Treatment of cardiomyopathy not associated with laminopathy

Abstract

The present disclosure relates to the treatment and prevention of diseases and conditions (e.g., cardiomyopathy) by LINC complex inhibition. LINC complex inhibitors are provided for use in methods of treating or preventing a disease or condition characterized by microtubule dysfunction and / or a disease or condition characterized by sarcomere defects or dysfunction.

Description

[0001] This application claims priority to GB 2312938.0, filed August 24, 2023, and GB2404416.6, filed March 27, 2024, the contents and elements of which are incorporated herein by reference for all purposes. Technical Field

[0002] This disclosure relates to the treatment and prevention of diseases and disorders through inhibition of the LINC complex. Background Technology

[0003] Encoding lamin proteins A and C LMNA Mutations in genes can lead to a range of diseases known as laminopathy. Recently, disruption of the nuclear skeleton and cytoskeleton junction (LINC) complex has been proposed as a therapeutic strategy for laminopathy – see, for example, Chai et al., Nature Communications (2021) 12: 4722.

[0004] Desmosomal plaques are important components of desmosomes, which form intercellular connections between adjacent cells. Desmosomes (also known as focal adhesions) are specialized structures that mediate lateral cell adhesion, particularly well-suited for tissues subjected to significant mechanical stress, such as cardiac muscle (as well as bladder tissue, gastrointestinal mucosa, and epithelial tissue). The structure and function of desmosomes have been reviewed, for example, in Delva et al., Cold Spring Harb Perspect Biol. (2009) 1(2): a002543 and Johnson et al., Cold Spring Harb Perspect Med. (2014) 4(11): a015297, which are incorporated herein by reference in their entirety.

[0005] The N-terminal globular domain of human desmosome plaque protein contains a 584-amino acid region essential for its interaction with plaquelin and plaqueglobin (see, for example, Stappenbeck et al., J Cell Biol. (1993) 123(3):691-705 and Kowalczyk et al., J Cell Biol. (1997) 139(3):773-84). Desmosome plaque protein binds to intermediate filaments via its C-terminus (see, for example, Stappenbeck and Green, J Cell Biol. (1992) 116(5):1197-209 and Stappenbeck et al., J Cell Biol. (1993) 123(3):691-705), which may be mediated by a glycine-serine-arginine repeat region at the C-terminus (see, for example, Fogl et al., Nat Commun. (2016) 7:10827).

[0006] Desmosome plaque protein (by) DSP Mutations in genes that encode cardiomyopathy can lead to cardiomyopathy, such as dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). DSP Mutations can also lead to skin diseases such as epidermolysis bullosa, and pleiotropic diseases such as dilated cardiomyopathy with piloerection and keratosis. These diseases are thought to be caused by the loss or reduction of desmosomal plaque protein expression levels, which results in a decrease in the number of desmosomes and / or suppression of desmosomal function.

[0007] Intercalated disks (ICDs) between adjacent cardiomyocytes are crucial for cell function and regulation, particularly facilitating the orderly arrangement of cells into organized layers, thereby maintaining tissue integrity and efficient electromechanical coupling. The basic components of intercellular connections can be broadly divided into mechanical coupling components involved in maintaining intercellular adhesion and electrocoupling components involved in promoting rapid intercellular ion transport, thus enabling the rapid transport of depolarization wavefronts (DJ Abrams, JE Saffitz, in Cardioskeletal Myopathies in Children and Young Adults, 2017). Mechanical coupling and intercellular adhesion are formed by adhesive junctions and... desmosomes Provided. Electrical coupling between cardiomyocytes mainly occurs through gap junctions.

[0008] The desmosomes of the ICD are composed of desmosome cadherin, which forms intercellular junctions and is connected to desmosome plaque protein within the cell via adaptor proteins plaqueglobin and plaquemin 2. Desmosome plaque protein then recruits desmin intermediate filaments.

[0009] A sarcomere is a single contractile unit of skeletal muscle cells, composed of actin, myosin, and related regulatory and scaffold proteins, which repeatedly form the basic rod-shaped organelle units of myofibrils—cardiomyocytes (Warner E. et al. Circulation Research. 2022;130:1723–1741). Actin filaments are attached to Z-discs. Z-disc-related proteins include α-actin, titin, nebulin, and intermediate filaments, such as desmin intermediate filaments.

[0010] Microtubules extend between Z-discs and appear to stabilize at the Z-lines, thus halting growth. Intermediate filaments such as desmin anchor detyrosinated microtubules to the Z-discs and crosslink the microtubules to the intermediate filament network, which increases resistance, forcing the microtubules to compress and adopt a flexed conformation during cardiomyocyte contraction (Warner E. et al., 2022). Therefore, desmin is thought to act as a mechanical anchor for microtubules, confining their distribution to specific spatial regions (Uchida K. et al., Annu.Rev. Physiol. 2022. 84:257–83). Summary of the Invention

[0011] In a first aspect, this disclosure provides a LINC complex inhibitor in a method for treating or preventing cardiomyopathy, wherein the cardiomyopathy is not a laminopathic cardiomyopathy.

[0012] This disclosure also provides the use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of cardiomyopathy, wherein the cardiomyopathy is not associated with lamininopathy.

[0013] This disclosure also provides a method for treating or preventing cardiomyopathy, comprising administering to a subject a therapeutically or preventively effective amount of a LINC complex inhibitor, wherein the cardiomyopathy is not a laminopathic cardiomyopathy.

[0014] In some embodiments, according to various aspects of this disclosure, cardiomyopathy is selected from dilated cardiomyopathy, arrhythmogenic cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy.

[0015] In some implementations, cardiomyopathy is associated with desmosome defects / deficiencies or dysfunction. In some implementations, cardiomyopathy is associated with mutations in genes encoding desmosome proteins.

[0016] In some implementations, cardiomyopathy is associated with mutations in genes selected from the following: DSP , DES , DSG2 , DSC2 , PKP2 and JUP .

[0017] In some implementation schemes, cardiomyopathy is characterized by microtubule dysfunction.

[0018] In some implementation schemes, cardiomyopathy is selected from dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy.

[0019] This disclosure also provides a LINC complex inhibitor in a method for treating or preventing a disease or disorder characterized by desmosome defects or dysfunction.

[0020] This disclosure also provides the use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of diseases or disorders characterized by desmosome defects or dysfunction.

[0021] This disclosure also provides a method for treating or preventing diseases or disorders characterized by desmosome defects or dysfunction, comprising administering to a subject a therapeutically or preventively effective amount of a LINC complex inhibitor.

[0022] In some embodiments, according to various aspects of this disclosure, diseases or disorders characterized by desmosome defects or dysfunction are associated with mutations in genes encoding desmosome proteins.

[0023] In some implementations, diseases or disorders characterized by desmosome defects or dysfunction are selected from... DSP , DES , DSG2 , DSC2 , PKP2 and JUP It is related to gene mutations.

[0024] In some implementations, diseases or disorders characterized by desmosome defects or dysfunction are characterized by cardiomyopathy.

[0025] In some implementations, diseases or disorders characterized by desmosome defects or dysfunction are characterized by microtubule dysfunction.

[0026] This disclosure also provides a LINC complex inhibitor in a method for treating or preventing a disease or disorder characterized by microtubule dysfunction.

[0027] This disclosure also provides the use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of diseases or disorders characterized by microtubule dysfunction.

[0028] This disclosure also provides a method for treating or preventing diseases or disorders characterized by microtubule dysfunction, comprising administering to a subject a therapeutically or preventively effective amount of a LINC complex inhibitor.

[0029] In some implementations, a disease or disorder characterized by microtubule dysfunction is cardiomyopathy or is characterized by cardiomyopathy.

[0030] This disclosure also provides a LINC complex inhibitor in a method for treating or preventing a disease or disorder characterized by sarcomere defects / deficiencies or dysfunctions.

[0031] This disclosure also provides the use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of diseases or disorders characterized by sarcomere defects / deficiencies or dysfunctions.

[0032] This disclosure also provides a method for treating or preventing diseases or disorders characterized by sarcomere defects / deficiencies or dysfunctions, comprising administering to a subject a therapeutically or preventively effective amount of a LINC complex inhibitor.

[0033] In some implementations, diseases or disorders characterized by sarcomere defects or dysfunction are associated with mutations in genes encoding sarcomere proteins.

[0034] In some implementations, diseases or disorders characterized by sarcomere defects or dysfunction are associated with mutations in genes selected from: TTN, MYH7, TNNT2, TNNI3, TNNC1, TPM1, MYBPC3, MYL2, MYL3, CSRP3, RBM20, ACTN2, TCAP, and ACTC1.

[0035] In some implementations, a disease or disorder characterized by sarcomere defects / deficiencies or dysfunction is cardiomyopathy or is characterized by cardiomyopathy.

[0036] In some implementations, diseases or disorders characterized by sarcomere defects or dysfunctions are characterized by microtubule dysfunction.

[0037] In some embodiments, according to various aspects of this disclosure, cardiomyopathy is selected from dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy.

[0038] In some implementations, according to various aspects of this disclosure, treatment or prevention includes administering to a subject a nucleic acid containing or encoding an inhibitor of the LINC complex.

[0039] In some implementations, treatment or prevention involves administering a nucleic acid encoding a LINC complex inhibitory peptide to the subject.

[0040] In some embodiments, the LINC complex inhibitory peptide comprises: (i) an inhibitory region comprising an α3 helix corresponding to the CC2 region of a protein containing a SUN domain and an amino acid sequence of the SUN domain, and (ii) an endoplasmic reticulum retention motif.

[0041] In some embodiments, the LINC complex inhibitory peptide comprises or is substantially composed of an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 99, 72, 95, 71, 69, 70 or 73.

[0042] In some implementations, the nucleic acid encoding a LINC complex inhibitory peptide targeting the subject is contained in an adeno-associated virus (AAV) vector. Detailed Implementation

[0043] This disclosure is based on the inventor's accidental discovery that LINC complex inhibition can be used for treatment / prevention. Apart from Diseases / conditions other than laminopathies, especially those caused by factors other than... LMNA Cardiomyopathy caused by factors other than mutations.

[0044] This disclosure is also based on the inventors’ accidental discovery that LINC complex inhibition can be used to treat / prevent diseases / conditions associated with abnormal microtubule function pathology.

[0045] The inventors here demonstrate that LINC complex inhibition can be used to treat / prevent diseases associated with desmosome defects / dysfunction, such as those caused by desmosome protein mutations. In particular, the inventors demonstrate that... DSP The pathology of mutation-related cardiomyopathy can be improved by inhibiting the LINC complex.

[0046] The inventors here demonstrate that LINC complex inhibition can be used to treat / prevent diseases associated with sarcoma dysfunction, such as those caused by mutations in sarcoma proteins. In particular, the inventors demonstrate that... TTN The pathology of mutation-related cardiomyopathy can be improved by inhibiting the LINC complex.

[0047] The specific cardiomyopathy model used in the experimental examples is due to different factors (i.e. LMNA, DSP or TTN This is caused by mutations in the LINC complex. Cardiomyopathy associated with these mutations, as well as acquired heart disease (i.e., non-hereditary cardiomyopathy) and other heart diseases, is characterized by abnormal microtubule organization, leading to alterations in microtubule load-bearing and force transmission. Therefore, the inventors unexpectedly demonstrated that inhibiting the LINC complex can reverse dysfunctional microtubule organization. Not wishing to be bound by any particular theory, the inventors infer that by disrupting the LINC complex to uncouple the nuclear capsule from the microtubule network, the anchoring points of the perinuclear microtubule population can be reduced, reversing the abnormal load beading and force transmission from the microtubule network to the nuclear capsule, thereby improving the pathology of cardiomyopathy and other diseases.

[0048] This disclosure provides a LINC complex inhibitor in a method for treating or preventing heart disease. This disclosure also provides the use of a LINC complex inhibitor in the manufacture of a medicament for treating or preventing heart disease. This disclosure further provides a method for treating or preventing heart disease, comprising administering a therapeutically or preventively effective amount of a LINC complex inhibitor to a subject. In some embodiments, the heart disease is unrelated to laminopathy.

[0049] Structure and function of LINC complexes The linker of the nuclear-cytoskeleton (LINC) complex is a polypeptide complex containing a protein with a SUN domain and a protein with a KASH domain. The structure of the LINC complex has been reviewed in, for example, Sosa et al., Curr Opin StructBiol. (2013) 23(2):285-91 and Hieda, Cells (2017) 6(1):3, which are incorporated herein by reference in their entirety.

[0050] The LINC complex connects the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope. Proteins containing the SUN domain span the INM, associating with laminins and chromatin-binding proteins on the nucleoplasmic side and with proteins containing the KASH domain on the perinuclear side. Proteins containing the KASH domain span the ONM, associating with cytoskeletal structural components such as actin filaments, microtubule motors, and intermediate filaments on the cytoplasmic side and with proteins containing the SUN domain on the perinuclear side. SUN domain proteins function as transluminal tethers for KASH domain proteins within the ONM.

[0051] In this article, "proteins containing the SUN domain" refers to any polypeptide containing the SUN domain. S ad1 and UN C-84 domain proteins are important INM components, containing a conserved C-terminal SUN domain located in the perinuclear space. The SUN domain contains approximately 175 residues and is situated at the end of the helical stalk region. The nucleoplasmic domain of proteins containing the SUN domain interacts with structural components of the nuclear skeleton.

[0052] The SUN domain may contain the amino acid sequence shown in SEQ ID NO: 8, 18, 27, 28, 29 or 30, or an amino acid sequence having at least 60%, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 8, 18, 27, 28, 29 or 30, or may be composed of said amino acid sequence.

[0053] In some embodiments, the protein containing the SUN domain is selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO. In some embodiments, the protein containing the SUN domain is SUN1 or SUN2.

[0054] In some embodiments, proteins containing the SUN domain can form LINC complexes. In some embodiments, proteins containing the SUN domain can interact with proteins containing the KASH domain and / or KASH domains.

[0055] Human SUN1 is a polypeptide identified by UniProtKB O94901, and its amino acid sequence is shown in SEQ ID NO:1. Human SUN2 is a polypeptide identified by UniProtKB Q9UH99, and its amino acid sequence is shown in SEQ ID NO:13. Human SUN3 is a polypeptide identified by UniProtKB Q8TAQ9, and its amino acid sequence is shown in SEQ ID NO:23. Human SUN5 is a polypeptide identified by UniProtKBA9Z1W8, and its amino acid sequence is shown in SEQ ID NO:24. Human SPAG4 is a polypeptide identified by UniProtKB Q9NPE6, and its amino acid sequence is shown in SEQ ID NO:25. Human SUCO is a polypeptide identified by UniProtKB Q9UBS9, and its amino acid sequence is shown in SEQ ID NO:26.

[0056] In this specification, “SUN1”, “SUN2”, “SUN3”, “SUN5”, “SPAG4” and “SUCO” refer to SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO from any species, including its isotypes, fragments, variants or homologs.

[0057] As used herein, a protein “fragment,” “variant,” or “homolog” may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with a reference protein (e.g., a reference isoform of the reference protein). In some embodiments, the fragment / variant / isoform / homolog is characterized by its ability to perform the functions performed by the reference protein.

[0058] A “fragment” typically refers to a portion of a reference protein. A “variant” typically refers to a protein having an amino acid sequence that includes one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of the reference protein, but retains a substantial degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. A “homotype” typically refers to a variant of a reference protein expressed by a species of the same species as the reference protein. A “homolog” typically refers to a variant of a reference protein produced by a species different from the reference protein species. Homologs include orthologs.

[0059] The “fragment” may be of any length (in terms of amino acid number), although it may optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment is derived), and the maximum length may be one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the reference protein.

[0060] Isotypes, fragments, variants, or homologs may optionally be functional isotypes, fragments, variants, or homologs, for example, having the functional properties / activities of a reference protein, as determined by appropriate assays of the functional properties / activities.

[0061] In this specification, “SUN1” refers to a protein, its fragments, variants, or homologs having the amino acid sequence shown in SEQ ID NO:1. In some embodiments, SUN1 comprises, or is composed of, an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity with SEQ ID NO:1.

[0062] In this specification, "SUN2" refers to a protein, its fragments, variants, or homologs having the amino acid sequence shown in SEQ ID NO:13. In some embodiments, SUN2 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:13.

[0063] In this specification, "SUN3" refers to a protein, its fragments, variants, or homologs having the amino acid sequence shown in SEQ ID NO:23. In some embodiments, SUN3 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:23.

[0064] In this specification, "SUN5" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:24. In some embodiments, SUN5 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:24.

[0065] In this specification, "SPAG4" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:25. In some embodiments, SPAG4 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:25.

[0066] In this specification, "SUCO" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:26. In some embodiments, SUCO comprises, or is composed of, an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity with, SEQ ID NO:26.

[0067] In this article, "proteins containing a KASH domain" refers to any polypeptide containing a KASH domain. KASH (klarschit, ANC-1, Syne homology) domain proteins are membrane proteins that target the C-terminus of the nuclear envelope. The 50-60 amino acid KASH domain is located at the C-terminus. The KASH domain is hydrophobic and contains a single membrane helix spanning the ONM and a region of approximately 30 amino acids extending into the perinuclear space.

[0068] The KASH domain may contain the amino acid sequence shown in SEQ ID NO: 37, 38, 39, 40, 41 or 42, or be composed of an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 37, 38, 39, 40, 41 or 42, or be composed of said amino acid sequence.

[0069] In some embodiments, the protein containing the KASH domain is selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4 (also known as SYNE1, SYNE2, SYNE3, and SYNE4), KASH5, and LRMP. In some embodiments, the protein containing the KASH domain is Nesprin-1, Nesprin-2, or Nesprin-3.

[0070] In some embodiments, proteins containing a KASH domain can form a LINC complex. In some embodiments, proteins containing a KASH domain can interact with a SUN domain and / or proteins containing a SUN domain.

[0071] Human Nesprin-1 is a polypeptide identified by UniProtKB Q8NF91, and its amino acid sequence is shown in SEQ ID NO:31. Human Nesprin-2 is a polypeptide identified by UniProtKB Q8WXH0, and its amino acid sequence is shown in SEQ ID NO:32. Human Nesprin-3 is a polypeptide identified by UniProtKB Q6ZMZ3, and its amino acid sequence is shown in SEQ ID NO:33. Human Nesprin-4 is a polypeptide identified by UniProtKB Q8N205, and its amino acid sequence is shown in SEQ ID NO:34. Human KASH5 is a polypeptide identified by UniProtKB Q8N6L0, and its amino acid sequence is shown in SEQ ID NO:35. Human LRMP is a polypeptide identified by UniProtKB Q12912, and its amino acid sequence is shown in SEQ ID NO:36.

[0072] In this specification, “Nesprin-1”, “Nesprin-2”, “Nesprin-3”, “Nesprin-4”, “KASH5” and “LRMP” refer to Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP from any species, including their isotypes, fragments, variants or homologs.

[0073] In this specification, "Nesprin-1" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:31. In some embodiments, Nesprin-1 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with, SEQ ID NO:31.

[0074] In this specification, "Nesprin-2" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:32. In some embodiments, Nesprin-2 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:32.

[0075] In this specification, "Nesprin-3" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:33. In some embodiments, Nesprin-3 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with, SEQ ID NO:33.

[0076] In this specification, "Nesprin-4" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:34. In some embodiments, Nesprin-4 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with, SEQ ID NO:34.

[0077] In this specification, "KASH5" refers to a protein, fragment thereof, variant thereof, or homolog having the amino acid sequence shown in SEQ ID NO:35. In some embodiments, KASH5 comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO:35.

[0078] In this specification, "LRMP" refers to a protein, its fragments, variants, or homologs having the amino acid sequence shown in SEQ ID NO:36. In some embodiments, the LRMP comprises, or consists of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with, SEQ ID NO:36.

[0079] As used in this article, "LINC complex" refers to a polypeptide complex containing a protein with a SUN domain and a protein with a KASH domain.

[0080] The LINC complex is formed by protein-protein interactions between a protein containing a SUN domain and a protein containing a KASH domain. The LINC complex may contain non-covalent and / or covalent interactions between the SUN and KASH domains. Non-covalent interactions include, for example, hydrogen bonds, ionic interactions, van der Waals forces, and hydrophobic bonds. Covalent interactions include, for example, disulfide bonds.

[0081] SUN domain proteins are thought to oligomerize and form trimers through interactions between stalk regions, thus forming a coiled triple helix (Zhou et al., J. Biol. Chem. (2012) 287: 5317-5326). The absence of the stalk region in SUN domain proteins has been shown to disrupt the formation of the LINC complex. The SUN domain exhibits a β-sandwich structure, with the trimer SUN domains interacting extensively through protruding β-sheets (called KASH caps); the KASH cap of one SUN domain partially overlaps with the β-sandwich portion of an adjacent SUN domain (Sosa et al., Cell (2012) 149:1035-1047).

[0082] KASH domain proteins can also oligomerize, which may involve protein-protein interactions between transmembrane helices. A single KASH domain interacts with two adjacent SUN domains along a groove formed between the KASH cap of one SUN domain and the upper region of the β-sandwich of the adjacent SUN domain. In this way, the SUNβ domain and KASH domain are thought to interact to form a 3:3 hexameric heterocomplex. Two to three proline residues preceding the C-terminus of the KASH domain are thought to be located in a deep pocket on the surface of the SUN domain. This region of KASH is crucial for the SUN-KASH interaction; extending the C-terminus by only one amino acid would disrupt the formation of the LINC complex. Conserved cysteine ​​residues in the SUN and KASH domains form disulfide bonds, further stabilizing the SUN-KASH complex. Disulfide bonds may be important for force transfer through the LINC complex (Jahed et al., Biophys. J. (2015) 109:501-509).

[0083] As mentioned above, the LINC complex is believed to be formed by the interaction of the SUN domain of three proteins containing the SUN domain with the KASH domain of three proteins containing the KASH domain. The interaction between SUN and KASH domain proteins is considered to be heterogeneous; SUN1 and SUN2 have been shown to interact with Nesprin-1, Nesprin-2, and Nesprin-3.

[0084] According to this disclosure, the LINC complex can include any protein containing a SUN domain and any protein containing a KASH domain. The SUN-containing proteins of the LINC complex can be the same or different. The KASH-containing proteins of the LINC complex can be the same or different.

[0085] The function of the LINC complex has been reviewed, for example, in Hieda, Cells (2017) 6(1):3 (incorporated above by reference) and Stroud, Biophys Rev. (2018) 10(4):1033–1051, which are incorporated herein by reference in their entirety.

[0086] The LINC complex has multiple functions, including providing structural support for the cell nucleus, shaping and positioning the cell nucleus, maintaining the connection between the centrosome and the cell nucleus and the spacing of the nuclear membrane, DNA repair, cell migration, and moving chromosomes within the cell nucleus during meiosis.

[0087] The LINC complex has mechanosensory functions, translating mechanical stimuli and changes in the extracellular matrix into signals that enable cells to adapt to their environment by regulating cytoskeleton organization, gene expression, nuclear organization, and structure. The microtubule cytoskeleton and the LINC complex play important roles in nuclear protection and the transfer of forces from the cell body / cytoplasm to the nucleus (Becker R. Cells 2020. 9(6): 1395).

[0088] Integrins mediate the transmission of forces from the external microenvironment to the intracellular cytoskeleton, and nuclear-cytoskeleton molecular connections transmit these forces to the chromosomal tissues within the nucleus. The nuclear lamina triggers nuclear structural deformation and initiates changes in gene regulation.

[0089] The nuclear envelope is a key structure in these processes. On the nucleoplasmic side of the nuclear internal membrane (INM), the nuclear lamina (composed of type A and type B laminins) forms a lattice structure that helps the nucleus resist mechanical stress, which is essential for the structural integrity of the nuclear envelope. Laminins participate in processes vital to cell function and survival, including maintaining nuclear integrity, regulating the cell cycle, mechanotransduction, cell signaling, and DNA repair.

[0090] Abnormal expression and / or function of nuclear envelope proteins, as well as abnormal expression and / or function of factors directly or indirectly related to the nuclear envelope, are associated with a variety of diseases, including muscular dystrophy, cardiomyopathy, lipodystrophy, progeria, cancer, and nervous system disorders.

[0091] Microtubules in cardiomyocytes Terminally differentiated cells, such as cardiomyocytes and skeletal muscle cells, have a highly organized microtubule network (MTN).

[0092] Given the physiological need for rhythmic pumping, cardiomyocytes possess a natural ability to sense mechanical stretch. Microtubules, along with myofilaments and intermediate filaments, form a complex cytoskeleton that controls cellular mechanics (Warner E. et al. 2022). Crosslinking between microtubules and other cytoskeleton components, such as desmin and actin, can significantly amplify intracellularly transduced mechanical forces (Warner E. et al. 2022). The microtubule cytoskeleton has high intrinsic stiffness, can withstand compressive stress, and can exhibit resistance to motion.

[0093] Cardiomyocytes are equipped with mechanisms for rapid force generation and movement, and microtubule tortuosity is estimated to contribute approximately 20% of cardiomyocyte contractile force. Factors influencing mechanical force include microtubule growth rate, microtubule length, microtubule post-translational modifications, and microtubule network (MTN) density (Warner E. et al. 2022). Microtubules connect the extracellular space and contractile apparatus to the nucleus via the LINC complex, thereby allowing the transmission of mechanical forces to regulate gene expression (Becker R. Cells 2020. 9(6):1395).

[0094] Microtubules are composed of heterodimers of α-tubulin and β-tubulin. These tubulin subunits form higher assemblies containing protofilaments arranged in hollow polymers. Microtubules nucleate from microtubule organizing centers (MTOCs) containing γ-tubulin, which serves as a structural template for efficient nucleation (Uchida K. et al., 2022).

[0095] In mitotic cells, the centrosome MTOC plays a continuous role in microtubule nucleation throughout the cell's lifespan. However, when cardiomyocytes transform into postmitotic cells shortly after birth, they simultaneously lose the integrity of the centrosome. Adult cardiomyocytes rely entirely on the noncentrosome MTOC, which decorates the nuclear envelope and the associated Golgi apparatus, for nucleation. Figure 5 A, and Uchida K. et al., 2022). Therefore, the entire cardiomyocyte nuclear capsule can be considered as the cellular MTOC.

[0096] Microtubule dysfunction is associated with various cardiac diseases, including changes in MTN density (Cheng G. et al., Am J Physiol Heart Circ Physiol. 2008;294:H2231–H2241), changes in microtubule post-translational modifications (Schuldt M. et al., Circ Heart Fail. 2021;14:e007022), and association with microtubule-associated proteins (MAPs) (Li L. et al., Front Physiol. 2020;11:1044. Doi: 10.3389 / fphys.2020.01044).

[0097] The most thoroughly studied post-translational modifications in cardiomyocytes are detyrosinening and acetylation (Warner E. et al., 2022). Detyrosinening enhances the biophysical rigidity of microtubules. TCP (tubulin carboxypeptidase) detyrosinening preferentially occurs on microtubule polymers rather than on tubulin dimers. Following detyrosinening, the C-terminal tyrosine residue of α-tubulin is removed.

[0098] Recent studies have shown that enhanced microtubule detyrosinization in muscle cells (through cross-linking of detyrosinated microtubules with desmin on Z-discs) is associated with increased mechanical resistance and impaired cell contraction under various pathophysiological conditions. In patients clinically diagnosed with hypertrophic and dilated cardiomyopathy (HCM and DCM, respectively), detyrosinated microtubules are significantly increased, along with a slight increase in total microtubule content (Robison P. et al., Science. 352, aaf0659 (2016)).

[0099] In cardiomyocytes, microtubules rich in acetylated α-tubulin increase cytoskeleton stiffness and viscoelastic resistance, independent of changes in MTN density or the level of detyrosinated microtubules (Coleman A. et al. J Gen Physiol. 2021; 153(7): e202012743). Macquart C. et al. Human Molecular Genetics, 2019, Vol.28, No. 24, 4043-4052 described... LMNA Decreased α-tubulin acetylation levels are observed in cardiomyopathy. The activity of histone deacetylase HDAC6 increases during cardiac stress. This enzyme regulates tubulin deacetylation, and HDAC inhibitors have been shown to improve stress overload hypertrophy and atrial fibrillation (Warner E. et al., 2022).

[0100] Currently, microtubule-targeted therapy has been used clinically to treat cancer and neurodegenerative diseases; however, the therapeutic intervention of microtubule-targeted therapy for cardiovascular diseases is limited to colchicine therapy, which is mainly related to the anti-inflammatory effect of colchicine at low doses, such as its use in the treatment of gout (Warner E. et al. 2022).

[0101] LINC complex inhibits This disclosure relates to LINC complex inhibition. As used herein, “LINC complex inhibition” includes inhibiting the formation of LINC complexes (i.e., inhibiting LINC complex assembly), disrupting / degrading LINC complexes, and inhibiting the activity / function of LINC complexes.

[0102] In some embodiments, the formation of the LINC complex can be inhibited by suppressing the expression of genes and / or proteins that constitute the LINC complex. The constituent proteins of the LINC complex include proteins containing a SUN domain and proteins containing a KASH domain. For the sake of brevity, in this disclosure, "constituent proteins of the LINC complex" may be simply referred to as "LINC complex proteins".

[0103] In some implementations, inhibiting the formation of the LINC complex includes one or more of the following: inhibiting the expression of a gene or protein encoding the LINC complex protein; modifying a gene encoding the LINC complex protein to reduce / prevent its expression; reducing the RNA level encoding the LINC complex protein; inhibiting the transcription of the nucleic acid encoding the LINC complex protein; increasing the degradation of the RNA encoding the LINC complex protein; reducing the level of the LINC complex protein; disrupting the normal post-transcriptional processing (e.g., splicing, translation, post-translational processing) of the RNA encoding the LINC complex protein; and increasing the degradation of the LINC complex protein.

[0104] Gene expression can be determined using methods familiar to technicians. RNA levels encoding LINC complex constituent proteins can be measured using techniques such as RT-qPCR and RNA blotting. Decreased RNA levels encoding LINC complex constituent proteins may be due to, for example, reduced transcription of the nucleic acid encoding the LINC complex protein or increased RNA degradation of the LINC complex protein.

[0105] Reduced transcription of nucleic acids encoding LINC complex proteins may be due to inhibition of the assembly and / or activity of factors required for DNA transcription of LINC complex proteins. Increased RNA degradation of LINC complex proteins may be due to increased enzymatic degradation of the RNA encoding LINC complex proteins, such as due to RNA interference (RNAi), and / or decreased RNA stability of the RNA encoding LINC complex proteins.

[0106] Protein expression can be determined using methods familiar to technicians. The levels of the constituent proteins of the LINC complex can be determined, for example, by antibody-based methods (including Western blotting, immunohistochemistry / cytochemistry, flow cytometry, ELISA) or by reporter gene-based methods.

[0107] Protein degradation can be assessed, for example, by detecting or analyzing the level / proportion of ubiquitinated proteins, their association with ubiquitin ligases, and / or proteosome localization.

[0108] The decrease in LINC complex protein levels may be a result of, for example, a decrease in the RNA level encoding the LINC complex protein, a reduction in post-transcriptional processing of the RNA encoding the LINC complex protein, or an increase in LINC complex protein degradation.

[0109] Disruption of the normal post-transcriptional processing of LINC complex proteins can, for example, reduce / alter the splicing of precursor mRNA encoding LINC complex proteins to mature mRNA, reduce the translation of mRNA encoding LINC complex proteins, or reduce / alter the post-translational processing of LINC complex proteins.

