably, treatment of the TM54 model for Dilated Cardiomyopathy with a ONM-Nb80 gene therapy vector improved cardiac function and decreased pathological remodeling. Thus, there appears to be promise in gene therapy targeting of perinuclear pARs.

[0282] Example 7

[0283] AIP

[0284] CaMKII regulates pathological cardiac myocyte hypertrophy

[0285] To demonstrate whether mAKAP -bound CaMKII regulates pathological cardiac myocyte hypertrophy, CaMKII was selectively activated (via expression of an AKAP185c- mCherry-nesprin) or inhibited (via expression of an AIP4-mCherry-nesprin peptide in the mAKAPP-nesprin- la perinuclear compartment. Norepinephrine-induced expression of the hypertrophy marker atrial natriuretic factor (ANF) was inhibited in neonatal cardiac myocytes by expression of ATP4-mCherry-nesprin (Fig. 35, Panel C, left graph). Conversely, expression of AKAP185c-mCherry-nesprin induced ANF expression in the absence of adrenergic stimulation. In addition, reduced local Ca2+concentration using mCherry-Parv- nesprin and inhibition of perinuclear CaMKII activity using AIP4-mCherry-nesprin protected against neonatal myocyte apoptosis induced by the pi AR-selective agonist denopamine (Fig. 36).

[0286] Materials and Methods

[0287] Animal Studies

[0288] Neonatal and adult rat cardiac myocytes were isolated from Sprague Dawley Rats purchased from Charles River. FVB / N-Tg(Myh6-Tpml*E54K) “TM54” mice were previously provided by Dr. Beata Wolska and Dr. David Wieczorek and are available as strain #035610 at the Jackson Laboratory (Bar Harbor, ME). Mice were genotyped by polymerase chain reaction using the following primers: Myh6 Forward: 5’-GCC CAC ACC AGA AAT GAC AGA-3’ (SEQ ID NOTO) and Tpml Reverse: 5’-TCC AGT TCA TCT TCA GTG CCC-3’ (SEQ ID NO:11) (236 bp product); Atpla2 internal control primers sense: 5’-AGC GAG CTC AGG ACA TTC TGG-3’ (SEQ ID NO:12) and antisense: 5’-CTC CTA ACC ACG CTC CTA GCA-3’ (SEQ ID NO: 13) (494 bp).4906-5655-6138 v.l 85Attorney Docket No. 65274.12WO01Customer No. 27683

[0289] Plasmids

[0290] All plasmids were constructed by Azenta Life Sciences (Genewiz, South Plainfield, NJ) or Vectorbuilder (Chicago, IL) using the methods of their choice. All plasmids were validated by sequencing, with most completely sequenced by next-generation sequencing, and by restriction digest before use.

[0291] pS-AKAR4-nesprin and pS-AKAR4 encoding 0NM-AKAR4 and parent AKAR4 were as previously described (Boczek, T., et al., J Neurosci 39, 5466-5480 (2019); incorporated by reference in its entirety). All pS series vectors direct cDNA expression under control of the cytomegalovirus immediate early promoter and contain LCeu I and PLSce I flanking sites for subcloning in the Adeno-X vector (Clontech Adeno-X Tet-Off Expression System 1). pTRE-GcAMP6S (containing the conditional TRE promoter and requiring coinfection with Adeno-tTA virus, Clontech Adeno-X Tet-Off Expression System 1), pS- GCaMP6s-nesprin, pS-CaNAR2, and pS-CaNAR2-nesprin were as previously described (Turcotte, M.G., et al., J Mol Cell Cardiol 172, 26-40 (2022), incorporated by reference). ONM-Epac2-camps was expressed using pS-Epac2-camps-nesprin that was identical to pS- CaNAR2-nesprin and pS-GCaMP6s-nesprin except for the substitution of a Flag-tagged Epac2-camps open reading frame from pCDNA3-epac2-camps. ONM-50-Epac2-camps was expressed using pS-Epac2-camps-EAAAK50-nesprin-l that contained a BssH II - Not I cassette encoding 50 EAAAK repeats between the Epac2-camps and nesprin-1 cDNAs. These sensor plasmids contain a myc-tagged human nesprin-1 cDNA (NCBI AF495910; bp 23997-26993), which is identical to the mRNA for nesprin-la2 (the major isoform in cardiac myocytes, NCBI AY184203) except for substitution of a 76 aa sequence from the longer nesprin-1 isoforms in lieu of the first 31 aa of la2 and for an exclusion of the DV23 alternatively spliced 23 aa exon (of unknown function) generally present in heart (Duong, N.T., et al., PLoS One 9, e94380 (2014); Randles, K.N., et al., Dev Dyn 239, 998-1009. 10.1002 / dvdy.22229 (2010); also incorporated by reference).

