Antagonists of camkii-delta 9 and uses thereof

Abstract

To provide methods for treating or preventing Ca2+ / calmodulin-dependent kinase II (CaMKII)-mediated diseases.SOLUTION: A method for treating or preventing a CaMKII-mediated disease in a subject, the method comprising administering to the subject an effective amount of an antagonist of CaMKII-δ9.SELECTED DRAWING: Figure 2-2

Description

[Technical Field]

[0001]

[0001] The present invention relates to the field of biomedicine. In particular, the present invention relates to an enzyme for CaMKII-δ9. agonists and their uses. [Background technology]

[0002]

[0002] Throughout the lifespan of an organism, the genome is constantly regulated by various internal and external stress signals. Excessive DNA damage compromises genome integrity and interferes with DNA replication and transcription (Campisi, J. & d'Adda di Fagagna,F.Nature reviews.Molecular cell Biology 8, 729-740, doi:10.1038 / nrm2233(2007)). Prophylactic DNA repair protects against harmful DNA damage and maintains genome stability and cell viability. Abnormal DNA repair causes accumulation of DNA damage and genomic instability, leading to cell death. Because mammalian cardiomyocytes have little or no regenerative capacity, loss of terminally differentiated cardiomyocytes is a common etiology of many types of cardiac disease, including myocardial infarction, cardiomyopathies, and heart failure. However, the mechanisms of DNA repair in cardiomyocytes remain largely unknown.

[0003]

[0003] Ca 2+ Calmodulin-dependent kinase II (CaMKII) is a multifunctional kinase CaMKII is a family of phospho / threonine protein kinases involved in regulating cardiac cell survival and cell death (Erickson, JR, He, BJ, Grumbach, IM & Anderson, ME Physiological reviews 91, 889-915, doi:10.1152 / physrev.00018.2010(2011)). CaMKII is encoded by four genes, CaMKII-α, β, γ, and δ, with CaMKII-δ being predominantly expressed in the heart. CaMKII-δ is alternatively spliced ​​at two variable domains, exons 13 to 17 (variable domain 1) and exons 20 to 22 (variable domain 2), to generate 11 different splice variants. In particular, CaMKII-δ2 (also called CaMKII-δC) and CaMKII-δ3 (or CaMKII-δB) have been previously identified as major cardiac splice variants, localized in the cytoplasm and nucleus, respectively. Emerging evidence suggests that CaMKII-δ2 and CaMKII-δ3 exert distinct, even antagonistic, effects on cardiomyocyte viability (Peng, W. et al., Circulation Research 106, 102-110, doi:10.1161 / CIRCRESAHA.109.210914 (2010)). However, little is known about the physiological and pathological functions of CaMKII-δ9 in the heart. Summary of the Invention

[0004] In the present invention, the present inventors have identified the major CaMKII-δ splice barrier in the human heart. We identified CaMKII-δ9 as a key regulator of cardiomyocyte DNA damage, rather than the well-studied CaMKII-δ2 and δ3. CaMKII-δ9 is upregulated in response to various stimuli and is far more potent than other splice variants (CaMKII-δ2 and δ3) in inducing DNA damage, genomic instability, and cardiac pathology in cardiomyocytes. We further demonstrated that a peptide encoded by exons 13-16-17, a characteristic sequence of CaMKII-δ9, confers splice variant-specific regulation of UBE2T phosphorylation and degradation.

[0005] In one aspect, the present invention provides a method for treating or preventing a CaMKII-mediated disorder in a subject. Disclosed is a method for treating a patient with CaMKII-δ9, the method comprising administering to the subject an effective amount of an antagonist of CaMKII-δ9.

[0006] In another aspect, the present invention provides a method of reducing cardiac injury in a subject, the method comprising administering to a subject a therapeutically effective amount of CaM Disclosed are methods comprising administering to the subject an effective amount of an antagonist of KII-δ9.

[0007] In yet another aspect, the present invention provides a method for determining the level of a ubiquitin-conjugating enzyme or discloses a method of stimulating activity, comprising administering to the subject an effective amount of an antagonist of CaMKII-δ9.

[0007]

[0008] In yet another aspect, the present invention provides a method for preventing the degradation of ubiquitin-conjugating enzymes in a subject. A method is disclosed, the method comprising administering to the subject an effective amount of an antagonist of CaMKII-δ9.

[0008]

[0009] In yet another aspect, the present invention provides a method for preventing cardiomyocyte death in a sample, the method comprising: Disclosed are methods that include contacting the sample with an effective amount of an antagonist of CaMKII-δ9.

[0009]

[0010] In yet another aspect, the present invention discloses a method of reducing DNA damage in a cell, comprising contacting the cell with an effective amount of an antagonist of CaMKII-δ9.

[0010]

[0011] In some embodiments, the CaMKII-δ9 antagonist disclosed herein can inhibit the activation of CaMKII-δ9 or inhibit the kinase activity of CaMKII-δ9. In some embodiments, CaMKII-δ9 is activated by phosphorylation and / or oxidation of CaMKII-δ9 itself. In some embodiments, the kinase activity of CaMKII-δ9 is expressed as its ability to phosphorylate its substrate, for example, a ubiquitin-conjugating enzyme, particularly ubiquitin-conjugating enzyme 2T (UBE2T). In some embodiments, the antagonist of the present invention inhibits the phosphorylation of a ubiquitin-conjugating enzyme. In some embodiments, the ubiquitin-conjugating enzyme is UBE2T. In some embodiments, the antagonist inhibits the phosphorylation of UBE2T at Ser110. In some embodiments, the antagonist is a specific antagonist of CaMKII-δ9. In some embodiments, the antagonist inhibits the levels or activity of CaMKII-δ9 but does not significantly inhibit the levels or activity of CaMKII-δ2 or CaMKII-δ3.

[0011]

[0012] In some embodiments, the antagonist is an antibody that specifically recognizes CaMKII-δ9, a small molecule compound that binds to CaMKII-δ9, an RNAi molecule that targets the coding sequence of CaMKII-δ9, an antisense nucleotide that targets the coding sequence of CaMKII-δ9, or an agent that competes with CaMKII-δ9 to bind to its substrate.

[0012]

[0013] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody. In some embodiments, the antibody binds to the amino acid sequence encoded by exon 16 of the CaMKII-δ gene.

[0013]

[0014] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a microRNA (miRNA). In some embodiments, the RNAi molecule has 10 to 100 bases. In some embodiments, the antisense nucleotides are modified to improve their stability. In some embodiments, In some embodiments, the RNAi molecule and antisense nucleotide bind to exon 16 of the CaMKII-δ gene. In some embodiments, the RNAi molecule and antisense nucleotide bind to exon 13 and exon 16 of the CaMKII-δ gene (also referred to as "exon 13-16" in the present invention), or to exon 16 and exon 17 of the CaMKII-δ gene (also referred to as "exon 16-17" in the present invention), or to exon 13, exon 16, and exon 17 of the CaMKII-δ gene (also referred to as "exon 13-16-17" in the present invention).

[0014]

[0015] In some embodiments, the agent that competes with CaMKII-δ9 and binds to its substrate is the vector that expresses CaMKII-δ9 that does not have the function of phosphorylation or oxidation.In some embodiments, the vector is adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus or plasmid.In some embodiments, AAV is AAV1, AAV2, AAV5, AAV8, AAV9 or AAVrhlO.

[0015]

[0016] In some embodiments, the subject is a human or non-human primate. In some embodiments, the non-human primate is a rhesus monkey. In some embodiments, the subject is a rodent, such as a rat or mouse.

[0016]

[0017] In some embodiments, the CaMKII-mediated disease is associated with elevated levels or activity of CaMKII-δ9. In some embodiments, the CaMKII-mediated disease is a cardiac disease or metabolic disease. In some embodiments, the cardiac disease is selected from the group consisting of cardiomyopathy, myocarditis, diabetic heart disease, myocardial ischemia, cardiac ischemia / reperfusion injury, myocardial infarction, heart failure, arrhythmia, cardiac rupture, angina pectoris, cardiac hypertrophy, cardiac damage, hypertensive heart disease, rheumatic heart disease, angina pectoris, myocarditis, coronary heart disease, and pericarditis. In some embodiments, the cardiac disease is hypertrophic cardiomyopathy. In some embodiments, the metabolic disease is selected from the group consisting of insulin resistance, obesity, diabetes, hypertension, dyslipidemia, diabetic cerebrovascular disease, diabetic eye complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, gout, and hyperuricemia.

[0017]

[0018] In another aspect, the present invention relates to a method for diagnosing a CaMKII-mediated disease in a subject, the method comprising the steps of: (a) obtaining a test biological sample from the subject; and (b) detecting a level or activity of CaMKII-δ9 in the test biological sample, wherein the level or activity of CaMKII-δ9 detected in the subject's test biological sample indicates that the subject has or has an increased likelihood of developing a CaMKII-mediated disease.

[0018]

[0019] In some embodiments, the level or activity of CaMKII-δ9 in a test biological sample is detected by contacting the sample with a reagent that specifically binds to CaMKII-δ9. In some embodiments, the level or activity of CaMKII-δ9 detected in the test biological sample is compared to a baseline level or activity of CaMKII-δ9 detected in a reference sample. In some embodiments, a higher level or activity of CaMKII-δ9 detected in the test biological sample than the baseline level or activity of CaMKII-δ9 indicates that the subject has developed or is at an increased risk of developing a CaMKII-mediated disease. In some embodiments, the reference sample is from a healthy subject, or a sample obtained from the same subject before or after the test biological sample. In some embodiments, the test biological sample is from the subject's heart. In some embodiments, the subject is a human or non-human primate.

[0019]

[0020] In another aspect, the present invention provides an antibody or antibody that specifically recognizes CaMKII-δ9. A kit for diagnosing a CaMKII-mediated disease in a subject is disclosed, comprising the body fragment.

[0020]

[0021] In another aspect, the present invention discloses a biomarker for diagnosing a CaMKII-mediated disease in a subject, the biomarker comprising the full-length protein sequence of CaMKII-δ9 or a fragment thereof. In some embodiments, the biomarker comprises the amino acid sequence set forth in SEQ ID NOs: 1-5.

[0021]

[0022] In another aspect, the present invention discloses the use of CaMKII-δ9 as a biomarker for diagnosing a CaMKII-mediated disease in a subject.

[0023] In another aspect, the present invention discloses a method for identifying a molecule that inhibits the activity of CaMKII-δ9, comprising contacting the molecule with CaMKII-δ9 and UBE2T and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that inhibits CaMKII-δ9.

[0022]

[0024] In yet another aspect, the present invention discloses a method for identifying a molecule that inhibits the phosphorylation ability of CaMKII-δ9, comprising contacting the molecule with CaMKII-δ9 and UBE2T and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that inhibits the phosphorylation ability of CaMKII-δ9.

[0023]

[0025] In yet another aspect, the present invention discloses a method for identifying a molecule that inhibits the phosphorylation and / or oxidation of CaMKII-δ9 itself, comprising the steps of contacting the molecule with CaMKII-δ9 and an antibody capable of detecting the phosphorylation and / or oxidation state of CaMKII-δ9, and determining whether the level of phosphorylated and / or oxidized CaMKII-δ9 is reduced, wherein a decrease in the level of phosphorylated and / or oxidized CaMKII-δ9 identifies a molecule that inhibits the phosphorylation and / or oxidation of CaMKII-δ9.

[0024]

[0026] In yet another aspect, the present invention discloses a method for identifying a molecule that treats or prevents a CaMKII-mediated disease, comprising contacting the molecule with CaMKII-δ9 and UBE2T and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that treats or prevents a CaMKII-mediated disease.

[0025]

[0027] In yet another aspect, the present invention discloses a method for identifying a molecule that treats or prevents a CaMKII-mediated disease, comprising contacting the molecule with CaMKII-δ9 and an antibody capable of detecting the phosphorylation and / or oxidation state of CaMKII-δ9, and determining whether the level of phosphorylated and / or oxidized CaMKII-δ9 is reduced, wherein a decrease in the level of phosphorylated and / or oxidized CaMKII-δ9 identifies a molecule that treats or prevents a CaMKII-mediated disease.

[0026]

[0028] In yet another aspect, the present invention discloses a method for identifying a molecule that alleviates cardiac damage, comprising contacting the molecule with CaMKII-δ9 and UBE2T and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that alleviates cardiac damage.

[0027]

[0029] In yet another aspect, the present invention provides a method for identifying molecules that mitigate cardiac damage. The present invention discloses a method for identifying a molecule in which a decrease in the level of phosphorylated and / or oxidized CaMKII-δ9 alleviates cardiac damage, the method comprising the steps of contacting the molecule with CaMKII-δ9 and an antibody capable of detecting the phosphorylation and / or oxidation state of CaMKII-δ9, and determining whether the level of phosphorylated and / or oxidized CaMKII-δ9 is reduced.

[0028]

[0030] In yet another aspect, the present invention discloses a method for identifying a molecule that prevents cardiomyocyte death, comprising contacting the molecule with CaMKII-δ9 and UBE2T and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that prevents cardiomyocyte death.

[0029]

[0031] In yet another aspect, the present invention discloses a method for identifying a molecule that prevents cardiomyocyte death, comprising contacting the molecule with CaMKII-δ9 and an antibody capable of detecting the phosphorylation and / or oxidation state of CaMKII-δ9, and determining whether the level of phosphorylated and / or oxidized CaMKII-δ9 is reduced, wherein a reduction in the level of phosphorylated and / or oxidized CaMKII-δ9 identifies a molecule that prevents cardiomyocyte death.

[0030]

[0032] In yet another aspect, the present invention discloses a method for identifying a molecule that reduces DNA damage, comprising contacting the molecule with CaMKII-δ9 and UBE2T and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that reduces DNA damage.

[0031]

[0033] In some embodiments, the phosphorylation of UBE2T is at Ser110.

[0034] In yet another aspect, the present invention discloses a method for identifying a molecule that reduces DNA damage, comprising contacting the molecule with CaMKII-δ9 and an antibody capable of detecting the phosphorylation and / or oxidation state of CaMKII-δ9, and determining whether the level of phosphorylated and / or oxidized CaMKII-δ9 is reduced, wherein a reduction in the level of phosphorylated and / or oxidized CaMKII-δ9 identifies a molecule that reduces DNA damage.

[0032]

[0035] In another aspect, the present invention discloses an isolated CaMKII-δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence set forth in SEQ ID NO: 2, the amino acid sequence set forth in SEQ ID NO: 3, the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 80% homology to the amino acid sequence set forth in SEQ ID NOs: 1 to 5.

[0033]

[0036] In yet another aspect, the present invention discloses an isolated CaMKII-δ nucleic acid comprising a nucleic acid sequence encoding a polypeptide of the present invention. In some embodiments, the CaMKII-δ nucleic acid comprises one of the nucleic acid sequences selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and nucleic acid sequences having at least 80% identity to SEQ ID NOs:6-19.

[0034]

[0037] In yet another aspect, the present invention discloses a CaMKII antagonist capable of inhibiting the level or activity of CaMKII-δ9. In some embodiments, the antagonist is an antagonist that inhibits the phosphorylation of a ubiquitin-conjugating enzyme. In some embodiments, the ubiquitin-conjugating enzyme is UBE2T. In some embodiments, the antagonist is an antagonist that inhibits the phosphorylation of UBE2T at Ser110. In some embodiments, the antagonist is a specific antagonist of CaMKII-δ9. In some embodiments, the antagonist inhibits the level or activity of CaMKII-δ9 but does not significantly inhibit the level or activity of CaMKII-δ2 or CaMKII-δ3. In some embodiments, the antagonist is an antibody that binds to the amino acid sequence encoded by exon 16 of the CaMKII-δ gene, an RNAi molecule that targets exon 16 of the CaMKII-δ gene, or an antisense nucleotide that targets exon 16 of the CaMKII-δ gene. In some embodiments, the antagonist is an antibody that binds to the amino acid sequence encoded by exon 13 and exon 16 of the CaMKII-δ gene, an RNAi molecule that targets exon 13 and exon 16 of the CaMKII-δ gene, or an antisense nucleotide that targets exon 13 and exon 16 of the CaMKII-δ gene. In some embodiments, the antagonist is an antibody that binds to the amino acid sequence encoded by exon 16 and exon 17 of the CaMKII-δ gene, an RNAi molecule that targets exon 16 and exon 17 of the CaMKII-δ gene, or an antisense nucleotide that targets exon 16 and exon 17 of the CaMKII-δ gene. In some embodiments, the antagonist is an antibody that binds to the amino acid sequence encoded by exon 13, exon 16, and exon 17 of the CaMKII-δ gene, an RNAi molecule that targets exon 13, exon 16, and exon 17 of the CaMKII-δ gene, or an antisense nucleotide that targets exon 13, exon 16, and exon 17 of the CaMKII-δ gene. In some embodiments, the antagonist is an antibody that binds to the amino acid sequence of full-length CaMKII-δ9, an RNAi molecule that targets the coding sequence of full-length CaMKII-δ9, or an antisense nucleotide that targets the coding sequence of full-length CaMKII-δ9.

[0035]

[0038] In yet another aspect, the present invention discloses a pharmaceutical composition comprising an antagonist of the present invention and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0036] [Figure 1]

[0039] Figure 1 shows that CaMKII-δ9 is an important cardiac cytosolic CaMKII-δ splice variant. Figure 1a shows the splicing landscape and expression levels of all CaMKII-δ splice variants in mouse, rat, rhesus monkey, and human hearts. The transcript map visualizes the 11 reported alternative splice variants (rows) obtained by SMRT sequencing. Exons (columns), if present, are black and numbered at the bottom (lower columns, UTR regions; black lines, exon junctions). The length of each box is proportional to the length of each exon. The percentage of each splice variant is shown in the bar graph (right panel, CaMKII-δ9 is gray), and the absolute read counts of some of the major variants are shown above the bars (three samples for each species are pooled). Figure 1b shows the reciprocal immunoprecipitation of total protein from mouse heart probed with exon 21 antibody and exon 16 antibody, and vice versa. The input lane is a longer exposure of the same membrane. n = 3 biologically independent samples. Figure 1c shows the relative peptide abundance of CaMKII-δ exon junctions in human hearts immunoprecipitated with anti-exon 21 or anti-exon 16 antibodies, as assayed by quantitative mass spectrometry. n = 3 (left panel) and n = 8 (right panel) biologically independent samples. Figure 1d shows confocal immunofluorescence microscopy images showing the cytosolic localization of Flag-CaMKII-δ9 (upper right panel) and HA-CaMKII-δ2 (lower left panel) in NRVM infected with Ad-Flag-CaMKII-δ9 and Ad-HA-CaMKII-δ2. Scale bar, 10 μm. n = 6 biologically independent samples. Figure 1e shows CaMKII-δ9 protein levels in NRVM exposed to 1 μM doxorubicin (24 h) (Figure 1e, n = 12 (vehicle) and 10 (Dox) biologically independent samples). Figure 1f shows CaMKII-δ9 protein levels in NRVMs exposed to 200 μM HO (24 h) (Figure 1f, n = 7 (vehicle) and 5 (HO) biologically independent samples). Figure 1g shows CaMKII-δ9 protein levels in hypertrophied cardiac mice (TAC for 2 weeks) along with corresponding controls (Figure 1g, n = 5 (sham) and 6 (TAC) biologically independent samples).Figure 1h shows CaMKII-δ9 protein levels in myocardial tissue from humans with hypertrophic cardiomyopathy (HCM) along with corresponding controls (Figure 1h, n = 7 (normal humans) and 6 (HCM) biologically independent samples). Data are mean ± SEM. One-way ANOVA (left panel of Figure 1c), two-tailed Student's t-test (right panel of Figure 1c, Figures 1e-h). [Figure 2-1]

[0040] Figure 2 shows that CaMKII-δ9 induces cardiomyocyte death by downregulating UBE2T. Figure 2a shows cellular caspase 3 / 7 activity in NRVMs treated with scrambled or CaMKII-δ9 siRNA in the presence or absence of HO (200 μM). Figure 2b shows cellular caspase 3 / 7 activity in NRVMs treated with scrambled or CaMKII-δ9 siRNA in the presence or absence of Dox (1 μM). n = 6 biologically independent samples. Figure 2c shows cellular caspase 3 / 7 activity in NRVMs infected with Ad-β-gal, Ad-CaMKII-δ9, and Ad-CaMKII-δ2 at the indicated MOIs for 48 h. n = 6 biologically independent samples (Ad-β-gal and Ad-CaMKII-δ9), n = 4 (Ad-CaMKII-δ2). Figure 2d shows averaged data on mRNA levels assayed by real-time PCR for three genes upregulated by CaMKII-δ9 but not CaMKII-δ2 in NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 50, 48 h). n = 14 biologically independent samples. Figure 2e shows representative Western blots and statistical data demonstrating that CaMKII-δ9, but not CaMKII-δ2, dose-dependently reduces UBE2T. n = 8 biologically independent samples. Figure 2f shows representative Western blots and statistical data demonstrating UBE2T expression in NRVM transfected with scrambled or CaMKII-δ9 siRNA. n = 5 biologically independent samples. Figure 2g shows cellular caspase 3 / 7 activity in NRVM infected with Ad-β-gal and Ad-CaMKII-δ9 (MOI 50, 48 h) with or without overexpression of UBE2T. n=5 biologically independent samples. Figure 2h shows cellular caspase 3 / 7 activity in NRVM treated with scrambled or UBE2T siRNA for 60 h. n=5 biologically independent samples.Figure 2i shows representative Western blots and statistical data showing UBE2T expression in NRVM with or without HO (200 μM). n = 5 biologically independent samples. Figure 2j shows cellular caspase 3 / 7 activity in NRVM exposed to HO (200 μM) with or without UBE2T overexpression. n = 8 biologically independent samples. Data are means ± sem. Two-way ANOVA (a, b, g, j), one-way ANOVA (c, d, e, h), or two-tailed Student's t-test (f, i). [Figure 2-2]