[0110] Reduced / altered splicing of precursor mRNA encoding the LINC complex protein to mature mRNA may be due to inhibition of the assembly and / or activity of factors required for normal splicing. Reduced translation of mRNA encoding the LINC complex protein may be due to inhibition of the assembly and / or activity of factors required for translation. Reduced / altered post-translational processing (e.g., enzymatic processing, folding) may be due to inhibition of the assembly and / or activity of factors required for normal post-translational processing of the LINC complex protein. Increased degradation of the LINC complex protein may be due to increased enzymatic (e.g., protease-mediated) protein degradation, which may be associated, for example, with protein misfolding.

[0111] In some embodiments, LINC complex formation can be inhibited by suppressing the transport of constituent proteins of the LINC complex (e.g., proteins containing the SUN domain and / or proteins containing the KASH domain) and / or disrupting their normal subcellular localization. In some embodiments, inhibiting LINC complex formation includes one or more of the following: reducing the level / proportion of LINC complex proteins localized to the nuclear envelope; reducing the level / proportion of SUN domain-containing proteins associated with the nuclear nuclear membrane; reducing the level / proportion of KASH domain-containing proteins associated with the outer nuclear membrane; increasing the retention of LINC complex proteins in the endoplasmic reticulum; and increasing the level / proportion of LINC complex proteins localized to the endoplasmic reticulum.

[0112] Subcellular localization of LINC complex constituent proteins within cells can be analyzed using techniques known to those skilled in the art. These techniques include, for example, analysis via immunocytochemistry and reporter molecule-based methods. For instance, Boni et al., J. Cell Biology (2015) 209(5):705-720 and Smoyer et al., J. Cell Biology (2016) 215(4):575-590 describe reporter systems that allow imaging of proteins in the ER, INM, and ONM. Such methods can be used to analyze the levels / proportions of LINC complex constituent proteins in the nuclear envelope, inner nuclear membrane, and outer nuclear membrane.

[0113] In some embodiments, the formation of the LINC complex can be inhibited by suppressing the interactions between the constituent proteins of the LINC complex (e.g., proteins containing a SUN domain and proteins containing a KASH domain). In some embodiments, the formation of the LINC complex can be inhibited by suppressing the interactions between the constituent proteins of the LINC complex and their interaction partners.

[0114] In some implementations, inhibiting the formation of the LINC complex includes one or more of the following: inhibiting the interaction between a protein containing a SUN domain and its interaction partner; inhibiting the interaction between a protein containing a KASH domain and its interaction partner; inhibiting the interaction between a protein containing a SUN domain and its KASH domain; inhibiting the interaction between a protein containing a SUN domain and a laminin; inhibiting the interaction between a protein containing a SUN domain and a chromatin-binding protein; inhibiting the interaction between a protein containing a KASH domain and a protein containing a SUN domain; and inhibiting the interaction between a protein containing a KASH domain and cytoskeleton components (e.g., microfilaments or components thereof (e.g., actin), microtubules or components thereof (e.g., tubulin), or intermediate filaments or components thereof).

[0115] In some implementations, the LINC complex inhibits the destruction / degradation of the LINC complex.

[0116] In some implementations, disruption / degradation of the LINC complex includes one or more of the following: inhibiting the interaction between a protein containing a SUN domain and its interaction partner; inhibiting the interaction between a protein containing a KASH domain and its interaction partner; inhibiting the interaction between a protein containing a SUN domain and its KASH domain; inhibiting the interaction between a protein containing a SUN domain and a laminin; inhibiting the interaction between a protein containing a SUN domain and a chromatin-binding protein; inhibiting the interaction between a protein containing a KASH domain and a protein containing a SUN domain; and inhibiting the interaction between a protein containing a KASH domain and cytoskeleton components (e.g., microfilaments or components thereof (e.g., actin), microtubules or components thereof (e.g., tubulin), or intermediate filaments or components thereof); increasing LINC complex disintegration; increasing LINC complex degradation; displacing LINC complex proteins from the LINC complex; and reducing the level of the LINC complex.

[0117] In this document, the interaction partner of a protein containing a SUN domain can be any molecule (e.g., protein / nucleic acid) that interacts with a protein containing a SUN domain. The interaction partner of a protein containing a SUN domain can be a protein capable of forming a complex with a protein containing a SUN domain through protein-protein interactions. In some embodiments, the interaction partner of a protein containing a SUN domain can be a protein containing a KASH domain (e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, or KASH5), a protein containing a SUN domain (e.g., SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO), a nucleoplasmic protein, a laminin (e.g., lamin A, lamin C, lamin B1, or lamin B2), or a chromatin-binding protein.

[0118] In this document, the interaction partner of a protein containing a KASH domain can be any molecule (e.g., protein / nucleic acid) that interacts with a protein containing a KASH domain. The interaction partner of a protein containing a KASH domain can be a protein capable of forming a complex with a protein containing a KASH domain through protein-protein interactions. In some embodiments, the interaction partner of a protein containing a KASH domain can be a protein containing a SUN domain (e.g., SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO), a protein containing a KASH domain (e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, or LRMP), a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, a microtubule, a tubulin, a microtubule motor, or an intermediate filament protein.

[0119] The interactions between the constituent proteins of the LINC complex and their interacting partners can be analyzed using techniques familiar to those skilled in the art, such as immunoprecipitation and resonance energy transfer (RET) assays using appropriately labeled substances. Inhibition of the interaction can be determined in such assays by detecting a decrease in the level of the interaction compared to a control under uninhibited conditions.

[0120] The LINC complex and its constituent proteins can be detected by methods known to those skilled in the art, such as antibody-based methods including Western blotting, immunohistochemistry / cytochemistry, flow cytometry, ELISA, or reporter gene-based methods.

[0121] LINC complex inhibition can be characterized by a decrease in the functional level of the LINC complex. In some embodiments, LINC complex inhibition can be determined by detecting a decrease in the levels of functionally relevant substances of the LINC complex.

[0122] In the specific implementation scheme envisioned herein, LINC complex inhibition is achieved through one or more of the following methods: modifying genes encoding proteins containing SUN domains to reduce / prevent their expression; modifying genes encoding proteins containing KASH domains to reduce / prevent their expression; inhibiting the expression of proteins containing SUN domains via RNAi; inhibiting the expression of proteins containing KASH domains via RNAi; or inhibiting the interaction between proteins containing SUN domains and proteins containing KASH domains.

[0123] In some implementations, site-specific nuclease (SSN) systems (e.g., CRISPR-based systems) targeting the relevant genes are used to modify genes encoding proteins containing a SUN domain or a KASH domain to reduce / prevent their expression. In some implementations, the inhibition of KASH domain-containing or SUN domain-containing protein expression by RNAi is achieved using siRNA, miRNA, or shRNA targeting the relevant proteins.

[0124] In some implementations, inhibition of the interaction between proteins containing the SUN domain and proteins containing the KASH domain is achieved by using small molecule inhibitors of the interaction between proteins containing the SUN domain and proteins containing the KASH domain, peptide mimics of the KASH domain, or peptide mimics of the SUN domain. It should be understood that the inhibition of the interaction occurs between endogenous proteins containing the SUN domain and endogenous proteins containing the KASH domain.

[0125] LINC complex inhibitors Certain aspects of this disclosure relate to the use of LINC complex inhibitors to inhibit LINC complexes. “LINC complex inhibitor” means any agent capable of achieving LINC complex inhibition. LINC complex inhibitors include agents capable of inhibiting LINC complex formation (i.e., inhibiting LINC complex assembly), disrupting / degrading LINC complexes, or inhibiting LINC complex function. Such agents may be effectors of the aforementioned LINC complex inhibition (i.e., may cause it directly or indirectly). LINC complex inhibitors may also be referred to herein as LINC complex antagonists.

[0126] LINC complex inhibitors are described, for example, in WO 2019 / 143300 A1, WO 2021 / 010898 A1, and WO2023 / 101607 A2. In some embodiments, the LINC complex inhibitors according to this disclosure are the LINC complex inhibitors described in WO 2019 / 143300 A1, WO 2021 / 010898 A1, and WO 2023 / 101607 A2, which are hereby incorporated in their entirety by reference.

[0127] In some implementations, LINC complex inhibitors can: inhibit LINC complex formation; disrupt / degrade LINC complexes; inhibit LINC complex activity; inhibit gene and / or protein expression of LINC complex proteins; modify genes encoding LINC complex proteins to reduce / prevent their expression; reduce RNA levels encoding LINC complex proteins; inhibit transcription of nucleic acids encoding LINC complex proteins; increase RNA degradation of LINC complex proteins; reduce LINC complex protein levels; disrupt normal post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding LINC complex proteins; increase LINC complex protein degradation; inhibit LINC complex protein transport and / or disrupt its normal subcellular localization; reduce the level / proportion of LINC complex proteins located on the nuclear envelope; reduce the level / proportion of KASH domain-containing proteins associated with the nuclear nuclear membrane; reduce the level / proportion of KASH domain-containing proteins associated with the outer nuclear membrane; increase the retention of LINC complex proteins in the endoplasmic reticulum; increase the level / proportion of LINC complex proteins located in the endoplasmic reticulum; inhibit Inhibits interactions between LINC complex constituent proteins; inhibits interactions between LINC complex proteins and their interaction partners; inhibits interactions between proteins containing the SUN domain and their interaction partners; inhibits interactions between proteins containing the SUN domain and their interaction partners containing the KASH domain; inhibits interactions between proteins containing the SUN domain and their KASH domain; inhibits interactions between proteins containing the SUN domain and their laminins; inhibits interactions between proteins containing the SUN domain and their chromatin-binding proteins; inhibits interactions between proteins containing the KASH domain and their SUN domain; inhibits interactions between proteins containing the KASH domain and their cytoskeleton components (e.g., microfilaments or their components (e.g., actin), microtubules or their components (e.g., tubulin), or intermediate filaments or their components); increases LINC complex disintegration; increases LINC complex degradation; displaces LINC complex proteins from the LINC complex; and / or decreases the level of the LINC complex.

[0128] It should be understood that a given LINC complex inhibitor may exhibit more than one of the properties listed in the foregoing paragraphs. Appropriate assays can be used to evaluate the properties listed in the foregoing paragraphs of a given agent. Assays can be, for example, in vitro assays, optionally cell-based assays, or cell-free assays. In the case of cell-based assays, the assay may include treating cells with the given test agent to determine whether the agent exhibits one or more of the listed properties. Assays may use endogenous or recombinantly expressed proteins and may use substances labeled with detectable entities to facilitate their detection.

[0129] Agents capable of reducing LINC complex protein gene expression (e.g., reducing RNA levels encoding LINC complex proteins, reducing nucleic acid transcription of LINC complex proteins, and / or increasing RNA degradation of LINC complex proteins) can be identified by assays (e.g., by RT-qPCR) that detect RNA levels encoding the relevant protein. Such assays may include treating cells / tissues with the agent and then comparing the RNA levels encoding the relevant protein in these cells / tissues with the RNA levels encoding the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / mediated cells / tissues). Assays for detecting reduced / altered pre-mRNA splicing of a given protein may include detecting and / or quantifying one or more isoforms of the relevant protein, or RNA encoding one or more of said isoforms.

[0130] Agents capable of reducing LINC complex protein expression (e.g., decreasing LINC complex protein levels, increasing LINC complex protein degradation) can be identified by assays that measure the levels of the relevant proteins, such as antibody / reporter gene-based methods (Western blotting, ELISA, immunohistochemistry / cytochemistry, etc.). Such assays may include treating cells / tissues with the agent and then comparing the levels of the relevant proteins in these cells / tissues to the levels of the relevant proteins in cells / tissues under appropriate control conditions (e.g., untreated / middle-treated cells / tissues). Assays of protein degradation may include assessing, for example, ubiquitination or proteasome localization of the relevant proteins, and / or the proportion of ubiquitinated or proteasome-localized relevant proteins.

[0131] Agents capable of inhibiting the transport of LINC complex proteins and / or disrupting their normal subcellular localization can be identified by assays that detect the presence or proportion of the relevant protein at specific subcellular locations, such as antibody / reporter gene-based methods (Western blotting, ELISA, immunohistochemistry / cytochemistry, etc.). Subcellular localization can be analyzed by, for example, immunocytochemistry or Western blotting of extracts prepared from different cell fractions, and can be performed using organelles labeled with and / or labeled with known subcellular localizations. Such assays may include treating cells / tissues with an agent and then comparing the subcellular localization of the relevant protein in such cells with the subcellular localization of the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / middle-treated cells / tissues).

[0132] Agents capable of inhibiting the interaction between the LINC complex protein and its interaction partner can be identified using assays that detect the level of interaction between the LINC complex protein and its interaction partner, such as antibody / reporter molecule-based methods. The level of interaction between the LINC complex protein and its interaction partner can be analyzed, for example, using resonance energy transfer techniques (e.g., FRET, BRET), immunoprecipitation, or methods analyzing interaction correlations (e.g., the function of the LINC complex). Assays may include treating cells / tissues with reagents and then comparing the interaction levels in these cells / tissues with the interaction levels in cells / tissues under appropriate control conditions (e.g., untreated / middled cells / tissues). Interactions between LINC complex proteins and their interacting partners can also be analyzed, for example using ELISA, surface plasmon resonance (SPR; see, for example, Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interference (see, for example, Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. Assays may include comparing interaction levels in the presence of the reagent with interaction levels under appropriate control conditions (e.g., in the absence of the reagent).

[0133] Agents capable of inhibiting the function of the LINC complex can be identified using assays that include detecting the functional correlation level of the LINC complex.

[0134] According to this disclosure, LINC complex inhibitors can be any agent / multiple agents that achieve the desired inhibitory activity. In some embodiments, LINC complex inhibitors can be or comprise peptides / peptides, small molecules, nucleic acids, or biomolecules.

[0135] In some implementations, the LINC complex inhibitor can bind to: the LINC complex, the LINC complex protein, or an interacting partner of the LINC complex protein.

[0136] LINC complex inhibitors may exhibit specific binding to relevant factors / complexes (i.e., LINC complex proteins or their interacting partners). As used herein, “specific binding” refers to selective binding and can be distinguished from nonspecific binding to non-target molecules. LINC complex inhibitors that specifically bind to LINC complex proteins or their interacting partners preferably bind to relevant factors with a higher affinity and / or longer duration than to other non-target molecules; such LINC complex inhibitors can be described as having “specificity” for the relevant factors.

[0137] In some embodiments, the LINC complex inhibitor can inhibit the interaction between the LINC complex protein and its interacting partner. In some embodiments, the LINC complex inhibitor can inhibit the function of the LINC complex. In some embodiments, the LINC complex inhibitor acts as a competitive inhibitor of the interaction between the LINC complex protein and its interacting partner. The LINC complex inhibitor may occupy the region required for the binding of the LINC complex protein to its interacting partner or otherwise reduce access to said region, or it may occupy the region required for the binding of the LINC complex protein to its interacting partner or otherwise reduce access to said region.

[0138] In some implementations, LINC complex inhibitors mimic the interacting partner of LINC complex proteins.

[0139] In some implementations, the LINC complex inhibitor suppresses the interaction between the SUN domain and the KASH domain.

[0140] In some embodiments, the LINC complex inhibitor inhibits binding between the C-terminal region of the KASH domain and the deep pocket on the surface of the SUN domain. In some embodiments, the LINC complex inhibitor binds to the SUN domain and inhibits the KASH domain from entering the deep pocket of the SUN domain. In some embodiments, the LINC complex inhibitor binds to the KASH domain and inhibits the KASH domain from approaching the deep pocket on the surface of the SUN domain.

[0141] In some implementations, the LINC complex inhibitor inhibits or disrupts the formation of disulfide bonds between the SUN and KASH domains.

[0142] In some implementations, the LINC complex inhibitor targets the proline-rich region at the C-terminus of a protein containing a KASH domain.

[0143] In some embodiments, the LINC complex inhibitor inhibits the oligomerization of proteins containing the SUN domain. In some embodiments, the LINC complex inhibitor targets the stalk region of proteins containing the SUN domain.

[0144] In some embodiments, the LINC complex inhibitor inhibits protein-protein interactions between the SUN domain and the KASH domain. In some embodiments, the LINC complex inhibitor inhibits protein-protein interactions between SUN1 and Nesprin-1, SUN2 and Nesprin-1, SUN1 and Nesprin-2, SUN1 and Nesprin-3, SUN2 and Nesprin-2, or SUN2 and Nesprin-3. In some embodiments, the LINC complex inhibitor inhibits protein-protein interactions between SUN1 and Nesprin-1.

[0145] The ability of a candidate LINC complex inhibitor to inhibit the interaction between the LINC complex protein and its interacting partner can be assessed by analyzing the interaction in the presence of the candidate LINC complex inhibitor or after incubation of one or both of the interacting partners with the candidate LINC complex inhibitor. An example of a suitable assay for determining whether a given binding agent can inhibit the interaction between the LINC complex protein and its interacting partner is a competitive ELISA.

[0146] In some implementations, molecules that bind to the LINC complex protein or its interaction partner inhibit the ability of the LINC complex protein to bind to its interaction partner.

[0147] In some implementations, LINC complex inhibitors are able to bind to the LINC complex protein or its interacting partner and inhibit the interaction between the LINC complex protein and its interacting partner.

[0148] LINC complex inhibitors, which bind to or inhibit the interaction between the LINC complex protein and its interacting partner, can be identified using any suitable assay of the binding of a relevant factor (i.e., the LINC complex protein or its interacting partner) and the inhibition of the interaction between the LINC complex protein and its interacting partner. Such assays may include, for example, detecting the formation of a complex between the relevant factor and the candidate inhibitor molecule, and / or detecting the formation of a complex between the LINC complex protein and its interacting partner.

[0149] In some embodiments, the LINC complex inhibitor, which is capable of binding to the LINC complex protein or its interacting partner and inhibiting the interaction between the LINC complex protein and its interacting partner, may be, for example, a peptide / peptide.

[0150] LINC complex inhibitors can be, for example, based on the interaction pair of the inhibitor-binding related factor (i.e., the LINC complex protein, or the interaction pair of the LINC complex protein).

[0151] As used herein, a peptide / polypeptide / amino acid sequence “based on” a reference protein comprises or consists of an amino acid sequence that has a high sequence identity (e.g., at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with all or part of the amino acids of the reference protein.

[0152] For example, a LINC complex inhibitor that binds to a LINC complex protein may comprise a peptide / peptide fragment of the LINC complex protein interaction partner, or be composed of said peptide / peptide fragment. Similarly, a LINC complex inhibitor that binds to a LINC complex protein interaction partner may comprise a peptide / peptide fragment of the LINC complex protein, or be composed of said peptide / peptide fragment.

[0153] Such inhibitors preferably have the ability to bind to the relevant factor, but are deficient in or exhibit a reduced level of one or more other properties of the protein on which they are based. For example, a LINC complex inhibitor may contain the amino acid sequence required for binding to the relevant factor, but may be deficient in the amino acid sequence required for one or more other properties of the protein on which it is based.

[0154] Such peptide / peptide LINC complex inhibitors can be referred to as “decoys,” “dominant-negative,” or “mimicking” forms of the proteins they are based on, and preferably exhibit competitive inhibition of the interaction between the LINC complex protein and its interaction partner. In this way, these peptide / peptide LINC complex inhibitors inhibit the formation of LINC complexes and / or disrupt existing LINC complexes by displacing endogenous interaction partners, resulting in non-functional or reduced-function complexes.

[0155] Crisp et al. described the dominant-negative form of the mouse Sun1 protein in J Cell Biol. (2006) 172(1): 41–53, while its humanized form was described in WO 2019 / 143300 A1. Other dominant-negative forms of SUN1 are described in WO 2023 / 101607 A2. Dominant-negative SUN2 is described, for example, in Stewart-Hutchinson et al. Exp Cell Res. (2008) 314(8):1892-905.

[0156] The dominant-negative form of KASH1 is described, for example, in Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8):1892-905, Grady et al., Proc. Natl. Acad. Sci. USA (2005) 102: 4359–4364, and Libotte et al., MBoC (2005) 16: 3411–3424. The dominant-negative form of KASH2 is described, for example, in Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8):1892-905, Libotte et al., MBoC (2005) 16: 3411–3424, Zhen et al., J. Cell. Sci. (2002) 115: 3207–3222, and Kim et al., Sci Transl Med (2018) 10. The dominant-negative forms of KASH3 are described, for example, by Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8):1892-905. The dominant-negative forms of KASH4 are described, for example, by Roux et al., Proc. Natl. Acad. Sci. USA (2009) 106: 2194–2199. The dominant-negative forms of KASH5 are described, for example, by Horn et al., J Cell Biol (2013) 202: 1023–1039.

[0157] In some embodiments, peptide / peptide LINC complex inhibitors are based on proteins containing a SUN domain (e.g., proteins containing a SUN domain as described herein). In some embodiments, peptide / peptide LINC complex inhibitors are decoy / dominant-negative forms of proteins containing a SUN domain.

[0158] LINC complex inhibitors based on proteins containing the SUN domain may bind to proteins containing the KASH domain (such as those described herein), but lack or exhibit a reduced level of one or more other properties of the SUN domain-based protein (e.g., binding to proteins containing the SUN domain, nucleoplasmic proteins, laminins, and / or chromatin-binding proteins).

[0159] LINC complex inhibitors based on proteins containing the SUN domain may bind to proteins containing the SUN domain (such as those described herein) but lack or exhibit a reduced level of one or more other properties of the SUN domain-based protein (e.g., binding to proteins containing the KASH domain, nucleoplasmic proteins, laminins, and / or chromatin-binding proteins).

[0160] LINC complex inhibitors based on proteins containing the SUN domain preferably contain the SUN domain. In some embodiments, the LINC complex is composed of or substantially composed of the SUN domain. The peptide / peptide may lack the amino acid sequence of a protein containing the SUN domain, which constitutes a protein domain other than the SUN domain. In addition to the properties mediated by the SUN domain, the peptide / peptide preferably lacks the properties of endogenous proteins containing the SUN domain. The peptide / peptide may exhibit dominant-negative peptide / peptide behavior, capable of inhibiting the interaction between endogenous proteins containing the SUN domain and their protein-interacting partners.

[0161] In some embodiments, the peptide / peptide LINC complex inhibitor is based on a KASH domain-containing protein (e.g., the KASH domain-containing protein described herein). In some embodiments, the peptide / peptide LINC complex inhibitor is a decoy / dominant-negative form of a KASH domain-containing protein, such as KASH1, KASH2, KASH3, KASH4, or KASH5.

[0162] LINC complex inhibitors based on KASH domain-containing proteins may exhibit binding to SUN domain-containing proteins (such as those described herein), but lack or show reduced levels of one or more other properties of the KASH domain-containing protein on which they are based (e.g., binding to KASH domain-containing proteins, cytoplasmic proteins, cytoskeletal proteins, microfilaments, actin, microtubules, tubulin, microtubule motors, or intermediate filament proteins).

[0163] LINC complex inhibitors based on KASH domain-containing proteins may exhibit binding to KASH domain-containing proteins (such as those described herein), but lack or show reduced levels of one or more other properties of the KASH domain-containing protein on which it is based (e.g., binding to proteins containing SUN domains, cytoplasmic proteins, cytoskeletal proteins, microfilaments, actin, microtubules, tubulin, microtubule motors, or intermediate filament proteins).

[0164] LINC complex inhibitors based on KASH-domain-containing proteins preferably contain a KASH domain. In some embodiments, the LINC complex consists of or is substantially composed of a KASH domain. The peptide / peptide may lack the amino acid sequence of a protein containing a KASH domain, which constitutes a protein domain other than the KASH domain. The peptide / peptide preferably lacks the properties of an endogenous KASH-domain-containing protein other than the properties mediated by the KASH domain. The peptide / peptide can act as a dominant-negative peptide / peptide, capable of inhibiting the interaction between an endogenous KASH-domain-containing protein and its interaction partner.

[0165] In some implementations, peptide / peptide LINC complex inhibitors capable of binding to the LINC complex / LINC complex protein / LINC complex protein interaction partner and inhibiting the interaction between the LINC complex protein and the LINC complex protein interaction partner and / or LINC complex function include, for example, peptide aptamers, thioredoxins, monoclonal antibodies, anticalcinins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affinity molecules, nanobodies (i.e., single-domain antibodies (sdAbs)), affinity proteins, armadillo repeats (ArmRP), OBodies, and fibronectins—for example, a review by Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082–1101, which is incorporated herein by reference (see also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48). Other peptide / peptide LINC complex inhibitors disclosed herein include antibodies (immunoglobulins), such as monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and their fragments and derivatives (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, biantibodies, triantibodies, scFv-Fc, mini antibodies, single-domain antibodies (e.g., VhH), etc.).

[0166] These peptide / peptide LINC complex inhibitors can be identified by screening libraries of relevant peptides / peptides for LINC complex inhibition.

[0167] The LINC complex inhibitory peptide will be described in more detail below.

[0168] In some implementations, the LINC complex inhibitor is a small molecule inhibitor of the LINC complex. As used herein, 'small molecule' refers to an organic compound with a low molecular weight (< 1000 Daltons, typically between ~300-700 Daltons).

[0169] Small molecule LINC complex inhibitors can bind to the LINC complex, the LINC complex protein, or its interacting partner. They can inhibit the interaction between the LINC complex protein and its interacting partner. Furthermore, they can bind to the LINC complex and inhibit its function.

[0170] Suitable small molecule LINC complex inhibitors can be identified, for example, by screening small molecule libraries, as described in Example 7 of WO2021 / 010898 A1.

[0171] In some implementations, the LINC complex inhibitor is or contains a nucleic acid.

[0172] Nucleic acids can bind to the LINC complex, LINC complex proteins, or LINC complex protein interaction partners. Nucleic acids can inhibit the interaction between the LINC complex protein and its interaction partners. Nucleic acids can bind to the LINC complex and inhibit its function.

[0173] For example, nucleic acid aptamers are reviewed in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202. They can be identified and / or produced by the exponential enrichment of ligands phylogenetic evolution (SELEX) method, or by developing SOMAmers (slow dissociation rate modified aptamers) (Gold L et al. (2010) PLoSONE 5(12):e15004). Aptamers and SELEX are described in Tuerk and Gold, Science (1990) 249(4968):505-10 and WO 91 / 19813.

[0174] Nucleic acid aptamers may contain DNA and / or RNA and may be single-stranded or double-stranded. They may contain chemically modified nucleic acids, such as sugars and / or phosphates and / or bases that are chemically modified. Such modifications can improve the stability of the aptamer or make it more resistant to degradation, and may include modifications at the 2' position of the ribose.

[0175] Nucleic acid aptamers can be synthesized chemically, for example, on a solid-phase support. Solid-phase synthesis can employ phosphoramidite chemistry. Briefly, the solid-phase-supported nucleotide is detrimethylated and then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite-triester bond. Capping may then occur, followed by oxidation of the phosphite-triester with an oxidizing agent (usually iodine). This cycle can then be repeated to assemble the aptamer (e.g., see Sinha, ND; Biernat, J.; McManus, J.; Köster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, SL; Lyer, RP (1992). Tetrahedron 48 (12): 2223).

[0176] In some embodiments, LINC complex inhibitors can reduce the expression of LINC complex proteins (e.g., gene and / or protein expression). In some embodiments, LINC complex inhibitors can reduce or prevent the expression of endogenous LINC complex proteins.

[0177] Inhibiting the expression of LINC complex proteins will result in a reduction in the amount of LINC complex proteins and / or the number of LINC complexes containing the constituent proteins in cells / tissues / organs / organ systems / subjects. For example, in a given cell, inhibiting the expression of LINC complex proteins will result in a reduction in the level of LINC complex proteins and / or the number of LINC complexes containing the constituent proteins in that cell compared to untreated cells.

[0178] Suppression can be partial. Preferred levels of suppression are at least 50%, more preferably at least one of 60%, 70%, 80%, 85%, or 90%. Suppression levels between 90% and 100% are considered "silencing" of expression or function. Gene and protein expression can be determined according to the methods described herein or by methods well known to those skilled in the art.

[0179] In some implementations, the LINC complex inhibitor reduces or blocks the expression of SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO. In some implementations, the LINC complex inhibitor reduces or blocks the expression of SUN1 or SUN2.

[0180] In some implementations, LINC complex inhibitors reduce or block the expression of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, or LRMP.

[0181] In some implementations, LINC complex inhibitors can target specific domains / regions of the LINC complex protein. In some implementations, LINC complex inhibitors reduce or prevent the expression of LINC complex protein isotypes containing one or more target domains / regions.

[0182] For example, the target domain or region may be or include: the SUN domain, the KASH domain, and / or the domain required for interaction with the interaction partner of the LINC complex protein. For example, the target domain or region may need to interact with proteins containing the KASH domain, proteins containing the SUN domain, nucleoplasmic proteins, laminins, chromatin-binding proteins, cytoplasmic proteins, cytoskeletal proteins, microfilaments, actin, microtubules, tubulin, microtubule motors, and / or intermediate filament proteins.

[0183] In some implementations, LINC complex inhibitors can alter the splicing of pre-mRNA encoding LINC complex proteins to increase the proportion of mature mRNA encoding the same type lacking the relevant domain / region, and / or decrease the proportion of mature mRNA encoding the same type containing the relevant domain / region.

[0184] In some implementations, LINC complex inhibitors may modify nucleic acids encoding LINC complex proteins to increase the expression of isoforms lacking the relevant domain / region, and / or may modify nucleic acids encoding LINC complex proteins to decrease the expression of isoforms containing the relevant domain / region of the protein.

[0185] In some embodiments, the LINC complex inhibitor is a repressive nucleic acid. In some embodiments, the repressive nucleic acid is an antisense nucleic acid. In some embodiments, the repressive nucleic acid is an antisense oligonucleotide (ASO). The antisense oligonucleotide may be single-stranded and may bind to the target oligonucleotide (e.g., mRNA) via complementary sequence binding.

[0186] ASO can be designed to inhibit / block the expression of LINC complex proteins or specific isoforms thereof.

[0187] Oligonucleotides designed to inhibit / block the expression of LINC complex proteins or specific isotypes can have basic sequence identity with a portion of the nucleic acid encoding the LINC complex protein / related isotype or its complementary sequence.

[0188] In some implementations, repressive nucleic acids reduce the expression of LINC complex proteins through RNA interference (RNAi). RNAi refers to the suppression of gene expression and translation by targeting and neutralizing mRNA molecules. In some implementations, the repressive nucleic acid is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

[0189] The mechanisms of RNAi and the role of small RNAs in epigenetic silencing targeting heterochromatin complexes and specific chromosomal loci have been established. Double-stranded RNA (dsRNA)-dependent posttranscriptional silencing, also known as RNA interference (RNAi), is the phenomenon where dsRNA complexes can target and silence homologous, specific genes over a short period. It acts as a signal to promote the degradation of mRNAs with sequence identity. 20-nt siRNAs are typically long enough to induce gene-specific silencing, yet short enough to evade host responses. The reduction in expression of the targeted gene product can be extensive; 90% of silencing is induced by a small number of siRNA molecules. RNAi-based therapies have progressed to Phase I, II, and III clinical trials for many indications. Nature January 22, 2009; 457(7228):426-433).

[0190] In this field, these RNA sequences are referred to as “short or small interfering RNA” (siRNA) or “microRNA” (miRNA) based on their origin. Both types of sequences can be used to downregulate gene expression by binding to complementary RNA and triggering mRNA elimination (RNAi) or preventing mRNA from being translated into protein. siRNA is obtained by processing long double-stranded RNA and is usually of exogenous origin when found in nature. MicroRNA (miRNA) is a small, endogenous, non-coding RNA obtained by processing short hairpins. Both siRNA and miRNA can inhibit the translation of mRNA with partially complementary target sequences without RNA cleavage and degrade mRNA with fully complementary sequences.

