Arrhythmogenic cardiomyopathy lipid pathogenesis

Abstract

Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease characterized by progressive myocardial fibrofatty infiltration. Compositions in the treatment of fibrofatty accumulation and its pathogenesis in ACM include activators of glycosphingolipid (GSL) glycosyltransferase, β-1,4-galactosyltransferase- V (β-1,4-GalT-V).

Description

PCT APPLICATION ARRHYTHMOGENIC CARDIOMYOPATHY LIPID PATHOGENESIS This application claims the benefit of U.S. provisional application no. 63 / 707,128 filed October 14, 2024, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under grant HL107153 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0002] Arrhythmogenic Cardiomyopathy (ACM) is an inherited heart disease with a prevalence of 1:2,000-1:5,000, although misdiagnosis as myocarditis could underestimate its prevalence.1,2It is characterized by increased mortality in the young, cardiac dysfunction, ventricular wall motion abnormalities and aneurisms, arrhythmias, myocardial inflammation, and progressive fibrofatty accumulation. Interestingly, desmosomal pathogenic gene variants often determine chamber-specific phenotypes.1

[0003] Inheritance is primarily due to an autosomal dominant pattern with reduced penetrance and variable expressivity, though some cases involve an autosomal recessive inheritance pattern.3ACM is often considered a “disease of the cardiac desmosome,” as over 50% of patient cases involve pathogenic variants in desmosomal genes.4Desmosomes, well- organized junctional complexes, are responsible for cell-cell adhesion, integrity, and electrical communication.5Pathogenic variants in genes encoding the desmosomal proteins, plakophilin-2 (PKP2), desmoglein-2 (DSG2), desmocollin-2 (DSC2), desmoplakin (DSP), and junctional plakoglobin (JUP) can lead to desmosome instability.6This instability reduces myocyte-myocyte adhesion and electrical signaling, causing abnormal connexin-43 (Cx43) localization at intercalated discs.6Prior studies have shown reduced Cx43 at the myocyte-myocyte intercalated disc in mice and patients with ACM.7-9This is observed even before overt functional phenotypes develop, as demonstrated in Dsp-deficient mice where early ultrastructural defects in desmosomes occur as early as 2.5 weeks of age.9163089693.1 178013745.1

[0004] Myocardial fibrotic remodeling has been observed in the natural progression of ACM,1yet little is known regarding the specific lipids that accumulate in the heart and / or their synthesis. SUMMARY

[0005] We have now discovered that oxidized-low-density lipoprotein (ox-LDL), β-1,4- Galactosyltransferase-V (β-1,4-GalT-V) / lactosylceramide (LacCer) dyslipidemia-axis are pathogenic contributors to cardiac dysfunction and calcification in ACM. Targeting β-1,4-GalT-V serves as a novel therapeutic strategy in mitigating disease onset and progression in ACM.

[0006] Accordingly, in one aspect, a method of diagnosing and treating a subject suffering from arrhythmogenic cardiomyopathy (ACM) comprises detecting increased amounts of lactosylceramide (LacCer) in a sample from a subject as compared to a normal baseline amount; administering to the subject a therapeutically effective amount of one or more inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, wherein the LacCer amounts in the subject are decreased as compared to a normal baseline amount; thereby diagnosing and treating a subject suffering from ACM.

[0007] In a preferred aspect, a method of diagnosing and treating a subject suffering from arrhythmogenic cardiomyopathy (ACM) comprises detecting increased amounts of lactosylceramide (LacCer) in a sample from a subject as compared to a normal baseline amount; administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of one or more inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, wherein the LacCer amounts in the subject are decreased as compared to a normal baseline amount; thereby diagnosing and treating a subject suffering from ACM.

[0008] In certain embodiments, diagnosing a subject with ACM further comprises detection of increased levels of tumor necrosis α (TNFα), muscle contraction signaling molecules or the combination thereof in a sample from the subject. In certain embodiments, muscle contraction signaling molecules comprise β-catenin, c-Src or the combination thereof. In certain embodiments, the β-galactosyltransferase is lactosylceramide synthase (LacCer synthase).2 178013745.1

[0009] In certain embodiments, an inhibitor of glycosphingolipid (GSL) synthesis, comprises D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (BPD) or analogs thereof. In certain embodiments, the BPD analog comprises D-threo- 1 -(3',4'-ethyleneidoxy) phenyl-2-palmitoylaminod-3 -pyrrolidino- 1 - propanol (EtDO-P4), D-threo-4'-hydroxy-l- phenyl-2-palmitoylamino-3-pyrrolidino-l- propanol (pOH-P4), D-threo-l-(3',4'- trimethylenedioxy)phenyl-2-palmitoylamino-3- pyrrolidino- 1 -propanol (trimethylenedioxy-P4), D-threo- 1 -(3',4'-methylenedioxy)phenyl-2- palmitoylamino-3 -pyrrolidino- 1 -propanol (methylenedioxy-P4), or adamantan-l-yl glucosyl ceramide,(1R,2R) nonanoic acid[2-3-(2',3'- dihydro-benzo[l,4]dioxin-6-'yl)-2-hydroxy-l- pyrrolidin-l-ylmethyl-ethyl]-l amide-L-tartaric acid salt (Genz-1223346). In certain embodiments, the one or more inhibitors of β- galactosyltransferase comprise a small molecule, an antibody, a protein, a peptide, or a nucleic acid. In certain embodiments, the composition comprising inhibitors of glycosphingolipid synthesis includes a therapeutically effective amount of the antibody which specifically binds to β1,4- Galactosyltransferase V (β-1,4-GalT-V), isoforms or peptides thereof. In certain embodiments, the one or more inhibitors are unencapsulated or encapsulated in a biodegradable polymer (BPD). In certain embodiments, the biodegradable polymer consists of polyethylene glycol and sebacic acid.

[0010] In certain embodiments, the subject is administered a pharmaceutical composition comprising a vector encoding a β-1,4-Galactosyltransferase-V (β-1,4-GalT-V), isoforms, mutants or variants thereof. In certain embodiments, the vector is a DNA plasmid. In certain embodiments, the vector comprises an inducible promoter. In certain embodiments the vector comprises a constitutive promoter. In certain embodiments, the vector comprises a tissue-specific promoter. In certain embodiments, the vector comprising β-1,4-GalT-V induces an immune response. In certain embodiments, the immune response induces an antibody response wherein the antibodies bind to β-1,4-GalT-V, thereby reducing the amount of β-1,4-GalT-V. In certain embodiments, the immune response is a T cell mediated response, whereby the immune response induces a specific β-1,4-GalT-V T cell response. In certain embodiments, an adjuvant is administered in combination with the vector encoding β-1,4-GalT-V.

[0011] In another aspect, a composition comprises vector comprising a nucleic acid sequence encoding a β-1,4-Galactosyltransferase-V (β-1,4-GalT-V), mutants or variants thereof.3 178013745.1In certain embodiments, the vector is a DNA plasmid. In certain embodiments, the vector comprises an inducible promoter. In certain embodiments, the vector comprises a constitutive promoter. In certain embodiments, the vector comprises a tissue-specific promoter.

[0012] In another aspect, a method of treating a subject suffering from arrhythmogenic cardiomyopathy (ACM) is provided comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of one or more inhibitors of β- galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, wherein the LacCer amounts in the subject are decreased as compared to a normal baseline amount; thereby diagnosing and treating a subject suffering from ACM. In certain embodiments, the method of treating a subject with ACM further comprises administering one or more inhibitors of tumor necrosis α (TNFα), muscle contraction signaling molecules or the combination thereof in a sample from the subject.

[0013] In certain embodiments, muscle contraction signaling molecules comprise β- catenin, c-Src or the combination thereof. In certain embodiments, the β-galactosyltransferase is lactosylceramide synthase (LacCer synthase). In certain embodiments, an inhibitor of glycosphingolipid (GSL) synthesis, comprises D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol (BPD) or analogs thereof. In certain embodiments, the BPD analog comprises D-threo- 1 -(3',4'-ethyleneidoxy) phenyl-2-palmitoylaminod-3 -pyrrolidino- 1 - propanol (EtDO-P4), D-threo-4'-hydroxy-l-phenyl-2-palmitoylamino-3-pyrrolidino-l- propanol (pOH-P4), D-threo-l-(3',4'-trimethylenedioxy)phenyl-2-palmitoylamino-3- pyrrolidino- 1 - propanol (trimethylenedioxy-P4), D-threo- 1 -(3',4'-methylenedioxy)phenyl-2- palmitoylamino-3 -pyrrolidino- 1 -propanol (methylenedioxy-P4), or adamantan-l-yl glucosyl ceramide,(1R,2R) nonanoic acid[2-3-(2',3'-dihydro-benzo[l,4]dioxin-6-'yl)-2-hydroxy-l- pyrrolidin-l-ylmethyl- ethyl]-l amide-L-tartaric acid salt (Genz-1223346). In certain embodiments, the one or more inhibitors of β-galactosyltransferase comprise a small molecule, an antibody, a protein, a peptide, or a nucleic acid. In certain embodiments, the one or more inhibitors are unencapsulated or encapsulated in a biodegradable polymer (BPD). In certain embodiments, the biodegradable polymer consists of polyethylene glycol and sebacic acid. In certain embodiments, the subject suffers from myocardial fibrosis. In certain embodiments, administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of one or more4 178013745.1inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, treats myocardial fibrosis.

[0014] In another aspect, a method of determining efficacy of one or more agents in the treatment of arrhythmogenic cardiomyopathy (ACM) comprises monitoring levels of lactosylceramide (LacCer), oxidized-low-density lipoprotein (ox-LDL), tumor necrosis factor alpha (TNFα), proto-oncogene tyrosine-protein kinase Src (c-Src), β-catenin, β-1,4- Galactosyltransferase-V (β-1,4-GalT-V), or combinations thereof, in a sample from a subject as compared to a normal baseline amount; wherein the levels of LacCer, ox-LDL, TNFα, c-Src, β- catenin, or β-1,4-GalT-V amounts in the subject are decreased as compared to a normal baseline amount; thereby determining efficacy of one or more agents in the treatment of ACM. In certain embodiments, the agent comprises vectors small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, Fab, Fab’, F(ab’)2 fragments, Fv fragments, single chain antibodies, antibody mimetics, peptoids, aptamers, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds.

[0015] In another aspect, a kit for diagnosing arrhythmogenic cardiomyopathy (ACM) comprises one or more agents detecting levels of lactosylceramide synthase (LacCer), tumor necrosis α (TNFα), β-catenin, c-Src or combinations thereof. In certain embodiments, the kit further comprises a pharmaceutical composition comprising one or more inhibitors of β- galactosyltransferases, one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof.

[0016] We also have now found that C24:0 and C24:1 fatty acid molecular species of LacCer are consistently increased in not only in human from myocardium (human myocardium tissue sample) but also in multiple ACM mutant mice myocardium(Dsg2 and DSp2).

[0017] We thus have now identified diagnostic biomarkers for arrhythmogenic cardiomyopathy (ACM). In aspects, C24:0 LacCer and / or C24:1 LacCer and optionally in combination one or more additional biomarkers including ox-LDL and genotyping can be used to diagnose ACM in subjects (including newborn subjects) as well as assess pathology of disease progression and therapy targeting GalT-V. A newborn subject includes a person that are less than 12, 11, 10, 8, 6, 4, 2 or 1 month in age.5 178013745.1

[0018] More particularly, in aspects, a method of diagnosing and treating arrhythmogenic cardiomyopathy (ACM) is provided that comprises: a) assaying to identify C24:0 LacCer and / or C24:1 LacCer in a sample obtained from a subject; b) diagnosing the subject as having arrhythmogenic cardiomyopathy (ACM) when the C24:0 / C24:1 LacCer level is different (e.g. increased) relative to the C24:0 / C24:1 LacCer level in a healthy subject; and optionally c) administering a therapy in subjects diagnosed with arrhythmogenic cardiomyopathy; thereby, diagnosing and treating a subject.

[0019] Accordingly, in a further aspect a method for diagnosing and / or treating for arrhythmogenic cardiomyopathy (ACM) in a subject suffering from or suspected to suffer from arrhythmogenic cardiomyopathy (ACM) is provided, the methos suitably comprising the steps of: a) determining the presence and / or amount of C24:0 LaCer and / or C24:1 LacCer in a sample of the subject; b) comparing the presence or determined amount to a reference; and c) diagnosing arrhythmogenic cardiomyopathy (ACM) based on the comparison to reference.

[0020] The diagnosed or identified subject also may be treated for arrhythmogenic cardiomyopathy (ACM). Assaying to identify C24:0 LacCer and / or C24:1 LacCer in a sample (e.g. myocardium tissue sample) obtained from a subject and determining the presence and / or amount of C24:0 LaCer and / or C24:1 LacCer in the sample of the subject can be performed by known methods including chromatography or spectroscopy (including mass spectroscopy of a sample). Such methods including both chromatography and spectroscopy can provide determinative including quantitative amounts of C24:0 LaCer and / or C24:1 LacCer in a test sample, including relative to amounts of C24:0 LaCer and / or C24:1 LacCer in a control sample (e.g. sample (e.g. myocardium tissue sample) from a subject known not to have or be susceptible to ACM). See Example 3 which follows for an exemplary diagnostic assay of C24:0 LaCer and / or C24:1 LacCer in a test sample.6 178013745.1

[0021] As referred to herein, C24:0 LacCer refers to C24:0 Lactosyl(β) Ceramide which is also known as D-lactosyl-β-1,1′ N-lignoceroyl-D-erythro-sphingosine, and in aspects may be characterized by molecular weight 974.39, CAS number 105087-85-2. The IUPAC name of C24:0 LacCer is (15Z)-N-[(2S,3R,4E)-1-{[4-O-(β-D-Galactopyranosyl)-β-D- mannopyranosyl]oxy}-3-hydroxy-4-octadecen-2-yl]-15-tetracosenamide. References to C24:0 LacCer also can include metabolized or other in vivo forms of C24:0 LacCer (such metabolized or other in vivo forms referred to as modified C24:0 LacCer ).

[0022] As referred to herein, C24:1 LacCer refers to C24:1 Lactosyl(β) Ceramide which is also known as D-lactosyl-β11’-N-nervonyl-D-rythro-sphingosine, , and in aspects may be characterized by molecular weight 972.38, Cas number 48330-7-1. References to C24:1 LacCer also can include metabolized or other in vivo forms of C24:1 LacCer (such metabolized or other in vivo forms referred to as modified C24:1 LacCer ).

[0023] We have found that the levels of C24:0 and / or C24:1 LacCer are markedly and consistently increased in human ACM myocardium and is also observed in myocardium from two mouse models of ACM. Without being bound by any theory, an increased level of C24:0 LaCer and / or C24:1 LacCer in ACM myocardium may point to a mechanistic explanation of an increased “oxidative stress “environment leading to produce more superoxides that can cross the cell membrane and oxidize LDL to form oxidized LDL. And that in turn can bind to its cognate receptor LOX-1 and downstream activate B -1,4GalT-V to generate more LacCer. Moreover, this cycle can be broken by inhibiting the activity of B-1,4GalT-V.

[0024] In aspects, the present methods provide that C24:0 LacCer and / or C24:1 LacCer can be used as one of the diagnostic biomarkers in ACM and in preferred aspects along with the measurement of oxidized LDL e.g. in blood and genotyping.

[0025] In particular results, LacCer fatty acid molecular species (FAMS) were assessed between ACM patient myocardium with healthy myocardium as well as two ACM mutant mice myocardium with wild type mice myocardium. It was found that the level of C24:0 LaCer and / or C24:1 LacCer is consistently increased for example 2-5 fold, respectively in mouse myocardium and human myocardium compared with healthy myocardium. We also noted that the level of C16:0 LacCer and C22:0 LacCer were also increased 3-8 fold only in human ACM myocardium and Dsg2 mice myocardium but not in Dsp2 mice myocardium. In contrast, the7 178013745.1level of several FAMS of Ceramides was decreased and glucosylceramide remained unchanged in human ACM myocardium compared to healthy myocardium. However, it was found that the level of C26:0 sphingomyelin increased in human ACM myocardium.

[0026] Definitions

[0027] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0028] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0029] Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a8 178013745.1range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0001] As used herein, an “adjuvant” refers to a substance that enhances the body's immune response to an antigen or a vaccine and may be added to the formulation that includes the immunizing agent. Adjuvants provide enhanced immune response even after administration of only a single dose of the vaccine. Adjuvants may include, for example, aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), non-metabolizable oil, mineral and / or plant / vegetable and / or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsion, oil- in-water emulsion, water-in-oil-in-water emulsion, HRA-3 (acrylic acid saccharide cross-linked polymer), HRA-3 with cottonseed oil (CSO), or an acrylic acid polyol cross-linked polymer. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri- (caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers comprise nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the PLURONICTMbrand products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.) John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). In a preferred embodiment the adjuvant is at a concentration of about 0.01 to about 50%, at a concentration of about 2% to 30%, at a concentration of about 5% to about 25%,9 178013745.1at a concentration of about 7% to about 22%, and at a concentration of about 10% to about 20% by volume of the final product. Examples of suitable adjuvants are described in U.S. Patent Application Publication No. US2004 / 0213817 A1. “Adjuvanted” refers to a composition that incorporates or is combined with an adjuvant.

[0030] As used herein, the term “agent” or “candidate therapeutic agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a dysfunction or other medical condition. The term includes small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fab’, F(ab’)2fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies), peptoids, aptamers; enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the disclosure at any stage during clinical trials, during pre-trial testing, or following FDA-approval.

[0031] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a dysfunction.

[0032] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0033] As used herein, the term “antibody” is used in reference to any immunoglobulin molecule that reacts with a specific antigen. It is intended that the term encompass any immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents, non-human primates, caprines, bovines, equines, ovines, etc.). Specific types / examples of antibodies include polyclonal, monoclonal, humanized, chimeric, human, or otherwise- human-suitable antibodies. “Antibodies” also includes any fragment or derivative of any of the herein described antibodies. In specific embodiments, antibodies may be raised against glycosyltransferases (e.g., glucosylceramide synthase and lactosylceramide synthase) and used as glycolipid synthesis inhibitors. In other embodiments, antibodies may be raised against10 178013745.1glycoyltransferase protein / peptides that have undergone post-translational modification(s) due to cardiac hypertrophy.

