Methods of modulating the subcellular compartmentalization of camkiid as a treatment for heart disease

Abstract

Methods of treating heart disease by modulating the cellular localization of the beta isoform calcium / calmodulin dependent protein kinase II delta (CaMKIIδ) via the disruption of post-transcriptional phosphorylation.

Description

[0001] METHODS OF MODULATING THE SUBCELLULAR COMPARTMENTALIZATION OF CAMKIID AS A TREATMENT FOR HEART DISEASE

[0002] STATEMENT OF GOVERNMENT INTEREST

[0003] This invention was made with government support under grant number RGM029090L awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This International PCT application claims the benefit of U.S. Provisional Application Serial No. 63 / 702,900, filed October 3, 2024, the specification, claims and drawings of which are incorporated herein by reference in their entirety.

[0006] SEQUENCE LISTING

[0007] The instant application contains contents of the electronic sequence listing (90245.01311- Sequence-Listing.xml; Size: 147,281 bytes; and Date of Creation: October 3, 2025) is herein incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] The present invention is directed to methods of treating heart disease, and in particular modulating the subcellular localization of calcium / calmodulin-dependent protein kinase II delta in cardiomyocytes via alternative splicing and inhibition of post-transcriptional modifications. BACKGROUND

[0010] Hyperactivation of Ca2+ / calmodulin-dependent kinase 118 (CaMKIIS or CAMK2D) contributes to heart disease development through the activation of cytosolic substrates associated with calcium signaling and inflammation. There have been multiple therapeutic efforts aimed at broadly inhibiting CaMKIIS activity. However, CaMKII8 has an important role in physiological functions in the heart, skeletal muscle, and brain, thus limiting the utility of broadly acting inhibitors. Notably, for patients with end-stage heart failure awaiting a new heart, left ventricular assist device (LVAD) therapy, which takes over cardiac pump function, is often lifesaving and serves as a “bridge to transplant.” About 10% of LVAD patients experience significant recovery from heart failure and in rare cases, the device can be removed without the heart returning to failure. This suggests that LVAD induces molecular changes in some patients that contribute to their recovery. With the goal of identifying new therapeutic targets for heart failure, Applicants used a multi-omics (RNA-seq, proteomics, phospho-proteomics) approach to examine human heart tissues from patients pre- and post-LVAD from LVAD responders and nonresponders. The most significant difference between patients who had reversal of heart failure and those who did not was distinct alternative splicing of the pre-mRNA transcript encoding CaMKIIo. Importantly, alternative splicing of this gene is tissue-specific with the human heart predominantly expressing three main isoforms. One isoform, CAMK2DB (also identified as C AMK2D-B), includes a nuclear localization signal (NLS) (KKRK which is adjacent to position 332-224) which sequesters CaMKIIS away from its heart disease-associated cytoplasmic targets. In patients who had no reversal of heart failure with LVAD, this isoform was hyperphosphorylated near the NLS which prevented it from translocating to the nucleus and was associated with increased pro-inflammatory cytokine expression. Follow-up targeted studies in cultured cardiac myocytes validated that phosphorylated CAMK2D-B was retained in the cytoplasm while the unphosphorylated version had nuclear localization.

[0011] These data support that targeting CAMK2D-B subcellular localization is a potential therapeutic strategy for heart failure. This approach overcomes the limitations of broad kinase inhibition as only a portion of the total CAMK2D-B would be targeted to the nucleus. CaMKIIo functions as a 12 to 14-member holoenzyme composed of all isoforms. Increasing the abundance of one isoform can shift the localization of the entire holoenzyme. So, increasing a nuclear-targeted version can increase the nuclear localization of all isoforms, thus limiting activation of pathological cytosolic pathways in a dose dependent manner.

[0012] SUMMARY OF THE INVENTION(S)

[0013] Increased activity of calcium / calmodulin-dependent protein kinase II delta (CaMKII6 or CaMK2D) is associated with heart disease development through activation of diverse cytoplasmic targets in cardiomyocytes. The present disclosure describes methods and composition to modulate the localization of isoform CAMK2D-B (also referred to as CAMK2DB) as a treatment for heart disease. Specifically, Applicants have identified that alternative splicing of the RNA transcript encoding CAMK2D-B modulates the localization of the kinase in human heart disease models. Moreover, in patients who display the worst functional outcomes with a medical device therapy, CAMK2D-B is modified post-translationally causing it to be retained in the cytosol and activate pathology-associated factors. Applicant’ s data from human heart tissue and in vitro studies support that targeting CAMK2D-B subcellular localization by mobilizing it from the cytoplasm to the nucleus, thereby sequestering it away from its targets in the cytoplasm, can be an effective therapeutic strategy for treating heart disease.

[0014] In a preferred embodiment, the present disclosure includes methods and composition to modulate the localization of calcium / calmodulin-dependent protein kinase II delta beta (CAMK2DB) isoform protein. In a preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at positions 332, 333, and / or 334 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a functional fragment thereof.

[0015] In another specific embodiment, the disclosure includes methods of treating heart disease by increasing nuclear localization of the beta isoform calcium / calmodulin dependent protein kinase II delta (CaMKIIS / y) via the disruption of post-transcriptional phosphorylation one or more of positions S332, S333, or S334 of exon 14. On one embodiment, the substitution mutation includes one or more mutations at positions selected from: S332, S333, or S334 of exon 14, with an amino acid reside incapable of being phosphorylated or acting as a phospho-mimetic. In a one embodiment, the substitution mutation includes one or more mutations selected from: S332A, S333A, and S334A according to SEQ ID NO. 1.

[0016] In another specific embodiment, the disclosure includes methods of treating heart disease in a subject in need thereof, the method comprising, obtaining a biological sample, such as preferably a cardiac cell, from the subject and determining the phosphorylation status of positions 332, 333, and 334 of CAMK2DB protein. A lack of phosphorylation at any of positions 332, 333, and 334 is indicative that the subject will benefit from LV assist device (LVAD) therapy, whereas and phosphorylation positions 332, 333, and 334 is indicative that the subject will not benefit from LV assist device (LVAD) therapy. Once this determination has been made, LVAD can be administered to the subject with a lack of phosphorylation at any of positions 332, 333, and 334.

[0017] Additional aspects of the invention may include one or more of the preferred embodiments set forth in the figures, specification, and claims provided below.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Fig. 1A-J. Partial myocardial recovery with mechanical circulatory support is linked to RNA alternative splicing. A-B. Left ventricular ejection fraction (LVEF) (A) and LV end diastolic dimension (LVEDD) (B) in pre- and post-LVAD in responders (R) and non-responders (NR); n = 10 R, 9 NR; paired two-tailed t-test. C-D. GSEA gene ontology (GO) Biological Process (BP) analysis of bulk RNA-seq data for genes with expression changes pre- to post-LVAD that were positively (C) or negatively (D) associated with a favorable response to LVAD. E. Volcano plot displaying quantitative proteomics results from post-LVAD responder (R) and non-responder (NR) myocardium; n = 9 / group. F. Gene ontology (GO) Biological Process pathway enrichment analysis of the significantly upregulated proteins in Responders. G. Heat map displaying the significantly differentially expressed RNA splicing proteins between post-LVAD R and NR. H. Venn diagram displaying the number of local splicing variations identified between HF (n = 19) and control (n = 5) by RNA-seq and the differential alternative splicing responses post-LVAD in R (n = 10) and NR (n = 9) patients. I. TTN exon 242 alternative splicing map and quantification between groups; displayed as median and range. J. CAMK2D exon 16 and 14 alternative splicing maps and quantification between groups; PSI = proportion spliced in; displayed as median and range; n = 5 control, 9 HF-NR and LVAD-NR, 10 HF-R and LVAD-R; *p < 0.05, **p < 0.01, ***p < 0.001 by an independent two-sample Mann- Whitney U-test (Wilcoxon test in MAJIQ).

[0020] FIG. 2A-G. Increased CAMK2D exon 14 inclusion in heart failure is reversed only in patients who experience functional recovery with LVAD. A. Graphical representation of cardiac CAMK2D splice variants and their respective variable region (exons 13-17) alternative splicing; NLS = nuclear localization signal. B. CAMK2D exon 14 proportion spliced in (PSI) in pre- and post-LVAD responders and non-responders from the RNA-seq data; data analysis by paired t-test within groups (pre- vs. post-LVAD) and unpaired t-test between groups (NR vs. R). C-D. Linear regression analysis of exon 14 PSI post-LVAD vs. absolute change in LVEF (C) and LVEDD (D) after LVAD therapy; confidence interval = 95%. E. Representative RT-PCR acrylamide gel for CAMK2D cardiac splice variants and PGK1 housekeeping gene in non-failing controls, HF, and post-LVAD R and NR; BP = base-pairs. F. Proportion CAMK2D splice variant expression from the RT-PCR; displayed as mean ± SEM; n = 6 Control, 10 HF and LVAD R, 9 HF and LVAD NR. G. Quantitative RT-PCR analysis of CAMK2D-B in Control, HF (pre-LVAD), and post-LVAD normalized to the housekeeping gene PGK1 and plotted as a fold-change vs. non-failing control expression; displayed as median and range; n = 10 R, 9 NR; one-way ANOVA with Tukey’s post- hoc test.

[0021] Fig. 3A-M. CAMK2D-B S332-S334 phosphorylation predicts poor functional recovery with LVAD therapy. A. Volcano plot displaying phospho-proteomics results from post-LVAD responder (R) and non-responder (NR) myocardium; n = 9 / group. B. CAMK2D-B S332, S333, and S334 phosphorylation between R and NR post-LVAD; two-tailed t-test; *p < 0.05, **p < 0.01, ****p < o oooi. C. Representative western blots for total CAMK2D, p-T287, and p-S332 in nonfailing control, pre-LVAD (HF) R and NR, and post-LVAD R and NR. D. p-S332 CAMK2D normalized to total protein; n = 6 Control, 10 HF and LVAD R, 9 HF and LVAD NR; one-way ANOVA with Tukey’s post-hoc test. E-F. Linear regression analysis of post-LVAD (E) and pre- LVAD (F) S332 phosphorylation vs. absolute change in LVEF on LVAD therapy; confidence interval = 95%. G. Representative images for NRVMs 24-hours post-transduction with GFP- CAMK2D-B adenoviruses ± PE (20 pM); Bsss = wildtype, BAAA = Serine 332-334 mutated to Alanine (Phospho-null), BDDD = Serine 332-334 mutated to Aspartic Acid (Phospho-mimetic). H. Nuclear GFP normalized to whole-cell GFP; Vehicle: n = 75 Bsss, 82 BAAA, and 72 BDDD. PE: n = 57 Bsss + PE, 72 BAAA, 91 BDDD from three biological replicates; displayed as median and range; two-way ANOVA with Tukey’s post-hoc test. I. Representative western blots for CAMK2D in subcellular fractions from post-LVAD responders and non-responders. J-M. CAMK2D expression in the cytosol (J), membrane (K), nucleus (L), and insoluble (M) fractions normalized to corresponding loading controls; ATP1A2 =Na+ / K+ATPase subunit a-2; NPM1 = nucleophosmin; two-tailed unpaired t-test. For all except I, the data are presented as the mean ± SEM.

[0022] Fig. 4A-H. Phosphorylation at S332-S334 prevents autoactivation-dependent CAMK2D- B nuclear translocation. A. Representative western blot for GFP-BAAA in the nuclear / insoluble and cytosolic fractions of NRVMs treated with vehicle or PE. B. Nuclear / insoluble GFP-BAAA normalized to Histone H3; n = 4 / group; two-tailed t-test. C. Representative western blot for T287 phosphorylated GFP-CAMK2D-B in adenovirus-transduced NRVMs ± PE. D. p-T287 GFP- CAMK2D-B normalized to GAPDH; n = 6 / group; two-way ANOVA with Tukey’s post-hoc test. E. Representative images for NRVMs 24-hours post-transduction with BAA or BAAA-T287A / D mutants. F. Nuclear GFP normalized to whole-cell GFP; Vehicle: n = 16 BAAA, 82 T287A, and 68 T287D. PE: n = 66 BAAA, 91 T287A, and 117 T287D from three biological replicates; two-way ANOVA with Tukey’s post-hoc test. G. Representative images for NRVMs 24-hours posttransduction with BDDD or BDDD-T287A / D mutants. H. Nuclear GFP normalized to whole-cell GFP; Vehicle: n = 52 BDDD, 40 T287A, and 82 T287D. PE: n = 47 BDDD, 67 T287A, and 83 T287D from three biological replicates. Data in B and D are presented as the mean ± SEM; data in F and H are plotted as the median and range. Scale bars for microscopy images = 15 pm. Fig. 5A-FT. Cytoplasm-restricted CAMK2D-B remodels the phospho-proteome and blunts cardiomyocyte calcium transients. A. Heat map depicting the differentially expressed phosphopeptides in NRVMs transduced with empty vector (E), BAAA, or BDDD ± PE; p-value < 0.01, log2 fold-change < -0.3, > 0.3. B. Volcano plot displaying differential phospho-peptide expression in NRVMs transduced with BAAA or BDDD. C. Volcano plot displaying differential phospho-peptide expression in NRVMs transduced with BAAA or BDDD and treated with PE. D-E. Reactome Pathway over-enrichment for the differentially expressed phospho-peptides in BAAA + PE (D) versus BDDD + PE (E). F. Representative images of EHTs 24 hours post-transduction with Empty vector, GFP- BAAA, or GFP-BDDD adenoviruses. G. Representative Ca2+transient traces for EHTs loaded with an intracellular calcium indicator (Cal 520, AM) and paced at 1 Hz and treated with vehicle or a combination of the a and adrenergic agonists PE (50 pM) and isoproterenol (ISO, 2 pM). Traces are plotted as % change in Cal 520, AM fluorescence (AF) over time. H. Normalized EHT mean calcium transient amplitude (AF / F at time 0, F0); displayed as median and range; Vehicle: n = 41 Empty vector, 43 BAAA, 41 BDDD EHTs from three biological replicates. ISO / PE: n = 14 EHTs / group from two biological replicates; two-way ANOVA with Tukey’s post-hoc test.

[0023] Fig. 6A-I. Mechanical circulatory support partially reverses transcriptomic features of heart failure. A-C. Volcano plots depicting differentially expressed genes identified by RNA-seq between heart failure (HF) and non-failing controls (A), LVAD and HF (B), and LVAD and controls (C). D-F. Significantly enriched GO: Biological Process Pathways for the upregulated and downregulated genes between the three comparisons; enriched pathways only reached significance for the upregulated genes in the HF vs. Control and LVAD vs. Control comparisons. G. Venn Diagrams depicting significantly differentially expressed genes that increased with HF and were reversed with LVAD. H. Venn Diagrams depicting significantly differentially expressed genes that decreased with HF and were reversed with LVAD. I. GO: Biological Process pathway enrichment for genes whose expression was restored by LVAD to Control expression levels. For all RNA-seq, n = 5 Control, n = 19 HF, n = 19 LVAD.

[0024] Fig. 7A-B. Responders and non-responders display similar gene expression profiles pre- and post-LVAD. A-B. Volcano plots depicting differentially expressed genes identified by RNA- seq between responders (R) and non-responders (NR) in heart failure (A) and post-LVAD (B). An FDR adjusted p-value cutoff of 0.05 was used; n = 9 NR, 10 R. X-axis represents the shrunken log2 fold-change using the apeglm algorithm. Fig. 8. Interactome of RNA splicing factors increased in LVAD responders. STRING analysis of the 11 RNA splicing factors increased in post-LVAD responders identified by quantitative proteomics.

[0025] Fig. 9A-D. Post-LVAD responder proteomes display increased RNA processing factors and decreased autophagosome membrane proteins. A-D. Pathway, biological processes, and cell components enriched in post-LVAD responder and non-responder proteomes; FDR-adjusted p- values were used to select pathways / processes / components that were significantly enriched. Blue color = increased in non-responders; Red color = increased in responders.

[0026] Fig. 10A-E. Some alternative splicing changes in heart failure are restored to healthy proportions with LVAD therapy. A. Venn Diagram depicting the number of significant local splicing variations (LSVs) identified by short-read RNA-sequencing analysis between HF and non-failing controls or post-LVAD. B-E. Examples of differential alternative splicing between control, HF (pre-LVAD), and post-LVAD patients. RBM20 splicing changes in HF that are reversed by LVAD include increased intron retention between exons 3 and 4 and decreased exon 4 inclusion (B). TTN exon 242 inclusion decreases in HF and is reversed by LVAD (C). CAMK2D exon 16 inclusion decreases in HF, while exon 14 inclusion increases; both events are partially reversed by LVAD therapy (D). VCL exon 19 inclusion decreases in heart failure. LVAD modestly reduces exon 19 skipping while increasing intron retention (E). n = 5 non-failing controls, n = 19 HF and LVAD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by an independent two- sample Mann-Whitney U-test (Wilcoxon test in MAJIQ). For B-E, all data are presented as the median and range.

[0027] Fig. 11. Types of alternative splicing changes with end-stage heart failure. Graphical representation of the alternative splicing modules that undergo differential splicing between HF and Control. The splicing events contained within those modules are shown. Modules were defined as changing if they contained a changing junction. See methods for definitions of significant changes. alt3: alternative 3’ splice site. alt5: alternative 5’ splice site. Mxe: mutually exclusive exons. Orphanjunc: orphan junction. Ale: alternative last exon. Afe: alternative first exon, ir: intron retention.

[0028] Fig. 12. Types of alternative splicing changes with LVAD. Graphical representation of the alternative splicing modules that undergo differential splicing between LVAD and HF. The splicing events contained within those modules are shown. Modules were defined as changing if they contained a changing junction. See methods for definitions of significant changes. alt3: alternative 3’ splice site. alt5: alternative 5’ splice site. Mxe: mutually exclusive exons. Orphanjunc: orphan junction. Ale: alternative last exon. Afe: alternative first exon, ir: intron retention.

[0029] Fig. 13A-C. Heart failure-associated protein features affected by cassette exon alternative splicing. A. Pie chart depicting the proportion of all HF-associated cassette exon splicing changes (vs. Control) impacting protein features. B. Types of protein features impacted by cassette exon alternative splicing in heart failure. C. REACTOME pathway over-enrichment of the pathways affected by cassette exon alternative splicing and subsequent changes in protein-protein interactions.

[0030] Fig. 14. Alternative splicing differences are predicted to alter protein-protein interactions in heart failure patients. Examples of four genes where heart-failure associated differential alternative splicing of cassette exons impacts protein domains that are known to mediate domaindomain interactions. Domains are shown as blue triangles, while proteins with predicted disrupted binding are shown as gray circles.

[0031] Fig. 15A-C. TTN exon 242 alternative splicing in heart failure and post-LVAD. A. Linear regression analysis of TTN exon 242 proportion spliced in (PSI) post-LVAD identified by RNA- seq versus absolute change in LVEF with LVAD therapy; confidence interval = 95%. B. qPCR quantification of exon 242 inclusion in control, HF, and LVAD using a forward primer spanning the exon-exon junction of exons 241 and 242; one-way ANOVA with Tukey’s post-hoc test for multiple independent pairwise comparisons; n = 6 control, 19 HF, 19 LVAD. C. Quantification of exon 242 inclusion between post-LVAD responders and non-responders from the qPCR analysis; n = 10 R, 9 NR. For B-C, the data are presented as the median and range.

[0032] Fig. 16. CAMK2D splice variant expression is impacted by heart failure and partially restored with mechanical circulatory support. Expression of total CAMK2D, CAMK2D-A (Exons 13-15), CAMK2D-B (Exons 13-14), CAMK2D-C (Exons 13-17), and CAMK2D-9 (Exons 13-16) by qPCR between non-failing controls (n = 6), heart failure / pre-LVAD (n = 19), and heart failure / post-LVAD (n = 19). For each, data were analyzed by one-way ANOVA with Tukey’s post-hoc test for multiple independent pairwise comparisons. Data presented as the median and range. Fig. 17. CAMK2D-B T287 phosphorylation is similar between LVAD responders and nonresponders. p-T287 CAMK2D normalized to total protein; n = 6 Control, 10 HF and LVAD R, 9 HF and LVAD NR; one-way ANOVA with Tukey’s post-hoc test.

[0033] Fig. 18A-B. Adrenergic and endothelin receptor agonists induce CAMK2D-BAAA nuclear translocation. A. Representative immunofluorescence microscopy images of NRVMs transduced with GFP-BAAA and treated with phenylephrine (PE), endothelin- 1 (ET-1), caffeine, or insulin-like growth factor-1 (IGF-1). B. Quantification of nuclear GFP signal to total cellular GFP signal; oneway ANOVA with Tukey’s post-hoc test for multiple independent pairwise comparisons; n = 73 Control, 85 PE, 53 ET-1, 55 Caffeine, and 45 IGF-1 from three independent biological replicates. Data are presented as the median and range.

[0034] Fig. 19. Subcellular fractionation of frozen human heart tissue. Representative western blot for CAMK2D in the cytosolic, membrane, nuclear, and insoluble (myofilament / chromatin- associated proteins) protein fractions isolated from human left ventricular tissue.

[0035] Fig. 20A-G. CAMKI and CAMK4 do not regulate the CAMK2D NLS in cardiomyocytes. A-C. Normalized transcripts per million (nTPM) from Protein Expression Atlas GTEx RNA-seq data for CAMK2D (A), CAMKI (B), and CAMK4 (C). Mean nTPMs denoted for the highest expressing tissue and heart (LV) in each comparison, n = 257 adrenal gland, 244 basal ganglia, 240 cerebellum, 254 cerebral cortex, 431 heart (LV), 802 skeletal muscle; data are presented as the median and range. D. Representative immunofluorescence images of NRVMs transduced with GFP-CAMK2D-Bsss and treated with various concentrations of the CAMKI inhibitor CS640 for 24 hours; scale bars = 15 pm; green = CAMK2D, blue = DAPI. E. CAMK2D NLS serine residue centralized sequences and top 3 PhosphoSite Plus kinase prediction. F-G. MoMo peptide motif enrichment (annotated with fold enrichment in top left comer) and top 5 PhosphoSite Plus kinase prediction (by percentile rank) for sites in non-responders (189 phospho-sites) (F) and responders (177 phospho-sites) (G) post-LVAD that were below an unadjusted p-value cut-off of 0.01; bolded kinases are those with >25 mean nTPM within hearts in the GTEx dataset.

[0036] Fig. 21. Both proposed CAMK2D NLS-regulating phosphatases are highly expressed in the human heart. A-B. Normalized transcripts per million (nTPM) from Protein Expression Atlas GTEx RNA-seq data for PPP1CC (A) and PPP3CC (calcineurin) (B). The top 6 tissues by RNA abundance are listed for each, n = 802 skeletal muscle, 105 retina, 361 testis, 431 heart (LV), 187 small intestine, 373 colon, 663 adipose, and 578 lung tissue samples. Data are presented as the median and range.

[0037] Fig. 22A-B. Pathway enrichment for phospho-peptides correlated with p-S332-334 CAMK2D post-LVAD. A. Reactome Pathway enrichment for phospho-peptides with r2> 0.50 vs. CAMK2D-B S332-S334 phosphorylation by linear regression. B. GO: Biological Process enrichment for phospho-peptides with r2> 0.50 vs. CAMK2D-B S332-S334 phosphorylation by linear regression.