[0191] siRNA is typically double-stranded, and to optimize the effectiveness of RNA-mediated downregulation of target gene function, it is preferable to select the length of the siRNA molecule to ensure that the RISC complex correctly recognizes the siRNA, which mediates the recognition of the mRNA target by the siRNA, and to make the siRNA short enough to reduce the host response.

[0192] miRNAs are typically single-stranded and have partially complementary regions that allow ligands to form hairpins. miRNAs are RNA genes transcribed from DNA but not translated into proteins. The DNA sequence encoding the miRNA gene is longer than the miRNA itself. This DNA sequence consists of the miRNA sequence and a nearly inversely complementary sequence. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence pairs with its inversely complementary base to form a partially double-stranded RNA segment. The design of microRNA sequences is exemplified by, for example, by John et al. PLoS Biology This was discussed in , 11(2), 1862-1879, 2004.

[0193] Typically, oligonucleotides designed to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or their synthetic analogues), more preferably between 17 and 30 ribonucleotides, even more preferably between 19 and 25 ribonucleotides, and most preferably between 21 and 23 ribonucleotides. In some embodiments of this disclosure using double-stranded siRNA, the molecule may have a symmetrical 3' overhang, such as a 3' overhang of one or two (ribo)nucleotides, typically a dT dT 3' overhang. Based on the disclosure provided herein, those skilled in the art can readily design suitable siRNA and miRNA sequences, for example using resources such as the Ambion siRNA finder. siRNA and miRNA sequences can be synthesized and exogenously added to induce gene downregulation, or generated using an expression system (e.g., a vector). In some embodiments, the siRNA is synthesized synthetically.

[0194] siRNA-mediated knockdown of LINC complex proteins and siRNAs used to achieve the same purpose are described in the following literature: for example, Östlund et al., Journal of Cell Science (2009) 122:4099-4108, Hatch and Hetzer, J Cell Biol. (2016) 215(1):27-36, Matsumoto et al., Nucleus. (2016) 7(1):68-83, Uzer et al., Stem Cells. (2015) 33(6):2063-76, Thakar et al., Mol BiolCell. (2017) 28(1): 182-191, Espigat-Georger et al., J Cell Sci. (2016) 129(22):4227-4237, Yang et al., Int J Mol Med. (2013) 32(4):805-12, Rajgor et al., PLoSOne. 2012;7(7):e40098, Zhang et al., Exp Cell Res. (2016) 345(2):168-179, Warren et al., J Biol Chem. (2010) 285(2):1311-20, and King et al., Cytoskeleton (Hoboken) (2014) 71(7):423-34, which are incorporated herein by reference in their entirety.

[0195] Longer double-stranded RNAs can be processed in cells to produce siRNAs (see, for example, Myers (2003) Nature Biotechnology 21:324-328). Longer dsRNA molecules may have symmetrical 3' or 5' overhangs, such as one or two (ribo)nucleotides, or may have blunt ends. Longer dsRNA molecules can be 25 nucleotides or longer. Preferably, the length of the longer dsRNA molecule is between 25 and 30 nucleotides. More preferably, the length of the longer dsRNA molecule is between 25 and 27 nucleotides. Most preferably, the length of the longer dsRNA molecule is 27 nucleotides. dsRNAs of 30 nucleotides or longer can be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

[0196] Another approach is to express short hairpin RNA molecules (shRNA) intracellularly. shRNA is more stable than synthetic siRNA. shRNA consists of short inverted repeat sequences separated by a small circular sequence. One inverted repeat sequence is complementary to the target gene. Intracellularly, shRNA is processed into siRNA via DICER, which degrades the target gene mRNA and inhibits its expression. In some embodiments, shRNA is produced intracellularly via vector transcription. shRNA can be produced intracellularly by transfecting a vector encoding the shRNA sequence into cells, the shRNA sequence being controlled by an RNA polymerase III promoter (e.g., human H1 or 7SK promoter) or an RNA polymerase II promoter. Alternatively, shRNA can be synthesized exogenously (in vitro) via transcription from a vector. The shRNA can then be introduced directly into cells. Preferably, the shRNA molecule contains a portion of the sequence of a gene encoding a LINC complex protein. Preferably, the length of the shRNA sequence is between 40 and 100 base pairs, more preferably between 40 and 70 base pairs. The hairpin stem is preferably between 19 and 30 base pairs in length. The stem may contain GU pairings to stabilize the hairpin structure.

[0197] shRNA-mediated knockdown of LINC complex proteins and shRNAs used to achieve the same purpose are described in the following literature: for example, Kelkar et al., Nucleus. (2015) 6(6): 479–489, Mroß et al., Nucleus. (2018) 9(1): 503–515, Xing et al., International Journal of Cell Biology (2017) ArticleID: 8607532, Arsenovic et al., Biophys J. (2016) 110(1):34-43, and Li et al., Scientific Reports (2017) 7: Article number: 9157, which are hereby incorporated in their entirety by reference.

[0198] In some implementations, the repressive nucleic acid is a splice-switching oligonucleotide (SSO). Splice-switching oligonucleotides have been reviewed, for example, in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549–6563, which is incorporated herein by reference in its entirety. SSOs disrupt the normal splicing of target RNA transcripts by blocking RNA-RNA base pairing and / or protein-RNA binding interactions between components of the splicing mechanism and the precursor mRNA. SSOs can be used to alter the quantity / ratio of mature mRNA transcripts encoding the LINC complex protein or a specific isotype thereof. SSOs can be programmed to target specific regions of the target transcript, such as skipping target exons, for example, exons encoding target domains / regions.

[0199] SSO typically includes modifications to the oligonucleotide sugar-phosphate backbone to prevent RNase H degradation, and may include, for example, phosphorylated diamine morpholino (PMO), peptide nucleic acid (PNA), locked nucleic acid (LNA), and / or 2′O-methyl (2′OMe) and 2′-methoxyethyl (MOE) ribose modifications.

[0200] Repressive nucleic acids can be recombinantly produced by transcription of nucleic acid sequences (e.g., nucleic acid sequences contained in a vector). Transcription can occur in cell-free transcription reactions or in cells containing nucleic acids encoding repressive nucleic acids. In some embodiments, the repressive nucleic acid is produced intracellularly, for example, by transcription via a vector. The vector encoding such a molecule can be introduced into the cell in any manner known in the art. Optionally, cell / tissue-specific promoters (e.g., heart, muscle, etc.) can be used to regulate nucleic acid expression.

[0201] Inhibitory nucleic acids can also be synthesized using standard solid-phase or liquid-phase synthesis techniques known in the art.

[0202] In some implementations, a LINC complex inhibitor is one or more molecules capable of modifying nucleic acids encoding LINC complex proteins to reduce / prevent the expression of LINC complex proteins or specific isoforms thereof.

[0203] Modifying the nucleic acid encoding the LINC complex protein may include modifying the gene encoding the LINC complex protein. In some embodiments, modifying the nucleic acid encoding the LINC complex protein includes introducing insertions, substitutions, or deletions into the nucleic acid sequence encoding the LINC complex protein.

[0204] In some embodiments, modifying the nucleic acid encoding the LINC complex protein includes modifying the nucleic acid to introduce an early stop codon into the sequence transcribed from the nucleic acid. In some embodiments, modifying the nucleic acid encoding the LINC complex protein includes modifying the nucleic acid to encode a truncated and / or non-functional form of the LINC complex protein. In some embodiments, modifying the nucleic acid encoding the LINC complex protein includes modifying the nucleic acid to encode a misfolded and / or degraded form of the LINC complex protein.

[0205] Modification can be a modification of nucleic acids in cells, such as modifications of endogenous nucleic acids encoding LINC complex proteins. Compared to unmodified cells, modifications result in reduced expression levels of genes and / or proteins of the LINC complex protein or its specific isotype in the cells.

[0206] In some embodiments, the modification may be a modification of a nucleic acid region encoding a LINC complex protein involved in (e.g., required to) the formation of the LINC complex. For example, in some embodiments, the modification may target a gene region encoding a protein containing a SUN domain. In some embodiments, the modification may target a gene region encoding a protein containing a KASH domain.

[0207] In some embodiments, the modification is performed in vitro or ex vivo. In some embodiments, the modification is performed in vivo.

[0208] Methods and agents for modifying nucleic acids encoding target proteins are well known in the art, including, for example, modifying target nucleic acids by homologous recombination and editing target nucleic acids using site-specific nucleases (SSNs). For example, the inventors demonstrated CRISPR / Cas9-mediated disruption of SUN1 and Nesprin-1 in experimental embodiments of WO 2021 / 010898 A1.

[0209] Suitable approaches can employ homologous recombination targeting, as reviewed in Mortensen Curr Protoc Neurosci. (2007), Chapter 4: Unit 4.29, and Vasquez et al., PNAS 2001, 98(15): 8403-8410, both of which are incorporated herein by reference. Homologous recombination targeting involves exchanging nucleic acid sequences through homologous sequence-guided exchange events.

[0210] In some implementations, the method employs target nucleic acid editing using SSNs. Gene editing using SSNs has been reviewed, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is incorporated herein by reference in its entirety. Enzymes capable of generating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into target nucleic acid sequences. DSBs can be repaired by error-prone non-homologous end joining (NHEJ), where the ends of the break are rejoined, typically with inserted or deleted nucleotides. Alternatively, DSBs can be repaired by homology-directed repair (HDR), where a DNA template with ends homologous to the break site is provided and introduced into the DSB site.

[0211] SSNs that can be engineered to produce target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced palindromic repeats / CRISPR-associated protein 9 (CRISPR / Cas9) systems.

[0212] ZFN systems have been reviewed, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is incorporated herein by reference in its entirety. ZFNs contain programmable zinc finger DNA-binding domains and DNA-cutting domains (e.g. Fok (I) Endonuclease domain. DNA-binding domains can be identified by screening zinc finger arrays that can bind to target nucleic acid sequences.

[0213] The TALEN system has been reviewed, for example, by Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is incorporated herein by reference in its entirety. TALEN comprises a programmable DNA-binding TALE domain and a DNA-cutting domain (e.g., Fok I. Endonuclease domain). TALE contains a repeating domain consisting of repeats of 33-39 amino acids, all identical except for the two residues at positions 12 and 13 of each repeat, which are repeat variable di residues (RVDs). Each RVD determines the binding of the repeat sequence to nucleotides in the target DNA sequence according to the following relationships: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501.).

[0214] CRISPR / Cas9 and related systems such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3 have been reviewed, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, which is incorporated herein by reference in its entirety. These systems comprise endonucleases (e.g., Cas9, Cpf1, etc.) and single guide RNA (sgRNA) molecules. sgRNAs can be engineered to target endonuclease activity to specific nucleic acid sequences.

[0215] In some embodiments, the LINC complex inhibitor employs a site-specific nuclease (SSN) system that targets the LINC complex protein. Therefore, in some embodiments, the LINC complex inhibitor comprises or is composed of an SSN system that targets the LINC complex protein. In some embodiments, the LINC complex inhibitor employs nucleic acids encoding an SSN system that targets the LINC complex protein.

[0216] In some embodiments, the SSN system targets nucleic acid regions of LINC complex proteins that participate in (e.g., require) the formation of the LINC complex. For example, in some embodiments, the SSN system can disrupt the expression of exons of genes encoding proteins containing SUN domains. In some embodiments, the SSN system can disrupt the expression of exons of genes encoding proteins containing KASH domains.

[0217] In a specific implementation, the SSN system can introduce insertions / deletions in the nucleic acid sequence of a gene encoding a protein containing a SUN domain, upstream of the sequence encoding a conserved tyrosine residue.

[0218] In some implementations, the SSN system can introduce an insertion / deletion into the nucleic acid sequence of a gene encoding a protein containing a SUN domain, the insertion / deletion being located upstream of the sequence encoding a tyrosine residue, corresponding to position 154 of SEQ ID NO:8 (i.e., position 153 of SEQ ID NO:18, position 153 of SEQ ID NO:27, position 151 of SEQ ID NO:28, and position 152 of SEQ ID NO:29).

[0219] In a specific implementation, the SSN system can introduce insertions / deletions in the nucleic acid sequence of a gene encoding a protein containing a SUN domain, upstream of the sequence encoding a conserved tyrosine residue.

[0220] In some implementations, the SSN system can introduce insertions / deletions in the nucleic acid sequence of a gene encoding a protein containing a KASH domain, upstream of a sequence encoding a C-terminal proline-rich region.

[0221] In some implementations, the SSN system can introduce an insertion / deletion into the nucleic acid sequence of a gene encoding a protein containing a KASH domain, the insertion / deletion being located upstream of the sequence encoding proline, corresponding to position 57 of SEQ ID NO:37 (i.e., position 57 of SEQ ID NO:38, position 56 of SEQ ID NO:39, and position 56 of SEQ ID NO:40).

[0222] In some implementations, the SSN system is a ZFN system, a TALEN system, a CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system, or a CRISPR / C2c3 system.

[0223] In some implementations, the SSN system is a CRISPR / Cas9 system. In such implementations, LINC complex inhibition can be achieved using nucleic acids encoding CRISPR RNA (crRNA), nucleic acids targeting LINC complex proteins, and trans-activating crRNA (tracrRNA) for processing the crRNA into its mature form. CRISPR / Cas9 systems for targeting and disrupting LINC complex proteins SUN1 and SUN2 are described, for example, in Schaller et al., J Virol. (2017) 91(19):pii: e00463-17, which is incorporated herein by reference in its entirety.

[0224] Another way to disrupt the LINC complex is to modify the endogenous SUN or KASH domains, rather than expressing components of proteins containing the SUN domain or KASH domain, to disrupt the LINC complex by competing with endogenous Nesprin (containing the KASH domain) or SUN1 and SUN2 (containing the SUN domain), so that they cannot bind to their homologous LINC complex binding partners, or their binding ability to their homologous LINC complex binding partners is reduced.

[0225] Since both the SUN and KASH domains are located at the C-terminus of their respective proteins, a method to generate modified SUN or KASH domains involves using a CRISPR / Cas system to modify the gene encoding the protein with the SUN or KASH domain, producing a premature stop codon at the 3' end of the respective protein sequence after CRISPR-induced non-homologous end joining. This results in a truncated protein with a mutated SUN or KASH domain at its C-terminus. The truncated protein is expressed and localized on the membrane but cannot interact with its homologous LINC complex partner.

[0226] Therefore, in some implementations, the LINC complex inhibitor is a CRISPR-Cas or other synthetic nuclease system that is capable of modifying nucleic acids encoding the SUN domain or KASH domain of endogenous SUN or Nesprin proteins, respectively.

[0227] In some implementations, the CRISPR-Cas system modifies the endogenous SUN domain or KASH domain of the SUN1 or Nesprin-1 protein, respectively, to disrupt the LINC complex. The respective nucleic acids are Sun1 and Syne1.

[0228] In some embodiments, the CRISPR-Cas system comprises a gRNA nucleic acid (e.g., SUN1) that targets the SUN domain of a protein containing the SUN domain. In some embodiments, the CRISPR-Cas system comprises a gRNA nucleic acid (e.g., Nesprin-1) that targets the KASH domain of a protein containing the KASH domain.

[0229] LINC complex inhibits peptides In some embodiments, the LINC complex inhibitor according to this disclosure is a LINC complex inhibitory peptide.

[0230] In this document, "LINC complex inhibitory peptide" refers to a peptide that inhibits the formation and / or function of the LINC complex. LINC complex inhibitory peptides are described, for example, in WO 2019 / 143300 A1, WO 2021 / 010898 A1, and WO 2023 / 101607A2. In some embodiments, the LINC complex inhibitor according to this disclosure is a LINC complex inhibitory peptide of any embodiment described in WO 2019 / 143300 A1, WO 2021 / 010898 A1, or WO 2023 / 101607 A2, all of which are hereby incorporated by reference in their entirety.

[0231] The LINC complex inhibitory peptide according to this disclosure preferably binds to a LINC complex protein. This binding can be characterized by a non-covalent protein-protein interaction between the LINC complex inhibitory peptide and the LINC complex protein. This interaction may include electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0232] In some embodiments, the LINC complex inhibitory peptide according to this disclosure binds to an interacting partner of a protein containing a SUN domain (e.g., a protein containing a KASH domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), with an affinity in the micromolar range, i.e., K D = 9.9 x 10 -4 Up to 1 x 10 -6 M. In some embodiments, the LINC complex inhibits the peptide from binding to an interaction partner of a protein containing a SUN domain (e.g., a protein containing a KASH domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), with an affinity in the sub-micromolar range, i.e., K D < 1 x 10 -6 M. In some embodiments, the LINC complex inhibits the peptide by binding to an interaction partner of a protein containing a SUN domain (e.g., a protein containing a KASH domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), with an affinity in the nanomolar range, i.e., K D = 9.9 x 10 -7 Up to 1 x 10 -9 M. In some embodiments, the LINC complex inhibits the peptide from binding to an interacting partner of a protein containing a SUN domain (e.g., a protein containing a KASH domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), with an affinity in the sub-nanomolar range, i.e., K D < 1 x 10 -9 M. In some embodiments, the LINC complex inhibits the peptide by binding to an interacting partner of a protein containing a SUN domain (e.g., a protein containing a KASH domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), with an affinity in the picomolar range, i.e., K D= 9.9 x 10 -10 to 1 x 10 -12 M. In some embodiments, the LINC complex inhibits the peptide from binding to an interacting partner of a protein containing a SUN domain (e.g., a protein containing a KASH domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), with an affinity in the sub-picomolar range, i.e., K... D < 1 x 10 -12 M.

[0233] The LINC complex inhibitory peptide according to this disclosure can bind to the interaction partner of a protein containing a SUN domain (e.g., a protein containing a SUN domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) in a manner similar to that of a protein containing a SUN domain (e.g., a protein containing a KASH domain selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO). In some embodiments, the LINC complex inhibitory peptide can bind to the same or overlapping region of the interaction partner of the protein containing the SUN domain, which is the same region to which the protein containing the SUN domain binds.

[0234] Those skilled in the art can use a variety of methods well known in the art to determine the region of a given target molecule to which a peptide binds, including X-ray cocrystal analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolysis-based “protection” methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which are incorporated herein by reference in their entirety.

[0235] In some embodiments, the LINC complex inhibitory peptide according to this disclosure inhibits the interaction between proteins containing a SUN domain (e.g., proteins containing a SUN domain selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) and their interacting partners (e.g., proteins containing a KASH domain, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). Inhibition of the interaction between proteins containing a SUN domain and their interacting partners includes inhibiting the binding between proteins containing a SUN domain and their interacting partners (thereby inhibiting the formation of complexes containing such proteins) and disrupting the complex composed of proteins containing a SUN domain and their interacting partners (e.g., by displacing the constituent proteins of such complexes, thereby causing the disintegration of such complexes).

[0236] In some embodiments, the LINC complex inhibiting peptide inhibits the binding of SUN domain-containing proteins (e.g., SUN domain-containing proteins selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) to the interaction partner of SUN domain-containing proteins (e.g., proteins containing KASH domains, such as Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In some embodiments, the LINC complex inhibiting peptide is a competitive inhibitor of the binding of SUN domain-containing proteins to their interaction partner. In some embodiments, the LINC complex inhibiting peptide blocks the binding of SUN domain-containing proteins to their interaction partner. In some embodiments, the LINC complex inhibiting peptide occupies the region of the SUN domain-containing protein interaction partner that binds to the SUN domain-containing protein, thereby inhibiting the interaction between the SUN domain-containing protein and its interaction partner. In some embodiments, the LINC complex inhibitory peptide displaces a protein containing a SUN domain from a complex containing both the SUN domain-containing protein and the interacting partner of the SUN domain-containing protein.

[0237] The ability of a given peptide to inhibit the interaction between two factors can be determined, for example, by analyzing the correlation of this interaction after incubation with the peptide in the presence of the peptide or with one or both of the interacting couples.

[0238] The analysis may include the detection of SUN domain-containing protein / SUN domain-containing protein interaction couples / complexes of SUN domain-containing proteins and SUN domain-containing protein interaction couples. These techniques are well known to those skilled in the art and include, for example, antibody / reporter gene methods (Western blotting, ELISA, immunohistochemistry / cytochemistry, etc.).

[0239] Peptides that inhibit a given interaction (e.g., the interaction between a protein containing a SUN domain and its interacting partner, such as a protein containing a KASH domain) are identified by observing a decrease / reduction in the level of the relevant substance of the interaction between the interacting partners, compared to the level observed in the absence of the peptide (or in the presence of a suitable control peptide known not to inhibit the interaction between the interacting partners), either in the presence of the peptide or after incubation of one or both of the interacting partners with the peptide. Appropriate analyses can be performed in vitro, for example using recombinant interacting partners or using cells expressing the interacting partners. Cells expressing the interacting partners can express them endogenously or by introducing nucleic acids into the cells. For such assays, one or both interacting partners and / or peptides can be labeled, or used in combination with a detectable entity, to detect and / or measure the level of the interaction.

[0240] The correlation between the interactions between two interacting partners can be, for example, the correlation of the complex formed by the interaction between the interacting partners, the functional properties of the complex formed by the interaction between the interacting partners, or the correlation of the downstream activities mediated by the complex formed by the interaction between the interacting partners.

[0241] Peptides that inhibit the interaction between proteins containing SUN domains (e.g., proteins containing SUN domains selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) and their interaction partners (e.g., proteins containing KASH domains, such as those selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), such as peptides that inhibit the formation of complexes containing proteins containing SUN domains and their interaction partners, and / or peptides that disrupt complexes containing proteins containing SUN domains and their interaction partners, can be observed by incubating the peptide in the presence of the peptide or with one or both of the interaction partners. Or a combination of the following conditions can be used to identify the levels observed compared to those observed in the absence of the peptide (or in the presence of a suitable control peptide known not to inhibit the interaction between interacting partners): (i) decreased levels of complexes containing both a protein with a SUN domain and an interacting partner containing a SUN domain; (ii) decreased levels of functional properties of complexes containing both a protein with a SUN domain and an interacting partner containing a SUN domain; (iii) decreased levels of correlation with downstream activities mediated by complexes containing both a protein with a SUN domain and an interacting partner containing a SUN domain; (iv) increased levels of free (i.e., unreintegrated) proteins containing SUN domains; and / or (v) increased levels of free (i.e., unreintegrated) interacting partners containing SUN domains.

[0242] According to this disclosure, the LINC complex inhibitory peptide can disrupt the normal subcellular localization of LINC complex proteins (e.g., proteins containing a SUN domain, or interacting partners of proteins containing a SUN domain).

[0243] In some embodiments, the LINC complex repressor peptide reduces the level / proportion of LINC complex proteins located on the nuclear envelope. In some embodiments, the LINC complex repressor peptide reduces the level / proportion of interacting partners of the LINC complex repressor peptide located on the nuclear envelope. In some embodiments, the LINC complex repressor peptide reduces the level / proportion of KASH domain-containing proteins associated with the outer nuclear membrane. In some embodiments, the LINC complex repressor peptide reduces the level / proportion of SUN domain-containing proteins associated with the inner nuclear membrane.

[0244] In some embodiments, the LINC complex repressor peptide increases the level / proportion of LINC complex proteins that are not limited to the nuclear envelope. In some embodiments, the LINC complex repressor peptide increases the level / proportion of KASH domain-containing proteins (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) that are independent of the outer nuclear membrane. In some embodiments, the LINC complex repressor peptide increases the level / proportion of SUN domain-containing proteins (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) that are independent of the inner nuclear membrane.

[0245] In some embodiments, the LINC complex repressor peptide increases the level / proportion of LINC complex proteins localized to the endoplasmic reticulum. In some embodiments, the LINC complex repressor peptide increases the level / proportion of KASH domain-containing proteins (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) localized to the endoplasmic reticulum. In some embodiments, the LINC complex repressor peptide increases the level / proportion of SUN domain-containing proteins (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) independent of the nuclear membrane.

[0246] Subcellular localization of LINC complex constituent proteins within cells can be analyzed using techniques known to those skilled in the art. These techniques include, for example, analysis via immunocytochemistry and reporter molecule-based methods. For instance, Boni et al., J. Cell Biology (2015) 209(5):705-720 and Smoyer et al., J. Cell Biology (2016) 215(4):575-590 describe reporter systems that allow imaging of proteins in the ER, INM, and ONM. Such methods can be used to analyze the levels / proportions of LINC complex constituent proteins in the nuclear envelope, inner nuclear membrane, and outer nuclear membrane.

[0247] LINC complex inhibitory peptides that disrupt the normal subcellular localization of LINC complex proteins can be identified using assays that include detecting the presence or proportion of the relevant protein at a given subcellular location, such as antibody / reporter molecule-based methods (Western blotting, ELISA, immunohistochemistry / cytochemistry, etc.). Subcellular localization can be analyzed, for example, by immunocytochemistry or by Western blotting of extracts prepared from different cell fractions, and can be performed using organelle markers and / or labeled proteins with known subcellular localization.

[0248] Assays may include expressing a putative LINC complex inhibitory peptide in cells (e.g., introducing a nucleic acid encoding the peptide into cells (e.g., via transfection / transduction, etc.)) and then comparing the subcellular localization of the relevant LINC complex protein in such cells with the subcellular localization observed in cells under appropriate control conditions (e.g., untransfected cells, cells transfected / transduced with an empty vector, or cells transfected / transduced with a nucleic acid encoding a peptide known not to affect the subcellular localization of the relevant LINC complex protein).

[0249] In some implementations, the ability of the peptide to inhibit the peptide as a LINC complex can be evaluated, which is essentially as described in Example 1 of WO 2023 / 101607 A2.

[0250] Nucleic acids for expressing a putative LINC complex repressive peptide can be introduced into cells (e.g., via transfection); these cells can then be evaluated to determine the subcellular localization of one or more LINC complex proteins (e.g., proteins containing the SUN domain (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO), and / or proteins containing the SUN domain (e.g., proteins containing the KASH domain, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP)) and their interacting partners. If, in a specific assay, the expression of a polypeptide is determined to reduce the level / proportion of LINC complex proteins localized to the nuclear envelope, or increase the level / proportion of LINC complex proteins not localized to the nuclear envelope (e.g., its expression is determined to reduce the level / proportion of KASH domain-containing proteins associated with the outer nuclear membrane, or increase the level / proportion of KASH domain-containing proteins not associated with the outer nuclear membrane, or increase the level / proportion of KASH domain-containing proteins localized to the endoplasmic reticulum), and such changes are relative to the levels / proportions observed in cells under appropriate control conditions (e.g., untransfected cells, cells transfected / transduced with an empty vector, or cells transfected / transduced with nucleic acids encoding polypeptides known not to affect the subcellular localization of the relevant LINC complex proteins), then the polypeptide can be identified as a LINC complex inhibitory polypeptide.

[0251] In some embodiments, the LINC complex according to this disclosure inhibits the cellular expression of peptides by: (i) reducing the level of interactions between LINC complex constituent proteins (e.g., interactions between proteins containing a SUN domain and proteins containing a KASH domain), (ii) reducing the level of the LINC complex (e.g., a LINC complex containing proteins with a SUN domain and proteins containing a KASH domain), (iii) reducing the level of indicators related to the function / activity of the LINC complex (e.g., a LINC complex containing proteins with a SUN domain and proteins containing a KASH domain), (iv) reducing the level / proportion of LINC complex proteins localized to the nuclear envelope (e.g., proteins containing a SUN domain or proteins containing a KASH domain), and / or (v) reducing the level of KASH domain-containing proteins localized to the nuclear envelope / outer nuclear membrane (e.g., from Nesprin-1, Nesprin-2, Nesprin- 3. The level / proportion of (selected from Nesprin-4, KASH5, and LRMP) is reduced to less than 1-fold / less than 100% compared to the level observed in a given assay without the LINC complex inhibiting peptide or under appropriate control conditions, for example, ≤0.99-fold / ≤99%, ≤0.95-fold / ≤95%, ≤0.9-fold / ≤90%, ≤0.85-fold / ≤85%, ≤0.8-fold / ≤80%, ≤0.75-fold / ≤75%, ≤0.7-fold / ≤80%. ≤70%, ≤0.65 times / ≤65%, ≤0.6 times / ≤60%, ≤0.55 times / ≤55%, ≤0.5 times / ≤50%, ≤0.45 times / ≤45%, ≤0.4 times / ≤40%, ≤0.35 times / ≤35%, ≤0.3 times / ≤30%, ≤0.25 times / ≤25%, ≤0.2 times / ≤20%, ≤0.15 times / ≤15%, ≤0.1 times / ≤10%, ≤0.05 times / ≤5%, or ≤0.01 times / ≤1%.

[0252] According to the preceding paragraph, the preferred reduction level is to reduce to less than 0.5 times / ≤50%, such as ≤0.4 times / ≤40%, ≤0.3 times / ≤30%, ≤0.2 times / ≤20%, ≤0.15 times / ≤15%, or ≤0.1 times / ≤10%.

[0253] In some embodiments, the LINC complex inhibits the cellular expression of peptides according to this disclosure by: (i) increasing the level / proportion of LINC complex proteins not localized to the nuclear envelope (e.g., proteins containing the SUN domain or the KASH domain), and / or (ii) increasing the level / proportion of LINC complex proteins localized to the endoplasmic reticulum (e.g., proteins containing the SUN domain or the KASH domain), compared to the level in a given assay without LINC complex inhibition. The level observed under the condition of peptide or appropriate control conditions increases by more than 1-fold, for example, ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, ≥10-fold, ≥50-fold, or ≥100-fold.

[0254] Certain functional properties of peptides can be evaluated in suitable in vivo models. For example, the in vivo therapeutic / preventive effects of peptides can be assessed in non-human animal models of the diseases / disorders described herein. In such assays, peptides can be delivered in the form of nucleic acids encoding proteins, for example using viral vectors with appropriate tropism for inhibiting LINC complexes, with the aim of obtaining therapeutic / preventive benefits.

[0255] Subject lifespan and survival can be assessed in this model by monitoring survival over time. Cardiac function and myocardial contractility can be assessed by measuring their correlation, and ejection fraction, fractional shortening, left ventricular posterior wall thickness, and / or left ventricular diameter can be measured by echocardiography / ultrasound.

[0256] It should be understood that it is preferable to assess the subject for one or more of the properties described in the preceding paragraph at a specific time point, such as after a period sufficient to observe the effect of peptide administration in the relevant model. For example, the subject may be assessed 7 days or more after administration of a viral vector encoding the putative LINC complex inhibitory peptide.

[0257] In some embodiments, the LINC complex inhibitory peptide according to this disclosure is administered to a subject with cardiomyopathy (e.g., with...). DSPMutation-associated cardiomyopathy (e.g., delivered via a viral vector encoding a peptide that inhibits the LINC complex) can improve the survival or lifespan of subjects and / or improve cardiac function, myocardial contractility, ejection fraction, fractional shortening, and / or left ventricular posterior wall thickness by more than 1-fold, such as ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, ≥10-fold, ≥50-fold, or ≥100-fold.