[0034] By “antisense oligonucleotides” or “antisense compound” is meant an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA. An antisense oligonucleotide can upregulate or downregulate expression and / or function of a particular polynucleotide. The definition is meant to include any foreign RNA or DNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, short, hairpin RNA (shRNA), therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0035] As used herein, the term “cardiac hypertrophy” is used in its ordinary meaning as understood by the medical community. It generally refers to the process in which adult cardiac myocytes respond to stress through hypertrophic growth. Such growth is characterized by cell size increases without cell division or proliferation, assembling of additional sarcomeres within the cell to maximize force generation, and an activation of a fetal cardiac gene program. Cardiac hypertrophy is often associated with increased risk of morbidity and mortality, and thus studies aimed at understanding the molecular mechanisms of cardiac hypertrophy could have a significant impact on human health.

[0036] As used herein, the term “cardiovascular disease” refers to diseases and disorders of the heart and circulatory system. Exemplary cardiovascular diseases, including cholesterol- or lipid-related disorders, include, but are not limited to arrhythmogenic cardiomyopathy (ACM), acute coronary syndrome, angina, arteriosclerosis, atherosclerosis, carotid atherosclerosis, cerebrovascular disease, cerebral infarction, congestive heart failure, congenital heart disease,11 178013745.1coronary heart disease, coronary artery disease, coronary plaque stabilization, dyslipidemias, dyslipoproteinemias, endothelium dysfunctions, familial hypercholeasterolemia, familial combined hyperlipidemia, hypoalphalipoproteinemia, hypertriglyceridemia, hyperbetalipoproteinemia, hypercholesterolemia, hypertension, hyperlipidemia, intermittent claudication, ischemia, ischemia reperfusion injury, ischemic heart diseases, cardiac ischemia, metabolic syndrome, multi-infarct dementia, myocardial infarction, obesity, peripheral vascular disease, reperfusion injury, restenosis, renal artery atherosclerosis, rheumatic heart disease, stroke, thrombotic disorder, and transitory ischemic attacks.

[0037] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements--or, as appropriate, equivalents thereof-and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0038] As used herein, a “controlled release dosage formulation” refers to a formulation of a drug that offers prolonged release at a specific controllable rate.

[0039] By “effective amount” or “therapeutically effective amount” is the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. Determination of a therapeutically effective amount, as well as other factors related to effective administration of a compound of the present invention to a subject of this invention, including dosage forms, routes of administration, and frequency of dosing, may depend upon the particulars of the condition that is encountered, including the subject and condition being treated or addressed, the severity of the condition in a particular subject, the particular compound being employed, the particular route of administration being employed, the frequency of dosing, and12 178013745.1the particular formulation being employed. Determination of a therapeutically effective treatment regimen for a subject of this invention is within the level of ordinary skill in the medical or veterinarian arts. In clinical use, an effective amount may be the amount that is recommended by the U.S. Food and Drug Administration, or an equivalent foreign agency. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the subject being treated and the particular mode of administration.

[0040] As used herein, the term “gene editing complex” refers to any complex such as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases or meganucleases. Each of these complexes can be targeted to specific sites by use of guide nucleic acid sequences, e.g. gRNAs, that are complementary to a desired target sequence. For example, a gene editing complex such as CRISPR comprises two RNAs and one protein component: a CRISPR RNA (crRNA), a short RNA that undergoes complementary binding with the foreign DNA; a tracrRNA that hybridizes with the crRNA; and a Cas9 enzyme that interacts with the DNA:RNA complexes and cleaves the DNA at a specific site.

[0041] The term “high affinity” for an antibody refers to an antibody having a KD of lxlO7M or less, more preferably 5xl08M or less, even more preferably lxl08M or less, even more preferably 5xl09M or less and even more preferably lxl09M or less for a target antigen. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype refers to an antibody having a KD of 106M or less, 107M or less, or 108M or less.

[0042] As used herein, the term “in combination” in the context of the administration of a therapy to a subject refers to the use of more than one therapy for therapeutic benefit. The term “in combination” in the context of the administration can also refer to the prophylactic use of a therapy to a subject when used with at least one additional therapy. The use of the term “in combination” does not restrict the order in which the therapies (e.g., a first and second therapy) are administered to a subject. A therapy can be administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 4813 178013745.1hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject which had, has, or is susceptible to cancer. The therapies are administered to a subject in a sequence and within a time interval such that the therapies can act together. In a particular embodiment, the therapies are administered to a subject in a sequence and within a time interval such that they provide an increased benefit than if they were administered otherwise. Any additional therapy can be administered in any order with the other additional therapy.

[0043] As used herein, an “inhibitor” of glycosphingolipid synthesis or of glucosylceramide synthesis inhibits the synthesis of these molecules including those associated in the cycle of the synthesis. The inhibition of synthesis of these molecules can be measured by any standard assay. See, for example, the methods in the examples section which follows.

[0044] As used herein, “inhibition” or “decrease” of β1 ,4-Galactosyltransl'erase V reduces the amount of b 1 ,4-Galactosyltransl'erase V in the cell by greater than about 20%, 40%, 60%, 80%, 85%, 90%, 95%, or 100%. The amount of bl,4-Galactosyltransferase V can be determined by well-known methods including, but are not limited to, densitometer, fluorometer, radiography, luminometer, antibody-based methods and activity measurements.

[0045] The term “inhibit,” “diminish,” “reduce” or “suppress” refers to a decrease in the specified parameter (e.g., at least about a l.l-fold, l.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 8-fold, l0 fold, twelve-fold, or even fifteen-fold or more increase) and / or a decrease or reduction in the specified activity of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%. These terms are intended to be relative to a reference or control.

[0046] The term “KasSoc” or “Ka,” as used herein, is intended to refer to the association rate of a particular antibody- antigen interaction, whereas the term “KdiS” or “Kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term14 178013745.1“KD,” as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kdto Ka(i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, for example, using a biosensor system such as a BIACORE™ system.

[0047] As used herein, “modulate,” “modulates” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., diminished, reduced or suppressed) of the specified activity.

[0048] As used herein, “nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides, ribonucleotides, and 2’-modified nucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness.

[0049] As used herein, the terms “prevent,” “preventing” and “prevention” in the context of the administration of a therapy to a subject refer to the prevention or inhibition of the recurrence, onset, and / or development of a disease or disorder or a symptom thereof in a subject resulting from the administration of a therapy (e.g., a prophylactic agent), or a combination of therapies (e.g., a combination of prophylactic agents).

[0050] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.15 178013745.1

[0051] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific or cell specific manner. A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0052] By “reduces” or “inhibits” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100% as compared to a reference.

[0053] By “reference” is meant a standard or control condition.

[0054] As used herein, an antibody that “specifically binds” to a polypeptide or epitope is intended to refer to an antibody that binds to a polypeptide or epitope with a KD of lxl07M or less, or 5xl08M or less, or 3xl08M or less, more preferably lxl08M or less, or 5xl09M or less. Therefore, the terms “specific binding” or “specifically binding” when used in reference to the interaction of a protein and an antibody or alternative protein scaffold or peptoid or aptamers, means that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. Thus, an antibody that” specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular16 178013745.1polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0055] As used herein, a “sustained release dosage formulation” is a formulation of a drug designed to release the drug at a predetermined rate in order to maintain a constant drug concentration for a specific period of time with minimum side effects. Optionally, the period of time is 30 minutes or more, e.g., 2-4 hours or more, e.g., 3-8 hours or more, e.g., 4-24 hours or more, e.g., 1-3 days or more, e.g., 2-7 days or more, e.g., 4-14 days or more, e.g., 7 days or more, e.g., 14 days to a month or more.

[0056] As used herein, a “subject” or “patient” means an individual and can include domesticated animals, (e.g., cats, dogs, etc.); livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds. In one aspect, the subject is a mammal such as a primate or a human. In particular, the term also includes mammals diagnosed with cardiac hypertrophy.

[0057] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse affect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease.

[0058] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Concentrations, amounts, cell counts, percentages and other numerical values may be presented herein in a range format. It is to be understood that such17 178013745.1range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0059] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0061] FIGS. 1A-1D are a series of plots demonstrating that LacCer myocardial levels are markedly increased in patients with ACM. FIGS. 1A-1D: Ceramide (Cer) and isoform- specific ceramides. FIG. 1C: Note, increased levels of LacCer in ACM myocardium compared to healthy controls. FIGS. 1A, 1B, 1D: Conversely, ceramide, glucosylceramide (GlcCer), and sphingomyelin (SM) levels were similar in myocardium samples from patients with ACM compared to healthy controls. Samples were analyzed in triplicate and measured in picomoles per mg tissue. *P<0.05 via two-tailed parametric t-test (n=4 / group); Data presented as mean±SEM.

[0062] FIGS. 2A-2F are a series of graphs, plots and an HPTLC plate demonstrating increased myocardial LacCer levels correlated with reduced cardiac function in Dsg2mut / mutmice. FIG. 2A: Representative HPTLC plate showing separation of myocardial samples from 16- week-old Dsg2mut / mutand wild-type (WT) hearts. Glycosphingolipid standards were run and separated simultaneously and served as positive (+) controls. FIG 2B: Reduced levels of myocardial LacCer in WT controls compared to Dsg2mut / mutmice. FIGS. 2C, 2D: Note, reduced percent ejection fraction (%EF) and fractional shortening (%FS) in ACM mice. FIGS. 2E, 2F: Cardiac function showed a strong, inverse relationship with LacCer levels. For B-D, a two-tailed parametric t-test was utilized. ***P<0.001; ****P<0.0001. E, F, used Pearson’s r correlation test; P-value inset. Data presented as mean±SEM; n=4 mice / group / parameter.18 178013745.1

[0063] FIGS. 3A-3E are a series of graphs demonstrating that elevated levels of plasma ox-LDL, myocardial LacCer synthase activity, and cardiac dysfunction are normalized in BPD- treated Dsg2mut / mutmice. FIG. 3A: plasma oxidized-LDL (Ox-LDL) levels were markedly increased in both DspS311A / S311Aand Dsg2mut / mutmice, the latter of which was attenuated after BPD treatment. FIG. 3B: LacCer synthase activity is increased in Dsg2mut / muthearts compared to WT controls, yet FIG. 3C, β-galactosidase activity was not. Treatment with 10mg / kg of the GSL synthase inhibitor (BPD) improved FIG. 3D, percent (%) ejection fraction, yet not FIG. 3E, fractional shortening in Dsg2mut / mutmice. Solid bar graphs, prior to treatment; hatched bar graphs, post treatment. Data presented as mean±SEM, n≥3 / group / parameter, *P<0.05, **P<0.01, ***P<0.001 using FIGS.3A-3C, One-way ANOVA and FIGS. 3D, 3E two-tailed parametric test.

[0064] FIGS. 4A-4F are a series of immunostains and plots demonstrating the reduced myocardial fibrosis, CD31 / PECAM-1, and junction plakoglobin (JUP) expression in Dsg2mut / mutmice following BPD treatment. FIG. 4A, 4D: Representative myocardium immunostained for Mason’s Trichrome, (FIGS. 4B, 4E) CD31, and (FIGS. 4C, 4F) JUP from placebo-and BPD- treated Dsg2mut / mutmice. Images are representative of n=4 mice / immunostain / cohort. (FIGS. 4E, 4F) Scale bar, 100µm. Data presented as mean ±SD, *P<0.05, **P<0.01, ***P<0.001,****P<0.00001 using (FIGS. 4A-4C) One-way ANOVA and (FIGS. 4D, 4E) two- tailed parametric test.

[0065] FIGS. 5A-5D are a series of images from in vivo18F-NaF PET / MRI and ex vivo BoneTag® NIRF and x-ray of murine hearts in placebo- BPD-treated mice. Representative images from FIG. 5A, PET / MRI, FIG. 5B, T1 MRI, FIG. 5C, ex vivo BoneTag NIRF, and FIG. 5D, Faxitron x-ray showed bright areas of calcification (white arrows). PET uptake and x-ray showed areas of ossified calcification in and around hearts of Placebo-treated mice.

[0066] FIG. 6 is a schematic depicting the hypothetical biochemical mechanism by which oxidized-low density lipoprotein (Ox-LDL) activates lactosylceramide synthase (β-1,4- GalT-V) to produce LacCer. In turn, LacCer activates multiple cell signaling pathways leading to cell junction proteins, such as platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular cell adhesion molecule-1 (ICAM-1), c-Src, and β-catenin contributing to cardiac19 178013745.1dysfunction and calcification. Treatment with a glycosyltransferase inhibitor (BPD) decreases β- 1,4-GalT-V activity and ox-LDL levels, leading to suppression of further cardiac dysfunction and myocardial calcification in a mouse model of ACM.

[0067] FIGS. 7A-7C are plots demonstrating that the levels of LacCer level were markedly increased in Dsg2mut / mutmouse myocardium. FIGS. 7A, 7B: Levels of ceramide and GlcCer species were comparable between Dsg2mut / mutand wild-type (WT) mice. FIG. 7C: Dsg2mut / mutmice displayed substantially elevated myocardial levels of LacCer than WT controls. Data presented as mean±SEM; n=3 WT and n=17 Dsg2mut / mutmice; *****P<.00005 via two- tailed t-test.

[0068] FIGS. 8A-8C are plots demonstrating the increased levels of LacCer species in DspS311A / S311Amouse myocardium. FIGS. 8A, 8B: Ceramide and GlcCer levels were comparable between DspS311A / S311Aand wild-type (WT) controls. FIG. 8C: DspS311A / S311Amice displayed elevated myocardial levels of LacCer compared to WT controls. Data presented as mean±SEM; n=5 mice / cohort; *P<.05 via two-tailed paired t-test.

[0069] FIGS. 9A-9D are plots demonstrating that treatment with Biopolymer- encapsulated-D-PDMP in Dsg2mut / mutmice (BPD) drastically decreased plasma levels of TNF-α, β-catenin, and C-Src. FIGS. 9A-9D: Enzyme-linked immunosorbent assays were performed on plasma samples from Dsg2mut / mutmice treated with or without BPD. FIGS. 9A-9C: Placebo- treated Dsg2mut / mutmice showed increased plasma levels of TNF-α, β-catenin, and C-Src compared to BPD-treated Dsg2mut / mutmice. FIG. 9D: Plasma levels of VE-Cadherin were not significantly different between BPD-treated and placebo-treated Dsg2mut / mutmice. All samples were analyzed in triplicate. Data presented as mean ±SEM; n=3-5 / cohort / parameter, *P<0.05, **P<0.01 via One-way ANOVA with Kruskal-Wallis comparisons.

[0070] FIGS. 10A-10D shows treatment with Biopolymer-encapsulated-D-PDMP in Dsg2mut / mutmice (BPD) drastically decreased TNF-α, β-catenin, and C-Src levels. In FIGS. 10A- D, Enzyme-linked immunosorbent assays were performed on serum samples from Dsg2mut / mutmice treated with or without BPD. In FIGS. 10A-C, Placebo-treated Dsg2mut / mutmice showed increased plasma levels of TNF-α, β-catenin and C-Src compared to BPD-treated Dsg2mut / mut20 178013745.1mice. FIG.10D: Plasma levels of VE-Cadherin were not significantly different between BPD- treated and placebo-treated Dsg2mut / mutmice. All samples were analyzed in triplicate. Data presented as mean±SEM; n=3-5 / cohort / parameter, *P<0.05, **P<0.01 via One-way ANOVA with Kruskal-Wallis comparisons.

[0071] FIG. 11 (includes FIGS. 11A-11C) shows C24:0 LactosylCeramide1 fatty acid molecular species common observation between Human and mouse heart. FIG. 11A shows Human ACM heart versus healthy heart; FIG.11B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 11C shows Dsp2 mutant mice versus healthy heart.

[0072] FIG. 12 (includes FIGS. 12A-12C) shows C24:1 LactosylCeramide1 fatty acid molecular species common observation between Human and mouse heart. FIG. 12A shows Human ACM heart versus healthy heart; FIG.11B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 11C shows Dsp2 mutant mice versus healthy heart.

[0073] FIG. 13 (includes FIGS. 13A-13C) shows C16:0 and C22:0 LactosylCeramide1 fatty acid molecular species common observation between Human and mouse heart. FIG. 13A shows Human heart versus healthy heart; FIG.11B shows Dsg2 ACM mutant mice versus healthy heart; FIG. 13C shows Dsp2 ACM Italian mutant mice hear ( .

[0074] FIG. 14 (includes FIGS. 14A-14C) shows C24:0 Glucosylceramide fatty acid molecular species common observation between Human and mouse heart. FIG. 14A shows Human ACM heart versus healthy heart; FIG.14B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 14C shows Dsp2 mutant mice versus healthy heart.

[0075] FIG. 15 (includes FIGS. 15A-15B) shows C24:1 Glucosylceramide fatty acid molecular species common observation between Human and mouse heart. FIG. 15A shows Human ACM heart versus healthy heart; and FIG.15B shows Dsg2 ACM mutant mice versus healthy heart.

[0076] FIG. 16 (includes FIGS. 16A-16C) shows C24:0 Ceramide fatty acid molecular species common observation between Human and mouse heart. FIG. 16A shows Human ACM heart versus healthy heart; FIG.16B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 16C shows Dsp2 mutant mice versus healthy heart.

[0077] FIG. 17 (includes FIGS. 17A-17C) shows C24:1 Ceramide fatty acid molecular species common observation between Human and mouse heart. FIG. 17A shows Human ACM21 178013745.1heart versus healthy heart; FIG.17B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 17C shows Dsp2 mutant mice versus healthy heart.

[0078] FIG. 18 (includes FIGS. 18A-18C) shows Ceramide fatty acid molecular species common observation between Human and mouse heart. FIG. 18A shows Human ACM heart versus healthy heart; FIG.18B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 18C shows Dsp2 mutant mice versus healthy heart.

[0079] FIG. 19 (includes FIGS. 19A-19C) shows C24:0 SM Sphingomyelin fatty acid molecular species common observation between Human and mouse heart. FIG. 19A shows Human ACM heart versus healthy heart; FIG.19B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 19C shows Dsp2 mutant mice versus healthy heart.

[0080] FIG. 20 (includes FIGS. 20A-209C) shows C16:0 SM Sphingomyelin fatty acid molecular species common observation between Human and mouse heart. FIG. 20A shows Human ACM heart versus healthy heart; FIG. 20B shows Dsg2 ACM mutant mice versus healthy heart; and FIG. 20C shows Dsp2 mutant mice versus healthy heart. DETAILED DESCRIPTION

[0081] In the studies herein, LacCer was a major lipid component in the fibro-fatty deposits in ACM in human and mouse hearts. LacCer serves as a central bioactive lipid second messenger, which transduces oxidized LDL-mediated calcification, cardiac dysfunction, and inflammation in the ACM heart. Additionally, these studies identified LacCer synthase as a therapeutic target to pharmacologically mitigate cardiac dysfunction and calcification in a mouse model of ACM.