[0038] Fig. 23A-F. Phospho-proteome differences between BDDD and BAAA transduced NRVMs. A. Heat map of significantly differentially phosphorylated protein residues between BAAA and BDDD transduced NRVMs identified by phospho-proteomics; p-value < 0.05 and log2-FC < -0.3, > 0.3. B-C. Significantly over-enriched Reactome Pathways in the BDDD (B) and BAAA (C) cells. D- E. Significantly over-enriched GO: Biological Processes in the BDDD (D) and BAAA (E) cells. F. GO Cell Component enrichment for the differentially expressed phospho-peptides.

[0039] Fig. 24A-D. Phospho-proteome differences between BDDD and BAAA transduced NRVMs with adrenergic agonism. A. Heat map of significantly differentially phosphorylated protein residues between BAAA and BDDD transduced NRVMs treated with PE identified by phospho- proteomics; p-value < 0.05 and log2-FC < -0.3, > 0.3. B-C. Significantly over-enriched GO: Biological Processes in the BDDD / PE (B) and BAAA / PE (C) cells. D. GO Cell Component enrichment for the differentially expressed phospho-peptides.

[0040] Fig. 25A-H. Phospho-proteome differences in BoDD-transduced NRVMs are shared in human heart failure non-responders. A-H. Quantification of four different phospho-peptides with shared expression features in transduced NRVMs and post-LVAD R vs. NR patients; NRVMs: n = 3 / group from 3 different experiments, one-way ANOVA with Tukey’s post-hoc test; post- LVAD: n = 9 / group, two-tailed t-test; amino acid position in humans used for rat peptides for simplicity. ANK3: ankyrin-3, ATP2B1 : ATPase Plasma Membrane Ca2+ Transporting 1, RPL19: Ribosomal protein L19, USP47: Ubiquitin-specific peptidase 47. Data are presented as the mean ± SEM.

[0041] Fig. 26A-D. Human engineered heart tissues (EHTs) transduced with BDDD have impaired calcium-mediated contractility compared to BAAA and empty vector-transduced EHTs. A-D. Area under the curve (AUC) (A), contraction speed (B), full-width at half max (FWHM) (C), and relaxation speed (D) for calcium transients in EHTs paced at 1Hz frequency. The data are plotted as a fold-change vs. the Empty vector-transduced / Vehicle-treated EHTs. Vehicle: n = 41 Empty vector, 43 BAAA, 41 BDDD EHTS from three biological replicates. ISO / PE: n = 14 EHTs / group from two biological replicates; two-way ANOVA with Tukey’s post-hoc test. Data are presented as the median and range.

[0042] Fig. 27A-C. CAMK2D exon 14 inclusion and S332 phosphorylation predict favorable response to LVAD using both our and previous definitions of a favorable response. A. Pre-LVAD and post-LVAD LVEF in ‘full’ responders (>40% LVEF and <6.0 cm LVEDD post-LVAD), ‘partial’ responders (patients who experienced >15% LVEF improvement and >15% reduction in LVEDD but did not reach LVEF of 40%), and non-responders. B. CAMK2D exon 14 proportion spliced in (PSI) across the three groups. C. CAMK2D-B S332 phosphorylation across the three groups. Data analysis by one-way ANOVA with Tukey’s post-hoc test for multiple independent pairwise comparisons. For B-C, data are presented as the mean ± SEM.

[0043] DETAILED DESCRIPTION OF THE INVENTION(S)

[0044] While the disclosure has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the disclosure and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. All references cited herein are incorporated in their entirety by reference. The terminology used herein is for describing particular embodiments and is not intended to be limiting. As used herein, the singular forms “a,” “and” and “the” include plural referents unless the content and context clearly dictate otherwise. Thus, for example, a reference to “a” or “the” reporter may include a combination of two or more such reporters. Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly used in the art to which they pertain.

[0045] In a preferred embodiment, the present disclosure includes methods and composition to modulate the localization of calcium / calmodulin-dependent protein kinase II delta beta (CAMK2DB) isoform protein. In a preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position 332, 333, and / or 334 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In another specific embodiment, the disclosure includes methods of treating heart disease by increasing nuclear localization of the beta isoform calcium / calmodulin dependent protein kinase II delta (CaMKIIS) via the disruption of post-transcriptional phosphorylation at position S332, S333, and / or S334 according to SEQ ID NO. 1, or a fragment thereof. Notably, in certain embodiments positions S332, S333, and / or S334 are each modified so as to disrupt phosphorylation, which in alternative preferred embodiment only one, or two of S332, S333, and / or S334 are modified so as to disrupt phosphorylation

[0046] In a preferred embodiment, the substitution mutation includes a S332A, S333A, and / or S334A mutation according to SEQ ID NO. 1 or a fragment thereof. Notably, in certain embodiments positions S332, S333, and / or S334 are each modified so as to disrupt phosphorylation, which in alternative preferred embodiment only one, or two of S332, S333, and / or S334 are modified so as to disrupt phosphorylation. In another preferred embodiment, the substitution mutation includes a S332X, S333X, and / or S334X mutation according to SEQ ID NO. 1 or a fragment thereof wherein X is a non-phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S332X, S333X, and / or S334X mutation according to SEQ ID NO. 1 or a fragment thereof wherein X is a non-phoshomimetic amino acid.

[0047] In one preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position S332 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In a preferred embodiment, the substitution mutation includes a S332A, mutation according to SEQ ID NO. 1 or a fragment thereof. In another preferred embodiment, the substitution mutation includes a S332X, mutation according to SEQ ID NO. 1 or a fragment thereof, wherein X is a non-phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S332X, mutation according to SEQ ID NO. 1 or a fragment thereof, wherein X is a non-phoshomimetic amino acid.

[0048] In one preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position S333 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In a preferred embodiment, the substitution mutation includes a S333 A, mutation according to SEQ ID NO. 1 or a fragment thereof. In another preferred embodiment, the substitution mutation includes a S333X, mutation according to SEQ ID NO. 1 or a fragment thereof, wherein X is a non-phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S333X, mutation according to SEQ ID NO. 1 or a fragment thereof, wherein X is a non-phoshomimetic amino acid.

[0049] In one preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position S334 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In a preferred embodiment, the substitution mutation includes a S334A, mutation according to SEQ ID NO. 1 or a fragment thereof. In another preferred embodiment, the substitution mutation includes a S334X, mutation according to SEQ ID NO. 1 or a fragment thereof, wherein X is a non-phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S334X, mutation according to SEQ ID NO. 1 or a fragment thereof, wherein X is a non-phoshomimetic amino acid.

[0050] As used herein, the term “fragment”: or “functional fragment” are interchangeable, and refers to a portion of a full-length protein that retains a specific biological activity or function of the original protein. Notably, the numbering of the amino acid sequence of the fragment can be retained from the wild type. So, for example in some embodiment the sequence for CAMK2DB can include a truncated functional fragment with a portion missing from the N- or C-terminal ends, or from an intervening section within the sequence, however positions 332, 333, and 334 can still be identified as conserved within the fragment by numbering the sequences as if they were present.

[0051] In a preferred embodiment, the present disclosure includes methods and composition to modulate the localization of calcium / calmodulin-dependent protein kinase II delta beta (CAMK2DB) isoform protein. In a preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position 332, 333, and / or 334 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions 332, 333, and / or 334 are conserved. In another specific embodiment, the disclosure includes methods of treating heart disease by increasing nuclear localization of the beta isoform calcium / calmodulin dependent protein kinase II delta (CaMKIIS) via the disruption of post- transcriptional phosphorylation at position S332, S333, and / or S334 according to SEQ ID NO. 1 or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions 332, 333, and / or 334 are conserved. In a preferred embodiment, the substitution mutation includes a S332A, S333A, and / or S334A mutation according to SEQ ID NO. 1 or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1 . Notably, in certain embodiments positions S332, S333, and / or S334 are each modified so as to disrupt phosphorylation, which in alternative preferred embodiment only one, or two of S332, S333, and / or S334 are modified so as to disrupt phosphorylation. In one preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position S332 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 332 is conserved.

[0052] In a preferred embodiment, the substitution mutation includes a S332A, S333A, and / or S334A mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions 332, 333, and / or 334 are conserved. Notably, in certain embodiments positions S332, S333, and / or S334 are each modified so as to disrupt phosphorylation, which in alternative preferred embodiment only one, or two of S332, S333, and / or S334 are modified so as to disrupt phosphorylation. In another preferred embodiment, the substitution mutation includes a S332X, S333X, and / or S334X mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions 332, 333, and / or 334 are conserved, and wherein X is a non-phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S332X, S333X, and / or S334X mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions 332, 333, and / or 334 are conserved, and wherein X is a non-phoshomimetic amino acid.

[0053] In one preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position S332 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 332 is conserved. In a preferred embodiment, the substitution mutation includes a S332A, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 332 is conserved. In another preferred embodiment, the substitution mutation includes a S332X, mutation according to SEQ ID NO. 1 or a fragment thereof, wherein X is a non- phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S332X, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 332 is conserved are conserved, and, wherein X is a non-phoshomimetic amino acid.

[0054] In one preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position S333 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 333 is conserved. In a preferred embodiment, the substitution mutation includes a S333A, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 333 is conserved. In another preferred embodiment, the substitution mutation includes a S333X, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 333 is conserved, and wherein X is a non-phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S333X, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position

[0055] 333 is conserved, and wherein X is a non-phoshomimetic amino acid.

[0056] In one preferred embodiment, the nuclear localization is promoted by disrupting phosphorylation at position S334 of CAMK2DB, wherein the position of the mutation corresponds to the amino acid sequence according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 334 is conserved. In a preferred embodiment, the substitution mutation includes a S334A, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 334 is conserved. In another preferred embodiment, the substitution mutation includes a S334X, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position 334 is conserved, and wherein X is a non-phosphorylatable amino acid. In another preferred embodiment, the substitution mutation includes a S334X, mutation according to SEQ ID NO. 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein position

[0057] 334 is conserved and wherein X is a non-phoshomimetic amino acid.

[0058] As noted above, in reference to a sequence that retains a certain level of sequence homology with SEQ ID NO. 1, as used herein this sequence can have variations within the sequence so as to retain its functions, while one or more of positions 332, 333, and / or 334 are conserved compared to the reference sequence according to SEQ ID NO. 1.

[0059] In another specific embodiment, the disclosure includes methods of treating heart disease in a subject in need thereof, the method comprising, obtaining a biological sample, such as preferably a cardiac cell, from the subject and determining the phosphorylation status of positions 332, 333, and 334 of CAMK2DB protein. A lack of phosphorylation at any of positions 332, 333, and 334 is indicative that the subject will benefit from LV assist device (LVAD) therapy, whereas and phosphorylation positions 332, 333, and 334 is indicative that the subject will not benefit from LV assist device (LVAD) therapy. Once this determination has been made, LVAD can be administered to the subject with a lack of phosphorylation at any of positions 332, 333, and 334.

[0060] Methods to quantify phosphorylation levels of proteins of a biological sample can include various analytical methods known by those of ordinary skill. Phosphorylation levels of proteins of a sample can be detected using MS, for example, PRM-MS. In some embodiments, the mass spectrometer is tuned to detect that the native protein has a different m / z ratio compared to the isotopically-enriched peptides of the invention. Phosphorylation levels of proteins of a test sample can also be determined using HPLC / MS / MS methods, phospho-specific antibodies, western blotting or enzyme-linked immunosorbent assays (ELISAs) that use antibodies specific to phosphorylated peptides or regions of a protein, chemiluminsecence, colorimetric detection methods, or horseradish peroxidase (HRP). Phosphorylation levels of proteins of a sample can be determined by arranging proteins in arrays and analyzing the samples using flow cytometry or by plating proteins on slides for detection, among others.

[0061] The disclosure includes methods and composition for treating heart disease in a subject in need thereof by promoting cellular localization of the beta isoform calcium / calmodulin dependent protein kinase II delta (CaMKIIS) to the nucleus, preferably a cardiac cell of a subject in need thereof to disrupt phosphorylation, via the disruption of post-transcriptional phosphorylation at one or more of position 332, 333, and / or 334, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof.

[0062] The disclosure includes methods and composition for treating heart disease in a subject in need thereof by promoting cellular localization of the beta isoform calcium / calmodulin dependent protein kinase II delta (CaMKII8) to the nucleus, preferably a cardiac cell of a subject in need thereof to disrupt phosphorylation, via the disruption of post-transcriptional phosphorylation at one or more of position 332, 333, and / or 334, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved.

[0063] In another preferred aspect, the disclosure includes methods and compositions to stably, or transiently express a recombinant CAMK2DB isoform, preferably a cardiac cell of a subject in need thereof, to disrupt phosphorylation, comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a functional fragment thereof.

[0064] In another preferred aspect, the disclosure includes methods and compositions to stably, or transiently express a recombinant CAMK2DB isoform, preferably a cardiac cell of a subject in need thereof, comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved.

[0065] In another preferred aspect, the disclosure includes methods and compositions to stably, or transiently express a recombinant CAMK2DB isoform, preferably a cardiac cell of a subject in need thereof, having one or more mutations selected from S332A, S333A, and / or S333A in a cell, and preferably a cardiac cell of a subject in need thereof to disrupt phosphorylation. In another preferred aspect, the disclosure includes expression vectors to stably, or transiently express a recombinant CAMK2DB isoform having one or more mutations selected from S332A, S333A, S333A mutation in a cell, and preferably a cardiac cell of a subject in need thereof to disrupt phosphorylation.

[0066] In another preferred aspect, the disclosure includes an isolated calcium / calmodulin- dependent protein kinase II delta beta (CAMK2DB) isoform protein comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non- phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved.

[0067] In another preferred aspect, the disclosure includes an isolated calcium / calmodulin- dependent protein kinase II delta beta (CAMK2DB) isoform protein comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non- phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof.

[0068] In another preferred aspect, the disclosure includes genetically modified cell expressing the CAMK2DB comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved. In this embodiment, the genetically modified cell is preferably modified by a CRISPR / Cas system, a transcription activator-like effector (TALE) system, a zinc-finger protein system, a synthetic polyamide system, or a meganuclease system.

[0069] In another preferred aspect, the disclosure includes an isolated nucleotide sequence encoding a calcium / calmodulin-dependent protein kinase II delta beta (CAMK2DB) isoform protein comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved.

[0070] In another preferred aspect, the disclosure includes genetically modified cell expressing the nucleotide sequence encoding a CAMK2DB protein comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved. In this embodiment, the genetically modified cell is preferably modified by a CRISPR / Cas system, a transcription activator-like effector (TALE) system, a zinc-finger protein system, a synthetic polyamide system, or a meganuclease system.

[0071] In another preferred aspect, the disclosure includes a method of treating heart disease, preferably in a human subject, includes contacting or expressing a therapeutically effective amount of the a calcium / calmodulin-dependent protein kinase II delta beta (CAMK2DB) isoform protein comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved with a cardiac cell of a subject in need thereof.

[0072] In another preferred aspect, the method of treating heart disease, preferably in a human subject, includes delivering a heterologous nucleotide sequence, operably linked to a promoter, encoding the CAMK2DB isoform protein, comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non- phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved, to the cardiac cell of a subject in need thereof, and further expressing said protein encoded by the nucleotide sequence. In a preferred aspect, the heterologous nucleotide sequence is delivered to the cell via a recombinant adeno-associated virus (rAAV) vector.

[0073] In another preferred aspect, the disclosure includes a composition comprising a gRNA that targets phosphorylation downstream of exon 14 of human calcium / calmodulin-dependent protein kinase II delta beta (CAMK2DB), and a base editor. In this preferred aspect, the gRNA disrupts phosphorylation at position 332, 333, and / or 334, wherein the position of the mutation corresponds to the CAMK2DB protein encoded by the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In a preferred aspect, the mutation comprises substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable and non- phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, and / or S334 of SEQ ID NO: 1, or a fragment thereof, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved. In another preferred aspect, the mutation comprises is selected from S332A, S333A, and S334A.

[0074] In a preferred aspect, the base editor is an adenine base editor (ABE), and more preferably a CRISPR / Cas nuclease linked to an adenosine deaminase. In another preferred embodiment, the CRISPR / Cas nuclease, and preferably the Cas nuclease is catalytically impaired, and even more preferably the Cas nuclease is a Cas9 nuclease. In alternative aspect, the composition includes a second gRNA that targets the splice sites of 14 of human calcium / calmodulin-dependent protein kinase II delta (CAMK2D) such that the gRNA causes increased production of the beta isoform of CAMK2D.

[0075] In another preferred aspect, the disclosure includes nucleic acid including: a sequence encoding a gRNA, operably linked to a promoter, which can preferably include a cardiac cellspecific promoter, and a sequence encoding a base editor, operably linked to a promoter; and wherein the gRNA disrupts phosphorylation downstream of exon 14 of CAMK2DB. In a preferred aspect, the gRNA disrupts phosphorylation at positions selected from 332, 333, and 334 of CAMK2DB, for example by substitution with a non-phosphorylatable or non-phosphomimetic amino acid, wherein the position of the mutation corresponds to the CAMK2DB protein encoded by the amino acid sequence according to SEQ ID NO. 1 , or a fragment thereof, and more preferably the mutation comprises a S332A, S333A, and S334A mutation. In a preferred aspect, the base editor is an adenine base editor (ABE), and more preferably a CRISPR / Cas nuclease linked to an adenosine deaminase. In another preferred embodiment, the CRISPR / Cas nuclease, and preferably the Cas nuclease is catalytically impaired, and even more preferably the Cas nuclease is a Cas9 nuclease. In alternative aspect, the composition includes a second gRNA that targets the splice sites of 14 of human calcium / calmodulin-dependent protein kinase II delta CAMK2D) such that the gRNA causes increased production of the beta isoform of CAMK2D.

[0076] In another preferred embodiment, the nucleic acid of the disclosures comprises a vector, such as a recombinant adeno-associated viral (aAAV) vector or an adenoviral vector which can include a viral or non-viral vector, such as a plasmid. In a preferred aspect, the AAV vector is replication-defective or conditionally replication defective. In one preferred aspect, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), or a combination thereof.

[0077] In another preferred embodiment, the vector of the disclosures can and optionally a pharmaceutically acceptable carrier, forming a pharmaceutical composition. In this aspect, the disclosure includes method for treating heart disease, the method comprising contacting a cell with a therapeutically effective amount of the composition of the disclosure under conditions suitable for expression of the first gRNA and the adenine base editor described above, wherein the gRNA forms a complex with the adenine base editor, wherein the complex modifies position 332, 333, and / or 334 of CAMK2DB, for example by substitution with a non-phosphorylatable or non- phosphomimetic amino acid, wherein the position of the mutation corresponds to the CAMK2DB protein encoded by the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In a preferred aspect, the modification results in a S332A, S333A, and / or S334A substitution to the CAMK2DB protein.

[0078] In another embodiment, the composition is administered prior to, during, and / or after LV assist device (LVAD) therapy locally. In alternative aspect, the composition is administered systemically, directly to a cardiac tissue, as an intramuscular infusion or injection, of as an intravenous infusion or injection. In a preferred aspect, following administration of the composition, the subject exhibits increased nuclear localization of CAMK2DB protein substitution with a non-phosphorylatable or non-phosphomimetic amino acid at positions 332, 333, and / or 334, and in a preferred embodiment a substitution selected from S332A, S333A, S334A.

[0079] As used herein, “calcium / calmodulin dependent protein kinase II delta,” used interchangeably with the term “CAMK2D” or “CAMKIID,” or “CaMKIId,” refers to a member of the serine / threonine protein kinase family and the Ca(2+) / calmodulin-dependent protein kinase subfamily. CAMK2D is involved in the regulation of Ca(2+) homeostatis and excitationcontraction coupling in heart by targeting ion channels, transporters and accessory proteins involved in Ca(2+) influx into the myocyte, Ca(2+) release from the sarcoplasmic reticulum (SR), SR Ca(2+) uptake and Na(+) and K(+) channel transport. CAMK2D also targets transcription factors and signaling molecules to regulate heart function. In its activated form, CAMK2D is involved in the pathogenesis of dilated cardiomyopathy and heart failure. CAMK2D contributes to cardiac decompensation and heart failure by regulating SR Ca(2+) release via direct phosphorylation of RYR2 Ca(2+) channel.

[0080] In the nucleus, CAMK2D phosphorylates the MEF2 repressor HDAC4, promoting its nuclear export and binding to 14-3-3 protein, and expression of MEF2 and genes involved in the hypertrophic program. CAMK2D is essential for left ventricular remodeling responses to myocardial infarction. In pathological myocardial remodeling, CAMK2D acts downstream of the beta-adrenergic receptor signaling cascade to regulate key proteins involved in excitationcontraction coupling. CAMK2D regulates Ca(2+) influx to myocytes by binding and phosphorylating the L-type Ca(2+) channel subunit beta-2 CACNB2. In addition to Ca(2+) channels, CAMK2D can target and regulate the cardiac sarcolemmal Na(+) channel Navi .5 / SCN5A and the K+ channel Kv4.3 / KCND3, which contribute to arrhythmogenesis in heart failure. CAMK2D phosphorylates phospholamban (PLN), an endogenous inhibitor of SERCA2A / ATP2A2, contributing to the enhancement of SR Ca(2+) uptake that may be important in frequency-dependent acceleration of relaxation and maintenance of contractile function during acidosis. CAMK2D may participate in the modulation of skeletal muscle function in response to exercise, by regulating SR Ca(2+) transport through phosphorylation of PLN and triadin, a ryanodine receptor-coupling factor.

[0081] CAMK2D is also known as Calcium / Calmodulin-Dependent Protein Kinase Type II Delta Chain, CaM Kinase II Delta Subunit, CaM Kinase II Subunit Delta, CAMKD, EC 2.7.11.17, or EC 2.7.11. An exemplary sequence of a human CAMK2D mRNA transcript can be found at, for example, GenBank Accession No. GI: 1519243899 (NM 001321571.2;). The sequence of mouse CAMK2D mRNA can be found at, for example, GenBank Accession No. GI: 654824235 (NM_001025439.2). The sequence of rat CAMK2D mRNA can be found at, for example, GenBank Accession No. GI: 144922682 (NM_012519.2). The sequence of Macaca mulatta CAMK2D mRNA can be found at, for example, GenBank Accession No. GI: 1622941163 (XM_015139100.2). Additional examples of CAMK2D mRNA sequences are readily available through publicly available databases, i.e., GenBank, UniProt, OMIM, UCSC Genome Browser, and the Macaca genome project web site. Further information on CAMK2D can be found, for example, at ncbi.nlm.nih.gov / gene / ?term= CAMK2D. The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference. The term CAMK2D, as used herein, also refers to variations of the CAMK2D gene including variants provided in the SNP database. In one preferred embodiment, CAMK2DB isoform includes the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof.

[0082] Phosphorylation levels of proteins of a test sample can also be determined using HPLC / MS / MS methods, phospho-specific antibodies, western blotting or enzyme-linked immunosorbent assays (ELISAs) that use antibodies specific to phosphorylated peptides or regions of a protein, chemiluminsecence, colorimetric detection methods, or horseradish peroxidase (HRP). Phosphorylation levels of proteins of a test sample can be determined by arranging proteins in arrays and analyzing the samples using flow cytometry or by plating proteins on slides for detection.