[0258] In some embodiments, administration of the LINC complex inhibitory peptide according to this disclosure to subjects with cardiomyopathy (e.g., cardiomyopathy associated with DSP mutations; e.g., delivery via a viral vector encoding the LINC complex inhibitory peptide) can reduce the subject's left ventricular diameter by less than 1-fold / less than 100%, for example, ≤0.99-fold / ≤99%, ≤0.95-fold / ≤95%, ≤0.9-fold / ≤90%, compared to levels observed in subjects not receiving such treatment or receiving an appropriate control peptide (e.g., a peptide known not to inhibit the LINC complex, e.g., delivered via the same viral vector). ≤0.85 times / ≤85%, ≤0.8 times / ≤80%, ≤0.75 times / ≤75%, ≤0.7 times / ≤70%, ≤0.65 times / ≤65%, ≤0.6 times / ≤60%, ≤0.55 times / ≤55%, ≤0.5 times / ≤50%, ≤0.45 times / ≤45%, ≤0.4 times / ≤40%, ≤0.35 times / ≤35%, ≤0.3 times / ≤30%, ≤0.25 times / ≤25%, ≤0.2 times / ≤20%, ≤0.15 times / ≤15%, ≤0.1 times / ≤10%, ≤0.05 times / ≤5%, or ≤0.01 times / ≤1%.

[0259] In some embodiments, the LINC complex according to this disclosure inhibits the cellular expression of peptides by: (i) reducing the level of interactions between LINC complex constituent proteins (e.g., interactions between proteins containing a SUN domain and proteins containing a KASH domain), (ii) reducing the level of the LINC complex (e.g., a LINC complex containing proteins with a SUN domain and proteins containing a KASH domain), (iii) reducing the level of indicators related to the function / activity of the LINC complex (e.g., a LINC complex containing proteins with a SUN domain and proteins containing a KASH domain), (iv) reducing the level / proportion of LINC complex proteins localized to the nuclear membrane (e.g., proteins containing a SUN domain or proteins containing a KASH domain), and / or (v) reducing the level of KASH domain-containing proteins localized to the nuclear envelope / outer nuclear membrane (e.g., from Nesprin-1, Nesprin-2, Nesprin- 3. The level / proportion of (selected from Nesprin-4, KASH5, and LRMP) decreased to less than 1-fold / less than 100% compared to the level observed in cells not expressing the LINC complex inhibitory peptide (e.g., similar cell types), for example ≤0.99-fold / ≤99%, ≤0.95-fold / ≤95%, ≤0.9-fold / ≤90%, ≤0.85-fold / ≤85%, ≤0.8-fold / ≤80%, ≤0.75-fold / ≤75%, ≤0.7-fold / ≤ One of the following: 70%, ≤0.65 times / ≤65%, ≤0.6 times / ≤60%, ≤0.55 times / ≤55%, ≤0.5 times / ≤50%, ≤0.45 times / ≤45%, ≤0.4 times / ≤40%, ≤0.35 times / ≤35%, ≤0.3 times / ≤30%, ≤0.25 times / ≤25%, ≤0.2 times / ≤20%, ≤0.15 times / ≤15%, ≤0.1 times / ≤10%, ≤0.05 times / ≤5%, or ≤0.01 times / ≤1%.

[0260] In some embodiments, the cellular expression of the LINC complex inhibitory peptide according to this disclosure is increased by: (i) increasing the level / proportion of LINC complex proteins (e.g., proteins containing a SUN domain or a KASH domain) not localized to the nuclear envelope, and / or (ii) increasing the level / proportion of LINC complex proteins (e.g., proteins containing a SUN domain or a KASH domain) localized to the endoplasmic reticulum, compared to cells not expressing the LINC complex inhibitory peptide. The level observed in cells (e.g., cells of the same type) increases by more than 1-fold, such as ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, ≥10-fold, ≥50-fold, or ≥100-fold.

[0261] In some embodiments, administering the LINC complex inhibitory peptide according to this disclosure to subjects with cardiomyopathy (e.g., cardiomyopathy associated with DSP mutations; delivered, for example, via a viral vector encoding the LINC complex inhibitory peptide) improves the subject's survival or lifespan, and / or improves cardiac function, myocardial contractility, ejection fraction, fractional shortening, and / or left ventricular posterior wall thickness by more than 1-fold compared to levels observed without administration, such as ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, ≥10-fold, ≥50-fold, or ≥100-fold.

[0262] In some embodiments, administration of the LINC complex inhibitory peptide according to this disclosure to a subject with cardiomyopathy (e.g., cardiomyopathy associated with DSP mutations; e.g., delivery via a viral vector encoding the LINC complex inhibitory peptide) reduces the subject's left ventricular diameter by less than 1-fold / less than 100% compared to the level observed without administration, for example ≤0.99-fold / ≤99%, ≤0.95-fold / ≤95%, ≤0.9-fold / ≤90%, ≤0.85-fold / ≤85%, ≤0.8-fold / ≤80%, ≤0 0.75 times / ≤75%, ≤0.7 times / ≤70%, ≤0.65 times / ≤65%, ≤0.6 times / ≤60%, ≤0.55 times / ≤55%, ≤0.5 times / ≤50%, ≤0.45 times / ≤45%, ≤0.4 times / ≤40%, ≤0.35 times / ≤35%, ≤0.3 times / ≤30%, ≤0.25 times / ≤25%, ≤0.2 times / ≤20%, ≤0.15 times / ≤15%, ≤0.1 times / ≤10%, ≤0.05 times / ≤5%, or ≤0.01 times / ≤1%.

[0263] The LINC complex inhibitory peptide according to this disclosure may be based on a protein containing a SUN domain, such as selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO. In some embodiments, the LINC complex inhibitory peptide according to this disclosure comprises an inhibitory region based on a protein containing a SUN domain (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO).

[0264] The "inhibition region" of a LINC complex-inhibiting polypeptide refers to a region within the polypeptide that achieves LINC complex inhibition. According to this disclosure, the inhibition region typically has a high sequence identity with a portion of the amino acid sequence of a protein containing a SUN domain. The inhibition region of a LINC complex-inhibiting polypeptide according to this disclosure can be a region where the LINC complex-inhibiting polypeptide binds to an interaction partner of a protein containing a SUN domain (e.g., a protein containing a KASH domain, such as one or more of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In aspects and embodiments of this disclosure, the inhibition region of the LINC complex-inhibiting polypeptide comprises an α3 helix corresponding to the CC2 region of a protein containing a SUN domain and the amino acid sequence of the SUN domain.

[0265] The LINC complex inhibitory polypeptide disclosed herein may comprise or consist substantially of an amino acid sequence that has a high sequence identity (e.g., at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with all or part of the amino acid sequence of a protein containing a SUN domain (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO). The LINC complex inhibitory polypeptide disclosed herein may include an inhibitory region comprising or consisting of an amino acid sequence having high sequence identity (e.g., at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with all or part of the amino acid sequence of a protein containing a SUN domain (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO).

[0266] Such LINC complex repressive peptides preferably (i) retain the ability of the SUN-domain-based protein on which they are based to bind to proteins containing KASH domains (e.g., one or more of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), but (ii) lack or exhibit a reduced level of one or more other properties of the SUN-domain-based protein on which they are based. In a preferred embodiment, compared to the SUN-domain-based protein on which they are based, the LINC complex repressive peptide lacks or exhibits a reduced ability to bind to laminins and / or chromatin-binding proteins, and / or lacks or exhibits a reduced ability to bind to (e.g., to) the nuclear membrane.

[0267] For example, according to this disclosure, the LINC complex inhibitory peptide / its inhibitory region may contain the amino acid sequence required to bind to a protein containing a KASH domain (e.g., one or more of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), and may lack the amino acid sequence required for one or more other properties of the protein on which it is based, containing a SUN domain (e.g., the amino acid sequence required to bind to laminins and / or chromatin-binding proteins, and / or the amino acid sequence required to bind to the nuclear membrane).

[0268] As used herein, a reference polypeptide / amino acid sequence / domain consisting “substantially” of an amino acid sequence (i) consists of a reference amino acid sequence, or (ii) contains a reference amino acid sequence, wherein the reference amino acid sequence constitutes at least 80% of the polypeptide / amino acid sequence (e.g., ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%). It should be understood that a polypeptide or an amino acid sequence consisting “substantially” of a reference amino acid sequence may contain the reference amino acid sequence and an additional amino acid at one or both of the N-terminus and C-terminus of the reference amino acid sequence, provided that the additional amino acid constitutes ≤20% of the polypeptide / amino acid sequence.

[0269] For example, a polypeptide consisting of the amino acid sequence of SEQ ID NO:74 is essentially composed of the amino acid sequence of SEQ ID NO:44. SEQ ID NO:74 contains the amino acid sequence of SEQ ID NO:44 (i.e., positions 1 to 197 of SEQ ID NO:74), and the amino acid sequence of SEQ ID NO:44 accounts for approximately 98% of the polypeptide (i.e., 197 / 201 residues). SEQ ID NO:74 contains the KDEL lingering motif (SEQ ID NO:77) at the C-terminus of the amino acid sequence of SEQ ID NO:44.

[0270] It should be understood that, in some embodiments, the LINC complex inhibitory peptides of this disclosure are substantially composed of a protein-binding fragment containing a KASH domain of a protein containing a SUN domain. It should be understood that, in some embodiments, the inhibitory region of the LINC complex inhibitory peptides according to this disclosure is composed of a protein-binding fragment containing a KASH domain of a protein containing a SUN domain. Such LINC complex inhibitory peptides may be referred to as “decoys,” “dominant-negative,” or “mimicking” forms of the SUN-domain-containing proteins on which they are based. That is, in aspects and embodiments of this disclosure, the LINC complex inhibitory peptide may be a peptide containing a dominant-negative SUN domain.

[0271] The LINC complex inhibitory peptides according to this disclosure preferably exhibit competitive inhibition of the interaction between proteins containing the SUN domain (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) and proteins containing the KASH domain (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). The decoy / dominant / negative / mimicking LINC complex inhibitory peptides preferably bind to proteins containing the KASH domain (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), occupying sites required for interaction with endogenous SUN domain-containing proteins, thereby preventing these proteins from binding to endogenous SUN domain-containing proteins.

[0272] Such LINC complex inhibitory peptides may inhibit LINC complex formation and / or disrupt existing LINC complexes by inhibiting the assembly and / or replacement of endogenous interacting partners. Decoy / dominant / negative / mimic LINC complex inhibitory peptides form nonfunctional LINC complexes, or LINC complexes with reduced functional levels compared to those formed by wild-type, endogenous SUN-domain-containing, and KASH-domain-containing proteins.

[0273] In aspects and embodiments according to this disclosure, the LINC complex repressive peptide comprises an α3 helix corresponding to the CC2 region of a protein containing a SUN domain and an amino acid sequence of the SUN domain. It should be understood that the amino acid sequence of the LINC complex repressive peptide described herein (corresponding to one or more regions of a protein containing a SUN domain) may be included within the repressive region of the LINC complex repressive peptide.

[0274] As used herein, the amino acid sequence “corresponding” to a specific region of a reference peptide or amino acid sequence has at least 60% sequence identity with the amino acid sequence of the peptide / amino acid sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The amino acid sequence “corresponding” to a specific region of a reference peptide or amino acid sequence can be identified by sequence alignment of the test sequence with the reference sequence, for example using sequence alignment software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960). For example, the amino acid sequence 522-717 of human SUN2 corresponds to the amino acid sequence 616-812 of human SUN1.

[0275] In some embodiments, the LINC complex inhibitory peptide according to this disclosure is substantially composed of a human amino acid sequence. In some embodiments, the inhibitory region of the LINC complex inhibitory peptide according to this disclosure is substantially composed of a human amino acid sequence. As used herein, "human amino acid sequence" refers to the amino acid sequence encoded by the nucleic acid of the human genome. That is, in some embodiments, the LINC complex inhibitory peptide or the inhibitory region of the LINC complex inhibitory peptide is substantially composed of an amino acid sequence having 100% amino acid sequence identity with the amino acid sequence encoded by the human subject's genome. It should be understood that in some embodiments, the amino acid sequence encoded by the human subject's genome is the amino acid sequence of a protein containing a human SUN domain (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO; e.g., SUN1 or SUN2). This embodiment of the LINC complex inhibitory peptide is particularly suitable for applications intended for administration to human subjects, such as therapeutic / preventive interventions according to this disclosure.

[0276] In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α3 helix of the CC2 region of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α3 helix of the CC2 region of human SUN1. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α3 helix of the CC2 region of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:22.

[0277] In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the SUN domain of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the SUN domain of human SUN1. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the SUN domain of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:8. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:18.

[0278] In some embodiments, the LINC complex repressive peptide comprises an α3 helix corresponding to the CC2 region of a protein containing a SUN domain and an amino acid sequence of the SUN domain. In some embodiments, the LINC complex repressive peptide comprises an α3 helix corresponding to the CC2 region of human SUN1 and an amino acid sequence of the SUN domain. In some embodiments, the LINC complex repressive peptide comprises a repressive region comprising an α3 helix corresponding to the CC2 region of human SUN2 and an amino acid sequence of the SUN domain. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:44. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:46.

[0279] In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α2 helix of the CC2 region of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α2 helix of the CC2 region of human SUN1. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α2 helix of the CC2 region of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:21.

[0280] In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α1 helix of the CC2 region of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α1 helix of the CC2 region of human SUN1. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the α1 helix of the CC2 region of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:20.

[0281] In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the CC1 region of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the CC1 region of human SUN1. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the CC1 region of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 9. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 19.

[0282] In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to the CC1 and CC2 regions of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to the CC1 and CC2 regions of human SUN1. In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to the CC1 and CC2 regions of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 88. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:91.

[0283] In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the CC2 region of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the CC2 region of human SUN1. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence corresponding to the CC2 region of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:89. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:92.

[0284] In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to the α2 and α3 helices of the CC2 region of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to the α2 and α3 helices of the CC2 region of human SUN1. In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to the α2 and α3 helices of the CC2 region of human SUN2. In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:90. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:93.

[0285] In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to the partially smart coil-and-coil 2, CC1, and CC2 regions of a protein containing a SUN domain. In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to positions 483 to 632 of human SUN1 (according to SEQ ID NO:1). In some embodiments, the LINC complex repressive peptide comprises amino acid sequences corresponding to positions 388 to 538 of human SUN2 (according to SEQ ID NO:13). In some embodiments, the LINC complex repressive peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:97. In some embodiments, the LINC complex inhibitory peptide comprises an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:98.

[0286] In some embodiments, the LINC complex inhibits the polypeptide or its inhibiting region, comprising or substantially consisting of an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 94, 95, 101, 102, 103, 104, 105, 106, 107, 108, or 109 (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0287] In some embodiments, the LINC complex inhibitory polypeptide according to this disclosure includes an inhibitory region that is substantially composed of an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 43, 66, 94, 65, 63, 64, or 67.

[0288] In some embodiments, the LINC complex inhibitory polypeptide according to this disclosure comprises an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 99, 72, 95, 71, 69, 70 or 73, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or is substantially composed of said amino acid sequence.

[0289] The LINC complex inhibitory peptide according to this disclosure may contain one or more additional amino acids or amino acid sequences. That is, in addition to containing the inhibitory region of the peptide (i.e., the region containing the amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a protein containing a SUN domain), the LINC complex inhibitory peptide may also contain one or more amino acids or amino acid sequences.

[0290] According to various aspects and embodiments of this disclosure, LINC complex-inhibiting peptides may include sequences that prevent the peptide from being secreted from cells expressing the peptide. In some embodiments, the peptide includes an endoplasmic reticulum (ER) retention motif. According to this disclosure, such sequences may be positioned at the C-terminus of the inhibitory region of the LINC complex-inhibiting peptide. In some embodiments, a sequence (e.g., an ER retention motif) for preventing the peptide from being secreted from cells expressing the peptide is provided at the C-terminus of the peptide's amino acid sequence. In some embodiments, the sequence for preventing the peptide from being secreted from cells expressing the peptide (e.g., an ER retention motif) does not follow any other amino acid at the C-terminus of the peptide.

[0291] Endoplasmic reticulum (ER) retention sequences are known in the art. In some embodiments, the ER retention motif is a KDEL sequence. In some embodiments, the ER retention motif is a KDEL motif or a variant thereof, which effectively retains the protein containing the motif at the C-terminus of the ER. KDEL variants may contain or consist of the following amino acid sequence conforming to the prosite motif: [K / R / H / Q / S / A]-[D / E / N / Q]-EL (e.g., Hulo et al., 2006, ). Nucleic acids research 34: As described in D227-D230), or Raykhel et al., 2007 ( J. Cell Biol The variant described in . 179(6):1193-1204) proposed an extended definition of the prosite motif. Raykhel et al. demonstrated endoplasmic reticulum retention with variants in which the 4th position (i.e., K position) contains F or WY as a substitute for KRHQSA, the 3rd position (i.e., D position) residues extend far beyond DENQ, the 1st position (L position) is F or M, and the 2nd position (i.e., E position) is D.

[0292] For example, variants of the KDEL motif can be, for example, one of CDEL, KCEL, or HVEL, as proposed by Raykhel et al. Therefore, in some aspects disclosed herein, the endoplasmic reticulum (ER) retention motif is KDEL or a variant thereof exhibiting ER retention activity.

[0293] In some embodiments, the ER retention motif comprises the amino acid sequence of SEQ ID NO: 77, or a variant comprising one or more (e.g., 1 or 2) substitutions for the amino acid sequence of SEQ ID NO: 77, or is composed of said amino acid sequence or variant.

[0294] In some embodiments, the LINC complex repressive polypeptide may include a signal peptide. According to this disclosure, the signal peptide may be provided at the N-terminus of the repressive region of the LINC complex repressive polypeptide. In some embodiments, the signal peptide is positioned at the N-terminus of the polypeptide's amino acid sequence. In some embodiments, the signal peptide is not preceded by any other amino acid at the N-terminus of the polypeptide. The signal peptide typically consists of a sequence of 5-30 hydrophobic amino acids forming a single α-helix. Secretory proteins and proteins expressed on the cell surface typically contain signal peptides. The signal peptide may be present at the N-terminus of a peptide / polypeptide or in newly synthesized peptides / polypeptides. The signal peptide is typically removed by cleavage and is therefore not included in mature peptides / polypeptides.

[0295] Many protein signal peptides are known and documented in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resources, Protein Databases, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176). In some embodiments, the N-terminal signal sequence is derived from secreted proteins or type I transmembrane proteins. In some embodiments, the secretory protein or type I transmembrane protein is selected from: human serum albumin, proinsulin, transferrin receptor, EGF receptor, proopiotropic melanocorticotropic hormone, carboxypeptidase, complement protein, fibrinogen, cytokines, chemokines, fibrinogen, pancreatic digestive enzymes (e.g., proteases, amylases, or lipases) or endoplasmic reticulum lumen proteins (e.g., protein disulfide isomerase or GRP94). In some embodiments, the N-terminal signal peptide is derived from human serum albumin.

[0296] In some embodiments, the signal peptide includes a signal peptidase cleavage site. The signal peptidase cleavage site provides for the removal of the signal peptide from the mature polypeptide. In some embodiments, the LINC complex-inhibiting polypeptide according to this disclosure includes a signal peptidase cleavage site derived from a secretory protein or a type I transmembrane protein, such as the secretory proteins or type I transmembrane proteins described above herein.

[0297] In some embodiments, the signal peptide comprises, or is composed of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 76. In some embodiments, the signal peptide comprises, or is composed of, an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 100.

[0298] In some implementations, the LINC complex inhibitory peptide may include a detectable moiety, such as a fluorescent label, phosphorescent label, luminescent label, immunodetectable label (e.g., epitope tag), radiolabel, chemical label, nucleic acid label, or enzyme label. The LINC complex inhibitory peptide may be covalently or non-covalently labeled with the detectable moiety.

[0299] In some embodiments, the detectable portion is located at the N-terminus of the polypeptide (before or after the signal peptidase treats the polypeptide to remove any signal peptide). In some embodiments, the detectable portion is located at the C-terminus of the polypeptide, for example downstream (after) a sequence used to prevent the polypeptide from being secreted from the cell expressing the polypeptide (e.g., downstream of an ER retention motif).

[0300] In some embodiments, the detectable portion is or includes an epitope tag. In some embodiments, the epitope tag is selected from: hemagglutinin A (HA), ALFA, histidine (His; e.g., 6XHis), c-Myc, glutathione S-transferase (GST), green fluorescent protein (GFP), maltose-binding protein (MBP), FLAG, E, biotin, protein A, protein G, streptavidin, T7, thioredoxin, V5, or vesicular stomatitis virus glycoprotein (VSV-G) tags. In some embodiments, the detectable portion is or includes a portion having detectable activity, such as enzymatic activity against a given substrate. Examples of such portions include, for example, horseradish peroxidase (HRP) and luciferase portions.

[0301] In some embodiments, the LINC complex inhibitory peptide according to this disclosure has one of the following structures: N-terminus - [signal peptide] - [inhibition region containing the α3 helix corresponding to the CC2 region of a protein containing the SUN domain and the amino acid sequence of the SUN domain] - [sequence that prevents the polypeptide from being secreted from the cell expressing the polypeptide] - C-terminus N-terminus - [Repressive region containing the α3 helix corresponding to the CC2 region of a protein containing the SUN domain and the amino acid sequence of the SUN domain] - [Sequence that prevents the polypeptide from being secreted from the cell expressing the polypeptide] - C-terminus In some embodiments, the LINC complex-repressed polypeptide according to this disclosure includes one or more linker sequences between amino acid sequences. In some embodiments, the linker sequence is 1-2, 1-3, 1-4, 1-5, or 1-10 amino acids in length. In some embodiments, the linker sequence may be located at one or both ends of one or more of the following: a repressive region comprising an α3 helix corresponding to the CC2 region of a protein containing a SUN domain and an amino acid sequence of the SUN domain; a signal peptide; a sequence that prevents the polypeptide from being secreted from the cell expressing the polypeptide; and / or a detectable entity of the LINC complex-repressed polypeptide.

[0302] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev. (2013) 65(10):1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences linked by the linker sequence. Flexible linkers are known to those skilled in the art, and several have been identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences typically contain a high proportion of glycine and / or serine residues. In some embodiments, the linker sequence contains at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues.

[0303] In a preferred embodiment, the size of the LINC complex repressor peptide (i.e., the number of amino acids that make up the LINC complex repressor peptide) allows the LINC complex repressor peptide to be delivered as a gene therapy, i.e., delivered in the form of nucleic acid encoding the peptide.

[0304] In some embodiments, the LINC complex inhibits the size of the polypeptide such that the size of the polynucleotide encoding the polypeptide (i.e., the number of nucleotides constituting the polynucleotide) is within the packaging limitations of the vector used to deliver the polynucleotide. In some embodiments, the LINC complex inhibits the size of the polypeptide such that the size of the polynucleotide encoding the polypeptide is within the packaging limitations of the vector described herein. In some embodiments, the LINC complex inhibits the size of the polypeptide such that the size of the polynucleotide encoding the polypeptide is within the packaging limitations of an adeno-associated virus (AAV) vector, such as the AAV vector described herein. In some embodiments, the LINC complex inhibits the size of the polypeptide such that the size of the polynucleotide encoding the polypeptide is within the packaging limitations of an scAAV vector.

[0305] In some embodiments, the LINC complex inhibitory peptide consists of an amino acid sequence containing fewer than 510 amino acids. In some embodiments, the LINC complex inhibitory peptide consists of an amino acid sequence containing fewer than 457 amino acids.

[0306] In some embodiments, the LINC complex inhibitory peptide consists of an amino acid sequence of less than 600 amino acids, such as one of ≤550, ≤500, ≤450, ≤400, ≤350, ≤340, ≤330, ≤320, ≤310, ≤300, ≤290, ≤280, ≤270, ≤260, ≤250, ≤240, ≤230, ≤220 or ≤210 amino acids.

[0307] The LINC complex inhibitory peptides according to this disclosure can be prepared according to peptide production methods known to those skilled in the art. The peptides can be prepared by chemical synthesis, such as liquid-phase or solid-phase synthesis. For example, peptides / peptides can be synthesized using methods described, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which are incorporated herein by reference in their entirety. Alternatively, antigen-binding molecules and peptides can be generated by recombinant expression. Molecular biology techniques suitable for recombinant peptide production are well known in the art, such as those listed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Press, 2012, and Nat Methods. (2008); 5(2):135-146, which are incorporated herein by reference in their entirety. The method for recombinantly generating antigen-binding molecules is also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451–3461, which are hereby incorporated in their entirety by reference.

[0308] cardiomyopathy Various aspects and embodiments of this disclosure relate to the treatment and prevention of cardiomyopathy. Experimental examples of this disclosure demonstrate that inhibition of the LINC complex improves the pathology of a mouse model of cardiomyopathy.

[0309] In this article, "cardiomyopathy" refers to a pathology affecting the structure (e.g., size, shape) and / or function of myocardial tissue (i.e., the heart muscle), characterized by a decline in cardiac function, i.e., a decrease in the heart's pumping capacity. For example, cardiomyopathy was reviewed by Ciarambino et al., Int J Mol Sci. (2021) 22(14): 7722, which is incorporated herein by reference in its entirety. The underlying causes of cardiomyopathy are diverse, including cardiovascular disease, genetic variations, infections, autoimmune diseases, and adverse drug reactions.

[0310] Cardiomyopathy can be broadly classified into five categories: dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM), arrhythmogenic cardiomyopathy (ACM; also known as arrhythmogenic right ventricular cardiomyopathy (ARVC) and arrhythmogenic right ventricular dysplasia (AVRD)), and unclassified cardiomyopathy (i.e., cardiomyopathy other than DCM, HCM, RCM, and ACM).

[0311] Dilated cardiomyopathy (DCM) is characterized by unilateral or bilateral ventricular enlargement (i.e., dilation) (usually affecting the left ventricle), leading to thinning and stretching of the ventricular walls. Thinner ventricular walls are less capable of contracting with sufficient force, resulting in impaired cardiac function. Patients with DCM may experience heart failure, with the most common symptoms being arrhythmias (ventricular / atrial) and sudden cardiac death. The causes of DCM are often hereditary, but it is also associated with myocarditis (e.g., related to viral infections), coronary artery disease, infiltrative diseases, pregnancy (perinatal cardiomyopathy), hypertension, connective tissue disorders, drug abuse, and doxorubicin treatment.

[0312] TTN Genetic variations (encoding the sarcomere myosin (identified by UniProt Q8WZ42), which connects actin and myosin) account for approximately 25% of dilated cardiomyopathy cases, particularly variations that cause myosin truncation, which are believed to be the cause of approximately 15–20% of dilated cardiomyopathy cases (Schaefer et al., Nat Genet. (2017) 49(1): 46–53). LMNA Gene mutations (the gene encodes lamins A and C) account for approximately 5% of dilated cardiomyopathy cases. PLN Mutations (encoding phosphoproteins) and FLNC Mutations in (encoding filamentin C) each account for 1-5% of dilated cardiomyopathy. Other genes with causal relationships to dilated cardiomyopathy include... DSP (encoding desmosome plaque protein) DMD (encoding dystrophin) ACTC (encoding cardiac actin) DES (encoding desmin) MYH7 (The cardiac subtype encoding the β-myosin heavy chain) TNNT2 (encoding troponin T) TNNI3 (encoding troponin I) SGCD (encoding δ-sarcosin) and SCN5A (Encoding V-type sodium channels).

[0313] Ischemic cardiomyopathy is a common type of dilated cardiomyopathy and a frequent cause of systolic heart failure. Ischemic cardiomyopathy (ICM) is a term referring to myocardial damage caused by ischemia (reduced blood supply to the myocardium), resulting in a decreased ability of the heart to pump blood normally. Ischemic cardiomyopathy is associated with reduced blood supply to the myocardium, caused by narrowing of the coronary arteries, such as due to coronary artery disease or a heart attack (called myocardial infarction). In patients with ischemic cardiomyopathy, the density and stability of microtubules are significantly increased (Chen C. et al., Nature Medicine (2018), 24, 1225–1233, which is incorporated herein by reference in its entirety).

[0314] Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the cardiac chamber walls (≥15 mm), most commonly the left ventricular wall. This thickening reduces the size of the cardiac chambers, decreasing the amount of blood they can hold and thus impairing cardiac function. Thicker walls are also typically rigid and cannot fully relax, thus failing to fill with sufficient blood. The etiology of HCM is usually hereditary and is often associated with mutations in genes encoding sarcomere proteins. However, some HCM patients have no genetic link and are negative for sarcomere mutations (Warner E. et al. 2022).

[0315] MYBPC3 A gene mutation encoding the intermediate filament protein myosin-binding protein C is believed to cause up to 40% of hypertrophic cardiomyopathy cases, while 15-25% of hypertrophic cardiomyopathy patients have [a specific genetic component]. MYH7 Gene mutation. TNNT2 Mutations account for 5-10% of cases, while TNNI3 Mutations in this gene are present in 4-8% of cases. Other genes whose mutations have been shown to be causally associated with hypertrophic cardiomyopathy include... MYL2 and MYL3 (encoding myosin light chain) TPM1 (encoding α-tropomyosin) and ACTC1 (Encodes α-cardiac actin).

[0316] A hallmark of hypertrophic cardiomyopathy is the upregulation of protein synthesis and the growth of cytoskeletal networks, including tubulin (“microtubule network densification”) and MAP4 (Chinnakkannu P. et al., J Biol Chem. 2010;285:21837–21848. doi: 10.1074 / jbc.M110.120709). MAP4 is a key microtubule-binding protein that promotes microtubule network stability and is upregulated in hypertrophic cardiomyocytes. This leads to the formation of a dense, stable microtubule network, interfering with cardiomyocyte contraction and microtubule-based transport.

[0317] In hypertrophic myocardium under stress overload, inducing microtubule depolymerization to reverse MTN density has been effective in reversing systolic dysfunction in animal models (Warner E. et al., 2022).

[0318] Restrictive cardiomyopathy is characterized by stiffening of the ventricular walls, leading to diastolic dysfunction, elevated end-diastolic pressure, and atrial dilation. The stiffened ventricles cannot dilate effectively to fill with blood, thus impairing cardiac function. Restrictive cardiomyopathy is relatively rare and can be idiopathic or caused by diseases / conditions affecting the heart.

[0319] Diseases / conditions that can cause restrictive cardiomyopathy include amyloidosis, sarcoidosis, primary hyperoxaluria, Fabry disease, Gaucher disease, hereditary hemochromatosis, glycogen storage diseases, type I mucopolysaccharidosis (Heller syndrome), type II mucopolysaccharidosis (Hunter syndrome), Niemann-Pick disease, diabetes, scleroderma, myofibril myopathy, pseudoxanthoma elastica, sarcopenic dysplasia, Werner syndrome, carcinoid heart disease, endocardial myocardial fibrosis, hypereosinophilic syndrome, cancer (e.g., metastatic cancer), chronic eosinophilic leukemia, and endocardial fibroelastosis. Restrictive cardiomyopathy can also be a side effect of certain medications (including anthracyclines, serotonin, methylergonovine, ergotamine, mercury, and busulfan), as well as radiation therapy.