[0040] Figure 2 shows that CaMKII-δ9 induces cardiomyocyte death by downregulating UBE2T. Figure 2a shows cellular caspase 3 / 7 activity in NRVMs treated with scrambled or CaMKII-δ9 siRNA in the presence or absence of HO (200 μM). Figure 2b shows cellular caspase 3 / 7 activity in NRVMs treated with scrambled or CaMKII-δ9 siRNA in the presence or absence of Dox (1 μM). n = 6 biologically independent samples. Figure 2c shows cellular caspase 3 / 7 activity in NRVMs infected with Ad-β-gal, Ad-CaMKII-δ9, and Ad-CaMKII-δ2 at the indicated MOIs (multiplicities of infection) for 48 h. n = 6 biologically independent samples (Ad-β-gal and Ad-CaMKII-δ9), n = 4 (Ad-CaMKII-δ2). Figure 2d shows averaged data on mRNA levels assayed by real-time PCR for three genes upregulated by CaMKII-δ9 but not CaMKII-δ2 in NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 50, 48 h). n = 14 biologically independent samples. Figure 2e shows representative Western blots and statistical data demonstrating that CaMKII-δ9, but not CaMKII-δ2, dose-dependently reduces UBE2T. n = 8 biologically independent samples. Figure 2f shows representative Western blots and statistical data demonstrating UBE2T expression in NRVM transfected with scrambled or CaMKII-δ9 siRNA. n = 5 biologically independent samples. Figure 2g shows cellular caspase 3 / 7 activity in NRVM infected with Ad-β-gal and Ad-CaMKII-δ9 (MOI 50, 48 h) with or without overexpression of UBE2T. n=5 biologically independent samples. Figure 2h shows cellular caspase 3 / 7 activity in NRVM treated with scrambled or UBE2T siRNA for 60 h. n=5 biologically independent samples.Figure 2i shows representative Western blots and statistical data showing UBE2T expression in NRVM with or without HO (200 μM). n = 5 biologically independent samples. Figure 2j shows cellular caspase 3 / 7 activity in NRVM exposed to HO (200 μM) with or without UBE2T overexpression. n = 8 biologically independent samples. Data are means ± sem. Two-way ANOVA (a, b, g, j), one-way ANOVA (c, d, e, h), or two-tailed Student's t-test (f, i). [Figure 2-3]

[0040] Figure 2 shows that CaMKII-δ9 induces cardiomyocyte death by downregulating UBE2T. Figure 2a shows cellular caspase 3 / 7 activity in NRVMs treated with scrambled or CaMKII-δ9 siRNA in the presence or absence of HO (200 μM). Figure 2b shows cellular caspase 3 / 7 activity in NRVMs treated with scrambled or CaMKII-δ9 siRNA in the presence or absence of Dox (1 μM). n = 6 biologically independent samples. Figure 2c shows cellular caspase 3 / 7 activity in NRVMs infected with Ad-β-gal, Ad-CaMKII-δ9, and Ad-CaMKII-δ2 at the indicated MOIs (multiplicities of infection) for 48 h. n = 6 biologically independent samples (Ad-β-gal and Ad-CaMKII-δ9), n = 4 (Ad-CaMKII-δ2). Figure 2d shows averaged data on mRNA levels assayed by real-time PCR for three genes upregulated by CaMKII-δ9 but not CaMKII-δ2 in NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 50, 48 h). n = 14 biologically independent samples. Figure 2e shows representative Western blots and statistical data demonstrating that CaMKII-δ9, but not CaMKII-δ2, dose-dependently reduces UBE2T. n = 8 biologically independent samples. Figure 2f shows representative Western blots and statistical data demonstrating UBE2T expression in NRVM transfected with scrambled or CaMKII-δ9 siRNA. n = 5 biologically independent samples. Figure 2g shows cellular caspase 3 / 7 activity in NRVM infected with Ad-β-gal and Ad-CaMKII-δ9 (MOI 50, 48 h) with or without overexpression of UBE2T. n=5 biologically independent samples. Figure 2h shows cellular caspase 3 / 7 activity in NRVM treated with scrambled or UBE2T siRNA for 60 h. n=5 biologically independent samples.Figure 2i shows representative Western blots and statistical data showing UBE2T expression in NRVM with or without HO (200 μM). n = 5 biologically independent samples. Figure 2j shows cellular caspase 3 / 7 activity in NRVM exposed to HO (200 μM) with or without UBE2T overexpression. n = 8 biologically independent samples. Data are means ± sem. Two-way ANOVA (a, b, g, j), one-way ANOVA (c, d, e, h), or two-tailed Student's t-test (f, i). [Figure 3]

[0041] Figure 3 shows that CaMKII-δ9 induces DNA damage and genomic instability in cardiomyocytes by disrupting UBE2T-mediated DNA repair. Figure 3a shows representative immunostaining and statistical data for γH2AX-positive NRVM infected with Ad-β-gal, Ad-CaMKII-δ2, or Ad-CaMKII-δ9 (MOI 50, 48 h). n = 6 independent biological samples. Arrows indicate γH2AX-positive nuclei. Scale bar, 20 μm. Figure 3b shows representative Western blots and statistical data showing that CaMKII-δ9 in NRVM increases γH2AX in a dose-dependent manner. n = 10 independent biological samples. Figure 3c shows DNA damage assessed by comet assay in NRVM infected with Ad-β-gal, Ad-CaMKII-δ2, or Ad-CaMKII-δ9 (MOI 50, 48 h). n = 6 independent biological samples. Arrows indicate nuclei with DNA damage. Scale bar, 20 μm. Figure 3d shows DNA damage assessed by γH2AX immunostaining in NRVMs treated with scrambled or CaMKII-δ9 siRNA, with or without HO (100 μM, 10 min). Figure 3e shows DNA damage assessed by comet assay in NRVMs treated with scrambled or CaMKII-δ9 siRNA, with or without HO (100 μM, 10 min). n = 6 biologically independent samples. Figure 3f shows DNA damage assessed by γH2AX immunostaining in NRVMs infected with Ad-β-gal or Ad-UBE2T (MOI 50, 48 h), with or without overexpression of CaMKII-δ9. Figure 3g shows DNA damage assessed by comet assay in NRVM infected with Ad-β-gal or Ad-UBE2T (MOI 50, 48 h) with or without CaMKII-δ9 overexpression. n = 6 biologically independent samples. Figure 3h shows DNA damage assessed by γH2AX immunostaining in NRVM infected with Ad-β-gal or Ad-UBE2T with or without HO (100 μM, 10 min).Figure 3i shows DNA damage assessed by comet assay in NRVM infected with Ad-β-gal or Ad-UBE2T with or without HO (100 μM, 10 min). n = 6 biologically independent samples. Figure 3j shows DNA damage assessed by γH2AX immunostaining in NRVM treated with scrambled or UBE2T siRNA for 60 h. Figure 3k shows DNA damage assessed by comet assay in NRVM treated with scrambled or UBE2T siRNA for 60 h. n = 6 biologically independent samples. Figure 3l shows representative Western blots and statistical data showing H2AX levels in NRVM infected with scrambled or FANCD2 siRNA. Figure 3m shows representative Western blots and statistical data showing H2AX levels in NRVM infected with scrambled or FANCI siRNA. n = 4 biologically independent samples. Data are mean ± sem. One-way ANOVA (a, b, c, j, k, l, m) or two-way ANOVA (d-i). [Figure 4]

[0042] Figure 4 shows enhanced CaMKII-δ9-UBE2T-DNA damage signaling in cardiomyopathy and heart failure. Figure 4a shows CaMKII-δ9 protein levels in myocardial tissue from wt and CaMKII-δ9 tg mice (n = 8 biologically independent animals). Figure 4b shows Kaplan-Meier survival curves for wt and CaMKII-δ9 tg mice (n = 20 (wt) and 22 (CaMKII-δ9 tg) biologically independent animals). Figure 4c shows cardiac macroscopic morphology for wt and CaMKII-δ9 tg mice (n = 15 (wt) and 9 (CaMKII-δ9 tg) biologically independent animals). Figure 4d shows cardiac TUNEL staining statistics for wt and CaMKII-δ9 tg mice (n = 5 biologically independent animals). Scale bar, 2 mm. Figure 4e shows representative echocardiographic images of wt and CaMKII-δ9 tg mice at 6 and 10 weeks of age. Figure 4f shows statistical data for wt and CaMKII-δ9 tg mice at 6 and 10 weeks of age. n = 13 (wt 6 weeks old), 15 (wt 10 weeks old), 15 (CaMKII-δ9 tg 6 weeks old), and 9 (CaMKII-δ9 tg 10 weeks old) biologically independent animals. EF, ejection fraction; FS, fractional shortening; LVIDd and LVIDs, left ventricular diastolic and systolic diameters; LVPWd and LVPWs, left ventricular posterior wall thickness at diastolic and systolic. Figure 4g shows statistical data for cardiac γH2AX staining in wt and CaMKII-δ9 tg mice at 10 weeks of age. n = 6 biologically independent animals. Figure 4h shows cardiac UBE2T protein levels in wt and CaMKII-δ9 tg mice at 10 weeks of age (n = 5 (wt) and n = 6 (CaMKII-δ9 tg) biologically independent animals). Figure 4i shows cardiac CaMKII-δ9 protein levels in wt and CaMKII-δ9 shRNA transgenic (shRNA tg) mice at 10 weeks of age (n = 14 (wt) and n = 11 (shRNA tg) biologically independent animals). Figure 4j shows statistical data on EF and FS in wt and shRNA tg mice 4 weeks after TAC surgery (n = 10 (sham), 16 (wt TAC), 9 (shRNA tg TAC) biologically independent animals).Figure 4k shows Kaplan-Meier survival curves for wt and shRNA tg mice 4 weeks after TAC surgery (n = 17 (wt) and 28 (shRNA tg) biologically independent animals). Figure 4l shows cardiac γH2AX levels for wt and shRNA tg mice 4 weeks after TAC surgery (n = 5 biologically independent animals). Figure 4m shows TUNEL staining for wt and shRNA tg mice 4 weeks after TAC surgery (n = 5 biologically independent animals). Data are means ± sem. Data were analyzed by Student's two-tailed t-test (a, d, g-i, l, m), log-rank (Mantel-Cox) test (b, k), or two-way analysis of variance (f, j). [Figure 5]

[0043] Figure 5 shows that overexpression of UBE2T attenuates CaMKII-δ9-induced DNA damage, cardiomyocyte death, and cardiomyopathy. Figure 5a is a schematic diagram of the construction of UBE2T tg mice. Figure 5b is a Kaplan-Meier survival curve of wt and CaMKII-δ9 tg mice crossed with wt and UBE2T tg mice. n = 6 (wt + wt), 5 (wt + UBE2T tg), 22 (CaMKII-δ9 tg + wt), and 8 (CaMKII-δ9 tg + UBE2T tg) biologically independent animals. Figure 5c is a graph of left ventricular ejection fraction (EF) and fractional shortening (FS) assessed by echocardiography in wt and CaMKII-δ9 tg mice crossed with wt and UBE2T tg mice. n = 6 (wt + wt), 5 (wt + UBE2T tg), 10 (CaMKII-δ9 tg + wt), and 7 (CaMKII-δ9 tg + UBE2T tg) biologically independent animals. Figure 5d shows statistics of cell death as indicated by TUNEL-positive cells in hearts from wt and CaMKII-δ9 tg mice crossed with wt and UBE2T tg mice. Figure 5e shows DNA damage as evidenced by γH2AX-positive cells in hearts from wt and CaMKII-δ9 tg mice crossed with wt and UBE2T tg mice. n = 5 biologically independent animals. Figure 5f shows representative Western blots and statistics showing UBE2T levels from hearts of wt and CaMKII-δ9 tg mice crossed with wt and UBE2T tg mice. n = 4 biologically independent animals. Data are mean ± sem. Log-rank (Mantel-Cox) test (b) or two-way ANOVA (c–f). [Figure 6]

[0044] Figure 6 shows enhanced CaMKII-δ9-UBE2T-DNA damage signaling in myocardium from patients with hypertrophic cardiomyopathy and in human cardiomyocytes treated with doxorubicin. Figure 6a shows representative Western blots and statistical data for cleaved caspase 3 in myocardial tissue from humans with hypertrophic cardiomyopathy (HCM) or normal controls. n = 4 (normal humans) and 8 (HCM) biologically independent samples. Figure 6b shows representative Western blots and statistical data for UBE2T in myocardial tissue from humans with hypertrophic cardiomyopathy (HCM) or normal controls. n = 4 (normal humans) and 8 (HCM) biologically independent samples. Figure 6c shows representative Western blots and statistical data for γH2AX in myocardial tissue from humans with hypertrophic cardiomyopathy (HCM) or normal controls. n = 4 (normal humans) and 8 (HCM) biologically independent samples. Figure 6d shows cell viability assayed by caspase 3 / 7 activity in human embryonic stem cell-derived cardiomyocytes infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 100, 48 h). n=4 biologically independent samples. Figure 6e shows representative Western blots and statistical data showing γH2AX levels in human embryonic stem cell-derived cardiomyocytes infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 100, 48 h). n=4 biologically independent samples. Figure 6f shows representative Western blots and statistical data showing UBE2T levels in human embryonic stem cell-derived cardiomyocytes infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 100, 48 h). n=4 biologically independent samples. Figure 6g shows cell viability assayed by caspase 3 / 7 activity in human embryonic stem cell-derived cardiomyocytes infected with scrambled or CaMKII-δ9 siRNA with or without doxorubicin (Dox) treatment (1 mM, 24 h) (n = 6 biologically independent samples).Figure 6h shows representative Western blots and statistical data showing the levels of γH2AX in human embryonic stem cell-derived cardiomyocytes infected with scrambled or CaMKII-δ9 siRNA, with or without doxorubicin (Dox) treatment (1 mM, 24 h) (n = 4 biologically independent samples). Figure 6i shows representative Western blots and statistical data showing the levels of UBE2T in human embryonic stem cell-derived cardiomyocytes infected with scrambled or CaMKII-δ9 siRNA, with or without doxorubicin (Dox) treatment (1 mM, 24 h) (n = 4 biologically independent samples). Data are means ± sem. Two-tailed Student's t-test (a-c), one-way ANOVA (d-f), or two-way ANOVA (g-i). [Figure 7]

[0045] Figure 7 shows that CaMKII-δ9 increases the phosphorylation of UBE2T at Ser110 and promotes its degradation. Figure 7a shows representative Western blots and statistical data demonstrating that the proteasome inhibitor β-lac prevents CaMKII-δ9-mediated UBE2T degradation and γH2AX upregulation in NRVM (5 μM, n = 8 biologically independent samples). Figure 7b shows representative Western blots and statistical data demonstrating that the proteasome inhibitor MG132 prevents CaMKII-δ9-mediated UBE2T degradation and γH2AX upregulation in NRVM (10 μM, n = 6 biologically independent samples). Figure 7c shows representative immunostaining images showing the localization of myc-tagged UBE2T in NRVM with or without MG132 (10 μM) in the absence and presence of CaMKII-δ9. n = 6 biologically independent samples. Scale bar, 20 μm. Figure 7d shows co-immunoprecipitation of CaMKII-δ9 by UBE2T in NRVM infected with Ad-Flag-CaMKII-δ9 and Ad-UBE2T-myc. Input represents 6% of the total cell lysate used for each immunoprecipitation. n=4 biologically independent samples. Figure 7e shows a representative Western blot demonstrating that CaMKII-δ9 increases the serine phosphorylation of UBE2T in NRVM. n=4 biologically independent samples. Figure 7f shows a representative Western blot demonstrating that CaMKII-δ9 does not increase the threonine phosphorylation in NRVM. n=4 biologically independent samples. Figure 7g shows a representative Western blot and average data demonstrating that the UBE2T-S110A mutant, but not WT UBE2T or the UBE2T-S193A mutant, resists CaMKII-δ9-mediated degradation (MOI 50, 48 h). n=4 biologically independent samples. Figure 7h shows representative Western blots and average data illustrating the serine phosphorylation and total levels of UBE2T recombinant protein in a cell-free system with or without co-incubation of CaMKII-δ9 or CaMKII-δ2 proteins, n=6 biologically independent samples.Figure 7i shows co-immunoprecipitation of UBE2T and CaMKII-δ2 in lysates from NRVM infected with Ad-UBE2T-myc and Ad-HA-CaMKII-δ2. n = 4 biologically independent samples. Data are means ± sem. Two-way ANOVA (a, b) or one-way ANOVA (g, h). [Figure 8]

[0046] Figure 8 shows the specific phosphorylation of UBE2T by CaMKII-δ9. Figure 8a shows the co-immunoprecipitation of CaMKII-δ1 by UBE2T in NRVM infected with Ad-Flag-CaMKII-δ1 and Ad-UBE2T-myc. Input represents 6% of the total cell lysate used for each immunoprecipitation. n=4 biologically independent samples. Figure 8b shows representative Western blots and statistical data showing that CaMKII-δ9, but not CaMKII-δ1, reduces UBE2T in NRVM. n=4 biologically independent samples. Figure 8c shows the co-immunoprecipitation of CaMKII-δ3 by UBE2T in NRVM infected with Ad-HA-CaMKII-δ3 and Ad-UBE2T-myc. Input represents 6% of the total cell lysate used for each immunoprecipitation. n=4 biologically independent samples. Figure 8d shows representative Western blots and statistical data demonstrating that CaMKII-δ9, but not CaMKII-δ3, reduces UBE2T but not CaMKII-δ3 in NRVM (n=6 independent biological samples). Figure 8e shows co-immunoprecipitation of UBE2T with peptides encoded by the corresponding exon junctions in HEK293 cells transfected with the CaMKII-δ9 exon junction (Flag-GFP tagged) at exons 13-16-17 or the UBE2T-myc plasmid (n=4 independent biological samples). Figure 8f shows a schematic diagram illustrating CaMKII-δ9-mediated cardiac DNA damage and cardiomyocyte cell death signaling. Under normal conditions, UBE2T protects the genome from various types of DNA damage to maintain cardiomyocyte survival. Upon cardiomyocyte injury, CaMKII-δ9 is upregulated and hyperactivated, leading to increased Ser110 phosphorylation and subsequent degradation of UBE2T. Reduced UBE2T levels impair DNA repair mechanisms, leading to accumulation of DNA damage, genomic instability, and cell death. Data are means ± sem. One-way ANOVA. [Figure 9]

[0047] Figure 9 shows that CaMKII-δ9 is present in the heart. Figure 9a is a schematic diagram of CaMKII-δ splice variants. CaMKII-δ undergoes alternative splicing events primarily in two variable domains (one between exon 13 and exon 17, and the other from exon 20 onward). Exons are numbered, full-sized boxes represent coding exons, small green boxes represent untranslated regions (UTRs), and special exons are highlighted. Figure 9b is a diagram of the strategy for SMRT sequencing of full-length CaMKII-δ transcripts. CaMKII-δ transcripts were reverse-transcribed from total RNA isolated from hearts of different species. Each cDNA was amplified with paired primers, the positions of which are indicated by different colored arrows. The forward primer (left) is located on exon 1, and the reverse primer (right) is located on exon 22. PCR products were concentrated and purified for SMRT sequencing. Figure 9c is a diagram of the percentage of exon junctions in variable domain 1 (between exon 13 and exon 17) of CaMKII-δ in human, rhesus monkey, dog, rat, and mouse hearts, as assayed by RNA-seq. Figure 9d is a diagram of the percentage of exon junctions in variable domain 1 (between exon 14 and exon 17) of CaMKII-δ in human, rhesus monkey, dog, rat, and mouse hearts, as assayed by RNA-seq. Figure 9e is a diagram of the percentage of exon junctions in variable domain 2 (between exon 20 and exon 22) of CaMKII-δ in human, rhesus monkey, dog, rat, and mouse hearts, as assayed by RNA-seq. In monkey and human hearts in panel e, exon 20b is 147 bases longer than the typical exon 20 and is a previously undescribed variant of exon 20. Data are means sem (n=8 (humans and rats), 7 (rhesus monkeys), 6 (dogs and mice) biologically independent samples). Data are means ± sem. One-way ANOVA. [Figure 10]

[0048] Figure 10 shows the identification of CaMKII-δ9 protein in the heart. Figure 10a shows the peptide sequences of exons 16 and 21 used as antigens to generate antibodies. Figure 10b shows immunoblots of NRVMs transfected with Ad-β-gal, Ad-HA-CaMKII-δ2, Ad-HA-CaMKII-δ3, or Ad-Flag-CaMKII-δ9 with serum containing anti-exon 16 or anti-exon 21. n=3 biologically independent samples. Figure 10c shows Western blots of Flag-tagged CaMKII-δ9 recombinant protein with anti-exon 16 at increasing ratios of the corresponding antigen peptide to CaMKII-δ9 protein. n=4 biologically independent samples. Mean ± sem. One-way ANOVA. Figure 10d shows Western blots of Flag-tagged CaMKII-δ9 recombinant protein with anti-exon 21 at increasing ratios of the corresponding antigen peptide to CaMKII-δ9 protein. n = 4 biologically independent samples. Mean ± sem. One-way analysis of variance. Figure 10e shows SDS-PAGE followed by Coomassie blue staining of 10-week-old mouse heart lysates immunoprecipitated with anti-exon 21. Figure 10f shows SDS-PAGE followed by Coomassie blue staining of 10-week-old mouse heart lysates immunoprecipitated with anti-exon 16. The approximately 50 kD band (boxed) was excised for MS analysis. Figure 10g shows LC-MS / MS spectra of peptides corresponding to the CaMKII-δ exon 13-16-17 junction from mouse heart immunoprecipitated with anti-exon 21. Figure 10h shows the LC-MS / MS spectrum of a peptide corresponding to the CaMKII-δ exon 20-21 junction from mouse heart immunoprecipitated with anti-exon 16. The upper peptide sequence is the corresponding exon junction, with the specific exon 16 and exon 21 shown in bold. The bn ion is a fragment containing the amino-terminal portion of the peptide at the nth peptide bond, and the yn ion contains the carboxy-terminal portion. NL, normalized intensity level (counts per second). Figure 10i shows the tissue distribution of CaMKII-δ9 in rhesus monkeys. n=4 biologically independent samples.Figure 10j shows the tissue distribution of CaMKII-δ9 in wt mice (n = 4 independent biological samples). Recombinant CaMKII-δ9 protein was used as a positive control (PC). In both species, a higher molecular weight band was detected in the brain, which was the brain-enriched splice variant CaMKII-δ1 (exons 13-15-16-17). Figure 10k shows confocal immunofluorescence microscopy images showing the cytosolic localization of endogenous CaMKII-δ9 (gray) in adult (left) and neonatal (right) ventricular cardiomyocytes of rats (n = 4 independent biological samples). Figure 10l shows confocal immunofluorescence microscopy images showing the nuclear localization of HA-CaMKII-δ3 (gray) in NRVM infected with Ad-HA-CaMKII-δ3 (n = 6 independent biological samples). Scale bar, 10 μm. Figure 10m shows CaMKII-δ9 protein levels in nuclear and cytosolic fractions of NRVM. n=6 biologically independent samples. [Figure 11]