[0083] In some embodiments, gene expression of CAMK2D is modified to express a CAMK2DB isoform having a mutation that disrupts phosphorylation at a position selected from: 332, 333, and / or 334, for example by substitution with a non-phosphorylatable or non-phosphomimetic amino acid, wherein the position of the mutation corresponds to the CAMK2DB protein encoded by the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In a preferred aspect, the modification results in a S332A, S333A, and / or S334A substitution to the CAMK2DB protein. In one embodiment, this modified gene expression at the transcriptional level by delivering to a cell, and preferably a human cardiac cells, one or more of a CRISPR / Cas system, a transcription activator-like effector (TALE) system, a zinc-finger protein system, a synthetic polyamide system, and a meganuclease system. In some embodiments, these systems are engineered to specifically target a CAMK2D.

[0084] In some embodiments, the agent for altering gene expression of CAMK2DB isoform having a mutation that disrupts phosphorylation at position 332, 333, and / or 334 as described herein is a zinc finger, or zinc finger nuclease or other equivalent. The term “zinc finger nuclease” or “zinc finger nuclease as used herein, refers to a nuclease comprising a nucleic acid cleavage domain conjugated to a binding domain that comprises a zinc finger array. In some embodiments, the cleavage domain is the cleavage domain of the type II restriction endonuclease Fokl. Zinc finger nucleases can be designed to target virtually any desired sequence in a given nucleic acid molecule for cleavage, and the possibility to design zinc finger binding domains to bind unique sites in the context of complex genomes allows for targeted cleavage of a single genomic site in living cells, for example, to achieve a targeted genomic alteration of therapeutic value. Targeting a double- strand break to a desired genomic locus can be used to introduce frame-shift mutations into the coding sequence of a gene due to the error-prone nature of the non-homologous DNA repair pathway.

[0085] Zinc finger nucleases can be generated to target a site of interest by methods well known to those of skill in the art. For example, zinc finger binding domains with a desired specificity can be designed by combining individual zinc finger motifs of known specificity. The structure of the zinc finger protein Zif268 bound to DNA has informed much of the work in this field and the concept of obtaining zinc fingers for each of the 64 possible base pair triplets and then mixing and matching these modular zinc fingers to design proteins with any desired sequence specificity has been described (Pavletich N P, Pabo Colo. (May 1991). “Zinc fmger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A”. Science 252 (5007): 809-17, the entire contents of which are incorporated herein).

[0086] In some embodiments, separate zinc fingers that each recognizes a 3 base pair DNA sequence are combined to generate 3-, 4-, 5-, or 6-finger arrays that recognize target sites ranging from 9 base pairs to 18 base pairs in length. In some embodiments, longer arrays are contemplated. In other embodiments, 2-finger modules recognizing 6-8 nucleotides are combined to generate 4- , 6-, or 8-zinc finger arrays. In some embodiments, bacterial or phage display is employed to develop a zinc finger domain that recognizes a desired nucleic acid sequence, for example, a desired nuclease target site of 3-30 bp in length.

[0087] Zinc finger nucleases, in some embodiments, comprise a zinc finger binding domain and a cleavage domain fused or otherwise conjugated to each other via a linker, for example, a polypeptide linker. The length of the linker determines the distance of the cut from the nucleic acid sequence bound by the zinc finger domain. If a shorter linker is used, the cleavage domain will cut the nucleic acid closer to the bound nucleic acid sequence, while a longer linker will result in a greater distance between the cut and the bound nucleic acid sequence. In some embodiments, the cleavage domain of a zinc finger nuclease has to dimerize in order to cut a bound nucleic acid. In some such embodiments, the dimer is a heterodimer of two monomers, each of which comprise a different zinc finger binding domain. For example, in some embodiments, the dimer may comprise one monomer comprising zinc finger domain A conjugated to a FokI cleavage domain, and one monomer comprising zinc finger domain B conjugated to a FokI cleavage domain. In this nonlimiting example, zinc finger domain A binds a nucleic acid sequence on one side of the target site, zinc finger domain B binds a nucleic acid sequence on the other side of the target site, and the dimerize FokI domain cuts the nucleic acid in between the zinc finger domain binding sites.

[0088] The term “zinc finger,” as used herein, refers to a small nucleic acid-binding protein structural motif characterized by a fold and the coordination of one or more zinc ions that stabilize the fold. Zinc fingers encompass a wide variety of differing protein structures (see, e.g., Klug A, Rhodes D (1987). “Zinc fingers: a novel protein fold for nucleic acid recognition”. Cold Spring Harb. Symp. Quant. Biol. 52: 473-82, the entire contents of which are incorporated herein by reference). Zinc fingers can be designed to bind a specific sequence of nucleotides, and zinc finger arrays comprising fusions of a series of zinc fingers, can be designed to bind virtually any desired target sequence. Such zinc finger arrays can form a binding domain of a protein, for example, of a nuclease, e.g., if conjugated to a nucleic acid cleavage domain. Different types of zinc finger motifs are known to those of skill in the art, including, but not limited to, Cys2His2, Gag knuckle, Treble clef, Zinc ribbon, Zn2 / Cys6, and TAZ2 domain-like motifs (see, e.g., Krishna S S, Majumdar I, Grishin N V (January 2003). “Structural classification of zinc fingers: survey and summary”. Nucleic Acids Res. 31 (2): 532-50). Typically, a single zinc finger motif binds 3 or 4 nucleotides of a nucleic acid molecule. Accordingly, a zinc finger domain comprising 2 zinc finger motifs may bind 6-8 nucleotides, a zinc finger domain comprising 3 zinc finger motifs may bind 9-12 nucleotides, a zinc finger domain comprising 4 zinc finger motifs may bind 12-16 nucleotides, and so forth. Any suitable protein engineering technique can be employed to alter the DNA-binding specificity of zinc fingers and / or design novel zinc finger fusions to bind virtually any desired target sequence from 3-30 nucleotides in length (see, e.g., Pabo C O, Peisach E, Grant R A (2001). “Design and selection of novel cys2H is2 Zinc finger proteins”. Annual Review of Biochemistry 70: 313-340; Jamieson A C, Miller J C, Pabo C O (2003). “Drug discovery with engineered zinc- finger proteins”. Nature Reviews Drug Discovery 2 (5): 361-368; and Liu Q, Segal D J, Ghiara J B, Barbas C F (May 1997). “Design of polydactyl zinc-finger proteins for unique addressing within complex genomes”. Proc. Natl. Acad. Sci. U.S.A. 94 (11); the entire contents of each of which are incorporated herein by reference).

[0089] Fusions between engineered zinc finger arrays and protein domains that cleave a nucleic acid can be used to generate a “zinc finger nuclease.” A zinc finger nuclease typically comprises a zinc finger domain that binds a specific target site within a nucleic acid molecule, and a nucleic acid cleavage domain that cuts the nucleic acid molecule within or in proximity to the target site bound by the binding domain. Typical engineered zinc finger nucleases comprise a binding domain having between 3 and 6 individual zinc finger motifs and binding target sites ranging from 9 base pairs to 18 base pairs in length. Longer target sites are particularly attractive in situations where it is desired to bind and cleave a target site that is unique in a given genome.

[0090] In some embodiments, the agent for altering the target gene is a TALE system or its equivalent. The term TALE or “Transcriptional Activator-Like Element Nuclease” or “TALE nuclease” as used herein, refers to an artificial nuclease comprising a transcriptional activator like effector DNA binding domain to a DNA cleavage domain, for example, a FokI domain. A number of modular assembly schemes for generating engineered TALE constructs have been reported (Zhang, Feng; et. al. (February 2011). “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription”. Nature Biotechnology 29 (2): 149-53; Geibler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011), Shiu, Shin-Han. ed. “Transcriptional Activators of Human Genes with Programmable DNA-Specificity”. PLoS ONE 6 (5): el9509; Cermak, T.; Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller, J. A.; Somia, N. V. et al. (2011). “Efficient design and assembly of custom TALE and other TAL effector-based constructs for DNA targeting”. Nucleic Acids Research; Morbitzer, R.; Elsaesser, J.; Hausner, J.; Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains by modular cloning”. Nucleic Acids Research; Li, T.; Huang, S.; Zhao, X.; Wright, D. A.; Carpenter, S.; Spalding, M. H.; Weeks, D. P.; Yang, B. (2011). “Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes”. Nucleic Acids Research.; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S. (2011). Bendahmane, Mohammed, ed. “Assembly of Designer TAL Effectors by Golden Gate Cloning”. PLoS ONE 6 (5): el9722; each of which is incorporated herein by reference).

[0091] Those of skill in the art will understand that TALE nucleases can be engineered to target virtually any genomic sequence with high specificity, and that such engineered nucleases can be used in embodiments of the present technology to manipulate the genome of a cell, e.g., by delivering the respective TALE via a method or strategy disclosed herein under circumstances suitable for the TALE to bind and cleave its target sequence within the genome of the cell. In some embodiments, the delivered TALE targets a gene or allele associated CAMK2DB isoform having a mutation that disrupts phosphorylation at positions 332, 333, and / or 334 with respect to SEQ ID NO. 1. In some embodiments, delivery of the TALE to a subject confers a therapeutic benefit to the subject, such as reducing, ameliorating or eliminating heart disease in a patient.

[0092] In some embodiments, the agent for altering gene expression of CAMK2DB isoform having a mutation that disrupts phosphorylation at positions 332, 333, and / or 334 with respect to SEQ ID NO. 1 is a CRISPR system. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (i.e. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus.

[0093] The CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (i.e., Cas9), with nuclease functionality (i.e., two nuclease domains). One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, i.e., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.

[0094] The CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions as discussed herein. In other embodiments, Cas9 variants, deemed “nickases,” are used to nick a single strand at the target site. Paired nickases can be used to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a base editing enzyme or a reverse transcriptase.

[0095] Base editors allow efficient installation of single base substitutions in DNA. For example, adenosine deaminases induce adenosine (A) to inosine (I) edits in single-stranded DNA that in turn result in A-to-G transitions after DNA repair or replication. Adenine base editors (ABEs) are fusions of programmable DNA-binding domains (i.e., catalytically impaired RNA-guided CRISPR / Cas nucleases) linked to an engineered adenosine deaminase. In instances where the programmable DNA-binding domain is a CRISPR / Cas nuclease, targeted adenines lie within an “editing window” in the single-stranded (ss) DNA bubble (R-loop) induced by the CRISPR-Cas RNA-protein complex. The most commonly used ABEs comprise an adenosine deaminase heterodimer consisting of E. coli TadA (wild type) fused to an engineered E. coli TadA variant (i.e., ABEmax) or a single engineered E. coli ^aAA variant (i.e., ABE8e, ABE8eV106W, or ABE8.20-m) as well as a nickase Cas9 and nuclear localization sequences (NLS). ABEs have been used successfully for installation of A-to-G substitutions in multiple cell types and organisms and could potentially reverse a large number of mutations known to be associated with human disease. Examples of ABEs include those described in U.S. Pub. No. US20200308571, PCT Pub. No. WO2020214842, and W02021025750, which are each incorporated herein by reference in their entirety.

[0096] Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a CRISPR system working in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the CRISPR system), wherein the prime editing system is programmed with a prime editing (pe) guide RNA (“pegRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (i.e., at the 5' or 3' end, or at an internal portion of a guide RNA). As such, prime editors allow for prime editing on a target nucleotide sequence in the presence of a pegRNA (or “extended guide RNA”). The term “prime editor” refers to fusion constructs comprising a Cas9 nickase and a reverse transcriptase. The term “prime editor” may refer to the fusion protein or to the fusion protein complexed with a pegRNA, and / or further complexed with a second strand nicking sgRNA. In some embodiments, the prime editor may also refer to the complex comprising a fusion protein (reverse transcriptase fused to a Cas9), a pegRNA, and a regular guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein. In other embodiments, the reverse transcriptase component of the “prime editor” may be provided in trans. Further examples of prime editors and their use are provided in PCT Pub. No. WO2020191249, which is incorporated by reference herein in its entirety.

[0097] In one aspect, a Cas nuclease and sgRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5' end of the gRNA target the Cas nuclease to the target site, i.e., the gene, using complementary base pairing. Target sites may be 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, NG, NAG, NNNRRT, or NNGG. In general, a CRTSPR system is characterized by elements that promote the formation of a CRTSPR complex at the site of a target sequence. Typically, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0098] The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence.” In one aspect, an exogenous template polynucleotide may be referred to as an editing template. In one aspect, the recombination is homologous recombination.

[0099] Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (i.e. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (i.e. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. The tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0100] One or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr- mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. The gRNA may be under the control of a constitutive promoter. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.

[0101] A vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.

[0102] The CRISPR enzyme can be Cas9 (i.e., from S. pyogenes or S. pneumonia or S. aureus or S. auricularis or X lugdunensis). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), i.e., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0103] In some embodiments, a Cas9 polypeptide can be a deactivated (i.e., mutated, dCAs9) Cas9 polypeptide, wherein the deactivated Cas9 does not comprise HNH and / or RuvC nickase activities. The HNH and RuvC motifs have been characterized in S. thermophilus (see, i.e., Sapranauskas et al. Nucleic Acids Res. 39:9275-9282 (2011)) and one of skill would be able to identify and mutate these motifs in Cas9 polypeptides from other organisms. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9. Notably, a Cas9 polypeptide in which the HNH motif and / or RuvC motif is / are specifically mutated so that the nickase activity is reduced, deactivated, and / or absent, can retain one or more of the other known Cas9 functions including DNA, RNA and PAM recognition and binding activities and thus remain functional with regard to these activities, while non-functional with regard to one or both nickase activities.

[0104] In an alternative embodiment, the CRISPR enzyme is a Cas protein, preferably Cas9 (having a nucleotide sequence of Genbank accession no NC_002737.2 and a protein sequence of Genbank accession no NP_269215.1). Again, the Cas9 protein may also be modified to improve activity. For example, the Cas9 protein may comprise the D10A amino acid substitution, this nickase cleaves only the DNA strand that is complementary to and recognized by the crRNA. In an alternative embodiment, the Cas9 protein may alternatively or additionally comprise the H840A amino acid substitution, this nickase cleaves only the DNA strand that does not interact with the sRNA. In this embodiment, Cas9 may be used with a pair (i.e. two) sgRNA molecules (or a construct expressing such a pair) and as a result can cleave the target region on the opposite DNA strand, with the possibility of improving specificity by 100-1500-fold. In a further embodiment, the Cas9 protein may comprise a D1135E substitution. The Cas 9 protein may also be the VQR variant. Alternatively, the Cas9 protein may be xCas9 (a Streptococcus pyogenes variant that can recognize a broad range of PAM sequences including NG, GAA and GAT). In other alternatives, the Cas9 variant is SpCas9-NG (with a relaxed preference to the third nucleotide of the PAM motif, such that the variant can recognize sequences where the PAM motif is NGN rather than NGG), SaCas9 (from S. aureus that can recognize NNGRR(T) PAM sequences; see Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191, doi : 10.1038 / nature 14299 (2015)), SaCas9-KKH (a variant from S. aureus that can recognize NNNRRT PAM sequences), SauCas9 (from S. auricularis that can recognize NNGG PAM sequences; Genbank accession no WP_107392933.1), or SlugCas9 (from S. lugdunensis M23590 that can recognize NNGG PAM sequences; Genbank accession no WP_002460848.1).

[0105] In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. Tn general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0106] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

[0107] Each of the guide sequences of the disclosure may further comprise additional nucleotides to form or encode a crRNA, i.e., using any known sequence appropriate for the Cas9 being used. In some embodiments, the crRNA comprises (5' to 3') at least a spacer sequence and a first complementarity domain. The first complementary domain is sufficiently complementary to a second complementarity domain, which may be part of the same molecule in the case of an sgRNA or in a tracrRNA in the case of a dual or modular gRNA, to form a duplex. See, i.e., US 2017 / 0007679 for detailed discussion of crRNA and gRNA domains, including first and second complementarity domains.

[0108] A single-molecule guide RNA (sgRNA) can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a singlemolecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and / or an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (i.e., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. In particular embodiments, the disclosure provides for an sgRNA comprising a spacer sequence and a tracrRNA sequence.

[0109] The guide RNA can be considered to comprise a scaffold sequence necessary for endonuclease binding and a spacer sequence required to bind to the genomic target sequence. An exemplary scaffold sequence suitable for use with SaCas9 to follow the guide sequence at its 3' end is:

[0110] GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAATGCC

[0111] GTGTTTATCTCGTCAACTTGTTGGCGAGA (SEQ ID NO. 133) in 5' to 3' orientation.

[0112] In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3' end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 133, or a sequence that differs from SEQ ID NO: 133 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0113] Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (i.e. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0114] The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid binding activity, base editing activity, or reverse transcription activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.

[0115] As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence (i.e., NGG or NG or NNNRRT or NNGG) it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because eukaryotic systems lack some of the proteins required to process CRISPR RNAs, the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type III promoter U6. Other promoters under the control of RNA Pol III include those for ribosomal 5S rRNA, tRNA and few other small RNAs, RNase P and RNase MRP RNA, 7SL RNA (the RNA component of the signal recognition particles), Vault RNAs, Y RNA, SINEs (short interspersed repetitive elements), 7SK RNA, two microRNAs, several small nucleolar RNAs and several few regulatory antisense RNAs. Synthetic gRNAs are slightly over 100 bp at the minimum length and contain a portion which is targets the 20 or 21 protospacer nucleotides immediately preceding the PAM sequence. The length of the sgRNA can also be shortened at the 5' with respect to its canonical length to meet specific criteria, i.e. the removal of a stretch of thymines that can inhibit the polymerase type III transcription activity. gRNAs do not contain the PAM sequence.

[0116] In some embodiments, the gRNA targets a site within a wildtype CAMK2D gene. In some embodiments, the gRNA targets a CAMK2D intron. In some embodiments, the gRNA targets a CAMK2D exon. In some embodiments, the gRNA targets a site in a CAMK2D exon that is expressed and is present in one or more CAMK2D isoforms, and preferably the CAMK2D« isoform. In embodiments, the gRNA targets a CAMK2D splice site, and preferably the splice sites implicating exon 14 that generate the CAMK2DB isoform. In some embodiments, the gRNA targets a splice donor site on the CAMK2D gene. In embodiments, the gRNA targets a splice acceptor site on the CAMK2D gene.

[0117] In some embodiments, gRNAs of the disclosure comprise a sequence that is complementary to a target sequence within a coding sequence or a non-coding sequence corresponding to the CAMK2D gene, and, therefore, hybridize to the target sequence. In a preferred embodiment, the target sequence includes all or part of the sequence of exon 14 of the CAMK2D gene. In some embodiments, a nucleic acid may comprise one or more sequences encoding a gRNA. In some embodiments, a nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 sequences encoding a gRNA. In some embodiments, all of the sequences encode the same gRNA. In some embodiments, all of the sequences encode different gRNAs. In some embodiments, at least 2 of the sequences encode the same gRNA, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or 21 of the sequences encode the same gRNA.

[0118] In a preferred embodiment, the gRNA is configured to modify expression in the cell to disrupts phosphorylation downstream of the nuclear localization sequence encoded by exon 14 of CAMK2DB. In a specific preferred embodiment, the gRNA is configured to modify expression in the cell to produce a (CAMK2D«) isoform having a mutation that disrupts phosphorylation at positions 332, 333, and / or 334 with respect to SEQ ID NO. 1 as described herein, wherein the position of the mutation corresponds to the CAMK2DB protein encoded by the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In a preferred embodiment, the gRNA is configured to modify expression in the cell, and specifically the gRNA targets the splice sites of exon 14 of human calcium / calmodulin-dependent protein kinase II delta (CAMK2D) such that the gRNA causes increased production of the beta isoform of CAMK2D.

[0119] In some embodiments, nucleotide gene editing may be performed in vitro or ex vivo. In some embodiments, cells are contacted in vitro or ex vivo with a nucleotide editing Cas9 and a gRNA that targets CAMK2D. In some embodiments, the cells are contacted with one or more nucleic acids encoding the Cas9 and the guide RNA. In some embodiments, the one or more nucleic acids are introduced into the cells using, for example, lipofection or electroporation. Nucleotide gene editing may also be performed in zygotes. In embodiments, zygotes may be injected with one or more nucleic acids encoding Cas9 and a gRNA that targets CAMK2D. The zygotes may subsequently be injected into a host. In some embodiments, the Cas9 is provided on a vector. In embodiments, the vector contains a Cas9 derived from S. pyogenes (SpCas9). In some embodiments, the vector contains a Cas9 derived from S. aureus (SaCas9). In some embodiments, the vector contains a Cas9 derived from S. auricularis (SauCas9). In some embodiments, the vector contains a Cas9 derived from S. lugdunensis (SlugCas9). In some embodiments, the Cas9 sequence is codon optimized for expression in human cells, and preferably human cardiac cells. In some embodiments, the vector further contains a sequence encoding a fluorescent protein, such as GFP, which allows Cas 9- expressing cells to be sorted using fluorescence activated cell sorting (FACS). In some embodiments, the vector is a viral vector such as an adeno-associated viral vector as described below.

[0120] In some embodiments, the gRNA is provided on a vector. In some embodiments, the vector is a viral vector such as an adeno-associated viral vector. In embodiments, the Cas9 and the guide RNA are provided on the same vector. In embodiments, the Cas9 and the guide RNA are provided on different vectors.

[0121] Any type of vector, such as any of those described herein, may be used. In some embodiments, the vector is a lipid nanoparticle. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a non-integrating viral vector (i.e., that does not insert sequence from the vector into a host chromosome). In some embodiments, the viral vector is an adeno-associated virus vector (AAV), a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the vector comprises a cardiac-specific promoter. In any of the foregoing embodiments, the vector may be an adeno-associated virus vector (AAV).

[0122] Where a vector is used, it may be a viral vector, such as a non-integrating viral vector. In some embodiments, the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the viral vector is an adeno- associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO (see, i.e., SEQ ID NO: 81 of U.S. Pat. No. 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, i.e., SEQ ID NO: 1 of U.S. Patent Publication No. 2015 / 0111955, which is incorporated by reference herein in its entirety), AAV9 vector, AAV9P vector (also known as AAVMYO, see, Weinmann et al., 2020, Nature Communications, 11 :5432), Myo-AAV vectors described in Tabebordbar et al.,

[0123] 2021, Cell, 184: 1-20 (i.e., MyoAAV 1A, 2A, 3A, 4A, 4C, or 4E), and AAV9-rh74-HB-Pl, AAV9- AAA-P1-SG vectors described in W02022053630. wherein the number following AAV indicates the AAV serotype. In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, i.e., McCarty et al., Gene Ther. 2001; 8: 1248-54, Naso et al., BioDrugs 2017; 31 :317-334, and references cited therein for detailed discussion of various AAV vectors. In some embodiments, the vector is an AAV9 vector.

[0124] Efficiency of in vitro or ex vivo nucleotide editing Cas9 may be assessed using techniques known to those of skill in the art, such as the T7 El assay or sequencing. Restoration of DMD expression may be confirmed using techniques known to those of skill in the art, such as RT-PCR, Western blotting, and immunocytochemistry.