[0082] Glycosphingolipids (GSLs) are a family of lipids composed of ceramide, which has its non-polar head group buried in the cell membrane, while the sugar moieties (e.g., glucose and galactose) project out of the cell membrane.10Prior studies have shown the lipoproteins, low-density (LDL) and high-density lipoproteins (HDL), are elevated in hyperlipidemic patients and are primary carriers of circulatory GSLs.10,11Increased levels of oxidized-LDL (ox-LDL), ox-phospholipids, and GSLs in atherosclerotic plaques is well known.12In cultured human arterial cells, ox-LDL and ox-phospholipids were shown to activate mitogen-activated protein22 178013745.1kinase, that, in turn, phosphorylated β-1,4-GalT-V at serine, threonine, and tyrosine residues, generating LacCer and inducing cell proliferation.13-15In sum, GSLs have been shown to play important roles in atherosclerosis and vascular biology by regulating critical signaling pathways and phenotypes such as cell proliferation, adhesion, immune cell migration, angiogenesis, apoptosis, and cardiac hypertrophy.16

[0083] Calcification of the aortic valve is well-known to be an age-related disease.17Furthermore, differentiation of valvular interstitial cells into osteoblasts contributes to the retention of calcium (Ca2+) and the formation of calcified lesions.18Additionally, vascular calcification and atherosclerosis, due to hypercholesterolemia and increased levels of ox-LDL and ox-phospholipids, has been shown to contribute to the mineralization of the aorta.19

[0084] According to the techniques herein, detection of increased activity of LacCer synthase was strongly correlated with cardiac dysfunction. This activity can be blocked by inhibitors of glycosyltransferase, for example, D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol (BPD), which dose-dependently decreased myocardial LacCer synthase activity and amounts.

[0085] Accordingly, in one aspect, a method of diagnosing and treating a subject suffering from arrhythmogenic cardiomyopathy (ACM) comprises detecting increased amounts of lactosylceramide (LacCer) in a sample from a subject as compared to a normal baseline amount; administering to the subject a pharmaceutical composition comprising a therapeutically effective amount one or more inhibitors of β-galactosyltransferases, one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, wherein the LacCer amounts in the subject are decreased as compared to a normal baseline amount; thereby diagnosing and treating a subject suffering from ACM. In certain embodiments, an inhibitor of glycosphingolipid (GSL) synthesis, comprises D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (BPD) or analogs thereof. In certain embodiments, the BPD analog comprises D-threo- 1 -(3',4'- ethyleneidoxy) phenyl-2-palmitoylaminod-3 -pyrrolidino- 1 - propanol (EtDO-P4), D-threo-4'- hydroxy-l-phenyl-2-palmitoylamino-3-pyrrolidino-l- propanol (pOH-P4), D-threo-l-(3',4'- trimethylenedioxy)phenyl-2-palmitoylamino-3- pyrrolidino- 1 -propanol (trimethylenedioxy-P4), D-threo- 1 -(3',4'-methylenedioxy)phenyl-2- palmitoylamino-3 -pyrrolidino- 1 -propanol23 178013745.1(methylenedioxy-P4), or adamantan-l-yl glucosyl ceramide,(1R,2R) nonanoic acid[2-3-(2',3'- dihydro-benzo[l,4]dioxin-6-'yl)-2-hydroxy-l- pyrrolidin-l-ylmethyl-ethyl]-l amide-L-tartaric acid salt (Genz-1223346). In certain embodiments, the one or more inhibitors of β- galactosyltransferase comprise a small molecule, an antibody, a protein, a peptide, or a nucleic acid. In certain embodiments, the composition comprising inhibitors of glycosphingolipid synthesis includes a therapeutically effective amount of the antibody which specifically binds to β1,4- Galactosyltransferase V (β-1,4-GalT-V), isoforms or peptides thereof. In certain embodiments, the one or more inhibitors are unencapsulated or encapsulated in a biodegradable polymer (BPD). In certain embodiments, the biodegradable polymer consists of polyethylene glycol and sebacic acid.

[0086] In certain embodiments, a pharmaceutical composition comprises a therapeutically effective amount of one or more inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof.

[0087] In certain embodiments, the glycosphingolipid synthesis inhibitor is D-threo-l- Phenyl-2-decanoylamino-3- morpholino-l-propanol HC1 (D-PDMP). D-PDMP is a glucosylceramide synthase and lactosylceramide synthase inhibitor. D-PDMP includes a molecular weight of 427.1 and is soluble in water. A group of inhibitors have been developed using PDMP as the lead compound. The parent compound of the new group of inhibitors, D- threo-l-phenyl-2-palmitoylamino-3-pyrrolidino-l-propanol (P4) can be used in the present disclosure. More specifically, the P4 analogs, D-threo-l-(3',4'- ethyleneidoxy) phenyl-2- palmitoylaminod-3-pyrrolidino-l-propanol (EtDO-P4) and D-threo- 4' -hydroxy- l-phenyl-2- palmitoylamino-3-pyrrolidino-l-propanol (pOH-P4) can be used (effective concentration (ID50) of about 0.1 μΜ). Other P4 derivatives include D-threo-1- (3 ' ,4 '-trimethylenedioxy)phenyl-2- palmitoylamino-3-pyrrolidino- 1 -propanol (trimethylenedioxy-P4), D-threo- 1 -(3 ' ,4 '- methylenedioxy)phenyl-2-palmitoylamino-3- pyrrolidino-l-propanol (methylenedioxy-P4). Dosages of P4 derivatives including EtDO-P4 are easily determined by the skilled artisan. Such dosages may range from about 0.5 mg / kg to about 50 mg / kg, from about 1 mg / kg to about 10 mg / kg by intraperitoneal or equivalent administration from one to five times daily. Such dosages may range from about 5 mg / kg to about 5 g / kg, from about 10 mg / kg to about 1 g / kg by oral or24 178013745.1equivalent administration from one to five times daily. A certainly oral dose range for a P4-like compound is from about 6 mg / kg / day to about 600 mg / kg / day.

[0088] In other embodiments, the glycosphingolipid synthesis inhibitor is l,5- (butylimino)-l,5- dideoxy-D-glucitol (also known as N-butyldeoxynojirimycin (NB-DNJ), Miglustat or Zavesca®) (effective concentration (ID5o of about 10-100 μΜ) (Actelion Pharmaceuticals U.S., Inc. (San Francisco, CA)). NB-DNJ is an inhibitor of glucosylceramide synthase, a glucosyl transferase enzyme that plays a role in the synthesis of many glycosphingo lipids. Miglustat is soluble in water. The molecular formula for Miglustat is C10H21NO4and has a molecular weight of 219.28. In other specific embodiments, N-nonyldeoxynojirimycin (NN- DNJ) can be used. In yet another embodiment, the galactose analogue N-butyl- deoxygalatconojirimycin (NB-DGJ) can be used. Another certain deoxynojirimycin derivative for use in the combination therapies of the disclosure is N-(5-adamantane-l-yl- methoxy)pentyl)- deoxynojirimycin (AMP-DNJ or AMP-DNM). Other inhibitors include adamantan-l-yl glucosyl ceramide,(lR,2R) nonanoic acid[2-3-(2',3'-dihydro- benzo[l ,4]dioxin-6-'yl)-2-hydroxy-l- pyrrolidin-l-ylmethyl-ethyl]-l amide-L-tartaric acid salt (Genz-1223346). Dosages of DNJ derivatives including NB-DNJ, NB-DGJ, AMP-DNJ in are also readily determined by the skilled artisan. Such dosages may range from about 0.01 mg / kg to about 1000 mg / kg, from about 0.1 mg / kg to about 100 mg / kg, more specifically from about 1 mg / kg to about 10 mg / kg, by intraperitoneal or equivalent administration from one to five times daily. Such dosages, when administered orally, may range from two- to twenty- fold greater. For example, NB-DNJ has been administered orally to humans in a 100 mg dose three times per day for twelve months, and a daily dose of up to 3 gm has been used. A certain oral dose range for a DNJ-like compound is from about 60 mg / kg / day to about 900 mg / kg / day.

[0089] In another aspect, a vector comprises one or more nucleic acid sequences encoding one or more inhibitors of β-1,4-GalT V. In certain embodiments, a vector comprises one or more mutations in an Sp1 binding site of β-1,4-GalT V.

[0090] In another aspect, a composition comprises vector comprising a nucleic acid sequence encoding a β-1,4-Galactosyltransferase-V (β-1,4-GalT-V), mutants or variants thereof. In certain embodiments, the vector is a DNA plasmid. In certain embodiments, the vector25 178013745.1comprises an inducible promoter. In certain embodiments, the vector comprises a constitutive promoter. In certain embodiments, the vector comprises a tissue-specific promoter.

[0091] The 2.3-kb 5′-flanking region of the human β-1,4-GalT V gene was previously cloned and the region –116 / –18 relative to the transcription start site was identified as that having promoter activity. The region was found to contain several putative binding sites for transcription factors, including AP2, AP4, N-Myc, Sp1, and upstream stimulatory factor. Electrophoretic mobility shift assay showed that Sp1 binds to nucleotide positions –81 / –69 of the promoter region. Mutations induced in the Sp1-binding site showed that the promoter activity of the β-1,4- GalT V gene is impaired completely in cancer cells. In contrast, the promoter activity increased significantly by the transfection of the Sp1 cDNA into A549 human lung carcinoma cells. Mithramycin A, which inhibits the binding of Sp1 to its binding site, reduced the promoter activation and expression of the β-1,4-GalT V gene in A549 cells (Sato, Takeshi et al. Transcriptional Regulation of the Human β-1,4-Galactosyltransferase V Gene in Cancer Cells, Journal of Biological Chemistry, Volume 279, Issue 38, 39574 - 39583).

[0092] In another aspect, the disclosure contemplates the use of antibodies specific for β- galactosyltransferases in the treatment and prevention of cardiovascular diseases. The phrases “binding specificity,” “binding specifically to, “specific binding” or otherwise any reference to an antibody to a glycosyltransferase, refers to a binding reaction that is determinative of the presence of the corresponding β-galactosyltransferases antigen to the antibody in a heterogeneous population of antigens and other biologics. The parameters required to achieve such specificity can be determined routinely, using conventional methods in the art including, but not limited to, competitive binding studies. The binding affinity of an antibody can also be readily determined, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51 : 660- 672, 1949). In some embodiments, the immunoglobulins of the present disclosure bind to a glycosyltransferase at least about 5, at least about 10, at least about 100, at least about 103, at least about 104, at least 105, and at least 106fold higher than to other proteins.

[0093] Various procedures known in the art may be used for the production of antibodies to a glycosyltransferase, glycosyltransferase family members or any subunit thereof, or a fragment, derivative, homolog or analog of the protein. Antibodies of the present disclosure26 178013745.1include, but are not limited to, synthetic antibodies, polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies

[0094] (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In particular, antibodies of the present disclosure include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules that contain an antigen binding site that immunospecifically binds to an antigen (e.g., one or more complementarity determining regions (CDRs) of an antibody).

[0095] Another embodiment for the preparation of antibodies according to the disclosure is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, “Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds., Chapman and Hall, New York (1993). The underlying rationale behind the use of peptide mimetics in rational design is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used to engineer second generation molecules having many of the natural properties of the targeting antibodies disclosed herein, but with altered and even improved characteristics. More specifically, under this rational design approach, peptide mapping may be used to determine “active” antigen recognition residues, and along with molecular modeling and molecular dynamics trajectory analysis, peptide mimic of the antibodies containing antigen contact residues from multiple CDRs may be prepared.

[0096] In some embodiments, an antibody specifically binds an epitope of the β- galactosyltransferase protein. It is to be understood that the peptide regions may not necessarily precisely map one epitope but may also contain a β-galactosyltransferase sequence that is not immunogenic. Methods of predicting other potential epitopes to which an immunoglobulin of the disclosure can bind are well-known to those of skill in the art and include, without limitation, Kyte-Doolittle Analysis (Kyte, J. and Dolittle, R. F., 157 J. MOL. BIOL. 105-32 (1982)); Hopp27 178013745.1and Woods Analysis (Hopp, T. P. and Woods, K. R., 78 P OC. NATL. ACAD. SCI. USA 3824- 28 (1981); Hopp, T. J. and Woods, K. R., 20 MOL. IMMUNOL. 483-89 (1983); Hopp, T. J., 88 J. IMMUNOL. METHODS 1-18 (1986)); Jameson-Wolf Analysis (Jameson, B. A. and Wolf, H., 4 COMPUT. APPL. BIOSCI. 181-86 (1988)); and Emini Analysis (Emini et al, 140 VIROLOGY 13-20 (1985)).

[0097] Amino acid sequence variants of the antibodies of the present disclosure may be prepared by introducing appropriate nucleotide changes into the polynucleotide that encodes the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct.