[0292] ONM-ICL3-9 and ONM-Nb80 were expressed using plasmids derived from the previously described control pS-mCherry-nesprin, in which a mCherry cDNA is 5’ to the same nesprin-1 cDNA in the biosensor plasmids (Boczek, T., et al., J Neurosci 39, 5466-5480 (2019)). For ONM-ICL3-9, a cDNA fragment encoding a myc tag, the ICL3-9 peptide GRFHVQNLSQVEQDGRTIGII (SEQ ID NO: 14) and a flexible (GGGGS)I3linker (SEQ ID NO: 15) was subcloned 5’ to the mCherry cDNA sequence. For ONM-Nb80, a cDNA4906-5655-6138 v.l 86Attorney Docket No. 65274.12WO01Customer No. 27683 fragment encoding a myc tag, the Nb80 nanobody and a flexible (GGGGS) linker was subcloned 5’ to the mCherry cDNA sequence. PM-ICL3-9 was expressed using pS- AKAP18(l-25)-ICL3-9-mCherry-Flag in which a cDNA encodes (1) the first 25 aa of AKAP18a (MGQLCCFPFSRDEGKISEKNGGEPD; SEQ ID NO: 16), (2) a flexible (GGGGS)B linker, (3) the ICL3-9 peptide, (4) mCherry, and (5) a C-terminal Flag tag. Endo-ICL3-9 was expressed using pS-FYVE-ICL3-9-mCherry-Flag in which a cDNA encodes (1) two direct repeats of mouse HGF-regulated tyrosine kinase substrate aa 147-223 (NCBI AAH03239) separated by QGQGS (SEQ ID NO: 17), (2) a flexible (GGGGS)B linker, (3) the ICL3-9 peptide, (4) mCherry, and (5) a C-terminal Flag tag. Golgi-ICL3-9 was expressed using pS-ICL3-9-GalT-mCherry-Flag in which a cDNA encodes (1) the ICL3-9 peptide, (2) a flexible (GGGGS)B linker, (3) Homo sapiens beta- 1 ,4-galactosyltransferase 1 aa 2-82 (NCBI CDJ98633),7 (4) mCherry, and (5) a C-terminal Flag tag. PM-ICL3-9, Endo- Nb80, and Golgi-Nb80 were expressed with the similar vectors pS-AKAP18(l-25)-Nb80- mCherry-Flag, pS-FYVE-Nb80-mCherry-Flag, and pS-Nb80-GalT-mCherry-Flag, respectively, in which the ICL3-9 peptide sequence was replaced with a Nb80 cDNA. AKAPIS-mCherry was expressed using pS-SuperAKAPIS-mCherry-Flag in which a cDNA encodes (1) the PKA binding peptide QIEYVAKQIVDYAIHQA (SEQ ID NO: 18), (2) a flexible (GGGGS)B linker, (3) mCherry, and (5) a C-terminal Flag tag. 4D3(E)-mCherry was expressed using pscS2-4D3(E)-mCherry-mh, as previously described (Boczek, T., et al., J Neurosci 39, 5466-5480 (2019)).

[0293] AAV shuttle plasmids were constructed with pAcTnTs that directs expression under the control of the 407 bp chicken cardiac troponin T promoter (Prasad, K.M., et al., Gene Ther 18, 43-52 (2011); incorporated by reference). OMN-Control AAV were generated using pAcTnT-myc-nesprin-1 that expresses the aforementioned myc-tagged nesprin-1 cDNA. OMN-Nb80 AAV were generated using pAcTnT-Nb80-myc-nesprin-l that encodes Nb80 and a flexible (GGGGS)B linker N-terminal to myc-tagged nesprin-1. Similarly, OMN- ICL3-9 AAV were generated using pAcTnT-ICL3-9-myc-nesprin-l that encodes the ICL3-9 peptide and a flexible (GGGGS) B linker N-terminal to myc-tagged nesprin-1. SRI -mCherry was expressed using a pS vector containing a cDNA for mAKAP aa 586-915 fused to Flag- tagged mCherry.

[0294] Adenoviruses4906-5655-6138 v.l 87Attorney Docket No. 65274.12WO01Customer No. 27683

[0295] NFATcl-GFP was expressed using adenovirus obtained from Seven Hills Bioreagents (Catalog no. JMAd-98). All adenoviruses were generated by transfection of HEK293 cells with Adeno-X plasmids (Clontech Adeno-X Tet-Off Expression System 1) into which genes of interest were subcloned using the I-Ceu I and Pl-Sce I restriction sites. Adenovirus were purified using Vivapure® AdenoPACK™ 20 kits (Sartorius), and titers were determined by end-point dilution method for HEK293 cell viability

[0296] Antibodies and Immunohistochemical Reagents

[0297] The antibodies and reagents in Table 1 were used for immunocytochemistry. Antibodies used in this manner were typically diluted to 1 :1000 of liquid stocks from supplier in phosphate-buffered saline (PBS) containing 0.2% BSA and 1% horse serum. SlowFade Diamond Antifade Mountant with DAPI (S36964, ThermoFisher) was used one drop per coverslip.Table 1

[0298] *The golgi images were not an antibody - transfected with GalT-GFP

[0299] Neonatal rat ventricular myocyte isolation and culture

[0300] 2-3 -day-old Sprague-Dawley rats were euthanized by decapitation. Heart tissue was gradually dissociated through several rounds of collagenase wash and trituration followed by serum neutralization. Heart cells were collected by centrifugation and strained4906-5655-6138 v.l 88Attorney Docket No. 65274.12WO01Customer No. 27683 via 70 pm mesh cell strainer. Next, a 2-hour pre-plating period maximally removed fibroblasts, and the remaining myocytes were collected by centrifugation and plated on 1% gelatin-coated plates (500,000 myocytes per 35 mm plate) in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12) supplemented with 1% penicillin / streptomycin (Gibco-BRL), 10% horse serum (HS), and 5% fetal bovine serum (FBS). The following day, cells were washed and cultured in serum-free DMEM / F12 containing antibiotic if being treated with adenovirus or DMEM / F12 containing 5% FBS if undergoing transfection. If applicable, 35 mm plates of RNV were transfected with Lipofectamine, 2 g of each plasmid DNA and if needed, adenovirus co-infection the following day.