[0125] In some embodiments, contacting the cell with the nucleotide editing Cas9 and the gRNA disrupts phosphorylation at positions 332, 333, and / or 334 of the CAMK2DB isoform, wherein the position of the mutation corresponds to the CAMK2DB protein encoded by the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof. In embodiments, cells which have been edited in vitro or ex vivo, or cells derived therefrom, show levels of modified CAMK2DB protein having a mutation at S332, S333, and / or S334 that disrupts phosphorylation at this position, and preferably an S332A mutation that is comparable to wildtype cells. In embodiments, the edited cells, or cells derived therefrom, express modified CAMK2DB protein having a S332, S333, and / or S334 mutation, and preferably an S332, S333A, and / or S334A mutation that at a level that is 20%, 30%, 40%, 50%, 60%, 70%o, 80%, 90%o, 95% or any percentage in between of wildtype CAMK2DB expression levels.

[0126] Provided herein are expression vectors which contain one or more nucleic acids encoding nucleotide editing Cas9 and at least one CAMK2DB guide RNA targeting exon 14, and in particular position S332, S333, and / or S334 of exon 14. In some embodiments, a nucleic acid encoding nucleotide editing Cas9 and a nucleic acid encoding at least one guide RNA are provided on the same vector. In further embodiments, a nucleic acid encoding nucleotide editing Cas9 and a nucleic acid encoding least one guide RNA are provided on separate vectors. Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

[0127] There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals.

[0128] One method for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express editing polynucleotide(s) that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

[0129] The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans. Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5 '-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

[0130] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El- deleted virus is incomplete. Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, i.e., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

[0131] The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.

[0132] The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome.

[0133] In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replicationdefective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells.

[0134] There are certain limitations to the use of retrovirus vectors in all aspects of the present disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes. Another concern with the use of defective retrovirus vectors is the potential appearance of wildtype replication-competent virus in the packaging cells. This can result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination.

[0135] Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

[0136] In embodiments, particular embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease. The virus causes a very mild immune response, lending further support to its apparent lack of pathogenicity. In many cases, AAV vectors integrate into the host cell genome, which can be important for certain applications, but can also have unwanted consequences. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur. These features make AAV an attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20 nm) replication-defective, nonenveloped virus.

[0137] Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. This feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNA of the vector. The desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (ITR) that aid in concatemer formation in the nucleus after the singlestranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at extremely low frequency. AAVs also present extremely low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human gene therapy.

[0138] Because of AAVs specialized gene therapy advantages, researchers have created an altered version of AAV termed self-complementary adeno-associated virus (scAAV). Whereas AAV packages a single strand of DNA and must wait for its second strand to be synthesized, scAAV packages two shorter strands that are complementary to each other. By avoiding second-strand synthesis, scAAV can express more quickly, although as a caveat, scAAV can only encode half of the already limited capacity of AAV. Recent reports suggest that scAAV vectors are more immunogenic than single stranded adenovirus vectors, inducing a stronger activation of cytotoxic T lymphocytes. The humoral immunity instigated by infection with the wild type is thought to be a common event. The associated neutralizing activity limits the usefulness of the most commonly used serotype AAV2 in certain applications. Accordingly, the majority of clinical trials currently under way involve delivery of AAV2 into the brain, a relatively immunologically privileged organ. In the brain, AAV2 is strongly neuron specific.

[0139] The AAV genome is built of single- stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

[0140] The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. The feature of these sequences that gives them this property is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles.

[0141] With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.

[0142] On the “left side” of the genome there are two promoters called p5 and pl 9, from which two overlapping messenger ribonucleic acids (mRNAs) of different length can be produced. Each of these contains an intron which can be either spliced out or not. Given these possibilities, four various mRNAs, and consequently four various Rep proteins with overlapping sequence can be synthesized. Their names depict their sizes in kilodaltons (kDa): Rep78, Rep68, Rep52 and Rep40. Rep78 and 68 can specifically bind the hairpin formed by the ITR in the self-priming act and cleave at a specific region, designated terminal resolution site, within the hairpin. They were also shown to be necessary for the AAVS1 -specific integration of the AAV genome. All four Rep proteins were shown to bind ATP and to possess helicase activity. It was also shown that they upregulate the transcription from the p40 promoter (mentioned below) but downregulate both p5 and pl9 promoters.

[0143] The right side of a positive-sensed AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2 and VP3, which start from one promoter, designated p40. The molecular weights of these proteins are 87, 72 and 62 kiloDaltons, respectively. The AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of 1 : 1 : 10, with an estimated size of 3.9 MegaDaltons.

[0144] The cap gene produces an additional, non-structural protein called the Assembly- Activating Protein (AAP). This protein is produced from ORF2 and is essential for the capsid- assembly process. The exact function of this protein in the assembly process and its structure have not been solved to date.

[0145] All three VPs are translated from one mRNA. After this mRNA is synthesized, it can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents the so-called “major splice”. In this form the first AUG codon, from which the synthesis of VP1 protein starts, is cut out, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon that remains in the major splice is the initiation codon for VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak context. This contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as is VP1.

[0146] Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation initiation signal, the ratio at which the AAV structural proteins are synthesized in vivo is about 1: 1 :20, which is the same as in the mature virus particle. The unique fragment at the N terminus of VP1 protein was shown to possess the phospholipase A2 (PLA2) activity, which is probably required for the releasing of AAV particles from late endosomes. Muralidhar et al. reported that VP2 and VP3 are crucial for correct virion assembly. More recently, however, Warrington et al. showed VP2 to be unnecessary for the complete virus particle formation and an efficient infectivity, and also presented that VP2 can tolerate large insertions in its N terminus, while VP1 cannot, probably because of the PLA2 domain presence.

[0147] The AAV vector may be replication-defective or conditionally replication defective. In embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof.

[0148] In some embodiments, a single viral vector is used to deliver a nucleic acid encoding a nucleotide editing Cas9 and at least one gRNA to a cell. In some embodiments, nucleotide editing Cas9 is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector. In some embodiment, the nucleotide editing Cas9 may use a split- intein dual AAV system which reconstitutes the full-length nucleotide editor by protein trans- splicing. In these systems, the Cas9 protein or the base editor is split into two sections, each fused with one part of an intein system (i.e., intein-N and intein-C encoded by dnaEn and dnaEc, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. See, U.S. Pat. Pub. No. US20180127780, which is incorporated by reference herein in its entirety.

[0149] Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.

[0150] Once the expression construct targeting positions S332, S333, and / or S334 of exon 14 of CAMK2D-B (SEQ ID NO. 1) has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement), or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

[0151] In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. DNA encoding a construct of interest, which preferably includes a CAMK2DB isoform protein according to SEQ ID NO. 1, or a fragment thereof, may also be transferred in a similar manner in vivo and express the gene product, wherein CAMK2DB isoform has been modified to disrupt phosphorylation at positions S332, S333, and / or S334 according to SEQ ID NO. 1. In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. In some embodiments, the expression construct is delivered directly to the cardiac tissue of a subject. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.

[0152] In a further embodiment, the expression construct, which preferably includes a CAMK2DB isoform protein according to SEQ ID NO. 1, comprising substitutions at one or more of positions 333, 334, and 335 that disrupt phosphorylation as described above, may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been successful. A reagent known as Lipofectamine 2000™ is widely used and commercially available. In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase. Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific.

[0153] In some embodiments, a Cas9 base editor or prime editor may be packaged into an AAV vector. In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. Exemplary AAV-Cas9 vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the Cas9 sequence. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and / or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,

[0154] 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or

[0155] 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104,

[0156] 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

[0157] 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,

[0158] 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110±10 base pairs. In some embodiments, the ITRs have a length of 120±10 base pairs. In some embodiments, the ITRs have a length of 130±10 base pairs. In some embodiments, the ITRs have a length of 140±10 base pairs. In some embodiments, the ITRs have a length of 150±10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, the AAV-Cas9 vector may contain one or more nuclear localization signals (NLS). In some embodiments, the AAV-Cas9 vector contains 1, 2, 3, 4, or 5 nuclear localization signals. Exemplary NLS include the c-myc NLS, the SV40 NLS, the hnRNPAI M9 NLS, the nucleoplasmin NLS, the sequence

[0159] RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO. 134) of the IBB domain from importin-alpha, the sequences VSRKRPRP (SEQ ID NO. 135) and PPKKARED (SEQ ID NO. 136) of the myoma T protein, the sequence PQPKKKPL (SEQ ID NO. 137) of human p53, the sequence SALIKKKKKMAP (SEQ ID NO. 138) of mouse c-abl IV, the sequences DRLRR (SEQ ID NO. 139) and PKQKKRK (SEQ ID NO. 140) of the influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO. 141) of the Hepatitis virus delta antigen and the sequence REKKKFLKRR (SEQ ID NO. 142) of the mouse Mxl protein. Further acceptable nuclear localization signals include bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO. 143) of the human poly(ADP -ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO. 144) of the steroid hormone receptors (human) glucocorticoid.

[0160] In some embodiments, the AAV-Cas9 vector may comprise additional elements to facilitate packaging of the vector and expression of the Cas9 constructs. In some embodiments, the AAV-Cas9 vector may comprise a polyA sequence. In some embodiments, the polyA sequence may be a mini-polyA sequence. In some embodiments, the AAV-CAs9 vector may comprise a transposable element. In some embodiments, the AAV-Cas9 vector may comprise a regulator element. In some embodiments, the regulator element is an activator or a repressor.

[0161] In some embodiments, the AAV-Cas9 may contain one or more promoters. In some embodiments, the one or more promoters drive expression of the Cas9. In some embodiments, the one or more promoters are cardiac-specific promoters.

[0162] In some embodiments, the AAV-Cas9 vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a bacculovirus expression system.

[0163] In some embodiments of the gene editing constructs of the disclosure, the construct comprises or consists of a promoter and a nuclease. In some embodiments, the construct comprises or consists of an CK8e promoter and a Cas9 nuclease. In some embodiments, the construct comprises or consists of an CK8e promoter and a Cas9 nuclease isolated or derived from Staphylococcus pyogenes (“SpCas9”). In some embodiments, the SpCas9 nuclease comprises or consists of a nucleotide sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCG ACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAA GAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAG AGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCA GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGA AGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCAC GAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACC TGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAAC CAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCA GACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGG CCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGC TGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCAT CCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATG ATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGC TGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGA CGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGC ACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACA ACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGA TTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCC TACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGG AAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCAT CGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTG CTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGA GAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAA CCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCC GTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGA AAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGT GCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCAC CTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGA GCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAA GTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAA GAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATC TGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGT GAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACC ACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGC TGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTA CCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTG TCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACA AGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGT GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTC GACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGA GACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAA CACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAG CTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACC ACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCT GGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGC GAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCA AGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGA AACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATG CCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCC TGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAG TCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCG AGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCAT CAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCC GGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGG CCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGA ACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATC CTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCA GAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTT CAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCC ACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAG GCGAC (SEQ ID NO. 145)

[0164] In some embodiments, the construct comprising a promoter, and a nuclease further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a promoter, and a nuclease further comprises at least two ITR sequences from isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a promoter, and a nuclease further comprises at least two ITR sequences each comprising or consisting of a nucleotide sequence of GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCC GACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA (SEQ ID NO. 146)

[0165] In some embodiments, the construct comprising a promoter, and a nuclease further comprises at least two ITR sequences, wherein the first ITR sequence comprises or consists of a nucleotide sequence of

[0166] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGT CGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGC CAACTCCATCACTAGGGGTTCCT (SEQ ID NO. 147) and the second ITR sequence comprises or consist of a nucleotide sequence of

[0167] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGA GCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO. 148)

[0168] In some embodiments, the construct comprises or consists of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR. In some embodiments, the construct comprises or consists of, from 5' to 3' a first AAV2 ITR, a sequence encoding an CK8e promoter, a sequence encoding a SpCas9 nuclease and a second AAV2 ITR. In some embodiments, the construct comprising or consisting of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR, further comprises a poly A sequence. In some embodiments, the polyA sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of

[0169] TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGGCGG

[0170] (SEQ ID NO. 149)

[0171] In some embodiments, the construct comprises or consists of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a minipoly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5' to 3' a first AAV2 ITR, a sequence encoding an CK8e promoter, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least one nuclear localization signal. In some embodiments, the construct comprising, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least two nuclear localization signals. Exemplary nuclear localization signals of the disclosure comprise or consist of a nucleotide sequence of

[0172] AAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAA (SEQ ID NO. 150) or a nucleotide sequence of ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO. 151)

[0173] In some embodiments, the construct comprises or consists of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR. In some embodiments, the construct comprising, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR, further comprises a stop codon. The stop codon may have a sequence of TAG, TAA, or TGA. In some embodiments, the construct comprises or consists of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR. In some embodiments, the construct comprising or consisting of, from 5' to 3' a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR, further comprises transposable element inverted repeats. Exemplary transposable element inverted repeats of the disclosure comprise or consist of a nucleotide sequence of

[0174] TGTGGGCGGACAAAATAGTTGGGAACTGGGAGGGGTGGAAATGGAGTTTTTAAGGATTA TTTAGGGAAGAGTGACAAAATAGATGGGAACTGGGTGTAGCGTCGTAAGCTAATACGAA AATTAAAAATGACAAAATAGTTTGGAACTAGATTTCACTTATCTGGTT (SEQ ID NO.

[0175] 152) and / or a nucleotide sequence of

[0176] GAATATAGTCTTTACCATGCCCTTGGCCACGCCCCTCTTTAATACGACGGGCAATTTGC ACTTCAGAAAATGAAGAGTTTGCTTTAGCCATAACAAAAGTCCAGTATGCTTTTTCACA GCATAACTGGACTGATTTCAGTTTACAACTATTCTGTCTAGTTTAAGACTTTATTGTCA TAGTTTAGATCTATTTTGTTCAGTTTAAGACTTTATTGTCCGCCCACA (SEQ ID NO.

[0177] 153)

[0178] In some embodiments, the construct comprises or consists of, from 5' to 3' a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprising or consisting of, from 5' to 3', a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat, further comprises a regulatory sequence. Exemplary regulatory sequences of the disclosure comprise or consist of a nucleotide sequence of

[0179] CATGCAAGCTGTAGCCAACCACTAGAACTATAGCTAGAGTCCTGGGCGA*ACAAACGATG CTCGCCTTCCAGAAAACCGAGGATGCGAACCACTTCATCCGGGGTCAGCACCACCGGCA AGCGCCGCGACGGCCGAGGTCTTCCGATCTCCTGAAGCCAGGGCAGATCCGTGCACAGC ACCTTGCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGAAAC CTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGCTGCCCAAGGTTG CCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGT TCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTT GACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTT TTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGA TGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGG GCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGG CTCGGCCCTGACCAAGTCAAATCCATGGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTC GGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCT CCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTC TCGCGGCTTACGTTCTGCCCAAGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGAT CTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCT CAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTG ACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTT GATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGC CGCGGAGTTGTTCGGTAAATTGTCACAACGCCG (SEQ ID NO. 154)

[0180] In some embodiments, the construct comprises or consists of, from 5' to 3' a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, a regulatory sequence and a second transposable element inverted repeat. In some embodiments, the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure have length from 1-1500 nucleotides, inclusive of all ranges therebetween. In some embodiments, the spacer sequences may be located either 5' to or 3' to an ITR, a promoter, a nuclear localization sequence, a nuclease, a stop codon, a polyA sequence, a transposable element inverted repeat, and / or a regulator element.

[0181] In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs are isolated or derived from an AAV vector of a first serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a second serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2 and the second serotype is AAV9.

[0182] Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the gRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, a first ITR is isolated or derived from an AAV vector of a first serotype, a second ITR is isolated or derived from an AAV vector of a second serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a third serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype, the second serotype, and the third serotype are the same. In some embodiments, the first serotype, the second serotype, and the third serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 . In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV9. Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and / or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,

[0183] 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,

[0184] 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,

[0185] 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,

[0186] 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110±10 base pairs. In some embodiments, the ITRs have a length of 120±10 base pairs. In some embodiments, the ITRs have a length of 130±10 base pairs. In some embodiments, the ITRs have a length of 140±10 base pairs. In some embodiments, the ITRs have a length of 150±10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs.

[0187] In some embodiments, the AAV-sgRNA vector may comprise additional elements to facilitate packaging of the vector and expression of the sgRNA. In some embodiments, the AAV- sgRNA vector may comprise a transposable element. In some embodiments, the AAV-sgRNA vector may comprise a regulatory element. In some embodiments, the regulatory element comprises an activator or a repressor. In some embodiments, the AAV-sgRNA sequence may comprise a non-functional or “stuffer” sequence. Exemplary stuffer sequences of the disclosure may have some (a non-zero percentage of) identity or homology to a genomic sequence of a mammal (including a human). Alternatively, exemplary stuffer sequences of the disclosure may have no identify or homology to a genomic sequence of a mammal (including a human). Exemplary stuffer sequences of the disclosure may comprise or consist of naturally occurring noncoding sequences or sequences that are neither transcribed nor translated following administration of the AAV vector to a subject.

[0188] For clinical applications, pharmaceutical compositions, which are also referred to herein as compositions, are prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. Appropriate salts and buffers are used to render drugs, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active ingredients of the present disclosure, its use in therapeutic compositions may be used. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.

[0189] In some embodiments, the active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.

[0190] The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

[0191] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0192] Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, i.e., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0193] In some embodiments, the compositions of the present disclosure are formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (i.e., hydrochloric or phosphoric acids, or from organic acids (i.e., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (i.e., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (i.e., isopropylamine, trimethylamine, histidine, procaine) and the like.

[0194] Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic, for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0195] In some embodiments, the nucleotide editing Cas9, and gRNAs described herein may be delivered to the patient using adoptive cell transfer (ACT). In adoptive cell transfer, one or more expression constructs are provided ex vivo to cells which have originated from the patient (autologous) or from one or more individual(s) other than the patient (allogeneic). The cells are subsequently introduced or reintroduced into the patient. Thus, in some embodiments, one or more nucleic acids encoding nucleotide editing Cas9 and a guide RNA that targets position S332, S333, and / or S334 of exon 14 of CAMK2D-B (SEQ ID NO. 1) are provided to a cell ex vivo before the cell is introduced or reintroduced to a patient.

[0196] Unless otherwise defined, 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 disclosure belongs. The terminology used in the detailed description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0197] All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are each incorporated by reference herein in their entirety.

[0198] The singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms “about” and “approximately” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount. Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0199] The term “nucleotide editing Cas9” refers to a Cas9 protein fused to a base editor or a prime editor. Non-limiting examples of Cas9 include SpCas9, SpCas9-NG, SaCas9, SaCas9-KKH, SauCas9, and SlugCas9. Non limiting examples of a base editor include ABEmax, ABE8e, ABE8eV106W, ABE8.20-m.

[0200] The terms “polynucleotide,” “nucleic acid” and “transgene” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, i.e., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides can include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (i.e., variant nucleic acid). Polynucleotides can be single stranded, double stranded, or triplex, linear or circular, and can be of any suitable length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95 / 32305), phosphor othioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, i.e., 2' methoxy or 2' halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (i.e., modified uridines such as 5- m ethoxyuridine, pseudouridine, or N1 -methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (i.e., N4-methyl deoxy guanosine, deaza- or aza-purines, deaza- or azapyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (i.e., 5- methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6- methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and PCT No. WO 93 / 13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., l lthed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (i.e., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42): 13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

[0201] A nucleic acid encoding a polypeptide often comprises an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions. Further disclosure of a DNA nucleotide sequence includes is corresponding RNA and amino acid sequence for which it encodes. Similarly, an amino acid sequence disclosures explicitly its corresponding RNA or DNA sequence. Finally, an RNA sequence includes is corresponding DNA and amino acid sequence for which it encodes.

[0202] Nucleic acids can include one or more expression control, or regulatory elements operably linked to the open reading frame, where the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (i.e., promoters, enhancers, a TATA box, and the like), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, and the like. Expression control / regulatory elements can be obtained from the genome of any suitable organism.

[0203] As used herein, “AAV” refers to an adeno-associated virus vector. As used herein, “AAV” refers to any AAV serotype and variant, including but not limited to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO (see, i.e., SEQ ID NO: 81 of U.S. Pat. No. 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, i.e., SEQ ID NO: 1 of US 2015 / 0111955, which is incorporated by reference herein in its entirety), AAV9 vector, AAV9P vector (also known as AAVMYO, see, Weinmann et al., 2020, Nature Communications, 11 :5432), and Myo-AAV vectors described in Tabebordbar et al., 2021, Cell, 184: 1-20 (i.e., MyoAAV 1A, 2A, 3A, 4A, 4C, or 4E), wherein the number following AAV indicates the AAV serotype. The term “AAV” can also refer to any known AAV (vector) system. In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, i.e., McCarty et al., Gene Ther. 2001; 8: 1248-54, Naso et al., BioDrugs 2017; 31 :317-334, and references cited therein for detailed discussion of various AAV vectors. Structurally, AAVs are small (25 nm), single-DNA stranded non-enveloped viruses with an icosahedral capsid. Naturally occurring or engineered AAV serotypes and variants that differ in the composition and structure of their capsid protein have varying tropism, i.e., ability to transduce different cell types. When combined with active promoters, this tropism defines the site of gene expression.

[0204] A “recombinant AAV” or “rAAV” is a DNAse-resistant viral particle containing two elements, an AAV capsid and a vector genome containing at least non-AAV coding sequences packaged within the AAV capsid. Unless otherwise specified, this term may be used interchangeably with the phrase “rAAV vector”. The rAAV is a “replication-defective virus” or “viral vector”, as it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny. In certain embodiments, the only AAV sequences are the AAV inverted terminal repeat sequences (ITRs), typically located at the extreme 5' and 3' ends of the vector genome in order to allow the gene and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0205] “Guide RNA”, “guide RNA”, and simply “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “guide RNA” refers to each type. The trRNA may be a naturally occurring sequence, or a trRNA sequence with modifications or variations compared to naturally occurring sequences. For clarity, the terms “guide RNA” or “guide” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. In general, in the case of a DNA nucleic acid construct encoding a guide RNA, the U residues in any of the RNA sequences described herein may be replaced with T residues, and in the case of a guide RNA construct encoded by any of the DNA sequences described herein, the T residues may be replaced with U residues.

[0206] Target sequences for Cas9s include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for a Cas9 is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (i.e., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0207] A “promoter” refers to a nucleotide sequence, usually upstream (5') of a coding sequence, which directs and / or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. “Promoter” includes a minimal promoter that is a short DNA sequence comprised of a TATA-box and optionally other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. An “enhancer” is a DNA sequence that can stimulate transcription activity and may be an innate element of the promoter or a heterologous element that enhances the level or tissue specificity of expression. It is capable of operating in either orientation (5'->3 ' or 3 '->5') and may be capable of functioning even when positioned either upstream or downstream of the promoter. Promoters and / or enhancers may be derived in their entirety from a native gene or be composed of different elements derived from different elements found in nature, or even be comprised of synthetic DNA segments. A promoter or enhancer may comprise DNA sequences that are involved in the binding of protein factors that modulate / control effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

[0208] The term “transduce” refers to introduction of a nucleic acid sequence into a cell or host organism by way of a vector (i.e., a viral particle). Introduction of a transgene into a cell by a viral particle is can therefore be referred to as “transduction” of the cell. The transgene may or may not be integrated into genomic nucleic acid of a transduced cell. If an introduced transgene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced transgene may exist in the recipient cell or host organism extra chromosomally, or only transiently. A “transduced cell” is therefore a cell into which the transgene has been introduced by way of transduction. Thus, a “transduced” cell is a cell into which, or a progeny thereof in which a transgene has been introduced. A transduced cell can be propagated, transgene transcribed, and the encoded inhibitory RNA or protein expressed. For gene therapy uses and methods, a transduced cell can be in a mammal.