[0098] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0099] Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of a polypeptide that increases the serum half-life of the antibody. Another type of antibody variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. For example, the sites of greatest interest for substitutional mutagenesis of antibodies include the hypervariable regions, but framework region (FR) alterations are also contemplated. [000100] A useful method for the identification of certain residues or regions of the β- galactosyltransferase antibodies that are preferred locations for substitution, i.e., mutagenesis, is alanine scanning mutagenesis. See Cunningham & Wells, 244 SCIENCE 1081-85 (1989). Briefly, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. The amino acid locations demonstrating functional sensitivity to the substitutions are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for28 178013745.1introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed antibody variants screened for the desired activity. [000101] Substantial modifications in the biological properties of the antibody can be accomplished by selecting substitutions that differ significantly in their effect on, maintaining (i) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (ii) the charge or hydrophobicity of the molecule at the target site, or (iii) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: [000102] (1) hydrophobic: norleucine, met, ala, val, leu, ile; [000103] (2) neutral hydrophilic: cys, ser, thr; [000104] (3) acidic: asp, glu; [000105] (4) basic: asn, gin, his, lys, arg; [000106] (5) residues that influence chain orientation: gly, pro; and [000107] (6) aromatic: trp, tyr, phe. [000108] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Conservative substitutions involve exchanging of amino acids within the same class. [000109] Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an immunoglobulin fragment such as an Fv fragment. [000110] Another type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s), i.e., functional equivalents as defined above, selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is by affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible29 178013745.1amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. [000111] In order to identify candidate hypervariable region sites for modification, alanine- scanning mutagenesis may be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antibody-antigen complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. [000112] Polynucleotide molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non- variant version of the anti-glycosyltransferase antibodies of the present disclosure. [000113] In one aspect of the present disclosure, the expression of glycosyltransferases may be inhibited by the use of RNA interference techniques (RNAi). RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in animals and plant cells. See Hutvagner and Zamore, 12 CURR. OPIN. GENET. DEV. 225-32 (2002); Hammond et al, 2 NATURE REV. GEN. 110- 19 (2001); Sharp, 15 GENES DEV. 485-90 (2001). RNAi can be triggered, for example, by nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al, 10 MOL. CELL. 549-61 (2002); Elbashir et al, 411 Nature 494-98 (2001)), micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in-vivo using DNA templates with RNA polymerase III promoters. See, e.g., Zeng et al, 9 MOL. CELL. 1327-33 (2002); BIOTECHNOL. 440-48 (2002); Yu et al, 99(9) PROC. NATL. ACAD. SCI. USA, 6047-52 (2002); McManus et al, 8 RNA 842-50 (2002); Sui et al, 99(6) PROC. NATL. ACAD. SCI. USA 5515- 20 (2002).30 178013745.1[000114] In certain embodiments, the present disclosure features “small interfering RNA molecules” (“siRNA molecules” or “siRNA”), methods of making siRNA molecules and methods for using siRNA molecules (e.g., research and / or therapeutic methods). The siRNAs of this disclosure encompass any siRNAs that can modulate the selective degradation of glycosyltransferase mRNA. [000115] In a specific embodiment, the siRNA of the present disclosure may comprise double- stranded small interfering RNA molecules (ds-siRNA). A ds-siRNA molecule of the present disclosure may be a duplex made up of a sense strand and a complementary antisense strand, the antisense strand being sufficiently complementary to a target glycosyltransferase mRNA to mediate RNAi. The siRNA molecule may comprise about 10 to about 50 or more nucleotides. More specifically, the siRNA molecule may comprise about 16 to about 30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand. The strands may be aligned such that there are at least , 2, or 3 bases at the end of the strands which do not align (e.g., for which no complementary bases occur in the opposing strand) such that an overhang of 1 , 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed. [000116] In an alternative embodiment, the siRNA of the present disclosure may comprise single-stranded small interfering RNA molecules (ss-siRNA). Similar to the ds-siRNA molecules, the ss-siRNA molecule may comprise about 10 to about 50 or more nucleotides. More specifically, the ss-siRNA molecule may comprise about 15 to about 45 or more nucleotides. Alternatively, the ss-siRNA molecule may comprise about 19 to about 40 nucleotides. The ss-siRNA molecules of the present disclosure comprise a sequence that is “sufficiently complementary” to a target mRNA sequence to direct target-specific RNA interference (RNAi), as defined herein, e.g., the ss-siRNA has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process. In one embodiment, the ss- siRNA molecule can be designed such that every residue is complementary to a residue in the target molecule. Alternatively, substitutions can be made within the molecule to increase stability and / or enhance processing activity of the molecule. Substitutions can be made within the strand or can be made to residues at the ends of the strand. In a specific embodiment, the 5 '-terminus31 178013745.1may be phosphorylated (e.g., comprises a phosphate, diphosphate, or triphosphate group). In another embodiment, the 3' end of an siRNA may be a hydroxyl group in order to facilitate RNAi, as there is no requirement for a 3 ' hydroxyl group when the active agent is a ss-siRNA molecule. In other instances, the 3' end (e.g., C3 of the 3' sugar) of ss-siRNA molecule may lack a hydroxyl group (e.g., ss-siRNA molecules lacking a 3' hydroxyl or C3 hydroxyl on the 3' sugar (e.g., ribose or deoxyribose). [000117] In another aspect, the siRNA molecules of the present disclosure may be modified to improve stability under in vitro and / or in vivo conditions, including, for example, in serum and in growth medium for cell cultures. In order to enhance the stability, the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, certainly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'- deoxythymidine is tolerated and does not affect the efficiency of RNA interference. For example, the absence of a 2' hydroxyl may significantly enhance the nuclease resistance of the siRNAs in tissue culture medium. [000118] Furthermore, the siRNAs of the present disclosure may include modifications to the sugar-phosphate backbone or nucleosides. These modifications can be tailored to promote selective genetic inhibition, while avoiding a general panic response reported to be generated by siRNA in some cells. In addition, modifications can be introduced in the bases to protect siRNAs from the action of one or more endogenous enzymes. [000119] In an embodiment of the present disclosure, the siRNA molecule may contain at least one modified nucleotide analogue. The nucleotide analogues may be located at positions where the target-specific activity, e.g., the RNAi mediating activity is not substantially effected, e.g., in a region at the 5 '-end and / or the 3 '-end of the RNA molecule. Certainly, the ends may be stabilized by incorporating modified nucleotide analogues. Examples of nucleotide analogues include sugar- and / or backbone-modified ribonucleotides (e.g., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In backbone-modified ribonucleotides, the phosphoester group connecting to adjacent ribonucleotides may be replaced32 178013745.1by a modified group, e.g., a phosphothioate group. In sugar- modified ribonucleotides, the 2' OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2or ON, wherein R is Ci-C6alkyl, alkenyl or alkynyl and halo is F, CI, Br or I. [000120] Nucleobase-modified ribonucleotides may also be utilized, e.g., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. [000121] Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7- deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined. [000122] Derivatives of siRNAs may also be utilized herein. For example, cross-linking can be employed to alter the pharmacokinetics of the composition, e.g., to increase half-life in the body. Thus, the present disclosure includes siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked. The present disclosure also includes siRNA derivatives having a non-nucleic acid moiety conjugated to its 3' terminus (e.g., a peptide), organic compositions (e.g., a dye), or the like. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA. [000123] The siRNAs of the present disclosure can be enzymatically produced or totally or partially synthesized. Moreover, the siRNAs can be synthesized in vivo or in vitro. For siRNAs that are biologically synthesized, an endogenous or a cloned exogenous RNA polymerase may be used for transcription in vivo, and a cloned RNA polymerase can be used in vitro. siRNAs that are chemically or enzymatically synthesized are preferably purified prior to the introduction into the cell.33 178013745.1[000124] Although one hundred percent (100%) sequence identity between the siRNA and the target region is preferred in certain embodiments, it is not required to practice the disclosure. siRNA molecules that contain some degree of modification in the sequence can also be adequately used for the purpose of this disclosure. Such modifications may include, but are not limited to, mutations, deletions or insertions, whether spontaneously occurring or intentionally introduced. [000125] Moreover, not all positions of a siRNA contribute equally to target recognition. In certain embodiments, for example, mismatches in the center of the siRNA may be critical and could essentially abolish target RNA cleavage. In other embodiments, the 3' nucleotides of the siRNA do not contribute significantly to specificity of the target recognition. In certain, residues 3 ' of the siRNA sequence which is complementary to the target RNA (e.g., the guide sequence) may not critical for target RNA cleavage. [000126] Sequence identity may be determined by sequence comparison and alignment algorithms known to those of ordinary skill in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology = # of identical positions / total # of positions x 100), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced. [000127] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (e.g., a local alignment).34 178013745.1[000128] In another embodiment, the alignment may optimized by introducing appropriate gaps and determining percent identity over the length of the aligned sequences (e.g., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 25(17) Nucleic Acids Res. 3389-3402 (1997). In another embodiment, the alignment may be optimized by introducing appropriate gaps and determining percent identity over the entire length of the sequences aligned (e.g., a global alignment). A non- limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. [000129] In certain embodiments, greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the siRNA and the portion of the target gene may be used. Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing). Additional hybridization conditions include, but are not limited to, hybridization at 70°C in lxSSC or 50°C in lxSSC, 50% formamide followed by washing at 70°C in 0.3xSSC or hybridization at 70°C in 4xSSC or 50°C in 4xSSC, 50% formamide followed by washing at 67°C in lxSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length can be about 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C) = 2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81.5+16.6(log 10[Na+])+0.41(% G+C)-(600 / N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC=0.165 M). Additional examples of stringency conditions for polynucleotideare provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current35 178013745.1Protocols in Molecular Biology, 1995, F. M. Ausubel et al, eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. The length of the identical nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 4750 or more bases. [000130] Antisense molecules can act in various stages of transcription, splicing and translation to block the expression of a target gene. Without being limited by theory, antisense molecules can inhibit the expression of a target gene by inhibiting transcription initiation by forming a triple strand, inhibiting transcription initiation by forming a hybrid at an RNA polymerase binding site, impeding transcription by hybridizing with an RNA molecule being synthesized, repressing splicing by hybridizing at the junction of an exon and an intron or at the spliceosome formation site, blocking the translocation of an mRNA from nucleus to cytoplasm by hybridization, repressing translation by hybridizing at the translation initiation factor binding site or ribosome biding site, inhibiting peptide chain elongation by hybridizing with the coding region or polysome binding site of an mRNA, or repressing gene expression by hybridizing at the sites of interaction between nucleic acids and proteins. An example of an antisense oligonucleotide of the present disclosure is a cDNA that, when introduced into a cell, transcribes into an RNA molecule having a sequence complementary to at least part of the glycosyltransferase mRNA. [000131] Furthermore, antisense oligonucleotides of the present disclosure include oligonucleotides having modified sugar-phosphodiester backbones or other sugar linkages, which can provide stability against endonuclease attacks. The present disclosure also encompasses antisense oligonucleotides that are covalently attached to an organic or other moiety that increase their affinity for a target nucleic acid sequence. For example, intercalating agents, alkylating agents, and metal complexes can be also attached to the antisense oligonucleotides of the present disclosure to modify their binding specificities. [000132] The present disclosure also provides ribozymes as a tool to inhibit glycosyltransferase expression. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The characteristics of ribozymes are well-known in the art. See, e.g., Rossi, 4 CURRENT BIOLOGY 469-71 (1994). Without being limited by theory, the36 178013745.1mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. In certain embodiments, the ribozyme molecules include one or more sequences complementary to the target gene mRNA, and include the well known catalytic sequence responsible for mRNA cleavage. See U.S. Patent No. 5,093,246. Using the known sequence of the target glycosyltransferase mRNA, a restriction enzyme-like ribozyme can be prepared using standard techniques. [000133] The expression of a glycosyltransferase gene can also be inhibited by using triple helix formation. Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription can be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base paring rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules that are purine-rich, e.g., containing a stretch of G residues, may be chosen. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex. [000134] Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5 '-3 ',3 '-5' manner, such that they base pair first with one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex. [000135] The expression of a glycosyltransferase may be also inhibited by what is referred to as “co-repression.” Co-repression refers to the phenomenon in which, when a gene having an identical or similar to the target sequence is introduced to a cell, expression of both introduced and endogenous genes becomes repressed. This phenomenon, although first observed in plant37 178013745.1system, has been observed in certain animal systems as well. The sequence of the gene to be introduced does not have to be identical to the target sequence, but sufficient homology allows the co-repression to occur. The determination of the extent of homology depends on individual cases, and is within the ordinary skill in the art. [000136] It would be readily apparent to one of ordinary skill in the art that other methods of gene expression inhibition that selectively target a glycosyltransferase DNA or mRNA can also be used in connection with this disclosure without departing from the spirit of the disclosure. In a specific embodiment, using techniques known to those of ordinary skill in the art, the present disclosure contemplates affecting the promoter region of a glycosyltransferase to effectively switch off transcription. [000137] In certain aspects, use of gene editing techniques are contemplated which target β- 1,4-GalT-V nucleic acid sequences. [000138] Accordingly, in certain embodiments, the compositions of the disclosure include at least one gene editing complex, comprising CRISPR-associated nucleases such as Cas9 and Cpf1 gRNAs, Argonaute family of endonucleases, clustered regularly interspaced short palindromic repeat (CRISPR) nucleases, zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, or combinations thereof. See Schiffer, 2012, J Virol 88(17):8920-8936, incorporated by reference. [000139] The composition can also include C2c2—the first naturally-occurring CRISPR system that targets only RNA. The Class 2 type VI-A CRISPR-Cas effector “C2c2” demonstrates an RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results demonstrate the capability of C2c2 as a new RNA-targeting tools.38 178013745.1[000140] C2c2 can be programmed to cleave particular RNA sequences in bacterial cells. The RNA-focused action of C2c2 complements the CRISPR-Cas9 system, which targets DNA, the genomic blueprint for cellular identity and function. The ability to target only RNA, which helps carry out the genomic instructions, offers the ability to specifically manipulate RNA in a high-throughput manner- and manipulate gene function more broadly. [000141] CRISPR / Cpf1 is a DNA-editing technology analogous to the CRISPR / Cas9 system, characterized in 2015 by Feng Zhang's group from the Broad Institute and MIT. Cpf1 is an RNA-guided endonuclease of a class II CRISPR / Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR / Cas9 system limitations. CRISPR / Cpf1 could have multiple applications, including treatment of genetic illnesses and degenerative conditions. As referenced above, Argonaute is another potential gene editing system. [000142] Argonautes are a family of endonucleases that use 5′ phosphorylated short single- stranded nucleic acids as guides to cleave targets (Swarts, D. C. et al. The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol. 21, 743-753 (2014)). Similar to Cas9, Argonautes have key roles in gene expression repression and defense against foreign nucleic acids (Swarts, D. C. et al. Nat. Struct. Mol. Biol. 21, 743-753 (2014); Makarova, K. S., et al. Biol. Direct 4, 29 (2009). Molloy, S. Nat. Rev. Microbiol.11, 743 (2013); Vogel, J. Science 344, 972-973 (2014). Swarts, D. C. et al. Nature 507, 258-261 (2014); Olovnikov, I., et al. Mol. Cell 51, 594-605 (2013)). However, Argonautes differ from Cas9 in many ways Swarts, D. C. et al. The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol. 21, 743-753 (2014)). Cas9 only exist in prokaryotes, whereas Argonautes are preserved through evolution and exist in virtually all organisms; although most Argonautes associate with single-stranded (ss)RNAs and have a central role in RNA silencing, some Argonautes bind ssDNAs and cleave target DNAs (Swarts, D. C. et al. Nature 507, 258-261 (2014); Swarts, D. C. et al. Nucleic Acids Res. 43, 5120-5129 (2015)). guide RNAs must have a 3′ RNA-RNA hybridization structure for correct Cas9 binding, whereas no specific consensus secondary structure of guides is required for39 178013745.1Argonaute binding; whereas Cas9 can only cleave a target upstream of a PAM, there is no specific sequence on targets required for Argonaute. Once Argonaute and guides bind, they affect the physicochemical characteristics of each other and work as a whole with kinetic properties more typical of nucleic-acid-binding proteins (Salomon, W. E., et al. Cell 162, 84-95 (2015)). [000143] Accordingly, in certain embodiments, Argonaute endonucleases comprise those which associate with single stranded RNA (ssRNA) or single stranded DNA (ssDNA). In certain embodiments, the Argonaute is derived from Natronobacterium gregoryi. In other embodiments. the Natronobacterium gregoryi Argonaute (NgAgo) is a wild type NgAgo, a modified NgAgo, or a fragment of a wild type or modified NgAgo. The NgAgo can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. For example, nuclease (e.g., DNase) domains of the NgAgo can be modified, deleted, or inactivated. [000144] The wild type NgAgo sequence can be modified. The NgAgo nucleotide sequence can be modified to encode biologically active variants of NgAgo, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type NgAgo by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations). One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of an NgAgo polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type NgAgo polypeptide. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine. The amino acid residues in the NgAgo amino acid sequence can be non-naturally occurring amino acid residues. Naturally occurring amino acid residues include those naturally encoded by the genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L- configuration). The present peptides can also include amino acid residues that are modified versions of standard residues (e.g. pyrrolysine can be used in place of lysine and selenocysteine40 178013745.1can be used in place of cysteine). Non-naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid and L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. For other examples, one can consult textbooks or the worldwide web (a site currently maintained by the California Institute of Technology displays structures of non-natural amino acids that have been successfully incorporated into functional proteins). [000145] Another gene editing complex is human WRN, a RecQ helicase encoded by the Werner syndrome gene. It is implicated in genome maintenance, including replication, recombination, excision repair and DNA damage response. These genetic processes and expression of WRN are concomitantly upregulated in many types of cancers. Therefore, it has been proposed that targeted destruction of this helicase could be useful for elimination of cancer cells. Reports have applied the external guide sequence (EGS) approach in directing an RNase P RNA to efficiently cleave the WRN mRNA in cultured human cell lines, thus abolishing translation and activity of this distinctive 3′-5′ DNA helicase-nuclease. RNase P RNA isanother potential endonuclease for use with the present disclosure. [000146] Gene editing complexes such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is found in bacteria and is believed to protect the bacteria from phage infection. It has recently been used as a means to alter gene expression in eukaryotic DNA, but has not been proposed as an anti-viral therapy or more broadly as a way to disrupt genomic material. Rather, it has been used to introduce insertions or deletions as a way of increasing or decreasing transcription in the DNA of a targeted cell or population of cells. See for example, Horvath et al., Science (2010) 327:167-170; Terns et al., Current Opinion in Microbiology (2011) 14:321-327; Bhaya et al., Annu Rev Genet (2011) 45:273-297; Wiedenheft et al., Nature (2012) 482:331-338); Jinek M et al., Science (2012) 337:816-821; Cong L et al., Science (2013) 339:819-823; Jinek M et al., (2013) eLife 2:e00471; Mali P et al. (2013) Science 339:823-826; Qi L S et al. (2013) Cell 152:1173-1183; Gilbert L A et al. (2013) Cell 154:442-451; Yang H et al. (2013) Cell 154:1370-1379; and Wang H et al. (2013) Cell 153:910-918).41 178013745.1[000147] CRISPR methodologies employ a nuclease, CRISPR-associated (Cas), that complexes with small RNAs as guides (gRNAs) to cleave DNA in a sequence-specific manner upstream of the protospacer adjacent motif (PAM) in any genomic location. CRISPR may use separate guide RNAs known as the crRNA and tracrRNA. These two separate RNAs have been combined into a single RNA to enable site-specific mammalian genome cutting through the design of a short guide RNA. Cas and guide RNA (gRNA) may be synthesized by known methods. Cas / guide-RNA (gRNA) uses a non-specific DNA cleavage protein Cas, and an RNA oligonucleotide to hybridize to target and recruit the Cas / gRNA complex. See Chang et al., 2013, Cell Res. 23:465-472; Hwang et al., 2013, Nat. Biotechnol. 