[0301] Adult rat ventricular myocyte isolation and culture

[0302] 2-3 -month-old male and female Sprague-Dawley rats were anti-coagulated by 300U heparin intraperitoneal injection. 20-30 minutes later rats were anesthetized with ketamine (80 mg / kg) and xylazine (8 mg / kg) for heart excision. Hearts were collected into chilled perfusion buffer (mmol / L: NaCl 120, KC1 5.4, Na2HPO 1.2, NaHCCh 20, MgCh 1.6, Taurine 5, Glucose 5.6, 2,3-Butanedione monoxime 10), equilibrated with 95% O2and 5% CO2). The aorta was cannulated and perfused using a Harvard Langendorff apparatus with buffer at 37 °C at a constant rate of 2.2 mL / min for 5 minutes, followed by perfusion for 45 minutes with 50 mL digestion buffer (perfusion buffer with 120 mg type IT collagenase (Worthington, 315 U / mg), 5 mg protease (Sigma type XIV), and 55 mg BSA), recycling enzyme once reduced to 30 mL remaining. Perfusion was terminated when the hearts were soft enough for trituration. Atria were removed, and the ventricles cut into pieces before suspension and trituration in 5 mL digestion buffer with a large bore pipette, followed by filtration using 150-200 pm nylon mesh. Cells were collected via centrifugation and subjected to gradual Ca2+stepwise addition (0.25, 0.5, and 1 mmol / L Ca2+). The remaining myocytes were plated (100,000 myocytes per 35 mm plate) on laminin (Corning, 10 pg laminin per dish) coated plates followed by washing 1.5 hour later and addition of ACCT medium [Medium 199, 5 mmol / L Creatine (Sigma), 2 mmol / L L-carnitine (Sigma), 5 mmol / L Taurine (Sigma), 25 mmol / L Hepes (Sigma), 10 mmol / L 2, 3-Butanedione monoxime (ACROS Organics), 1% penicillin / streptomycin, 0.2% BSA (fatty acid free, Sigma), 0.1% ITS], Adult myocytes were infected with adenovirus the same day as preparation, with drug treatments occurring the next day.4906-5655-6138 v.l 89Attorney Docket No. 65274.12WO01Customer No. 27683

[0303] Live Cell Imaging

[0304] For imaging of both parent and ONM-targeted Epac2-camps, CaNAR2 and AKAR4 Forster resonance energy transfer (FRET) sensors and GCaMP6s intensiometric sensors, adult and neonatal myocytes were infected with adenovirus (multiplicity of infection [MOI] 5-50). Cells were imaged within two days after infection. For imaging, cells are washed and imaged in Hanks’ Balanced Salt Solution (Gibco, mmol / L: KH2PO40.44; Na2HPO40.34; NaHCO34.2; NaCl 138, KC1 5.3; D-glucose 5.6; CaCl21.3; MgCl20.49; MgSO40.41). All imaging was done on a Zeiss Pascal confocal microscope using a 40x / 1.2 numerical aperture objective, a 440 nm laser (Toptica Photonics), and HQ535 / 50M and HQ480 / 40M emission and 510DCLP dichroic filters (Chroma Technology). For AKAR4 studies involving acute treatment with NE and serial imaging, images were acquired at 15 second intervals. For other sensors, images were acquired every 5 seconds. Due to a minimum required ~25 minutes for CaN to become active, cells expressing CaNAR2 or CaNAR2-nesprin were treated with drugs as indicated for at least 30 minutes prior to imaging. FRET for regions of interest was quantified using background-subtracted images and Image I, with statistical analyses performed using Graphpad Prism 8. FRET ratio “R” was defined as net FRET - donor signal and normalized to Ro (ratio for time=0) for experiments involving acute treatment. For Epac2-camps, which has decreased FRET signal upon cAMP binding, 1 / R was reported. For intensiometric GCaMP6s and GcaMP6s-nesprin sensors, fluorescent signal reflecting Ca2+binding was calculated for regions of interest using background- subtracted images and Image J with normalization to the fluorescence intensity of control samples. For each experiment, traces or single time point images were obtained for cells obtained from at least 3 different myocyte preparations.

[0305] The following drugs were used in this study: Norepinephrine (10 nM), Sotalol (20 pM), Propranolol (1 pM), Corticosterone (20 pM), H-89 (1 pM). All pharmacological interventions were added directly to the media of cells before initiation of live cell imaging, with corticosterone administered at least 20 minutes prior to imaging.

[0306] Immunocytochemistry and cell-based assays

[0307] Myocytes were fixed using 3.7% formaldehyde in PBS for 10 minutes for neonatal and 1 hour for adult myocytes. Cells were permeabilized with 0.3% Triton X-100 in PBS and then washed and blocked using PBS containing 0.2% BSA and 1% horse serum for4906-5655-6138 v.l 90Attorney Docket No. 65274.12WO01Customer No. 2768330 minutes. Slides were incubated for 1 hour with primary antibodies, followed by 1-hour incubation with Alexa fluorescent dye-conjugated specific-secondary antibodies. Blocking buffer was used for antibody dilution and washes following primary and secondary antibody additions. Slides were mounted with SlowFade Diamond Mountant with DAPI. Widefield fluorescent images were acquired by a Zeiss Observer Z1 fluorescent microscope with an Axiocam camera.NFATcl-GFP Localization and ANF expression assays: Neonatal myocytes were infected and / or transfected the day after plating as indicated. Cells were washed with maintenance medium and drugs added the following day with endpoint at 48 hours. For each slide, at least 50 cells were examined for NFATcl-GFP localization or 100 for perinuclear ANF staining. The relative localization of NFATcl-GFP in myocytes was measured as the ratio of nuclear vs cytosolic fluorescence intensity.

[0308] Myocyte Hypertrophy Assay

[0309] Adult myocytes were infected with adenovirus as indicated for 24 hours (MOI 5- 50), followed by washing with ACCT media and treatment with drug as indicated for 48 hours. For each slide, at least 100 cells were measured for maximum length and width.