[0320] Amyloid mutations associated with restrictive cardiomyopathy include APOA1 Mutations and TTR Mutations such as V122I, I68L, L111M, T60A, S23N, P24S, W41L, V30M, and V20I. Mutations associated with Fabry disease and restrictive cardiomyopathy include... GLA Mutations, and those associated with Gaucher disease GBA Mutations are also associated with restrictive cardiomyopathy. Hereditary hemochromatosis mutations associated with restrictive cardiomyopathy include... HAMP , HFE , HFE2 , HJV , PNPLA3 , SLC40A1 and TfR2 Mutations associated with type I mucopolysaccharidosis and restrictive cardiomyopathy include... IDUA Mutations associated with type II mucopolysaccharidosis IDS Mutations are also associated with restrictive cardiomyopathy. Niemann-Pick disease mutations associated with restrictive cardiomyopathy include... NPC1, NPC2 and SMPD1 Mutations. Myofibrilogenic mutations associated with restrictive cardiomyopathy include BAG3, CRYAB DES, DNAJB6, FHL1, FLNC, LDB3 and MYOT Mutations in sarcoproteins associated with restrictive cardiomyopathy include... ACTC, β-MHC, TNNT2, TNNI3, TNNC1, DES, MYH, MYL3, CRYAB Mutations associated with elastic pseudoxanthoma in restrictive cardiomyopathy include... ABCC6 Mutations, and those associated with Werner syndrome WRN Mutations are also associated with restrictive cardiomyopathy. Endocardial fibroelastosis-related mutations associated with restrictive cardiomyopathy include... BMP5, BMP7 and TAZ mutation.

[0321] Arrhythmogenic cardiomyopathy (ACM) is another rare type of cardiomyopathy in which the muscular tissue of the ventricles is replaced by fatty and / or fibrous tissue, often leading to arrhythmias such as ventricular tachycardia. ACM was previously known as arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic right ventricular dysplasia (ARVD) because it was thought to affect only the right ventricle; however, further research has shown that the right ventricle, left ventricle, or both ventricles can be affected. The cause of arrhythmogenic cardiomyopathy is usually hereditary.

[0322] PKP2 Mutations in (encoding phenanthrene protein 2) are believed to account for 34%-74% of ACM cases, while DSG2 Mutations in (encoding desmosome core protein 2) account for 5-26% of ACM cases. DSP Mutations in these genes account for 2-39% of ACM cases. Other mutated genes that cause ACM include... DSC2 , JUP , TMEM43 , DES and PLN .

[0323] Diabetic cardiomyopathy is another form of cardiomyopathy in which diabetes leads to myocardial remodeling, loss of left ventricular (LV) pumping function, and ultimately heart failure. Diabetic cardiomyopathy results from diabetes-related glucose and lipid metabolism disorders that increase oxidative stress and activate multiple inflammatory pathways mediating cellular and extracellular damage, pathological cardiac remodeling, and diastolic and systolic dysfunction (Tan Y. et al., Nature Reviews Cardiology (2020) 17; 585-607). Abnormally increased microtubule stability contributes to cardiac dysfunction in diabetic cardiomyopathy (Warner E. et al. 2022, and Table A below). Microtubule acetylation is also involved in the pathogenesis of diabetic cardiomyopathy, where hyperglycemia leads to the formation of advanced glycation end products (AGEs). AGEs promote microtubule stability by inhibiting the SIRT2 / acetylated α-tubulin signaling pathway (Warner E. et al. 2022).

[0324] The most common form of the nucleotide sequence of a given gene can be referred to as the wild-type allele of that gene. The nucleotide sequence form of a given gene that contains a “gene variation” or “mutation” relative to the wild-type allele can be referred to as the “variant” or “mutated” allele of that gene. As used herein, a “gene variation” or “gene mutation” refers to an insertion, deletion, substitution, or a larger-scale translocation / rearrangement in the nucleotide sequence of a gene relative to a reference allele (e.g., the “unmutated” or “wild-type” allele) that does not contain that gene variation / mutation. It is understood that the nucleotide sequence of a variant / mutated allele of a given gene is different from the nucleotide sequence of the wild-type allele.

[0325] When a given disease / condition is described herein as being “associated” with a given gene variant / mutation or disease / condition, the gene variant / mutation / disease / condition may be positively correlated with the onset, development, or progression of the given disease / condition and / or the severity of one or more symptoms of the given disease / condition. The gene variant / mutation or disease / condition may be a risk factor for the development or progression of a particular disease / condition. In some embodiments, the disease / condition associated with a given gene variant / mutation or disease / condition may be caused by or aggravated by the gene variant / mutation / disease / condition. If a disease / condition is described herein as being “caused” by a certain gene variant / mutation or disease / condition, then that gene variant / mutation / disease / condition may be directly or indirectly related to the pathology of that disease / condition.

[0326] Genetic variations / mutations can lead to changes in the expression levels and / or activity of gene products (e.g., RNA and / or peptides) encoded by wild-type alleles of a related gene. Genetic variations / mutations can lead to decreased levels of gene products encoded by wild-type alleles of a related gene. Genetic variations / mutations can lead to increased levels of peptides encoded by disease-related alleles of a related gene. Genetic variations / mutations can lead to the production of truncated forms of peptides encoded by wild-type alleles of a related gene. Genetic variations / mutations can lead to the production of peptide forms encoded by wild-type alleles of a related gene that are misfolded and / or degraded. Genetic variations / mutations can lead to the production of peptides with altered activities relative to those encoded by wild-type alleles of a related gene, such as decreased or increased activity levels of the peptide encoded by the wild-type allele, and / or acquired activities not present in the peptide encoded by the wild-type allele.

[0327] In some implementations, the cardiomyopathy according to this disclosure is dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, or unclassified cardiomyopathy.

[0328] In some embodiments, the cardiomyopathy according to this disclosure is dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic cardiomyopathy, diabetic cardiomyopathy, ischemic cardiomyopathy, or unclassified cardiomyopathy.

[0329] In some embodiments, the cardiomyopathy of this disclosure is dilated cardiomyopathy. In some embodiments, cardiomyopathy refers to cardiomyopathy (e.g., dilated cardiomyopathy) associated with factors such as: genetic mutations, infectious diseases, viral infections, myocarditis, coronary artery disease, invasive disease, pregnancy, hypertension, connective tissue disease, drug abuse, or doxorubicin treatment. In some embodiments, cardiomyopathy is cardiomyopathy (e.g., dilated cardiomyopathy) associated with the following gene mutations: TTN , LMNA , PLN , FLNC , DSP , DMD , ACTC , DES , MYH7 , TNNT2 , TNNI3 , SGCD and SCN5A In some implementations, cardiomyopathy is defined as cardiomyopathy associated with mutations in the following genes (e.g., dilated cardiomyopathy): TTN , PLN , FLNC , DSP , DMD , ACTC , DES , MYH7 , TNNT2 , TNNI3 , SGCD and SCN5A In some implementation schemes, cardiomyopathy is associated with... DSP or DES Mutation-related cardiomyopathy (e.g., dilated cardiomyopathy). In some implementations, cardiomyopathy is associated with... DSP Mutation-related cardiomyopathy (e.g., dilated cardiomyopathy). In some implementations, cardiomyopathy is associated with... DMD Mutation-related cardiomyopathy (e.g., dilated cardiomyopathy).

[0330] In some embodiments, cardiomyopathy is cardiomyopathy associated with mutations in genes encoding sarcomeric proteins (e.g., dilated cardiomyopathy). As used herein, “sarcomeric protein” refers to the constituent polypeptides of sarcomeres. The constituent polypeptides of sarcomeres, i.e., sarcomeric proteins, include titin, troponin T, troponin I, troponin C1, cardiac myosin-binding C, α-tropomyosin, α-cardiac actin, myosin, and filamentin C. In some embodiments, cardiomyopathy is cardiomyopathy associated with mutations in genes selected from: TTN , TNNT2, TNNI3, TNNC1 MYBPC3, MYH7, TPM1, ACTC1, MYL2 , MYL3 and FLNC. In some implementations, cardiomyopathy is cardiomyopathy associated with gene mutations selected from the following: TTN , TNNT2, TNNI3, TNNC1, MYBPC3, MYH7, TPM1, ACTC1, MYL2 and MYL3. In some implementations, cardiomyopathy is cardiomyopathy associated with TTN mutations (e.g., dilated cardiomyopathy).

[0331] In some embodiments, the cardiomyopathy according to this disclosure is hypertrophic cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a gene mutation (e.g., hypertrophic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a gene mutation selected from: MYBPC3 , MYH7, TNNT2, TNNI3, MYL2 , MYL3 , TPM1 and ACTC1 .

[0332] In some embodiments, the cardiomyopathy according to this disclosure is restrictive cardiomyopathy. In some embodiments, cardiomyopathy refers to cardiomyopathy (e.g., restrictive cardiomyopathy) associated with the following factors: gene mutations, amyloidosis, sarcoidosis, primary hyperoxaluria, Fabry disease, Gaucher disease, hereditary hemochromatosis, glycogen storage disease, type I mucopolysaccharidosis (Heller syndrome), type II mucopolysaccharidosis (Hunter syndrome), Niemann-Pick disease, diabetes, scleroderma, myofibril myopathy, pseudoxanthoma elastica, sarcopenic dysplasia, Werner syndrome, carcinoid heart disease, endocardial myocardial fibrosis, hypereosinophilic syndrome, cancer (e.g., metastatic cancer), chronic eosinophilic leukemia, endocardial fibroelastosis, chemotherapy, radiotherapy, or treatment with anthracyclines, serotonin, methylergonovine, ergotamine, mercury, or busulfan. In some embodiments, cardiomyopathy is cardiomyopathy (e.g., restrictive cardiomyopathy) associated with the following gene mutations: APOA1 , TTR (e.g., V122I, I68L, L111M, T60A, S23N, P24S, W41L, V30M, or V20I) GLA , GBAHAMP , HFE , HFE2 , HJV , PNPLA3 , SLC40A1 , TfR2 , IDUA , IDS , NPC1 , NPC2 , SMPD1 , BAG3 , CRYAB , DNAJB6 , FHL1 , FLNC , LDB3 , MYOT , ACTC , β-MHC , TNNT2 , TNNI3 , TNNC1 , DES , MYH , MYL3 , CRYAB , ABCC6 , WRN , BMP5 , BMP7 and TAZ. In some embodiments, the cardiomyopathy is cardiomyopathy associated with DES mutations (e.g., restrictive cardiomyopathy). In some embodiments, the cardiomyopathy is cardiomyopathy associated with FLNC mutations (e.g., restrictive cardiomyopathy). In some embodiments, the cardiomyopathy is associated with FLNC mutations... irrelevant Cardiomyopathy (e.g., restrictive cardiomyopathy).

[0333] In some embodiments, the cardiomyopathy according to this disclosure is arrhythmogenic cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a gene mutation (e.g., arrhythmogenic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a gene mutation (e.g., arrhythmogenic cardiomyopathy): PKP2 , DSG2 , DSP , DSC2 , JUP , TMEM43 , DES and PLN In some implementations, cardiomyopathy is cardiomyopathy associated with gene mutations selected from the following (e.g., arrhythmogenic cardiomyopathy): PKP2 , DSG2 , DES , DSC2 , JUP and DES In some implementation schemes, cardiomyopathy is associated with... DSPMutation-related cardiomyopathy (e.g., arrhythmogenic cardiomyopathy). In some implementations, cardiomyopathy is associated with... PKP2 Mutation-related cardiomyopathy (e.g., arrhythmogenic cardiomyopathy).

[0334] In some implementations, cardiomyopathy is cardiomyopathy associated with gene mutations selected from the following (e.g., dilated cardiomyopathy): DSP , TTN, FLNC, and PKP2. In some implementations, cardiomyopathy is cardiomyopathy associated with gene mutations selected from the following (e.g., dilated cardiomyopathy): DSP , TTN and PKP2.

[0335] In some implementation schemes, cardiomyopathy no Cardiomyopathy associated with laminopathy. Cardiomyopathy associated with laminopathy is a type of cardiomyopathy caused by mutations in the gene encoding lamin. In some implementations, cardiomyopathy refers to any cardiomyopathy other than that associated with laminopathy. As used herein, “laminopathy” refers to a disease / pathological condition associated with a mutation in the gene encoding lamin. Laminopathy has been reviewed, for example, in Burke and Stewart, Nat Rev Mol Cell Biol. (2013) 14(1):13-24 and Hah and Kim, Cells (2019) 8(3): 231, which are incorporated herein by reference in their entirety. Laminopathy is typically associated with nuclear-level load-bearing defects that may reduce the cell’s tolerance to physical forces.

[0336] Genes encoding laminin include LMNA (encoding lamins A and C) and LMNB1 , LMNB2 (Encoding laminins B1 and B2). In some implementations, cardiomyopathy no and LMNA Cardiomyopathy associated with gene mutations. In some implementations, cardiomyopathy is... Apart from and LMNA Cardiomyopathy other than mutation-related cardiomyopathy. In some implementations, cardiomyopathy no and LMNA , LMNB1 and LMNB2 Cardiomyopathy associated with any mutation in (i.e., no Depend on LMNA Cardiomyopathy caused by mutation no and LMNB1 Mutation-related cardiomyopathy, also no and LMNB2 (Mutation-related cardiomyopathy). In some implementations, cardiomyopathy Apart from and LMNA , LMNB1 and LMNB2 Apart from cardiomyopathy associated with any mutation in the blood.

[0337] In some implementation schemes, cardiomyopathy no Cardiomyopathy associated with nuclear envelope disease. Nuclear envelope disease is a disease / pathological condition associated with mutations in genes encoding nuclear envelope proteins (i.e., proteins contained in or directly / indirectly related to the ONM, perinuclear space, or INM). Nuclear envelope disease has been reviewed, for example, by Chi et al., Journal of Biomedical Science (2009) 16:96, which is incorporated herein by reference in its entirety. Nuclear envelope disease includes cardiomyopathy associated with nuclear envelope disease. LMNA , LMNB1 , LMNB2 , EMD , LAP2 , LBR , ZMPSTE24 , SYNE-1 and NUP62 Diseases / pathological conditions related to gene mutations. In some implementations, cardiomyopathy... no and LMNA , LMNB1 , LMNB2 , EMD , LAP2 , LBR , ZMPSTE24 , SYNE-1 and NUP62 Cardiomyopathy associated with any mutation in the blood.

[0338] In some embodiments, the cardiomyopathy according to this disclosure is diabetic cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with one or more of the following (e.g., diabetic cardiomyopathy): type 1 diabetes, type 2 diabetes, prediabetes, metabolic syndrome, pregnancy-related hyperglycemia (i.e., gestational diabetes), obesity, and hyperglycemia.

[0339] In some embodiments, the cardiomyopathy according to this disclosure is ischemic cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy (e.g., ischemic cardiomyopathy) associated with one or more of the following: coronary artery disease, myocardial infarction, diabetes, prediabetes, metabolic syndrome, pregnancy-related hyperglycemia (i.e., gestational diabetes), hyperglycemia, amyloidosis, hypertension, obesity, and hypercholesterolemia.

[0340] In some implementations, cardiomyopathy is cardiomyopathy that is not related to gene variations (such as those described herein) (e.g., dilated cardiomyopathy).

[0341] In some embodiments, the cardiomyopathy according to this disclosure is cardiomyopathy associated with desmosome defects / deficiencies or dysfunction.

[0342] In some embodiments, the cardiomyopathy according to this disclosure is a cardiomyopathy associated with mutations in genes encoding desmosome proteins. As used herein, "desmosome protein" refers to the constituent polypeptides of desmosomes. The constituent polypeptides of desmosomes include desmosome plaque proteins, desmin, desmosome core proteins (e.g., desmosome core proteins 1, 2, 3, 4), desmosome glial proteins (e.g., desmosome glial proteins 1, 2, 3), plaque proteins (e.g., plaque proteins 1, 2, 3), and plaque globin. In some embodiments, the gene encoding desmosome proteins is selected from... DSP , DES , DSG1 , DSG2 , DSG3 , DSG4 , DSC1 , DSC2 , DSC3 , PKP1 , PKP2 , PKP3 and JUP .

[0343] In some embodiments, the cardiomyopathy according to this disclosure is a cardiomyopathy associated with gene mutations encoding the constituent polypeptide of the desmosome-intermediate filament complex (DIFC).

[0344] Desmosomes (also known as focal adhesions) are specialized structures that mediate transverse cell adhesion between cells, particularly well-suited for tissues subjected to significant mechanical stress, such as myocardial tissue, bladder tissue, gastrointestinal mucosa, and epithelial tissue. The structure and function of desmosomes have been reviewed, for example, in Delva et al., Cold Spring Harb Perspect Biol. (2009) 1(2): a002543 and Johnson et al., Cold Spring Harb Perspect Med. (2014) 4(11): a015297, which are incorporated herein by reference in their entirety. Desmosomes consist of the desmosome-intermediate filament complex (DIFC), a network of cadherins, connexins, and intermediate filaments. Desmosomes possess an extracellular region called the desmoglea, composed of cadherins (e.g., desmoglobin and desmoglycin, which bind via heterologous interactions between their N-termini). At their C-termini, cadherins bind to plaqueglobin 1 and 2 and plaqueglobin in the outer dense plaque (ODP). Banflin 1 and 2, along with globin, are linked to internal dense plaques (IDPs) via desmosome plaques, which are composed of intermediate filaments.

[0345] In some embodiments, the cardiomyopathy according to this disclosure is a cardiomyopathy associated with gene mutations selected from: DSP , DES , DSG2 , DSC2 , PKP2 and JUP .

[0346] In some embodiments, the cardiomyopathy according to this disclosure is a cardiomyopathy associated with desmosome plaque protein deficiency / insufficiency or dysfunction.

[0347] In some embodiments, cardiomyopathy according to this disclosure is related to DSP Mutation-related cardiomyopathy. (and) DSP Mutation-associated cardiomyopathy is even considered to constitute a separate category of cardiomyopathy, namely the so-called "demosome-associated cardiomyopathy"—see Smith et al., Circulation (2020) 141(23):1872-1884.

[0348] Desmosome plaque protein (also known as DSP) is a protein identified by UniProt P15924. The structure and function of desmosome plaque protein are described in, for example, Yuan et al., Chin Med J (Engl). (2021) 134(15):1771-1779, Müller et al., Front. Cell Dev. Biol. (2021) 9:745670, and Mohammed and Chidgey, Journal of Structural Biology (2021) 213(3):107749, all of which are incorporated herein by reference in their entirety.

[0349] Desmosomes are important components of desmosomes, which form intercellular connections between adjacent cells. Desmosomes bind to intermediate filaments of the cytoskeleton via their C-termini (see, for example, Stappenbeck and Green, J Cell Biol. (1992) 116(5):1197-209 and Stappenbeck et al., J Cell Biol. (1993) 123(3):691-705), and this binding varies with cell type. In cardiomyocytes, desmosomes bind to desmosomes; in endothelial and epithelial cells, they bind to cytokeratin-type intermediate filaments; and in dendritic cells, they bind to vimentin intermediate filaments. At the N-terminus, desmosomes bind to armadillo family proteins (such as desmosomesin 1 and 2 and desmosome globin), which in turn bind to desmosome cadherins (particularly desmosome glialin and desmosome corein). Therefore, desmosome plaque protein acts as a linker protein, connecting the intermediate filament to the cadherin of the desmosome.

[0350] people DSP Gene mutations can lead to various cardiomyopathies, including dilated cardiomyopathy and arrhythmogenic cardiomyopathy. DSP This mutation is described, for example, in Mohammed and Chidgey, Journal of Structural Biology (2021) 213(3): 107749 (this article is incorporated here in its entirety by reference; see details). Figure 4e), and includes G2056R, D2757H, K2689T, R2834H, T2595I, R2541K, R2541S, R2639Q, R2366H, R2366C, D230N, S299R, N375I, S442F, I445V, N458Y, K470E, S507F, N287D, N287S, A206T, W207C, E422K, I533T, A556T, N661I, Y787C, R808C, R808H, R1255K, R1255T, L1348R, T1373A, L1654P, R1775I, R1838H, Q198P, D203N, R222L, D241N, E290K, I305F, A306T, R315C, R315P, M316V, A349P, Y350H, T356K, I368T, E384Q, K401N, N408K, R451G, R451H, I461V, C482Y, Y494F, P515L, L540P, T587N, N593S, M599R, R606W, E721K, Y895H, I870M, I870T, R907H, R908H, L933F, G939S, S987P, T1960P, Y1981C, L1995S, A2019S, A2019V, P2061S, H2062R, D2070N, V2107L, R2083C, K2103E, A2148T, T2177N, R2189Q, T2196I, I2263T, T2267S, A2294G, Q2295H, G2338R, E2343K, I2347V, D2579H, I2593S, T2664N, G2666D, A2674T, M2684V, M2707T, A2709T, R2759S, P2777H, I2797T, M2819L, G2844V, I2869V, K1094N, Y1169N, L1178R, L1178V, Y1188H, R1207K, T1217M, D1251N, D1258E, A1274P, K1288Q, T1319I, R1341H, E1357D, R1392Q, R1392W, E1407D, A1435P, R1458G, Y1512C, L1514P, L1535P, K1537C, K1537H, T1557M, K1581E, K1592R, M1601I, S1623C, V1639M, S1658F, R1666Q, R1666W, H1684R, E1721D, R1738Q, E1740K, E1833V, R1852H, R2366H, T2770C,G2832V, S2616L, G2647D, R1838H, T13I, E21K, V30M, R44Q, C81Y, Q90R, R105Q, E109K, M118K, I125F, C174F, and A566T. Associated with erythrokeratosis-cardiomyopathy syndrome. DSP Mutations include L622P, Q616P, and H618P. These are associated with cardiocutaneous syndromes such as Naxos disease and Cavallariar syndrome (characterized by dilated cardiomyopathy, hyperkeratosis, and woolly trichomes). DSP Mutations include I560F, T564I, L583P, S597L, E2193K, L2329P, Q2371K, G2375R, and A2655D.

[0351] In some embodiments, cardiomyopathy according to this disclosure is related to DSPCardiomyopathy associated with one or more of the following mutations: G2056R, D2757H, K2689T, R2834H, T2595I, R2541K, R2541S, R2639Q, R2366H, R2366C, D230N, S299R, N375I, S442F, I445V, N458Y, K470E, S507F, N287D, N287S, A206T, W207C, E422K, I533T, A556T, N661I, Y787C, R808C, R808H, R1255K, R1255T, L1348R, T1373A, L1654P, R1775I, R1838H , Q198P, D203N, R222L, D241N, E290K, I305F, A306T, R315C, R315P, M316V, A 349P, Y350H, T356K, I368T, E384Q, K401N, N408K, R451G, R451H, I461V, C48 2Y, Y494F, P515L, L540P, T587N, N593S, M599R, R606W, E721K, Y895H, I870M , I870T, R907H, R908H, L933F, G939S, S987P, T1960P, Y1981C, L1995S, A2019 S, A2019V, P2061S, H2062R, D2070N, V2107L, R2083C, K2103E, A2148T, T217 7N, R2189Q, T2196I, I2263T, T2267S, A2294G, Q2295H, G2338R, E2343K, I23 47V, D2579H, I2593S, T2664N, G2666D, A2674T, M2684V, M2707T, A2709T, R2 759S, P2777H, I2797T, M2819L, G2844V, I2869V, K1094N, Y1169N, L1178R, L1 178V, Y1188H, R1207K, T1217M, D1251N, D1258E, A1274P, K1288Q, T1319I, R 1341H, E1357D, R1392Q, R1392W, E1407D, A1435P, R1458G, Y1512C, L1514P, L 1535P, K1537C, K1537H, T1557M, K1581E, K1592R, M1601I, S1623C, V1639M, S1658F, R1666Q, R1666W, H1684R, E1721D, R1738Q, E1740K, E1833V, R1852H,R2366H, T2770C, G2832V, S2616L, G2647D, R1838H, T13I, E21K, V30M, R44Q, C81Y, Q90R, R105Q, E109K, M118K, I125F, C174F, A566T, L622P, Q616P, H618P, I560F, T564I, L583P, S597L, E2193K, L2329P, Q2371K, G2375R, or A2655D.

[0352] In some embodiments of various aspects of this disclosure, the cardiomyopathy according to this disclosure is a cardiomyopathy characterized by microtubule dysfunction.

[0353] Diseases / disorders characterized by microtubule dysfunction The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and disorders characterized by microtubule dysfunction. Experimental examples of this disclosure demonstrate that inhibition of the LINC complex improves pathology in mouse models of diseases characterized by microtubule network dysfunction.

[0354] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition in which microtubule dysfunction or deficiency / insufficiency is pathologically related, such as a disease / condition in which microtubule dysfunction or deficiency / insufficiency is positively correlated with the onset, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition. In some embodiments, microtubule dysfunction or deficiency / insufficiency may be a risk factor for the onset, development, or progression of the disease / condition.

[0355] As mentioned above, microtubule dysfunction is associated with the heart / cardiac disease (Warner E. et al., 2022, see Table A below). In some implementations, the disease / disorder characterized by microtubule dysfunction is cardiac disease.

[0356] Microtubule dysfunction is commonly associated with cardiomyopathy. In some implementations, the disease / disorder characterized by microtubule dysfunction is cardiomyopathy.

[0357] In some implementations, a disease or disorder characterized by microtubule dysfunction is characterized by cardiomyopathy. That is, in some implementations, cardiomyopathy is a symptom of a disease or disorder characterized by microtubule dysfunction.

[0358] In some implementations, cardiomyopathy is the cardiomyopathy described herein. In some implementations, cardiomyopathy is a non-hereditary cardiomyopathy.

[0359] Warner E. et al. (2022), Circulation Research. 2022;130:1723–1741, reviewed various diseases characterized by microtubule dysfunction, which are incorporated herein by reference in their entirety. For example, in patients with cardiomyopathies such as ischemic cardiomyopathy, hypertrophic cardiomyopathy, diabetic cardiomyopathy, and dilated cardiomyopathy, microtubule density and stability are dramatically increased. Muscular dystrophy cardiomyopathy is caused by… DMD Cardiomyopathy caused by mutations in (the protein encoding dystrophin) is also associated with microtubule dysfunction (see Table A below).

[0360] In some implementations, the disease / disorder characterized by microtubule dysfunction is ischemic cardiomyopathy, hypertrophic cardiomyopathy, diabetic cardiomyopathy, and dilated cardiomyopathy, or is characterized by ischemic cardiomyopathy, hypertrophic cardiomyopathy, diabetic cardiomyopathy, and dilated cardiomyopathy. In some implementations, the disease / disorder characterized by microtubule dysfunction is malnutrition cardiomyopathy or is characterized by malnutrition cardiomyopathy.

[0361] Heart failure occurs when the heart muscle stiffens and is unable to relax properly (diastolic dysfunction). In failing cardiomyocytes, increased microtubule network (MTN) stiffness leads to systolic dysfunction. This increase in MTN stiffness is typically due to enhanced interaction between detyrosine-treated microtubules and intermediate filaments at the Z-discs of the sarcomere (Warner E. et al., 2022). In other words, detyrosine-treated microtubules provide mechanical resistance that can impede the movement of contracting cardiomyocytes (Chen C. et al., Nature Medicine (2018) volume 24, 1225–1233).

[0362] Failed cardiomyocytes are often characterized by increased microtubule network density, which is associated with increased cardiomyocyte stiffness and impaired contractility (Chen C. et al., Nature Medicine (2018) volume 24, 1225–1233). In the hearts of patients with ischemic cardiomyopathy, hypertrophic cardiomyopathy, and dilated cardiomyopathy, increased MTN density is accompanied by a significant upregulation of total tubulin.

[0363] Compared with cardiomyocytes isolated from mice that underwent sham surgery, cardiomyocytes isolated from ischemic hearts showed significantly higher levels of detyrosine-derived α-tubulin, while this was not the case for other cell populations tested (e.g., immune cells, fibroblasts, and endothelial cells) (Yu X. et al. 2021 Nature: 594, 560–565, which is incorporated herein by reference in its entirety).

[0364] In some implementations, the disease / disorder characterized by microtubule dysfunction is heart failure. In some implementations, heart failure is associated with ischemic injury. That is, in some implementations, heart failure is ischemic heart failure.

[0365] According to reports, various models of heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) are characterized by microtubule dysfunction, as summarized in Table A below.

[0366] Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome in which patients present with signs and symptoms of heart failure due to high left ventricular (LV) filling pressure, even though the LV ejection fraction (LVEF; ≥50%) is normal or near normal (Harper A. et al., Clin Med (Lond). 2018; 18(Supplement 2): s24–s29). Heart failure with reduced ejection fraction (HFrEF) is heart failure characterized by reduced left ventricular ejection fraction (LVEF <40%). In some cases, hypertrophic cardiomyopathy can lead to either heart failure with preserved ejection fraction (HFpEF) or heart failure with reduced ejection fraction (HFrEF).

[0367] In some implementations, the disease / disorder characterized by microtubule dysfunction is heart failure with preserved ejection fraction (HFpEF). In some implementations, the disease / disorder characterized by microtubule dysfunction is heart failure with reduced ejection fraction (HFrEF). In some implementations, HFpEF and / or HFrEF are associated with hypertrophic cardiomyopathy.

[0368] Aortic stenosis is characterized by microtubule dysfunction, as described, for example, in Zile MR et al. J Am Coll Cardiol. 2001 Mar 15;37(4):1080-4 (see Table A below). In some implementations, the disease / disorder characterized by microtubule dysfunction is aortic stenosis.

[0369] Stones R. et al., J Mol Cell Cardiol. 2013; 56: 91–96, describe microtubule proliferation as a common response to pulmonary hypertension in right ventricular failure (see Table A below). Prins K. et al., J Am Heart Assoc. 2017;6(6):e006195, describe pulmonary hypertension (PAH) characterized by abnormal microtubule structures in right ventricular (RV) cardiomyocytes, which impair RV function (see Table A below). Drug-induced pulmonary hypertension (D-PAH) is a type of pulmonary hypertension (PH) characterized by severe small vessel loss and obstructive vascular disease leading to progressive right heart failure and death (Orcholski M et al., Am J Physiol Lung Cell Mol Physiol. 2018; 314(6): L967–L983).

[0370] In some implementations, the disease / disorder characterized by microtubule dysfunction is pulmonary hypertension (PAH). In some implementations, the disease / disorder characterized by microtubule dysfunction is pulmonary hypertension (PH). In some implementations, the disease / disorder characterized by microtubule dysfunction is drug-induced pulmonary hypertension or hypoxia-induced pulmonary hypertension (e.g., hypoxia-induced neonatal pulmonary hypertension).

[0371] Microtubule dysfunction is also described as a characteristic of right ventricular pressure overload and left ventricular pressure overload (summarized in Table A below). In some implementations, the disease / disorder characterized by microtubule dysfunction is right ventricular (RV) pressure overload. In some implementations, the disease / disorder characterized by microtubule dysfunction is left ventricular (LV) pressure overload.

[0372] Myocardial infarction (also known as a heart attack) is associated with increased phosphorylation of microtubule-associated protein 4 (MAP4) (see Table A below). In some implementations, a disease / disorder characterized by microtubule dysfunction is myocardial infarction, such as acute myocardial infarction.

[0373] As used in this article, “microtubule dysfunction” or “microtubule network dysfunction” refers to a state of impaired / abnormal / insufficient normal function of the microtubule cytoskeleton, resulting in subjects exhibiting abnormal function of the relevant tissue in specific tissues (e.g., cardiac tissue). Normal function and microtubule dysfunction of the microtubule network are reviewed, for example, by Warner E. et al. (2022).

[0374] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition characterized by abnormal tissue of microtubule networks (e.g., in cells), for example, compared to tissue observed when the disease / condition is not present (e.g., in healthy subjects or in equivalent non-pathological cells).

[0375] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by an increased density of microtubule networks (e.g., in cells such as cardiomyocytes), for example, compared to the density observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells). In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by an increased density of perinuclear microtubule networks (e.g., in cells such as cardiomyocytes), for example, compared to the density observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells).