31:227-229; Xiao et al., 2013, Nucl. Acids Res.1-11. [000148] In general, the CRISPR / Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with guide RNAs. CRISPR / Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNase domains, protein-protein interaction domains, dimerization domains, as well as other domains. [000149] In certain embodiments, three variants found have the fewest off-target effects: SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (a.k.a. eSpCas91.0), and SpCas9 (K848A / K1003A / R1060A) (a.k.a. eSPCas91.1) are employed in the compositions. The disclosure is by no means limited to these variants, and also encompasses all Cas9 variants (Slaymaker, I. M. et al. Science. 2016 Jan. 1; 351(6268):84-8. doi: 10.1126 / science.aad5227. Epub 2015 Dec. 1). The present disclosure also includes another type of enhanced specificity Cas9 variant, “high fidelity” spCas9 variants (HF-Cas9). Examples of high fidelity variants include SpCas9-HF1 (N497A / R661A / Q695A / Q926A), SpCas9-HF2 (N497A / R661A / Q695A / Q926A / D1135E), SpCas9-HF3 (N497A / R661A / Q695A / Q926A / L169A), SpCas9-HF4 (N497A / R661A / Q695A / Q926A / Y450A). Also included are all SpCas9 variants bearing all possible single, double, triple and quadruple combinations of N497A, R661A, Q695A, Q926A or any other substitutions (Kleinstiver, B. P. et al., 2016, Nature. DOI: 10.1038 / nature16526).42 178013745.1[000150] As used herein, the term “Cas” is meant to include all Cas molecules comprising variants, mutants, orthologues, high-fidelity variants and the like. [000151] In one embodiment, the endonuclease is derived from a type II CRISPR / Cas system. In other embodiments, the endonuclease is derived from a Cas9 protein and includes Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, ARMAN 1, ARMAN 4, mutants, variants, high- fidelity variants, orthologs, analogs, fragments, or combinations thereof. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina. Included are Cas9 proteins encoded in genomes of the nanoarchaea ARMAN-1 (Candidatus Micrarchaeum acidiphilum ARMAN-1) and ARMAN-4 (Candidatus Parvarchaeum acidiphilum ARMAN-4), CasY (Kerfeldbacteria, Vogelbacteria, Komeilibacteria, Katanobacteria), CasX (Planctomycetes, Deltaproteobacteria). [000152] Guide RNA sequences according to the present disclosure can be sense or anti- sense sequences. The guide RNA sequence generally includes a proto-spacer adjacent motif (PAM). The sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S. pyogenes, the target43 178013745.1DNA typically immediately precedes a 5′-NGG proto-spacer adjacent motif (PAM). Thus, for the S. pyogenes Cas9, the PAM sequence can be AGG, TGG, CGG or GGG. Other Cas9 orthologs may have different PAM specificities. For example, Cas9 from S. thermophilus requires 5′-NNAGAA for CRISPR 1 and 5′-NGGNG for CRISPR 3) and Neiseria meningitidis requires 5′-NNNNGATT). [000153] The guide RNA sequence can be configured as a single sequence or as a combination of one or more different sequences, e.g., a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs. [000154] The gRNA sequences can include additional 5′ and / or 3′ sequences that may or may not be complementary to a target sequence. They can have less than 100% complementarity to a target sequence, for example 75% complementarity. The gRNA sequences can be employed as a combination of one or more different sequences, e.g., a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs. [000155] In some embodiments, the RNA molecules e.g. crRNA, tracrRNA, gRNA are engineered to comprise one or more modified nucleobases. For example, known modifications of RNA molecules can be found, for example, in Genes VI, Chapter 9 (“Interpreting the Genetic Code”), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington D.C.). Modified RNA components include the following: 2′-O-methylcytidine; N4-methylcytidine; N4-2′-O- dimethylcytidine; N4-acetylcytidine; 5-methylcytidine; 5,2′-O-dimethylcytidine; 5- hydroxymethylcytidine; 5-formylcytidine; 2′-O-methyl-5-formaylcytidine; 3-methylcytidine; 2- thiocytidine; lysidine; 2′-O-methyluridine; 2-thiouridine; 2-thio-2′-O-methyluri dine; 3,2′-O- dimethyluridine; 3-(3-amino-3-carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2′-O- dimethyluridine; 5-methyl-2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine; uridine 5- oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine; 5- methoxycarbonylmethyluridine; 5-methoxycarbonylmethyl-2′-O-methyluridine; 5- methoxycarbonylmethyl-2′-thiouridine; 5-carbamoylmethyluridine; 5-carbamoylmethyl-2′-O-44 178013745.1methyluridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl) uridinemethyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methylaminomethyl-2- thiouridine; 5-methylaminomethyl-2-selenouridine; 5-carboxymethylaminomethyluridine; 5- carboxymethylaminomethyl-2′-O-methyl-uridine; 5-carboxymethylaminomethyl-2-thiouridine; dihydrouridine; dihydroribosylthymine; 2′-methyladenosine; 2-methyladenosine; N6Nmethyladenosine; N6,N6-dimethyladenosine; N6,2′-O-trimethyladenosine; 2 methylthio- N6Nisopentenyladenosine; N6-(cis-hydroxyisopentenyl)-adenosine; 2-methylthio-N6-(cis- hydroxyisopentenyl)-adenosine; N6-glycinylcarbamoyl)adenosine; N6threonylcarbamoyl adenosine; N6-methyl-N6-threonylcarbamoyl adenosine; 2-methylthio-N6-methyl-N6- threonylcarbamoyl adenosine; N6-hydroxynorvalylcarbamoyl adenosine; 2-methylthio-N6- hydroxnorvalylcarbamoyl adenosine; 2′-O-ribosyladenosine (phosphate); inosine; 2′O-methyl inosine; 1-methyl inosine; 1,2′-O-dimethyl inosine; 2′-O-methyl guanosine; 1-methyl guanosine; N2-methyl guanosine; N2,N2-dimethyl guanosine; N2,2′-O-dimethyl guanosine; N2,N2,2′-O- trimethyl guanosine; 2′-O-ribosyl guanosine (phosphate); 7-methyl guanosine; N2,7-dimethyl guanosine; N2,N2;7-trimethyl guanosine; wyosine; methylwyosine; under-modified hydroxywybutosine; wybutosine; hydroxywybutosine; peroxywybutosine; queuosine; epoxyqueuosine; galactosyl-queuosine; mannosyl-queuosine; 7-cyano-7-deazaguanosine; arachaeosine [also called 7-formamido-7-deazaguanosine]; and 7-aminomethyl-7- deazaguanosine. [000156] Other examples that target include chimeric antigen receptor T cells (CAR-T cells), drug therapies, and the like. See, also Chatterjee S, et al. which details various therapies and is incorporated in its entirety herein by reference. (Chatterjee S, Yuan R, Thapa S, Talwar R. Central Role of β-1,4-GalT-V in Cancer Signaling, Inflammation, and Other Disease-Centric Pathways. Int J Mol Sci. 2023 Dec 29;25(1):483. doi: 10.3390 / ijms25010483. PMID: 38203654; PMCID: PMC10778672) which is incorporated herein by reference in its entirety. [000157] Combination Therapies. [000158] In certain embodiments, the method of treating a subject with ACM further comprises administering one or more inhibitors of tumor necrosis α (TNFα), muscle contraction signaling molecules or the combination thereof in a sample from the subject. In certain45 178013745.1embodiments, muscle contraction signaling molecules comprise β-catenin, c-Src or the combination thereof. [000159] In certain embodiments an inhibitor of TNFα comprises one or more of Etanercept (Enbrel), Infliximab (Remicade), Adalimumab (Humira), Certolizumab pegol (Cimzia), Golimumab (Simponi, Simponi Aria), lenalidomide, pomalidomide or combinations thereof. [000160] In certain embodiments, the subject is administered a pharmaceutical composition comprising a vector encoding a β-1,4-Galactosyltransferase-V (β-1,4-GalT-V), isoforms, mutants or variants thereof. In certain embodiments, the vector is a DNA plasmid. In certain embodiments, the vector comprises an inducible promoter. In certain embodiments the vector comprises a constitutive promoter. In certain embodiments, the vector comprises a tissue-specific promoter. In certain embodiments, the vector comprising β-1,4-GalT-V induces an immune response. In certain embodiments, the immune response induces an antibody response wherein the antibodies bind to β-1,4-GalT-V, thereby reducing the amount of β-1,4-GalT-V. In certain embodiments, the immune response is a T cell mediated response, whereby the immune response induces a specific β-1,4-GalT-V T cell response. In certain embodiments, an adjuvant is administered in combination with the vector encoding β-1,4-GalT-V. [000161] In certain embodiments, β-catenin inhibitors comprise XAV 939 (tankyrase inhibitor), ICG 001 (inhibits TCF / β-catenin-mediated transcription) IWP 4 (potent inhibitor of Wnt / β-catenin signaling), NLS-StAx-h (Wnt signaling inhibitor; inhibits β-catenin-transcription factor interactions), TAK 715 (potent p38 MAPK inhibitor; anti-inflammatory), WIKI4 (tankyrase inhibitor), WIC1 (inhibitor of Wnt / β-catenin signaling), WNTinib (inhibitor of mutant β-catenin signaling in hepatocellular carcinoma). Other examples include:46 178013745.1Michael A. McCoy, et al., Journal of Medicinal Chemistry 202265 (10), 7246-7261, DOI:47 178013745.110.1021 / acs.jmedchem.2c00228. See, also, Dev A Jr, Vachher M, Prasad CP. β-catenin inhibitors in cancer therapeutics: intricacies and way forward. Bioengineered. 2023 Dec;14(1):2251696. doi: 10.1080 / 21655979.2023.2251696. PMID: 37655825; PMCID: PMC10478749). [000163] PHARMACEUTICAL COMPOSITIONS [000164] The present disclosure also provides pharmaceutical compositions. Such compositions comprise a glycosphingolipid synthesis inhibitor of the present disclosure. The composition further comprises a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the glycolipid synthesis inhibitor is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water may be a carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose may be carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried slim milk, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. [000165] The pharmaceutical compositions of the present disclosure can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. In a specific embodiment, a pharmaceutical composition comprises an effective amount of a glycolipid synthesis inhibitor together with a suitable amount48 178013745.1of a pharmaceutically acceptable carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. [000166] Furthermore, a glycolipid synthesis inhibitor of the present disclosure can be administered with compounds that facilitate uptake of the glycosphingolipid synthesis inhibitor by target cells or otherwise enhance transport of an inhibitor to a particular site for action. [000167] Absorption promoters, detergents and chemical irritants (e.g., keratinolytic agents) can enhance transmission of an agent into a target tissue (e.g., through the skin). For general principles regarding absorption promoters and detergents which have been used with success in mucosal delivery of organic and peptide -based drugs, see, e.g., Chien, Novel Drug Delivery Systems, Ch. 4 (Marcel Dekker, 1992). Suitable agents for use in the methods of the present disclosure for mucosal / nasal delivery are also described in Chang, et al., Nasal Drug Delivery, “Treatise on Controlled Drug Delivery”, Ch. 9 and Tables 3-4B thereof, (Marcel Dekker, 1992). Suitable agents which are known to enhance absorption of drugs through skin are described in Sloan, Use of Solubility Parameters from Regular Solution Theory to Describe Partitioning-Driven Processes, Ch. 5, “Prodrugs: Topical and Ocular Drug Delivery” (Marcel Dekker, 1992), and at places elsewhere in the text. All of these references are incorporated herein for the sole purpose of illustrating the level of knowledge and skill in the art concerning drug delivery techniques. [000168] In other embodiments, a colloidal dispersion system may be used for targeted delivery of the glycolipid synthesis inhibitor to specific issue. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. [000169] The pharmaceutical compositions of the present disclosure may be administered by any particular route of administration including, but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intraosseous, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary,49 178013745.1intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means, or transdermal means. [000170] In general, the pharmaceutical compositions disclosed herein may be used alone or in concert with other therapeutic agents at appropriate dosages defined by routine testing in order to obtain optimal efficacy while minimizing any potential toxicity. The dosage regimen utilizing a pharmaceutical composition of the present disclosure may be selected in accordance with a variety of factors including type, species, age, weight, sex, medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular pharmaceutical composition employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the pharmaceutical composition (and potentially other agents including therapeutic agents) required to prevent, counter, or arrest the progress of the condition. [000171] Optimal precision in achieving concentrations of the therapeutic regimen (e.g., a pharmaceutical composition comprising a glycosphingolipid synthesis inhibitor in combination with another therapeutic agent) within the range that yields maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the pharmaceutical composition's availability to one or more target sites. Distribution, equilibrium, and elimination of a pharmaceutical composition may be considered when determining the optimal concentration for a treatment regimen. The dosages of a pharmaceutical composition disclosed herein may be adjusted when combined to achieve desired effects. On the other hand, dosages of the pharmaceutical composition and various therapeutic agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either were used alone. [000172] In particular, toxicity and therapeutic efficacy of a pharmaceutical composition disclosed herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index and it may be expressed as the ratio50 178013745.1LD50 / ED50. Pharmaceutical compositions exhibiting large therapeutic indices are preferred except when cytotoxicity of the composition is the activity or therapeutic outcome that is desired. Although pharmaceutical compositions that exhibit toxic side effects may be used, a delivery system can target such compositions to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. Generally, the pharmaceutical compositions of the present disclosure may be administered in a manner that maximizes efficacy and minimizes toxicity. [000173] Data obtained from cell culture assays and animal studies may be used in formulating a range of dosages for use in humans. The dosages of such compositions lie preferably within a range of circulating concentrations that include the ED5o with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the methods of the disclosure, the therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50(the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. [000174] Moreover, the dosage administration of the compositions of the present disclosure may be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen and a pharmacokinetic / pharmacodynamic model may be used to determine the pharmacokinetic / pharmacodynamic profile of one or more dosage regimens. Next, one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic / pharmacodynamic response based on the particular pharmacokinetic / pharmacodynamic profile. [000175] More specifically, the pharmaceutical compositions may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily. In the case of oral administration, the daily dosage of the compositions may be varied over a wide range from about 0.1 ng to about 1 ,000 mg per patient, per day. The range51 178013745.1may more particularly be from about 0.001 ng / kg to 10 mg / kg of body weight per day, about 0.1- 100 μg, about 1.0-50 μg or about 1.0-20 mg per day for adults (at about 60 kg). [000176] The daily dosage of the pharmaceutical compositions may be varied over a wide range from about 0.1 ng to about 1000 mg per adult human per day. For oral administration, the compositions may be provided in the form of tablets containing from about 0.1 ng to about 1000 mg ofthe composition or 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 milligrams of the composition for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the pharmaceutical composition is ordinarily supplied at a dosage level of from about 0.1 ng / kg to about 20 mg / kg of body weight per day. In one embodiment, the range is from about 0.2 ng / kg to about 10 mg / kg of body weight per day. In another embodiment, the range is from about 0.5 ng / kg to about 10 mg / kg of body weight per day. The pharmaceutical compositions may be administered on a regimen of about 1 to about 10 times per day. [000177] In the case of injections, it is usually convenient to give by an intravenous route in an amount of about 0.000 ^g-30 mg, about 0.01 μg-20 mg or about 0.01-10 mg per day to adults (at about 60 kg). In the case of other animals, the dose calculated for 60 kg may be administered as well. [000178] Doses of a pharmaceutical composition of the present disclosure can optionally include 0.0001 μg to 1,000 mg / kg / administration, or 0.001 μg to 100.0 mg / kg / administration, from 0.01 μg to 10 mg / kg / administration, from 0.1 μg to 10 mg / kg / administration, including, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and / or 100-500 mg / kg / administration or any range, value or fraction thereof, or to achieve a serum concentration of 0.1 , 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0,52 178013745.113.5, 13.9, 14, 14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and / or 5000 μg / ml serum concentration per single or multiple administration or any range, value or fraction thereof. [000179] As a non- limiting example, treatment of humans or animals can be provided as a onetime or periodic dosage of a composition of the present disclosure 0.1 ng to 100 mg / kg such as 0.0001, 0.001, 0.01, 0.10.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, or alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof, using single, infusion or repeated doses. [000180] Specifically, the pharmaceutical compositions of the present disclosure may be administered at least once a week over the course of several weeks. In one embodiment, the pharmaceutical compositions are administered at least once a week over several weeks to several months. In another embodiment, the pharmaceutical compositions are administered once a week over four to eight weeks. In yet another embodiment, the pharmaceutical compositions are administered once a week over four weeks. [000181] More specifically, the pharmaceutical compositions may be administered at least once a day for about 2 days, at least once a day for about 3 days, at least once a day for about 4 days, at least once a day for about 5 days, at least once a day for about 6 days, at least once a day for about 7 days, at least once a day for about 8 days, at least once a day for about 9 days, at least once a day for about 10 days, at least once a day for about 11 days, at least once a day for about 12 days, at least once a day for about 13 days, at least once a day for about 14 days, at least once a day for about 15 days, at least once a day for about 16 days, at least once a day for about 1753 178013745.1days, at least once a day for about 18 days, at least once a day for about 19 days, at least once a day for about 20 days, at least once a day for about 21 days, at least once a day for about 22 days, at least once a day for about 23 days, at least once a day for about 24 days, at least once a day for about 25 days, at least once a day for about 26 days, at least once a day for about 27 days, at least once a day for about 28 days, at least once a day for about 29 days, at least once a day for about 30 days, or at least once a day for about 31 days. [000182] Alternatively, the pharmaceutical compositions may be administered about once every day, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every 7 days, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 14 days, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, about once every 30 days, or about once every 31 days. The pharmaceutical compositions of the present disclosure may alternatively be administered about once every week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about once every 9 weeks, about once every 10 weeks, about once every 11 weeks, about once every 12 weeks, about once every 13 weeks, about once every 14 weeks, about once every 15 weeks, about once every 16 weeks, about once every 17 weeks, about once every 18 weeks, about once every 19 weeks, about once every 20 weeks. [000183] Alternatively, the pharmaceutical compositions of the present disclosure may be administered about once every month, about once every 2 months, about once every 3 months, about once every 4 months, about once every 5 months, about once every 6 months, about once every 7 months, about once every 8 months, about once every 9 months, about once every 10 months, about once every 11 months, or about once every 12 months.54 178013745.1[000184] Alternatively, the pharmaceutical compositions may be administered at least once a week for about 2 weeks, at least once a week for about 3 weeks, at least once a week for about 4 weeks, at least once a week for about 5 weeks, at least once a week for about 6 weeks, at least once a week for about 7 weeks, at least once a week for about 8 weeks, at least once a week for about 9 weeks, at least once a week for about 10 weeks, at least once a week for about 11 weeks, at least once a week for about 12 weeks, at least once a week for about 13 weeks, at least once a week for about 14 weeks, at least once a week for about 15 weeks, at least once a week for about 16 weeks, at least once a week for about 17 weeks, at least once a week for about 18 weeks, at least once a week for about 19 weeks, or at least once a week for about 20 weeks. [000185] Alternatively the pharmaceutical compositions may be administered at least once a week for about 1 month, at least once a week for about 2 months, at least once a week for about 3 months, at least once a week for about 4 months, at least once a week for about 5 months, at least once a week for about 6 months, at least once a week for about 7 months, at least once a week for about 8 months, at least once a week for about 9 months, at least once a week for about 10 months, at least once a week for about 11 months, or at least once a week for about 12 months. [000186] KITS [000187] In certain embodiments, diagnosing a subject with ACM further comprises detection of increased levels of tumor necrosis α (TNFα), muscle contraction signaling molecules or the combination thereof in a sample from the subject. In certain embodiments, muscle contraction signaling molecules comprise β-catenin, c-Src or the combination thereof. [000188] Accordingly, the disclosure provides kits for diagnosing arrhythmogenic cardiomyopathy (ACM) comprises one or more agents detecting levels of lactosylceramide synthase (LacCer), tumor necrosis α (TNFα), β-catenin, c-Src or combinations thereof. In certain embodiments, the kit further comprises a pharmaceutical composition comprising one or more inhibitors of β-galactosyltransferases, one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof. [000189] Diagnosis and Treatment for Arrhythmogenic Cardiomyopathy (ACM)55 178013745.1[000190] Provided herein are methods of identifying a subject suffering from or susceptible to arrhythmogenic cardiomyopathy (ACM) is provided that comprises: a) assaying to identify C24:0 LaCer and / or C24:1 LacCer in a sample obtained from a subject; b) diagnosing the subject as having arrhythmogenic cardiomyopathy (ACM) when the C24:0 LaCer and / or C24:1 