[0310] Adeno- associated virus

[0311] Serotype 9 AAV were produced by the University of Pennsylvania Vector Core and titered by ddPCR. AAV9 was injected via the tail vein (5xl0nvg i.p.) into 6-8 week old male C57BL / 6NJ mice (The Jackson Laboratory Strain #005304) or male and female TM54 and wildtype littermate mice as indicated. Masking of cohorts was provided by assigning a random number to each mouse by ear tag, such that identification of mouse cohort was not revealed until in vivo and post-mortem analyses were complete. A formal power analysis was not performed for the studies in this project. Mice were not selected for AAV delivery or expression before analysis.

[0312] Echocardiography

[0313] Mice, minimally anesthetized with 1-3% isoflurane, were studied by transthoracic echocardiography using a Vevo 3100 High- Resolution Imaging System (VisualSonics, Toronto, ON, Canada). A Visualsonic MX400 (20-46 MHz, 50 pm axial resolution) linear array transducer was used for all image acquisitions. For M-mode echocardiography,4906-5655-6138 v.l 91Attorney Docket No. 65274.12WO01Customer No. 27683 calculated parameters from at least three cardiac cycles were as follows: FS, fractional shortening = (LVID;d - LVID;s) / (LVID;d) in which LVID, LVAW, and LVPW are left ventricular interior diameter, anterior wall thickness and posterior wall thickness, respectively, and d and s refer to diastole and systole, respectively. For 4D image acquisition, the step motor was positioned just below the apex and the motor aligned to acquire concentric short axis images in 0.2 mm steps. At each position, a complete cardiac cycle was recorded using automated ECG and respiratory gating. 4D images were constructed using Vevo 4D image software. Image analysis was performed by blinded sonographer. 4D measurement of ejection fraction (EF) were calculated directly from volumetric measurements for end- diastolic (EDV) and end-systolic volume (ESV) based on operator-defined edge-tracing using Vevo 4D imaging software (VevoLAB, VisualSonics).

[0314] Histochemistry

[0315] Heart tissue was fixed in 3.7% formaldehyde. De-paraffinized 6 pm tissue sections were stained using the Picrosirius Red Stain Kit (Polysciences, Warrington. PA) and Alexa Fluor 555 wheat germ agglutinin conjugate (Invitrogen, Waltham, MA), as previously described.15For each mouse, the cross-section area of a total of >150 myocytes in >3 distinct regions of the left ventricle were measured using the wheat germ agglutinin-stained sections following fluorescent imaging at 200x using a Leica DM4000 microscope and a DFC3000G camera. Collagen content in myocardium, excluding the area around blood vessels, was assayed using Picrosirius Red stained sections and imaging of the entire left ventricle by circularly polarized light microscopy at 200x magnification using a Leica DM4000 microscope and a DMC2900 color camera, with illumination and exposure time settings optimized to differentiate bright collagen birefringence and the dark background.Measurements were acquired using NIH Image J or Leica LAS software.

[0316] Statistical analysis

[0317] Statistics were computed using Prism 10 (Graphpad, San Diego, California). All data are expressed as mean ± s.e.m. Unpaired, two-tailed t-tests and one-way or two-way ANOVA (with or without matching) followed by Tukey’s (comparison with more than 2 groups for 1-way ANOVA or more than 2 groups in a row for 2-way ANOVA), Dunnett’s (comparison of multiple groups with common control for 1-way ANOVA), or Uncorrected Fisher's LSD (comparisons of cell means with others in its row and its column for 2x24906-5655-6138 v.l 92Attorney Docket No. 65274.12WO01Customer No. 27683 design) post-hoc testing were performed as appropriate. Brown-Forsythe test was used to determine if there was a significant difference in standard deviation among multiple groups for 1-way ANOVA (as apparent by visual inspection for many of the imaging studies), and, if so, Brown-Forsythe and Welch ANOVA tests were used for ANOVA, with Dunnett’s T3 test used for post-hoc testing. Likewise, Welch’s correction was used for t-tests with significant differences in variances (F-test). Repeated symbols used as follows: * p < 0.05; ** p < 0.01 ; *** / ? < 0.001 ; **** p < 0.0001; ns - p > 0.05. n refers to the number of individual mice, myocyte preparations, or live cell traces.

[0318] All statements herein reciting principles, aspects, and embodiments of the disclosure, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure.

[0319] Various other components may be included and called upon for providing for aspects of the teachings herein. For example, additional materials, combinations of materials and / or omission of materials may be used to provide for added embodiments that are within the scope of the teachings herein. Adequacy of any particular element for practice of the teachings herein is to be judged from the perspective of a designer, manufacturer, seller, user, system operator or other similarly interested party, and such limitations are to be perceived according to the standards of the interested party.4906-5655-6138 v.l 93

Claims

Attorney Docket No. 65274.12WO01Customer No. 27683CLAIMSWhat is claimed is:

1. A composition comprising a viral-based gene therapy vector encoding a fusion protein comprising ( 1 ) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-l a amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 80% identical to amino acids 916-975 of SEQ ID NO:7.

2. A composition comprising a viral-based gene therapy vector encoding a fusion protein comprising (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin- 1 a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80% identical to amino acids 193-1209 of SEQ ID NO:5, or at least 80% identical to amino acids 916-975 of SEQ ID NO:7.

3. A composition comprising a viral-based gene therapy vector encoding a fusion protein comprising (1) one or more Autocam tide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein each present AIP amino acid sequence is at least 80% identical to amino acids 3-15 of SEQ ID NO:1, and (2) a nesprin- 1 a amino acid sequence, wherein the nesprin- la sequence is at least 80% identical to amino acids 125-1141 of SEQ ID NO: 1, or at least 80% identical to amino acids 916-975 of SEQ ID NO:7.