[0376] As used herein, “microtubule density” refers to the number of microtubules as a function of the cross-sectional area of ​​the cell body. Microtubule density can be determined by immunostaining, as described herein and, for example, by Chen C. et al., Nature Medicine (2018), Vol. 24, 1225–1233, which is incorporated herein by reference in its entirety.

[0377] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition characterized by increased mechanical resistance of microtubule networks (e.g., in cells such as cardiomyocytes), for example, compared to the increased mechanical resistance observed in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells).

[0378] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by an increase in mechanical force transmitted to the cell nucleus / nuclear envelope (e.g., in cells such as cardiomyocytes), for example, compared to the mechanical force observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells).

[0379] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition characterized by impaired contractility / contractility of cardiac tissue or cardiomyocytes, for example, compared to the contractility / contractility of cardiac tissue or cardiomyocytes in the absence of disease / condition (e.g., in healthy subjects). Chen C. et al., Nature Medicine (2018) volume 24, 1225–1233 describes a method for measuring cellular contractility.

[0380] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by increased levels / expression of tubulin (e.g., in cells such as cardiomyocytes), compared to levels / expression in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells). In some embodiments, the disease / condition is characterized by increased levels / expression of α-tubulin. In some embodiments, the disease / condition is characterized by increased levels / expression of β-tubulin.

[0381] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by an increase in the level / proportion of detyrosine-treated tubulin (e.g., in cells such as cardiomyocytes), for example, compared to the level / expression in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells). The proportion of detyrosine-treated tubulin refers to the ratio of detyrosine-treated α-tubulin to total tubulin (e.g., in cells). "Total tubulin" may refer to the protein levels of α-tubulin and / or β-tubulin, which are major components of microtubules.

[0382] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by an increased level / ratio of acetylated tubulin (e.g., in cells such as cardiomyocytes), for example, compared to the level / expression in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells). In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by a decreased level / ratio of acetylated tubulin (e.g., in cells such as cardiomyocytes), for example, compared to the level / expression in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-pathological cells). The ratio of acetylated tubulin refers to the ratio of acetylated α-tubulin to total tubulin (e.g., in cells). "Total tubulin" may refer to the protein levels of α-tubulin and / or β-tubulin, which are major components of microtubules.

[0383] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition characterized by an increase in the density of detyrosinated microtubules (e.g., in cells such as cardiomyocytes), for example, compared to the detyrosinated microtubule populations when the disease / condition is not present (e.g., in healthy subjects or in equivalent non-pathological cells).

[0384] In some implementations, microtubule dysfunction refers to perinuclear microtubule dysfunction. That is, as mentioned above, dysfunction is related to the number / density / function / detyrosinization level of perinuclear microtubules, etc.

[0385] Pathologically, diseases / disorders related to microtubule dysfunction may have one or more of the following characteristics: Impaired cell contractility (e.g., cardiomyocytes), for example, compared to the contractility observed when there is no disease / condition; Abnormal organization of microtubule networks (e.g., within cells), such as compared to tissues observed without disease / condition; Increased mechanical forces transmitted to the cell nucleus / nuclear envelope, for example, compared to mechanical force levels in the absence of disease / condition; Abnormal tissue of sarcomeres (e.g., within cells), such as compared to tissue observed when there is no disease / condition; The mechanical resistance of the microtubule network increases, for example, compared to levels before disease / condition; The amplitude of myocardial cell contraction decreases, for example, compared to the amplitude of contraction when there is no disease / condition; Increased density of microtubule networks, such as perinuclear microtubules, interfibrillary microtubules and / or cortical microtubules, compared to the density before disease / condition; Increased levels / expression of tubulin (e.g., α-tubulin and / or β-tubulin), for example, compared to levels / expression without disease / condition. Increased levels / proportions of detyrosinated tubulin, for example, compared to levels / proportions without disease / condition; Increased levels / proportions of microtubules interacting with intermediate filaments (e.g., desmin); for example, compared to levels / proportions before disease / condition; and Increased levels / proportions of acetylated tubulin, for example, compared to levels / proportions before disease / condition; The level / proportion of acetylated tubulin is reduced, for example, compared to the level / proportion before the disease / condition; Abnormal organization of perinuclear microtubule networks (e.g., in cells), such as compared to tissues observed without disease / condition; Increased mechanical forces transmitted to the cell nucleus / nuclear envelope via the perinuclear microtubule network, for example, compared to mechanical force levels in the absence of disease / condition; The mechanical resistance of the perinuclear microtubule network increases, for example, compared to levels in the absence of disease / condition; The density of the perinuclear microtubule network increases, for example, compared to the density before disease / condition; Increased levels / expression of perinuclear tubulin (e.g., α-tubulin and / or β-tubulin), for example, compared to levels / expression in the absence of disease / condition. Increased levels / proportions of detyrosine-derived perinuclear tubulin, for example, compared to levels / proportions in the absence of disease / condition; and The level / proportion of perinuclear microtubules that interact with intermediate filaments (e.g., desmin) is increased; for example, compared to the level / proportion in the absence of disease / condition.

[0386] Disease models observing microtubule dysfunction Chen C. and colleagues conducted functional tests on human cardiomyocytes to support the potential for treating a range of heart diseases by modulating microtubule proliferation and dysfunction. Chen C. et al. noted that earlier studies have confirmed the presence of microtubule proliferation in patients with aortic stenosis, as well as in various animal models of left ventricular pressure overload, right ventricular pressure overload, drug-induced pulmonary hypertension, malnutrition-related cardiomyopathy, and diabetic cardiomyopathy, as shown in Table A below.

[0387] Other evidence suggests a link between microtubule dysfunction and heart disease. HDAC6 inhibitors have been shown to increase acetylated tubulin protein levels and have therefore been proposed as a treatment for HFpEF; microtubule affinity-regulated kinase 4 (MARK4) deficiency reduces left ventricular ejection fraction after acute myocardial infarction in mice; and elevated phosphorylation levels of microtubule-associated protein 4 (MAP4) have been found in mouse models of myocardial infarction (MI) and transverse aortic coarctation (TAC). Furthermore, elevated levels of tubulin and detyrosine-treated tubulin were observed in ZSF1 obese rats (HFpEF model).

[0388] Table A. Disease models in which microtubule dysfunction was observed (Source: Chen C. et al., Nature Medicine (2018), 24, 1225–1233 or as described below) Diseases / disorders characterized by defects / dysfunctions of desmosomes / desmosomal plaque proteins. The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and disorders characterized by desmosome defects / deficiencies or dysfunctions. Experimental examples of this disclosure demonstrate that inhibition of the LINC complex improves pathology in mouse models of diseases characterized by desmosome defects / deficiencies.

[0389] In some embodiments, the disease / disorder to be treated / prevented according to this disclosure is a disease / disorder in which desmosome dysfunction or deficiency / insufficiency is pathologically related, such as a disease / disorder in which desmosome dysfunction or deficiency / insufficiency is positively correlated with the onset, development, or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder. In some embodiments, desmosome dysfunction or deficiency / insufficiency may be a risk factor for the onset, development, or progression of the disease / disorder.

[0390] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition characterized by a reduced number of desmosomes (e.g., formation between adjacent cells in a given tissue), for example, compared to the number observed when the disease / condition is not present (e.g., in a healthy subject or in an equivalent non-lesion tissue).

[0391] Diseases / disorders characterized by desmosome defects / deficiencies or dysfunctions include those characterized by reduced desmosome protein expression / activity (e.g., diseases / disorders characterized by reduced expression / activity of the constituent peptides of the desmosome-intermediate filament complex (DIFC)).

[0392] Diseases / disorders characterized by desmosome defects / deficiencies or dysfunction include those associated with mutations in genes encoding desmosome proteins. In some embodiments, the disease / disorder to be treated / prevented according to this disclosure is a disease / disorder associated with mutations in genes encoding constituent polypeptides of the desmosome-intermediate filament complex (DIFC). In some embodiments, the disease / disorder is related to a protein selected from... DSP , DES , DSG1 , DSG2 , DSG3 , DSG4 , DSC1 , DSC2, DSC3 , PKP1 , PKP2 , PKP3 and JUP The disease / condition is associated with gene mutations. In some implementations, the disease / condition is related to genes selected from [the specific gene]. DSP , DES , DSG2 , DSC2 , PKP2 and JUP It is related to gene mutations.

[0393] The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and disorders including / characterized by desmosome plaque protein deficiency / insufficiency or dysfunction. Experimental examples of this disclosure demonstrate that inhibition of the LINC complex improves the pathology in mouse models of diseases caused by desmosome plaque protein deficiency / insufficiency.

[0394] In some implementations, a disease or disorder characterized by a deficiency / deficiency or dysfunction of desmosomes / desmosomal plaques is characterized by cardiomyopathy. That is, in some implementations, cardiomyopathy is a symptom of a disease or disorder characterized by a deficiency / deficiency or dysfunction of desmosomes / desmosomal plaques.

[0395] As mentioned above, mutations in the gene encoding desmosome protein can lead to cardiomyopathy. DSP The mutation is associated with dilated cardiomyopathy and arrhythmogenic cardiomyopathy. DESThe mutation is associated with dilated cardiomyopathy, arrhythmogenic cardiomyopathy, and restrictive cardiomyopathy. DSG2 , PKP2 and JUP All mutations are associated with arrhythmogenic cardiomyopathy.

[0396] DSP Mutations can lead to cardiomyopathy (particularly dilated cardiomyopathy and arrhythmogenic cardiomyopathy) and diseases / disorders characterized by cardiomyopathy, such as erythrokeratosis-cardiomyopathy syndrome and cardiocutaneous syndromes, such as Naxos disease and Cavallary syndrome. DSP Mutations in this protein are also associated with a variety of other diseases / conditions, including skin fragility (epidermolysis bullosa) and palmoplantar keratosis. Autoantibodies against desmosome protein are a hallmark of the autoimmune disease paraneoplastic pemphigus. Decreased desmosome protein expression has also been observed in patients with oropharyngeal cancer and breast cancer; it is believed that this decreased expression may alter intercellular adhesion properties and promote metastasis.

[0397] In some embodiments, the disease / disorder to be treated / prevented according to this disclosure may be a disease / disorder involving a deficiency / insufficiency or dysfunction of desmosome protein, such as a disease / disorder in which a deficiency / insufficiency or dysfunction of desmosome protein is positively correlated with the onset, development, or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder. In some embodiments, a deficiency / insufficiency or dysfunction of desmosome protein may be a risk factor for the onset, development, or progression of the disease / disorder.

[0398] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is characterized by a decrease in the expression / activity of desmosome plaque protein, for example, compared to the expression / activity level in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-lesion tissue). In some embodiments, the disease / condition is characterized by a reduction in the number of desmosomes (e.g., desmosomes formed between adjacent cells in a given tissue), for example, compared to the number observed in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-lesion tissue).

[0399] Diseases / disorders characterized by defects / deficiencies or dysfunctions of desmosomes / desmosomal plaque proteins include those related to DSP Mutation-related diseases / conditions.

[0400] In some implementation schemes, it is known or predicted DSPThe mutation reduces the level of desmosome isoforms encoded by wild-type alleles (e.g., DPI, DPII, and / or DSPIa). In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is known or predicted to result in the production of a truncated form of the desmosome isoform encoded by the wild-type allele. In some embodiments, the mutation is known or predicted to result in the production of a dysfunctional and / or nonfunctional desmosome isoform. In some embodiments, the mutation is known or predicted to result in the production of a desmosome isoform that exhibits reduced desmosome-mediated function (e.g., DPI, DPII, and / or DSPIa) compared to the associated functional level exhibited by the desmosome isoform encoded by the wild-type allele. In some embodiments, the mutation is known or predicted to result in the production of misfolded and / or degraded desmosome isoforms. In some embodiments, the mutation is known or predicted to increase the level of disease-associated desmosome variants. In some embodiments, the mutation is known or predicted to increase the level of desmosome variants encoded by wild-type alleles. DSP The level of desmosome plaque protein isotype encoded by disease-related alleles.

[0401] and DSP Mutation-related diseases / disorders are described in, for example, Yuan et al., Chin Med J (Engl) (2021) 134(15):1771-1779, Smith et al., Circulation (2020) 141(23):1872-1884, and Mohammed and Chidgey, Journal of Structural Biology (2021) 213(3):107749. All references are incorporated herein by reference. Disease-related DSPMissense mutations include G2056R, D2757H, K2689T, R2834H, T2595I, R2541K, R2541S, R2639Q, R2366H, R2366C, D230N, S299R, N375I, S442F, I445V, N458Y, K470E, S507F, and N. 287D, N287S, A206T, W207C, E422K, I533T, A556T, N661I, Y787C, R808C, R80 8H, R1255K, R1255T, L1348R, T1373A, L1654P, R1775I, R1838H, Q198P, D203N , R222L, D241N, E290K, I305F, A306T, R315C, R315P, M316V, A349P, Y350H, T 356K, I368T, E384Q, K401N, N408K, R451G, R451H, I461V, C482Y, Y494F, P515 L, L540P, T587N, N593S, M599R, R606W, E721K, Y895H, I870M, I870T, R907H, R908H, L933F, G939S, S987P, T1960P, Y1981C, L1995S, A2019S, A2019V, P206 1S, H2062R, D2070N, V2107L, R2083C, K2103E, A2148T, T2177N, R2189Q, T21 96I, I2263T, T2267S, A2294G, Q2295H, G2338R, E2343K, I2347V, D2579H, I25 93S, T2664N, G2666D, A2674T, M2684V, M2707T, A2709T, R2759S, P2777H, I2 797T, M2819L, G2844V, I2869V, K1094N, Y1169N, L1178R, L1178V, Y1188H, R1 207K, T1217M, D1251N, D1258E, A1274P, K1288Q, T1319I, R1341H, E1357D, R 1392Q, R1392W, E1407D, A1435P, R1458G, Y1512C, L1514P, L1535P, K1537C, K 1537H, T1557M, K1581E, K1592R, M1601I, S1623C, V1639M, S1658F, R1666Q, R1666W, H1684R, E1721D, R1738Q, E1740K, E1833V, R1852H, R2366H, T2770C,G2832V, S2616L, G2647D, R1838H, T13I, E21K, V30M, R44Q, C81Y, Q90R, R105Q, E109K, M118K, I125F, C174F, A566T, I560F, T564I, L583P, S597L, E2193K, L2329P, Q2371K, G2375R, A2655D, N287K, R2366H, R2366C, L622P, Q616P, H618P, P498L, R1184W, S610P, H586P, and H586Y.

[0402] In some implementation schemes, DSP The mutation is one that is associated with heart diseases such as cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic cardiomyopathy. DSPSuch mutations include G2056R, D2757H, K2689T, R2834H, T2595I, R2541K, R2541S, R2639Q, R2366H, R2366C, D230N, S299R, N375I, S442F, I445V, N458Y, K470E, S507F, N287D, N287S, A206T, W207C, E422K, I533T, A556T, N661I, Y787C, R808C, R8 08H, R1255K, R1255T, L1348R, T1373A, L1654P, R1775I, R1838H, Q198P, D203 N, R222L, D241N, E290K, I305F, A306T, R315C, R315P, M316V, A349P, Y350H, T356K, I368T, E384Q, K401N, N408K, R451G, R451H, I461V, C482Y, Y494F, P51 5L, L540P, T587N, N593S, M599R, R606W, E721K, Y895H, I870M, I870T, R907H , R908H, L933F, G939S, S987P, T1960P, Y1981C, L1995S, A2019S, A2019V, P20 61S, H2062R, D2070N, V2107L, R2083C, K2103E, A2148T, T2177N, R2189Q, T2 196I, I2263T, T2267S, A2294G, Q2295H, G2338R, E2343K, I2347V, D2579H, I2 593S, T2664N, G2666D, A2674T, M2684V, M2707T, A2709T, R2759S, P2777H, I 2797T, M2819L, G2844V, I2869V, K1094N, Y1169N, L1178R, L1178V, Y1188H, R 1207K, T1217M, D1251N, D1258E, A1274P, K1288Q, T1319I, R1341H, E1357D, R1392Q, R1392W, E1407D, A1435P, R1458G, Y1512C, L1514P, L1535P, K1537C, K1537H, T1557M, K1581E, K1592R, M1601I, S1623C, V1639M, S1658F, R1666Q, R1666W, H1684R, E1721D, R1738Q, E1740K, E1833V, R1852H, R2366H, T2770C,G2832V, S2616L, G2647D, R1838H, T13I, E21K, V30M, R44Q, C81Y, Q90R, R105Q, E109K, M118K, I125F, C174F, and A566T.

[0403] In some implementation schemes, DSP The mutation is associated with cardiodermal syndrome (CCS). For example, it has been reported in subjects with CCS characterized by palmoplantar keratoderma and arrhythmogenic cardiomyopathy. DSP The deletion variant c.825_827del (Çimen et al., J Clin Med. (2023) 12(3): 913). Cardiocutaneous syndromes include Naxos disease and Cavallar syndrome.

[0404] In some implementation schemes, DSP The mutation is one associated with Naxos disease or Kavayal syndrome. DSP Such mutations include I560F, T564I, L583P, S597L, E2193K, L2329P, Q2371K, G2375R, and A2655D.

[0405] In some implementation schemes, DSP The mutation is associated with skin fragility-woolly hair syndrome. DSP Such mutations include N287K, R2366H, and R2366C.

[0406] In some implementation schemes, DSP The mutation is associated with erythematous keratosis-cardiomyopathy syndrome. DSP Such mutations include L622P, Q616P, and H618P.

[0407] In some implementation schemes, DSP The mutation is one that is associated with non-syndromic alopecia. DSP Such mutations include P498L.

[0408] In some implementation schemes, DSP The mutation is associated with palmoplantar keratoderma. DSP Such mutations include R1184W.

[0409] In some implementation schemes, DSP The mutation is associated with severe dermatitis, polydipsia, and metabolic wasting syndrome (SAM). DSP Such mutations include S610P, H586P, and H586Y.

[0410] In all aspects and embodiments of this disclosure, the disease / condition to be treated / prevented is selected from: cardiomyopathy, dilated cardiomyopathy, arrhythmogenic cardiomyopathy, disease / condition pathologically involving desmosome defects / deficiencies or dysfunction, disease / condition characterized by a reduction in the number of desmosomes, and disease / condition associated with mutations in genes encoding desmosome proteins (e.g., selected from...). DSP , DES , DSG2 , DSC2 , PKP2 and JUP Diseases / disorders characterized by decreased desmosome protein expression / activity; pathologically involving desmosome plaque protein deficiency / insufficiency or dysfunction; diseases / disorders characterized by decreased desmosome plaque protein expression / activity; and related to... DSP Mutation-related diseases / disorders, skin diseases, cardiodermal syndrome (CCS), Naxos disease, Kavayal syndrome, keratosis, palmoplantar keratosis, epidermolysis bullosa, fragile skin, woolly hair syndrome, erythrokeratosis-cardiomyopathy syndrome, nonsyndromic alopecia, palmoplantar keratosis with severe dermatitis, multiple allergies and metabolic consumption (SAM) syndrome.

[0411] Diseases / disorders characterized by sarcomere / connectin deficiency / function The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and disorders characterized by sarcomere / tipin deficiency / insufficiency or dysfunction. Experimental examples of this disclosure demonstrate that inhibition of the LINC complex improves the pathology in mouse models of diseases characterized by sarcomere / tipin deficiency / insufficiency.

[0412] In some embodiments, the disease / disorder to be treated / prevented according to this disclosure is a pathologically related disease / disorder, such as one in which sarcomere / tipin dysfunction or deficiency / insufficiency is positively correlated with the onset, development, or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder. In some embodiments, sarcomere / tipin dysfunction or deficiency / insufficiency may be a risk factor for the onset, development, or progression of the disease / disorder.

[0413] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition characterized by the breakdown of sarcomeres, which results in a decrease in contractility, for example, compared to the tissue of sarcomeres observed when the disease / condition is not present (e.g., in healthy subjects or in equivalent non-lesion tissue).

[0414] In some embodiments, the disease / condition to be treated / prevented according to this disclosure is a disease / condition characterized by increased metabolic stress, for example, compared to cardiac metabolism observed in the absence of disease / condition (e.g., in healthy subjects or in equivalent non-lesion tissues).

[0415] Diseases / disorders characterized by titin deficiency / insufficiency or dysfunction include diseases / disorders characterized by reduced titin expression / activity (e.g., diseases / disorders characterized by reduced titin peptide expression / activity).

[0416] Diseases / disorders characterized by sarcomere defects / deficiencies or dysfunctions include those associated with gene mutations encoding sarcomere proteins. In some embodiments, the disease / disorder to be treated / prevented according to this disclosure is a disease / disorder associated with gene mutations encoding sarcomere constituent polypeptides. In some embodiments, the disease / disorder is related to a protein selected from... TTN, MYH7, TNNT2, TNNI3, TNNC1, TPM1, MYBPC3, MYL2, MYL3, CSRP3, RBM20, ACTN2, TCAP and ACTC1 The disease / condition is associated with gene mutations. In some implementations, the disease / condition is related to genes selected from [the specific gene]. TTN, TNNT2, TPM1, MYH7 and MYBPC3 It is related to gene mutations.

[0417] The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and disorders including / characterized by titin deficiency / insufficiency or dysfunction. Experimental examples of this disclosure demonstrate that inhibition of the LINC complex improves the pathology in mouse models of diseases caused by titin deficiency / insufficiency.

[0418] In some implementations, a disease or disorder characterized by insufficient / deficient or impaired sarcomere / tibin is characterized by cardiomyopathy. That is, in some implementations, cardiomyopathy is a symptom of a disease or disorder characterized by insufficient / deficient or impaired sarcomere / tibin.

[0419] As mentioned above, mutations in genes encoding sarcoproteins can cause cardiomyopathy. TTN The mutation is associated with dilated cardiomyopathy, arrhythmogenic cardiomyopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy. TNNT2 The mutation is associated with dilated cardiomyopathy and hypertrophic cardiomyopathy. TPM1 The mutation is associated with dilated cardiomyopathy. MYH7 The mutation is associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, and left ventricular noncompaction cardiomyopathy. MYBPC3 The mutations are associated with hypertrophic cardiomyopathy and left ventricular noncompaction cardiomyopathy.

[0420] TTNMutations lead to cardiomyopathy (particularly dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic cardiomyopathy) and diseases / disorders characterized by cardiomyopathy, such as early-onset adult-onset recessive distal titinopathy, early-onset myopathy with fatal cardiomyopathy, multimicronucleus disease with heart disease, and Emory-Dreyfus muscular dystrophy (EDMD). Mutations in titin are also associated with a variety of other diseases / disorders, including purely skeletal muscle myopathy: limb-girdle muscular dystrophy type 2J (LGMD2J), late-onset autosomal dominant tibial muscular dystrophy (TMD), hereditary myopathy with early respiratory failure (HMERF), and congenital central nucleus myopathy (CNM).

[0421] In some embodiments, the disease / disorder to be treated / prevented according to this disclosure may be a disease / disorder in which a titin deficiency / insufficiency or dysfunction is pathologically related, such as a disease / disorder in which a titin deficiency / insufficiency or dysfunction is positively correlated with the onset, development, or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder. In some embodiments, a titin deficiency / insufficiency or dysfunction may be a risk factor for the onset, development, or progression of the disease / disorder.

[0422] In some embodiments, the disease / disorder to be treated / prevented according to this disclosure is characterized by a decrease in the expression / activity of titin, for example, compared to the expression / activity level in the absence of disease / disorder (e.g., in healthy subjects or in equivalent non-lesion tissue). In some embodiments, the disease / disorder is characterized by sarcomere disorder, resulting in a decrease in contractility, for example, compared to the tissue structure of sarcomeres observed in the absence of disease / disorder (e.g., in healthy subjects or in equivalent non-lesion tissue). In some embodiments, the disease / disorder is characterized by an increase in metabolic stress, for example, compared to cardiac metabolism observed in the absence of disease / disorder (e.g., in healthy subjects or in equivalent non-lesion tissue).

[0423] Diseases / disorders characterized by sarcomere / connectin deficiency / insufficiency or dysfunction include those related to TTN Mutation-related diseases / conditions.

[0424] In some implementation schemes, it is known or predicted TTNThe mutation reduces the level of the myosin isoforms encoded by wild-type alleles (e.g., inferred complete isoforms (IC), N2BA-G, N2BA-N2, N2BA-A1, N2BA-A2, N2B, Novex-1, Novex-2, Novex-3, N2A-solecis, and N2A-psoas). In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is known or predicted to result in the production of truncated forms of the myosin isoforms encoded by wild-type alleles. In some embodiments, the mutation is known or predicted to result in the production of dysfunctional and / or nonfunctional myosin isoforms. In some embodiments, the mutation is known or predicted to result in the production of a titin isoform exhibiting a reduced level of titin-mediated function compared to the associated functional levels shown by the titin isoforms encoded by the wild-type allele (e.g., inferred complete isoforms (IC), N2BA-G, N2BA-N2, N2BA-A1, N2BA-A2, N2B, Novex-1, Novex-2, Novex-3, N2A-soleus, and N2A-psoas). In some embodiments, the mutation is known or predicted to result in the production of a misfolded and / or degraded titin isoform. In some embodiments, the mutation is known or predicted to increase the levels of disease-associated titin variants. In some embodiments, the mutation is known or predicted to increase the levels of titin isoforms encoded by disease-associated alleles of TTN.

[0425] Diseases / disorders associated with TTN gene mutations are described, for example, by Laddach, A., M. Gautel and F. Fraternali (2017). Bioinformatics 33(21): 3482–3485 and Wolfgang A. Linke and Nazha Hamdani (2014). Circulation Research; 114:1052–1068, all of which are incorporated herein by reference in their entirety. Disease-associated TTN missense mutations (UniProt: Q8WZ42-1) includes Val54Met, Arg740Leu, Ala743Val, Trp976Arg, Val1034Met, Arg2083fs, Thr2896lle, Glu2989GlufsX4, Ser3799Tyr, Gln4053X, Gln4249X, Ser4465Asn, Leu5782Phe, Ser6395fs, Glu7004Lys, Gly793 3fs, Asp7971_Ile7976del, Tyr8958Cys, Arg9427His, Arg9531Gln, His9775Tyr, Gly10159, Val11396, 114 77InsPro, Val11879, Val11879, Val11879Leu, Ala12675, Ala12873fs, Pro13298_Thr17642dup, Cys13771 X、Glu14779fs、Asp14909、Tyr15045Cys、Phe15108fs、Asp16122、Gly16189X、Trp16359X、Lys16782、Arg1 7295X, Ala17342Thr, Arg17470X, Glu17715fs, Glu17783X, Ile17876Thr, Lys17898fs, Glu17978fs, Glu18 141. Asp18235, His18335, Cys18789X, Arg18858X, Arg18985X, Ala19506Thr, Arg19560X, Pro20049fs, Ala 20236Ser、Val20591Glu、Gln20809、Arg20858X、Thr21135、Tyr21301、Gly21497、Lys21640fs、Truncation afterIle21923、Glu21956fs、Ala22353fs、Pro22582fs、Asn23824fs、Gln24059fs、Ser24241fs、Gln24281X、Gln25689X、Trp26632X、Arg26949X、Lys27016X、Ser27052IfsX1、Trp27147X、Ser27179fs、Glu27300、Tyr27567X、Tyr28326fs、Glu28386LysfsX10、Trp29318X、Arg29415X、Glu29510X、Thr29725fs、Pro30068Arg、Cys30071Arg、Gln30081X、Trp30088Arg、Trp30088Cys、Trp30088Leu、Pro30091Leu、Asn30145Lys、Gly30150Asp、Thr30165、Asn30348fs、Thr30513fs、Arg30857、Arg31126fs、Arg31195X、Lys31371X、Pro31774Leu、Ser31841X、Asn32379ThrfsX9、Trp32431Arg、Arg32450Trp、Arg32534X、Pro32976fs、Arg33041Gly、His33534fs、Gln33637X、Gly33698fs、Ser33828SerfsX11、Lys33915fs、Met34218Thr、Ser34242GlnfsX10、Glu34286_Trp34289delinsValLysGluLys、His34305Pro、Iso34306Asp、Leu34315Pro、Lys34322AsnfsX9、Lys34322fs、Gln34323X。

[0426] In some implementations, TTN mutations are mutations associated with heart diseases such as cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic cardiomyopathy. Such TTN mutations include Val54Met, Arg740Leu, Ala743Val, Trp976Arg, Val1034Met, Arg2083fs, Thr2896lle, Glu2989GlufsX4, Ser3799Tyr, Gln4053X, Gln4249X, Ser4465Asn, Leu5782Phe, Ser6395fs, Glu7004Lys, and Gly7933f. s, Asp7971_Ile7976del, Tyr8958Cys, Arg9427His, Arg9531Gln, His9775Tyr, Gly10159, Val11396, 1147 7InsPro, Val11879, Val11879, Val11879Leu, Ala12675, Ala12873fs, Pro13298_Thr17642dup, Cys13771X , Glu14779fs, Asp14909, Tyr15045Cys, Phe15108fs, Asp16122, Gly16189X, Trp16359X, Lys16782, Arg17 295X、Ala17342Thr、Arg17470X、Glu17715fs、Glu17783X、Ile17876Thr、Lys17898fs、Glu17978fs、Glu181 41. Asp18235, His18335, Cys18789X, Arg18858X, Arg18985X, Ala19506Thr, Arg19560X, Pro20049fs, Ala 20236Ser、Val20591Glu、Gln20809、Arg20858X、Thr21135、Tyr21301、Gly21497、Lys21640fs、Truncation afterIle21923、Glu21956fs、Ala22353fs、Pro22582fs、Asn23824fs、Gln24059fs、Ser24241fs、Gln24281X、Gln25689X、Trp26632X、Arg26949X、Lys27016X、Ser27052IfsX1、Trp27147X、Ser27179fs、Glu27300、Tyr27567X、Tyr28326fs、Glu28386LysfsX10、Trp29318X、Arg29415X、Glu29510X、Thr29725fs、Pro30068Arg、Cys30071Arg、Gln30081X、Trp30088Arg、Trp30088Cys、Trp30088Leu、Pro30091Leu、Asn30145Lys、Gly30150Asp、Thr30165、Asn30348fs、Thr30513fs、Arg30857、Arg31126fs、Arg31195X、Lys31371X、Pro31774Leu、Ser31841X、Asn32379ThrfsX9、Trp32431Arg、Arg32450Trp、Arg32534X、Pro32976fs、Arg33041Gly、His33534fs、Gln33637X、Gly33698fs、Ser33828SerfsX11、Lys33915fs、Met34218Thr、Ser34242GlnfsX10、Glu34286_Trp34289delinsValLysGluLys、His34305Pro、Iso34306Asp、Leu34315Pro、Lys34322AsnfsX9、Lys34322fs、Gln34323X。

[0427] In some implementations, TTN mutations are mutations associated with diseases / disorders characterized by cardiomyopathy, such as early-onset myopathy (EOMFC) with fatal cardiomyopathy, characterized by dilated cardiomyopathy, motor developmental delay, and generalized muscle weakness primarily affecting the proximal and distal lower limbs. The myosin deletion variants include K35556fs, K26491fs, K26616fs, K26683fs, K33915fs, K32988fs, D26449fs, D26574fs, D26641fs, D32946fs, D33873fs, D35514fs, Q35176fs, Q26303fs, Q32608fs, Q33535fs, Q26111fs, Q26236fs, P12918fs, P13845fs, P15486fs, P6421fs, and P6546fs. s, P6613fs, Y13414fs, P10340fs, P11641fs, P11267fs, E16226Ter, E17867Ter, E8802Ter, E15299Ter, E8927Ter, E8994Ter, T211 29fs, T13897fs, T20202fs, T22770fs, T13705fs, T13830fs, R17915Ter, R15347Ter, R9042Ter, R8975Ter, R16274Ter, R8850Ter.