LacCer level is different (e.g. increased) relative to the C24:0 LaCer and / or C24:1 LacCer level in a healthy subject; and optionally c) administering a therapy in subjects diagnosed with arrhythmogenic cardiomyopathy; thereby, diagnosing and treating a subject. [000191] In aspects, the subject may be identified at as having arrhythmogenic cardiomyopathy (ACM) when the C24:0 LacCer and / or C24:1 LacCer level is increased by at least 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 10, 120, 140, 160, 180, 200, 300, 400, 500 percent or more relative to the C24:0 LacCer and / or C24:1 LacCer levels in a healthy subject. C24:0 LacCer and / or C24:1 LacCer level may be assessed in amounts for example of pmole or nmole / mg. In aspects, the subject may be identified at as having arrhythmogenic cardiomyopathy (ACM) when the C24:0 LacCer and / or C24:1 LacCer level is increased by a detectable increased level relative to a control such as up to or at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 pmole or nmole / mgof C24:0 LacCer and / or C24:1 LacCer. Suitable methods to determine C24:0 LacCer and / or C24:1 LacCer levels are known and include chromatography and spectroscopy (including mass spectroscopy). An exemplary preferred protocol to determine C24:0 LacCer and / or C24:1 LacCer levels is set forth in Example 3 which follows. [000192] Detection of an alteration relative to a normal, reference sample can be used as a diagnostic indicator of a disease, particularly arrhythmogenic cardiomyopathy (ACM). In some embodiments, altered methylation of a particular gene is correlated with a particular disease. [000193] The present disclosure also features diagnostic assays for the detection of a disease or the propensity to develop such a condition. In one embodiment, the level of C24:0 LacCer and / or C24:1 LacCer level is measured once or in some aspects at least two separate occasions and an increase in the level is an indication of disease progression. [000194] The diagnostic methods described herein can be used to provide a diagnosis individually or to confirm the results of another diagnostic method. Additionally, the methods described herein can be used with any other diagnostic method described herein for a more56 178013745.1accurate diagnosis of the presence or severity of a disease, particularly arrhythmogenic cardiomyopathy (ACM). [000195] In aspects, The disclosure also provides for methods where C24:0 / C24:1 LacCer levels are measured before and after subject management. In these cases, the methods are used to monitor the status of esophageal cancer, e.g., a response to treatment, or progression of arrhythmogenic cardiomyopathy (ACM). [000196] In some embodiments, the present methods can be used in monitoring responses to therapy. [000197] Kits and Compositions [000198] In another embodiment, kits and compositions are provided that advantageously allow for the detection of where C24:0 LaCer and / or C24:1 LacCer in a subject sample. [000199] Therapy [000200] Once the subject has been diagnosed with arrhythmogenic cardiomyopathy (ACM), health care professionals can administer arrhythmogenic cardiomyopathy (ACM) therapy. Preferred methods of treatment include administering to the subject a therapeutically effective amount of one or more inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, wherein the LacCer amounts in the subject are decreased as compared to a normal baseline amount a disclosed herein. [000201] Additional therapeutics include suppressing suppress abnormal heart rhythms and prevent ventricular tachycardia (VT). Beta-blockers therapeutics may be administered To slow the heart rate and reduce the risk of arrhythmias. Diuretics may be administered to remove excess fluid and improve heart function. Aldosterone antagonists may be administered To reduce fluid retention and improve heart function. [000202] Additional treatment may include implanting a cardioverter-defibrillator (ICD), e.g. implanted under a subject’s skin that monitors heart rhythm and delivers an electric shock if a life-threatening arrhythmia occurs. A catheter ablation procedure e.g. to destroy heart tissue that is causing the abnormal rhythms.57 178013745.1EXAMPLES [000203] Example 1: [000204] A new mechanism of GSL-mediated dyslipidemia in CM is shown. By investigating GSL levels and β-1,4-GalT-V activity in ACM and wild-type mice, extensive calcification was found in the aortic valves and right ventricles and increased levels of LacCer. The latter of which was the result of increased activity of LacCer synthase and was strongly correlated with cardiac dysfunction. Moreover, oral delivery of the GSL glycosyltransferase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (BPD), dose- dependently decreased myocardial LacCer synthase activity and LacCer levels in ACM mice. Findings that were accompanied by improved heart function. Additionally increased levels of LacCer in cardiac explants, endomyocardial biopsies in patients with ACM, and two mouse models of ACM (Dsg2mut / mutand DspS311A / S311Amice) was observed. Blocking β-1,4-GalT-V activity and LacCer generation, via BPD, markedly reduced cardiac and aortic calcification and improved cardiovascular outcomes. [000205] METHODS [000206] Animal studies [000207] All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH publication #85-23, revised in 1996) and were approved by the Johns Hopkins University School of Medicine Animal Care and Use Committee. Dsg2mut / mutand DspS311A / S311Amice were fed standard mouse chow and water ad libitum. A detailed description of the Dsg2mut / mutmice used in this study is described in Chelko, SP et al.7Frozen tissues from DspS311A / S311Amice were collected by Dr. Tania Zaglia and colleagues from the University of Padua and delivered to Dr. Chatterjee’s lab on dry ice. [000208] Echocardiography and in vivo drug treatment [000209] Cardiac function was assessed via transthoracic echocardiography, as previously described,7,20using a Vevo 2100 Visual Sonic imaging system (FUJIFILM Visualsonic, Inc., Toronto, ON Canada) on non-anesthetized mice. Echocardiographic measurements followed the guidelines of the American Society of Echocardiography.20Baseline echocardiograms were58 178013745.1performed before BPD treatment (14 weeks of age) and at study endpoint (20 weeks of age). BPD was dissolved in 5% Tween-80 in 1X PBS at 1 mg / ml and delivered at 10 mg / kg of BPD via oral gavage, 5 days / week for 6 weeks. (+)-D-threo-PDMP (D-PDMP, a sphingolipid biosynthesis inhibitor) was purchased from Matreya LLC Lipids and Biochemicals (Cat. No. 1719) and encapsulated within a biodegradable polymer.21[000210] Immunohistochemistry and immunofluorescence [000211] Dsg2mut / muthearts were fixed in 10% formalin overnight then embedded in paraffin blocks. Thin sections (5 µm) were used in various hematology and immunohistochemistry and immunofluorescent (IHC / IF) studies. Masson’s Trichrome was used to determine percent fibrosis and various antibodies were utilized to determine antigen distribution in myocardial sections (e.g., platelet endothelial cell adhesion molecule-1 [PECAM- 1]). A detailed list of antibodies and their respective vendor, catalog number, and dilutions can be found in Table 2. Mouse anti-β-1,4-GalT-V antibody was used at a 1:10,000. Immunostains were performed and standardized by the Johns Hopkins Cytopathology laboratory, and images were captured at 100X. [000212] ImageJ was utilized to quantitatively analyze immunostains. The image files were converted to RGB format to facilitate accurate color analysis, maintaining the integrity of the color information needed for further downstream analyses. Color deconvolution was then performed to separate the visible colors, allowing for more precise identification of the specific components of interest. Threshold values were set manually following antibody optimization and test images acquired, thus, minimizing inclusion of extraneous elements. To further refine the analysis, a minimum particle size threshold was defined. This parameter was crucial in eliminating noise, preventing false positives, and enhancing the specificity and reliability of the quantitative analysis of immunostains. The Analyze Particle and Measure tools in ImageJ were used for this analysis. This approach yielded several key metrics: mean area, standard deviation with variability in the size of the stained regions, and histograms of maximum and minimum fluorophore intensity vs fluorophore distribution.59 178013745.1[000213] These data points were used to calculate the percentage of positively immunostained tissue. Lastly, number of positively immunostained cells and total cell count was determined for each image via The Cell Counter Plug-in software within ImageJ. [000214] Liquid chromatography tandem mass spectrometry (LCMSMS) [000215] Appropriate internal standards were added to tissue before extracting total lipids. As previously described, we subjected samples to hydrophilic interaction liquid chromatography (HILIC).22We counted individual sphingolipids using reference standard curves and deuterium- labeled sphingolipids. [000216] Liquid Chromatography Tandem Mass Spectrometry, LCMSMS, Analysis of Sphingolipids [000217] Chemicals, reagents, and materials: MilliQ water, filtered deionized water; Methanol, optima grade was obtained from Thermo Fisher Scientific (Fair Lawn, NJ). Chloroform, HPLC grade was from Burdick & Jackson (Muskegan, MI), Acetonitrile, HPLC grade, Formic acid and ammonium formate, and reagent grade were purchased from Sigma- Aldrich (St. Louis, MO) [000218] Formalin-fixed human heart samples and frozen mouse heart tissues (0.1 to 0.2 g) were extracted with chloroform: methanol 2:1, v / v according to Folch et al. (1). The following internal standards were added at time of extraction: (i) Ceramide (d31-C16:0), (ii) Lactosyl (β)Ceramide (d7-24:1), (iii) Sphingomyelin (SM, d9-18:1), (iv) Glucosyl(β) C12 Ceramide, and (v) C12-Ceramide1-P, all purchased from Avanti Polar Lipids Inc. [000219] Lower phase lipids were dried under N2and weighed. Approximately 0.1mg lipid from each sample was dissolved in methanol and was analyzed by LCMSMS on a 3200 mass spectrometer (AB Sciex) equipped with a Schimadzu HPLC. Data was collected using multiple reaction monitoring in the positive ion mode. Curtain gas was set at 30, ion spray voltage at 5500, and ion source was heated to 6000C Collision energy was optimized for each lipid class, and was 40 for Cer and DH Cer, 45 for GlcCer, 55 for LacCer, 45 for sphingomyelins and 50 for Cer-1-P, Table 1. The LCMSMS data acquisition and management was processed using AB- Sciex Analyst 2.1 mass spectrometer software. The same software integration parameters were60 178013745.1used to integrate the areas under the curve for both the internal standards and the analytes. Standard curves were made for each sphingolipid species. [000220] Separation of lipids was achieved by analysis on 2 separate columns, straight phase and reverse phase in order to confirm the identity of the sphingolipids The first column was a hydrophilic interaction liquid chromatography, a Kinetex HILIC column, 150 x 3mm, particle size 2.6um (Phenomenex, Torrance, CA) by a modification of the method of Scherer et al. (2) The injection volume was 10ul and the column temperature was maintained at 370C. The mobile phase A was 100% MilliQ water containing 53uM formic acid and 60mM ammonium formate. Mobile phase B was 100% acetonitrile containing 53uM formic acid. Flow rate was 0.35ml per minute. The lipids were separated with a linear gradient, of time 0, 90% B to 70%B at 12 minutes stop at 13 minutes with return to 90%B and equilibration for 2 min. before the next injection. The second column, reverse phase, was an Acquity BEH C18, 2.1 x 50 mm column, particle size 1.7um (Waters, Milford, MA) by a modification of the method of Anroedh et al. (3) The injection volume was 10ul and the column temperature was maintained at 370C . Mobile phase A was 100% MilliQ water containing 0.2% formic acid and 2mM ammonium formate. Mobile phase B was 100% acetonitrile with 0.2% formic acid and 1mM ammonium formate. Flow rate was 0.4ml per minute with 80%B at time zero increasing to 90%B from 3.0 min. to 6 min. and a linear increase to 99% at 10min. with stop at 13min. and return to 80% B for 2min. before the next injection. [000221] The measurement of the N-acyl sphingolipids by LCMSMS was done to confirm the identity of and to quantitate the amounts of the sphingolipids that had been found by analyzing the heart tissue lipids by thin layer chromatography. Note: Only the non-hydroxy sphingolipids are reported in this manuscript. [000222] Myocardial LacCer synthase and β-galactosidase activity, and circulating biomarkers, cholesterol, and triglyceride levels [000223] Ventricular LacCer synthase activity was measured using the radioactive substrate [3H]UDP -galactose as substrate, as previously described,23from untreated and BPD-treated61 178013745.1Dsg2mut / mutmice. β-galactosidase activity was measured using an artificial substrate, as previously described.24[000224] The levels of cholesterol and triglycerides were measured in serum samples from wild-type (WT) and ACM mice using Wako Lab assay kits (Cat. No., LCHM-23E1, LTGM- 23F1) per the manufacturer’s protocol and normalized using cholesterol and triglyceride standards (Fuji Wako Shibayagi Corporation, Japan). Enzyme-linked immunosorbent assays were used to determine plasma levels of ox-LDL, β-catenin, TNFα, c-Src, and VE-cadherin. [000225] Positron emission tomography and MR imaging [000226] Each mouse was injected with 4.1 ± 0.74 MBq (110 ± 20 µCi) of18F-NaF (PETNET Solutions, Los Angeles, CA) diluted in physiologic saline. One-hour post-injection uptake, mice were scanned simultaneously by a PET-MRI (7T pre-clinical PET-MRI, Bruker BioSpec, 70 / 30 USR, Germany) with and without a navigator. In this procedure, whole-body coronal Turbo RARE T2-weighted scan was first collected to localize the cardiac structure using the flowing parameters: (i) TE: 45 ms, (ii) TR: 3000 ms, (iii) slice thickness: 1 mm, (iv) matrix size: 400 X 200 mm, and (v) field of view (FOV): 100 X 60 mm. A total of 4 recordings were taken per mouse and averaged. [000227] Cardiac MRI data were collected using the following parameters: (i) TE: 4.8 ms; (ii) TR: 11.25 ms; (iii) FA: 10 degrees / Santosh; (iv) movie frame: 14 total frames / Santosh with 0.8 mm slice thickness with no slice gap; (v) matrix size: 256 X 256 mm; (vi) and FOV: 25 X 25 mm with 0.098 mm resolution using an in-slice navigator position. Total scan time for cardiac imaging was 10 minutes. [000228] PET imaging was performed using a 3-ring PET insert with a spatial resolution of 0.7 mm and a FOV of 80 x 148 mm. 10min static scans were used that were simultaneous with MRI imaging 60 minutes post-18F-NaF injection for PET data collection. The PET data was reconstructed using maximum likelihood expectation maximization (MLEM) with a 0.5 mm preset in 18 iterations. After quality assessment, reconstructed cardiac imaging data were spatially co-registered with PET data using PV 360. Analyses were performed using PMOD software (PMOD Technologies, LLC; Faellanden, Switzerland).62 178013745.1[000229] Near-infrared fluorescence (NIRF) imaging [000230] Concurrent with radio-fluoride injection, each mouse was intravenously injected with 2 nmol of BoneTag® (LI-COR Biosciences, Lincoln, Nebraska) for calcium density imaging. Mice underwent a 1 hour uptake time followed by PET / MRI scanning. Mice were then euthanized by cervical dislocation, thoracic cavities were exposed, and hearts were imaged using a Pearl Impulse near-IR imager (LI-COR Biosciences) for in situ imaging. Following in situ imaging, hearts containing the coronary artery, and the roots of the greater vessels were harvested and underwent high resolution near-IR fluorescence imaging. [000231] Statistical analysis [000232] Statistical analyses used are described in each figure legend. Data are presented as mean±SEM and Pearson’s r correlations. Differences between measured variables underwent parametric t-tests, Mann-Whitney, or One-Way ANOVA with Tukey’s posthoc analysis. GraphPad Prism 9 was used to perform statistical analyses and prepare figures. A P-value of <0.05 was deemed statistically significant and differences between P-values are distinguished according to *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. [000233] RESULTS [000234] Myocardium from ACM subjects harbor elevated levels of lactosylceramide [000235] Sphingolipids play critical roles in regulating multiple cell responses, organ systems and phenotypes.25,26In certain, LacCer is associated with atherosclerosis and inflammation,10,11and the inventors recently highlighted the impact of myocardial inflammation on disease progression in ACM.27Therefore, it was investigated whether LacCer or other sphingolipid metabolites play a role in ACM myocardial inflammation, dyslipidemia, or cardiac function. The levels of various sphingolipids were determined using HPTLC, and then sophisticated mass spectrometric methods were employed to validate the initial findings. Using HILIC column LCMSMS analysis, it was observed that the levels of total ceramide (Cer) and glucosylceramide (GlcCer) were unchanged in myocardium from patients with ACM compared to healthy subjects (FIGS. 1A, 1B). In contrast, the total levels of LacCer were increased by 2.3- fold in myocardial tissue from patients with ACM (FIG. 1C). In contrast, the level of63 178013745.1sphingomyelin (SM), the predominant sphingolipid, was similar in myocardium from patients with ACM and healthy subjects (FIG. 1D). [000236] Hearts from Dsg2mut / mutmice additionally showed elevated LacCer levels, which strongly correlated with decreased cardiac function (FIGS. 2A-2F). HPTLC analysis of myocardial lipid extracts from 16-week-old Dsg2mut / mutmice showed a marked increase in the level of LacCer compared to wild-type mice (FIGS. 2A, 2B). Specifically, echocardiographic data demonstrated a strong inverse relationship between LacCer levels and percent ejection fraction (%EF, r = -0.9456) and percent fractional shortening (%FS, r = -0.9432) (FIGS. 2C-2F). [000237] Dsg2mut / mutand DspS311A / S311Amice display elevated levels of myocardial lactosylceramide [000238] To confirm the findings in samples from patients with ACM and Dsg2mut / mutmice, myocardial sphingolipid levels were assessed by MSMS analysis in two different mouse models of ACM, Dsg2mut / mutand DspS311A / S311Amice. Pathogenic variants in DSG2 and DSP give rise to left-dominant disease, yet both Dsg2mut / mutand DspS311A / S311Amice show biventricular fibrosis and dysfunction. Both mice exhibit age-onset diminution in cardiac function and biventricular fibrosis, yet disease onset is more aggressive in Dsg2mut / mutmice (2 months of age)7compared to DspS311A / S311Amice (4 months of age). Previously, we have shown that feeding a high fat and cholesterol diet to ApoE- / -mice for 20 weeks contributes to cardiac hypertrophy.21In contrast, Dsg2mut / mutmice display a steady decline in cardiac function from age 8 weeks.7LCMSMS analysis revealed a ~5.0-fold increase in myocardial levels of LacCer in Dsg2mut / mutmice compared to wild-type controls at age 12 weeks (FIGS. 7A-7C). Notably, cardiac tissue from DspS311A / S311Amice only showed a ~3-fold increase in LacCer levels (FIGS. 8A-8C). Myocardial ceramide and glucosylceramide levels were not different among the two groups of mouse myocardial tissue. (FIGS. 8A-8C). Collectively, a dyslipidemic phenotype in ACM appears to be a consistent theme, as our findings demonstrated cardiac samples from patients with ACM and two different mouse models of ACM all showed elevated levels of myocardial LacCer. Whether the higher levels of LacCer observed in Dsg2mut / mutmice contribute to the earlier and more aggressive disease onset compared with DspS311A / S311Amice remains to be established.64 178013745.1[000239] Inhibiting glycosphingolipid synthesis reduced elevated plasma levels of ox- LDL in Dsg2mut / mutmice and lactosylceramide synthase [000240] It was previously observed in human arterial smooth muscle cells that ox-LDL and / or oxidized phospholipids (e.g., POVPC) can activate LacCer synthase via the phosphorylation of its threonine, serine, and tyrosine residues via activating protein tyrosine kinases.11It was also found that the glycosphingolipid (GSL) synthesis inhibitor BPD blocked the activation of LacCer synthase. Hence the mass of ox-LDL was measured in plasma from Dsg2mut / mut, DspS311A / S311A, and WT mice. As shown in FIGS. 3A, 3B, plasma levels of ox-LDL were increased in both Dsg2mut / mutand DspS311A / S311Amice. However, when Dsg2mut / mutmice were treated for 6 weeks with BPD, these mice showed reduced plasma levels of ox-LDL compared to WT counterparts (FIG. 3A). [000241] Two major enzymatic activities govern the cellular level of LacCer. The first is LacCer synthase, which converts glucosylceramide to LacCer via the transfer of galactose from UDP-galactose. The other enzyme is a β-galactosidase that cleaves the terminal β-1,4 galactose linked to glucose in LacCer to form glucosylceramide. To determine if any of these enzymes contributed to an increase in the level of LacCer in Dsg2mut / mutmice myocardium, the activity of LacCer synthase and β-galactosidase was measured. It was observed that the activity of myocardial LacCer synthase was markedly elevated (P<0.0097) in Dsg2mut / mutmice compared to WT controls (FIG. 3B). In contrast, the enzymatic activity of β-galactosidase was similar between cohorts (FIG. 3C). [000242] In placebo-treated Dsg2mut / mutmice, a stark decline (P<0.0157) in cardiac function occurred from 14 to 20 weeks of age (FIGS. 3D, 3E). In contrast, the difference in cardiac function was insignificant in mice treated with 1 mg / kg of BPD (FIGS. 3D, 3E). In contrast, Dsg2mut / mutmice administered 10 mg / kg of BPD during this period resulted in further cardiac dysfunction (FIGS. 3D, 3E). Also, measuring fractional shortening in the placebo group of mice revealed that this decreased significantly (P<.0158) in 20-week-old mice vs. 14-week-old mice. Conversely, feeding these mice 1 mpk or 10 mpk of BPD daily for 6 weeks maintained the fractional shortening as the difference was insignificant (FIG. 3E). Thus, these studies reveal that65 178013745.1lactosylceramide synthase may well be a novel therapeutic target to improve cardiac function in the Dsg2mut / mutmice. [000243] Dsg2mut / mutis accompanied by increased plasma levels of TNFα, β-catenin, and c-Src expression; these levels upon treatment with BPD [000244] Plasma levels of a powerful pro-inflammatory cytokine, TNFα, was significantly increased in placebo-treated Dsg2mut / mutmice compared to wild-type mice plasma (FIG. 9A). However, treatment with BPD decreased TNFα level to within the normal range (FIG. 9A). Similarly, the plasma level of critical signaling molecules in muscle contraction, e.g., β-catenin and c-Src but not VE-Cadherin, were also increased in Dsg2mut / mutmice and were decreased upon treatment (FIGS. 9B, 9C, 9D). These studies show that Dsg2mut / mutmice are in an inflammatory / pro-oxidant state. This state can improve remarkably when blocking β-1,4-GalT-V activity and LacCer generation with BPD. [000245] Dsg2mut / mutmice have increased fibrosis, PECAM-1, and ^^-catenin, and this is mitigated by BPD treatment [000246] Myocardial fibrotic lesions were abundant from placebo-Dsg2mut / mutmice (FIG. 4A), a finding that was reduced in 1mg / kg BPD-treated Dsg2mut / mutmice (FIGS. 4A-4F). A strong reaction to an antibody against PECAM-1 / CD31 was noted in placebo mice (FIGS. 4A- 4F). This reaction was significantly reduced by treatment with 1 BPD and 10 BPD (FIGS. 4F, 4G). ^^-catenin is a major cell junctional protein in cardiomyocytes, implicated in muscle contraction. A statistically significant increase was observed in ^^-catenin in placebo mice (FIG. 4I), markedly reduced upon treatment with BPD (FIGS. 4J, 4K). [000247] Dsg2mut / mutmice have increased aortic and myocardial calcification; this is mitigated by treatment with BPD [000248] A cohort of mice were imaged using 10 min static PET acquisitions followed by standard T1 imaging to provide anatomic background of the thoracic cavity (FIGS. 5A, 5B). Other mice were also scanned using a 10 min static PET acquisition but co-registered to MRI anatomy acquired using the Navigator protocol, yielding