4. A composition comprising a viral-based gene therapy vector encoding a fusion protein comprising one of (i) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-27 of SEQ ID NO:3, (ii) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-122 of SEQ ID NO:5, or (iii) one or more AIP amino acid sequences, wherein the AIP amino acid sequence(s) is / (are) at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-15 of SEQ ID NO:1 ; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin- 1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of4906-5655-6138 v.l 94Attorney Docket No. 65274.12WO01Customer No. 27683 nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

5. A composition comprising a viral-based gene therapy vector encoding a fusion protein comprising a fragment of a RyR inhibitor comprising spinophilin, sorcin, calsequestrin, junctin, FKBP12 / 12.6 (calstabin), homer, natrin, triadin or calmodulin, wherein the amino acid sequence of each is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin- 4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

6. A composition comprising a viral-based gene therapy vector encoding a fusion protein comprising (i) a P-AR nanobody amino acid sequence according to Table 1 of US 9,453,065 (incorporated herein by reference in its entirety), wherein the P-AR nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acid sequences described in US 9,453,065, or (ii) a Nb80 peptide mimetic, such as those described in C. Martin, et al., Chem. Eur. J. 2017, 23, 9632 (incorporated by reference in its entirety); and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.4906-5655-6138 v.l 95Attorney Docket No. 65274.12WO01Customer No. 276837. A composition comprising a viral-based gene therapy vector encoding a fusion protein comprising one or more fragments of a CaMKII inhibitor comprising one or more Autocamtide-3 inhibitor (AC3-I), CN27 (also known as CaMKIINtide), CN21 , and CN19o amino acid sequences, wherein the AC3-I, CaMKIINtide, CN21, and CN19o amino acid sequences are at least 80%, 90%, 95%, 99% or 100% identical to their canonical sequences; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin- 4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

8. The composition of any one of claims 1, 4 or 5, wherein the composition inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2-mediated Ca2+release.

9. The composition of any one of claims 2, 4, or 6, wherein the composition inhibits adenylyl cyclase activity, binds to P-AR, or suppresses perinuclear cAMP production.

10. The composition of any one of claims 3, 4, or 7, wherein the composition inhibits calmodulin-dependent protein kinase II (CaMKII) bound to mAKAP , inhibits CaMKII activity, or binds to CaMKII.

11. The composition of any one of claims 1 , 4, or 5, wherein the fragment of Bcl-2 is at least 90% identical to amino acids 3-27 of SEQ ID NO:3, and the nesprin- la amino acid sequence is at least 90% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 90% identical to amino acids 916-975 of SEQ ID NO:7.

12. The composition of any one of claims 1 , 4 or 5, wherein the fragment of Bcl-2 is at least 95% identical to amino acids 3-27 of SEQ ID NO:3, and the nesprin- 1 a amino acid sequence is at least 95% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 95% identical to amino acids 916-975 of SEQ ID NO:7.4906-5655-6138 v.l 96Attorney Docket No. 65274.12WO01Customer No. 2768313. The composition of any one of claims 1, 4 or 5, wherein the fragment of Bcl-2 is at least 99% identical to amino acids 3-27 of SEQ ID NO:3, and the nesprin- 1 a amino acid sequence is at least 99% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 99% identical to amino acids 916-975 of SEQ ID NO:7.

14. The composition of any one of claims 1 , 4, or 5, wherein the fragment of Bcl-2 is amino acids 3-27 of SEQ ID NO:3, and the nesprin- la amino acid sequence is amino acids 98-1114 of SEQ ID NO:3, or amino acids 916-975 of SEQ ID NO:7.

15. The composition of any one of claims 2, 4, or 6, wherein the Nb80 nanobody amino acid sequence is at least 90% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin- la amino acid sequence is at least 90% identical to amino acids 193-1209 of SEQ ID NO:5.

16. The composition of any one of claims 2, 4 or 6, wherein the Nb80 nanobody amino acid sequence is at least 95% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin-la amino acid sequence is at least 95% identical to amino acids 193-1209 of SEQ ID NO:5.

17. The composition of any one of claims 2, 4, or 6, wherein the Nb80 nanobody amino acid sequence is at least 99% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin- 1 a amino acid sequence is at least 99% identical to amino acids 193-1209 of SEQ ID NO:5.

18. The composition of any one of claims 2, 4 or 6, wherein the Nb80 nanobody amino acid sequence is amino acids 3-122 of SEQ ID NO:5, and the nesprin-la amino acid sequence is amino acids 193-1209 of SEQ ID NO:5.

19. The composition of any one of claims 3, 5, or 7, wherein the AIP amino acid sequence is at least 90% identical to amino acids 3-54 of SEQ ID NO: 1, and the nesprin-la amino acid sequence is at least 90% identical to amino acids 125-1141 of SEQ ID NO: 1.

20. The composition of any one of claims 3, 5, or 7, wherein the AIP amino acid sequence is at least 95% identical to amino acids 3-54 of SEQ ID NO: 1, and the nesprin-la amino acid sequence is at least 95% identical to amino acids 125-1141 of SEQ ID NO:1.4906-5655-6138 v.l 97Attorney Docket No. 65274.12WO01Customer No. 2768321. The composition of any one of claims 3, 5, or 7, wherein the AIP amino acid sequence is at least 99% identical to amino acids 3-54 of SEQ ID NO: 1, and the nesprin-la amino acid sequence is at least 99% identical to amino acids 125-1141 of SEQ ID NO: 1.

22. The composition of any one of claims 3, 5, or 7, wherein the AIP amino acid sequence is amino acids 3-54 of SEQ ID NO:1, and the nesprin- 1 a amino acid sequence is amino acids 125-1141 of SEQ ID NO:1.