[0049] Figure 11 shows the pathological relevance of CaMKII-δ9 in the heart. Figure 11a is a Western blot showing the phosphorylation level of CaMKII-δ9 in NRVM with or without Dox treatment (1 μM, 30 and 60 min) (n=8 biologically independent samples). Figure 11b is a Western blot showing the oxidation level of CaMKII-δ9 in NRVM with or without Dox treatment (1 μM, 30 and 60 min) (n=7 biologically independent samples). NRVM were infected with Ad-Flag-CaMKII-δ9, and lysates were immunoprecipitated with Flag antibody and analyzed by Western blot analysis. Figure 11c is a representative Western blot and statistical data showing the phosphorylation level of CaMKII-δ9 in perfused mouse hearts with or without I / R injury (30 min ischemia followed by 60 min reperfusion). n=6 biologically independent samples. Heart lysates were immunoprecipitated with exon 16 antibody and subjected to Western blot analysis. Figure 11d shows representative Western blots and statistical data showing the oxidation levels of CaMKII-δ9 in perfused mouse hearts with or without I / R injury (30 minutes of ischemia followed by 60 minutes of reperfusion). n = 6 biologically independent samples. Heart lysates were immunoprecipitated with exon 16 antibody and subjected to Western blot analysis. Figure 11e shows the sequence of CaMKII-δ9 siRNA. The black sequence is the siRNA target in exon 16 of CaMKII-δ9. The siRNA sequence is shown in gray underneath. Figure 11f shows the knockdown efficiency of CaMKII-δ9 siRNA confirmed by mRNA level (n = 5 (scrambled) and 8 (CaMKII-δ9 siRNA) biologically independent samples). Figure 11g shows the knockdown efficiency of CaMKII-δ9 siRNA confirmed by protein level (n = 3 biologically independent samples). Figure 11h shows averaged data of CaMKII-δ2 and CaMKII-δ3 mRNA levels assayed by real-time PCR in NRVM infected with scrambled or CaMKII-δ9 siRNA, n=18 (CaMKII-δ2) and n=15 (CaMKII-δ3) biologically independent samples.Figure 11i shows cell viability, as assessed by LDH concentration in the culture medium of NRVMs treated with scrambled or CaMKII-δ9 siRNA, with or without HO (200 μM). n=6 biologically independent samples. Figure 11j shows cell viability, as assessed by LDH concentration in the culture medium of NRVMs treated with scrambled or CaMKII-δ9 siRNA, with or without Dox (1 μM). n=6 biologically independent samples. Figure 11k shows cell viability, as assessed by LDH concentration in the culture medium of NRVMs infected with the indicated MOIs of Ad-β-gal, Ad-CaMKII-δ9, and Ad-CaMKII-δ2 for 48 h. n=10 biologically independent samples. Figure 11l shows representative Western blots and statistical data for CaMKII-δ expression in NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 50, 48 h). n = 3 biologically independent samples. Data are mean ± sem. One-way ANOVA (a, b, g, k), two-tailed Student's t-test (c, d, f, h, l), or two-way ANOVA (i, j). [Figure 12]

[0050] Figure 12 shows RNA-seq analysis of gene expression profiles of CaMKII-δ9 and CaMKII-δ2. Figure 12a shows a heatmap depicting gene expression signatures based on differentially expressed genes between Ad-β-gal and Ad-CaMKII-δ9 among NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 50, 48 h). Expression values ​​for each gene were calculated as fragments per kilobase of transcript per million mapped fragments (FPKM). Seventy-seven genes are listed. Expression of these genes was differentially and significantly altered in cells infected with Ad-CaMKII-δ9 compared to cells infected with Ad-β-gal (>1.5-fold or <0.67-fold, n = 3 biologically independent samples). The 15 differentially regulated genes in Ad-CaMKII-δ9 and Ad-CaMKII-δ2 are shown in gray. Heat maps were generated using the R package "Peatmap" with the option "scale=row," which means that the expression values ​​of each gene were Z-score normalized by the FPKM value. Figure 12b shows data on mRNA levels assayed by real-time PCR for 12 of the 15 genes identified by RNA-seq as regulated by CaMKII-δ9 but not CaMKII-δ2 in NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (MOI 50, 48 h). n = 14 biologically independent samples. Figure 12c shows protein levels of COX-2 in cultured NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (indicated doses, 48 ​​h). n = 4 biologically independent samples. Figure 12d shows PAI-2 protein levels in cultured NRVM infected with Ad-β-gal, Ad-CaMKII-δ9, or Ad-CaMKII-δ2 (dose indicated, 48 h). n=4 biologically independent samples. Figure 12e shows representative Western blots and averaged data showing COX-2 protein levels in NRVM transfected with scrambled or COX-2 siRNA. n=4 biologically independent samples.Figure 12f shows cell viability, as measured by caspase 3 / 7 activity, in NRVM infected with Ad-β-gal or Ad-CaMKII-δ9 in the presence or absence of COX-2 siRNA (n=3 biologically independent samples). Figure 12g shows LDH concentrations in the culture medium in NRVM infected with Ad-β-gal or Ad-CaMKII-δ9 in the presence or absence of COX-2 siRNA (n=6 biologically independent samples). Figure 12h shows representative Western blots and averaged data showing UBE2T protein levels in NRVM transfected with scrambled or UBE2T siRNA (n=4 biologically independent samples). Data are means ± sem. One-way ANOVA (b, c, d, e, h) or two-way ANOVA (f, g). [Figure 13]

[0051] Figure 13 shows that CaMKII-δ9 induced cardiomyocyte DNA damage. Figure 13a shows representative immunostaining images of γH2AX in NRVM infected with Ad-β-gal, Ad-CaMKII-δ2, or Ad-CaMKII-δ9 (MOI 50, 48 h). Scale bar, 20 μm. Figure 13b shows representative comet assay images in NRVM infected with Ad-β-gal, Ad-CaMKII-δ2, or Ad-CaMKII-δ9 (MOI 50, 48 h). Scale bar, 20 μm. Representative images and averaged data are shown in Figures 3a and 3c. Figure 13c shows representative Western blots and statistical data showing FANCD2 levels in NRVM infected with scrambled and FANCD2 siRNA (n = 4 biologically independent samples). Figure 13d is a representative Western blot and statistical data showing FANCI levels in NRVM infected with scrambled, FANCI siRNA (n=4 biologically independent samples). Figure 13e is a statistical data showing caspase 3 / 7 activity in NRVM infected with scrambled, FANCD2 siRNA (n=5 biologically independent samples). Figure 13f is a statistical data showing caspase 3 / 7 activity in NRVM infected with scrambled, FANCI siRNA (n=5 biologically independent samples). Data are mean ± sem. One-way ANOVA. [Figure 14]

[0052] Figure 14 shows the role of CaMKII-δ9-UBE2T signaling in cardiac pathophysiology. Figure 14a is a schematic diagram of the construction of CaMKII-δ9 tg mice. Figure 14b is a diagram of PCR genotyping of wt and CaMKII-δ9 tg mice. Figure 14c is a diagram of the heart weight-to-body weight ratio of 10-week-old wt and CaMKII-δ9 tg mice (n = 15 (wt) and 9 (CaMKII-δ9 tg) biologically independent samples). Figure 14d is a diagram of ventricular gene expression of 10-week-old wt and CaMKII-δ9 tg mice (n = 7 (wt) and 6 (CaMKII-δ9 tg) biologically independent samples). Figure 14e is a diagram of cardiac γH2AX immunostaining of 10-week-old wt and CaMKII-δ9 tg mice. Arrows indicate γH2AX-positive cells. Right window: Enlarged image of the indicated field. Averaged data are in Figure 4g. Scale bar, 20 μm. Figure 4f is a schematic diagram of the construction of CaMKII-δ9 shRNA transgenic (shRNA tg) mice. Figure 14g is a diagram of PCR genotyping of wt and shRNA tg mice. Figure 14h is a diagram of cardiac exon 21 protein levels in 10-week-old wt and shRNA tg mice (n = 4 biologically independent samples). Figure 14i is a diagram of heart weight-to-body weight ratios in wt and shRNA tg mice 4 weeks after TAC surgery (n = 16 (wt) and 8 (shRNA tg) biologically independent samples). Figure 14j is a diagram of cardiac UBE2T protein levels in wt and shRNA tg mice 4 weeks after TAC surgery (n = 4 biologically independent samples). Figure 14k shows cardiac CaMKII-δ protein levels in 10-week-old CaMKII-δ9 tg mice and CaMKII-δ2 tg mice (n=4 biologically independent samples). Figure 14l shows cardiac TUNEL staining from wt, CaMKII-δ9 tg, and CaMKII-δ2 tg mice (n=8 biologically independent samples). Figure 14m shows echocardiography from wt, CaMKII-δ9 tg, and CaMKII-δ2 tg mice (n=8 biologically independent samples).Figure 14n shows the heart weight-to-body weight ratio from wt, CaMKII-δ9 tg, and CaMKII-δ2 tg mice (n = 8 biologically independent animals). Figure 14o shows Kaplan-Meier survival curves from wt, CaMKII-δ9 tg, and CaMKII-δ2 tg mice (n = 10 (wt), 19 (CaMKII-δ9 tg), and 12 (CaMKII-δ2) biologically independent samples). Figure 14p shows cardiac γH2AX staining from wt, CaMKII-δ9 tg, and CaMKII-δ2 tg mice (n = 8 biologically independent samples). Figure 14q shows cardiac UBE2T protein levels from wt, CaMKII-δ9 tg, and CaMKII-δ2 tg mice (n = 4 biologically independent samples). Data are mean ± sem. Two-tailed Student's t-test (c, d, i), one-way ANOVA (k-n, p, q), two-way ANOVA (j), or log-rank (Mantel-Cox) test (o). [Figure 15]

[0053] Figure 15 shows that CAMKII-δ9 phosphorylates UBE2T at the Ser110 site. Figure 15a depicts mass spectrometry data showing two potential phosphorylation sites of UBE2T (Ser110 and Ser193, circles) mediated by CAMKII-δ9 (n = 3 biologically independent samples). HEK293 cells were transfected with myc-tagged UBE2T in the presence of control vector or Flag-tagged CAMKII-δ9, and whole-cell lysates were immunoprecipitated with myc antibody, followed by post-translational modification mass spectrometry of the immune complexes. Figure 15b depicts a sequence alignment of UBE2T proteins across 12 species, showing the conservation of the Ser110 site (arrow), but not the Ser193 site. Figure 15c shows representative Western blots and averaged data showing the serine phosphorylation and total levels of UBE2T and UBE2T-S110A recombinant proteins in a cell-free system with or without co-incubation with CaMKII-δ9 protein (n = 4 biologically independent samples). Two-way ANOVA. Figure 15d shows representative immunofluorescence images of wt UBE2T, UBE2T-S110A, and UBE2T-S193A (all myc-tagged) in NRVM infected with Ad-UBE2T, Ad-UBE2T-S110A, or Ad-UBE2T-S193A (n = 6 biologically independent samples). Note that UBE2T-S110A was resistant to degradation and distributed to both the cytoplasm and nucleus, indicating that the Ser110 site of UBE2T is responsible for its degradation but not its subcellular distribution. Scale bar, 10 μm. Data are means ± sem. [Figure 16]

[0054] Figure 16 shows the interaction between peptides encoded by CAMKII-δ exon junctions and UBE2T. Figure 16a shows the co-immunoprecipitation of peptides encoded by the corresponding exon junctions by UBE2T in HEK293 cells transfected with the CAMKII-δ exon junctions of exons 13-17 (tagged with Flag-GFP) or UBE2T-myc plasmid (n=4 biologically independent samples). Figure 16b shows the co-immunoprecipitation of peptides encoded by the corresponding exon junctions by UBE2T in HEK293 cells transfected with the CAMKII-δ exon junctions of exons 13-14-17 (tagged with Flag-GFP) or UBE2T-myc plasmid (n=4 biologically independent samples). Figure 16c shows co-immunoprecipitation of peptides encoded by the corresponding exon junctions of CAMKII-δ exons 13-15-16-17 (tagged with Flag-GFP) by UBE2T in HEK293 cells transfected with the corresponding exon junctions or UBE2T-myc plasmid (n=4 biologically independent samples). [Figure 17]

[0055] Figure 17 shows the amino acid sequence of exon 16 of the CAMKII-δ gene (sequence number 1), the amino acid sequence of exons 13-16 of the CAMKII-δ gene (sequence number 2), the amino acid sequence of exons 16-17 of the CAMKII-δ gene (sequence number 3), the amino acid sequence of exons 13-16-17 of the CAMKII-δ gene (sequence number 4), and the full-length amino acid sequence of CAMKII-δ9 (sequence number 5). [Figure 18]

[0056] FIG. 18 shows the nucleic acid sequences of human and rat exon 16 of the CAMKII-δ gene (SEQ ID NO: 6), the nucleic acid sequence of mouse exon 16 of the CAMKII-δ gene (SEQ ID NO: 7), the nucleic acid sequence of human exons 13-16 of the CAMKII-δ gene (SEQ ID NO: 8), the nucleic acid sequence of rat exons 13-16 of the CAMKII-δ gene (SEQ ID NO: 9), the nucleic acid sequence of mouse exons 13-16 of the CAMKII-δ gene (SEQ ID NO: 10), and the nucleic acid sequence of human exons 16-17 of the CAMKII-δ gene. The nucleic acid sequence of exons 16-17 of the CAMKII-δ gene in rats (SEQ ID NO: 11), the nucleic acid sequence of exons 16-17 of the CAMKII-δ gene in mouse (SEQ ID NO: 13), the nucleic acid sequence of exons 13-16-17 of the CAMKII-δ gene in humans (SEQ ID NO: 14), the nucleic acid sequence of exons 13-16-17 of the CAMKII-δ gene in rats (SEQ ID NO: 15), and the nucleic acid sequence of exons 13-16-17 of the CAMKII-δ gene in mouse (SEQ ID NO: 16) are shown. [Figure 19]

[0057] FIG. 19 shows the nucleic acid sequence of full-length human CAMKII-δ9 (SEQ ID NO: 17). [Figure 20]

[0058] FIG. 20 shows the nucleic acid sequence of full-length rat CAMKII-δ9 (SEQ ID NO: 18). [Figure 21]

[0059] FIG. 21 shows the nucleic acid sequence of full-length mouse CAMKII-δ9 (SEQ ID NO: 19). DETAILED DESCRIPTION OF THE INVENTION

[0037]

[0060] Before describing the present invention in more detail, it is understood that this disclosure is not limited to the particular embodiments described, which, as such, may, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intervening value within that stated range, is included within the scope of the disclosure. The upper and lower limits of those smaller ranges may independently be included in the smaller ranges and are also included within the scope of the invention, subject to any specifically excluded ranges in the stated ranges. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0038]

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Edition, J. Wiley & Sons (New New York, NY 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th ed., John Wiley & Sons (New York, NY 1992) will provide one of ordinary skill in the art with a general guide to many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0039]

[0062] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publication is cited. The citation of any publication should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of its disclosure prior to the filing date. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0040]

[0063] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated or combined with the features of any of several other embodiments without departing from the scope and spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0041]

[0064] Embodiments of the present invention employ, unless otherwise specified, techniques of chemistry, solid state chemistry, inorganic chemistry, organic chemistry, physical chemistry, analytical chemistry, materials chemistry, biochemistry, biology, molecular biology, recombinant DNA technology, pharmacology, imaging, and the like, which are within the skill of the art. The techniques are fully explained in the literature, e.g., "Molecular Cloning: A Laboratory Manual," 2nd ed. (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" series (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M.Ausubel et al., eds., 1987, and periodically updated); and "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994). Primers, polynucleotides, and polypeptides used in the present invention can be produced using standard techniques known in the art.

[0042]

[0065] The following embodiments are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the biomarkers and kits disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for.

[0043]

[0066] As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include plural forms of the same unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of compounds. In this specification and the claims that follow, reference will be made to a number of terms, which will be defined to have the following meanings unless a contrary intention is apparent.

[0044]

[0067] In one aspect, the present invention discloses a method for treating or preventing a CaMKII-mediated disease in a subject, the method comprising administering to the subject an effective amount of a CaMKII-δ9 antagonist. In another aspect, the present invention discloses the use of a CaMKII-δ9 antagonist in the manufacture of a medicament for treating or preventing a CaMKII-mediated disease in a subject. In yet another aspect, the present invention discloses a CaMKII-δ9 antagonist for use in treating or preventing a CaMKII-mediated disease in a subject.

[0045]

[0068] As used herein, the terms "treat," "treating," and "treatment" refer to clinical intervention in an attempt to alter the natural course of a disease, condition, or disorder in the subject being treated, and may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include prevention of the onset or recurrence of the disease, condition, or disorder, alleviation of symptoms, mitigation of any direct or indirect pathological consequences of the disease, condition, or disorder, slowing the rate of progression of the disease, condition, or disorder, amelioration or palliation of the pathology, and remission or improved prognosis.

[0046]

[0069] As used herein, the terms "prevent", "preventing" and "prevention" relate to reducing the likelihood of developing a disease, condition or disorder in a subject who does not have the disease, condition or disorder but is at risk of or susceptible to developing the disease, condition or disorder.

[0047]

[0070] As used herein, the term "CaMKII-mediated disease" refers to a disease, condition, or disorder associated with abnormal levels and / or activity of CaMKII, caused or promoted by abnormally high or low levels and / or activity of CaMKII due to abnormal activation or disruption of CaMKII in a subject. In some embodiments, the CaMKII-mediated disease is associated with elevated levels and / or activity of CaMKII-δ9. In some embodiments, the CaMKII-mediated disease is a cardiac disease or metabolic disease. In some embodiments, the cardiac disease is cardiomyopathy, myocarditis, diabetic heart disease, myocardial ischemia, cardiac ischemia / reperfusion injury, myocardial infarction, heart failure, arrhythmia, cardiac rupture, angina pectoris, cardiac hypertrophy, cardiac damage, hypertensive heart disease, rheumatic heart disease, angina pectoris, myocarditis, coronary heart disease, and pericarditis. In some embodiments, the cardiac disease is hypertrophic cardiomyopathy. In some embodiments, the metabolic disease is selected from the group consisting of insulin resistance, obesity, diabetes, hypertension, dyslipidemia, diabetic cerebrovascular disease, diabetic eye complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, gout, and hyperuricemia.

[0048]

[0071] As used herein, the terms "administer," "administering," "administered," and "administration" refer to the delivery of a substance to a subject in need thereof. The route of administration can be topical, oral, intranasal, parenteral, enteral, rectal, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, buccal, sublingual, or ocular. In some embodiments, a substance may be administered to a subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.

[0049]

[0072] As used herein, the term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals. A "subject" can be a domestic animal, e.g., a cow, pig, sheep, poultry, and horse, or a rodent, e.g., a rat, a mouse, or a non-human primate, e.g., an ape, monkey, or rhesus monkey, or a domesticated animal, e.g., a dog or a cat. The term "subject" is not intended to be limiting in any respect and can be of any age, sex, and health status, e.g., male or female, elderly, adult, adolescent, child, or infant. A human "subject" can be Caucasian, African, Asian, Jewish, or of another ethnic background, or a mixture of the aforementioned ethnic backgrounds. In some embodiments, the subject is a human or a non-human primate. In some embodiments, the non-human primate is a rhesus monkey. In some embodiments, the subject is a human.

[0050]

[0073] As used herein, the term "effective amount" refers to the amount of a pharmaceutical agent that achieves a therapeutic or prophylactic effect by inhibiting or alleviating a disease, condition, or disorder in a subject, or by prophylactically inhibiting or preventing the onset of a disease, disorder, or symptom. An effective amount can be an amount of pharmaceutical agent that relieves to some extent one or more symptoms of a disease or disorder in a subject; partially or completely restores to normal levels one or more physiological or biochemical parameters associated with or causing the disease or disorder; and / or reduces the likelihood of the onset of the disease or disorder. A skilled clinician can determine the effective amount of a pharmaceutical agent for treating or preventing a particular disease or disorder at the time of administration. The exact amount of pharmaceutical agent required to be effective depends on numerous factors, such as the specific activity of the active agent, the delivery device used, the physical properties of the agent, and the purpose of administration, in addition to many patient-specific considerations. Determining the amount of pharmaceutical agent that should be administered to be effective is routine in the art and within the skill of a skilled clinician.

[0051]

[0074] The term "antagonist" as used herein refers to a molecule that reduces the amount, formation, function and / or downstream signaling of a protein, polypeptide or peptide by suppressing its expression level or activity. For example, an "antagonist of CaMKII-δ9" of the present invention refers to a molecule that reduces the amount, formation, function and / or downstream signaling of CaMKII-δ9 by suppressing its expression level or activity.

[0052]

[0075] A molecule is considered to inhibit the expression level or activity of CaMKII-δ9 if it causes a significant decrease in the expression level (at either the transcriptional or translational level) or activity of CaMKII-δ9. Similarly, a molecule is considered to inhibit the binding between CaMKII-δ9 and its substrate if it causes a significant decrease in the binding between CaMKII-δ9 and its substrate, which causes a significant decrease in downstream signaling and function mediated by CaMKII-δ9 (e.g., ubiquitination). A decrease is considered significant if it is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0053]

[0076] Binding antagonists can act in two ways. In some embodiments, the binding antagonists of the present invention compete with CaMKII-δ9 to bind to its substrate, thereby interfering with, blocking, or preventing CaMKII-δ9 from binding to its substrate. This type of antagonist, which binds to a substrate but does not induce potential signal transduction, is also known as a "competitive antagonist" and may include, for example, a vector expressing CaMKII-δ9 that does not have the function of phosphorylation or oxidation. In another embodiment, the binding antagonists of the present invention can bind to and sequester CaMKII-δ9 with sufficient affinity and specificity to substantially interfere with, block, or prevent CaMKII-δ9 from binding to its substrate. This type of antagonist is also known as a "neutralizing antagonist" and may include, for example, an antibody or aptamer against CaMKII-δ9 that specifically binds to CaMKII-δ9.

[0054]

[0077] In some embodiments, the antagonist is an antagonist that inhibits phosphorylation of a ubiquitin-conjugating enzyme. In some embodiments, the ubiquitin-conjugating enzyme is UBE2T. In some embodiments, the antagonist is an antagonist that inhibits phosphorylation of UBE2T at Ser110.