[0209] A nucleic acid / transgene is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding and RNAi or a polypeptide, or a nucleic acid directing expression of a polypeptide may include an inducible promoter, or a tissue-specific promoter for controlling transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element can also be referred to as an expression cassette.

[0210] As used herein, the terms “modify” or “modified” means that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, i.e., a missense or nonsense mutation.

[0211] As used herein, “phosphomimetic,” refers to an amino acid reside that mimics phosphorylated at a specific site of a protein. In some embodiment, a phosphomimetic substation modification refers to an amino acid substitution with a phosphomimetic amino acid selected from glutamate (E) and aspartate (A). As used herein, “non-phosphomimetic,” refers to an amino acid reside that is not defined as a phosphomimetic mimetic amino acid.

[0212] As used herein, “non-phosphorylatable,” refers to an amino acid reside that cannot, or is rarely phosphorylated, for example due to instability in the bond between the amino acid and phosphate group. Examples of non-phosphorylatable amino acids can be selected from: Alanine, Arginine, Asparagine, Cysteine, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine. In a preferred embodiment, the non-phosphorylatable amino acids can preferably be selected from: Alanine or Glycine.

[0213] As used herein, the term “disrupts phosphorylation” means to genetically modify a protein, or its underlying nucleotide sequence to include a substitution mutation that replaces a phosphorylatable or a phosphomimetic amino acid reside with a non-phosphorylatable amino acid reside.

[0214] In additional embodiments, non-phosphorylatable amino acids can include non-canonical amino acids that cannot, or are rarely phosphorylated, for example due to instability in the bond between the non-canonical amino acid, which are know this those of ordinary skill in the art.

[0215] Examples of phosphorylatable amino acids include serine, tyrosine, or threonine.

[0216] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (i.e. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA- processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus.

[0217] As used herein, a “spacer sequence,” sometimes also referred to herein and in the literature as a “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for cleavage by a Cas9. For clarity, the terms “spacer sequence”, “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof.

[0218] A “nucleic acid” or “polynucleotide” variant refers to a modified sequence which has been genetically altered compared to wild type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without altering the amino acids of a protein encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes the amino acids of a protein encoded thereby.

[0219] The terms “protein” and “polypeptide” are used interchangeably herein. The “polypeptides” encoded by a “nucleic acid”, or “polynucleotide” or “transgene” disclosed herein include partial or full-length native sequences, as with naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, so long as the polypeptide retains some degree of function or activity. Accordingly, in methods and uses of the disclosure, such polypeptides encoded by nucleic acid sequences are not required to be identical to the endogenous protein that is defective, or whose activity, function, or expression is insufficient, deficient or absent in a treated mammal.

[0220] An example of an amino acid modification is a conservative amino acid substitution or a deletion. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of the unmodified sequence (i.e., wild-type sequence).

[0221] Another example of an amino acid modification is a targeting peptide introduced into a capsid protein of a viral particle. Peptides have been identified that target recombinant viral vectors or nanoparticles to various organs and tissues. A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the disclosure will have at least 40%, 50%, 60%, to 70%, i.e., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, i.e., 81%-84%, at least 85%, i.e., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of activity or function of wild type).

[0222] “Conservative variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations,” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein that encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill in the art will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0223] The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%.

[0224] The term “substantial identity” in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.

[0225] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994).

[0226] Moreover, in some embodiments, a substitution mutation can include one or more conservative substitutions. Such conservative amino acid substitutions involve replacing a residue by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) 35 acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0227] The terms “treat,” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, inhibit, reduce, or decrease an undesired physiological change or disorder, such as the development, progression or worsening of the disorder. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizing a (i.e., not worsening or progressing) symptom or adverse effect of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed (i.e., as determined by a genetic assay).

[0228] “Subject” refers to a mammal, and preferably a human.

[0229] The disclosure now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. EXAMPLES

[0230] Example 1 : Background, Experimental Overview, and Results.

[0231] Heart failure is one of the leading causes of morbidity and mortality in the developed world; however, many current therapies treat heart failure symptoms and do not target the underlying molecular causes of the disease. Thus, while patients with chronic heart failure are living longer than ever before, their quality of life remains poor and the burden of heart failure on the healthcare system is increasing. The only current ‘cure’ for heart failure is cardiac transplant, access to which is limited by a shortage of healthy donor hearts. However, substantial clinical evidence suggests that heart failure may be at least partially reversible, as a subset of patients treated with guideline- directed therapies experience modest recovery from heart failure, which is characterized by reduced left ventricular (LV) dilatation and improved LV systolic function (i.e., reverse remodeling). Reverse remodeling is associated with dramatic improvements in patient survival and quality of life. However, despite considerable interest from both clinical and basic science perspectives in identifying therapeutic targets for reverse remodeling and recovery, the molecular mechanisms that regulate this process remain poorly understood.

[0232] Left ventricular assist device (LVAD) therapy, which mechanically unloads the LV and assumes control of cardiac circulatory function, is the treatment that is most often associated with reverse remodeling and partial functional recovery. LVAD therapy also necessitates the removal of some LV myocardium during device placement, thus generating a pre-treatment sample for future paired comparisons. A recent investigation into the molecular biology of recovery by bulk RNA-sequencing found few pre-LVAD gene expression differences between patients who went on to recover and those who did not, indicating a striking transcriptional similarity between response sub-groups before treatment. This finding suggested that molecular features of a favorable response might only manifest after treatment (e.g., they are induced by LVAD). In support of this, single-nucleus RNA-sequencing of pre- and post-LVAD myocardium from responders and non-responders identified cell type-specific recovery signatures post-LVAD, where decreased expression of pro-inflammatory genes in cardiac macrophages and fibroblasts were the most discriminating features. However, as is true with inflammation and heart failure, whether changes in pro-inflammatory gene expression are a cause or consequence of the partial functional recovery induced by LVAD is not clear. Further, this earlier study found that, despite improved gross cardiac contractility denoted by increased systolic function in responders post- LVAD, cardiomyocytes did not revert to a ‘healthy’ transcriptional state in recovery. These findings support the conclusion that gene expression changes alone cannot explain the different patient outcomes with LVAD. Further, they suggest that other molecular factors contribute to improved outcomes in the subset of advanced heart failure patients who experience reverse remodeling and partial functional recovery.

[0233] Partial restoration of cardiac function in heart failure, while rare and unpredictable, does occur in a small subset of patients treated with current therapies. Given this evident reversibility of what was long thought to be a static disease state, there is considerable interest in identifying new therapeutic targets that promote reverse remodeling and functional recovery. However, a lack of human heart tissue studies paired with molecular mechanistic investigations has resulted in the underlying biology that mediates reverse remodeling and recovery remaining poorly understood. Previous investigations using multi-omics analyses to identify molecular features of partial myocardial recovery with LVAD identified modest differences in cardiac cell type proportions and gene expression between responders and non-responders. Applicants RNA-seq analysis of pre- and post-LVAD samples further corroborates the findings of these previous studies indicating that the cardiac transcriptome is remarkably similar between these response sub-groups, although modest differential expression of inflammatory genes was observed, in line with previous findings. However, a deeper analysis of protein and splice variant expression identified dramatic changes in RNA alternative splicing in heart failure and unique alternative splice variant expression changes in patients who experienced partial functional recovery with LVAD. Among these, alternative splicing and phosphorylation of CAMK2D were strong predictors of LV structural and functional outcomes with LVAD therapy. Notably, this was true whether Applicants used Applicants criteria for reverse remodeling / recovery or the criteria established previously by other groups who implanted LVADs at earlier stages of systolic heart failure (Fig. 27). Further, Applicants mechanistic investigations using primary rodent cardiomyocytes and human EHTs identified that increased cytoplasm-restricted CAMK2D-B expression in cardiomyocytes, mimicking the nonresponder state, dramatically remodeled the phospho-proteome and blunted cardiomyocyte Ca2+transients. The reduced Ca2+transients are consistent with previous observations with hyperactivation of cytoplasmic CAMK2D, which resulted from increased SR Ca2+leak.

[0234] Two previously mouse studies from separate groups published confirmed the significance of CAMK2D activation for heart failure progression that had been implied from findings in the human heart failure myocardium, as CAMK2D knockout mice were found to be protected from heart failure with chronic pressure overload. Given this link between CAMK2D and heart disease progression, the development of therapeutic strategies that inhibit CAMK2D has long been a goal of heart disease research. However, despite the clear role of CAMK2D in driving cardiac pathology, small molecule inhibitors have yet to emerge in clinical practice. Some reasons for this may include off-target inhibition of this ubiquitously expressed kinase in other tissues, inhibition of beneficial cardiac CAMK2 functions, and failure to discriminate between different CAMK2D splice variants or CAMK2 gene isoforms. Appropriate patient stratification is likely another factor influencing the varied clinical outcomes with CAMK2 inhibitors and our findings suggest that LVAD non-responders are one sub-population that might be particularly well-suited for a CAMK2 inhibitor. Thus, this study may help to inform future clinical trial design to select appropriate heart failure patient sub-populations for CAMK2 inhibitors.

[0235] Nevertheless, alternative strategies to regulate CAMK2 may prove more effective than inhibitors due to their above-described drawbacks, although development of such therapies is admittedly time-consuming and high-risk. For example, modulating CAMK2D subcellular localization may represent a new therapeutic strategy for advanced heart failure, particularly in patients with non-ischemic dilated cardiomyopathy receiving mechanical circulatory support. The specificity of such an approach would also mitigate the off-target inhibition concerns posed by current therapeutic strategies, as the B splice variant of CAMK2D is almost exclusively expressed in cardiomyocytes.

[0236] Alternative splicing can generate at least 11 different splice variants from the CAMK2D gene, three of which are highly expressed in cardiomyocytes: B, C, and 9. In the human heart, the B and 9 splice variants comprise -90% of total CAMK2D. The C and 9 splice variants localize to the cytoplasm and regulate the activity of proteins involved in Ca2+-handling and inflammation. Overexpression of the C and 9 splice variants in the mouse heart induces rapid heart failure onset. In contrast, multiple studies have shown that the B splice variant, which is the only variant that includes the NLS encoded by exon 14, has cardioprotective properties. These include prevention of cardiomyocyte apoptosis, inhibition of pro-inflammatory signaling, and induction of mitochondrial Ca2+uptake in conditions of Ca2+overload. Overexpression of CAMK2D-B does induce hypertrophic cardiac remodeling in mice, but disease onset occurs much later than with overexpression of the cytoplasmic splice variants. However, considering the findings, it should be noted that simply overexpressing CAMK2D-B does not ensure nuclear translocation in cardiomyocytes. Increased CAMK2D-B expression in human heart failure, identified herein and in one previous study, suggests a compensatory shift in alternative splicing to a protective splice variant. However, Applicants findings show that hyperphosphorylation at the NLS in LVAD therapy non-responsive patients prevented CAMK2D-B from reaching the nucleus, resulting in increased cytoplasmic CAMK2D. Meanwhile, in heart failure patients who experienced partial functional recovery with LVAD support, exon 14 inclusion decreased to healthy control levels. Applicants speculate that this is because these patients were no longer in the dysfunctional state that triggered this attempted cardioprotective post-transcriptional response. Applicants findings suggest that nuclear targeting of CAMK2D-B represents a molecular strategy to sequester the kinase away from cytoplasmic targets associated with cardiac pathology and, moreover, that restricting this splice variant to the cytoplasm may prevent functional recovery from advanced heart failure in patients receiving mechanical circulatory support.

[0237] Example 2: Ventricular transcriptome remodeling with mechanical circulatory support

[0238] Among 41 available paired pre- and post-LVAD patient samples, Applicants identified 10 responders and 9 non-responders based on LV structural and functional outcomes obtained by echocardiography (see methods) (Fig. 1A-B). It is important to note that the LV structural and functional improvements exhibited by the responder group, while significant, represent partial myocardial recovery that was insufficient to obviate the need for cardiac transplant. All patients had non-ischemic dilated cardiomyopathy and were classified as having NYHA class IV (end stage) heart failure at the time of LVAD placement. Patients in both outcome groups displayed similar age ranges, medication histories, and clinical characteristics before starting therapy (Table 1, Table 2), suggesting that molecular differences likely contributed to their bifurcated functional responses to LVAD therapy. To identify potential transcriptional differences, Applicants performed bulk RNA sequencing on heart failure (pre-LVAD), post-LVAD, and non-failing control samples. Applicants found that mechanical circulatory support partially reversed heart failure-associated gene expression changes to non-failing control levels (Fig. 6), which included reduced pro-inflammatory gene expression (Fig. 16). However, few genes were significantly differentially expressed between responders and non-responders pre-LVAD, in agreement with previous work, or post-LVAD (Fig. 7). Although total gene expression levels were not different between groups, Applicants reasoned that a paired analysis of gene expression changes from pre- to post-LVAD might reveal transcriptional signatures of recovery. Applicants therefore analyzed the RNA-seq data for LVAD treatment effects between responders and non-responders. Gene set enrichment analysis revealed that increased RNA splicing factor gene expression was positively associated with recovery (Fig. 1C), while genes involved in the immune response were negatively associated (Fig. ID).

[0239] Example 3: Partial functional recovery with LVAD is associated with unique RNA alternative splicing changes.

[0240] The generation of multiple transcripts from a single gene by alternative splicing increases proteome diversity and thus serves as a mechanism to modulate cellular function. Over 90% of the -20,000 protein-coding genes in the human genome undergo alternative splicing, which increases potential proteome complexity by an order of magnitude. To examine whether the transcriptional differences in RNA splicing factor expression translated to meaningful changes at the protein level, Applicants performed tandem-mass-tag (TMT) quantitative proteomics on post-LVAD responder and non-responder samples. This approach identified 154 differentially expressed proteins between the groups, including increased abundance of RNA splicing factors in responders (Fig. 1E-G, Fig. 8-9). Given this support for the role of alternative splicing in heart failure development and recovery, Applicants next mined the RNA-seq dataset to detect and quantify local splicing variations (LSVs). Applicants identified 1,235 LSVs that were significantly impacted in heart failure versus control samples and 411 that changed with mechanical unloading (Fig. 10-12). Among the heart failure-associated splicing changes, 34% of altered cassette exon events (i.e., exon-skipping) were predicted to affect protein domains involved in cellular signal transduction (Fig. 13-14, Table 3), suggesting that alternative splicing contributes to cell functional changes in heart failure. To determine if unique alternative splicing changes were induced in LVAD responders, Applicants examined LSVs in the responder and non-responder groups compared to heart failure. This analysis revealed that responders and non-responders had distinct alternative splicing responses to LVAD, with 351 and 236 LSVs unique to each group, respectively (Fig. 1H).

[0241] Among 73 heart failure-associated LSVs that were modified in responders, one notable example was exon 242 inclusion in TIN (Fig. II). TIN encodes the giant protein titin, which regulates myofibril passive tension. Exon 242 encodes an 89 amino acid immunoglobulin domain that mediates protein-protein interactions (Fig. 14) in the I-band region of titin, which is the primary determinant of titin’s elastic properties. Thus, alternative splicing in this region is expected to impact titin-based myofibril passive tension. Our follow-up analysis of this event using targeted assays revealed that, while exon 242 inclusion increased across all patients after LVAD compared with heart failure, variability between responders and non-responders contributed to a modest, but non-significant change between these groups as measured by exon-specific qPCR (Fig. 15).

[0242] Example 4: Partial myocardial functional recovery with LVAD coincides with decreased CAMK2D exon 14 inclusion.

[0243] Among the most significant recovery-associated splicing changes identified in the RNA sequencing dataset were increased exon 16 and decreased exon 14 inclusion in CAMK2D (Fig. 1 J), which encodes Ca2+ / calmodulin-dependent protein kinase 115. CAMK2D is a serine / threonine kinase that regulates Ca2+-dependent contractility and pro-inflammatory signaling in cardiomyocytes and its chronic hyperactivation is implicated in multiple forms of heart disease. Alternative splicing of CAMK2D exons 14-16 generates four cardiac splice variants (Fig. 2A), which differ in their subcellular localization. Exon-specific qPCR analysis corroborated the RNA- seq finding that the B splice variant, which includes a nuclear localization signal (NLS) encoded by exon 14, increased in heart failure (Fig. 16). Notably, exon 14 inclusion was increased in nonresponder patients both pre- and post-LVAD (Fig. 2B) and displayed a strong inverse correlation with LV functional and structural outcomes (Fig. 2C-D). Targeted RT-PCR and qPCR analyses further validated that there was a shift in the dominant CAMK2D splice variant from 9 (exons 13- 16-17) in non-failing controls to B (exons 13-14-17) in heart failure, which was reversed only in patients who exhibited partial functional recovery after receiving LVAD support (Fig. 2E-G). Example 5: CAMK2D-B NLS phosphorylation correlates with LVAD-induced partial functional recovery.

[0244] To identify potential additional molecular factors uniquely regulated in recovery, Applicants performed TMT quantitative phospho-proteomics on the post-LVAD responder and non-responder samples. Remarkably, the most significantly differentially phosphorylated sites identified were three serine residues in CAMK2D (S332, S333, and S334), which increased in non-responders (Fig. 3A-B). These residues are located immediately downstream of the NLS encoded within exon 14 (Fig. 3A) and their phosphorylation is identified as a mechanism for regulating nuclear localization of CAMK2D. Western blot analyses did not identify differences in CAMK2D regulatory domain T287 autophosphorylation between groups (Fig. 3C, Fig. 17), suggesting similar kinase activity between responders and non-responders. However, in agreement with the phospho-proteomics findings, Applicants found S332 phosphorylation increased in heart failure and then was fully restored to non-failing control levels in patients who exhibited functional recovery (Fig. 3C, 3D). In non-responders, pre-LVAD p-S332 levels were also ~2-fold higher than in responders and remained elevated following mechanical unloading (Fig. 3C, 3D). Pre- and post- LVAD S332 phosphorylation displayed significant inverse correlations with recovery of LV function (Fig. 3E-F), supporting the potential utility of this event as a biomarker to predict which patients are poised for partial recovery from heart failure with LVAD.

[0245] Example 6: Regulation of CAMK2D-B subcellular localization.

[0246] Since phosphorylation at S332-S334 had previously been shown to prevent nuclear translocation of CAMK2D-B in other cell types, Applicants next sought to determine the role of phosphorylation at these sites in cardiomyocytes. Applicants generated adenovirus vectors expressing GFP-tagged wildtype (Bsss), phospho-null (serine to alanine mutations at 332-334: BAAA), or phospho-mimetic (serine to aspartate mutations at 332-334: BDDD) CAMK2D-B and transduced neonatal rat ventricular myocytes (NRVMs). As expected, BDDD displayed cytoplasmic localization (Fig. 3G-H). However, unlike in non-cardiomyocytes where phospho-null mutations alone led to a complete translocation of CAMK2D-B to the nucleus, the BAAA construct displayed only modest nuclear localization in NRVMs (Fig. 3G-H). Notably, when NRVMs were treated with the adrenergic agonist phenylephrine (PE), nuclear localization of BAAA was potently induced, while BDDD remained cytoplasmic (Fig. 3G-H). Nuclear translocation of BAAA was also induced with endothelin-1, but not caffeine or insulin-like growth factor-1 (Fig. 18). To determine whether subcellular localization of CAMK2D was also altered in the hearts of LVAD non-responder patients, Applicants performed subcellular fractionation on post-LVAD samples to isolate the cytosol, membrane, nucleus (soluble / nucleoplasm), and insoluble (myofilament and chromatin- associated proteins) fractions (Fig. 19). Western blot for CAMK2D within these fractions revealed that non-responder patients had modestly increased cytosolic CAMK2D, similar membrane- associated CAMK2D, and reduced CAMK2D abundance in the nuclear and insoluble fractions compared to responders (Fig. 3I-M). These findings suggest that the increased CAMK2D-B in non-responders failed to translocate to the nucleus due to the NLS phosphorylation, which led to increased total cytosolic CAMK2D. Analyses of subcellular fractions from the NRVMs transduced with BAAA and treated with PE further corroborated the finding that NLS de-phosphorylated CAMK2D-B translocated to the nucleus with adrenergic agonism (Fig. 4A-B). Given the above finding that adrenergic and endothelin receptor agonism induced nuclear translocation of the BAAA construct, Applicants hypothesized that both NLS dephosphorylation and CAMK2D constitutive activity were required for nuclear translocation in cardiomyocytes, as activation of these receptors increases cytosolic Ca2+, leading to CAMK2D activation by autophosphorylation at T287. Applicants measured CAMK2D T287 phosphorylation of the GFP- tagged CAMK2D constructs by western blot and found it expectedly increased with PE treatment (Fig. 4C-D). Next, to test the hypothesis that both T287 autophosphorylation and S332-334 dephosphorylation were required for nuclear translocation, Applicants generated adenovirus expression vectors from the BAAA and BDDD constructs where phospho-null (T287A) or phosphomimetic (T287D) mutations were introduced in the regulatory domain. As hypothesized, BAAA- T287D displayed near-complete nuclear localization that was PE-independent (Fig. 4E-F). Meanwhile, BDDD localized to the cytoplasm and was unaffected by T287A / D modifications or PE treatment (Fig. 4G-H), indicating that NLS de-phosphoryl ati on is a pre-requisite for autoactivation-dependent nuclear translocation of CAMK2D-B. Interestingly, the BAAA-T287A construct did exhibit a small, but significant, increase in nuclear localization with PE treatment, although the effect was decreased compared to BAAA alone (Fig. 4E-F). The reason for this is unknown, but Applicants hypothesize that exposure of the CAMK2D-B NLS sequence may be dependent on structural changes associated with the binding of calmodulin, which should still be able to bind the T287A mutant, albeit with much lesser affinity than in the T287 phosphorylated or phospho-mimetic (T287D) states.

[0247] A previous study of CAMK2D-B NLS regulation in a non-cardiomyocyte cell line identified that CAMKI and CAMKIV could phosphorylate the NLS regulatory serine residues. To determine if these kinases might be implicated in the dysregulation of CAMK2D-B localization in non-responder patients, Applicants first examined their tissue-specific RNA expression across hundreds human samples in the GTEx dataset. While CAMKI and CAMKIV are highly expressed in the brain, where CAMK2D plays important roles in memory consolidation and synaptic plasticity, Applicants found that they were expressed lowly (CAMKI) or not at all (CAMKIV) in the heart (Fig. 20A-C). To examine whether CAMKI might still regulate CAMK2D-B despite its low expression, Applicants treated NRVMs with a CAMKI inhibitor during viral transduction with the GFP-B sss construct and examined localization via microscopy. CAMKI inhibition had no effect on nuclear GFP signal, with or without PE treatment, suggesting that this kinase does not regulate the CAMK2D-B NLS in cardiomyocytes (Fig. 20D). Previous studies have identified that protein phosphatase 1 and calcineurin can each de-phosphorylate the S332 site of CAMK2D-B and both are highly expressed in the heart (Fig. 21), suggesting they may have overlapping functions with respect to CAMK2D-B NLS regulation. Identification of the upstream kinase(s) in cardiomyocytes is an important area for future investigation.