23. The composition of any one of claims 1-22, wherein the viral vector is adeno-associated virus (AAV).

24. The composition of any one of claims 1-23, wherein the viral vector is AAV2.

25. The composition of any one of claims 1-23, wherein the viral vector is AAV9.

26. A composition comprising fusion protein that inhibits RyR2 activity, binds to RyR2, or reduces RyR2-mediated Ca2+release.

27. A composition comprising fusion protein that binds to P-AR, or suppresses perinuclear cAMP production.

28. A composition comprising fusion protein that inhibits CaMKII activity, or binds to CaMKII.

29. A composition for treating or preventing a heart disease comprising fusion protein that binds to mAKAP and inhibits RyR2 activity, binds to RyR2, or reduces RyR2-mediated Ca2+release.

30. A composition for treating or preventing a heart disease comprising fusion protein that binds to mAKAPP and inhibits adenylyl cyclase activity, binds to P-AR, or suppresses perinuclear cAMP production.

31. A composition for treating or preventing a heart disease comprising fusion protein that inhibits CaMKII bound to mAKAPP, inhibits CaMKII activity, or binds to CaMKII.

32. A composition comprising fusion protein, which fusion protein comprises one of (i) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%,4906-5655-6138 v.l 98Attorney Docket No. 65274.12WO01Customer No. 2768399% or 100% identical to amino acids 3-27 of SEQ ID NO:3, (ii) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-122 of SEQ ID NO:5, or (iii) one or more Autocamtide-2- Related Inhibitory Peptide (AIP) amino acid sequences, wherein the AIP amino acid sequence(s) is / (are) at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-15 of SEQ ID NO: 1 ; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916- 975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

33. A composition comprising fusion protein, which fusion protein comprises a fragment of a RyR inhibitor comprising spinophilin, sorcin, calsequestrin, junctin, FKBP12 / 12.6 (calstabin), homer, natrin, triadin or calmodulin, wherein the amino acid sequence of each is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

34. A composition comprising fusion protein, which fusion protein comprises (i) a [1-AR nanobody amino acid sequence according to Table 1 of US 9,453,065, wherein the P-AR nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acid sequences described in US 9,453,065, or (ii) a Nb80 peptide mimetic; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or4906-5655-6138 v.l 99Attorney Docket No. 65274.12WO01Customer No. 27683 more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

35. A composition comprising fusion protein, which fusion protein comprises one or more fragments of a CaMKII inhibitor comprising one or more Autocamtide-3 inhibitor (AC3-I), CN27 (also known as CaMKIINtide), CN21, and CN19o amino acid sequences, or combinations thereof, wherein the AC3-1, CaMKIINtide, CN21, and CN19o amino acid sequences are at least 80%, 90%, 95%, 99% or 100% identical to their canonical sequences; and (a) one or more of a fragment of a nesprin- 1 a amino acid sequence, wherein the nesprin- la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7, (b) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin- 4, KASH5, or lymphocyte-restricted membrane protein (LRMP), (c) one or more nuclear envelope targeting sequences, (d) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or (e) combinations thereof.

36. The composition of any one of claims 26, 29, 32, or 33, wherein the fusion protein comprises (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-1 a amino acid sequence, wherein the nesprin- la sequence is at least 80% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 80% identical to amino acids 916-975 of SEQ ID NO:7.

37. The composition of any one of claims 26, 29, 32, or 33, wherein the fusion protein comprises (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 90% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin- la amino acid sequence, wherein the nesprin-la sequence is at least 90% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 90% identical to amino acids 916-975 of SEQ ID NO:7.

38. The composition of any one of claims 26, 29, 32, or 33, wherein the fragment is at least 95% identical to amino acids 3-27 of SEQ ID NO:3, and the nesprin-la amino acid4906-5655-6138 v.l 100Attorney Docket No. 65274.12WO01Customer No. 27683 sequence is at least 95% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 95% identical to amino acids 916-975 of SEQ ID NO:7.

39. The composition of any one of claims 26, 29, 32, or 33, wherein the fragment is at least 99% identical to amino acids 3-27 of SEQ ID NO:3, and the nesprin-la amino acid sequence is at least 99% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 99% identical to amino acids 916-975 of SEQ ID NO:7.

40. The composition of any one of claims 26, 29, 32, or 33, wherein the fragment is amino acids 3-27 of SEQ ID NO:3, and the nesprin- 1 a amino acid sequence is amino acids 98- 1 114 of SEQ ID NO:3, or comprises amino acids 916-975 of SEQ ID NO:7.

41. The composition of any one of claims 27, 30, 32, or 34, wherein the fusion protein comprises ( 1 ) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin- l a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80% identical to amino acids 193-1209 of SEQ ID NO:5, or at least 80% identical to amino acids 916-975 of SEQ ID NO:7.

42. The composition of any one of claims 27, 30, 32, or 34, wherein the Nb80 nanobody amino acid sequence is at least 90% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin-la amino acid sequence is at least 90% identical to amino acids 193-1209 of SEQ ID NO:5, or at least 90% identical to amino acids 916-975 of SEQ ID NO:7.

43. The composition of any one of claims 27, 30, 32, or 34, wherein the Nb80 nanobody amino acid sequence is at least 95% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin- 1 a amino acid sequence is at least 95% identical to amino acids 193-1209 of SEQ ID NO:5, or at least 95% identical to amino acids 916-975 of SEQ ID NO:7.

44. The composition of any one of claims 27, 30, 32, or 34, wherein the Nb80 nanobody amino acid sequence is at least 99% identical to amino acids 3-122 of SEQ ID NO:5, and the nesprin-la amino acid sequence is at least 99% identical to amino acids 193-1209 of SEQ ID NO:5, or at least 99% identical to amino acids 916-975 of SEQ ID NO:7.