[0055]

[0078] Ubiquitination regulates the degradation of cellular proteins by the ubiquitin-proteasome system, controlling protein half-life and expression levels. This process involves the sequential action of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Ubiquitin-conjugating enzymes perform the second step in the ubiquitination reaction, targeting proteins for degradation by the proteasome. Phosphorylation is a biochemical reaction in which a phosphate group is added to a serine (Ser), threonine (Thr), or tyrosine (Tyr) residue of a protein and is catalyzed by a protein kinase enzyme. For example, CaMKII-δ9 phosphorylates UBE2T at Ser110. Phosphorylation usually modifies the function of target proteins and frequently leads to their activation. As part of cellular homeostasis, phosphorylation is a transient process that is reversed by other enzymes called phosphatases. Therefore, protein phosphorylation levels change over time and can be assessed in a variety of well-known ways, for example, by immunological approaches. For example, the amount of phosphorylated UBE2T is measured by an immunoassay using a reagent that specifically binds to phosphorylated UBE2T. Such immunoassays can have a variety of well-known forms, including, but not limited to, radioimmunoassay, Western blot assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay, or slot blot assay.

[0056]

[0079] In some embodiments, the enzyme immunoassay is a sandwich enzyme immunoassay that uses a capture antibody or fragment thereof that specifically binds to UBE2T and a detection antibody or fragment thereof that specifically binds to phosphorylated UBE2T. Such enzyme immunoassays are particularly advantageous because the identification of protein level differences between related kinase family members or isoforms provides a relatively high homology between the kinases themselves and their phosphorylated forms.

[0057]

[0080] Immunoreagents for identifying both phosphorylated and non-phosphorylated forms of UBE2T, as well as for detecting CaMKII-δ9, are well known in the art and can be prepared using standard techniques, e.g., suitable antibodies for UBE2T, for generating anti-CaMKII-δ9 antibodies, anti-UBE2T antibodies, and / or anti-phospho-UBE2T antibodies (e.g., monoclonal antibodies). Antibodies can be produced by inoculating a host animal with the fragments. Such anti-CaMKII-δ9 antibody, anti-UBE2T antibody, and / or anti-phospho-UBE2T antibody reagents can be used, for example, to isolate and / or determine the amount of each protein in a cell lysate. Such reagents can further be used to monitor protein levels in cells or tissues, e.g., white blood cells or lymphocytes, as part of a clinical testing method, e.g., to monitor the optimal dose of an inhibitor. Detection can be facilitated by coupling (e.g., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S or 3 Contains H.

[0058]

[0081] In some embodiments, the antagonist is a specific antagonist of CaMKII-δ9.

[0082] The term "specific antagonist" as used herein means that the antagonist does not significantly inhibit any peptide, polypeptide, or substance other than CaMKII-δ9. In some embodiments, the specific antagonist has an inhibitory effect on CaMKII-δ9 that is at least 3-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than that on any other related peptide or polypeptide. For example, the antagonist has an inhibitory effect on CaMKII-δ9 that is at least 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater than that on CaMKII-δ2 or CaMKII-δ3. In some embodiments, the antagonist inhibits the level or activity of CaMKII-δ9 but does not significantly inhibit the level or activity of CaMKII-δ2 or CaMKII-δ3. The term "significantly" as used herein relates to a statistically significant difference or a notable difference that would be recognizable to one skilled in the art.

[0059]

[0083] In some embodiments, the antagonist is an antibody that specifically recognizes CaMKII-δ9.

[0084] Unless otherwise specified herein, the term "antibody" broadly includes naturally occurring antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric antibodies, humanized antibodies, and multispecific antibodies, as well as all fragments and derivatives of the foregoing, wherein the fragments and derivatives have at least one antigen-binding site. Antibody derivatives may include proteins or chemical moieties attached to the antibody.

[0060]

[0085] As used herein, the term "antibody" further includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). As used herein, the term "antigen-binding portion" relates to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or a fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region ( Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that generates the VL and VH domains as a single protein chain in which the VL and VH domains pair to form a monovalent polypeptide (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL nucleic acid sequence of a specific scFv can be joined to cDNA or genomic sequences of human immunoglobulin constant regions to generate an expression vector encoding a complete IgG polypeptide or another isotype. The VH and VL can further be used in the generation of Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other types of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the use of a linker that is too short to allow pairing between the two domains on the same chain forces them to pair with complementary domains on another chain, creating two antigen-binding sites (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2:1121-1123).

[0061]

[0086] Furthermore, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion polypeptide formed by covalent or noncovalent association of the antibody or antibody portion with one or more additional proteins or peptides. Examples of such immunoadhesion polypeptides include the use of the streptavidin core region to generate tetrameric scFv polypeptides (Kipriyanov, SM et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, biomarker peptides, and C-terminal polyhistidine tags to generate bivalent biotinylated scFv polypeptides (Kipriyanov, SM et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab fragments and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques as described herein.

[0062]

[0087] The antibody can be a polyclonal or monoclonal antibody; a xenoantibody, an alloantibody, or a syngeneic antibody; or a modified version thereof (e.g., humanized, chimeric, etc.). The antibody can also be fully human. In some embodiments, the antibody of the invention specifically or substantially specifically binds to CaMKII-δ9 or a fragment thereof.

[0063]

[0088] As used herein, the term "monoclonal antibody" refers to a population of antibody polypeptides containing only one type of antigen-binding site capable of immunoreacting with a particular epitope of an antigen, and the term "polyclonal antibody" refers to a population of antibody polypeptides containing multiple types of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody typically exhibits a single binding affinity for a particular antigen with which it immunoreacts. In some embodiments, a monoclonal antibody typically comprises an antibody comprising a polypeptide sequence that binds to a target, provided that the target-binding polypeptide sequence is obtained by a process comprising selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be selection of a unique clone from a pool of multiple clones, e.g., hybridoma clones, phage clones, or recombinant DNA clones. The selected target-binding sequence can be selected by, for example, improving affinity for the target, humanizing the target-binding sequence, or by enhancing its production in cell culture. Antibodies that can be further modified to improve activity, reduce their immunogenicity in vivo, create multispecific antibodies, etc., and that contain altered target binding sequences are also understood to be monoclonal antibodies of the invention. To screen for antibodies that bind to an epitope on the antigen bound by an antibody of interest, a routine cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.

[0064]

[0089] Antibodies may also be "humanized," which is intended to include antibodies produced in non-human cells with variable and constant regions altered to more closely resemble antibodies produced in human cells. For example, the amino acid sequence of a non-human antibody is altered to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the present invention may contain amino acid residues, for example, in the CDRs, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0065]

[0090] As used herein, the term "specifically recognize" means that the antibody does not substantially bind ("cross-react") to other peptides, polypeptides, or substances. In some embodiments, the antibody binds to the specifically recognized peptide or polypeptide with at least 3-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater affinity than any other related peptide or polypeptide. For example, the antagonist specifically binds to CaMKII-δ9 with at least 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold greater affinity than CaMKII-δ2 or CaMKII-δ3. In some embodiments, the antagonist inhibits the activity of CaMKII-δ9 but does not significantly inhibit the activity of CaMKII-δ2 or CaMKII-δ3.

[0066]

[0091] As used herein, the terms "inhibit," "inhibiting," and "inhibition" refer to a reduction in the baseline activity of a biological activity or biological process. "Inhibition of CaMKII-δ9 activity" refers to a reduction in the level or activity of CaMKII-δ9 as a direct or indirect response to the presence of an antagonist of the present invention, compared to the level or activity of CaMKII-δ9 in the absence of the antagonist. In some embodiments, the activity of CaMKII-δ9 includes the phosphorylation and / or oxidation activity of CaMKII-δ9, which can be measured by a person skilled in the art.

[0067]

[0092] In some embodiments, the antibody binds to the amino acid sequence encoded by exon 16 of the CaMKII-δ gene. In some embodiments, the antibody binds to the amino acid sequence encoded by exons 13-16 of the CaMKII-δ gene. In some embodiments, the antibody binds to the amino acid sequence encoded by exons 16-17 of the CaMKII-δ gene. In some embodiments, the antibody binds to the amino acid sequence encoded by exons 13-16-17 of the CaMKII-δ gene. In some embodiments, the antibody binds to the amino acid sequence of full-length CaMKII-δ9.

[0068]

[0093] As used herein, the term "amino acid" in its broadest sense relates to any compound and / or substance that can be incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid, in some embodiments, the amino acid is a D-amino acid, in some embodiments, the amino acid is an L-amino acid, in some embodiments, the amino acid is a standard amino acid, and in some embodiments, the amino acid is a non-standard amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in natural peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including the carboxy- and / or amino-terminal amino acids within a polypeptide, may contain a structural modification compared to the general structure described above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, and / or substitution compared to the general structure. In some embodiments, such a modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to a polypeptide containing an otherwise identical, unmodified amino acid. In some embodiments, such a modification does not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to a polypeptide containing an otherwise identical, unmodified amino acid. As will be clear from the context, in some embodiments the term "amino acid" is used in reference to free amino acids, and in some embodiments in reference to amino acid residues of polypeptides. Amino acid names are further represented in this disclosure as standard one-letter or three-letter codes, summarized below.

[0069] [Table 1]

[0070]

[0094] As used herein, the terms "encoded" or "encoding" refer to the ability to be transcribed into mRNA and / or translated into a peptide or protein. The term "coding sequence" or "gene" relates to a polynucleotide sequence that encodes a peptide or protein. These two terms can be used interchangeably in the present invention. In some embodiments, a coding sequence is a complementary DNA (cDNA) sequence that is reverse transcribed from messenger RNA (mRNA). In some embodiments, a coding sequence is mRNA.

[0071]

[0095] In some embodiments, the nucleic acid sequences of exon 16, exon 13-16, exon 16-17 and exon 13-16-17 of the CaMKII-δ gene, and full-length CaMKII-δ9 comprise nucleic acid sequences having at least 70% homology, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology, to any one of the nucleic acid sequences set forth in SEQ ID NOs: 6-19, and further encode one of the amino acid sequences set forth in SEQ ID NOs: 1-5.

[0072]

[0096] In some embodiments, CaMKII-δ9 has the amino acid sequence set forth in SEQ ID NOs: 1-5. In some embodiments, the coding sequence for CaMKII-δ9 has the nucleic acid sequence set forth in SEQ ID NOs: 6-19. In some embodiments, the invention provides nucleic acid sequences encoding SEQ ID NOs: 1-5, but which sequences differ from any one of the nucleic acid sequences set forth in SEQ ID NOs: 6-19 due to degeneracy of the genetic code.

[0073]

[0097] The term "degeneracy of the genetic code" as used herein refers to the phenomenon in which one amino acid has two or more corresponding genetic codes. For example, proline has four synonymous codons: CCU, CCC, CCA, and CCG. It is well known in the art that due to the degeneracy of the genetic code, a nucleic acid at a specific position can be substituted with any nucleic acid sequence without changing the encoded amino acid sequence. Substitution of the degeneracy of the genetic code, for example, by site-directed mutagenesis of bases, is commonplace for those skilled in the art. Different organisms have developed different preferences for different codons. To express the polypeptides of the present invention in a selected biological cell, the preferred codons of the biological cell can be selected to obtain the corresponding coding sequence, and the amino acid sequences of the present invention (e.g., SEQ ID NOS: 1-5) can be obtained by recombinant expression.

[0074]

[0098] In some embodiments, the antagonist is a small molecule compound that binds to CaMKII-δ9.

[0099] As used herein, the term "small molecule compound" refers to a low molecular weight compound that can function as an enzyme substrate or regulator of a biological process. Generally, a "small molecule compound" is a molecule less than about 5 kilodaltons (kD) in size. In some embodiments, the small molecule compound is less than about 4 kD, about 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule compound is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, the small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, the small molecule is non-polymeric. In some embodiments, in accordance with the present invention, a small molecule is not a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, polysaccharide, glycoprotein, proteoglycan, etc. In some embodiments, the small molecule is a therapeutic agent. In some embodiments, the small molecule is an adjuvant. In some embodiments, the small molecule is a drug.

[0075]

[0100] In some embodiments, the antagonist inhibits the coding sequence of CaMKII-δ9. In some embodiments, the RNAi molecule is a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a microRNA (miRNA).

[0076]

[0101] The RNAi of the present invention is one or more RNAi having specificity for CaMKII-δ9. It may contain two or more types of nucleic acid molecules. For example, A single type of siRNA may be used to regulate CaMKII-δ9 expression, two types of siRNA (e.g., having different sequences) may be used in combination to regulate the expression level of CaMKII-δ9, or an antisense oligonucleotide may be combined with an siRNA to reduce the level of CaMKII-δ9.

[0077]

[0102] The RNAi of the present invention inhibits CaMKII-δ9 at various levels, for example, at the post-transcriptional level. Downregulate at the level, pre-transcriptional level or epigenetic level. In a non-limiting example, epigenetic regulation of gene expression by the RNAi molecules of the present invention can be due to the RNAi molecule-mediated modification of chromatin structure, which alters gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

[0078]

[0103] The RNAi molecules of the present invention can be double-stranded or single-stranded. In this case, one strand is a sense strand and the other is an antisense strand.Antisense strand comprises the nucleotide sequence complementary to the coding sequence of CaMKII-δ9 or a part thereof, and sense strand comprises the nucleotide sequence corresponding to the coding sequence of CaMKII-δ9 or a part thereof.Instead, RNAi molecule is assembled from a single oligonucleotide, and in this case, the self-complementary sense region and the antisense region of RNAi molecule are linked using a nucleic acid-based or non-nucleic acid-based linker.RNAi molecule can be a polynucleotide with double-stranded, asymmetric double-stranded, hairpin secondary structure or asymmetric hairpin secondary structure.RNAi can be a circular single-stranded polynucleotide with a stem comprising two or more loop structures and self-complementary sense region and antisense region.The circular polynucleotide can be processed either in vivo or in vitro to generate active RNAi molecule.

[0079]

[0104] In some embodiments, the RNAi molecule has 10-100 bases, e.g. The length may be about 10 to about 100 bases, about 15 to about 90 bases, about 20 to about 80 bases, about 25 to about 70 bases, about 30 to about 60 bases, or about 35 to about 50 bases.

[0080]

[0105] Small interfering RNA (siRNA) suppresses the expression of genes that share homology. The siRNA is a double-stranded RNA molecule that can enhance or reduce the cleavage of ribosomal proteins. Each strand of the siRNA can have a length of about 10 to about 100 bases, about 15 to about 90 bases, about 20 to about 80 bases, about 25 to about 70 bases, about 30 to about 60 bases, or about 35 to about 50 bases. The double-stranded siRNA can have a length of about 10 to about 50 base pairs, about 12 to about 45 base pairs, about 15 to about 40 base pairs, about 20 to about 35 base pairs, about 20 to about 30 base pairs, or about 20 to about 25 base pairs.

[0081]

[0106] Small hairpin RNAs (shRNAs) suppress target gene expression through RNA interference. shRNAs are artificial RNA molecules with tight hairpin turns that can be used to induce transcription. In some embodiments, expression of shRNAs in cells is achieved by delivery of a plasmid or a viral or bacterial vector. shRNAs typically have a length of about 10 to 100 base pairs, e.g., about 10 to about 100 base pairs, about 15 to about 90 base pairs, about 20 to about 80 base pairs, about 25 to about 70 base pairs, about 30 to about 60 base pairs, or about 35 to about 50 base pairs.

[0082]

[0107] MicroRNAs (miRNAs) mediate the degradation of their target mRNAs and / or their Small non-coding RNA molecules involved in the control of gene expression at the post-transcriptional level by repressing translation of miRNAs are of type A. miRNAs typically have a length of about 10 to 100 bases, for example, about 10 to 100 bases, about 15 to 90 bases, about 20 to 80 bases, about 25 to 70 bases, about 30 to 60 bases, or about 35 to 50 bases.

[0083]

[0108] As used herein, the term "antisense nucleotide" refers to a nucleotide that binds to a target molecule via hydrogen bonds. It refers to an oligomeric compound that can hybridize to a target nucleic acid. For example, an "antisense nucleotide targeted to the coding sequence of CaMKII-δ9" refers to a nucleotide that can hybridize to the coding sequence of CaMKII-δ9 or a portion thereof.

[0084]

[0109] In some embodiments, the antisense nucleotides are used to improve their stability. Antisense nucleotides can be modified to achieve their intended effect. Modifications to antisense nucleotides include substitutions or changes to the internucleoside linkage, sugar moiety, or nucleobase. Modified antisense nucleotides are often preferred over natural forms due to desirable properties, such as increased cellular uptake, increased affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0085]

[0110] In some embodiments, the RNAi molecule or antisense nucleotide is The RNAi molecule or antisense nucleotide is complementary to exon 16 of the CaMKII-δ gene. In some embodiments, the RNAi molecule or antisense nucleotide is complementary to exons 13-16 of the CaMKII-δ gene. In some embodiments, the RNAi molecule or antisense nucleotide is complementary to exons 16-17 of the CaMKII-δ gene. In some embodiments, the RNAi molecule or antisense nucleotide is complementary to exons 13-17 of the CaMKII-δ gene. In some embodiments, the RNAi molecule or antisense nucleotide is complementary to the coding sequence of full-length CaMKII-δ9.

[0086]

[0111] As used herein, the term "complementary" or "complementarity" refers to a nucleic acid of a first nucleic acid. It relates to the pairing ability between the base and the nucleobase of a second nucleic acid.In some embodiments, the RNAi molecule or antisense nucleotide provided herein is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the target nucleic acid, wherein the target nucleic acid is selected from the group consisting of exon 16 of CaMKII-δ gene, exon 13-16 of CaMKII-δ gene, exon 16-17 of CaMKII-δ gene, exon 13-17 of CaMKII-δ gene and the coding sequence of full-length CaMKII-δ9.The complementarity percentage of the RNAi molecule or antisense nucleotide and the target nucleic acid can be determined by routine methods in the art.

[0087]

[0112] In some embodiments, the antagonist competes with CaMKII-δ9. It is a drug that binds to its substrate.

[0113] As used herein, the term "compete" or "compete with" refers to The present invention relates to an agent that partially or completely inhibits the effect of CaMKII-δ9 by competing with CaMKII-δ9 for binding to its substrate. Inhibition of CaMKII-δ9 binding to its substrate reduces or alters the normal level or type of cell signaling that occurs when CaMKII-δ9 binds to its substrate without such inhibition. Inhibition is further intended to include any measurable decrease, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, in CaMKII-δ9 binding to its substrate in the presence of the antagonist disclosed herein, compared to CaMKII-δ9 not contacted with the antagonist.

[0088]

[0114] In some embodiments, the substrates compete with CaMKII-δ9 for binding to its substrate. The agent is a vector expressing CaMKII-δ9 that does not have the function of phosphorylation or oxidation. In some embodiments, the vector of the present invention can be any gene transfer vector known in the art. In some embodiments, the vector of the present invention can contain a foreign gene that includes, consists essentially of, or consists of a CaMKII-δ9 coding gene. In some embodiments, the CaMKII-δ9 expressed by the vector provided herein does not have the function of phosphorylation or oxidation. This is because the coding gene responsible for the phosphorylation or oxidation function of CaMKII-δ9 is mutated, silenced, or deleted. In some embodiments, the CaMKII-δ9 expressed by the vector provided herein does not have the function of phosphorylation or oxidation. This is because the CaMKII-δ9 undergoes post-translational processing, thereby losing its phosphorylation or oxidation function. In some embodiments, the vector is an adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, or plasmid.

[0089]

[0115] AAVs are members of the Parvoviridae family and are approximately AAV contains a linear, single-stranded DNA genome of less than 1,000 nucleotides. For efficient replication, AAV requires co-infection with a helper virus (i.e., adenovirus or helper virus) or expression of helper genes. AAV vectors used to administer therapeutic nucleic acids typically have approximately 96% of the parent genome deleted, leaving only the terminal repeats (ITRs), which contain recognition signals for DNA replication and packaging. This eliminates immunological or toxic side effects due to the expression of the viral genome. Furthermore, delivery of specific AAV proteins to producer cells allows the AAV vector containing AAV ITRs to be integrated into specific regions of the cellular genome, if desired (see, e.g., U.S. Pat. Nos. 6,342,390 and 6,821,511). Host cells containing the integrated AAV genome do not exhibit changes in cell growth or morphology (see, e.g., U.S. Pat. No. 4,797,368).

[0090]

[0116] AAV vectors can be generated using any AAV serotype known in the art. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or human or non-human primate tissues (reviewed, for example, in Wu et al., Molecular Therapy, 14(3):316-327 (2006)). Generally, AAV serotypes have genome sequences that are significantly homologous at the nucleic acid and amino acid sequence levels, so that different serotypes have the same set of genetic functions and produce virions that are essentially physically and functionally equivalent and replicate and assemble by virtually the same mechanisms. In some embodiments, the AAV of the present invention is AAV1, AAV2, AAV5, AAV8, AAV9, or AAVrhlO.

[0091]

[0117] In another aspect, the present invention provides a method of reducing cardiac injury in a subject, comprising administering Ca The present invention also discloses a method for alleviating cardiac damage in a subject, comprising administering to the subject an effective amount of an antagonist of CaMKII-δ9. In yet another aspect, the present invention discloses the use of an antagonist of CaMKII-δ9 in the manufacture of a medicament for alleviating cardiac damage in a subject. In yet another aspect, the present invention discloses an antagonist of CaMKII-δ9 for use in alleviating cardiac damage in a subject.

[0092]

[0118] As used herein, the terms "mitigate," "mitigating," or "mitigating" mean For example, it includes reducing, restricting, or interfering with a specific action, function, or interaction. In some embodiments, cardiac damage is alleviated when at least one symptom of cardiac damage is terminated, slowed, or prevented. In some embodiments, cardiac damage is alleviated when the level or activity of a protein that can cause cardiac damage (e.g., CaMKII-δ9) is reduced compared to a baseline state. Such alleviation can be partial or complete.

[0093]

[0119] In another aspect, the present invention provides a method for determining the level or activity of a ubiquitin-conjugating enzyme in a subject. The present invention discloses a method for stimulating ubiquitin-conjugating enzyme activity in a subject, the method comprising administering to the subject an effective amount of a CaMKII-δ9 antagonist. In yet another aspect, the present invention discloses the use of a CaMKII-δ9 antagonist in the manufacture of a medicament for stimulating the level or activity of a ubiquitin-conjugating enzyme in a subject. In yet another aspect, the present invention discloses a CaMKII-δ9 antagonist for use in stimulating the level or activity of a ubiquitin-conjugating enzyme in a subject. The "level or activity of a ubiquitin-conjugating enzyme" relates to the amount of a ubiquitin-conjugating enzyme in a subject, or the ability of a ubiquitin-conjugating enzyme in a subject to target proteins for degradation by the proteasome during the ubiquitination reaction.