[0248] Example 7: Cellular signaling and functional consequences of cytoplasmic CAMK2D-B,

[0249] Due to the variability associated with human samples and the stringent statistical cutoffs employed, Applicants phospho-proteomics analysis identified few significant hits, thus limiting insight into potential downstream cellular signaling consequences of increased cytoplasmic CAMK2D-B in heart failure. However, Applicants reasoned that the phosphorylation events mediated (directly or indirectly) by cytoplasmic CAMK2D-B would correlate with p-S332-S334 levels. Applicants therefore performed linear regression analysis for phospho-peptide intensities across the entire phospho-proteomics dataset versus p-S332-S334 CAMK2D-B, which identified several previously established CAMK2D substrates among the strongest correlated events (Table 4). Pathway over-enrichment analysis of phospho-sites with an r2> 0.50 versus p-S332-S334 CAMK2D-B identified Rho GTPase signaling, cardiac conduction, and regulation of cardiac hypertrophy as the top affected pathways (Fig. 22).

[0250] To further explore the cellular signaling changes regulated by cytoplasmic CAMK2D-B using a more systematic approach, Applicants performed TMT quantitative phospho-proteomics on NRVMs transduced with BAAA or BDDD and treated with PE or vehicle. This approach identified the baseline phospho-proteome effects of nuclear-competent and cytoplasm-restricted CAMK2D- B, as well as activation-dependent effects (Fig. 5A-E). At baseline, phosphorylation events that increased in NRVMs transduced with BAAA were enriched with cell surface and cytoskeletal proteins, while the downregulated phosphorylation events included nuclear proteins involved in alternative splicing (Fig. 23). With PE treatment, BAAA led to increased phosphorylation of nuclear-localized RNA processing factors (Fig. 5D, Fig. 24). PE-dependent phosphorylation events that increased in BoDD-transduced cells were enriched with proteins involved in Rho GTPase regulation (Fig. 5E), matching the findings in the human phospho-proteome (Fig. 22). Additionally, when Applicants mined the human and NRVM phospho-proteomics datasets for shared features, Applicants identified multiple individual peptide examples with similar phosphorylation differences between non-responders / BoDD and responders / B vw, including proteins involved plasma membrane calcium transport (ATP2B1), plasma membrane ion channel anchoring (ANK3), and protein homeostasis (RPL19 and USP47) (Fig. 25). These findings further support the conclusion that increased cytoplasm-restricted CAMK2D-B remodeled the phospho- proteome of non-responders.

[0251] To test for potential functional consequences of increased cytoplasmic CAMK2D-B in human cardiomyocytes, Applicants employed a human induced pluripotent stem cell (iPSC)- derived cardiomyocyte engineered heart tissue (EHT) model. EHTs were transduced with Empty vector control, BAAA, or BDDD adenoviral vectors (Fig. 5F) and then Ca2+transients were measured in the absence and presence of adrenergic stimulation. EHTs transduced with BDDD had reduced Ca2+transient amplitude compared to BAAA at baseline (Fig. 5G-H). As expected, adrenergic agonism caused a significant increase in Ca2+transient amplitude in EHTs transduced with the Empty vector control (Fig. 5G-H). While the Ca2+transient amplitude in BAAA EHTS did not increase to the same extent as with Empty vector, the response to adrenergic agonism was significantly improved compared with BDDD EHTS, which were completely unresponsive to the stimulus (Fig. 5G-H). Other parameters were also significantly impacted by BDDD, including the area under the curve of the Ca2+transient, contraction speed, and relaxation speed (Fig. 26). Given the previously identified positive relationship between Ca2+transient amplitude and contraction force at physiological beat-rate frequencies in other human iPSC-CM EHT models, these data support the conclusion that increased cytoplasmic CAMK2D-B impairs cardiomyocyte contractility through dysregulation of Ca2+transient kinetics.

[0252] Example 8: Materials and Methods.

[0253] Clinical Definition of Responders and Non-responders

[0254] Among 41 available paired pre- and post-LVAD patient samples in the University of Colorado Anschutz Medical Center biobank at the time of the study, responder (R) classification (n = 10) was assigned if both of the following criteria were met: 1. The LV ejection fraction (LVEF) increased by an absolute value of > 15 (e.g., from 20% to 35%), and 2. The LV end diastolic dimension (LVEDD) decreased by > 15% following mechanical circulatory support. Non-responder (NR) classification (n = 9) was assigned if both of the following criteria were met: 1. LV ejection fraction increased by an absolute value < 5 or decreased, and 2. LV end diastolic dimension decreased by < 15% or increased following mechanical circulatory support. These definitions were specified prior to downstream analyses. Patients whose responses did not meet these criteria (n = 22) were considered intermediate responders and were not included in the study. The echocardiographic measures of LV structure and function were obtained as part of standard clinical care and reflect the clinical condition of the patient at the time of the study. The pre-LVAD echocardiograms were obtained at the closest time frame prior to LVAD implantation. The post- LVAD echocardiograms were obtained at the time closest to LVAD explant (i.e., at the time of cardiac transplant). The post LVAD echocardiograms for all patients in the study were obtained with the LVAD settings clinically required for the patient and reflect an unloaded condition.

[0255] Human Cardiac Tissue Procurement and Ethical Oversight

[0256] Paired pre-LVAD cores and post-LVAD explanted hearts were obtained with informed consent at the University of Colorado Anschutz Medical Center. All patients receiving LVADs were in New York Heart Association (NYHA) Class IV heart failure (end-stage) at the time of device placement. In all cases, LVAD therapy was used as bridge to transplant. Non-failing control LV tissue (n = 6) was obtained from age- and sex-matched donor hearts without a history of coronary artery disease that were deemed unsuitable for transplant due to size or other incompatibility. Following cardiac explant in the operating room, the samples were flash frozen in liquid nitrogen and then stored at -80°C prior to experimentation. De-identified patient clinical data was stored in a secure REDCap database. Heart failure patient samples were sequenced to identify variants in cardiomyopathy-associated genes, which are reported in Table 5. The collection, storage, and experimentation on these samples were approved by the Institutional Review Board of the University of Colorado Anschutz Medical Center.

[0257] Neonatal Rat Ventricular Myocyte Isolation

[0258] NRVMs were isolated from 1-2-day-old Sprague-Dawley rat pups as previously described. In brief, rat pups were dipped in ethanol, decapitated, and their hearts rapidly removed and placed in calcium and bicarbonate-free HEPES-buffered Hanks’ Solution (CBFHH). The atria were then removed and the ventricles from all rats pooled in a beaker containing 10 mL warm CBFHH, Trypsin (3 mg / mL), and 100 pl DNase. The solution was placed on a stir plate and incubated for 15 minutes. Cardiomyocytes were dissociated by multiple sequential incubations in this solution paired with mechanical dissociation by pipetting up and down. When the cells were fully dissociated, they were suspended in recovery media (MEM, IX PB 12, IX HEPES, 5% calf serum) and pre-plated for 90 minutes to remove adherent cells (e.g., fibroblasts, endothelial cells). The suspended cells (cardiomyocytes) were then plated on gelatin-coated tissue culture plates and incubated at 37°C, 1.5% CO2. The day after isolation, the media was switched to experimental medium (MEM, 1X PB12, 1X HEPES, IX BrdU, Pen-Strep: 500 mL MEM, 1 mL PB12 stock, 10 mL IM HEPES pH 7.3, 1 mL BrdU, 2 mL Pen-Strep stock) and experiments were started two days post-plating.

[0259] RNA Extraction, cDNA Synthesis, and qPCR Analysis

[0260] Frozen left ventricular tissue (~40 mg) was added to 1 mL of Trizol, homogenized with a mechanical homogenizer, and incubated at room temperature for 5-10 minutes. Following this incubation, the homogenate was transferred to a 1.5 mL Eppendorf tube, chloroform added at 1 :5 vol / vol, the tubes shaken vigorously for ~15 seconds, and then incubated at room temperature for 15 minutes. The samples were then centrifuged for 15 minutes at 12,000 x g, 4°C and the upper aqueous phase collected and transferred to a clean tube. An equal volume of isopropanol (500 pl) was added, the tubes briefly vortexed, and then incubated at -20°C for 15 minutes followed by centrifugation at 12,000 x g, 4°C for 8 minutes to pellet the RNA. The supernatant was discarded, and RNA pellet washed twice with ice-cold 75% ethanol. After removal of the final ethanol wash, the RNA was dried for 5 minutes by uncapping the tubes and then reconstituted in Milli-Q H2O. RNA concentrations were determined by Nanodrop. 500 ng of RNA was used to synthesize complementary DNA (cDNA) with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. The cDNA was then diluted to 2.5 ng / pl and 5 ng total cDNA used for quantitative real-time PCR with the primers listed in Table 6. Gene expression analysis was performed using the AA-CT method with PGK1, chosen based on stable high expression across groups in RNA-seq analysis, as the housekeeping gene.

[0261] RT-PCR and DNA Acrylamide Gel Visualization

[0262] PCR was performed using Taq PCR Master Mix (Qiagen). Reactions were 25 pl total volume, including 30 ng of cDNA template. 30 cycles of PCR with the following conditions were conducted: Denaturation - 95°C, 30 seconds; Annealing - 5°C below primer Tm, 30 seconds; Extension - 72°C, 1 minute. The primer sequences are listed in Table 7. Results were visualized by adding 5 pl of Hi-Density TBE Sample Buffer (Novex) and separating PCR products via electrophoresis on a 10% TBE acrylamide gel at 145V for 1.5 hours. The gel was incubated in IX ethidium bromide in milliQ-H20 for 5 minutes and then imaged using the UV setting on an IQ- 800 imager (Cytiva).

[0263] Bulk RNA Sequencing and Alternative Splicing Analysis RNA was extracted from frozen left ventricular tissue as described above. mRNA was purified by poly-A enrichment, cDNA libraries synthesized, and bulk RNA-seq performed using Illumina NovaSeq with 15O-bp paired-end reads (Novogene Corporation, Sacramento, CA). The minimum sequencing depth was 50 million read pairs. Differential gene expression analysis was performed using DESeq2 version 1.46.0. For analysis between non-failing controls, HF, and LVAD groups, the dataset was prefiltered to remove genes that had less than 5 samples (size of the smallest group: controls) with 10 or more counts. The apeglm shrinkage estimator was used to produce shrunken log fold changes for visualization. Genes were considered significantly differentially expressed if they displayed >1.0, <-1.0 shrunken log2 fold-change and an FDR- adjusted p-value < 0.05. For comparison of responders and non-responders at pre- and post-LVAD timepoints, genes were considered significantly differentially expressed if they displayed FDR- adjusted p-value < 0.05. To contrast the responses to LVAD treatments between responders and non-responders, a model matrix with the following design was constructed: response + response:patient.n + response:timepoint (see DESeq2 vignette for further details). The dds object was prefiltered to remove genes that had less than 9 samples (size of the smallest group: non- responders) with 10 or more counts. DESeq2 was used to determine differences in the LVAD treatment effect between responders and non-responders, considering the paired nature of the samples. Pathway over-enrichment analysis on the significantly differentially expressed genes was performed using Enrichr. Pathways with FDR q-values < 0.05 were considered significantly enriched. Gene Set Enrichment Analysis was performed with GSEA version 4.3.3 on pre-ranked gene lists ranked by the negative log of the p-value multiplied by the sign of the fold-change. Majiq HET and Voila version 2.4.dev3+g85d0781 were used to analyze the short-read data for alternative splicing differences. Modules were extracted via the Voila Modulize command. Local splicing variations (LSVs) were considered changed if they had a dpsi change greater than 0.15 between conditions and a Wilcoxon p-value < 0.05. For defining modules as changing, default values were used except fore\ the following flags: — changing-between-group-dpsi 0.15 — changing-between- group-dpsi-secondary 0.0. DIGGER (https: / / exbio.wzw.tum.de / digger / nease_analysis) was used run NEASE analysis on cassette exons that underwent differential splicing (defined as a cassette exon event from Voila Modulize with a changing junction, dpsi > 0.15 and Wilcoxon p-value < 0.05). Majiq HET output was manually converted to the standard input format for DIGGER NEASE analysis. Protein Extraction for Proteomics Studies

[0264] Tissue. Post-LVAD left ventricular tissue (n = 9 responders - one responder sample was excluded due to a maximum multiplexing capacity of 18; 9 non-responders) was pulverized while frozen, solubilized in a 5% SDS, 0.75% sodium deoxycholate, 50 mM Tris pH 8 buffer containing IX protease and phosphatase inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, Thermo) and immediately heated for 10 minutes 95°C. The samples were then centrifuged at 12,000 x g for 10 minutes to remove any remaining insoluble material and a BCA assay was performed on the soluble portion to determine protein concentration. Cells. NRVMs on 10-cm tissue culture plates (5.5 million cells per plate) were washed twice with PBS and then rapidly frozen at -80°C before thawing and lysing in 5% SDS, 0.75% sodium deoxycholate, 50 mM Tris pH 8 buffer containing IX protease and phosphatase inhibitors by scraping. Lysates from two 10- cm plates were pooled to ensure >1 mg of total protein and make up one biological replicate. Collected lysates were then immediately heated for 10 minutes 95°C. The samples were then centrifuged at 12,000 x g for 10 minutes to remove any remaining insoluble material and a BCA assay was performed on the soluble portion to determine protein concentration.

[0265] Tandem Mass Tag (TMT) Quantitative Proteomics and Phospho-proteomics

[0266] Human heart tissue protein lysates (1300-1800 ug) and NRVM protein lysates (350-550 pg) were reduced and alkylated with the addition of 10 mM tris(2-carboxy ethylphosphine) (TCEP), 40 mM 2-chloroacetamide, incubated at 70°C for 10 minutes and then shaking at 2000 rpm at 37°C for 30 minutes. Lysates were digested using the SP3 method. Briefly, 1000 pg carboxylate-functionalized speedbeads (Cytiva Life Sciences) were added to protein lysates. Acetonitrile was added to 80% (v / v) to bind proteins to the beads, then washed twice with 80% (v / v) ethanol and twice with 100% acetonitrile. Proteins were digested in 50 mM Tris-HCl buffer, pH 8.5, with Lys-C / Trypsin (Promega) and incubated at 37°C overnight. Tryptic peptides were desalted using HLB Oasis cartridges (Waters) according to the manufacturer’s instructions and dried in a speedvac vacuum centrifuge. Phosphopeptides were serially enriched using the High- Select TiO2 phosphoenrichment kit (Thermo Scientific) followed by the High-Select Fe-NTA phosphoenrichment kit (Thermo Scientific). Briefly, samples were dissolved in Binding / Equilibration buffer, loaded on the TiO2 column, washed and eluted. The TiO2 elutions and unretained fraction were dried immediately using a speedvac vacuum centrifuge. The TiO2 unretained fraction was then dissolved in Fe-NTA Binding / Wash Buffer, loaded on the Fe-NTA column, washed and eluted. The TiO2 and Fe-NTA elutions were combined and cleaned up then labeled with TMT-Pro reagents (Thermo Scientific) along with proteome samples from human hearts according to the manufacturer’s instructions. Multiplexed samples were immediately cleaned up with HLB Oasis cartridge and dried in a speedvac vacuum centrifuge. To reduce sample complexity, multiplexed peptides were fractionated using a high pH reversed-phase Cl 8 UPLC with a 0.5 mm X 150 mm custom packed rpC18 1.9 pm 120A (Dr. Maisch Gmbh) column with mobile phases 0.1% (v / v) aqueous ammonia, pHlO in water and acetonitrile (ACN). Peptides were gradient eluted at 20 pL / minute from 2 to 50% ACN in 50 minutes (proteome) or 2 to 20% ACN in 50 minutes (phosphoproteome) concatenating for a total of 12 fractions using a Waters M-class UPLC (Waters). Peptide fractions were then lyophilized in a speedvac vacuum centrifuge and stored at -20°C until analysis. Fractionated peptides were suspended in 3% (v / v) ACN, 0.1% (v / v) trifluoroacetic acid (TFA) and directly injected onto a reversed-phase C18 1.7 pm, 130 A, 75 mm X 250 mm M-class column (Waters), using either an Ultimate 3000 nanoUPLC coupled to a Q Exactive HF-X (human heart tissues) or a Vanquish Neo nanoUPLC coupled to an Orbitrap Exploris 480 (NRVMs) (Thermo Scientific). Multiplexed peptides were eluted into the mass spectrometers at 300 nL / minute with a gradient from 4% to 16% ACN in 120 minutes then to 40% ACN in 5 minutes (human hearts) or 4% to 20% ACN in 120 minutes then to 40% ACN in 5 minutes (NRVMs). Precursor mass spectra (MSI) were acquired at a resolution of 120,000 from 350 to 1500 m / z with an automatic gain control (AGC) target of 3E6 and a maximum injection time of 50 milliseconds (human hearts) or Standard AGC Target and Auto Maximum injection time (NRVMs). Precursor peptide ion isolation width for MS2 fragment scans was 0.7 m / z, and the top 15 most intense ions were sequenced (human hearts) or a cycle time of 3 seconds (NRVMs). MS2 spectra were either acquired at a resolution of 45,000 with higher energy collision dissociation (HCD) at 30% normalized collision energy (human hearts) or at 30,000 resolution using TurboTMT with HCD at 32%. An AGC target of 1E5 and 120 milliseconds maximum injection time (human hearts) or 200% Normalized AGC Target and Auto maximum injection time (NRVMs) were used. Dynamic exclusion was set for 30 seconds. Rawfdes were searched against either the Human database (UP000005640) using MaxQuant v.2.0.3.0 or the Rattus norvegicus database (UP000002494) using MaxQuant v.2.6.3.0. Cysteine carbamidomethylation was considered a fixed modification, while methionine oxidation, protein N-terminal acetylation and phosphorylation of serine, threonine, or tyrosine were searched as variable modifications. All peptide and protein identifications were thresholded at a 1% false discovery rate (FDR). TMT reporter ion intensities were Cyclic loess normalized and log2 fold changes and p-values were calculated with limma (Bioconductor.com) using an R-script. Pathway over-enrichment analysis on the significantly differentially expressed proteins was performed using Enrichr . Pathways with FDR q-values < 0.05 were considered significantly enriched.

[0267] Subcellular Fractionation

[0268] Tissue. Approximately 120 mg of frozen LV tissue was added to a glass vial containing 1 mL of cytosol extraction buffer (125 mM NaCl, 10 mM HEPES, 25 pg / mL digitonin, pH 7.4, + protease / phosphatase inhibitors) and minced on ice using small dissection scissors. The slurry was then added to a 2 mL Dounce homogenizer (Pyrex, No. 7727-02) and homogenized on ice until there were no longer large tissue pieces (~3-5 minutes). The solution was then filtered through a 70 pm filter and transferred to a 1.5 mL Eppendorf tube. Insoluble material was pelleted by centrifugation at 7,000 RCF, 4°C, 15 minutes. The supernatant was collected as the crude cytosolic fraction and then further clarified by centrifugation at 15,000 RCF, 4°C, 15 minutes. The pellet was washed once by re-suspension in ice-cold PBS and then resuspended by pipetting up and down in 150 pL of membrane extraction buffer (125 mM NaCl, 10 mM HEPES, 1% NP-40, pH 7.4, + protease / phosphatase inhibitors). Membrane proteins were extracted by incubation at 4°C for 30 minutes with end-over-end rotation. The samples were then centrifuged at 7,000 RCF, 4°C, 5 minutes and the supernatant collected as the membrane fraction. The pellet was washed once by re-suspension in ice-cold PBS and then resuspended in 125 pL of nuclear extraction buffer (125 mM NaCl, 10 mM HEPES, 5 mM MgCh, 1% triton X-100, 0.5% (w / v) sodium deoxycholate, 0.1% (w / v) SDS, pH 8.0, + protease / phosphatase inhibitors) and incubated at room temperature for 10 minutes with end-over-end rotation. The samples were then centrifuged at 15,000 RCF, 4°C, 10 minutes to pellet insoluble material. The supernatant was collected as the nuclear fraction. The pellet was resuspended in 150 pL of insoluble protein buffer (500 mM NaCl, 8M Urea, 2% (w / v) SDS, 10% glycerol - deionized with mix-bed resin and pH to 6.8 + protease / phosphatase inhibitors), incubated at room temperature for 10 minutes with end-over-end rotation, and then boiled for 10 minutes. The samples were then centrifuged at 15,000 RCF, room temp, 10 minutes and the supernatant collected as the insoluble fraction. Cells. NRVMs on 60-mm tissue culture plates were washed twice with PBS and then 300 pl of buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCE, 0.3% NP-40, IX protease / phosphatase inhibitors) added and cells collected by scraping. The cells in buffer were added to Eppendorf tubes and incubated on ice for 3 minutes. Insoluble material was then pelleted by centrifugation at 800 x g for 5 minutes. The supernatant was collected as the crude cytosolic fraction, which was then clarified by centrifugation at 12,000 x g for 10 minutes. The insoluble pellet was washed twice in buffer without NP-40 and then resuspended in 5% SDS, 0.75% deoxycholate buffer and heated at 95°C for 10 minutes to solubilize nuclei and myofilament components.

[0269] Protein Extraction for Western Blot Analyses

[0270] Tissue: Left ventricular tissue was pulverized while frozen, added to lysis buffer (5% SDS, 0.75% sodium deoxycholate, 50 mM Tris pH 8) containing IX protease and phosphatase inhibitors, and then immediately heated for 10 minutes 95°C. Samples were then centrifuged at 12,000 x g for 10 minutes, 4°C and the supernatant collected. Cells: NRVMs on 6-well tissue culture plates were washed twice with PBS and then plates were immediately frozen at -80°C. Plates were brought to room temperature and then 150 pl lysis buffer (5% SDS, 0.75% deoxycholate, as above) containing IX protease and phosphatase inhibitors added to the wells. Cells were lysed with a cell scraper and then the collected material immediately heat at 95°C for 10 minutes. Samples were then centrifuged at 12,000 x g for 10 minutes, 4°C and the supernatant collected.

[0271] Western Blot

[0272] Protein concentration was determined by BCA assay and then 20 pg of protein was added to equal volume loading buffer (SDS Tris Glycine buffer + Bolt Reducing Agent) and heated at 95°C for 8 minutes. Proteins were loaded onto 4-12% Bis-Tris gradient gels (Invitrogen) for separation by electrophoresis and then transferred onto nitrocellulose membrane. To assess loading and quantify total protein prior to blocking, membranes were incubated in Revert Total Protein stain (LICOR) for 5 minutes followed by image acquisition on an IQ-800 imager (Cytiva) using the IR-short setting with automatic exposure. Membranes were blocked in 5% milk TBS-T for 1 hour at room temperature and then primary antibodies were added in 5% BSA TBS-T and incubated overnight at 4°C with agitation. Primary antibodies and dilutions: CAMK2D (Thermo Fisher, PA5-22168, 1 : 1000), Phospho-CAMK2D T287 (Cell Signaling, 12716, 1 : 1000), Phospho- CAMK2D S332 (a gift from Dr. Wang Wang, University of Washington, 1 : 100), Histone H3 (Cell Signaling, 4499, 1: 1000), GAPDH (Cell Signaling, 2118, 1 : 1000), Myosin heavy chain (Developmental Studies Hybridoma Bank, F59, 1 : 150), ATP1A2 (ProteinTech, 16836-1-AP, 1 : 1000), a / p-tubulin (Cell Signaling, 2148S, 1 : 1000), B23 / NPM1 (ProteinTech, 60096-1-Ig, 1 : 1000). Following overnight incubation, primary antibodies were removed, the membranes washed three times for 5 minutes each with TBS-T, and secondary antibodies (IRDye CW800 goat anti-rabbit, LICOR) added in 5% Milk TBS-T at 1 :4000 dilution and incubated for 1 hour at room temperature with agitation. Secondary antibodies were then removed, and the membranes washed three times for 5 minutes each with TBS. Blots were imaged on an IQ-800 imager using the IR- long setting and quantification of signal intensity was performed using Fiji.