both anatomy and T1 weighted contrast showing blood within the heart and vessels (FIG. 5A).18F-NaF PET imaging revealed putative66 178013745.1calcifications in the large vessels (FIGS. 5A, 5B, white arrows) and heart wall (FIG. 5A, white arrows) of untreated controls and some treated but non-responder mice. [000249] Following PET imaging, mice were injected with a fluorescent tetracycline derivative (Bone TagTM), which detects calcifications and de- / re-mineralization of Ca2+ions.28As with the PET uptake data, the NIRF imaging showed calcifications within large vessels and even heart walls (FIGS. 5A, 5B white arrows) in untreated mice and mice responsive to treatment. [000250] DISCUSSION [000251] The study herein identifies LacCer as a bioactive signaling molecule in cardiac function, aortic calcification, and myocardial calcification in a mouse model of ACM (FIG. 6). First, lipids in myocardium were examined from deceased and discarded tissues from patients with ACM and compared them with healthy tissues. The studies herein focused on glycosphingolipids, as ACM is associated with extensive myocardial inflammation. Further, lactosylceramide, a member of the large family of GSL, exhibits inflammatory properties via superoxide generation and facilitation of circulating neutrophil infiltration into the sub- endothelial space.29, ,30This is because LacCer induces the expression of cell adhesion molecules, e.g., intercellular cell adhesion molecule-1 (ICAM-1) and platelet / endothelial cell adhesion molecule-1 (PECAM-1) in human arterial endothelial cells and human umbilical vein endothelial cells.31,32,33LacCer also induces the expression of CD11b / Mac-1 on the surface of human neutrophils and monocytes.34,35As ICAM-1 serves as a cognate receptor to its ligand CD11b / Mac-1, it results in cell-cell adhesion followed by the trans-endothelial migration of neutrophils and monocytes. As neutrophils carry the bulk of LacCer among all blood cells, a neutrophil burst may release a LacCer load in the heart muscle. Additionally, in human neutrophils, LacCer activates cytosolic phospholipase A-2 (cPLA2), an enzyme that cleaves phosphatidylcholine to generate arachidonic acid — a precursor to eicosanoids and prostaglandins, which are intimately involved in inflammation.34In human neutrophils, where LacCer is predominantly localized on the cell surface, LacCer forms GSL-enriched microdomains involving the Src family of kinases.36Studies show that CD11b / CD18 integrin can cause rearrangement of the actin cytoskeleton, allowing neutrophil infiltration.37Additionally, as PECAM-1 is one of the integral components of cell-cell junctions, its regulation by LacCer may67 178013745.1well impact muscle contraction mediated by various muscle proteins (e.g., desmoglein-2 and desmocollin-2) and Ca2+flux.32,33[000252] The LCMSMS studies herein revealed an enriched total mass of LacCer in myocardium from patients with ACM and Dsg2mut / mutmice compared to control counterparts. Previous Langmuir film balance and calorimetry studies reveal that very long chain fatty acid (VLCFA) refer LacCer imparts a condensed and chain-ordered phase behavior.39This physicality of VLCFA-LacCer may provide easy access to caveolar proteins, e.g. Lyn, Src. This tenet is supported by the observation that LacCer prepared from prepared from human atherosclerotic plaques produced more O2-than bovine erythrocyte membrane LacCer or stearoyl LacCer.40,41Thus, it was speculated that the abundance of LacCer in human and Dsg2mut / mutmouse heart tissue itself renders a “pro-oxidant” environment by generating large amounts of superoxides which can participate in oxidizing a variety of biologically critical molecules, including LDL. In turn, ox-LDL can activate β-1,4-GalT-V to generate more LacCer to continue the cycle of reactions building upon the “pro-oxidant” environment and ox-LDL in the heart. The inventors have shown the mechanism by which ox-LDL activate various phosphokinases, which in turn activate LacCer synthase via phosphorylating serine, threonine, and tryptophan residues in LacCer synthase.14[000253] No differences were observed in the ceramide mass in ACM patient myocardium tissue and two ACM mutant mouse RV tissues. In light of these observations, a presumptive role of Cer in the apoptotic death of cardiomyocytes in ACM needs to be examined in detail. However, the level of glucosylceramide was similar between the ACM and healthy heart RV (FIG. 1B). A possible explanation for this observation is that since LacCer synthase activity in ACM heart RV was significantly higher than WT mice heart (FIG. 3A), it utilized glucosylceramide (GlcCer) to synthesize more LacCer. [000254] In plasma, levels of ox-LDL and its cognate receptor, CD35, were higher in ACM mice. Thus, ox-LDL has been proposed as a pathogenic trigger in ACM.42Here, adipogenesis in ACM was attributed to over-expression of PPAR-^^, a major effector in lipid accumulation. This study used a heterozygous mouse model of ACM fed a Western diet. Thus, hyperlipidemia was induced by the consumption of a Western diet. In contrast, this study used two different68 178013745.1homozygous ACM mouse models. The mice were fed normal mouse chow and elevated ox-LDL levels were observed, which may be more causally related to cardiovascular events than elevated LDL cholesterol levels. This is because total plasma cholesterol levels in the Dsg2mut / mutand DspS311A / S311Amutant mice were modestly lower than in wild-type mice. In a retrospective study using 6,881 patients, high triglyceride / HDL and LDL / HDL ratios were observed to be predictive of arrhythmias and diabetes. Thus, patients with low HDL had a greater risk of developing cardiac arrhythmias.43Whether increased plasma levels of ox-LDL alone are predictive / associated with arrhythmias in patients with ACM needs to be explored. [000255] As reported previously, the Dsg2mut / mutmice exhibit an age-onset decline in cardiac function by way of decreased ejection fraction and fractional shortening, which was reproduced in the present study (compare 14 weeks vs 20 weeks (solid and hatched bars in FIGS. 3D, 3E, respectively).7When siblings from the same cohort of ACM mice were fed BPD by oral gavage, we observed no significant decline in ejection fraction and fractional shortening. Moreover, treatment decreased both the plasma level of ox-LDL (FIGS. 3A-3E) and LacCer synthase activity (FIG. 3B). Our immunohistochemistry studies revealed that treatment also reduced fibrosis (FIG. 4A). PECAM-1 (CD-31) (FIG. 4B) and ^^-catenin (FIG. 5C) in cardiac tissue in Dsg2mut / mutmice. Thus, blocking LacCer synthase activity is a novel approach to mitigate cardiac dysfunction in a Dsg2mut / mutmouse model of ACM. [000256] Previous studies have shown that oxidized / modified LDL is enriched in atherosclerotic plaques and promotes inflammation, inflammatory cytokine production, and aortic valve calcification.44,45Another study showed that β-catenin overexpression increased TNF-^^ and IL-8 in cardio-myoblasts and hypertensive neonatal rat cardiomyocytes.46Treatment reduced β-catenin and TNF-^^ (FIG. 9A) and β-catenin (FIG. 9B). Additionally, these studies identify LacCer accumulation in the fibro-fatty deposits in the RV or LV as a critical bioactive signaling molecule that generates O2-, thus providing a “pro-oxidant” environment allowing a cycle of oxidation of LDL, ox-LDL induced β-1,4-GalT-V activation, and LacCer generation (FIG. 6). LacCer is a pro-inflammatory molecule, activating c-PLA2 and catabolizing phosphatidylcholine to produce arachidonic acid, a precursor to many pro-inflammatory molecules.69 178013745.1[000257] A previous study on Dsg2mut / mutmice suggests a central role for inflammation in the pathophysiology of this disease.27This was mitigated downstream using Bay 11-7082, a small molecule inhibitor of NFĸB signaling. Since we and others have documented LacCer as a crucial pro-inflammatory molecule that is a conduit to TNFα induced phenotypes, the present study identifies LacCer accumulation in ACM as a causative factor contributing to inflammation. Treatment can mitigate inflammation by reducing LacCer synthase activity, which may also block the glycosylation of N-linked glycoproteins with the GlcNac 6-Gal sequence. [000258] The calcification studies in ACM evolved while homogenizing heart tissue that revealed tissue mineralization. That led us to use PET-MRI and imaging studies in live ACM mice treated with and without BPD. A previous study showed that cardiac arrhythmias could be due to heritable mutations in the myocardium associated with defects in Ca2+handling genes, as in the case of atrial fibrillation and ventricular arrhythmias in heart patients.47This may be the case in this study with ACM mice. Both in vivo PET-MRI imaging and subsequent ex vivo NIRF imaging showed significant calcifications in untreated and some treated mice. Calcification was observed of not only the brachiocephalic artery, ascending aorta, and aortic valve but also, surprisingly, in the right ventricle walls in Dsg2mut / mutmice (FIGS. 5A, 5B). Treatment with BPD markedly reduced calcification in these tissues (FIGS. 5C, 5D). [000259] Inflammation and atherosclerosis have been collectively observed in several studies of ACM. However, vascular calcification and inflammation have also been reported in patients with chronic kidney disease.48Calcification also causes stenosis and aortic valve disease in older people.49Increased oxidized phospholipids (components of oxidized LDL) and lipoprotein(a) in patients and ApoE- / -mice have also been associated with aortic valve calcification.50Non-invasive PET imaging using18F-NaF PET is widely clinically available. It can monitor treatment progress in mice and patients, allowing changes to the treatment regimen before physical symptoms appear. At present, it was speculated that trans-differentiation of cardiovascular cells into bone-forming osteoblasts due to increased levels of ox-LDL and TNF-^^ may well contribute to increased calcification. Alternatively, BMDC migration to sites of inflammation where the local cytokine mixes steers cells to differentiate into an osteoblast phenotype may be another possible avenue for calcification to occur. Moreover, in the70 178013745.1Dsg2mut / mutmice, as both ox-LDL and TNF-^^ rise in the plasma, β-1,4-GalT-V activity increases and consequently raises cardiac LacCer levels. Conversely, BPD, which targets β-1,4-GalT-V, reduces plasma levels of ox-LDL and plasma levels of TNF-^^, improves cardiovascular health, and reduces calcification. [000260] Table 1. API 3200 mass spectrometer settings for complex sphingolipids.71 178013745.1Sphingolipid N-Acyl Q1 m / z Q3 m / z DP(V) CE(V) d31-C16:0 569.40 265.3 40 40178013745.1[000261] Table 2. Antibody Specifics Antibody Dilution Vendor Catalog # Species VE-Cadherin 1:3,000 (ELISA), 1:2,000 Santa Cruz sc-52751 MouseREFERENCES 1. Pilichou K, Thiene G, Bauce B, Rigato I, Lazzarini E, Migliore F, Perazzolo Marra M, Rizzo S, Zorzi A, Daliento L, et al. Arrhythmogenic cardiomyopathy. Orphanet journal of rare diseases.2016; 11:1-7. 2. Scheel III PJ, Murray B, Tichnell C, James CA, Tandri H, Calkins H, Chelko SP, Gilotra NA. Arrhythmogenic right ventricular cardiomyopathy presenting as clinical myocarditis in women. The American journal of cardiology.2021; 145:128-34. 3. Wlodarska EK, Konka M, Zaleska T, Ploski R, Cedro K, Pucilowska B, Bekiesinska-Figatowska M, Rydlewska-Sadowska W, Ruzyllo W, Hoffman P. Arrhythmogenic right ventricular cardiomyopathy in two pairs of monozygotic twins. International journal of cardiology.2005;105(2):126-33.8. 4. Poloni G, De Bortoli M, Calore M, Rampazzo A, Lorenzon A. Arrhythmogenic right-ventricular cardiomyopathy: molecular genetics into clinical practice in the era of next generation sequencing. Journal of Cardiovascular Medicine 17(6):p 399-407, June 2016. | DOI: 10.2459 / JCM.0000000000000385 5. Kowalczyk AP, Green KJ. Structure, function, and regulation of desmosomes. Progress in molecular biology and translational science.2013; 116:95-118. 6. Vimalanathan AK, Ehler E, Gehmlich K. Genetics of and pathogenic mechanisms in arrhythmogenic right ventricular cardiomyopathy. Biophysical Reviews.2018;10(4):973–82. 7. Chelko SP, Asimaki A, Andersen P, Bedja D, Amat-Alarcon N, DeMazumder D, Jasti R, MacRae CA, Leber R, Kleber AG, et al. Central role for GSK3β in the pathogenesis of arrhythmogenic cardiomyopathy. JCI Insight.2016;1(5). 8. Asimaki A, Tandri H, Huang H, Halushka MK, Gautam S, Basso C, Thiene G, Tsatsopoulou A, Protonotarios N, McKenna WJ, Calkins H, Saffitz JE. A new diagnostic test for arrhythmogenic right ventricular cardiomyopathy. N Engl J Med.2009 Mar 12;360(11):1075-84. doi: 10.1056 / NEJMoa0808138. PMID: 19279339.73 178013745.19. Lyon RC, Mezzano V, Wright AT, Pfeiffer E, Chuang J, Banares K, Castaneda A, Ouyang K, Cui L, Contu R, et al. Connexin defects underlie arrhythmogenic right ventricular cardiomyopathy in a novel mouse model. Hum Mol Genet.2014; 23:1134-50. 10. Chatterjee S. Sphingolipids in Atherosclerosis and vascular biology. Arterioscler Thromb Vasc Biol. 1998; 18:1523-33. 11. Chatterjee S, Balram A, Li W. Convergence: Lactosylceramide-Centric Signaling Pathways Induce Inflammation, Oxidative Stress, and Other Phenotypic Outcomes. Int. J. Mol. Sci.2021;22(4):1816. 12. Navab M, Berliner JA, Watson AD, Hama SY, Territo MC, Lusis AJ, Shih DM, Van Lenten BJ, Frank JS, Demer LL, et al. The yin and yang of oxidation in the development of the fatty streak: a review based on the 1994 George Lyman Duff Memorial Lecture. Arteriosclerosis, thrombosis, and vascular biology. 1996;16(7):831-42. 13. Chatterjee S, Berliner JA, Subbanagounder GG, Bhunia AK, Koh S. Identification of a biologically active component in minimally oxidized low-density lipoprotein (MM-LDL) responsible for aortic smooth muscle cell proliferation. Glycoconjugate journal.2003; 20:331-8. 14. Chatterjee S, Bhunia AK, Snowden A, Han H. Oxidized low-density lipoproteins stimulate galactosyltransferase activity, ras activation, p44 mitogen-activated protein kinase and c-fos expression in aortic smooth muscle cells. Glycobiology.1997;7(5):703-10.19. 15. Chatterjee, S. Role of oxidized human plasma low-density lipoproteins in atherosclerosis: Effects on smooth muscle cell proliferation. Mol. Cell Biochem.1992; 111:143–147. 16. Chatterjee S. Pathophysiology and Vascular Biology in Atherosclerosis. The Johns Hopkins Textbook of Dyslipidemia, 1st ed. Baltimore, MD: Lippincott, Williams & Wilkins.2010:48-57. 17. Chakrabarti A, Goldstein DR, Sutton NR. Age-associated arterial calcification: the current pursuit of aggravating and mitigating factors. Curr Opin Lipidol.2020;31(5):265-272. 18. Rajamannan NM, Evans FJ, Aikawa E, Grande-Allen KJ, Demer LL, Heistad DD, Simmons CA, Masters KS, Mathieu P, O’Brien KD, et al. Calcific aortic valve disease: not simply a degenerative process: A review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group. Circulation.2011;124(16):1783-91. 19. Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol. 1997;17(4):680-7. 20. Syed F, Diwan A, Hahn HS. Murine echocardiography: A practical approach for phenotyping genetically manipulated and surgically modeled mice. Journal of the American Society of Echocardiography. 2005;18(9):982–90. doi:10.1016 / j.echo.2005.05.001 21. Mishra S, Bedja D, Amuzie C, Avolio A, Chatterjee S. Prevention of cardiac hypertrophy by the use of a glycosphingolipid synthesis inhibitor in ApoE- / - mice. Biochem Biophys Res Commun. 2015 Sep 11;465(1):159-64. 22. Scherer M, Leuthäuser-Jaschinski K, Ecker J, Schmitz G, Liebisch G. A rapid and quantitative LC- MS / MS method to profile sphingolipids. Journal of Lipid Research.2010; 51(7):2001-11. 23. Chatterjee S. Assay of lactosylceramide synthase and comments on its potential role in signal transduction. Methods Enzymol.2000;311:73-81. 24. Chatterjee S. Velicer LF, Sweeley, CC. Glycosphingolipid glycosyl hydrolases and glycosidases of synchronized human KB cells. Journal of Biological Chemistry.1975;250(13):4972-9. 25. Spiegel, S. Sphingosine-1-phosphate: From insipid lipid to a key regulator. J. Biol. Chem.2020, 295:3371–3384. 26. Hannun YA, Obeid LM. Sphingolipids and their metabolism in physiology and disease. Nature reviews Molecular cell biology.2018;19(3):175-91. 27. Chelko SP, Asimaki A, Lowenthal J, Bueno-Beti C, Bedja D, Scalco A, Amat-Alarcon N, Andersen P, Judge DP, Tung L, et al. Therapeutic modulation of the immune response in arrhythmogenic cardiomyopathy. Circulation.2019;140(18):1491-505. 28. Jung JS, Jo D, Jo G, Hyun H. Near-Infrared Contrast Agents for Bone-Targeted Imaging. Tissue Eng Regen Med.2019;16(5):443-450.74 178013745.129. Bhunia AK, Han H, Snowden A, Chatterjee S. Lactosylceramide Stimulates Ras-GTP Loading, Kinases (MEK, Raf), p44 Mitogen-activated Protein Kinase, and c-fos Expression in Human Aortic Smooth Muscle Cells (∗). Journal of Biological Chemistry.1996;271(18):10660-6.37. 30. Bedja D, Yan W, Lad V, Iocco D, Sivakumar N, Bandaru VV, Chatterjee S. Inhibition of glycosphingolipid synthesis reverses skin inflammation and hair loss in ApoE− / − mice fed western diet. Scientific reports. 2018;8(1):11463. 31. Bhunia AK, Arai T, Bulkley G, Chatterjee S. Lactosylceramide mediates tumor necrosis factor-α-induced intercellular adhesion molecule-1 (ICAM-1) expression and the adhesion of neutrophil in human umbilical vein endothelial cells. Journal of Biological Chemistry.1998;273(51):34349-57. 32. Rajesh M, Kolmakova A, Chatterjee S. Novel Role of Lactosylceramide in Vascular Endothelial Growth Factor–Mediated Angiogenesis in Human Endothelial Cells. Circulation research.2005;97(8):796-804. 33. Kolmakova A, Rajesh M, Zang D, Pili R, Chatterjee S. VEGF recruits lactosylceramide to induce endothelial cell adhesion molecule expression and angiogenesis in vitro and in vivo. Glycoconjugate journal.2009;26:547-58. 34. Arai T, Bhunia AK, Chatterjee S, Bulkley GB. Lactosylceramide stimulates human neutrophils to upregulate Mac-1, adhere to endothelium, and generate reactive oxygen metabolites in vitro. Circulation research.1998;82(5):540-7.40. 35. Gong N, Wei H, Chowdhury SH, Chatterjee S. Lactosylceramide recruits PKCalpha / epsilon and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and adhesion to endothelial cells. Proc Natl Acad Sci USA.2004;101(17):6490-5. 36. Iwabuchi K, Nagaoka I. Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils. Blood.2002;100(4):1454-64. 37. Nakayama H, Yoshizaki F, Prinetti A, Sonnino S, Mauri L, Takamori K, Ogawa H, Iwabuchi K. Lyn- coupled LacCer-enriched lipid rafts are required for CD11b / CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms. J Leukoc Biol.2008;83(3):728-41. 38. Nakamura H, Moriyama Y, Watanabe K, Tomizawa S, Yamazaki R, Takahashi H, Murayama T. Lactosylceramide-induced phosphorylation signaling to group IVA phospholipase A2 via reactive oxygen species in tumor necrosis factor-α-treated cells. Journal of cellular biochemistry.2017;118(12):4370-82. 39. Li XM, Momsen MM, Brockman HL, Brown RE. Lactosylceramide: effect of acyl chain structure on phase behavior and molecular packing. Biophys J.2002;83(3):1535-46. 40. Bhunia AK, Han H, Snowden A, Chatterjee S. Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. J Biol Chem.1997;272(25):15642-9. 41. Iwabuchi K, Prinetti A, Sonnino S, Mauri L, Kobayashi T, Ishii K, Kaga N, Murayama K, Kurihara H, Nakayama H, et al. Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide- mediated superoxide generation and migration in neutrophils. Glycoconj J.2008;25(4):357-74. 42. Sommariva E, Stadiotti I, Casella M, Catto V, Dello Russo A, Carbucicchio C, Arnaboldi L, De Metrio S, Milano G, Scopece A, et al. Oxidized LDL-dependent pathway as new pathogenic trigger in arrhythmogenic cardiomyopathy. EMBO Mol Med.2021;13(9):e14365. 43. Boudi FB, Kalayeh N, Movahed MR. High-Density Lipoprotein Cholesterol (HDL-C) Levels Independently Correlates With Cardiac Arrhythmias and Atrial Fibrillation. J Intensive Care Med. 2020;35(5):438-444. 44. Berliner JA, Watson AD. A role for oxidized phospholipids in atherosclerosis. N Engl J Med. 2005;353(1):9-11. 45. Pohle K, Mäffert R, Ropers D, Moshage W, Stilianakis N, Daniel WG, Achenbach S. Progression of aortic valve calcification: association with coronary atherosclerosis and cardiovascular risk factors. Circulation.2001;104(16):1927-32. 46. Lin JC, Chang RL, Chen YF, Yang JJ, Baskaran R, Chung LC, Chen RJ, Day CH, Vijaya Padma V, Huang CY. β-Catenin overexpression causes an increase in inflammatory cytokines and NF-κB activation in cardiomyocytes. Cellular and Molecular Biology (Noisy-le-grand).2016;63(1):17-22. 47. Landstrom AP, Dobrev D, Wehrens XHT. Calcium Signaling and Cardiac Arrhythmias. Circulation Research.2017;120(12):1969-93.75 178013745.148. Rapa SF, Di Iorio BR, Campiglia P, Heidland A, Marzocco S. Inflammation and Oxidative Stress in Chronic Kidney Disease-Potential Therapeutic Role of Minerals, Vitamins and Plant-Derived Metabolites. International Journal of Molecular Sciences.2019;21(1):263. 49. Danielsen R, Aspelund T, Harris TB, Gudnason V. The prevalence of aortic stenosis in the elderly in Iceland and predictions for the coming decades: the AGES-Reykjavík study. Int J Cardiol.2014;176(3):916- 22. 50. Zheng KH, Tsimikas S, Pawade T, Kroon J, Jenkins WSA, Doris MK, White AC, Timmers NKLM, Hjortnaes J, Rogers MA, et al. Lipoprotein(a) and Oxidized Phospholipids Promote Valve Calcification in Patients With Aortic Stenosis. J Am Coll Cardiol.2019; 73(17):2150-2162. 51. Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem.226, 497–509. 52. Scherer M, Leuthäuser-Jaschinski K, Ecker J, Schmitz G, Liebisch G. A rapid and quantitative LC- MS / MS method to profile sphingolipids. J Lipid Res.2010 Jul; 51(7):2001-11. 53. Anroedh S, Hilvo M, Akkerhuis KM, Kauhanen D, Koistinen K, Oemrawsingh R, Serruys P, van Geuns RJ, Boersma E, Laaksonen R, Kardys I. Plasma concentrations of molecular lipid species predict long-term clinical outcome in coronary artery disease patients. J Lipid Res.2018 Sep; 59 (9):1729-1737. doi: 10.1194 / jlr.P081281. Epub 2018 Jun 1. PMID: 29858423; PMCID: PMC6121931. [000262] Example 2 [000263] Methods of Example 1 were followed to treat Dsg2mut / mutmice (BPD) with TNF- α, β-catenin, and C-Src levels. Subsequent administration of biopolymer-encapsulated-D-PDMP (BPD) drastically decreased TNF-α, β-catenin, and C-Src levels. Enzyme-linked immunosorbent assays were performed on serum samples from Dsg2mut / mutmice treated with or without BPD. Placebo-treated Dsg2mut / mutmice showed increased plasma levels of TNF-α, β-catenin and C-Src compared to BPD-treated Dsg2mut / mutmice. Plasma levels of VE-Cadherin were not significantly different between BPD-treated and placebo-treated Dsg2mut / mutmice. Results are further shown in FIGS. 10A-10D. [000264] While TNF-A is a biomarker of inflammation, B catenin and VE cadherin are key regulatory proteins implicated in muscle contraction, and C-Src is implicated in the regulation of angiogenesis that can be activated with LacCer. Interestingly, these proteins constitute the B- 1,4GalT-V interactome-i.e. the network of genes / proteins and lipids with which B-1, GalT-V interacts (doi.org / 10.3390 / ijms26168088). In sum, mitigating B-1,4GalT-V with oral delivery of BPD altered the expression of TNF-A, the most pro-inflammatory molecule as well improved cardiac health.76 178013745.1[000265] Example 3: Quantification of fatty acid molecular species of sphingolipids (C24:0 LacCer / C24:1 LacCer) in myocardium from human patients with arrhythmogenic cardiomyopathy(ACM) and two mouse models of ACM [000266] Tissue samples were obtained from human Appropriate internal standards were added to tissue before extracting total lipids. The total lipid extracts were dried, resuspended in methanol. Suitable aliquots were analyzed by hydrophilic interaction liquid chromatography (HILIC) using procedures set forth in Scherer M, Leuthäuser-Jaschinski K, Ecker J, Schmitz G, Liebisch G. A rapid and quantitative LC-MS / MS method to profile sphingolipids. Journal of Lipid Research. 2010; 51(7):2001-11. Individual sphingolipids were counted using reference standard curves and deuterium-labeled sphingolipids. The data was expressed as pmole or nmole / mg tissue).77 178013745.1OTHER EMBODIMENTS [000267] While the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. [000268] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.78 178013745.1