[0094]

[0120] In some embodiments, the level of a ubiquitin-conjugating enzyme or The activity is compared with the reference level or activity of ubiquitin-conjugating enzyme in a reference sample. As used herein, the term "reference level or activity" refers to the threshold level or activity of a substance in a subject. For example, if the level or activity of ubiquitin-conjugating enzyme in a test biological sample from a subject who has received a CaMKII-δ9 antagonist is higher than the reference level or activity of ubiquitin-conjugating enzyme in a reference sample, the CaMKII-δ9 antagonist can be considered to stimulate the level or activity of ubiquitin-conjugating enzyme in the subject. The reference level or activity of ubiquitin-conjugating enzyme can be derived from one or more reference samples, where the reference level or activity is obtained from an experiment conducted in parallel with the experiment for testing the sample of interest. Alternatively, the reference level or activity can be obtained from a reference database containing a data collection, a standard, levels or activities from one or more reference samples or disease reference samples. In some embodiments, such data collection, standard, level or activity is normalized, so that it can be used for comparison with data from one or more samples. "Normalizing" or "normalization" refers to the process of converting raw measurement data into data that can be directly compared with other such normalized data. Normalization is used to overcome assay-specific errors caused by factors that may vary from assay to assay, such as variations in loading, binding efficiency, detection sensitivity, and other various errors. In certain embodiments, a reference database includes the concentration and / or other laboratory and clinical data of ubiquitin-conjugating enzymes from one or more reference samples. In some embodiments, the reference database includes the levels or activities of ubiquitin-conjugating enzymes, each of which is normalized as a percentage of the level or activity (e.g., a known amount or activity of ubiquitin-conjugating enzymes) of the reference sample tested under the same conditions as the reference sample. To compare with such normalized levels or activities of ubiquitin-conjugating enzymes, the levels or activities of the ubiquitin-conjugating enzymes of the test biological sample are also measured and calculated as a percentage of the level or activity of the ubiquitin-conjugating enzymes of the reference sample tested under the same conditions as the test sample.

[0095]

[0121] Without being bound by any theory, the expression of ubiquitin-conjugating enzymes in a subject Increased level or activity is considered beneficial to the subject.In some embodiments, the level or activity of ubiquitin-conjugating enzyme detected in test biological sample is at least twice the baseline level or activity of ubiquitin-conjugating enzyme.In some embodiments, the level or activity of ubiquitin-conjugating enzyme detected in test biological sample is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times the baseline level or activity of ubiquitin-conjugating enzyme.

[0096]

[0122] In some embodiments, the reference sample is from a healthy subject or the same A sample obtained from a subject either before or after the test biological sample.

[0123] As used herein, the term "healthy subject" refers to a subject in need of administering the methods or compositions of the present invention to the subject. known to be free of or not suffering from the disease, condition, or disorder used in the definition In some embodiments, the reference sample is obtained from a healthy part of the body of the same subject in whom a disease or condition has been identified using a method or composition of the invention. In some embodiments, the test biological sample is from the heart of the subject. In some embodiments, the subject is a human or non-human primate.

[0097]

[0124] In another aspect, the present invention provides a method for preventing degradation of a ubiquitin-conjugating enzyme in a subject. The present invention discloses a method for preventing the degradation of ubiquitin-conjugating enzymes in a subject, the method comprising administering to the subject an effective amount of a CaMKII-δ9 antagonist. In yet another aspect, the present invention discloses the use of a CaMKII-δ9 antagonist in the manufacture of a medicament for preventing the degradation of ubiquitin-conjugating enzymes in a subject. In yet another aspect, the present invention discloses a CaMKII-δ9 antagonist for use in preventing the degradation of ubiquitin-conjugating enzymes in a subject.

[0098]

[0125] As used herein, "degradation of ubiquitin-conjugating enzymes" refers to the degradation of ubiquitin in a test biological sample. The level or activity of chitin-conjugating enzyme is reduced compared to the standard level or activity of ubiquitin-conjugating enzyme in reference sample.Without being bound by any theory, it is believed that the degradation of ubiquitin-conjugating enzyme in subject is harmful to the subject.In some embodiments, the antagonist of CaMKII-δ9 disclosed herein can, for example, prevent at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% degradation of the amount or activity of ubiquitin-conjugating enzyme in test biological sample.

[0099]

[0126] In another aspect, the present invention provides a method for preventing cardiomyocyte death in a sample, the method comprising: with an effective amount of a CaMKII-δ9 antagonist. In yet another aspect, the present invention discloses the use of a CaMKII-δ9 antagonist in the manufacture of a medicament for preventing cardiomyocyte death in a sample. In yet another aspect, the present invention discloses a CaMKII-δ9 antagonist for use in preventing cardiomyocyte death in a sample. In some embodiments, the method or use provided herein can prevent, for example, the death of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of cardiomyocytes in a test sample.

[0100]

[0127] In another aspect, the present invention provides a method for reducing DNA damage in a cell, the method comprising: The present invention discloses a method comprising contacting cell with an effective amount of CaMKII-δ9 antagonist.In yet another aspect, the present invention discloses the use of CaMKII-δ9 antagonist in the manufacture of a medicine for reducing DNA damage in cell.In yet another aspect, the present invention discloses the CaMKII-δ9 antagonist for use in reducing DNA damage in cell.As used herein, the term "DNA damage" refers to changes in the chemical structure of DNA, for example, breaks in DNA strand, base deletion from DNA backbone, or chemically altered base.

[0101]

[0128] Cells are exposed to factors that can cause DNA damage, such as intracellular reactive species and the environment. DNA is constantly exposed to factors. The potential mutational consequences of DNA damage are minimized by DNA repair pathways, which are generally characterized by three types: base excision repair (BER), mismatch repair (MMR), and nucleotide excision repair (NER) (Wood et al., Science, 291:1284-1289 (2001)). In some embodiments, the antagonist of the present invention can activate DNA repair pathways, for example, by suppressing the level and / or activity of CaMKII-δ9 in cells.

[0102]

[0129] In another aspect, the present invention provides a method for diagnosing a CaMKII-mediated disease in a subject. (b) detecting a level or activity of CaMKII-δ9 in the test biological sample from the subject, wherein the level or activity of CaMKII-δ9 detected in the test biological sample from the subject indicates that the subject is suffering from or has an increased likelihood of developing a CaMKII-mediated disease.

[0103]

[0130] In yet another aspect, the present invention provides a method for diagnosing a CaMKII-mediated disease in a subject. The present invention discloses the use of an agent in the manufacture of a medicine for diagnosing a CaMKII-mediated disease, wherein the diagnosis comprises the steps of: (a) obtaining a test biological sample from the subject; and (b) detecting a level or activity of CaMKII-δ9 in the test biological sample, wherein the level or activity of CaMKII-δ9 detected in the test biological sample from the subject indicates that the subject is suffering from or has an increased likelihood of developing a CaMKII-mediated disease.

[0104]

[0131] In yet another aspect, the present invention provides a method for diagnosing a CaMKII-mediated disorder in a subject. The present invention discloses an agent for diagnosing a CaMKII-δ9-mediated disease, the diagnosis comprising the steps of: (a) obtaining a test biological sample from the subject; and (b) detecting a level or activity of CaMKII-δ9 in the test biological sample, wherein the level or activity of CaMKII-δ9 detected in the test biological sample from the subject indicates that the subject is suffering from or has an increased likelihood of developing a CaMKII-mediated disease.

[0105]

[0132] As used herein, the term "diagnosis" or "to diagnose" refers to the diagnosis of a pathological condition, disease, Or it relates to the identification of a condition, for example, the identification of a CaMKII-mediated disease, or the identification of a subject suffering from a CaMKII-mediated disease that can benefit from a specific treatment plan. In some embodiments, the diagnosis comprises the identification of an abnormal level or activity of CaMKII-δ9. In some embodiments, the diagnosis relates to the identification of cardiac disease or metabolic disease in a subject.

[0106]

[0133] As used herein, the term "biological sample" refers to a sample that has been analyzed, e.g., for its physical, biochemical, chemical, or biological properties. The present invention relates to a biological composition obtained from or derived from a subject of interest, containing a cellular entity and / or another molecular entity to be characterized and / or identified based on its chemical and / or physiological properties. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those skilled in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, serum, mucus (including nasal discharge and sputum), peritoneal fluid, pleural effusion, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, ascites fluid, peritoneal fluid, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lung, kidney, skin, or blood vessels of the subject. In some embodiments, the biological sample is obtained from the heart of the subject.

[0107]

[0134] In accordance with the present invention, a peptide of interest (e.g., a ubiquitin-conjugated peptide) is isolated from a test biological sample. Detection of the level or activity of a peptide (e.g., an enzyme, CaMKII-δ9) in a sample can be accomplished by any suitable means for determining the level or activity of the peptide in the sample. In some embodiments, detection methods include immunoassay devices and methods that can utilize labeled molecules in a variety of sandwich, competitive, or other assay formats. The assay produces a signal indicative of the presence or absence of the peptide of interest. Furthermore, the signal strength can be directly or indirectly (e.g., inversely) correlated with the amount of the peptide of interest present in the sample. Further suitable methods include measuring physical or chemical properties specific to the peptide of interest, such as its precise molecular weight or NMR spectrum. Suitable detection methods further include biosensors, optical devices coupled with immunoassays, biochips, analytical devices, such as mass spectrometers, NMR analyzers, or chromatographic devices. Furthermore, suitable detection methods include: These methods may include microplate ELISA-based methods, fully automated or robotic immunoassays (e.g., available on ELECSYS analyzers), CBA (enzymatic cobalt-binding assays, e.g., available on Roche-Hitachi analyzers), and latex agglutination assays (e.g., available on Roche-Hitachi analyzers). In some embodiments, the level or activity of the peptide of interest is detected by measuring the specific intensity signal obtained from the peptide in a sample. As noted above, such a signal may be the signal intensity observed at an m / z (mass-to-charge ratio) variable specific to the peptide of interest, as observed in a mass spectrum or NMR spectrum specific to the peptide of interest.

[0108]

[0135] In some embodiments, the level of CaMKII-δ9 or CaMKII-δ9 activity is detected by contacting the sample with a reagent that specifically binds to CaMKII-δ9. The reagent generates an intensity signal. Binding according to the present invention includes both covalent and non-covalent binding. A reagent that binds to CaMKII-δ9 according to the present invention can be any compound that binds to CaMKII-δ9 described herein, such as a peptide, polypeptide, nucleic acid, or small molecule. In some embodiments, the reagent includes an antibody, nucleic acid, peptide, or polypeptide, e.g., a receptor or binding partner for the peptide or a fragment thereof containing a binding domain for the peptide, and an aptamer, e.g., a nucleic acid aptamer or peptide aptamer. Methods for preparing such reagents are well known in the art. For example, the identification and production of suitable antibodies or aptamers are also provided by commercial suppliers. Those skilled in the art are familiar with methods for developing derivatives of such reagents with higher affinity or specificity. For example, random mutations can be introduced into the nucleic acid, peptide, or polypeptide. These derivatives can then be tested for binding by screening methods known in the art, such as phage display.

[0109]

[0136] In some embodiments, non-specific binding can be measured by, for example, measuring the binding activity on a Western blot.It is acceptable for the binding of a reagent to still be clearly distinguishable and measurable due to its size or its relatively high abundance in the sample. The binding of the reagent can be measured by any method known in the art. In some embodiments, the method is semi-quantitative or quantitative. Suitable methods include: (1) the binding of the reagent can be measured directly, for example, by NMR or surface plasmon resonance; (2) if the reagent is further used as a substrate for the enzymatic activity of the peptide of interest, the enzymatic reaction product can be measured (e.g., the amount of cleaved substrate can be measured, for example, on a Western blot, to measure the amount of protease). Alternatively, the reagent may itself exhibit enzymatic properties, and the peptide-bound reagent can be contacted with an appropriate substrate to allow detection by generating an intensity signal. In some embodiments, the amount of substrate is saturated with respect to the measurement of the enzymatic reaction product. The substrate may also be labeled with a detectable label prior to the reaction. In some embodiments, the sample is contacted with the substrate for a suitable period of time. The suitable period of time relates to the time required to produce a detectable or measurable amount of product. Instead of measuring the amount of product, the time required for the appearance of any (e.g., detectable) amount of product may be measured; (3) the reagent may be covalently or noncovalently attached to a label that allows for detection and measurement of the reagent. Labeling can be performed by direct or indirect methods. Direct labeling involves directly (covalently or noncovalently) coupling a label to the reagent. Indirect labeling involves binding (covalently or noncovalently) a secondary reagent to the primary reagent. The secondary reagent should specifically bind to the primary reagent. The secondary reagent may be conjugated to an appropriate label and / or may be a target (receptor) for a tertiary reagent that binds to the secondary reagent. The use of secondary, tertiary, and even higher-order reagents is often to increase signal intensity. Suitable secondary and higher-order reagents may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The reagent or substrate may also be "tagged" with one or more tags known in the art. Such tags can then be targets for higher order reagents. Suitable tags are biotin, digoxigenin. , His tag, glutathione S-transferase, FLAG, GFP, myc tag, influenza A virus, hemagglutinin (HA), maltose binding protein, etc. In the case of a peptide or polypeptide, the tag may be at the N-terminus and / or C-terminus.

[0110]

[0137] In some embodiments, the reagent that specifically binds to CaMKII-δ9 is an antibody In some embodiments, the antibody is a monoclonal antibody.

[0111]

[0138] In some embodiments, the level of CaMKII-δ9 detected in the test biological sample The level or activity is compared to a reference level or activity of CaMKII-δ9 detected in a reference sample.

[0112]

[0139] As used herein, the term "compared" refers to a comparison of the amount of a test biological sample being analyzed. It relates to the comparison of the level or activity of target protein (e.g., ubiquitin-conjugating enzyme, CaMKII-δ9, etc.) with the level or activity of an appropriate reference sample. The terms used herein are understood to relate to the comparison of corresponding parameters or values, for example, the absolute amount is compared with the absolute reference amount, the concentration is compared with the reference concentration, or the intensity signal obtained from the test sample is compared with the similar intensity signal of the reference sample. The comparison can be performed manually or computer-assisted. In computer-assisted comparison, the determined amount value can be compared with the value corresponding to an appropriate standard stored in a database by a computer program. The computer program can further evaluate the result of the comparison and automatically provide the desired evaluation in an appropriate output format. Based on the comparison of the detected CaMKII-δ9 level or activity with an appropriate reference level, the CaMKII-mediated disease in the subject can be diagnosed.

[0113]

[0140] In some embodiments, the level of CaMKII-δ9 detected in the test biological sample A CaMKII-δ9 level or activity higher than the baseline level or activity indicates that the subject has or is at an increased likelihood of developing a CaMKII-mediated disease. Preferably, the CaMKII-δ9 level or activity detected in the test biological sample is at least twice the baseline level or activity of CaMKII-δ9. More preferably, the CaMKII-δ9 level or activity detected in the test biological sample is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 times the baseline level or activity of CaMKII-δ9.

[0114]

[0141] As used herein, the term "increased likelihood" refers to the likelihood that a subject has CaMKII-mediated The term relates to a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 1-fold, 2-fold, 3-fold, 5-fold, 8-fold, 10-fold, 20-fold, 50-fold or greater overall increase in the level of likelihood of developing a presenting disease compared to the subject from whom the reference sample was derived.

[0115]

[0142] In some embodiments, the reference sample is from a healthy subject or an identical pair. A sample obtained from an elephant either before or after the test biosample.

[0143] In yet another aspect, the present invention provides an antibody that specifically recognizes CaMKII-δ9. The present invention also provides a kit for diagnosing a CaMKII-mediated disease in a subject, comprising an antibody or antibody fragment thereof. In some embodiments, the antibody or antibody fragment specifically binds to an amino acid sequence encoded by exon 16 of the CaMKII-δ gene. The kit may use any suitable means for detecting the content or activity of CaMKII-δ9 in a sample.

[0116]

[0144] In some embodiments, the kit further comprises a method for detecting a subject as defined herein. The kit may contain a user manual for interpreting the results of any measurement with respect to diagnosing a CaMKII-mediated disease. In particular, such a manual may include information on which determined levels correspond to which diagnoses. Furthermore, such a user manual may provide instructions on the correct use of the components of the kit to detect CaMKII-δ9 levels. In some embodiments, the detection means and instructions for the kit are provided in a single container.

[0117]

[0145] In one aspect, the present invention identifies molecules that inhibit the activity of CaMKII-δ9. The present invention discloses a method for identifying a molecule that inhibits CaMKII-δ9, the method comprising contacting the molecule with a sample containing (i) CaMKII-δ9 and (ii) UBE2T, and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that inhibits CaMKII-δ9.

[0118]

[0146] In another aspect, the present invention provides a molecule that inhibits the phosphorylation ability of CaMKII-δ9. A method for identifying a molecule is disclosed, the method comprising contacting the molecule with a sample containing (i) CaMKII-δ9 and (ii) UBE2T, and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that inhibits the ability of CaMKII-δ9 to phosphorylate.

[0119]

[0147] In yet another aspect, the present invention provides a method for treating or preventing a CaMKII-mediated disease. Disclosed is a method for identifying a molecule that treats or prevents a CaMKII-mediated disease, the method comprising contacting the molecule with a sample containing (i) CaMKII-δ9 and (ii) UBE2T, and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that treats or prevents a CaMKII-mediated disease.

[0120]

[0148] In yet another aspect, the present invention provides a method for identifying molecules that mitigate cardiac damage. The present invention discloses a method for identifying a molecule in which inhibition of UBE2T phosphorylation alleviates cardiac damage, the method comprising contacting the molecule with a sample containing (i) CaMKII-δ9 and (ii) UBE2T, and determining whether phosphorylation of UBE2T is inhibited.

[0121]

[0149] In yet another aspect, the present invention provides a method for identifying molecules that prevent cardiomyocyte death. The present invention discloses a method for identifying a molecule that prevents cardiomyocyte death, the method comprising contacting the molecule with a sample containing (i) CaMKII-δ9 and (ii) UBE2T, and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that prevents cardiomyocyte death.

[0122]

[0150] In yet another aspect, the present invention provides a method for identifying molecules that reduce DNA damage. A method is disclosed, comprising contacting the molecule with a sample containing (i) CaMKII-δ9 and (ii) UBE2T, and determining whether phosphorylation of UBE2T is inhibited, wherein inhibition of phosphorylation of UBE2T identifies a molecule that reduces DNA damage.

[0123]

[0151] These methods are further referred to herein as drug screening assays, Typically, this involves screening candidate / test molecules for their ability to interact with (e.g., bind to) CaMKII-δ9, to modulate the phosphorylation of UBE2T by CaMKII-δ9, and / or to modulate the interaction of phosphorylatable residues of UBE2T with CaMKII-δ9-mediated intracellular signaling targets.

[0124]

[0152] In some embodiments, the method further comprises: The method includes determining whether the antibody directly binds to the target protein.

[0153] In some embodiments, the amount of phosphorylated UBE2T in the sample is compared to a control. In some embodiments, inhibition of UBE2T phosphorylation is determined by comparing the ratio of the amount of phosphorylated UBE2T in the sample to the total amount of UBE2T with the control. In some embodiments, the control is the ratio of the amount of phosphorylated UBE2T in the sample to the total amount of UBE2T with the control. In some embodiments, the control is the ratio of the amount of phosphorylated UBE2T in the sample to the ratio of the amount of phosphorylated UBE2T in the absence of the molecule or to the ratio of the amount of phosphorylated UBE2T in the sample to the absence of the molecule or to the early time point after contacting the sample with the molecule.

[0125]

[0154] In some embodiments, the sample may be an in vitro sample, an ex vivo sample, or In some embodiments, the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scraping, saliva, cerebrospinal fluid, urine, stool, and bone marrow. In some embodiments, the sample comprises cells (e.g., cardiac cells). In some embodiments, the cells are obtained from a patient. In some embodiments, the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scraping, saliva, cerebrospinal fluid, urine, stool, and bone marrow.

[0126]

[0155] In some embodiments, the candidate / test for use in a drug screening assay The molecule is a small molecule compound or an antibody or antigen-binding fragment thereof. In some embodiments, the candidate / test molecule reduces the amount of phosphorylated UBE2T by at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%.

[0127]

[0156] The candidate / test molecules of the present invention that are to be identified are, at least in part, biological These libraries can be obtained using any of a number of techniques in combinatorial library methods known in the art, including libraries, spatially addressable parallel solid-phase or solution-phase libraries, synthetic library methods requiring deconvolution, "one-bead-one-compound" library methods, and synthetic library methods using affinity chromatography selection. While the biological library method is limited to peptide libraries, the other four techniques can be applied to peptide libraries, non-peptide oligomer libraries, or small molecule libraries of compounds (Lam, KS (1997) Anticancer Drug Des. 12:145).

[0128]

[0157] Examples of methods for synthesizing molecular libraries can be found in the art, for example, in De (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[0129]

[0158] Libraries of compounds can be prepared in solution (e.g., Houghten (1992) Bi otechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,233,409), spores (Ladner U.S. Pat. No. 5,233,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phages (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6379). 382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra)).

[0130]

[0159] In some embodiments, phosphorylation of UBE2T is achieved by cleaving a serine at any position (S In some embodiments, phosphorylation of UBE2T is at Ser5, Ser81, Ser82, Ser101, Ser110, Ser129, Ser130, Ser165, Ser166, Ser172, Ser174, Ser176, Ser177, Ser193, or Ser204. In some embodiments, phosphorylation of UBE2T is at Ser110. In some embodiments, phosphorylation of UBE2T is at Thr23, Thr44, Thr52, Thr72, Thr106, Thr109, Thr144, Thr177, or Thr178. In some embodiments, phosphorylation of UBE2T is at Tyr46, Tyr61, Tyr74, or Tyr134. The amino acid positions referred to herein relate to the serine, threonine and tyrosine positions of, for example, human, mouse and rat wild-type UBE2T.

[0131]

[0160] In one aspect, the present invention provides a method for the production of a medicament comprising administering to a subject the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, Disclosed is an isolated CaMKII-δ polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or an amino acid sequence having at least 80% homology to an amino acid sequence set forth in SEQ ID NOs: 1-5. In some embodiments, the isolated CaMKII-δ polypeptide is not a full-length naturally occurring polypeptide. In some embodiments, the isolated CaMKII-δ polypeptide is 14 amino acids, 27 amino acids, 30 amino acids, 43 amino acids, or 513 amino acids in length.