[0273] Cloning and Adenovirus Production

[0274] The Human CAMK2D Gene ORF cDNA clone expression plasmid (Catalog #: HG29766- NF), encoding the CAMK2D (minus exons 14-16 and 21-22), was purchased from Sino Biological. Molecular cloning with Gibson Assembly was used to add a synthetic double-stranded oligo encoding exons 21 and 22 to the C-terminus and an N-terminal EGFP. The resulting EGFP- CAMK2D-full-length ORF was assembled in the pShuttle-CMV shuttle vector by Gibson Assembly. Cloning was confirmed by sequencing. The pShuttle-CMV-EGFP-CAMK2D plasmid was then used as a PCR template with Gibson Assembly to generate the wild-type CAMK2D-B splice variant (addition of 33 base pairs encoding exon 14), phospho-null CAMK2D-B-AAA (AAA - serine to alanine mutations at amino acids 332, 333, and 334), phospho-mimetic CAMK2D-B-DDD (DDD - serine to aspartate mutations at amino acids 332, 333, and 334). The B-AAA and B-DDD constructs were then used as PCR templates with Gibson Assembly to generate the T287AZD mutants. pShuttle-CMV empty vector was prepared as a negative control. All cloning was confirmed by whole plasmid sequencing (Plasmidsaurus). pShuttle constructs were then subcloned into the pAdEasy backbone and used to transfect HEK293 cells. Ten days after transfection, the virus was passaged onto new plates and then amplified in subsequent larger scale infections. Purified viruses for each were obtained from 72 two-day post-infection 10-cm plates by overlaying concentrated freeze-thawed cell lysates on CsCl gradients and ultracentrifuging. Adenoviral particles were extracted from Beckman centrifuge tubes by sidepuncture with an 18-gauge needle and stored in 1:4 ratio in Falck-Pedersen viral storage buffer at -20°C. A multiplicity of infection (MOI) of 20 was used for in vitro experiments with these viruses in NRVMs and iPSC-CM EHTs.

[0275] Adenovirus Transduction Experiments in NRVMs Two days after their isolation, NRVMs were transduced with adenoviral constructs expressing empty vector or EGFP-tagged B-SSS, B-AAA, B-DDD, B-AAA-T287A, B-AAA- T287D, B-DDD-T287A, or B-DDD-T287D at 20 viral particles per cell (20 MOI). Phenylephrine (PE) was added to the media at this timepoint at a final concentration of 20 pM. For the CAMKI inhibition studies, the CAMKI inhibitor CS640 was added to the media at the time of viral transduction / PE treatment and incubated for 24 hours. 24 hours after viral transduction / PE / CS640 treatment, protein lysates were collected for western blot / phospho-proteomic analyses or cells were fixed for immunofluorescence microscopy analysis of GFP-CAMK2D-B localization. For western blot experiments, NRVMs were plated on 6-well dishes at 500,000 cells per well. For immunofluorescence microscopy experiments, NRVMs were plated on 24-well glass-bottomed dishes at 100,000 cells per well. For phospho-proteomics experiments, 5.5 million cells were plated on 10-cm tissue culture plates.

[0276] Immunofluorescence Microscopy

[0277] Cells or EHTs were washed twice with PBS and then fixed in ice-cold 100% methanol for 1 minute, followed by 4% paraformaldehyde for 5 minutes. Permeabilization was performed using 0.5% triton in PBS for 20 minutes at room temperature. Antigen retrieval was then conducted by incubation in 100 mM glycine, pH 3.5, for 30 minutes at room temperature. The wells were washed three times with PBS and then incubated in blocking buffer (5% BSA in PBS - 0.25 pm filtered) for 1 hour at room temperature. Mouse a-actinin primary antibody (Clone EA-53, Thermo) was added in fresh blocking buffer at 1 :300 dilution and incubated overnight at 4°C. The following day, the wells were washed three times with PBS and Alexa-Fluor goat anti-mouse 555 secondary antibody (4409, Cell Signaling) added in fresh blocking buffer at 1: 1000 dilution. The plate was then incubated for 1 hour at room temperature protected from the light. Following this incubation, the wells were washed twice with PBS and then DAPI (D3571, Thermo) added in PBS and incubated for 15 minutes at room temperature. Two additional washes were performed and then 0.5 m PBS added per well prior to imaging on a Nikon Ti-E spinning disc confocal microscope. Images were acquired using constant laser intensity and photomultiplier gain settings. Intensity of nuclear and whole-cell GFP signal was measured using Fiji (Image J). Nuclear GFP-CaMKII8-B intensity was normalized to total GFP signal from the same cell for quantification.

[0278] Peptide motif enrichment analysis and kinase prediction. To identify peptide motifs within the up and downregulated phospho-peptides between responders and non-responders post-LVAD, Applicants used the MEME suite (version 5.5.8) MoMo peptide motif enrichment software on the 13 amino acid phospho-site centralized sequence (for all peptides with an unadjusted p value < 0.01 between groups. The top two significantly enriched peptide motifs (sorted by fold enrichment) were then plugged into the Cell Signaling PhosphositePlus Kinase Prediction tool to identify potential kinases. The kinases were sorted by percentile rank and the top five kinases for each motif were listed. iPSC-derived Cardiomyocyte Differentiation and Culture

[0279] Human induced pluripotent stem cells (WTC11) were grown in E8 medium on Matrigel- coated plates and split twice before media switch to RPMI 1640 for induction of cardiomyocyte differentiation. Mesoderm differentiation was induced in RPMI containing B27 minus insulin, 50 pg / mL ascorbic acid, and 7 pM CHIR-99021 (GSK3 inhibitor) for 48 hours. The cells were allowed to recover in RPMI / B27 minus insulin / ascorbic acid medium for one day and then cardiac mesoderm specification was induced using 5 pM IWR1 (Wnt inhibitor) in RPMI / B27 minus insulin / ascorbic acid medium for 48 hours. The cells were allowed to recover for one day, as above, then media was changed to RPMI with B27 plus insulin. Beating cells were observed on day 7-8 after GSK3 inhibition. The cultures were maintained in RPMI / B27 plus insulin medium until day 12 when they were collected and seeded into engineered heart tissue (EHT) molds.

[0280] Human Engineered Heart Tissue (EHT) Generation

[0281] Hydrogel micropillar molds were 3D printed using a custom poly(ethylene glycol) diacrylate resin on a Lumen Alpha DLP printer (Volumetric Inc., USA) with predefined light exposure settings (7.5 s per layer, 100 pm layer thickness, 20 mW cm-2) as described previously. Following 3D printing, hydrogel micropillar molds were washed in DPBS (with multiple rinses over 3-5 days to remove unreacted reagents), sterilized with immersion in 70% ethanol for 30 minutes followed by UV germicidal lamp for another 30 minutes, and stored in sterile DPBS (IX, Gibco) at 4°C until use. The stiffness of the micropillar was determined to be 2.096 pN pm'1via static stress simulation of micropillar bending using Fusion 360 (Autodesk, USA). To form EHTs, hydrogel micropillar molds were seeded with iPSC-derived cardiomyocytes (cell age: day 12, density: 10 million cells mL'1). Briefly, acid-solubilized type I bovine telocollagen (Advanced BioMatrix, USA) was mixed with prechilled, sterile DPBS (10X), distilled water, Matrigel® (basement membrane matrix growth factor reduced, 10% v / v, Corning), and neutralized with NaOH (0.1 N) to achieve a final collagen concentration of 1 mg ml1. Cells were added to the mixture, the resultant precursor (6 pL) was transferred into micropillar wells under ice-cold conditions, and the molds were incubated (37°C, 5% CO2 incubator) for 15 min to enable collagen gelation. After incubation, the molds containing cell-laden gels were immersed in recovery medium comprising RPMI 1640 (Gibco) supplemented with B-27 (IX, Thermo Scientific), FBS (20% v / v), and penicillin-streptomycin (1% v / v). Two days after seeding, the recovery medium was replaced with growth medium consisting of RPMI 1640 with B-27 (IX) and penicillinstreptomycin (1% v / v) with regular media changes every other day. After growth phase (day 10 after seeding), the media was switched to maturation media consisting of low glucose RPMI supplemented with palmitic acid (PA, 50 pM), oleic acid (OA, 100 pM), galactose (10 mM), B27 (IX), and penicillin-streptomycin (1% v / v). EHTs were cultured in maturation media until day 28 (i.e., cell age: day 40) before initiating treatment.

[0282] Calcium / Contractility Analysis in Engineered Heart Tissues

[0283] On day 28 after seeding (i.e., cell age: day 40), EHTs were transduced with adenoviruses at 20 MOI. One day later, a subset of EHTs were treated with a combination of the adrenergic agonists phenylephrine (PE, 50 pM) and isoproterenol (ISO, 2 pM). 48 hours after viral transduction, and 24 hours after agonist addition, EHTs were washed with sterile DPBS (IX) and incubated (37°C, 5% CO2 incubator, 90 min) with intracellular calcium indicator (Cal 520, AAT Bioquest, 4 pM) in sterile Tyrode’s salt solution (Sigma) supplemented with Pluronic F-127 (0.04 wt.%, Sigma) to enhance cellular uptake as described previously. Following dye loading, EHTs were washed and incubated in fresh Tyrode’s solution to remove the staining solution. Live cell videos of EHT contractility were recorded on a Nikon Ti2 Eclipse with AXR laser scanning confocal microscope (2X objective, 105 Hz frame rate, 37°C with 5% CO2) under electrical stimulation (1 Hz, 10 V cm'1, carbon electrodes with Myopacer setup, lonOptix). All EHTs were first allowed to reach equilibrium by subjecting to stimulation frequency for 5 min before recording any measurements. Calcium transient traces as recorded by the temporal change in fluorescence intensity upon stimulation were used for all measurements. Analysis of parameters (i.e., mean calcium amplitude) was performed using an automated software (Beatprofiler) as described previously.

[0284] Statistical Analysis and Data Presentation Sample size calculations were not performed for deciding on the analysis of patient groups as this was limited to the number of samples available. All available samples that met the predefined criteria for responders and non-responders were included in the study. Sample sizes for NRVM and EHT experiments were calculated based on previous experience using these models. NRVMs from at least three independent biological replicates were used in these studies. Groups were blinded to the experimenter for the proteomics, phospho-proteomics, and RNA-seq sample preparation and data collection. Groups were otherwise not blinded for the remainder of the experiments. Comparisons of two groups were performed by paired or unpaired two-tailed t-test, as described in the figure legends. Paired analyses were performed for pre- versus post-LVAD comparisons and unpaired analyses for pre- versus pre-LVAD and post- versus post-LVAD comparisons pertaining to the responder and non-responder groups. Comparisons of more than two groups were performed by one- or two-way ANOVA, as described in the figure legends. Comparisons of categorical data between responders and non-responders pre- and post-LVAD were performed using the Chi-squared test. All data analyses and graphical representations were performed using GraphPad Prism version 10. The data throughout the manuscript are presented as the mean ± standard error, mean ± SD, or median and range as detailed in the figure legends and table footnotes. For the RNA-seq and proteomics data and the corresponding gene ontology enrichment analyses, an adjusted p-value of < 0.05 was considered statistically significant. For all other data analyses, a p-value of < 0.05 was considered statistically significant.

[0285] TABLES

[0286] Table 1. Patient clinical characteristics.

[0287] Data are presented as the mean ± SD. All clinical data were analyzed by two-tailed paired t-test.

[0288] Categorical data (Sex, Race, Medications) were analyzed by Chi-squared test.

[0289] ‘Values not available for 4 pre-LVAD and 4 post-LVAD patients

[0290] Table 2. Clinical characteristics of responders and non-responders pre- and post-LVAD.

[0291]

[0292] Data are presented as the mean ± SD.

[0293] Categorical data were analyzed by Chi-squared test. All other data were analyzed by two-tailed unpaired t-test

[0294] #Values not available for 1 pre-LVAD and 1 post-LVAD patient

[0295] ‘Values not available for 4 pre-LVAD and 4 post-LVAD patients

[0296] Table 3. Domain interactions altered by alternative splicing in heart failure.

[0297] Table 4. Top phospho-sites correlated with CAMK2D-B NLS phosphorylation in post-LVAD human hearts. Previously identified CAMK2 substrates / binding partners are denoted by bold italics.

[0298] Table 5. Identification of likely pathogenic variants in human heart failure samples.

[0299] Table 6. Primers for quantitative real-time PCR.

[0300] Table 7. Primers for RT-PCR and acrylamide gel visualization.

[0301] REFERENCES

[0302] 1. Savarese G, Becher PM, Lund LH, Seferovic P, Rosano GMC, Coats AJS. Global burden of heart failure: a comprehensive and updated review of epidemiology. Cardiovasc Res. 2022; 118.

[0303] 2. Bozkurt B. Contemporary pharmacological treatment and management of heart failure. Nat Rev Cardiol. 2024;21.

[0304] 3. Kavalieratos D, Gelfman LP, Tycon LE, Riegel B, Bekelman DB, Ikejiani DZ, Goldstein N, Kimmel SE, Bakitas MA, Arnold RM. Palliative Care in Heart Failure: Rationale, Evidence, and Future Priorities. J Am Coll Cardiol. 2017;70.

[0305] 4. Sapna F, Raveena F, Chandio M, Bai K, Sayyar M, Varrassi G, Khatri M, Kumar S, Mohamad T. Advancements in Heart Failure Management: A Comprehensive Narrative Review of Emerging Therapies. Cureus. 2023;

[0306] 5. Jones NR, Roalfe AK, Adoki I, Hobbs FDR, Taylor CJ. Survival of patients with chronic heart failure in the community: a systematic review and meta-analysis. Eur J Heart Fail. 2019;21.

[0307] 6. Yan T, Zhu S, Yin X, Xie C, Xue J, Zhu M, Weng F, Zhu S, Xiang B, Zhou X, Liu G, Ming Y, Zhu K, Wang C, Guo C. Burden, Trends, and Inequalities of Heart Failure Globally, 1990 to 2019: A Secondary Analysis Based on the Global Burden of Disease 2019 Study. J Am Heart Assoc. 2023; 12.

[0308] 7. Heidenreich PA, Fonarow GC, Opsha Y, Sandhu AT, Sweitzer NK, Warraich HJ, Butler J, Hsieh E, Pressler SB, Shah K, Taylor K, Sabe M, Ng T. Economic Issues in Heart Failure in the United States. J Card Fail. 2022;28.

[0309] 8. Awad MA, Shah A, Griffith BP. Current status and outcomes in heart transplantation: a narrative review. Rev Cardiovasc Med. 2022;23.

[0310] 9. Martin TG, Juarros MA, Leinwand LA. Regression of cardiac hypertrophy in health and disease: mechanisms and therapeutic potential. Nat Rev Cardiol. 2023;20:343-367.

[0311] 10. Kanwar MK, Selzman CH, Ton VK, Mi era O, Cornwell WK, Antaki J, Drakos S, Shah P. Clinical myocardial recovery in advanced heart failure with long term left ventricular assist device support. Journal of Heart and Lung Transplantation. 2022;41 : 1324-1334.

[0312] 11. Shepherd CW, While AE. Cardiac rehabilitation and quality of life: A systematic review. Int J Nurs Stud. 2012;49. 12. Chaudhry SP, Devore AD, Vidula H, Nassif M, Mudy K, Birati EY, Gong T, Atluri P, Pham D, Sun B, Bansal A, Najjar SS. Left Ventricular Assist Devices: A Primer For the General Cardiologist. J Am Heart Assoc. 2022; 11.

[0313] 13. Martin TG, Juarros MA, Cleveland JC, Bristow MR, Ambardekar A V., Buttrick PM, Leinwand LA. Assessment of Autophagy Markers Suggests Increased Activity Following LVAD Therapy. JACC Basic Transl Sci. 2023;8: 1043-1056.

[0314] 14. Drakos SG, Badolia R, Makaju A, Kyriakopoulos CP, Wever-Pinzon O, Tracy CM, Bakhtina A, Bia R, Parnell T, Taleb I, Ramadurai DKA, Navankasattusas S, Dranow E, Hanff TC, Tseliou E, Shankar TS, Visker J, Hamouche R, Stauder EL, Caine WT, Alharethi R, Selzman CH, Franklin S. Distinct Transcriptomic and Proteomic Profile Specifies Patients Who Have Heart Failure With Potential of Myocardial Recovery on Mechanical Unloading and Circulatory Support. Circulation. 2022;147:409-424.

[0315] 15. Amrute JM, Lai L, Ma P, Koenig AL, Kamimoto K, Bredemeyer A, Shankar TS, Kuppe C, Kadyrov FF, Schulte LJ, Stoutenburg D, Kopecky BJ, Navankasattusas S, Visker J, Morris SA, Kramann R, Leuschner F, Mann DL, Drakos SG, Lavine KJ. Defining cardiac functional recovery in end-stage heart failure at single-cell resolution. Nature Cardiovascular Research. 2023 ;2.

[0316] 16. Van Linthout S, Tschope C. Inflammation - Cause or Consequence of Heart Failure or Both? Curr Heart Fail Rep. 2017; 14.

[0317] 17. Perez-Riverol Y, Bandla C, Kundu DJ, Kamatchinathan S, Bai J, Hewapathirana S, John NS, Prakash A, Walzer M, Wang S, Vizcaino JA. The PRIDE database at 20 years: 2025 update. Nucleic Acids Res. 2025;53:D543-D553.

[0318] 18. Gotthardt M, Badillo-Lisakowski V, Parikh VN, Ashley E, Furtado M, Carmo- Fonseca M, Schudy S, Meder B, Grosch M, Steinmetz L, Crocini C, Leinwand L. Cardiac splicing as a diagnostic and therapeutic target. Nat Rev Cardiol. 2023;20:517-530.

[0319] 19. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008;40: 1413-1415.

[0320] 20. Green CJ, Gazzara MR, Barash Y. MAJIQ-SPEL: Web-Tool to interrogate classical and complex splicing variations from RNA-Seq data. Bioinformatics. 2018;34:300-302. 21 . Herzog W. The multiple roles of titin in muscle contraction and force production. Biophys Rev. 2018;10.

[0321] 22. Dauksaite V, Gotthardt M. Molecular basis of titin exon exclusion by RBM20 and the novel titin splice regulator PTB4. Nucleic Acids Res. 2018;46.

[0322] 23. Beckendorf J, van den Hoogenhof MMG, Backs J. Physiological and unappreciated roles of CaMKII in the heart. Basic Res Cardiol. 2018; 113:29.

[0323] 24. Reyes Gaido OE, Nkashama LJ, Schole KL, Wang Q, Umapathi P, Mesubi 00, Konstantinidis K, Luczak ED, Anderson ME. CaMKII as a Therapeutic Target in Cardiovascular Disease. Annu Rev Pharmacol Toxicol. 2023;63:249-272.

[0324] 25. Lebek S, Chemello F, Caravia XM, Tan W, Li H, Chen K, Xu L, Liu N, Bassel- Duby R, Olson EN. Ablation of CaMKIId oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease. Science (1979). 2023;379: 179-185.

[0325] 26. Duran J, Nickel L, Estrada M, Backs J, van den Hoogenhof MMG. CaMKIId Splice Variants in the Healthy and Diseased Heart. Front Cell Dev Biol. 2021;9.

[0326] 27. Xu X, Yang D, Ding JH, Wang W, Chu PH, Dalton ND, Wang HY, Bermingham JR, Ye Z, Liu F, Rosenfeld MG, Manley JL, Ross J, Chen J, Xiao RP, Cheng H, Fu XD. ASF / SF2- regulated CaMKIId alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle. Cell. 2005; 120.

[0327] 28. Heist EK, Srinivasan M, Schulman H. Phosphorylation at the nuclear localization signal of Ca2+ / calmodulin- dependent protein kinase II blocks its nuclear targeting. Journal of Biological Chemistry. 1998;273: 19763-19771.

[0328] 29. Backs J, Song K, Bezprozvannaya S, Chang S, Olson EN. CaM kinase II selectively signals to histone deacetylase 4 during cardiomyocyte hypertrophy. Journal of Clinical Investigation. 2006; 116: 1853-1864.

[0329] 30. Srinivasan M, Edman CF, Schulman H. Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus. Journal of Cell Biology. 1994;126:839-852.

[0330] 31. Erickson JR. Mechanisms of CaMKII activation in the heart. Front Pharmacol. 2014; 5 APR. 32. Higazi DR, Feamley CJ, Drawnel FM, Talasila A, Corps EM, Ritter 0, McDonald

[0331] F, Mikoshiba K, Bootman MD, Roderick HL. Endothelin-1 -Stimulated InsP3-Induced Ca2+ Release Is a Nexus for Hypertrophic Signaling in Cardiac Myocytes. Mol Cell. 2009;33.

[0332] 33. Zhu W, Zou Y, Shiojima I, Kudoh S, Aikawa R, Hayashi D, Mizukami M, Toko H, Shibasaki F, Yazaki Y, Nagai R, Komuro I. Ca2+ / calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1 -induced cardiomyocyte hypertrophy. Journal of Biological Chemistry. 2000;275.

[0333] 34. Grimm M, Brown JH. P-Adrenergic receptor signaling in the heart: Role of CaMKII. J Mol Cell Cardiol. 2010;48.

[0334] 35. Simon M, Ko CY, Rebbeck RT, Baidar S, Cornea RL, Bers DM. CaMKIIo post- translational modifications increase affinity for calmodulin inside cardiac ventricular myocytes. J Mol Cell Cardiol. 2021; 161.

[0335] 36. Lonsdale J, Thomas J, Salvatore M, Phillips R, Lo E, Shad S, Hasz R, Walters G, Garcia F, Young N, Foster B, Moser M, Karasik E, Gillard B, Ramsey K, Sullivan S, Bridge J, Magazine H, Syron J, Fleming J, Siminoff L, Traino H, Mosavel M, Barker L, Jewell S, Rohrer D, Maxim D, Filkins D, Harbach P, Cortadillo E, Berghuis B, Turner L, Hudson E, Feenstra K, Sobin L, Robb J, Branton P, Korzeniewski G, Shive C, Tabor D, Qi L, Groch K, Nampally S, Buia S, Zimmerman A, Smith A, Burges R, Robinson K, Valentino K, Bradbury D, Cosentino M, DiazMayoral N, Kennedy M, Engel T, Williams P, Erickson K, Ardlie K, Winckler W, Getz G, DeLuca D, Daniel MacArthur, Kellis M, Thomson A, Young T, Gelfand E, Donovan M, Meng Y, Grant

[0336] G, Mash D, Marcus Y, Basile M, Liu J, Zhu J, Tu Z, Cox NJ, Nicolae DL, Gamazon ER, Im HK, Konkashbaev A, Pritchard J, Stevens M, Flutre T, Wen X, Dermitzakis ET, Lappalainen T, Guigo R, Monlong J, Sammeth M, Koller D, Battle A, Mostafavi S, McCarthy M, Rivas M, Mailer J, Rusyn I, Nobel A, Wright F, Shabalin A, et al. The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013;45.