45. The composition of any one of claims 27, 30, 32, or 34, wherein the Nb80 nanobody amino acid sequence is amino acids 3-122 of SEQ ID NO:5, and the nesprin- l a amino acid4906-5655-6138 v.l 101Attorney Docket No. 65274.12WO01Customer No. 27683 sequence is amino acids 193-1209 of SEQ ID NO:5, or comprises amino acids 916-975 of SEQ ID NO:7.

46. The composition of any one of claims 28, 31, 32, or 35, wherein the fusion protein comprises (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80% identical to amino acids 3-54 of SEQ ID NO: 1, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80% identical to amino acids 125-1141 of SEQ ID NO: 1, or at least 80% identical to amino acids 916-975 of SEQ ID NO:7.

47. The composition of claim 37, wherein the AIP amino acid sequence is at least 90% identical to amino acids 3-54 of SEQ ID NO:1, and the nesprin-la amino acid sequence is at least 90% identical to amino acids 125-1141 of SEQ ID NO:1, or at least 90% identical to amino acids 916-975 of SEQ ID NO:7.

48. The composition of claim 37, wherein the AIP amino acid sequence is at least 95% identical to amino acids 3-54 of SEQ ID NO:1, and the nesprin-la amino acid sequence is at least 95% identical to amino acids 125- 1141 of SEQ ID NO: 1 , or at least 95% identical to amino acids 916-975 of SEQ ID NO:7.

49. The composition of claim 37, wherein the AIP amino acid sequence is at least 99% identical to amino acids 3-54 of SEQ ID NO: 1, and the nesprin-la amino acid sequence is at least 99% identical to amino acids 125-1141 of SEQ ID NO:1, or at least 99% identical to amino acids 916-975 of SEQ ID NO:7.

50. The composition of claim 37, wherein the AIP amino acid sequence is amino acids 3- 54 of SEQ ID NO:1, and the nesprin-la amino acid sequence is amino acids 125-1141 of SEQ ID NO:1, or comprises amino acids 916-975 of SEQ ID NO:7.

51. The composition of any one of claims 36-50, wherein the composition comprises (a) one or more KASH domain fragments of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, (b) one or more nuclear envelope targeting sequences, (c) one or more nesprin binding partners, e.g., pericentrin or AKAP9, or4906-5655-6138 v.l 102Attorney Docket No. 65274.12WO01Customer No. 27683(d) combinations thereof in place of the nesprin-1 a amino acid sequence.

52. A nuclear envelope targeting composition comprising:(la) one or more of a fragment of a nesprin-1 a amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 916-975 of SEQ ID NO:7;(lb) one or more KASH domain fragments of nesprin-1 , nesprin-2, nesprin-3, nesprin- 4, KASH5, or lymphocyte-restricted membrane protein (LRMP) wherein the KASH domain fragment of nesprin-1, nesprin-2, nesprin-3, nesprin-4, KASH5, or LRMP sequence is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence;(lc) one or more nuclear envelope targeting sequences;(ld) one or more nesprin binding partners, e.g., pericentrin or AKAP9; or(le) combinations thereof; and(Ila) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-27 of SEQ ID NO:3; a P-AR nanobody amino acid sequence according to Table 1 of US 9,453,065, wherein the P-AR nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acid sequences described in US 9,453,065, or (ii) a Nb80 peptide mimetic;(lib) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-122 of SEQ ID NO:5, a fragment of a RyR inhibitor comprising spinophilin, sorcin, calsequestrin, junctin, FKBP12 / 12.6 (calstabin), homer, natrin, triadin or calmodulin, wherein the amino acid sequence of each is at least 80%, 90%, 95%, 99% or 100% identical to its canonical sequence, or(lie) one or more AIP amino acid sequences, wherein the AIP amino acid sequence(s) is / (are) at least 80%, 90%, 95%, 99% or 100% identical to amino acids 3-15 of SEQ ID NO: 1 ; a -AR nanobody amino acid sequence according to Table 1 of US 9,453,065, wherein the P-AR nanobody amino acid sequence is at least 80%, 90%, 95%, 99% or 100% identical to amino acid sequences described in US 9,453,065,4906-5655-6138 v.l 103Attorney Docket No. 65274.12WO01Customer No. 27683 or a Nb80 peptide mimetic; wherein the composition targets the nuclear envelope.

53. The composition of any one of claims 26-52, wherein the fusion protein is expressed using a viral vector.

54. The composition of claim 53, wherein the vector is adeno-associated virus (AAV).

55. The composition of claim 54, wherein the viral vector is AAV2.

56. The composition of claim 54, wherein the viral vector is AAV9.

57. The composition of any one of claims 26-52, wherein the fusion protein is formulated as a pharmaceutically acceptable salt with; (1) an acid selected from the group consisting of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and mandelic acid; (2) an inorganic base selected from the group consisting of sodium, potassium, ammonium, calcium, and ferric hydroxide; or (3) an organic base selected from the group consisting of isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.

58. The composition of any one of claims 26-52, wherein the fragment is modified with a cell membrane -penetrating sequence.

59. The composition of claim 58, wherein the cell-penetrating sequence is a transactivator of transcription (TAT) polypeptide, polyarginine peptide, or a penetratin peptide.

60. The composition of claim 59, wherein the fragment is modified with a polyarginine tail.

61. The composition of any one of claims 26-52, wherein the fragment is modified with a lipid-derived group.

62. The composition of claim 61, wherein the lipid-derived group is a stearate.

63. A method of treating or preventing a heart disease by administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that is localized to the nuclear envelope and inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2-mediated Ca2+release.4906-5655-6138 v.l 104Attorney Docket No. 65274.12WO01Customer No. 2768364. A method of treating or preventing a heart disease by administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that is localized to the nuclear envelope, binds to -AR, or suppresses perinuclear cAMP production.