[0132]

[0161] In some embodiments, the present invention provides a method for the production of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, Disclosed is an isolated CaMKII-δ polypeptide having an amino acid sequence having at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 2, the amino acid sequence set forth in SEQ ID NO: 3, the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, or the amino acid sequence set forth in SEQ ID NOs: 1 to 5.

[0133]

[0162] As used herein, the term "isolated" refers to a substance (e.g., a polypeptide or The term relates to a nucleic acid (or nucleic acid) being separated from the environment in which it normally occurs in nature or being in an environment that is different from the environment in which it is normally found in nature.

[0134]

[0163] As used herein, percent (%) "sequence identity" refers to an amino acid sequence. For nucleotide sequences, it refers to the percentage identity between two nucleotide sequences after aligning a candidate sequence with a reference sequence and introducing gaps, if necessary, to achieve the maximum number of identical nucleotides.

[0135]

[0164] The percentage of homology can be determined by various methods well known in the art, for example Sequence comparisons were performed using the following publicly available tools: BLASTp software (available from the National Center for Biotechnology Information (NCBI) website http: / / blast.ncbi.nlm.nih.gov / Blast.cgi; see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute) Available from the EBI website http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ; see Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)) and TCoffee (available from the Swiss Institute of Bioinformatics website, Poirot (See also O. et al., Nucleic Acids Res., 31(13):3503-6 (2003); Notredame C. et al., J. Mol. Boil., 302(1):205-17 (2000)). When aligning sequences using software, the default parameters available in the software may be used, or the parameters may be customized to suit the alignment purpose. All of these are within the knowledge of those skilled in the art.

[0136]

[0165] Conservative substitutions of amino acid residues are substitutions between amino acids with similar properties, e.g. , substitutions between polar amino acids (e.g., substitutions between glutamine and asparagine), substitutions between hydrophobic amino acids (e.g., substitutions between arginine, isoleucine, methionine, and valine), and substitutions between amino acids with the same charge (e.g., substitutions between arginine, lysine, and histidine, or substitutions between glutamine and aspartate).

[0137]

[0166] In another aspect, the present invention provides a method for the preparation of a nucleic acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, The present invention discloses an isolated CaMKII-δ nucleic acid comprising a nucleic acid sequence encoding a CaMKII-δ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence set forth in SEQ ID NO: 3, the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 80% homology to the amino acid sequence set forth in SEQ ID NOs: 1 to 5.

[0138]

[0167] As used herein, the term "nucleic acid" or "polynucleotide" refers to a ribonucleic acid ( The term "nucleic acid" refers to a nucleic acid or polynucleotide that is a single-stranded or double-stranded DNA, RNA, or DNA-RNA hybrid. The nucleic acid or polynucleotide may be linear or circular.

[0139]

[0168] In some embodiments, the CaMKII-δ nucleic acid is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and nucleic acid sequences having at least 80% homology to SEQ ID NOs: 6 to 19.

[0140]

[0169] In some embodiments, the isolated CaMKII-δ nucleic acids provided herein a nucleic acid sequence having at least 70% homology, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology, to any one of the nucleic acid sequences set forth in SEQ ID NOs: 6 to 19, and further encoding a CaMKII-δ polypeptide having an amino acid sequence having at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence set forth in SEQ ID NO: 2, the amino acid sequence set forth in SEQ ID NO: 3, the amino acid sequence set forth in SEQ ID NO: 4, the amino acid sequence set forth in SEQ ID NO: 5, or the amino acid sequence set forth in SEQ ID NOs: 1 to 5.

[0141]

[0170] In some embodiments, the present invention provides a method for the production of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, The amino acid sequence set forth in SEQ ID NO: 2, the amino acid sequence set forth in SEQ ID NO: 3, the amino acid sequence set forth in SEQ ID NO: The present invention provides a CaMKII-δ nucleic acid sequence encoding a CaMKII-δ polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4, an amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 80% homology to an amino acid sequence set forth in SEQ ID NO: 1 to 5, but which sequence differs from any one of the nucleic acid sequences set forth in SEQ ID NO: 6 to 19 due to degeneracy of the genetic code.

[0142]

[0171] In another aspect, the present invention provides a CaMKII-δ9 inhibitor capable of inhibiting the activity of CaMKII-δ9. I antagonists are disclosed.

[0172] In some embodiments, the antagonist inhibits ubiquitination by CaMKII-δ9. In some embodiments, the antagonist inhibits phosphorylation of a chitin-conjugating enzyme. In some embodiments, the ubiquitin-conjugating enzyme is UBE2T. In some embodiments, the antagonist inhibits phosphorylation of UBE2T at Ser110.

[0143]

[0173] In some embodiments, the antagonist inhibits the expression of the CaMKII-δ gene. an antibody that binds to an amino acid sequence encoded by exon 16; an RNAi molecule that targets exon 16 of the CaMKII-δ gene; and an antisense nucleotide that targets exon 16 of the CaMKII-δ gene.

[0144]

[0174] In another aspect, the present invention provides a method for inhibiting the activity of CaMKII-δ9 disclosed in the present invention. A pharmaceutical composition comprising an antagonist capable of inhibiting the action of a steroid hormone is disclosed.

[0175] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. .

[0145]

[0176] As used herein, the expression "pharmaceutically acceptable" means any substance that is within the scope of sound medical judgment. Pharmaceutically acceptable compounds, materials, compositions, and / or dosage forms refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, without excessive toxicity, inflammation, allergic response, or other problems or complications, and at a reasonable benefit / risk ratio. In some embodiments, pharmaceutically acceptable compounds, materials, compositions, and / or dosage forms refer to those that have been approved by a regulatory agency (e.g., the U.S. Food and Drug Administration, the China Food and Drug Administration, or the European Medicines Agency) or are listed in a generally recognized pharmacopoeia (e.g., the United States Pharmacopoeia, the Chinese Pharmacopoeia, or the European Pharmacopoeia) for use in animals, particularly humans.

[0146]

[0177] The pharmaceutically acceptable carrier used in the pharmaceutical composition of the present invention is, for example, a pharmaceutically acceptable liquid, gel, or solid carriers to be administered, aqueous vehicles (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, Ringer's injection of glucose and lactate), non-aqueous vehicles (e.g., fixed vegetable oils, cottonseed oil, corn oil, sesame oil, or peanut oil), antibacterial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate buffer or citrate buffer), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispending agents (e.g., carboxymethylcellulose, The composition may include, but is not limited to, a chelating agent (e.g., sodium cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), a chelating agent (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), an emulsifier (e.g., polysorbate 80 (TWEEN®-80)), a diluent, adjuvant, excipient, or non-toxic auxiliary, another ingredient known in the art, or various combinations thereof. Suitable ingredients may include, for example, a filler, binder, disintegrant, buffer, preservative, lubricant, flavoring, thickener, colorant, or emulsifier.

[0147]

[0178] In some embodiments, the pharmaceutical composition is an oral formulation. Capsules, cachets, pills, tablets, lozenges (if flavored, usually sucrose or and acacia or tragacanth), powders, granules, or aqueous or non-aqueous solutions or suspensions, or water-in-oil emulsions or oil-in-water emulsions, or elixirs, or syrups, or confectionery lozenges (for inert bases, e.g., gelatin and glycerin, or sucrose or acacia) and / or mouthwashes and the like.

[0148]

[0179] In some embodiments, oral solid dosage forms (e.g., capsules, tablets, pills, dragees, Tablets, powders, granules, etc.) contain an active agent and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) cutting agents, such as agar-agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) retarder solutions, such as paraffin; and (6) absorption enhancers. (7) lubricants, such as acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium sulfate, and mixtures thereof; and (10) colorants.

[0149]

[0180] In some embodiments, the oral liquid is a pharmaceutically acceptable emulsion, microemulsion, or the like. These include formulations such as infusions, solutions, suspensions, syrups, elixirs, and the like. In addition to the active ingredient, liquid dosage forms may further contain conventional inert diluents, such as water or another solvent, solubilizing and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzene (meth)acrylate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol fatty acid esters, and mixtures thereof. Besides inert diluents, oral compositions may further contain adjuvants, such as wetting agents, emulsifying agents, suspending agents, sweeteners, flavorings, coloring agents, flavorings, and preservatives.

[0150]

[0181] In some embodiments, the pharmaceutical composition is a sterile aqueous solution, dispersion, suspension or The injectable preparation may be an injectable preparation containing an emulsion or emulsion. In either case, the injectable preparation is sterile and liquid to facilitate injection. The pharmaceutical composition is stable under the conditions of manufacture and storage and is resistant to infection by microorganisms (e.g., bacteria and fungi). The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof, and / or vegetable oils. The injectable preparation should maintain appropriate fluidity, which can be maintained in various ways, for example, using a coating such as lecithin, using surfactants, etc. Antibacterial contamination can occur due to the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).

[0151]

[0182] In some embodiments, the pharmaceutical composition is an oral spray formulation or a nasal spray. Such spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposomal formulations, or solid particle formulations. Aqueous aerosols are prepared by combining an aqueous solution or suspension of the drug with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers may vary depending on the needs of the particular compound, but generally include non-ionic surfactants (Tween® or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars, or the like. The aerosols are usually prepared from isotonic solutions and can be delivered by nebulizers.

[0152]

[0183] In some embodiments, the pharmaceutical composition is administered in combination with one or more additional drugs. In some embodiments, the composition includes at least one additional drug, which in some embodiments is a cardiovascular drug, a drug for treating renal disease, a drug for cell membrane repair, or the like.

[0153]

[0184] In some embodiments, the pharmaceutical compositions can be administered by oral routes, injection routes (e.g., intravenous). The pharmaceutical composition may be delivered to a subject by any suitable route, including, but not limited to, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal, etc.), mucosal routes (e.g., intranasal administration, oral administration, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary routes. In some embodiments, the pharmaceutical composition may be administered by injection. [Example]

[0154] Embodiment

[0185] Biological materials, various clones, and expression plasmids used in all examples The culture media, enzymes, buffers, and various culturing methods, protein extraction and purification methods, and other molecular biological manipulation methods are all well known to those skilled in the art. For further details, see "Molecular Cloning: A Laboratory Manual" edited by Sambrook et al. (Cold Spring Harbor, 1989) and "Short Protocols in Molecular Biology" (Frederick M. Ausubel et al., translated by Yan Ziying et al., Science Press (Beijing), 1998).

[0155]

[0186] General methods and materials

[0187] 1.1 Animals

[0188] Animals were kept in the Laboratory Animal Center of Peking University (Beijing, China) (Laboratory Animal Care Assessment and Certification). Animals were housed in a laboratory animal facility certified by the Peking University Association. Animals were randomly assigned to experimental groups. Only males were used. No inclusion or exclusion parameters were used in our study. Investigators were not blinded to treatments, but no subjective evaluations were performed. All procedures involving experimental animals (mice, rats, and rhesus monkeys) followed protocols approved by the Peking University Animal Care and Use Committee and compliant with the Guide for the Care and Use of Laboratory Animals.

[0156]

[0189] Adult C57BL / 6 mice and Sprague-Dawley rats were Rhesus macaques were from a previously reported in-house cohort (Zhang, X. et al., Circulation 124, 77-86, doi:10.1161 / CIRCULATIONAHA.110.990333 (2011)). Animals were euthanized by intravenous injection of an overdose of sodium pentobarbital, and tissues were quickly frozen in liquid nitrogen for protein and total RNA extraction.

[0157]

[0190] 1.2 Animal surgery and treatment

[0191] Transverse aortic constriction (TAC) was performed in 6-week-old male mice. Mice were anesthetized with soflurane, the chest was opened, the aortic arch was visualized, and a 7-0 silk suture was passed below the arch between the innominate artery and the left common carotid artery. The suture was secured around both the aorta and a 28-gauge needle, the needle was removed, the chest was closed, and the mice were extubated. Sham-operated mice underwent the same procedure, except for the aortic ligation. Mice were given buprenorphine intraperitoneally during the recovery period.

[0158]

[0192] 1.3 Library preparation, sequencing, and sequencing for SMRT sequencing and data collection

[0193] RNeasy Fibrous Tissue Mini Kit (Qiagen, Catalog No.: 74704 Total RNA was prepared from the left ventricle of normal mice, rats, and rhesus monkeys using a PCR product purification kit (Qiagen, Catalog No. 28004). Total RNA from normal adult left ventricles was obtained from MY Biosource (Cat. No. MBS537570), Biological (Cat. No. T5595-7325), and Biochain (Cat. No. R1234138-50). Two micrograms of total RNA was used for first-strand synthesis. CaMKII-δ-specific primers annealing to the first and last coding exons were used for cDNA amplification (Figure 9b). PCR product corresponding to full-length CaMKII-δ (approximately 1600 bp) was enriched using a PCR product purification kit (Qiagen, Catalog No. 28004). SMRTbell sequencing libraries were prepared according to standard PacBio guidelines. Sequencing was performed at the Wuhan Institute of Biotechnology Public Technology Service Platform using a Pacific Biosciences real-time sequencer and C2 sequencing reagents. Subread filtering was performed using Pacific Biosciences' SMRT analysis software (v2.2). FASTQ-formatted circular consensus sequence (CCS) reads from all sample species were mapped to the CaMKII-δ locus using GMAP (version 2014-09-29) with the following parameters: format=1, batch=2, nthreads=6, trimendexons=4, ordered. Meanwhile, primer / barcode detection was performed using flexbar (version 2.5) with the following parameters: threads 6, barcode-min-overlap 8, barcode-threshold 2, log-level TAB-barcode-keep-barcode-unassigned. Alignments were kept as best-mapped and indicated primers only if they were from the same species. The following analysis was adapted from Treutlein et al., PNAS 111, E1291-1299, doi:10.1073 / pnas.1403244111 (2014). Alignments were analyzed for splice junctions (i.e., variant structures) with a gap tolerance of 3 bp.Only CCS reads analyzed for unambiguous and concordant splice junctions were kept. Splice junction frequencies were then calculated.

[0159]

[0194] The primers are as follows:

[0160] [Table 2]

[0161]

[0195] 1.4 Human samples

[0196] Human ventricular tissues from patients with hypertrophic cardiomyopathy were collected from the Fuwai Hospital (Beijing, China). Ventricular septal tissue was obtained during myectomy. The protocol was approved by the Ethics Committees of Fuwai Hospital, the Chinese Academy of Medical Sciences, and Peking Union Medical College. This study complied with all ethical regulations. All patients provided written informed consent. Normal human ventricular tissue was obtained from the NIH NeuroBioBank at the University of Maryland (Baltimore, MD).

[0162]

[0197] 1.5 RNA-seq (second-generation sequencing) and CaMKII-δ Exon junction analysis

[0198] Total RNA from mouse tissues was prepared using the RNeasy Mini Kit (Qiagen, catalogue number 1011111). RNA-seq data for different species were obtained from the National Center for Biotechnology Information (NCBI) Sequence Read Archive database as follows: human left ventricle (SRR830965, SRR830966, SRR830967, SRR830968, SRR830969, SRR830970, SRR830971, and SRR830972), rhesus monkey heart (SRX196319, SRX196328, SRX196337, SRX081927), and rhesus monkey heart (SRX196319, SRX196328, SRX196337, SRX081927). , SRX081928, SRX494639, and SRX066573), canine left ventricle (SRR1735880, SRR1735881, SRR1735882, SRR1735883, SRR1735884, and SRR1735885), and rat heart (SRX471444, SRX471445, SRX471446, SRX471447, SRX471460, SRX471461, SRX471462, and SRX471463). The exon structures of CaMKII-δ splice variants shown in Figure 9a were compiled from the RefSeq, Uniprot, and UCSC KnownGene databases. The classification of CaMKII-δ was based on Mayer's work (Mayer, P. et al., The Biochemical Journal 298, Part 3, 757-758 (1994)). The structure of mouse CaMKII-δ was searched using LiftOver with default parameters. In multiple sequence alignment, the inventors found two major alternative splicing domains: one between exon 13 and exon 17, and the other between exon 20 and exon 22. RNA-seq reads were mapped to the mouse (mm9), rat (rn4), dog (canFam2), rhesus monkey (rheMac2), or human genome (hg19) using TopHat-2.0.8 with default parameters.All potential junction reads were counted and normalized by the size factor (ratio of reads that mapped to only one location in the target tissue to tissues containing minimal sequencing).

[0163]

[0199] 1.6 Antibodies specific to exon 16 or exon 21 of CaMKII-δ Generation of

[0200] Peptides were commercially synthesized and purified ( (Abcam, USA). The antigen epitope contained the following amino acid sequences: PPCIPNGKENFSGGTSLW (SEQ ID NO: 28), corresponding to exon 21, the C-terminus unique to one subclass of splice variants of CaMKII-δ, and CEPQTTVIHNPDGNK (SEQ ID NO: 29), corresponding to exon 16 of CaMKII-δ. Internal cysteines were used for conjugation to three different carrier proteins. Each peptide was conjugated to keyhole limpet hemocyanin or ovalbumin to immunize two 3-month-old New Zealand white rabbits. The rabbits were immunized using a customized protocol of 5 to 6 injections. To prepare the affinity column for purification, the original antigen was bound to a pre-activated matrix (agarose beads, 1.5 ml). All antisera were collected and loaded onto the prepared peptide affinity column, followed by elution with elution buffer (p The eluted polyantibody was collected and neutralized based on UV280 absorption.

[0164]

[0201] 1.7 Absolute quantification of CaMKII-δ splice variants by mass spectrometry

[0202] Human left ventricles were homogenized in RIPA buffer and the lysate was collected at 13,000 r The mixture was centrifuged at 37°C for 10 min. CaMKII-δ was immunoprecipitated with antibodies recognizing exon 21 or exon 16, and the agarose pellet was washed and eluted. The eluted proteins were subjected to SDS-PAGE, and the band corresponding to CaMKII-δ was excised for mass spectrometry analysis. Absolute quantification of CaMKII-δ splicing variants was performed as previously reported (Gerber, SA et al., Proceedings of the National Academy of Sciences of the United States of America 100, 6940-6945, doi:10.1073 / pnas.0832254100 (2003); Kawakami, H. et al. Journal of Pharmaceutical Sciences 100, 341-352, doi:10.1002 / jps.22255(2011)). Specifically, to test the best operating conditions, in-gel digestion was performed using trypsin (400 ng) for different times (0.5 h, 1 h, 2 h, or 4 h) followed by mass spectrometry. A 2-h trypsin digestion was chosen because the highest number of unique peptides was observed under these conditions. Under these conditions, several unique peptides from each target variant were selected as standard peptides for quantification (Table 1). The synthetic peptides contained 13C- and 15N-labeled lysine. A calibration curve was generated by mixing serial dilutions of the synthetic peptides (0.78, 1.56, 3.31, 6.25, 12.5, 25, 50, 100, and 200 fmol) with trypsin-digested and immunoprecipitated heart samples followed by mass spectrometry analysis. Based on the loading amount and peak area, linear calibration curves with R2 >0.992 were obtained for all synthetic peptides. Quantification values ​​for each target peptide were calculated by calculating the ratio of the peak area to that of the isotope-labeled peptide (standard spiked with 50 fmol). The average peptide amounts in the exon 21 immunoprecipitation samples were 2.32 ± 0.27 fmol for exons 13-17, 4.02 ± 1.50 fmol for exons 13-14, and 9.52 ± 0.88 fmol for exons 13-16. The average peptide amounts in the exon 16 immunoprecipitation samples were 11.87 ± 3.73 fmol for exons 20-21, and 1.73 ± 0.71 fmol for exons 20-22. Relative data are shown in Figure 1c.

[0165] [Table 3]

[0166]

[0203] Easy-nLC1000 liquid chromatography system (Thermo) All samples were analyzed using a Q-Exactive HF (Thermo) mass spectrometer with a HPLC system. Peptides were eluted from a 100 μm x 2 cm C18 trap column and separated on a 150 μm x 15 cm homemade column (C18 resin, 1.9 μm, 120 Å, Dr. Maisch GmbH) at 600 nL / min using a 75-minute linear gradient of 5 to 35% acetonitrile. MS analysis of Q-Exactive HF was performed using a full scan (300–1400 m / z, R = 120,000 at 200 m / z) with an automatic gain control target of 3e6 ions, followed by up to 20 data-dependent MS / MS scans using higher-energy collisional dissociation (AGC target of 2e4 ions, maximum allowed injection time of 40 ms, isolation window of 1.6 m / z, normalized collision energy of 27%) and detection in an Orbitrap (R = 15,000 at 200 m / z). Dynamic exclusion of pre-acquired precursor ions was allowed for 12 s.

[0167]

[0204] Peptides were analyzed using Mascot software to achieve a false discovery rate of <1%. Identification was performed using Proteome Discoverer software (version 1.4.1.14, Thermo) adapted with Proteome Discoverer software (version 2.3.01, Matrix Science). Mass tolerance was set to 20 ppm for precursors and 50 mmu for product ions. The following modifications were selected: oxidation (M), acetyl (protein N-terminus), and destreak (C), with two uncleaved ions by trypsin allowed.

[0168]

[0205] 1.8 Mass spectrometry

[0206] For analysis of UBE2T phosphorylation, HEK293 cells were transfected with control vector or f Cells were transfected with myc-tagged UBE2T in the presence of lag-tagged CaMKII-δ9 plasmid. 12 h before cell harvest, the proteasome inhibitor MG132 was added, and total protein was extracted with RIPA buffer. Total UBE2T was immunoprecipitated with anti-myc antibody, followed by washing and elution of the agarose pellet. The eluted protein was subjected to SDS-PAGE, and the band corresponding to UBE2T was excised for mass spectrometry analysis.

[0169]

[0207] For LC-MS / MS analysis, a Thermo LTQ Orbitrap Velocity Digestion products were separated using a Dionex 3000 nano-HPLC system directly interfaced with an IOS Pro mass spectrometer using a 65-minute gradient elution at a flow rate of 0.3 μL / min. The analytical column was a fused silica capillary column (75 μm internal diameter, 150 mm length, packed with C18 resin). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 80% acetonitrile and 0.08% formic acid. The mass spectrometer was operated in data-dependent acquisition mode using Xcalibur 2.1.3 software, with a single full-scan mass spectrum on the Orbitrap (400–1800 m / z, 30,000 resolution) followed by 10 data-dependent MS / MS scans. MS / MS spectra from each LC-MS / MS run were searched against selected databases using the Proteome Discovery search algorithm (version 1.3).