[0428] In some implementations, TTN mutations are mutations associated with autosomal recessive limb-girdle muscular dystrophy type 2J. Such TTN mutations include Y22311Ter, Y22436Ter, Y22503Ter, Y28808Ter, Y29735Ter, Y31376Ter, K17301Ter, K17426Ter, K17493Ter, K23798Ter, K24725Ter, K26366Ter, E21058Ter, E23626Ter, E21985Ter, E14561Ter, E14686Ter, E14753Ter, E10603Ter, E11530Ter, E13037Ter, E10380Ter, and E11681Ter. , E11307Ter, D2143fs, D2189fs, K25643fs, K32015fs, K25518fs, K2571 0fs, K32942fs, K34583fs, M25640fs, M25707fs, M34580fs, M32939fs, M2 5515fs, M32012fs, E19273fs, E19398fs, E19465fs, E25770fs, E26697fs , E28338fs, E18442fs, E27315fs, E18375fs, E25674fs, E18250fs, E2474 7fs, I16818fs, I16943fs, I17010fs, I23315fs, I24242fs, I25883fs, P 15942fs, P24815fs, P15875fs, P23174fs, P15750fs, P22247fs, D888fs, D842fs, T25284fs, T25476fs, T34349fs, T31781fs, T32708fs, T25409fs , C31712R, C30071R, C29144R, C22647R, C22772R, C22839R, Q13385Ter, Q 13510Ter, Q13577Ter, Q19882Ter, Q20809Ter, Q22450Ter, C10433fs, C10500fs, C10308fs, C16805fs, C17732fs, C19373fs, Q24894Ter, Q26535T er, Q17470Ter, Q17662Ter, Q17595Ter, Q23967Ter, R15103Ter, R12535Ter, R13462Ter, R6163Ter, R6230Ter, R6038Ter, K19784Ter, K26089Ter,K19592Ter、K28657Ter、K19717Ter、K27016Ter、Q32829Ter、Q33756Ter、Q26457Ter、Q26524Ter、Q35397Ter、Q26332Ter、K26507Ter、K32812Ter、K35380Ter、K26440Ter、K26315Ter、K33739Ter、L24957Ter、L26598Ter、L17658Ter、L17725Ter、L17533Ter、L24030Ter、W23785Ter、W25426Ter、W16361Ter、W16486Ter、W22858Ter、W16553Ter、D15446fs、D21751fs、D24319fs、D15254fs、D15379fs、D22678fs、Q21931Ter、Q23572Ter、Q14699Ter、Q21004Ter、Q14507Ter、Q14632Ter、Q13337Ter、Q20569Ter、Q19642Ter、Q22210Ter、Q13145Ter、Q13270Ter、K16846fs、K9614fs、K15919fs、K18487fs、K9422fs、K9547fs、F14486fs、F7254fs、F13559fs、F16127fs、F7187fs、F7062fs、R35252Ter、R32684Ter、R33611Ter、R26312Ter、R26379Ter、R26187Ter、V13897fs、V13964fs、V22837fs、V13772fs、V20269fs、V21196fs、R23351Ter、R14286Ter、R14411Ter、R14478Ter、R20783Ter、R21710Ter、C22206Ter、C19638Ter、C13141Ter、C13266Ter、C20565Ter、C13333Ter、I33379N、I34306N、I35947N、I27007N、I27074N、I26882N、I21018fs、I12145fs、I19377fs、I11953fs、I12078fs、I18450fs、Y32573Ter、Y35141Ter、Y26268Ter、Y33500Ter、Y26076Ter、Y26201Ter、R26234fs、R35174fs、R33533fs、R26109fs、R26301fs、R32606fs、E12913Ter、Q26794Ter、Q17921Ter、Q24226Ter、Q17854Ter、Q17729Ter、Q25153Ter、 In some implementations, the TTN mutation is a mutation associated with tibial muscular dystrophy. Such mutations in TTN include I33379N, I34306N, I35947N, I27007N, I27074N, I26882N, I21018fs, I12145fs, I19377fs, I11953fs, I12078fs, I18450fs, Y32573Ter, Y35141Ter, Y26268Ter, Y33500Ter, Y26076Ter, Y26201Ter, Q26794Ter, Q17921Ter, Q24226Ter, Q17854Ter, Q17729Ter, Q25153Ter, K27023fs, K27090fs, and K26898fs. K35963fs, K33395fs, K34322fs, R33703Ter, R32062Ter, R24638Ter, R31135Ter, R24763Ter, R24830Ter, P 31732L, P29164L, P22792L, P22859L, P30091L, P22667L, R22499Ter, R20858Ter, R19931Ter, R13626Ter, R 13434Ter, R13559Ter, R23868Ter, R21300Ter, R22227Ter, R14803Ter, R14995Ter, R14928Ter, L29811fs, L23439fs, L30738fs, L23314fs, L23506fs, L32379fs, R18878Ter, R20519Ter, R11579Ter, R11646Ter, R179 51Ter, R11454Ter, P22521fs, P28826fs, P29753fs, P31394fs, P22329fs, P22454fs, Y24539Ter, Y27107Te r, Y25466Ter, Y18042Ter, Y18167Ter, Y18234Ter, N20902fs, N21094fs, N21027fs, N28326fs, N27399fs, N2 9967fs, R23469Ter, R25110Ter, R16237Ter, R16045Ter, R16170Ter, R22542Ter, R28364Ter, R19299Ter, R 25796Ter, R19424Ter, R26723Ter, R19491Ter, E24419Ter, E26060Ter, E23492Ter, E16995Ter, E17120Ter,E17187Ter、R31056Ter、R29415Ter、R21991Ter、R28488Ter、R22116Ter、R22183Ter、R20626Ter R18058Ter、R18985Ter、R11561Ter、R11686Ter、R11753Ter、R32102Ter、R33743Ter、R24678Ter、R31175Ter、R24803Ter、R24870Ter、R22975Ter、R24616Ter、R15676Ter、R15743Ter、R15551Ter、R22048Ter、R20026Ter、R21667Ter、R12727Ter、R19099Ter、R12602Ter、R12794Ter、R25552Ter、R22984Ter、R16487Ter、R16679Ter R16612Ter、R23911Ter、G25912Ter、G27553Ter、G24985Ter、G18488Ter、G18613Ter、G18680Ter、R16290Ter、R9918Ter、R17217Ter、R18858Ter、R9793Ter、R9985Ter、R19856fs、R19923fs、R26228fs、R19731fs、R28796fs、A27689fs、A18624fs、A25121fs、A26048fs、A18749fs、A18816fs、W23158Ter、W23283Ter W23350Ter、W29655Ter、W30582Ter、W32223Ter。、

[0429] In some implementations, specifically in certain embodiments, the TTN mutation is a mutation associated with myofibril myopathy type 9 with early respiratory failure. Such mutations in TTN include C31712R, C30071R, C29144R, C22647R, C22772R, C22839R, I21018fs, I12145fs, I19377fs, I11953fs, I12078fs, I18450fs, Y32573Ter, Y35141Ter, Y26268Ter, Y33500Ter, Y26076Ter, Y26201Ter, Q26794Ter, Q17921Ter, Q24226Ter, Q17854Ter, Q17729Ter, Q25153Ter, K270 23fs, K27090fs, K26898fs, K35963fs, K33395fs, K34322fs, R33703Ter, R32062Ter, R24638Ter, R31135Ter, R24763Ter, R24830Ter, P31732L, P29164L, P22792L, P22859L, P30091L, P22667L, R22499Ter, R20858Ter, R19931Ter, R13626Ter, R13434Ter, R13559Ter, R23868Ter, R21300Ter , R22227Ter, R14803Ter, R14995Ter, R14928Ter, L29811fs, L23439fs, L30738fs, L23314fs, L23506fs, L32379fs, R18878Ter, R20519Ter, R11 579Ter, R11646Ter, R17951Ter, R11454Ter, P22521fs, P28826fs, P29753fs, P31394fs, P22329fs, P22454fs, Y24539Ter, Y27107Ter, Y25466T er, Y18042Ter, Y18167Ter, Y18234Ter, N20902fs, N21094fs, N21027fs, N28326fs, N27399fs, N29967fs, R23469Ter, R25110Ter, R16237Ter, R 16045Ter, R16170Ter, R22542Ter, R28364Ter, R19299Ter, R25796Ter, R19424Ter, R26723Ter, R19491Ter, E24419Ter, E26060Ter, E23492Ter,E16995Ter, E17120Ter, E17187Ter, R31056Ter, R29415Ter, R21991Ter, R28488Ter, R22116Ter, R22 183Ter, R20626Ter, R18058Ter, R18985Ter, R11561Ter, R11686Ter, R11753Ter, R32102Ter, R33743 Ter, R24678Ter, R31175Ter, R24803Ter, R24870Ter, R22975Ter, R24616Ter, R15676Ter, R15743Ter , R15551Ter, R22048Ter, R20026Ter, R21667Ter, R12727Ter, R19099Ter, R12602Ter, R12794Ter, R25 552Ter, R22984Ter, R16487Ter, R16679Ter, R16612Ter, R23911Ter, G25912Ter, G27553Ter, G24985 Ter, G18488Ter, G18613Ter, G18680Ter, R16290Ter, R9918Ter, R17217Ter, R18858Ter, R9793Ter, R 9985Ter, R19856fs, R19923fs, R26228fs, R19731fs, R28796fs, A27689fs, A18624fs, A25121fs, A26 048fs, A18749fs, A18816fs, W23158Ter, W23283Ter, W23350Ter, W29655Ter, W30582Ter, W32223Ter. ,

[0430] In all aspects and embodiments of this disclosure, the diseases / conditions to be treated / prevented are selected from: cardiomyopathy, dilated cardiomyopathy, arrhythmogenic cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, diseases / conditions pathologically involving sarcomere defects / deficiencies or dysfunctions, diseases / conditions characterized by sarcomere structural disorder leading to weakened contractility, and diseases / conditions associated with mutations in genes encoding sarcomere proteins (e.g., selected from...). TTN, TNNT2, TPM1, MYH7 and MYBPC3 Diseases / disorders characterized by decreased sarcoprotein expression / activity; diseases / disorders pathologically involving titin deficiency / insufficiency or dysfunction; diseases / disorders characterized by decreased titin expression / activity; and related to TTNDiseases / disorders related to gene mutations, myopathy, autosomal recessive limb-girdle muscular dystrophy type 2J, myofibril myopathy type 9 with early respiratory failure, early-onset myopathy with fatal cardiomyopathy, and tibial muscular dystrophy.

[0431] Treatment / prevention of diseases / conditions through the inhibition of LINC complex This disclosure provides methods and articles (pharmaceuticals and compositions) for treating and / or preventing diseases / conditions by inhibiting LINC complexes. The treatment / prevention of diseases is achieved by inhibiting LINC complexes, for example, in cells, tissues / organs / organ systems / subjects.

[0432] This disclosure relates to the treatment / prevention of the diseases / conditions described herein through inhibition of the LINC complex.

[0433] These methods may aim to: delay / prevent the onset of disease / disorder symptoms; reduce (i.e. alleviate) the severity of disease / disorder symptoms; reduce the levels of disease / disorder pathogenesis-related factors; reverse disease / disorder symptoms; reduce the morbidity of subjects with the disease / disorder; improve the survival rate of subjects with the disease / disorder; prolong the lifespan of subjects with the disease / disorder; reduce the mortality rate of subjects with the disease / disorder; and / or delay / prevent the progression of the disease / disorder (e.g., progression to later stages).

[0434] In some implementation schemes, the diseases / conditions to be treated / prevented according to this disclosure no Laminarinopathy. In some implementations, the disease / disorder is related to... LMNA Mutation is irrelevant. In some implementations, the disease / condition is related to... LMNA , LMNB1 and LMNB2 Mutation of any one of them irrelevant In some implementations, disease / condition is related to LMNA , LMNB1 and LMNB2 Mutation of any one of them irrelevant .

[0435] In some implementation schemes, the disease / condition to be treated / prevented according to this disclosure no Nuclear envelope disease. In some implementations, the disease / disorder is related to... LMNA , LMNB1 , LMNB2 , EMD , LAP2 , LBR , ZMPSTE24 , SYNE-1 and NUP62 Mutation of any one of them irrelevant .

[0436] According to this disclosure, LINC complex inhibitors can be provided in the form of nucleic acids containing or encoding LINC complex inhibitors. Such nucleic acids contain or are composed of DNA and / or RNA, and may be or contain polynucleotides.

[0437] When the LINC complex inhibitor is a nucleic acid, it can be provided in the form of a larger nucleic acid containing the LINC complex inhibitor. When the LINC complex inhibitor is a peptide / peptide or SSN system targeting the LINC complex protein, it can be provided in the form of a nucleic acid encoding the LINC complex inhibitor.

[0438] In some implementations, the LINC complex inhibitor is administered in the form of a nucleic acid encoding a factor required for the production of the LINC complex inhibitor (e.g., a nucleic acid encoding a precursor of the LINC complex inhibitor and / or a nucleic acid encoding a factor required for the production / assembly of the LINC complex inhibitor). For example, the LINC complex inhibitor may be administered in the form of a nucleic acid encoding a factor required to produce a small molecule or biomolecular LINC complex inhibitor.

[0439] The nucleic acid may include a nucleotide sequence encoding a LINC complex repressor polypeptide, and may also include one or more non-peptide-coding nucleotide sequences. Non-peptide-coding nucleotide sequences may be, for example, 5' cap, 5' UTR, 3' UTR, and / or PolyA tail sequences.

[0440] Nucleic acids can be vectors, or can be contained within vectors. As used herein, "vector" refers to a nucleic acid used as a medium for transferring exogenous nucleic acids into cells. A vector can be a carrier for expressing nucleic acids in cells (i.e., a vector can be an expression vector). Such a vector may include a promoter sequence effectively linked to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express peptides or polypeptides from the vectors disclosed herein.

[0441] The term "operably linked" can include cases where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a way that the expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thus forming an expression cassette). Therefore, if the regulatory sequence can influence the transcription of the nucleic acid sequence, the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into a polypeptide, such as a LINC complex repressive polypeptide.

[0442] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma-retroviral vectors (e.g., vectors derived from murine leukemia virus (MLV)), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described in, for example, Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4, 9, both of which are incorporated herein by reference in their entirety. In a preferred embodiment, the vector is an adeno-associated virus vector or a lentiviral vector.

[0443] In some embodiments, the vector is selected based on the tropism of the cell type / tissue / organ to which the nucleic acid containing / encoding a LINC complex inhibitor is desired to be delivered. For example, according to this disclosure, it may be necessary to deliver nucleic acids or express LINC complex inhibitors in cell types, tissues, or organs affected by a disease to be treated or prevented (e.g., in cell types, tissues, or organs where symptoms of the disease are present).

[0444] In some embodiments, it is necessary to deliver nucleic acids encoding LINC complex inhibitors to muscle cells / tissues (e.g., cardiac and / or skeletal muscle cells / tissues), and vectors that are oriented towards these types of cells / tissues can be used in such embodiments. In some embodiments, the vector may be centripetal. In some embodiments, the vector may be myotropic.

[0445] In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector. AAV vectors and their applications in vector gene therapy have been reviewed, for example, in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378 and Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272, which are incorporated herein by reference in their entirety. In some embodiments, the vector may be the AAV vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378. In some embodiments, the vector may be the AAV vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272.

[0446] In some implementations, the vector is a self-complementary adeno-associated virus (scAAV) vector. Self-complementary adeno-associated virus vectors are described, for example, in McCarty, Mol Ther. (2008) 16(10):1648-56, which is incorporated herein by reference. Conventional AAVs have a single-stranded DNA genome and require DNA replication mechanisms in the transduced cell to synthesize the complementary strand, thus delaying transgene expression. In contrast, scAAVs contain complementary sequences that anneal spontaneously after infection, eliminating the need for DNA synthesis in the transduced host cell. Compared to classic single-stranded AAV vectors, scAAV vectors have been shown to provide accelerated initiation of transgene expression and increased transgene expression levels.

[0447] In some embodiments, the vector may be an adeno-associated virus vector of one of the following serotypes: AAV1, AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV9, AAV9.45, AAV10, or AAVrh74. In some embodiments, the vector is an AAV9 vector. In some embodiments, the vector is an AAV6 vector.

[0448] In some embodiments, the vector may be a centripetal adeno-associated virus vector. In some embodiments, the vector may be an adeno-associated virus vector of one of the following serotypes: AAV1, AAV8, AAV9, or AAV9.45.

[0449] In some embodiments, the vector may be a skeletal muscle tropism adeno-associated virus vector. In some embodiments, the vector may be an adeno-associated virus vector of one of the following serotypes: AAV1, AAV6, AAV7, AAV8, AAV9, AAV9.45.

[0450] In some embodiments, the vector includes modifications to increase binding and / or transduction with the target cell type (i.e., compared to the binding / transduction level of an unmodified vector). In some embodiments, the modification is targeted at the capsid protein.

[0451] In some embodiments, the carrier comprises a capsid protein containing a cell-targeting peptide. In some embodiments, the cell-targeting peptide is the cell-targeting peptide described in Büning and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248-265, which is incorporated herein by reference, for example, the cell-targeting peptides shown in Tables 1, 2, 3 or 4.

[0452] In some embodiments, the carrier comprises a capsid protein containing the substitution of one or more tyrosine residues (e.g., one or more surface-exposed tyrosine residues). In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments, the carrier comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid, as described in Iida et al., Biomed Res Int. (2013) 2013: 974819, which is incorporated herein by reference in its entirety.

[0453] In some embodiments, the vector may be the adeno-associated virus vector described above by Büning and Srivastava. In some embodiments, the vector may be the adeno-associated virus vector described above by Iida et al.

[0454] In some embodiments, the nucleic acid / vector contains one or more sequences for controlling nucleic acid expression. Therefore, in some embodiments, the nucleic acid / vector contains control elements for inducible nucleic acid expression. The sequences for controlling nucleic acid expression can provide nucleic acid expression through specific cell or tissue types. For example, expression can be controlled by cell type or tissue-specific promoters.

[0455] The promoter for cell type or tissue-specific expression of the nucleic acid according to this disclosure can be selected based on the disease to be treated / prevented. For example, the promoter can drive expression in cell types / tissues / organs affected by the disease (e.g., cell types / tissues / organs where disease symptoms are present).

[0456] In some embodiments, the promoter can provide expression in muscle cells / tissues (e.g., cardiac and / or skeletal muscle cells / tissues). In some embodiments, the promoter can be a cardiac or cardiomyocyte-specific promoter (e.g., cTNT, α-MHC, or MLC2v promoter). In some embodiments, the promoter can be a skeletal muscle / striated muscle cell-specific promoter (e.g., MCK, MHCK7, or desmin promoter). In some embodiments, the promoter is cTNT.

[0457] In some embodiments, the promoter may be a vascular endothelial cell-specific promoter (e.g., the Tie2 promoter). In some embodiments, the promoter may be a vascular smooth muscle cell-specific promoter (e.g., the SM22a promoter). In some embodiments, the promoter may be a monocyte / macrophage-specific promoter (e.g., the LysM promoter).

[0458] The sequence used to control nucleic acid expression can provide nucleic acid expression in response to a given agent / signal. For example, expression can be under the control of an inducible promoter. The agent can provide inducible expression of nucleic acids in vivo by administering the agent to a subject who has been treated with modified cells according to this disclosure, or by administering the agent to cells cultured in vitro or in vitro, thereby providing inducible expression of nucleic acids in vitro or in vitro.

[0459] In some embodiments, the nucleic acid or vector of this disclosure can be used with a conditional expression system to control the expression of a nucleic acid containing / encoding a LINC complex inhibitor in cells containing the nucleic acid / vector. As used herein, “conditional expression” may also be referred to as “induced expression”, meaning expression dependent on certain conditions (e.g., the presence of specific factors). Conditional expression systems are well known in the art, for example, as reviewed by Ryding et al. in the Journal of Endocrinology (2001) 171, 1-14, which is incorporated herein by reference in its entirety.

[0460] In a preferred embodiment, the size of the nucleic acid allows it to be delivered as a gene therapy, i.e., delivered via a suitable vector.

[0461] In some embodiments, the nucleic acid containing / encoding a LINC complex inhibitor consists of a nucleotide sequence whose size is within the packaging limits of a vector for delivering the polynucleotide. In some embodiments, the nucleic acid consists of a nucleotide sequence whose size is within the packaging limits of a vector described herein. In some embodiments, the nucleic acid consists of a nucleotide sequence whose size is within the packaging limits of an adeno-associated virus (AAV) vector (e.g., the AAV vector described herein). In some embodiments, the nucleic acid consists of a nucleotide sequence whose size is within the packaging limits of an scAAV vector. In some embodiments, the nucleotide sequence of the nucleic acid consists of fewer than 6,000 nucleotides, for example, one of ≤5,000, ≤4,500, ≤4,000, ≤3,500, ≤3,000, ≤2,500, ≤2,400, or ≤2,300 nucleotides.

[0462] The LINC complex inhibitors according to this disclosure can be produced intracellularly. For example, in the case where the LINC complex inhibitor is a LINC complex repressive polypeptide, it may be produced intracellularly by transcription from the nucleic acid encoding the polypeptide, followed by translation of the transcribed RNA.

[0463] The LINC complex inhibitors according to this disclosure can be provided in the form of compositions comprising LINC complex inhibitors and / or compositions comprising nucleic acids comprising / encoding LINC complex inhibitors. Such compositions may be incorporated into formulations suitable for clinical use, including related articles (i.e., LINC complex inhibitory peptides / nucleic acids / carriers).

[0464] The composition may contain one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (e.g., vitamin A, cellulose E, cellulose C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silica, stearic acid, vegetable lipids), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (such as titanium dioxide).

[0465] As used herein, the term "pharmaceutical acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissues of the subject in question (e.g., a human subject) without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener in the compositions according to this disclosure must also be "acceptable" in the sense of compatibility with the other components of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners can be found in pharmacy textbooks, such as Remington's "The Science and Practice of Pharmacy" (edited by A. Adejare), 23rd edition (2020), Academic Press.

[0466] The articles of this disclosure can be formulated for administration to a subject, for example, via a route of administration suitable to the nature of the therapeutic agent and the disease to be treated / prevented. Administration of the articles of this disclosure can be parenteral, systemic, local, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral, or transdermal. In some embodiments, administration can be by injection or infusion, or by ingestion.

[0467] In some aspects and embodiments, the articles of this disclosure can be applied to target tissues / organs (e.g., tissues / organs affected by disease / condition (e.g., tissues / organs exhibiting symptoms of disease / condition)). In some aspects and embodiments, the articles of this disclosure can be administered into the bloodstream by injection or infusion (e.g., via cannula) (i.e., intravenous / arterial administration) or subcutaneously or orally. The specific mode of administration and / or site can be selected based on the location where therapeutic / preventive effects are desired, such as cardiomyocytes or myocardial tissue.

[0468] Pharmaceutical compositions / pharmaceuticals may contain LINC complex inhibitors / nucleic acids / carriers in a sterile or isotonic medium. Pharmaceutical compositions / pharmaceuticals may be provided in fluid form (including gel form). Fluid formulations may be formulated for administration to selected areas of the human or animal body via injection or infusion (e.g., through a cannula). Pharmaceutical compositions / pharmaceuticals may be provided in solid form, such as lyophilized form.

[0469] The LINC complex inhibitors, nucleic acids, vectors, cells, and compositions disclosed herein can be modified and / or formulated to facilitate delivery to and / or uptake by target cells / tissues (e.g., cardiomyocytes / tissues). Strategies for targeted delivery to such species have been reviewed, for example, in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are incorporated herein by reference in their entirety.

[0470] In some embodiments, the articles of this disclosure may be encapsulated in nanoparticles or liposomes. In some embodiments, the articles of this disclosure may be (covalently or non-covalently) bound to cell-penetrating peptides (e.g., protein transduction domains, trojan peptides, arginine-rich peptides, Vectocell peptides), cationic polymers, cationic lipids, or viral vectors.

[0471] Nanoparticles can be organic, such as micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendritic polymers, and polymeric therapeutic agents. Nanoparticles can also be inorganic, such as nanotubes or metal particles, optionally with the addition of organic molecules. In some embodiments, the nanoparticles are those described in Chen et al., Mol Ther Methods ClinDev. (2016) 3:16023, which is incorporated herein by reference. In some embodiments, the nanoparticles are PLGA, peptides, poly(β-amino esters), DOPE, β-cyclodextrin-containing polycationic, linear PEI, PAMAM dendritic polymers, branched PEI, chitosan, or polyphosphate nanoparticles.

[0472] In some embodiments, the LINC complex inhibitor, nucleic acid, and vector according to this disclosure include modifications to introduce one or more portions that promote delivery to and / or absorption by a target cell type or tissue (e.g., cardiac and / or skeletal muscle cells / tissue). In some embodiments, the LINC complex inhibitor, nucleic acid, and vector according to this disclosure are linked to one or more portions that promote delivery to and / or absorption by a target cell type or tissue (e.g., via chemical conjugation).

[0473] Parts that facilitate delivery to and / or uptake by target cell types or tissues are described, for example, in Benizri et al., Bioconjug Chem. (2019) 30(2): 366–383, which is incorporated herein by reference in its entirety. Such parts include, for example, N-acetylgalactosamine (GalNAc), α-tocopherol, cell-penetrating peptides, nucleic acid aptamers, antibodies and their antigen-binding fragments / derivatives, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g., palmitic acid), and nucleoli.

[0474] The articles of this disclosure can be formulated into sustained-release delivery systems to release LINC complex inhibitors, nucleic acids, carriers, cells, or compositions at a predetermined rate. The sustained-release drug delivery system can maintain a constant drug / therapeutic / preventative concentration over a specific time period. In some embodiments, the articles of this disclosure are formulated as liposomes, gels, implants, devices, or drug-polymer conjugates (e.g., hydrogels).

[0475] According to this disclosure, the administration of a LINC complex inhibitor to a subject is preferably in a "therapeuticly effective" or "preventively effective" amount, sufficient to demonstrate a therapeutic / preventive benefit to the subject. Administration of the LINC complex inhibitor preferably results in the modification of one or more cells to contain the LINC complex inhibitor described herein.

[0476] The actual amount, rate of administration, and timing of administration will depend on the nature and severity of the disease to be treated / prevented, and the nature of the LINC complex inhibitor. Treatment prescriptions (e.g., dosage decisions) are the responsibility of general practitioners and other physicians, and will typically take into account the disease / condition to be treated, the individual subject's condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols mentioned above can be found in Remington, “The Science and Practice of Pharmacy” (edited by A. Adejare), 23rd edition (2020), AcademicPress.

[0477] In a preferred embodiment, a nucleic acid / vector, or a composition comprising a nucleic acid / vector according to the present disclosure, is administered. In a preferred embodiment, a modification resulting in one or more cells is administered to comprise / express a nucleic acid / vector, and / or comprise / express a polypeptide according to the present disclosure. That is, the nucleic acid / vector / composition is used as a gene therapy.

[0478] In some embodiments, the treatment or preventive intervention according to this disclosure may also include administration of another agent for treating / preventing the associated disease / condition. The administration of the LINC complex inhibitor / nucleic acid / carrier / composition described herein may be done alone or in combination with other treatment methods, and may be administered simultaneously or sequentially, depending on the disease to be treated. Simultaneous administration means administration with another therapeutic agent, such as as a pharmaceutical composition comprising two drugs (a combination formulation), or administration immediately after each other, and optionally via the same route of administration (e.g., to the same tissue, artery, vein, or other blood vessel). Sequential administration means administration of one agent, followed by administration of another agent alone after a given time interval. While this is the case in some embodiments, it is not required that the two agents be administered via the same route. The time interval can be any time interval.

[0479] Multiple doses of LINC complex inhibitors / nucleic acids / carriers / compositions are available. One or more doses, or each dose, may be administered concurrently or sequentially with another therapeutic / preventive agent. Multiple doses may be spaced at predetermined time intervals, selectable as 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, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, a dose may be given every 7, 14, 21, or 28 days (with or without 3, 2, or 1 day).

[0480] According to various aspects of this disclosure, methods for treating and / or preventing diseases / conditions (e.g., cardiomyopathy) may include one or more of the following: promoting normal contraction / contractile force of cells (e.g., cardiomyocytes); promoting normal organization of microtubule networks (e.g., intracellularly); promoting normal organization of sarcomeres (e.g., intracellularly); reducing mechanical forces transmitted to the cell nucleus / nuclear envelope; reducing mechanical resistance of microtubule networks; increasing the contractile amplitude of cardiomyocytes; reducing the density of microtubule networks; reducing the level of tubulin (e.g., α-tubulin and / or β-tubulin) (e.g., intracellularly); reducing the level / proportion of detyrosinated tubulin; increasing acetylcholine levels; and increasing the level of acetylcholine. Acylated tubulin levels / proportions; reduced acetylated tubulin levels / proportions; reduced microtubule levels / proportions interacting with intermediate filaments (e.g., desmin); promoted normal organization of perinuclear microtubule networks (e.g., in cells); reduced mechanical forces transmitted from perinuclear microtubule networks to the nucleus / nuclear membrane; reduced mechanical resistance of perinuclear microtubule networks; reduced perinuclear microtubule network density; reduced perinuclear tubulin (e.g., α-tubulin and / or β-tubulin) levels (e.g., in cells); reduced detyrosineized perinuclear tubulin levels / proportions; and reduced microtubule levels / proportions interacting with intermediate filaments (e.g., desmin).

[0481] This disclosure further provides the use of the LINC complex inhibitor according to this disclosure for: promoting normal contractility / contractile force of cells (e.g., cardiomyocytes); promoting normal organization of microtubule networks (e.g., intracellularly); promoting normal organization of sarcomeres (e.g., intracellularly); reducing mechanical forces transmitted to the nucleus / nuclear membrane; reducing mechanical resistance of microtubule networks; increasing the contractile amplitude of cardiomyocytes; reducing the density of microtubule networks; reducing the level of tubulin (e.g., α-tubulin and / or β-tubulin) (e.g., intracellularly); reducing the level / proportion of detyrosine-treated tubulin; increasing the water content of acetylated tubulin. Equal / proportion; reduced levels / proportion of acetylated tubulin; reduced levels / proportion of microtubules interacting with intermediate filaments (e.g., desmin); promoted normal organization of the perinuclear microtubule network (e.g., in cells); reduced mechanical forces transmitted from the perinuclear microtubule network to the nucleus / nuclear membrane; reduced mechanical resistance of the perinuclear microtubule network; reduced density of the perinuclear microtubule network; reduced levels of perinuclear tubulin (e.g., α-tubulin and / or β-tubulin) (e.g., in cells); and / or reduced levels / proportion of detyrosine-treated perinuclear tubulin and reduced levels / proportion of microtubules interacting with intermediate filaments (e.g., desmin).