Claims

What is claimed:

1. A method of diagnosing and treating a subject suffering from arrhythmogenic cardiomyopathy (ACM) comprising: detecting increased amounts of lactosylceramide (LacCer) in a sample from a subject as compared to a normal baseline amount; administering to the subject a therapeutically effective amount of one or more inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, wherein the LacCer amounts in the subject are decreased as compared to a normal baseline amount; thereby diagnosing and treating a subject suffering from ACM.

2. The method of claim 1, wherein diagnosing a subject with ACM further comprises detection of increased levels of tumor necrosis α (TNFα), muscle contraction signaling molecules or the combination thereof in a sample from the subject.

3. The method of claim 2, wherein muscle contraction signaling molecules comprise β-catenin, c-Src or the combination thereof.

4. The method of claim 1, wherein the β-galactosyltransferase is lactosylceramide synthase (LacCer synthase).

5. The method of claim 1, wherein an inhibitor of glycosphingolipid (GSL) synthesis, comprises D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (BPD) or analogs thereof.

6. The method of claim 5, wherein the BPD analog comprises D-threo- 1 -(3',4'- ethyleneidoxy) phenyl-2-palmitoylaminod-3 -pyrrolidino- 1 - propanol (EtDO-P4), D-threo-4'- hydroxy-l-phenyl-2-palmitoylamino-3-pyrrolidino-l- propanol (pOH-P4), D-threo-l-(3',4'- trimethylenedioxy)phenyl-2-palmitoylamino-3- pyrrolidino- 1 -propanol (trimethylenedioxy-P4), D-threo- 1 -(3',4'-methylenedioxy)phenyl-2- palmitoylamino-3 -pyrrolidino- 1 -propanol (methylenedioxy-P4), or adamantan-l-yl glucosyl ceramide,(1R,2R) nonanoic acid[2-3-(2',3'-79 178013745.1dihydro-benzo[l,4]dioxin-6-'yl)-2-hydroxy-l- pyrrolidin-l-ylmethyl-ethyl]-l amide-L-tartaric acid salt (Genz-1223346).

7. The method of claim 1, wherein the one or more inhibitors of β- galactosyltransferase comprise a small molecule, an antibody, a protein, a peptide, a nucleic acid, a vector comprising one or more nucleic acid sequences encoding one or more inhibitors of β- 1,4-GalT V, a vector comprising one or more mutations in an Sp1 binding site of β-1,4-GalT V, or combinations thereof.

8. The method of claim 1, wherein the one or more inhibitors are unencapsulated or encapsulated in a biodegradable polymer (BPD).

9. The method of claim 8, wherein the biodegradable polymer consists of polyethylene glycol and sebacic acid.

10. The method of claim 1, wherein the subject suffers from myocardial fibrosis.

11. The method of claim 10, wherein administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of one or more inhibitors of β- galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, treats myocardial fibrosis.

12. A method of treating a subject suffering from arrhythmogenic cardiomyopathy (ACM) comprising: administering to the subject a therapeutically effective amount of one or more inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, wherein the LacCer amounts in the subject are decreased as compared to a normal baseline amount; thereby treating a subject suffering from ACM.

13. The method of claim 12, wherein treating a subject with ACM further comprises administering one or more inhibitors of tumor necrosis α (TNFα), muscle contraction signaling molecules or the combination thereof in a sample from the subject.80 178013745.

114. The method of claim 13, wherein muscle contraction signaling molecules comprise β-catenin, c-Src or the combination thereof.

15. The method of claim 12, wherein the β-galactosyltransferase is lactosylceramide synthase (LacCer synthase).

16. The method of claim 12, wherein an inhibitor of glycosphingolipid (GSL) synthesis, comprises D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (BPD) or analogs thereof.

17. The method of claim 16, wherein the BPD analog comprises D-threo- 1 -(3',4'- ethyleneidoxy) phenyl-2-palmitoylaminod-3 -pyrrolidino- 1 - propanol (EtDO-P4), D-threo-4'- hydroxy-l-phenyl-2-palmitoylamino-3-pyrrolidino-l- propanol (pOH-P4), D-threo-l-(3',4'- trimethylenedioxy)phenyl-2-palmitoylamino-3- pyrrolidino- 1 -propanol (trimethylenedioxy-P4), D-threo- 1 -(3',4'-methylenedioxy)phenyl-2- palmitoylamino-3 -pyrrolidino- 1 -propanol (methylenedioxy-P4), or adamantan-l-yl glucosyl ceramide,(1R,2R) nonanoic acid[2-3-(2',3'- dihydro-benzo[l,4]dioxin-6-'yl)-2-hydroxy-l- pyrrolidin-l-ylmethyl-ethyl]-l amide-L-tartaric acid salt (Genz-1223346).

18. The method of claim 12, wherein the one or more inhibitors of β- galactosyltransferase comprise a small molecule, an antibody, a protein, a peptide, a nucleic acid, a vector comprising one or more nucleic acid sequences encoding one or more inhibitors of β- 1,4-GalT V, a vector comprising one or more mutations in an Sp1 binding site of β-1,4-GalT V, or combinations thereof.

19. The method of claim 12, wherein the one or more inhibitors are unencapsulated or encapsulated in a biodegradable polymer (BPD).

20. The method of claim 19, wherein the biodegradable polymer consists of polyethylene glycol and sebacic acid.

21. The method of claim 19, wherein the subject suffers from myocardial fibrosis.

22. The method of claim 21, wherein administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of one or more inhibitors of β-81 178013745.1galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof, treats myocardial fibrosis.

23. A method of determining efficacy of one or more agents in the treatment of arrhythmogenic cardiomyopathy (ACM) comprising monitoring levels of lactosylceramide (LacCer), oxidized-low-density lipoprotein (ox-LDL), tumor necrosis factor alpha (TNFα), proto-oncogene tyrosine-protein kinase Src (c-Src), β-catenin, β-1,4-Galactosyltransferase-V (β- 1,4-GalT-V), or combinations thereof, in a sample from a subject as compared to a normal baseline amount; wherein the levels of LacCer, ox-LDL, TNFα, c-Src, β-catenin, or β-1,4-GalT-V amounts in the subject are decreased as compared to a normal baseline amount; thereby determining efficacy of one or more agents in the treatment of ACM.

24. The method of claim 23, wherein the agent comprises vectors expressing one or more β-1,4-GalT-V inhibitors, small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, Fab, Fab’, F(ab’)2fragments, Fv fragments, single chain antibodies, antibody mimetics, peptoids, aptamers, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds.

25. A kit for diagnosing arrhythmogenic cardiomyopathy (ACM) comprising one or more agents detecting levels of lactosylceramide synthase (LacCer), tumor necrosis α (TNFα), β- catenin, c-Src or combinations thereof.

26. The kit of claim 25, wherein the kit further comprises a pharmaceutical composition comprising one or more inhibitors of β-galactosyltransferases, one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof.

27. A method of diagnosing and treating arrhythmogenic cardiomyopathy (ACM), comprising: a) assaying to identify C24:0 LaCer and / or C24:1 LacCer in a sample obtained from a subject;82 178013745.1b) diagnosing the subject as having or susceptible to arrhythmogenic cardiomyopathy (ACM) when the C24:0 LacCer and / or C24:1 LacCer level is increased relative to the C24:0 LacCer and / or C24:1 LacCer level in a healthy subject; and optionally c) administering a therapy in subjects diagnosed with arrhythmogenic cardiomyopathy; thereby, diagnosing and treating a subject.

28. The method of claim 27 wherein the therapy is administration of one or more therapeutic agent or medical device, or a surgical procedure.

29. The method of claim 27 wherein a therapeutically effective amount of one or more inhibitors of β-galactosyltransferases, a therapeutically effective amount of one or more inhibitors of glycosphingolipid (GSL) synthesis, or combinations thereof are administered to the subject diagnosed with arrhythmogenic cardiomyopathy.

30. The method of any one of claims 27 to 29 wherein the C24:0 LacCer is modified or unmodified and the C24:1 LacCer is modified or unmodified.83 178013745.1

Images