[0170]

[0208] 1.9 Comet assay

[0209] Cultured neonatal rat ventricular cardiomyocytes (NRVMs) were treated as indicated and then After washing with cold PBS, cells were incubated under alkaline conditions according to the protocol of the Comet Assay Kit (Trevigen, catalog number 4250-050-K). Mean tail moment was quantified using the Comet Assay Software Project (v1.2.3b1) for 300–500 cells per sample in each experiment.

[0171]

[0210] 1.10 Generation of CaMKII-δ9 tg mice

[0211] Full-length human CaMKII-δ9 cDNA coding sequence with an N-terminal Flag tag The sequence was cloned into the HindIII and EcoRV sites of an expression vector containing the αMHC promoter. After linearization with XhoI and NotI, gel purification was performed. The construct was microinjected into the pronuclei of C57BL / 6 mouse fertilized eggs. PCR was used for genotyping. The primer sequences were 5'-GTATCGATAAGCTTGCCACCATGG-3' (forward) (SEQ ID NO: 35) and 5'-CATGAAGTCGCACAATATTAGG-3' (reverse) (SEQ ID NO: 36).

[0172]

[0212] 1.11 Generation of CaMKII-δ9 shRNA tg mice

[0213] The sequence of the miR30-based CaMKII-δ9 shRNA is 5'-GAA The shRNA with an N-terminal EGFP tag and a C-terminal BGH poly(A) sequence was cloned into the pLKO.1 plasmid, which contains a U6 promoter. After linearization, gel purification was performed. The construct was microinjected into the pronuclei of C57BL / 6 mouse fertilized eggs. PCR was used for genotyping. The primer sequences were 5'-CTTCACCGAGGGCCTATTTCC-3' (forward) (SEQ ID NO: 38) and 5'-CCGTAGGTGGCATCGCCCTC-3' (reverse) (SEQ ID NO: 39).

[0173]

[0214] 1.12 Generation of CaMKII-δ2 tg mice

[0215] The full-length human CaMKII-δ2 cDNA coding sequence with an N-terminal HA tag was The construct was cloned into the HindIII and EcoRV sites of an expression vector containing the αMHC promoter. After linearization with XhoI and NotI, gel purification was performed. The construct was microinjected into the pronuclei of C57BL / 6 mouse fertilized eggs. PCR was used for genotyping. The primer sequences were 5'-CGGTATCGATAAGCTTGGCC-3' (forward) (SEQ ID NO: 40) and 5'-TCACAATATTGGGGTGCTTC-3' (reverse) (SEQ ID NO: 41).

[0174]

[0216] 1.13 Generation of UBE2T tg mice

[0217] The full-length rat UBE2T cDNA coding sequence with a C-terminal myc tag was cloned into α The construct was cloned into the HindIII and EcoRV sites of an expression vector containing an MHC promoter. After linearization with XhoI and NotI, gel purification was performed. The construct was microinjected into the pronuclei of C57BL / 6 mouse fertilized eggs. PCR was used for genotyping. The primer sequences were 5'-ATAGAAGCCTAGCCCACACC-3' (forward) (SEQ ID NO: 42) and 5'-GATCTGTGGTGGCTCAAATG-3' (reverse) (SEQ ID NO: 43).

[0175]

[0218] 1.14 Echocardiography

[0219] At 6 and 10 weeks of age, mice were incubated under 1% isoflurane with a Vevo2100 digital microscope. Echocardiographic analysis was performed using midventricular M- and B-mode measurements acquired in the parasternal short-axis view at the level of the papillary muscles using a digital imaging system (Visual Sonics, Toronto, ON, Canada). Once the mice were familiar with the procedure, images were digitally stored on a magneto-optical disk for examination and analysis. LV end-diastolic internal diameter (LVIDd) was measured at the time of maximum apparent left ventricular dilation, and LV end-systolic internal diameter (LVIDs) was measured at the time of maximum posterior wall systolic anterior deflection. LV ejection fraction (EF) was calculated using the cubic method: LVEF (%) = {(LVIDd)}3 -(LVIDs) 3} / (LVIDd) 3 x 100, and LV fractional shortening (FS) was calculated as FS (%) = (LVIDd - LVIDs) / LVIDd x 100. Data were averaged from 5 cardiac cycles.

[0176]

[0220] 1.15 Histological analysis

[0221] Histological analysis of cardiac tissue was previously described (Zhang, T. et al. Nature 2014). re medicine 22, 175-182, doi:10.1038 / nm.4017(2016)). TUNEL staining was performed as previously described (Zhang, T. et al. Nature Medicine 22, 175-182, doi:10.1038 / nm.4017(2016)) and CardioTACSTM in situ apoptosis detection kit (Roche Applied Science, catalog number: 11684795910) were used.

[0177]

[0222] 1.16 Gene Expression Analysis and Primers

[0223] The primer pairs used for quantitative real-time PCR are listed in Table 2. Amplification was performed as follows: 95°C for 30 s, 40 cycles of 95°C for 15 s and 60°C for 30 s. Data are the average of at least three independent experiments.

[0178] [Table 4-1]

[0179] [Table 4-2]

[0180]

[0224] 1.17 Plasmid construction

[0225] A vector expressing CaMKII-δ9 was cloned from human left ventricular cDNA. Rat UBE2T was cloned from rat heart cDNA, and plasmids carrying the S110A or S913A point mutation of rat UBE2T were generated using the Stratagene QuikChange II site-directed mutagenesis kit. Plasmids expressing the CaMKII-δ1, δ2, δ3, and δ9 signature sequences (exons 13-15-16-17, 13-17, 13-14-17, and 13-16-17), respectively, were cloned into the pcDNA5 / flag plasmid with a GFP tag. HEK293 cells were transfected with the plasmids when they reached 80% confluence.

[0181]

[0226] Human CaMKII-δ9, rat UBE2T, rat UBE2T-S110A Adenoviral vectors expressing rat UBE2T-S193A were obtained from Sino The adenoviral vector expressing CaMKII-δ2 was constructed by Geno Max Co., Ltd. The adenoviral vector expressing CaMKII-δ2 was previously described (Zhu, W. et al., The Journal of Biological Chemistry 282, 10833-10839, doi:10.1074 / jbc.M611507200(2007)).

[0182]

[0227] The plasmid expressing human UBE2T was purchased from OriGene (catalog no. T The S110A point mutation was generated using the Stratagene QuikChange II site-directed mutagenesis kit (expression plasmid for P300748). Positive clones carrying the S110A point mutation were confirmed by sequencing, and the plasmid was then amplified for protein purification.

[0183]

[0228] 1.18 Ventricular myocyte isolation, culture, and adenoviral infection

[0229] NRVMs were isolated from 1-day-old Sprague-Dawley rats and Viral-mediated gene transfer was performed using a previously described method (Zhang, T. et al., Nature Medicine 22, 175-182, doi:10.1038 / nm.4017(2016)). NRVMs were exposed to HO (200 μM) or Dox (1 μM) for 24 h.

[0184]

[0230] 1.19 Isolated Mouse Heart Perfusion

[0231] Adult mice (10-12 weeks old) were treated with pentobarbital (70 mg / kg). The rats were anesthetized by intraperitoneal injection. The heart was excised and then ventilated at 55 mmH using a Langendorff apparatus. Cardiac tissue was perfused at a constant pressure of 1000 kJ / cm². The buffer was continuously gassed with 95% O / 5% CO (pH 7.4) and warmed with a heat bath / circulator. Cardiac temperature was continuously monitored and maintained at 37 ± 0.5°C. Global ischemia was induced by interrupting perfusion for 30 min followed by reperfusion.

[0185]

[0232] 1.20 Subcellular fractionation

[0233] Nuclear / cytoplasmic fractionation kit (Biovision Research Products) Cytoplasmic and nuclear proteins were separated using a centrifuge tube (Central Microscopy, Catalog No. K266, USA) according to the manufacturer's instructions.

[0186]

[0234] 1.21 Cell viability analysis

[0235] Cardiomyocyte viability was measured as previously described (Zhang, T. et al., Nature e Medicine 22, 175-182, doi:10.1038 / nm.4017(2016)), caspase 3 / 7 activity and LDH concentration in the culture medium were assayed. Caspase 3 / 7 activity was measured using a Promega kit (catalog number: G8091) according to the manufacturer's instructions. LDH concentration in the culture medium was assayed spectrophotometrically using a Sigma kit (catalog number: MAK066).

[0187]

[0236] 1.22 Histology of the Heart and Cardiomyocytes

[0237] Hearts were fixed overnight in 4% paraformaldehyde (pH 7.4) and paraffinized. The tissue was embedded in PBS and serially sectioned at 5 μm, and standard hematoxylin-eosin staining or immunohistochemistry was performed on the sections.

[0188]

[0238] Immunofluorescence of cardiomyocytes was measured as previously described (Erickson, JR et al., Physiological reviews 91, 889-915, doi:10.1152 / physrev.00018.2010(2011)).

[0189]

[0239] 1.23 Western blot and co-immunoprecipitation

[0240] Western blots and co-immunoprecipitations were performed as previously described (Zh ang, T. et al. Nature medicine 22, 175-182, doi:10.1038 / nm.4017(2016)).

[0190]

[0241] 1.24 RNA interference-mediated gene silencing

[0242] For gene silencing assays, please refer to the Invitrogen website. Using the siRNA, 19 nucleotides in length with a dTdT overhang at the 3' end was designed. Cardiomyocytes were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions (Erickson, JR et al., Physiological reviews 91, 889-915, doi:10.1152 / physrev.00018.2010(2011)). Gene knockdown efficiency was assessed by Western blot 72 h after siRNA transfection. The sequences of the siRNAs are listed in Table 3.

[0191] [Table 5]

[0192]

[0243] 1.25 Cell-free kinase assay of CaMKII-δ

[0244] Human CaMKII-δ2 protein was obtained from Abcam (catalog number: ab845 52), human UBE2T protein was from OriGene (catalog number: TP300748), and human CaMKII-δ9 and UBE2T-S110A proteins were produced by OriGene. Cell-free kinase assays were performed in kinase buffer containing 100 mM Tris-HCl, pH 7.5, 20 mM MgCl2, and 4 mM DTT. CaMKII-δ9 or δ2 proteins were incubated with 200 μM CaCl2 and 1 μM CaM (Sigma) on ice for 1 min and then exposed to 1 mM ATP in the presence of UBE2T protein at 30°C for 30 min. The reaction was stopped by adding SDS loading buffer. Samples were boiled for 5 min and resolved on SDS-PAGE. Commercially available antibodies against phosphoserine and UBE2T were used for immunoblot analysis.

[0193]

[0245] 1.26 Human embryonic stem cells induced into cardiomyocytes

[0246] Human embryonic stem cells H9 were cultured as previously reported (Burridge, PW (Nature Methods 11, 855–860, doi:10.1038 / nmeth.2999 (2014)) used a chemically defined, xeno-free, small molecule-based method to differentiate into cardiomyocytes. Briefly, H9 cells were maintained in E8 medium (Life Technologies, catalog number: A1517001) on 6-well plates precoated with Matrigel (BD Biosciences, catalog number: 354277). When cells reached 70% confluence, the medium was replaced with basal medium supplemented with 6 μM CHIR99021 (Selleckchem, catalog number: S1263-25mg). After 48 h, the medium was replaced with basal medium supplemented with 2 μM Wnt-C59 (Biorbyt, catalog number: orb181132) for the next 48 h. The cells were then maintained in basal medium and replaced with fresh medium every 48 hours. Beating cardiomyocytes appeared on approximately day 8. On day 10, the basal medium was replaced with glucose-free RPMI1640 (Life Technologies, Catalog No. 11879020) to purify cardiomyocytes. Two days later, the cardiomyocytes were mixed with TrypLE™ expression enzyme (Life Technologies) for 10 minutes and transferred to a Matrigel-precoated well plate.

[0194]

[0247] 1.27 Materials

[0248] Antibodies against the following proteins were used: rat / human UBE2T and p- Threonine (Cell Signaling Technology, 12992 (Lot No.: 1, 1:1000) and 9381 (Lot No.: 22, 1:1000)); mouse UBE2T (Aviva Systems Biology, ARP-43145 (Lot No.: QC13585-40506, 1:1000)); p-CaMKII (Thermo, MA1-047 (Lot No.: QC207772, 1:1000)), t-CaMKII-δ (GeneTex, GTX111401 (Lot No.: 40058, 1:1000)), Myc and Flag (Sigma, SAB4700447 (Lot No.: 522137, 1:5000 for Western blot and 1:200 for immunohistochemistry)), and F1804 (Lot No.: S LBR7936V, 1:5000 for Western blotting and 1:200 for immunohistochemistry), γH2AX, p-serine, and ox-CaMKII (Millipore, 05-636, clone JBW301 (lot number: 2884537, 1:1000 for Western blotting and 1:200 for immunohistochemistry), 05-1000, clone 4A4 (lot number: 2691195, 1:1000), and 07-1387 (lot number: 2739150, 1:1000)), COX-2, HA, and lamin A / C (Santa Cruz, sc-1747 (Lot No.: H1911, 1:1000), sc-7392 (Lot No.: L1115, 1:1000 for Western blotting and 1:200 for immunohistochemistry), and sc-6215 (Lot No.: J2615, 1:1000)), PAI-2 (Bioworld, BS3702 (Lot No.: CA36131, 1:1000)), β-actin (EARTHO X, E021070-01 (Lot No.: 0a1401, 1:100 for immunohistochemistry)), FANCD2 and FANCI (abcam, ab108928 (Lot No.: GR130039-32, 1:1000) and ab74332 (Lot No.: GR251812-8, 1:1000)), and GAPDH (EASYBIO, BE0023, clone 2B8 (1:10000)).MG132, clasto-lactacystin β-lactone (β-lac), doxorubicin, and H2O2 were from Sigma-Aldrich.

[0195]

[0249] 1.28 Statistics and Reproducibility

[0250] Data are expressed as mean ± sem. Statistical analysis was performed using GraphPad Pro. The analysis was performed using the Statistical Modeling System (SSM) version 5.01 (GraphPad Software, Inc.) and the SPSS 18.0 software package (SPSS, Inc.). The normality of the distribution of the data sets was tested using the Kolmogorov-Smirnov test. Data sets with normal distribution (two groups) were compared using the Student's unpaired two-tailed t-test. For nonparametric data, the Mann-Whitney U test was used. Comparisons between multiple groups were evaluated using one-way or two-way analysis of variance with Tukey's multiple comparison test. No statistical methods were used for a given sample size.

[0196]

[0251] Example 1 Presence of CaMKII-δ9 in the human heart

[0252] The present inventors have used a method to detect the concentration of CaMKII-δ splice variants. Single-molecule real-time (SMRT) sequencing (Pacific Biosciences) was performed on cardiac tissue from mice, rats, rhesus monkeys, and humans (Sharon, D. et al., Nature biotechnology 31, 1009-1014, doi:10.1038 / nbt.2705(2013)). Library preparation, sequencing, and data collection for SMRT sequencing were described in Section 1.3 of the General Methods and Materials section. Quantification of CaMKII-δ splice variants was performed according to the method described in Section 1.7 of the General Methods and Materials section.

[0197]

[0253] Surprisingly, the inventors discovered that the well-studied splice variant We found that CaMKII-δ2 accounted for 29% and 22.5% of total cardiac CaMKII-δ transcripts in mice and rats, respectively, but was significantly underrepresented in rhesus (3.1%) and human (6.3%) hearts. The previously reported but functionally overlooked CaMKII-δ9 emerged as a highly abundant splice variant, accounting for 33.5%, 31.9%, 32.9%, and 14.9% of total cardiac CaMKII-δ transcripts in rhesus, humans, mice, and rats, respectively, comparable in abundance to CaMKII-δ3 in all cases and significantly more abundant than δ2 in primates (Fig. 1a).

[0198]

[0254] Furthermore, cardiac CaMKII-δ9 expression at the protein level was analyzed using the The peptides were detected using two custom antibodies (anti-exon 16 and anti-exon 21) that react with peptides corresponding to exons 16 and 21. The antibodies were prepared according to the method described in Section 1.6 of the General Methods and Materials section. Immunoprecipitation of mouse cardiac proteins with one antibody followed by immunoblotting with the other antibody revealed a single band of approximately 50 kD. Mass spectrometry (MS) of the approximately 50 kD band immunoprecipitated from mouse heart with anti-exon 21 identified a peptide encoded by exons 13-16-17, a characteristic sequence of CaMKII-δ9 (Fig. 10e, g). Instead, a peptide encoded by exons 20-21 was detected in samples immunoprecipitated with anti-exon 16 (Fig. 10f, h). Importantly, the inventors performed absolute quantitative MS analysis of human cardiac tissue (Gerber, SA et al., Proceedings of the National Academy of Sciences of the United States of America 100, 6940-6945, doi:10.1073 / pnas.0832254100 (2003); Kawakami, H. et al., Journal of Pharmaceutical Sciences 100, 341–352, doi:10.1002 / jps.22255(2011)) (Table 1). When immunoprecipitated with anti-exon 21, we found that the levels of peptides containing exons 13–16 (δ9) and 13–14 (δ3 and δ11) were 4.1- and 1.7-fold higher than those of exon 13–17 (δ2) (Fig. 1c). Similarly, when immunoprecipitated with anti-exon 16, the levels of peptides encoded by exons 20–21 (δ1, δ9, and δ11) were 9.2-fold higher than those encoded by exons 20–22 (δ5 and δ10) (Fig. 1c), distinguishing CaMKII-δ9 from δ10. Therefore, the present inventors conclude that CaMKII-δ9 constitutes the major cardiac CaMKII-δ splice variant at both the mRNA and protein levels in mammals, particularly non-human primates and humans.

[0199]

[0255] To identify the tissue distribution of CaMKII-δ9, we used anti-exon 1 Using 6, we found that CaMKII-δ9 was detected in both cardiac and skeletal striated muscle of rhesus monkeys (Fig. 10i). However, in mice, CaMKII-δ9 was specifically expressed in the heart (Fig. 10j). Immunofluorescence imaging revealed a cytoplasmic distribution pattern of CaMKII-δ9 in cardiomyocytes that partially overlapped with that of CaMKII-δ2 (Fig. 1d and Fig. 10k), whereas CaMKII-δ3 was enriched in the nuclear fraction (Fig. 10l). The cytoplasmic distribution of CaMKII-δ9 was confirmed by Western blot of subcellular fractions of cultured neonatal rat ventricular cardiomyocytes (NRVMs) (performed according to the method described in Section 1.20 of the General Methods and Materials section) (Fig. 10m). Therefore, we conclude that CaMKII-δ9 constitutes an important CaMKII-δ splice variant with cytoplasmic distribution in mammalian hearts, particularly nonhuman primates and humans.

[0200]

[0256] Example 2 Upregulation of CaMKII-δ9 is associated with various cardiac diseases

[0257] To investigate the pathological relevance of CaMKII-δ9, the inventors first investigated Its levels were evaluated in several cardiac injury models. The inventors performed transverse thoracic constriction (TAC) surgery in mice to mimic hemodynamic pressure overload. The experimental methods, animals, and materials used in this example are described in Sections 1.1, 1.2, 1.4, 1.15, 1.18, 1.19, 1.26, and 1.27 of the General Methods and Materials section.

[0201]

[0258] CaMKII-δ9 expression was significantly increased by the chemotherapy drug doxorubicin (Dox, 1 μM). CaMKII-δ9 was significantly elevated in NRVMs exposed to oxidative stress induced by HO (200 μM) or TAC (Fig. 1e, f). Protein levels of CaMKII-δ9 were significantly elevated in TAC hearts compared with sham controls (Fig. 1g). More importantly, CaMKII-δ9 was highly elevated in cardiac tissue from patients with hypertrophic cardiomyopathy (HCM) compared with controls (Fig. 1h). CaMKII is known to be activated by both phosphorylation and oxidation (Erickson, JR et al., Physiological reviews 2014). 91, 889-915, doi:10.1152 / physrev.00018.2010 (2011); Erickson, JR et al., Cell 133, 462-474, doi:10.1016 / j.cell.2008.02.048 (2008)). Acute Dox treatment increased both the phosphorylation and oxidation levels of CaMKII-δ9 in cardiomyocytes (Fig. 11a, b). Furthermore, CaMKII-δ9 phosphorylation and oxidation were also elevated in mouse hearts subjected to acute ischemia / reperfusion injury (30 min of ischemia followed by 60 min of reperfusion) (Fig. 11c, d). Thus, cardiac CaMKII-δ9 is upregulated and hyperactivated in response to various pathological stresses, highlighting the pathological relevance of CaMKII-δ9 in the heart.

[0202]

[0259] Example 3 Enhanced CaMKII-δ9 signaling induces cardiomyocyte death

[0260] Next, the inventors investigated the possible role of CaMKII-δ9 in the regulation of cardiac cell fate. We sought to identify the mechanism underlying this. We designed siRNA targeting exon 16 of CaMKII-δ to specifically reduce CaMKII-δ9 levels (Fig. 11e-g) without altering the expression levels of CaMKII-δ2 or δ3 in cardiomyocytes (Fig. 11h). The siRNA was designed according to the method described in Section 1.24 of the General Methods and Materials. Cell viability analysis was performed according to the method described in Section 1.21 of the General Methods and Materials.

[0203]

[0261] Knockdown of CaMKII-δ9 significantly reduced caspase 3 / 7 activity and the culture medium As indicated by lactate dehydrogenase (LDH) levels in the cells (Fig. 2a, b and Fig. 11i, j), CaMKII-δ9 significantly attenuated both HO- and Dox-induced cardiomyocyte death, suggesting its involvement in oxidative stress and Dox-induced cardiac injury. Furthermore, adenoviral gene transfer of CaMKII-δ9 was sufficient to induce robust cardiomyocyte death in a titer-dependent manner (Fig. 11k). Furthermore, when overexpressed at comparable levels (Fig. 11l), CaMKII-δ9 was much more potent than CaMKII-δ2 in inducing cardiomyocyte death (Fig. 11k), characterizing CaMKII-δ9 as a key pathogenic factor involved in oxidative damage and hypertrophic cardiomyopathy.

[0204]

[0262] Example 4 CaMKII-δ9 inhibits DNA damage in cardiomyocytes by downregulating UBE2T. Induces injury and cell death

[0263] To elucidate the mechanism responsible for CaMKII-δ9-induced cardiomyocyte death and to To distinguish the CaMKII-δ9 mechanism from the CaMKII-δ2 mechanism, we performed RNA-seq analysis using cultured NRVMs overexpressing CaMKII-δ9 or CaMKII-δ2 at comparable protein levels. RNA-seq analysis was performed according to the method described in Section 1.5 of the General Methods and Materials section.