65. A method of treating or preventing a heart disease by administering to a patient at risk of heart disease a pharmaceutically effective amount of a composition that is localized to the nuclear envelope and inhibits CaMKII bound to mAKAP , inhibits CaMKII activity, or binds to CaMKII.

66. The method of any one of claims 63-65, wherein the heart disease comprises an eccentric cardiac hypertrophy, concentric cardiac hypertrophy, ischemic dilated cardiomyopathy, or non-ischemic dilated cardiomyopathy.

67. The method of any one of claims 63-65, wherein the heart disease is ventricular myocyte hypertrophy or ventricular hypertrophy.

68. The method of any one of claims 63-65, wherein the heart disease is heart failure.

69. A method for treating or preventing pathological cardiac remodeling by administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that is localized to the nuclear envelope, inhibits ryanodine receptor (RyR2) activity, binds to RyR2, or reduces RyR2-mediated Ca2+release.

70. A method of treating or preventing pathological cardiac remodeling by administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that is localized to the nuclear envelope and binds to P-AR, or suppresses perinuclear cAMP production.

71. A method of treating or preventing pathological cardiac remodeling by administering to a patient at risk of pathological cardiac remodeling a pharmaceutically effective amount of a composition that is localized to the nuclear envelope and inhibits CaMKII activity, or binds to CaMKII.

72. The method of any one of claims 69-71, wherein pathological cardiac remodeling comprises eccentric or concentric cardiac hypertrophy.4906-5655-6138 v.l 105Attorney Docket No. 65274.12WO01Customer No. 2768373. The method of any one of claims 69-71, wherein the pathological cardiac remodeling is ventricular myocyte hypertrophy or ventricular hypertrophy.

74. The method of any one of claims 69-71, wherein the pathological cardiac remodeling is an ischemic dilated cardiomyopathy or a non-ischemic dilated cardiomyopathy.

75. The method of any one of claims 69-71 , wherein the pathological cardiac remodeling is heart failure.

76. A method of treating or preventing a disease or condition associated with an increase in perinuclear Ca2+release, comprising delivering to a patient in need thereof, an amount of the composition of any one of claims 1 -62 sufficient to inhibit the perinuclear Ca2+release in the patient.

77. The method of claim 76, wherein the disease or condition affects the heart.

78. The method of claim 76, wherein the disease or condition comprises eccentric or concentric cardiac hypertrophy.

79. The method of claim 76, wherein the disease or condition is ventricular myocyte hypertrophy or ventricular hypertrophy.

80. The method of claim 76, wherein the disease or condition is heart failure.

81. The method of claim 76, wherein said disease or condition is caused by hypertension, coronary artery disease, myocardial infarction, valvular disease, primary cardiomyopathy, congenital heart disease, arrhythmia, pulmonary disease, diabetes, anemia, or hyperthyroidism.

82. The method of any one of claims 63, 69, or 76, wherein the composition comprises a viral-based gene therapy vector encoding a fusion protein comprising (1) a fragment of a Bcl- 2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 916-975 of SEQ ID NO:7.

83. The method of claim 64 or claim 70, wherein the composition comprises a viral-based4906-5655-6138 v.l 106Attorney Docket No. 65274.12WO01Customer No. 27683 gene therapy vector encoding a fusion protein comprising (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin-la amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 193-1209 of SEQ ID NO:5, or at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 916-975 of SEQ ID NO:7.

84. The method of claim 65 or claim 71, wherein the composition comprises a viral-based gene therapy vector encoding a fusion protein comprising (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-54 of SEQ ID NO:1, and (2) a nesprin-l amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 125-1141 of SEQ ID NO:1, or at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 916-975 of SEQ ID NO:7.

85. The method of any one of claims 82-84, wherein the vector is an adeno-associated virus (AAV).

86. The method of claim 85, wherein the viral vector is AAV2.

87. The method of claim 85, wherein the viral vector is AAV9.

88. The method of any one of claims 63, 69, or 76, wherein the composition comprises a fusion protein that comprises (1) a fragment of a Bcl-2 amino acid sequence, wherein the fragment is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-27 of SEQ ID NO:3, and (2) a nesprin- 1 a amino acid sequence, wherein the nesprin- 1 a sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 98-1114 of SEQ ID NO:3, or at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 916-975 of SEQ ID NO:7.

89. The method of claim 64 or claim 70, wherein the composition comprises a fusion protein that comprises (1) a Nb80 nanobody amino acid sequence, wherein the Nb80 nanobody amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-122 of SEQ ID NO:5, and (2) a nesprin- 1 a amino acid sequence, wherein the nesprin- la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 193-1209 of SEQ ID NO:5, or at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 916-975 of SEQ ID NO:7.4906-5655-6138 v.l 107Attorney Docket No. 65274.12WO01Customer No. 2768390. The method of claim 65 or claim 71, wherein the composition comprises a fusion protein that comprises (1) an Autocamtide-2-Related Inhibitory Peptide (AIP) amino acid sequence, wherein the AIP amino acid sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 3-54 of SEQ ID NO:1, and (2) a nesprin-l amino acid sequence, wherein the nesprin-la sequence is at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 125-1141 of SEQ ID NO: 1, or at least 80%, 90%, 95%, 99%, or 100% identical to amino acids 916-975 of SEQ ID NO:7.

91. The method of any one of claims 88-90, wherein the fusion protein is administered directly.

92. The method of claim 91 , wherein the fusion protein is administered using a viral vector.

93. The method of claim 92, wherein the fusion protein is delivered by intracellular expression via a viral-based gene therapy vector.4906-5655-6138 v.l 108

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