[0337] 37. Rigter PMF, de Konink C, Dunn MJ, Proietti Onori M, Humberson JB, Thomas M, Barnes C, Prada CE, Weaver KN, Ryan TD, Caluseriu O, Conway J, Calamaro E, Fong CT, Wuyts W, Meuwissen M, Hordijk E, Jonkers CN, Anderson L, Yuseinova B, Polonia S, Beysen D, Stark Z, Savva E, Poulton C, McKenzie F, Bhoj E, Bupp CP, Bezieau S, Mercier S, Blevins A, Wentzensen IM, Xia F, Rosenfeld JA, Hsieh TC, Krawitz PM, Elbracht M, Veenma DCM, Schulman H, Stratton MM, Kiiry S, van Woerden GM. Role of CAMK2D in neurodevelopment and associated conditions. Am J Hum Genet. 2024; 111.

[0338] 38. Shioda N, Sawai M, Ishizuka Y, Shirao T, Fukunaga K. Nuclear translocation of calcium / calmodulin-dependent protein kinase 1153 promoted by protein phosphatase-1 enhances brain-derived neurotrophic factor expression in dopaminergic neurons. Journal of Biological Chemistry. 2015;290.

[0339] 39. Wang P, Xu S, Xu J, Xin Y, Lu Y, Zhang H, Zhou B, Xu H, Sheu SS, Tian R, Wang W. Elevated MCU Expression by CaMKIIoB Limits Pathological Cardiac Remodeling. Circulation. 2022;145: 1067-1083.

[0340] 40. Dhand AP, Juarros MA, Martin TG, Rodriguez-Rivera GJ, Hunt DR, Obenreder MC, Crosby CO, Meurer-Zeman B, McAfee Q, Valle-Ayala H, Zlotnick HM, Goddard DN, Ebmeier CC, Burdick JA, Leinwand LA. Digital Light Processing 3D Printing enables High Throughput Fabrication of Human Engineered Heart Tissues for Disease Modeling. bioRxiv [Internet], 2024;Available from: http: / / biorxiv.org / lookup / doi / 10.1101 / 2024.10.01.616163

[0341] 41. Saleem U, Mannhardt I, Braren I, Denning C, Eschenhagen T, Hansen A. Force and Calcium Transients Analysis in Human Engineered Heart Tissues Reveals Positive Force- Frequency Relation at Physiological Frequency. Stem Cell Reports. 2020; 14.

[0342] 42. Kyriakopoulos CP, Kapelios CJ, Stauder EL, Taleb I, Hamouche R, Sideris K, Koliopoulou AG, Bonios MJ, Drakos SG. LVAD as a Bridge to Remission from Advanced Heart Failure: Current Data and Opportunities for Improvement. J Clin Med. 2022; 11.

[0343] 43. Taleb I, Tseliou E, Fang JC, Drakos SG. A Mechanical Bridge to Recovery as a Bridge to Discovery: Learning From Few and Applying to Many. Circulation. 2022; 145.

[0344] 44. Chrysakis N, Xanthopoulos A, Magouliotis D, Starling RC, Drakos SG, Triposkiadis F, Skoularigis J. Myocardial Recovery. Diagnostics. 2023; 13.

[0345] 45. Kim GH, Uriel N, Burkhoff D. Reverse remodelling and myocardial recovery in heart failure. Nat Rev Cardiol. 2018; 15.

[0346] 46. Pereira L, Metrich M, Fernandez-velasco M, Lucas A, Leroy J, Perrier R, Morel E, Fischmeister R, Richard S, Benitah JP, Lezoualc’h F, Gomez AM. The cAMP binding protein Epac modulates Ca2+ sparks by a Ca2+ / calmodulin kinase signalling pathway in rat cardiac myocytes. Journal of Physiology. 2007; 583. 47. Hoch B, Meyer R, Hetzer R, Krause EG, Karczewski P. Identification and expression of 8-isoforms of the multifunctional Ca2+ / calmodulin-dependent protein kinase in failing and nonfailing human myocardium. Circ Res. 1999;84:713-721.

[0347] 48. Kirchhefer U, Schmitz W, Scholz H, Neumann J. Activity of cAMP-dependent protein kinase and Ca2+ / calmodulin- dependent protein kinase in failing and nonfailing human hearts. Cardiovasc Res. 1999;42.

[0348] 49. Ling H, Zhang T, Pereira L, Means CK, Cheng H, Gu Y, Dalton ND, Peterson KL, Chen J, Bers D, Brown JH. Requirement for Ca2+ / calmodulin-dependent kinase II in the transition from pressure overload-induced cardiac hypertrophy to heart failure in mice. Journal of Clinical Investigation. 2009;119.

[0349] 50. Backs J, Backs T, Neef S, Kreusser MM, Lehmann LH, Patrick DM, Grueter CE, Qi X, Richardson JA, Hill JA, Katus HA, Bassel-Duby R, Maier LS, Olson EN. The 8 isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload. Proc Natl Acad Sci U S A. 2009; 106.

[0350] 51. Lebek S, Caravia XM, Chemello F, Tan W, McAnally JR, Chen K, Xu L, Liu N, Bassel-Duby R, Olson EN. Elimination of CaMKIIS Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice. Circulation. 2023;148: 1490-1504.

[0351] 52. Lebek S, Caravia XM, Straub LG, Alzhanov D, Tan W, Li H, McAnally JR, Chen K, Xu L, Scherer PE, Liu N, Bassel-Duby R, Olson EN. CRISPR-Cas9 base editing of pathogenic CaMKIIS improves cardiac function in a humanized mouse model. Journal of Clinical Investigation. 2024; 134:el75164.

[0352] 53. Zhang T, Johnson EN, Gu Y, Morissette MR, Sah VP, Gigena MS, Belke DD, Dillmann WH, Rogers TB, Schulman H, Ross J, Brown JH. The cardiac-specific nuclear SB isoform of Ca2+ / calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity. Journal of Biological Chemistry. 2002;277.

[0353] 54. Zhang M, Gao H, Liu D, Zhong X, Shi X, Yu P, Jin L, Liu Y, Tang Y, Song Y, Liu J, Hu X, Li CY, Song L, Qin J, Wu F, Lan F, Zhang Y, Xiao RP. CaMKII-89 promotes cardiomyopathy through disrupting UBE2T-dependent DNA repair. Nat Cell Biol. 2019;21. 55. Gray CBB, Brown JH. CaMKIIdelta subtypes: Localization and function. Front

[0354] Pharmacol. 2014; 5 FEB.

[0355] 56. Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J, Bers DM, Brown JH. The 5c isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ Res. 2003;92:912-919.

[0356] 57. Ljubojevic-Holzer S, Herren AW, Djalinac N, Voglhuber J, Morotti S, Holzer M, Wood BM, Abdellatif M, Matzer I, Sacherer M, Radulovic S, Wallner M, Ivanov M, Wagner S, Sossalla S, Von Lewinski D, Pieske B, Brown JH, Sedej S, Bossuyt J, Bers DM. CaMKIKC Drives Early Adaptive Ca2+Change and Late Eccentric Cardiac Hypertrophy. Circ Res. 2020; 127.

[0357] 58. Zhang M, Zhang J, Zhang W, Hu Q, Jin L, Xie P, Zheng W, Shang H, Zhang Y. CaMKIL59 Induces Cardiomyocyte Death to Promote Cardiomyopathy and Heart Failure. Front Cardiovasc Med. 2021;8.

[0358] 59. Peng W, Zhang Y, Zheng M, Cheng H, Zhu W, Cao CM, Xiao RP. Cardioprotection by CaMKII-5B is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70. Circ Res. 2010; 106.

[0359] 60. Quijada P, Hariharan N, Cubillo JD, Bala KM, Emathinger JM, Wang BJ, Ormachea L, Bers DM, Sussman MA, Poizat C. Nuclear calcium / calmodulin-dependent protein kinase II signaling enhances cardiac progenitor cell survival and cardiac lineage commitment. Journal of Biological Chemistry. 2015;290:25411-25426.

[0360] 61. Little GH, Saw A, Bai Y, Dow J, Marjoram P, Simkhovich B, Leeka J, Kedes L, Kloner RA, Poizat C. Critical role of nuclear calcium / calmodulin-dependent protein kinase II5B in cardiomyocyte survival in cardiomyopathy. Journal of Biological Chemistry. 2009;284:24857- 24868.

[0361] 62. Gray CBB, Suetomi T, Xiang S, Blackwood EA, Glembotski CC, Miyamoto S, Westenbrink BD, Brown JH. CaMKIIS subtypes differentially regulate infarct formation following ex vivo myocardial ischemia / reperfusion through NF-KB and TNF-a. J Mol Cell Cardiol. 2017;103.

[0362] SUPPLEMENTAL REFERENCES

[0363] 1. Maass AH, Buvoli M. Cardiomyocyte preparation, culture, and gene transfer. Methods in Molecular Biology . 2007;366. 2. Love MT, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.

[0364] 3. Zhu A, Ibrahim JG, Love MI. Heavy-Tailed prior distributions for sequence count data: Removing the noise and preserving large differences. Bioinformatics. 2019;35.

[0365] 4. Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles G V., Clark NR, Ma’ayan A. Enrichr: Interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013 ; 14.

[0366] 5. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P, Spiegelman B, Lander ES, Hirschhom JN, Altshuler D, Groop LC. PGC- la-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34.

[0367] 6. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102.

[0368] 7. Vaquero-Garcia J, Aicher JK, Jewell S, Gazzara MR, Radens CM, Jha A, Norton SS, Lahens NF, Grant GR, Barash Y. RNA splicing analysis using heterogeneous and large RNA- seq datasets. Nat Commun. 2023; 14.

[0369] 8. Louadi Z, Yuan K, Gress A, Tsoy O, Kalinina O V., Baumbach J, Kacprowski T, List M. DIGGER: Exploring the functional role of alternative splicing in protein interactions. Nucleic Acids Res. 2021 ;49.

[0370] 9. Louadi Z, Elkjaer ML, Klug M, Lio CT, Fenn A, Illes Z, Bongiovanni D, Baumbach J, Kacprowski T, List M, Tsoy O. Functional enrichment of alternative splicing events with NEASE reveals insights into tissue identity and diseases. Genome Biol. 2021;22.

[0371] 10. Hughes CS, Foehr S, Garfield DA, Furlong EE, Steinmetz LM, Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol. 2014;10:757.

[0372] 11. Wang P, Xu S, Xu J, Xin Y, Lu Y, Zhang H, Zhou B, Xu H, Sheu SS, Tian R, Wang W. Elevated MCU Expression by CaMKIIbB Limits Pathological Cardiac Remodeling. Circulation. 2022; 145:1067-1083. 12. Cheng A, Grant CE, Noble WS, Bailey TL. MoMo: Discovery of statistically significant post-translational modification motifs. Bioinformatics. 2019;35.

[0373] 13. Hornbeck P V., Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B, Latham V, Sullivan M. PhosphoSitePlus: A comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 2012;40.

[0374] 14. Dhand AP, Davidson MD, Galarraga JH, Qazi TH, Locke RC, Mauck RL, Burdick JA. Simultaneous One-Pot Interpenetrating Network Formation to Expand 3D Processing Capabilities. Advanced Materials . 2022;34.

[0375] 15. Dhand AP, Juarros MA, Martin TG, Rodriguez -Rivera GJ, Hunt DR, Obenreder MC, Crosby CO, Meurer-Zeman B, McAfee Q, Valle-Ayala H, Zlotnick HM, Goddard DN, Ebmeier CC, Burdick JA, Leinwand LA. Digital Light Processing 3D Printing enables High Throughput Fabrication of Human Engineered Heart Tissues for Disease Modeling. bioRxiv [Internet], 2024;Available from: http: / / biorxiv.org / lookup / doi / 10.1101 / 2024.10.01.616163

[0376] 16. Daly AC, Davidson MD, Burdick JA. 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels. Nat Commun. 2021;12.

[0377] 17. Kim Y, Wang K, Lock RI, Nash TR, Fleischer S, Wang BZ, Fine BM, Vunjak- Novakovic G. BeatProfiler: Multimodal in Vitro Analysis of Cardiac Function Enables Machine Learning Classification of Diseases and Drugs. IEEE Open J Eng Med Biol. 2024;5:238-249.

Claims

CLAIMSWhat is claimed is1. A modified calcium / calmodulin-dependent protein kinase II delta beta (CAMK2DB) protein comprising substitution of one or more, two or more, or all three phosphorylatable amino acid residues with a non-phosphorylatable or a non-phosphomimetic amino acid residue, wherein the one or more, two or more, or all three phosphorylatable amino acid residues correspond to amino acids S332, S333, or S334 of SEQ ID NO: 1.

2. The modified protein of claim 1, wherein the non-phosphorylatable amino acid residue is selected from: alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.

3. The modified protein of claim 1, wherein said non-phosphorylatable amino acid residue is alanine.

4. The modified protein of claim 1, wherein the non-phosphorylatable amino acid residue comprises a non-phosphorylatable non-canonical amino acid reside5. The modified protein of claim 1, wherein the non-phosphomimetic amino acid residue comprises a non-phosphorylatable amino acid other than glutamate and aspartate.

6. The modified protein of claim 1, wherein the wherein the substation mutation is selected from: a S332A mutation, a S333A mutation, a S334A mutation, or any combination of a S332A mutation, a S333A mutation, or S334A mutation.

7. The modified protein of any of claim 1-6, or a functional fragment thereof, or a sequence having between 70%-99% or more sequence homology with SEQ ID NO. 1, and wherein positions S332, S333, and / or S334 are conserved.

8. The modified protein of claim 1, wherein the modified CAMK2DB protein comprises a protein with at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or atleast 99% identity to the amino acid sequence according to SEQ ID NO: 1 , or a functional fragment, thereof, and wherein at least one, at least two, or all three of the positions S332, S333, and S334 are conserved.

9. The modified protein of any of claims 1-8, wherein the modified CAMK2DB protein induces nuclear localization, preferably in a cardiac cell.

10. The modified protein of claim 9, wherein the cardiac cell comprise a human cardiac cell.

11. A method of treating heart disease, the method comprising contacting a therapeutically effective amount of the modified CAMK2DB protein of any of claims 1 - 10 with a cardiac cell of a subject in need thereof.

12. The method of claim 11, wherein the subject is a human.

13. The method of claim 11, wherein the step of contacting comprises delivering a heterologous nucleotide sequence, operably linked to a promoter, encoding the modified CAMK2DB protein of any of claims 1 - 10, to the cardiac cell of a subject in need thereof, and further expressing said protein encoded by the nucleotide sequence.

14. A pharmaceutical composition comprising the modified CAMK2DB protein of any of claims 1 - 10, and a pharmaceutically acceptable carrier.

15. A method of treating heart disease, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 14 to a subject in need thereof.

16. The method of claim 15, wherein the subject is a human.

17. An isolated nucleotide sequence encoding the modified CAMK2DB protein of any of claims18. An expression vector encoding the nucleotide sequence of claim 17, operably linked to a promoter.

19. The expression vector of claim 18, wherein the vector comprises a recombinant adeno- associated virus (rAAV) vector.

20. A method of treating heart disease, the method comprising introducing the expression vector of any of claims 18-19 to the cardiac cell of a subject in need thereof, and further expressing the expression vector encoding the modified CAMK2DB protein.

21. A method of treating heart disease, the method comprising introducing the nucleotide sequence of claim 17 to a cardiac cell of a subject in need thereof, and further expressing the nucleotide sequence encoding the modified CAMK2DB protein.

22. A method claim 21, wherein the subject is a human.

23. A method claim 21, wherein the nucleotide sequence comprises a DNA or RNA sequence encoding the modified CAMK2DB protein.

24. A method claim 23, wherein the RNA sequence comprises an mRNA encoding the modified CAMK2DB protein.

25. A recombinant cell expressing the expression vector of any of claim 18-19.

26. A recombinant cell expressing a heterologous nucleotide sequence, operably linked to a promoter, encoding a modified CAMK2DB protein of any of claims 1-10.

27. The recombinant cell of any of claims 25-26, wherein the recombinant cell comprises a cardiac cell.

28. The recombinant cell of any of claims 25-27, wherein the heterologous nucleotide sequence comprises a DNA or RNA sequence encoding the modified CAMK2DB protein.

29. The recombinant cell of claim 28, wherein the RNA sequence comprises an mRNA encoding the modified CAMK2DB protein.

30. The recombinant cell of claim 27, wherein the cardiac cell comprise a human cardiac cell.

31. A composition comprising a gRNA that targets phosphorylation downstream of exon 14 of CAMK2DB, and a base editor.

32. The composition of claim 31, wherein the gRNA disrupts phosphorylation at one or more of positions S332, S333, or S334, according to SEQ ID NO. 1 or a functional fragment, thereof, and wherein at least one, at least two, or all three of the positions S332, S333, and S334 are conserved.

33. The composition of claim 32, wherein the mutation is selected from: a S332A mutation, a S333A mutation, a S334A mutation, or any combination of a S332A mutation, a S333A mutation, or S334A mutation.

34. The composition of claim 31, wherein the base editor is an adenine base editor (ABE).

35. The composition of claim 31, wherein the base editor comprises a CRISPR / Cas nuclease linked to an adenosine deaminase.

36. The composition of claim 35, wherein the CRISPR / Cas nuclease is catalytically impaired.

37. The composition of claim 36, wherein the CRISPR / Cas nuclease is a Cas9 nuclease.

38. The composition of any of claims 31-37, further comprising a second gRNA that targets the splice sites of 14 of human calcium / calmodulin-dependent protein kinase II delta (CAMK2D) such that the gRNA causes increased production of the beta isoform of CAMK2D.

39. A composition comprising a gRNA that targets the splice sites of intron 14 of human calcium / calmodulin-dependent protein kinase II delta (CAMK2D) such that the gRNA causes increased production of the beta isoform of CAMK2D, and a base editor.

40. The composition of claim 39, wherein the base editor is an adenine base editor (ABE).

41. The composition of claim 40, wherein the base editor comprises a CRISPR / Cas nuclease linked to an adenosine deaminase.

42. The composition of claim 41, wherein the CRISPR / Cas nuclease is catalytically impaired.

43. The composition of claim 41, wherein the CRISPR / Cas nuclease is a Cas9 nuclease.

44. The composition of any of claims 39-43, further comprising a second gRNA that targets phosphorylation downstream of exon 14 of CAMK2DB, and a base editor.

45. The composition of claim 44, wherein the second gRNA disrupts phosphorylation at one or more of positions S332, S333, or S334, according to SEQ ID NO. 1 or a functional fragment, thereof, and wherein at least one, at least two, or all three of the positions S332, S333, and S334 are conserved.

46. The composition of claim 45, wherein the mutation comprises a S332A mutation, a S333A mutation, a S334A mutation, or any combination of a S332A mutation, a S333A mutation, and S334A mutation.

47. A nucleic acid comprising a nucleotide sequence encoding a gRNA, operably linked to a promoter, and a second nucleotide sequence encoding a base editor, operably linked to a promoter, wherein the gRNA disrupts phosphorylation downstream of exon 14 of CAMK2DB.I l l48. The nucleic acid of claim 47, wherein the gRNA disrupts phosphorylation at one or more of positions S332, S333, or S334, according to SEQ ID NO. 1 or a functional fragment, thereof, and wherein at least one, at least two, or all three of the positions S332, S333, and S334 are conserved.

49. The nucleic acid of claim 48, wherein the mutation comprises a S332A mutation, a S333A mutation, a S334A mutation, or any combination of a S332A mutation, a S333A mutation, and S334A mutation.

50. The nucleic acid of claim 47, wherein the base editor is an adenine base editor (ABE).

51. The nucleic acid of claim 50, wherein the base editor comprises a CRISPR / Cas nuclease linked to an adenosine deaminase.

52. The nucleic acid of claim 51, wherein the CRISPR / Cas nuclease is catalytically impaired.

53. The nucleic acid of claim 51, wherein the CRISPR / Cas nuclease is a Cas9 nuclease.

54. The nucleic acid of claim 47, wherein the promoter comprises a cell-type specific promoter.

55. The nucleic acid of claim 54, wherein cell-type specific promoter is a cardiac cell-specific promoter.

56. The nucleic acid of claim 47, wherein the nucleotide sequence comprises a DNA sequence.

57. The nucleic acid of claim 47, wherein the nucleotide sequence comprises an RNA sequence.

58. A cell comprising the nucleic acid of any of claims 47-57.

59. The cell of claim 58, wherein the cell is a human cardiac cell.

60. An expression vector comprising the nucleic acid of any of claims 47-57.

61. The expression vector of claim 60, wherein the vector is a non-viral vector.

62. The expression vector of claim 61, wherein the non-viral vector is a plasmid.

63. The expression vector of claim 60, wherein the vector is a viral vector.

64. The expression vector of claim 63, wherein the viral vector is an adeno-associated viral (AAV) vector or an adenoviral vector.

65. The expression vector of claim 64, wherein the AAV vector is replication-defective or conditionally replication defective.

66. The expression vector of claim 64, wherein the AAV vector is a recombinant AAV vector.

67. The expression vector of claim 64, wherein the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), or a combination thereof.

68. A composition comprising the expression vector of any of claims 60-67, and a pharmaceutically acceptable carrier.

69. A method for treating heart disease, the method comprising contacting a cell with a therapeutically effective amount of the pharmaceutical composition of claim 68 or the cell of claim 59 under conditions suitable for expression of the first gRNA and the adenine base editor, wherein the gRNA forms a complex with the adenine base editor, wherein the complex and disrupts phosphorylation of one or more of positions S332, S333, or S334 of CAMK2DB, wherein the position of the one or more mutations corresponds to the CAMK2DB protein encoded by the amino acid sequence according to SEQ ID NO. 1, or a fragment thereof.

70. The method of claim 69, wherein the mutation comprises a S332A mutation, a S333A mutation, a S334A mutation, or any combination of a S332A mutation, a S333A mutation, and S334A mutation.

70. A method of treating heart disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of claim 68 or the cell of claim 59.

71. The method of claim 70, wherein the composition is administered prior to, during, or after LV assist device (LVAD) therapy locally.

72. The method of claim 70, wherein the composition is administered directly to a cardiac tissue.

73. The method of claim 70, wherein the composition is administered by an intramuscular infusion or injection.

74. The method of claim 70, wherein the composition is administered systemically.

75. The method of claim 74, wherein the composition is administered by an intravenous infusion or injection.

76. The method of claim 70, wherein following administration of the composition, the subject exhibits increased nuclear localization of CAMK2DB protein.

77. A method of treating heart disease in a subject in need thereof, the method comprising:- obtaining a biological sample from the subject;- determining the phosphorylation status of positions 332, 333, and 334 of CAMK2DB protein according to SEQ ID NO. 1;- wherein lack of phosphorylation at any of positions 332, 333, and 334 is indicative that the subject will benefit from LV assist device (LVAD) therapy; and- administering the LVAD therapy to the subject with a lack of phosphorylation at any of positions 332, 333, and 334.

78. The method of claim 77, wherein the subject is a human.

79. The method of claim 77, wherein the biological sample is a cardiac cell.

80. The method of claim 77, wherein the positions 332, 333, and 334 of CAMK2DB protein according to SEQ ID NO. 1, comprise S332, S333, and S334.

Images