[0205]

[0264] After normalization to the control group (Ad-β-gal), overexpression of CaMKII-δ9 Fifteen genes were altered by CaMKII-δ9 but not by δ2 (Fig. 12a and Table 4). Using real-time PCR (performed according to the method described in Section 1.16 of General Methods and Materials), we verified the differential regulation of gene expression by CaMKII-δ9 versus CaMKII-δ2. In particular, UBE2T (ubiquitin-conjugating enzyme E2T), COX-2 (prostaglandin G / H synthase 2), and PAI-2 (plasminogen activator inhibitor 2 type A) were upregulated by CaMKII-δ9 but not by δ2 (Fig. 2d and Fig. 12b). At the protein level, COX-2 was elevated by CaMKII-δ9 but not by δ2, consistent with its mRNA level (Fig. 12c), whereas PAI-2 protein levels were unchanged by either CaMKII-δ9 or δ2 overexpression (Fig. 12d).

[0206] [Table 6-1]

[0207] [Table 6-2]

[0208]

[0265] Overexpression of CaMKII-δ9 inhibits the UBE2T protein in cultured cardiomyocytes. CaMKII-δ9 significantly reduced UBE2T levels (Figure 2e), despite elevated mRNA levels (Figure 2d), and knockdown of CaMKII-δ9 could increase UBE2T protein levels (Figure 2f). In contrast, overexpression of another cytoplasmic splice variant, CaMKII-δ2, did not alter UBE2T protein levels in the same experimental setting (Figure 2e). Furthermore, UBE2T overexpression rescued cardiomyocytes from CaMKII-δ9-induced cell death, as evidenced by caspase 3 / 7 activity (Figure 2g), whereas UBE2T knockdown was sufficient to induce cardiac cell death (Figures 2h and 12h). Furthermore, UBE2T protein levels were reduced in NRVMs subjected to oxidative stress (Figure 2i), and UBE2T overexpression reduced H2O2-induced cardiomyocyte death (Figure 2j). Thus, the present inventors provide multiple lines of evidence that downregulation of UBE2T plays a critical role in mediating CaMKII-δ9-induced cardiac cell death.

[0209]

[0266] UBE2T is a ubiquitin-binding protein involved in the Fanconi anemia (FA) DNA repair pathway. CaMKII-δ9 is a synthase (E2) required for FANCL-mediated monoubiquitination of FANCD2 and FANCI and subsequent DNA repair. We investigated whether CaMKII-δ9 regulation of cardiac cell fate is due to impaired UBE2T-dependent DNA repair pathways and the resulting DNA damage. DNA damage was assessed by comet assay, as described in Section 1.9 of the General Methods and Materials section.

[0210]

[0267] Increased DNA double-strand break marker, γH2AX (Kuo, LJ & Ya As demonstrated by the comet assay (Olive, PL & Banath, JP Nature protocols 1, 23-29, doi:10.1038 / nprot.2006.5(2006)), overexpression of CaMKII-δ9, but not δ2, was found to induce DNA damage in cardiomyocytes (Fig. 3a-c and Fig. S13a, b). In contrast, knockdown of CaMKII-δ9 attenuated oxidative stress-induced DNA damage in cultured cardiomyocytes (Fig. 3d, e), and CaMKII-δ9 deficiency also reduced cardiac cell death (Fig. 2a, b). Furthermore, UBE2T overexpression reduced cardiac genomic instability induced by CaMKII-δ9 and oxidative stress (Fig. 3f-i), whereas knockdown of UBE2T resulted in significant DNA damage in cardiomyocytes (Fig. 3j, k). Furthermore, we found that silencing of two downstream molecules of UBE2T, FANCD2 and FANCI, by siRNA significantly enhanced DNA damage and increased cardiomyocyte death (Fig. 3l, m and Fig. 13c-f). Thus, we demonstrated that CaMKII-δ9-mediated regulation of cardiac cell fate is primarily due to downregulation of UBE2T and subsequent increased DNA damage. .

[0211]

[0268] Example 5 Enhanced CaMKII-δ9-UBE2T-DNA damage signaling in cardiomyopathy and heart failure

[0269] To further evaluate the role of CaMKII-δ9 in cardiac injury and heart failure Next, we generated transgenic mice with cardiac-specific overexpression of CaMKII-δ9 (CaMKII-δ9 tg) (Fig. 14a, b). tg mice and UBE2T tg mice were generated according to the methods described in General Methods and Materials, Sections 1.10 and 1.13, respectively.

[0212]

[0270] CaMKII-δ9 protein levels were significantly higher in tg mice than in wild-type (wt) mice. The expression level of tg mice was elevated approximately 8-fold compared to littermates (Fig. 4a), similar to the elevation of its splice variant in patients with HCM. tg mice began to die at 2 weeks of age and all had died by 15 weeks, whereas none of the wt mice died during the same period (Fig. 4b). At 10 weeks of age, tg mice exhibited significant cardiomyopathy, manifested by cardiac hypertrophy, ventricular dilation, and cardiomyocyte death (Fig. 4c, d). Cardiomyopathy was also indicated by elevated heart weight-to-body weight ratios and a hypertrophic gene expression profile (Fig. 4c, d). Consequently, cardiac function in tg mice declined over time (Fig. 4e, f). By 10 weeks of age, tg mice developed severe heart failure, manifested by significantly reduced ejection fraction (EF) and fractional shortening (FS) compared to wt mice (Fig. 4e, f). Furthermore, ventricular dilation and cardiac wall thinning in tg mice were confirmed by echocardiography (Figures 4e and 4f, performed according to the method described in Section 1.14 of the General Methods and Materials section). Importantly, DNA damage was clearly elevated in the hearts of CaMKII-δ9 tg mice (Figures 4g and 14e), and this was accompanied by reduced UBE2T protein abundance compared to wt mice (Figure 4h). In contrast, cardiac-specific knockdown of CaMKII-δ9 by transgenic expression of shRNA targeting CaMKII-δ exon 16 (performed according to the method described in Section 1.11 of the General Methods and Materials section) significantly attenuated TAC-induced cardiac hypertrophy, systolic dysfunction, and early death of mice (Figures 4i-k and 14f-i). TAC-induced cardiac DNA damage and cardiomyocyte death were also reduced by CaMKII-δ9 knockdown (Figures 4l and 4m). Concurrently, UBE2T protein abundance was increased in the hearts of CaMKII-δ9-deficient mice (Fig. S4j). Although cardiac-specific overexpression of UBE2T by itself did not result in any macroscopic phenotype (Fig. 5), crossing it with CaMKII-δ9 tg mice effectively ameliorated CaMKII-δ9-induced cardiac injury and dysfunction and significantly improved mouse survival (Fig. 5).Thus, CaMKII-δ9-induced downregulation of UBE2T is a major mechanism underlying cardiac DNA damage, cell death, and cardiomyopathy induced by multiple damaging stimuli, leading to heart failure and death in mice.

[0213]

[0271] Functions and signal transduction mechanisms of CaMKII-δ9 and CaMKII-δ2 To further compare and contrast CaMKII-δ9 in vivo, the present inventors As in the case of tg mice, we constructed transgenic mice with cardiac-specific overexpression of CaMKII-δ2 approximately 8-fold higher than that in wt mice (Fig. 14k).The CaMKII-δ2 tg mice were generated according to the method described in Section 1.12 of the General Methods and Materials.

[0214]

[0272] CaMKII-δ2 tg mice exhibit cardiomyocyte death, cardiac hypertrophy, cardiac dysfunction, and Although the results showed that the CaMKII-δ9 tg mice died, the adverse effects, as judged by all parameters, were much less severe than those in the CaMKII-δ9 tg mice ( (Fig. 14l-o). Furthermore, we did not detect a decrease in UBE2T abundance or an increase in DNA damage in CaMKII-δ2 tg hearts (Fig. 14p, q). These results indicate that the two cytoplasmic CaMKII splice variants activate distinct signaling pathways and play distinct roles in cardiac physiology and pathology.

[0215]

[0273] In myocardium from HCM patients, elevated CaMKII-δ9 (Fig. 1h) was associated with UB The decrease in E2T abundance and the increase in DNA damage (as indicated by γH2AX) were accompanied by increased cardiomyocyte apoptosis (as indicated by cleaved caspase 3) (Fig. 6a-c). Furthermore, in human embryonic stem cell-derived cardiomyocytes (obtained according to the method described in Section 1.26 of the General Methods and Materials), overexpression of CaMKII-δ9 resulted in more severe cell death than overexpression of CaMKII-δ2 (Fig. 6d). Concomitantly, CaMKII-δ9, but not δ2, induced degradation of UBE2T and subsequent DNA damage (Fig. 6e, f). In contrast, knockdown of CaMKII-δ9 effectively ameliorated Dox-induced UBE2T degradation, DNA damage, and cell death in these human cells (Fig. 6g-i).

[0216]

[0274] Example 6 CaMKII-δ9 phosphorylates UBE2T at Ser110 and promotes its degradation

[0275] Overexpression of CaMKII-δ9 increased UBE2T mRNA levels. Therefore, downregulation of UBE2T at the protein level may be mediated by increased proteolysis. To test this hypothesis, we used the proteasome inhibitors β-lac and MG132 and found that both completely abolished the CaMKII-δ9-induced decrease in UBE2T protein levels (Fig. 7a, b). UBE2T is primarily localized in the nucleus of cardiomyocytes (Fig. 7c). In cells overexpressing CaMKII-δ9, UBE2T was still enriched in the nucleus, but its abundance was reduced (Fig. 7c). Notably, the proteasome inhibitor MG132 caused UBE2T to be present in both the nuclear and cytoplasmic fractions (Fig. 7c). These results suggest that UBE2T is distributed in both the cytoplasm and nucleus of cardiomyocytes and that its apparent enrichment in the nucleus is the result of CaMKII-δ9-mediated degradation of UBET2 in the cytoplasm. The CaMKII-δ9-induced increase in UBE2T mRNA levels is likely due to cellular compensation for the decrease in its protein abundance.

[0217]

[0276] The present inventors have investigated the potential interactions between CaMKII-δ9 and UBE2T in cardiomyocytes. We investigated the physical interaction between CaMKII-δ9 and UBE2T. Co-immunoprecipitation assays revealed that CaMKII-δ9 and UBE2T form a protein complex (Fig. 7d). Overexpression of CaMKII-δ9 in NRVMs specifically enhanced the serine phosphorylation of UBE2T (Fig. 7e), but not the threonine phosphorylation (Fig. 7f). To delineate the phosphorylation site of UBE2T on CaMKII-δ9, we transfected CaMKII-δ9-free HEK293 cells (human embryonic kidney cells) with a myc-tagged UBE2T plasmid and a CaMKII-δ9 plasmid. Cell lysates were immunoprecipitated with myc antibody and then subjected to MS analysis. MS analysis was performed according to the method described in Section 1.8 of the General Methods and Materials section. Two serine sites (Ser110 and Ser193) of UBE2T were identified as potential targets of CaMKII-δ9 (Fig. 15a). We found that the UBE2T-S110A mutant, but not the UBE2T-S193A mutant, was resistant to CaMKII-δ9-mediated degradation (Fig. 7g), indicating that phosphorylation of UBE2T at Ser110, a highly conserved site across multiple species (Fig. 15b), is essential for CaMKII-δ9-mediated UBE2T degradation.

[0218]

[0277] Next, the present inventors performed a cell-free kinase assay (General Methods and Materials We used recombinant UBE2T protein in the presence or absence of CaMKII-δ9 (performed according to the method described in Section 1.25) to determine whether UBE2T is a direct substrate of CaMKII-δ9. The presence of recombinant CaMKII-δ9 protein significantly enhanced the serine phosphorylation level of wtUBE2T, but not its S110A mutant (Fig. 7h and Fig. 15c). This result provides direct evidence that CaMKII-δ9 mediates the phosphorylation of UBE2T at Ser110. Furthermore, disruption of phosphorylation at S110 (UBE2T-S110A), but not at S193 (UBE2T-S193A), resulted in UBE2T localized in both the cytoplasm and nucleus (Fig. 15d), confirming that CaMKII-δ9 phosphorylates UBE2T at Ser110 and promotes its proteasome-dependent degradation in the cytoplasm, leading to its enrichment in the nucleus. Collectively, our in vivo and in vitro data demonstrate that after cardiac injury, CaMKII-δ9 is activated and upregulated, promoting Ser110 phosphorylation and subsequent degradation of UBE2T, ultimately leading to myocardial DNA damage, genomic instability, and cell death.

[0219]

[0278] Example 7 UBE2T is not regulated by CaMKII-δ1, δ2, or δ3

[0279] The minor cytoplasmic CaMKII-δ splice variant, CaMKII CaMKII-δ2 did not interact with UBE2T (Fig. 7i) or increase its serine phosphorylation (Fig. 7h), suggesting that UBE2T is a specific substrate of CaMKII-δ9, but not CaMKII-δ2. Furthermore, neither CaMKII-δ1 nor CaMKII-δ3 interacted with or induced its degradation (Fig. 8a–d), reaffirming that UBE2T is a selective target of CaMKII-δ9. To pinpoint the molecular basis for splice variant-specific regulation of UBE2T by CaMKII-δ9, we constructed plasmids expressing peptides encoded by exons 13-15-16-17, 13-17, 13-14-17, and 13-16-17, which are characteristic sequences of CaMKII-δ1, δ2, δ3, and δ9, respectively (Fig. 9a). The plasmids were constructed according to the method described in Section 1.17 of the General Methods and Materials section. A peptide encoded by exons 13-16-17 interacted with UBE2T, but other peptides did not (Fig. 8e and Fig. 16), suggesting that the characteristic sequence of CaMKII-δ9 (exons 13-16-17) is responsible for its substrate selectivity.

Claims

1. A method for treating or preventing a CaMKII-mediated disease in a subject, comprising administering to said subject an effective amount of an antagonist of CaMKII-δ9.

2. A method of alleviating cardiac damage in a subject, comprising administering to said subject an effective amount of an antagonist of CaMKII-δ9.

3. A method of stimulating the level or activity of a ubiquitin-conjugating enzyme in a subject, comprising administering to said subject an effective amount of an antagonist of CaMKII-δ9.

4. A method for preventing the degradation of a ubiquitin-conjugating enzyme in a subject, comprising administering to said subject an effective amount of an antagonist of CaMKII-δ9.

5. A method of preventing cardiomyocyte cell death in a sample, comprising contacting said sample with an effective amount of an antagonist of CaMKII-δ9.

6. A method of reducing DNA damage in a cell, comprising contacting said cell with an effective amount of an antagonist of CaMKII-δ9.

7. The method of any one of claims 1 to 6, wherein the antagonist is an antagonist that inhibits phosphorylation of a ubiquitin-conjugating enzyme.

8. The method of claim 7, wherein the ubiquitin-conjugating enzyme is ubiquitin-conjugating enzyme 2T.

9. The method of claim 8, wherein the antagonist is an antagonist that inhibits phosphorylation of ubiquitin-conjugating enzyme 2T at Ser110.

10. The method of any one of claims 1 to 6, wherein the antagonist is a specific antagonist of CaMKII-δ9.

11. 7. The method of any one of claims 1 to 6, wherein the antagonist inhibits the level or activity of CaMKII-δ9 but does not significantly inhibit the level or activity of CaMKII-δ2 or CaMKII-δ3.

12. The method of any one of claims 1 to 6, wherein the antagonist is an antibody that specifically recognizes CaMKII-δ9, a small molecule compound that binds to CaMKII-δ9, an RNAi molecule that targets the coding sequence of CaMKII-δ9, an antisense nucleotide that targets the coding sequence of CaMKII-δ9, or an agent that competes with CaMKII-δ9 to bind to its substrate.

13. The method of claim 12, wherein the antibody is a monoclonal or polyclonal antibody.

14. 13. The method of claim 12, wherein the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody.

15. The method of any one of claims 12 to 14, wherein the antibody binds to an amino acid sequence encoded by exon 16 of the CaMKII-δ gene.

16. 13. The method of claim 12, wherein the RNAi molecule is a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a microRNA (miRNA).

17. The method of claim 16, wherein the RNAi molecule has 10 to 100 bases.

18. 13. The method of claim 12, wherein the antisense nucleotide is modified to improve its stability.

19. The method of claim 12, wherein the RNAi molecule and the antisense nucleotide bind to exon 16 of the CaMKII-δ gene.

20. The method of claim 19, wherein the RNAi molecule and the antisense nucleotide bind to exon 13 and exon 16 of the CaMKII-δ gene, or exon 16 and exon 17 of the CaMKII-δ gene, or exon 13, exon 16, and exon 17 of the CaMKII-δ gene.

21. The method of claim 12, wherein the agent that competes with CaMKII-δ9 to bind to its substrate is a vector that expresses CaMKII-δ9 that does not have phosphorylation or oxidation functions.

22. 22. The method of claim 21, wherein the vector is an adeno-associated virus (AAV), an adenovirus, a lentivirus, a retrovirus, or a plasmid.

23. 23. The method of claim 22, wherein the AAV is AAV1, AAV2, AAV5, AAV8, AAV9 or AAVrhlO.

24. 5. The method of any one of claims 1 to 4, wherein the subject is a human or non-human primate.

25. 25. The method of claim 24, wherein the non-human primate is a rhesus monkey.

26. The method of claim 1, wherein the CaMKII-mediated disease is associated with elevated levels or activity of CaMKII-δ9.

27. The method of claim 1, wherein the CaMKII-mediated disease is a cardiac disease or a metabolic disease.

28. 28. The method of claim 27, wherein the cardiac disease is selected from the group consisting of cardiomyopathy, myocarditis, diabetic heart disease, myocardial ischemia, cardiac ischemia / reperfusion injury, myocardial infarction, heart failure, arrhythmia, cardiac rupture, angina pectoris, cardiac hypertrophy, cardiac damage, hypertensive heart disease, rheumatic heart disease, angina pectoris, myocarditis, coronary heart disease, and pericarditis.

29. 28. The method of claim 27, wherein the metabolic disease is selected from the group consisting of insulin resistance, obesity, diabetes, hypertension, dyslipidemia, diabetic cerebrovascular disease, diabetic eye complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, gout, and hyperuricemia.

30. 1. A method of diagnosing a CaMKII-mediated disease in a subject, comprising: (a) obtaining a test biological sample from the subject; (b) detecting the level or activity of CaMKII-δ9 in the test biological sample. wherein the level or activity of CaMKII-δ9 detected in the test biological sample of the subject indicates that the subject is suffering from or has an increased likelihood of developing a CaMKII-mediated disease. A method comprising:

31. 31. The method of claim 30, wherein the level or activity of CaMKII-δ9 in the test biological sample is detected by contacting the sample with a reagent that specifically binds to CaMKII-δ9.

32. 31. The method of claim 30, wherein the level or activity of CaMKII-δ9 detected in the test biological sample is compared to a reference level or activity of CaMKII-δ9 detected in a reference sample.

33. 33. The method of claim 32, wherein a level or activity of CaMKII-δ9 detected in the test biological sample that is higher than the reference level or activity of CaMKII-δ9 indicates that the subject has or has an increased likelihood of developing a CaMKII-mediated disease.

34. 33. The method of claim 32, wherein the reference sample is from a healthy subject or is a sample obtained from the same subject before or after the test biological sample.

35. 35. The method of any one of claims 30 to 34, wherein the test biological sample is from the heart of the subject.

36. 36. The method of claim 35, wherein the subject is a human or non-human primate.

37. A kit for diagnosing a CaMKII-mediated disease in a subject, comprising an antibody or antibody fragment that specifically recognizes CaMKII-δ9.

38. A method for identifying a molecule that inhibits the activity of CaMKII-δ9, comprising contacting the molecule with CaMKII-δ9 and ubiquitin-conjugating enzyme 2T, and determining whether phosphorylation of ubiquitin-conjugating enzyme 2T is inhibited, wherein inhibition of phosphorylation of ubiquitin-conjugating enzyme 2T identifies a molecule that inhibits CaMKII-δ9.

39. A method for identifying a molecule that inhibits the phosphorylation of CaMKII-δ9, comprising contacting the molecule with CaMKII-δ9 and ubiquitin-conjugating enzyme 2T, and determining whether phosphorylation of ubiquitin-conjugating enzyme 2T is inhibited, wherein inhibition of phosphorylation of ubiquitin-conjugating enzyme 2T identifies a molecule that inhibits CaMKII-δ9.

40. A method for identifying a molecule that treats or prevents a CaMKII-mediated disease, comprising contacting the molecule with CaMKII-δ9 and ubiquitin-conjugating enzyme 2T, and determining whether phosphorylation of ubiquitin-conjugating enzyme 2T is inhibited, wherein inhibition of phosphorylation of ubiquitin-conjugating enzyme 2T identifies a molecule that inhibits CaMKII-δ9.

41. A method for identifying a molecule that alleviates cardiac damage, comprising contacting the molecule with CaMKII-δ9 and ubiquitin-conjugating enzyme 2T, and determining whether phosphorylation of ubiquitin-conjugating enzyme 2T is inhibited, wherein inhibition of phosphorylation of ubiquitin-conjugating enzyme 2T identifies a molecule that inhibits CaMKII-δ9.

42. A method for identifying a molecule that prevents cardiomyocyte death, comprising contacting the molecule with CaMKII-δ9 and ubiquitin-conjugating enzyme 2T, and determining whether phosphorylation of ubiquitin-conjugating enzyme 2T is inhibited, wherein inhibition of phosphorylation of ubiquitin-conjugating enzyme 2T identifies a molecule that inhibits CaMKII-δ9.

43. A method for identifying a molecule that reduces DNA damage, comprising contacting the molecule with CaMKII-δ9 and ubiquitin-conjugating enzyme 2T, and determining whether phosphorylation of ubiquitin-conjugating enzyme 2T is inhibited, wherein inhibition of phosphorylation of ubiquitin-conjugating enzyme 2T identifies a molecule that inhibits CaMKII-δ9.

44. 44. The method of any one of claims 38 to 43, wherein the phosphorylation of ubiquitin-conjugating enzyme 2T is at Ser110.

45. A biomarker for diagnosing a CaMKII-mediated disease in a subject, comprising the full-length protein sequence of CaMKII-δ9 or a fragment thereof.

46. The biomarker of claim 45, comprising an amino acid sequence set forth in SEQ ID NOs: 1 to 5.

47. Use of CaMKII-δ9 as a biomarker for diagnosing CaMKII-mediated diseases in a subject.

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