[0482] This disclosure further provides methods for: promoting normal contraction / contractile force of cells (e.g., cardiomyocytes); promoting normal organization of microtubule networks (e.g., intracellularly); promoting normal organization of sarcomeres (e.g., intracellularly); reducing mechanical forces transmitted to the cell nucleus / nuclear membrane; reducing mechanical resistance of microtubule networks; increasing the contractile amplitude of cardiomyocytes; reducing the density of microtubule networks; reducing the level of tubulin (e.g., α-tubulin and / or β-tubulin) (e.g., intracellularly); reducing the level / proportion of detyrosine-treated tubulin; increasing the level / proportion of acetylated tubulin; and reducing the level / proportion of acetylated tubulin. The following are achieved using LINC complex inhibitors: reducing the level / proportion of microtubules interacting with intermediate filaments (e.g., desmin); promoting normal organization of perinuclear microtubule networks (e.g., in cells); reducing the mechanical forces transmitted from the perinuclear microtubule network to the nucleus / nuclear membrane; reducing the mechanical resistance of the perinuclear microtubule network; reducing the density of the perinuclear microtubule network; reducing the level of perinuclear tubulin (e.g., α-tubulin and / or β-tubulin) (e.g., in cells); and / or reducing the level / proportion of detyrosine-depleted perinuclear tubulin and reducing the level / proportion of microtubules interacting with intermediate filaments (e.g., desmin); all of the above are achieved using LINC complex inhibitors according to this disclosure.

[0483] Therefore, this disclosure provides methods for: promoting normal contractility / contraction of cells (e.g., cardiomyocytes); promoting normal assembly of microtubule networks (e.g., intracellularly); promoting normal assembly of sarcomeres (e.g., intracellularly); reducing mechanical forces transmitted to the nucleus / nuclear membrane; reducing mechanical resistance of microtubule networks; increasing the amplitude of cardiomyocyte contraction; reducing the density of microtubule networks; reducing the level of tubulin (e.g., α-tubulin and / or β-tubulin) (e.g., intracellularly); reducing the level / proportion of detyrosine-treated tubulin; and reducing interactions with intermediate filaments (e.g., desmin). The method includes: reducing the level / proportion of microtubules; promoting normal assembly of perinuclear microtubule networks (e.g., intracellularly); reducing the mechanical forces transmitted from the perinuclear microtubule network to the nucleus / nuclear membrane; reducing the mechanical resistance of the perinuclear microtubule network; reducing the density of the perinuclear microtubule network; reducing the level of perinuclear tubulin (e.g., α-tubulin and / or β-tubulin) (e.g., intracellularly); reducing the level / proportion of detyrosineized perinuclear tubulin; and / or reducing the level / proportion of microtubules interacting with intermediate filaments (e.g., desmin); the method comprising administering a LINC complex inhibitor according to the present disclosure to a subject.

[0484] Subjects According to various aspects of this disclosure, the subject can be any animal or human. Therapeutic and preventative applications can be made in humans or animals (for veterinary use).

[0485] The subject to administer the products of this disclosure (e.g., according to a treatment or preventative intervention) may be a subject requiring such intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient.

[0486] Subjects may have (e.g., may have been diagnosed with) the diseases or disorders described herein (e.g., cardiomyopathy or a disease or disorder characterized by desmosome dysfunction / deficiency / insufficiency, myosin dysfunction / deficiency / insufficiency, or microtubule dysfunction), may be suspected of having such diseases / disorders, or may be at risk of developing / being infected with such diseases / disorders. In embodiments according to this disclosure, subjects may be selected for treatment based on a method (e.g., as described above) characterized by one or more biomarkers of such disease / disorder. Subjects may be suspected of having the diseases / disorders described herein based on the presence of other symptoms in the subject's cells / tissues / organs indicating the disease / disorder. Subjects may be considered at risk of developing the disease / disorder due to genetic susceptibility or other risk factors for the disease.

[0487] In some implementations, the subject contains mutations according to any of the implementations described herein. In some implementations, the subject contains mutations that cause the disease / condition described herein.

[0488] In some implementation schemes, subjects No Includes laminopathies. In some implementations, subjects... No Including disease-related LMNA Mutations. In some implementations, the subject does not contain... LMNA Mutation (i.e., for) LMNA (The wild-type allele is homozygous). In some implementation schemes, the subjects... No Include LMNA , LMNB1 and LMNB2 Any of the disease-related mutations. In some implementations, the subject does not contain... LMNA , LMNB1 and LMNB2 Any mutation in any of them (i.e., for) LMNA , LMNB1 and LMNB2 (The wild-type allele is homozygous). In some implementation schemes, the subjects... No Includes nuclear envelope disease. In some implementations, subjects... No Include LMNA , LMNB1 , LMNB2 , EMD , LAP2 , LBR , ZMPSTE24 , SYNE-1 and NUP62 Any of the disease-related mutations. In some implementations, the subject does not contain... LMNA , LMNB1 , LMNB2 , EMD , LAP2 , LBR , ZMPSTE24 , SYNE-1 and NUP62 Any mutation in any of them (i.e., for) LMNA , LMNB1 , LMNB2 , EMD , LAP2 , LBR , ZMPSTE24 , SYNE-1 and NUP62 The wild-type allele is homozygous.

[0489] In some embodiments, the method according to this disclosure may include determining whether a subject has the disease / condition described herein. In some embodiments, the method includes diagnosing the disease / condition described herein. Determining whether a subject has the disease / condition described herein may include analyzing one or more symptoms / associated symptoms of the subject's disease / condition. Genetic factors may be determined by methods known to those skilled in the art, including PCR-based and sequencing-based assays. By detecting the presence of genetic factors (e.g., in samples obtained from the subject), a disease may be diagnosed, and / or the subject may be classified as being at risk of having the disease / condition described herein, and / or the subject may be identified as suitable for treatment with the LINC complex inhibitor / nucleic acid / vector / composition described herein.

[0490] The assay can be performed in vitro on a sample obtained from the subject, or after processing the sample obtained from the subject. The sample obtained from the subject can be of any kind. Biological samples can be taken from any tissue or body fluid, such as blood samples, blood-derived samples, serum samples, lymph samples, semen samples, saliva samples, or synovial fluid samples. Blood-derived samples can be selected portions of a patient's blood, such as selected cellular portions or plasma or serum portions. Samples can include tissue samples or biopsy samples; or cells isolated from the subject.

[0491] Sequence identity As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in the subject sequence that are identical to those in the reference sequence after alignment and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity between sequences. For the purpose of determining the percentage of identity between two or more amino acid or nucleic acid sequences, pairwise and multiple sequence alignments can be performed in various ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772–780). When using this software, it is preferable to use default parameters, such as open space penalty and extension penalty.

[0492] sequence *** This disclosure includes combinations of the described aspects and preferred features, except where such combinations are expressly not permitted or should be expressly avoided.

[0493] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0494] Aspects and embodiments of this disclosure will now be described, for example, with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0495] Throughout the specification, including the appended claims, unless the context otherwise requires, the word “comprising” and its variations (such as “including” and “containing”) shall be understood to implicitly include the indicated integer or step or group of integers or steps, but not exclude any other integer or step or group of integers or steps.

[0496] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used in this specification and the appended claims include a plural referent. A range may be expressed herein as “about” a particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another embodiment.

[0497] The methods described herein can preferably be performed in vitro. The term 'in vitro' is intended to cover procedures performed with cultured cells, while the term 'in vivo' is intended to cover procedures performed using / on a complete multicellular organism. Attached Figure Description

[0498] The implementation schemes and experiments illustrating the principles of this disclosure will now be discussed with reference to the accompanying drawings.

[0499] Figure 1 A to 1D. Functional rescue of LMNA N195K iPSC-CM by DNSUN1. (1A) Schematic diagram of the experimental setup for functional measurements using the CardioExcyte system. (1B) Snapshots of EFP of the control group, LMNA N195K iPSC-CM, and LMNA N195K iPSC-CM transduced with DNSUN1. (1C) The figure illustrates the phenotypes observed in LMNA N195K iPSC-CM during EFP measurements: increased coefficient of variation (indicating arrhythmia), increased amplitude, and increased beat frequency. Induction by DNSUN1 resulted in the rescue or relief of these phenotypes. (1D) A graph showing the decrease in amplitude observed in LMNA N195K iPSC-CM during systolic measurements and the rescue of this phenotype by the introduction of DNSUN1. Each point corresponds to one well.

[0500] Figure 2A and 2B. DNSUN1 reduces microtubule networks in cardiomyocytes. (2A) Immunostaining of untransduced and DNSUN1-transduced CMs using a specified anti-tubulin antibody and the nuclear dye DAPI. The top image includes a magnified view of the inset in Tyr-alpha-Tub. The treated cells exhibited weaker and heterogeneous perinuclear microtubule signal. (2B) Quantification of microtubule intensity, normalized relative to DAPI, with each point representing a distinct imaging region. Statistical significance was determined using the Mann-Whitney U test.

[0501] Figure 3 A graph showing the BioID (BioIn Vivo Identifier) ​​experimental setup.

[0502] Figure 4 A graph showing the desmin proximity network in diseased cardiomyocytes associated with healthy and DSP-related conditions. Proximity scores (represented by gray gradients) reflect the proximity of proteins belonging to different functional units (boxes) to desmin decoys. Decoys (DES) are clearly marked with continuous black borders. Among acquired interactions, the most striking finding is the association between desmin and MTOC (microtubule organizing center) in the disease state. Proteins significantly affected by the disease state are marked with black dashed lines. IF, intermediate filament. AJ, adhesion junction.

[0503] Figure 5 A, 5B, and 5C. Schematic diagram of microtubules and MTOCs in adult cardiomyocytes. (5A) Shortly after birth, cardiomyocytes transition from mitotic (i) to postmitotic (ii), altering the localization of microtubule nucleation factors (e.g., microtubule organizing centers (MTOCs)) and thus changing the polarity of the network. Mature cardiomyocytes consist of three groups of microtubules. ： Interfibrillary microtubules, cortical microtubules, and perinuclear microtubules. (5B) Magnified view of microtubule-desin and Z-disc interaction sites. Each myofibril in a cardiomyocyte contains many independent units called sarcomeres. Sarcomeres are mainly composed of actin and myosin. α-actin is used to anchor actin to the Z-disc. Desin cross-linked by Plectin is also present at the Z-disc and reinforces microtubules there. (5C) Magnified view of microtubule-desin and nuclear envelope interaction sites. The rectangle highlights the enhanced interaction between desin and the subdistal appendages of MTOC in disease states. In addition, the schematic diagram depicts the action site of DNSUN1 (SUN1-2), which dismantles the connection between the nuclear envelope and the cytoskeleton. Figure 5 A and 5B are adapted from Uchida, K., Scarborough, EA, & Prosser, BL (2021).

[0504] Figure 6 A and 6B. Elevated levels of total tubulin and detyrosine-treated tubulin in shDSP-affected hearts. (6A) Western blot analysis compared the levels of total tubulin and detyrosine-treated tubulin in healthy controls and shDSP-affected hearts, using β-actin as a loading control. (6B) Quantitative analysis of fold changes in total tubulin and detyrosine-treated tubulin, standardized relative to controls (shLacZ), with bar height representing the mean and error bars representing the standard deviation. Two-way ANOVA was used for statistical analysis. * P ≤ 0.05; ** P ≤ 0.01 Figure 7A and 7B . Dsp Cardiac function in mice after knockdown (KD) and DNSUN1 rescue. (7A) A plot showing the design and timeline of the in vivo rescue experiment. Ctrl shLacZ - Control AAV expressing shRNA targeting LacZ; Ctrl cTnT-EGFP - Control AAV expressing EGFP under the control of the cardiac troponin promoter; shDSP - AAV expressing shRNA targeting Dsp; cTnT-DNSUN1 - AAV expressing dominant-negative SUN1 under the control of the cardiac troponin promoter. (7B) Dsp Both ejection fraction (EF) and fractional shortening (FS) were decreased in the KD mice, while EF and FS were similar to those in the rescue group. (****P < 0.0001; ***P < 0.001; one-way ANOVA, mean ± standard deviation). Starting on day 84, group 4 consisted of 3 mice because one mouse died during echocardiography preparation due to overexposure to anesthesia. Starting on day 133, group 2 consisted of 4 mice because one mouse died during parallel experiments due to accidental hypoxia and asphyxia caused by the use of an ECG tunnel that was too small.

[0505] Figure 8A and 8B The second batch of 20 mice... Dsp Cardiac function after knockdown (KD) and DNSUN1 rescue. (8A) Graph showing the design and timeline of the in vivo rescue experiment. Ctrl shLacZ - Control AAV expressing shRNA targeting LacZ; Ctrl cTnT-EGFP - Control AAV expressing EGFP driven by the cardiac troponin promoter; shDSP - AAV expressing shRNA targeting Dsp; cTnT-DNSUN1 - AAV expressing dominant-negative SUN1 driven by the cardiac troponin promoter. (8B) DspBoth EF% and FS% were reduced in KD mice. Data analysis was performed on the total number of treated animals (N) in each group, as shown in (A). (****P < 0.0001; one-way ANOVA, mean ± standard deviation).

[0506] Figure 9A and 9B . TTN Cardiac function in mice after knockdown (KD) and cardiac function after DNSUN1 rescue. Compared with the control group, mice with... TTN KD mice showed decreased ejection fraction (EF) (9A) and fractional shortening (FS) (9B). Mice treated with DNSUN1 had statistically significantly higher EF and FS compared to the disease group. (****P < 0.0001; ***P < 0.001; one-way ANOVA, mean ± standard deviation). Ctrl – control AAV expressing shRNA targeting LacZ; EGFP – AAV expressing EGFP; shTTN – AAV expressing shRNA targeting titin; DNSUN1 – AAV expressing dominant-negative SUN1.

[0507] Figure 10A , 10B Treatment with DNSun1 reduced tubulin levels and improved cardiac function in a mouse model of LMNA-DCM. (10A) Tubulin levels were compared in healthy controls (WT), diseased hearts of LMNA-DCM patients, and LMNA-DCM mice treated with DNSun1 by Western blotting, with GAPDH as a loading control. (10B) Fold changes in tubulin levels were quantitatively analyzed and normalized relative to controls (WT). Bar height represents the mean, and error bars represent the standard deviation. Statistical analysis was performed using ordinary one-way ANOVA. **P ≤ 0.01; ****P ≤ 0.0001. (10C) LVEF (EF %) was negatively correlated with normalized tubulin expression in control (WT), diseased (LMNA-DCM), and treated (LMNA-DCM + DNSun1) mice.

[0508] Figure 11A and 11BMicrotubule levels were elevated in shTTN-affected hearts. (11A) Western blot analysis compared microtubule levels in healthy controls and shTTN-affected hearts, using histone H2B as a loading control. Western blotting was performed using specific antibodies against α-tubulin and histone H2B according to the standard protocol available on the Bio-Rad website. (11B) Quantification of microtubule fold changes, normalized relative to controls (shLacZ), with bar height representing the mean and error bars representing the standard deviation. Statistical analysis was performed using unpaired t-tests. * P ≤ 0.05.

[0509] Example Example 1: Reversal of pathological changes in human iPSC-derived cardiomyocytes using DNSUN1 to uncouple the LINC complex. microtubules DNSUN1 competes with endogenous SUN protein for binding to KASH protein in the perinuclear space of cardiomyocytes, thereby uncoupling the nucleus from the cytoskeleton (Crisp, M. et al. J. Cell Biol. 172, 41–53 (2006)).

[0510] Based on the above findings, and given that many heart diseases (such as cardiomyopathy—whether of hereditary or non-hereditary origin, as described above) share a common feature: a significant and maladaptive increase in microtubule density within cardiomyocytes, the inventors conducted research to investigate whether DNSUN1 could potentially serve as an effective therapy for a variety of such diseases by reversing the formation of pathological microtubule networks.

[0511] The inventors initially tested their hypothesis using an in vitro model of LMNA cardiomyopathy. This decision was based on substantial evidence demonstrating the effectiveness of DNSUN1 in treating LMNA cardiomyopathy in mouse models (Chai, RJ, Werner, H., Li, PY et al. Disrupting the LINC complex by AAV-mediated gene transduction prevents progression of Lamin-induced cardiomyopathy. Nat Commun 12, 4722 (2021). https: / / doi.org / 10.1038 / s41467-021-24849-4, WO 2019 / 143300 A1 and WO2021 / 010898 A1).

[0512] In vitro model specifically refers to LMNAN195K cardiomyopathy. In the type A lamin variant, the asparagine (N) at amino acid position 195 is replaced by lysine (K), resulting in dilated cardiomyopathy (DCM) in humans (Fatkin et al. (1999) N. Engl. J. Med., 341, 1715–1724).

[0513] Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with LMNA-N195K mutations exhibit electrophysiological and nuclear morphological abnormalities. Therefore, they are suitable models for rescue experiments. (Shemer Y et al. (2021) Int JMol Sci. 23;22(15):7874).

[0514] 1.1 DNSUN1-mediated uncoupling of the LINC complex reversed the electrophysiological abnormalities of LMNA N195K iPSC-CM. The CardioExcyte system was used for electrophysiological studies of LMNA N195K iPSC-CM.

[0515] The CardioExcyte system enables simultaneous measurement of impedance and extracellular field potential (EFP) from a single well. An electrode located at the bottom of the well measures the impedance (resistance) introduced by the monolayer above it. The conformation of the cells forming the monolayer affects impedance; flattened cells (e.g., relaxed CMs) result in high impedance, while round cells (e.g., contracted CMs) result in low impedance. Cell contractility can be assessed by capturing impedance changes at high temporal resolution. Furthermore, fluctuations in the membrane potential of beating cardiomyocytes affect the concentration of ions trapped between the monolayer membrane and the detection electrode. Therefore, measuring ion concentration (i.e., EFP) at high temporal resolution can serve as a proxy for changes in CM membrane potential.

[0516] EFP analysis of LMNA N195K iPSC-CM showed that, compared with normal homologous CM (control), its arrhythmia phenotype, along with beat frequency and amplitude, was increased. Figure 1 B and 1C). Shrinkage measurements indicate that the amplitude is reduced compared to normal homologous CM. Figure 1 D). Notably, treatment of LMNA N195K iPSC-CM with AAV6-DNSUN1 resulted in the rescue or reduction of all these disease phenotypes ( Figure 1 B, 1C, and 1D), namely the reversal of the arrhythmia phenotype (manifested as a decrease in the coefficient of variation, a decrease in membrane potential amplitude and beat frequency, such as... Figure 1 As shown in C), and the reversal of impaired contractile force (manifested as an increase in impedance amplitude).

[0517] The rescue of electrophysiological abnormalities by DNSUN1 observed in LMNA N195K iPSC-CM makes this in vitro system a suitable tool for explaining the mechanism of action of DNSUN1.

[0518] 1.2 DNSUN1 can reverse pathological microtubule tissue in iPSC-CM To examine the effect of DNSUN1 on the microtubule network in iPSC-CM, immunostaining was performed using antibodies targeting tyrosine- and detyrosine-treated microtubules. In untreated LMNA N195K iPSC-CM, a distinct microtubule network was observed around the nucleus. Conversely, compared to untreated LMNA N195K iPSC-CM, DNSUN1-treated cardiomyocytes exhibited a more uniform distribution pattern in the cytoplasm, and the microtubule density in the cytoplasm was reduced. Figure 2 These findings indicate that DNSUN1 can reverse pathological microtubule tissue found in disease states.

[0519] Based on the findings described in this embodiment, the inventors proposed inhibiting the LINC complex (e.g., via DNSUN1) to salvage cardiomyopathy (e.g.) LMNA A novel mechanism for the development of cardiomyopathy: This mechanism is achieved by mitigating the pathological effects of dysfunctional microtubules. Two potential additional protective effects can be expected: 1. Protects the cell nucleus from excessive forces caused by microtubule structural disorder, thereby reducing the risk of DNA damage.

[0520] 2. Protects sarcomeres from impaired contractile properties caused by changes in microtubule load and force transmission. DNSUN1 may reduce the anchoring points of perinuclear microtubule populations and thus decrease their stability by disrupting the uncoupling of the nuclear envelope from the cytoskeleton mediated by the LINC complex.

[0521] Current data suggest that inhibiting LINC complexes (e.g., via DNSUN1) holds promise as an effective treatment for a variety of diseases associated with abnormal microtubule density or microtubule organization within cardiomyocytes.

[0522] 1.3 Materials and Methods The generation of AAV6 AAV6 was generated using a triple transfection method and purified using methods adapted from Arden et al., J Biol Methods. 2016;3(2):e38 or Strobel et al., Hum Gene Ther Methods. 2015;26(4):147-57.

[0523] iPSC-CM Experiment Human iPSC lines derived from PGP1 donors (Personal Genome Project) were engineered using the CRISPR / Cas9 system to carry a homozygous point mutation in LMNA (N195K). The iPSC cell lines were then guided to differentiate into beating cardiomyocytes (CM) and cultured according to a published protocol (Lian, X., et al. PNAS. (2012) 109(27), E1848-57).

[0524] For functional testing ( Figure 1 A) Cells were seeded in each CardioExcyte well (Nanion Technologies). One day after seeding, LMNA-N195K iPSC-CM and control (WT) cells were treated with AAV6-DNSUN1 (encoding DNSUN1, SEQ ID NO:112) or AAV6-GFP. Seeded cells were stimulated with endothelin 1. EFP and contractility were then assessed according to the manufacturer's protocol. Statistical analysis was performed using one-way ANOVA with GraphPad Prism software. The experiment was repeated three times. Figure 1 Each point in C and 1D represents a hole.

[0525] For immunofluorescence experiments, CM cells were seeded into 96-well plastic phnoplates coated with Matrigel. One day after seeding, treated cells were transduced using AAV6 carrying DNSUN1. Seven days after transduction, cells were fixed with 4% paraformaldehyde and immunostained using a specific antibody against microtubules according to standard protocol. Images were acquired from treated and untreated wells using a Perkin Elmer Opera Phenix (high-content) microscope system, with three images acquired from each well. The mean fluorescence intensity of microtubules in the target region was analyzed using IMARIS 8.2 software (Bitplane) and normalized relative to the mean nuclear intensity. For statistical analysis, the Mann-Whitney U test was performed using Prism software.

[0526] Example 2: BioID (BioIn Vivo) Marker Identifies Abnormal Microtubules in DSP-Linked Cardiomyopathy.

[0527] 2.1 Identifying changes in the desmin neighbor network between healthy hearts and DSP-related diseased hearts To identify potential cytoskeletal rearrangements associated with desmosomal plaque cardiomyopathy, an in vivo comparison of the desmin interactomes of healthy and shDSP-induced diseased hearts was performed. Figure 3 ).

[0528] The method used to identify proteins that interact with desmin is BioID. BioID is described in Roux K. et al., Curr Protoc Protein Sci. 2018, 91: 19.23.1-19.23.5, which is incorporated herein by reference. This method screens for physiologically relevant protein interactions occurring in living cells. The technique utilizes a broad-spectrum biotin ligase to biotinylate proteins based on proximity. The ligase is fused to the target protein and expressed in the cell, leading to the biotinylation of proximal endogenous proteins. Because this is a rare protein modification in nature, the biotinylation of these endogenous proteins by the BioID fusion protein allows for their selective separation and identification using standard biotin affinity capture.

[0529] Desmin is a component of the intermediate filament and acts as a key protein bridging the intercalated disc (by directly binding to desmosomal plaque protein) to the nuclear envelope (by interacting with the LINC complex via Plectin).

[0530] The inventors identified 18 proteins with differential proximity to desmin in pathological samples associated with DSP, compared to healthy heart tissue samples. Of these, 9 showed increased proximity, while 9 showed decreased proximity. Among the proteins showing decreased proximity, DSP represents a quality control, as shRNA reduced the overall level of this protein to induce the disease state.

[0531] The most relevant change observed in the pathological state was an enhanced association between desmin and microtubule organizing centers (MTOC). Figure 4 Specifically, in the disease state, desmin exhibits increased proximity to Ccdc61, Cc2d2a, Nhsl2, Plekha6, and Dvl3.

[0532] Centrosome MTOC, as a key factor in microtubule nucleation, plays a crucial role in shaping the polarity of the microtubule network. Adult cardiomyocytes lose their typical centrosome MTOC and instead rely on non-centrosome MTOC located around the nuclear envelope for microtubule nucleation. Figure 5 A). In this way, the entire nuclear capsule of the cardiomyocyte is used as the microtubule organizing center (MTOC). In adult cardiomyocytes, there are three distinct groups of microtubules: interfibrous microtubules, cortical microtubules, and perinuclear microtubules. Figure 5 A, ii). Desmin is thought to function as a mechanical anchor for microtubules, confining their distribution to specific spatial regions (Uchida K., et al. (2022)).

[0533] The observed enhanced association between intermediate filaments (detin) and MTOC suggests a pathological link leading to microtubule dysfunction in DSP-associated cardiomyopathy.

[0534] 2.2 Materials and Methods Example 4.2 of this article describes the methods for shRNA vector design (shLacZ and shDSP) and AAV9 production.

[0535] Bio-ID (Bio-Proximity Marker) study Following the method described by Roux et al. (Curr Protoc Protein Sci. 2018, 91: 19.23.1-19.23.5), a desmin (Uniprot accession number P17661) was linked to BirA and Alfa tags to create an AAV9 vector. The AAV9 virus was injected into C57BL6 mice via retro-orbital administration. Animals were randomly assigned to different experimental groups. Mice hearts were harvested after injection following app...

Claims

1. A LINC complex inhibitor in a method for treating or preventing cardiomyopathy, wherein the cardiomyopathy is not a laminopathic cardiomyopathy.

2. Use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of cardiomyopathy, wherein the cardiomyopathy is not associated with lamininopathy.

3. A method for treating or preventing cardiomyopathy, the method comprising administering to a subject a therapeutically or preventively effective amount of a LINC complex inhibitor, wherein the cardiomyopathy is not a cardiomyopathy associated with lamininopathy.

4. The LINC complex inhibitor used according to claim 1, the use according to claim 2, or the method according to claim 3, wherein the cardiomyopathy is selected from dilated cardiomyopathy, arrhythmogenic cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy.

5. The LINC complex inhibitor used according to claim 1 or claim 4, the use according to claim 2 or claim 4, or the method according to claim 3 or claim 4, wherein the cardiomyopathy is associated with a mutation in the gene encoding desmosome protein.

6. The LINC complex inhibitor used according to any one of claims 1, 4, or 5, the use according to any one of claims 2, 4, or 5, or the method according to any one of claims 3 to 5, wherein the cardiomyopathy is associated with mutations in genes selected from: DSP , DES , DSG2 , DSC2 , PKP2 and JUP .

7. The LINC complex inhibitor used according to any one of claims 1 to 6, the use according to any one of claims 2 to 4 to 6, or the method according to any one of claims 3 to 6, wherein the cardiomyopathy is characterized by microtubule dysfunction.

8. A LINC complex inhibitor used in a method of treating or preventing a disease or disorder characterized by desmosome defects or dysfunction.

9. Use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of diseases or disorders characterized by desmosome defects or dysfunction.

10. A method for treating or preventing a disease or disorder characterized by desmosome defects or dysfunction, the method comprising administering to the subject a therapeutically or preventively effective amount of a LINC complex inhibitor.

11. The LINC complex inhibitor of claim 8, the use of claim 9, or the method of claim 10, wherein the disease or disorder characterized by desmosome defects or dysfunction is associated with a mutation in the gene encoding desmosome proteins.

12. The LINC complex inhibitor used according to claim 8 or claim 11, the use according to claim 9 or claim 11, or the method according to claim 10 or claim 11, wherein the disease or disorder characterized by desmosome defect or dysfunction is associated with mutations in genes selected from: DSP , DES , DSG2 , DSC2 , PKP2 and JUP .

13. The LINC complex inhibitor used according to any one of claims 8, 11 or 12, the use according to any one of claims 9, 11 or 12, or the method according to any one of claims 10 to 12, wherein the disease or disorder characterized by desmosome defects or dysfunction is characterized by cardiomyopathy.

14. The LINC complex inhibitor used according to any one of claims 8 and 11 to 13, the use according to any one of claims 9 and 11 to 13, or the method according to any one of claims 10 to 13, wherein the disease or disorder characterized by desmosome defects or dysfunction is characterized by microtubule dysfunction.

15. A LINC complex inhibitor used in a method of treating or preventing a disease or disorder characterized by microtubule dysfunction.

16. Use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of diseases or disorders characterized by microtubule dysfunction.

17. A method for treating or preventing a disease or disorder characterized by microtubule dysfunction, the method comprising administering to a subject a therapeutically or preventively effective amount of a LINC complex inhibitor.

18. The LINC complex inhibitor of claim 15, the use of claim 16, or the method of claim 17, wherein the disease or disorder characterized by microtubule dysfunction is cardiomyopathy or is characterized by cardiomyopathy.

19. The LINC complex inhibitor used according to claim 1 or claim 18, the use according to claim 2 or claim 18, or the method according to claim 3 or claim 18, wherein the cardiomyopathy is selected from dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy.

20. The LINC complex inhibitor used according to any one of claims 1, 4, 5, 7, 8, 11, 12, 14, 15, 18 or 19, the use according to any one of claims 2, 4, 5, 7, 9, 11, 12, 14, 16, 18 or 19, or the method according to any one of claims 3 to 5, 7, 10 to 12, 14 or 17 to 19, wherein the treatment or prevention comprises administering to a subject a nucleic acid containing or encoding a LINC complex inhibitor.

21. The LINC complex inhibitor used according to any one of claims 1, 4, 5, 7, 8, 11, 12, 14, 15, 18, 19 or 20, the use according to any one of claims 2, 4, 5, 7, 9, 11, 12, 14, 16, 18, 19 or 20, or the method according to any one of claims 3 to 5, 7, 10 to 12, 14 or 17 to 20, wherein the treatment or prevention comprises administering to the subject a nucleic acid encoding a LINC complex inhibitory polypeptide.

22. The LINC complex inhibitor of claim 21, the use of claim 21, or the method of claim 21, wherein the LINC complex inhibitory polypeptide comprises: (i) an inhibitory region comprising an α3 helix corresponding to the CC2 region of a protein containing a SUN domain and an amino acid sequence of the SUN domain, and (ii) an endoplasmic reticulum retention motif.

23. The LINC complex inhibitor of claim 21 or claim 22, the use of the inhibitor of claim 21 or claim 22, or the method of claim 21 or claim 22, wherein the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 99, 72, 95, 71, 69, 70, or 73, or is substantially composed of said amino acid sequence.

24. The LINC complex inhibitor used according to any one of claims 21 to 23, the use according to any one of claims 21 to 23, or the method according to any one of claims 21 to 23, wherein the nucleic acid encoding the LINC complex inhibitory polypeptide against the subject is contained in an adeno-associated virus (AAV) vector.

25. A LINC complex inhibitor used in a method of treating or preventing a disease or disorder characterized by sarcomere defects or dysfunction.

26. Use of LINC complex inhibitors in the manufacture of medicaments for the treatment or prevention of diseases or disorders characterized by sarcomere defects or dysfunctions.

27. A method for treating or preventing a disease or disorder characterized by sarcomere defects or dysfunction, the method comprising administering to a subject a therapeutically or preventively effective amount of a LINC complex inhibitor.

28. The LINC complex inhibitor of claim 25, the use of claim 26, or the method of claim 27, wherein the disease or disorder characterized by sarcomere defects or dysfunction is associated with a mutation in a gene encoding a sarcomere protein.

29. The LINC complex inhibitor used according to claim 25 or claim 28, the use according to claim 26 or claim 28, or the method according to claim 27 or claim 28, wherein the disease or disorder characterized by sarcomere defects or dysfunction is associated with mutations in genes selected from: TTN, MYH7, TNNT2, TNNI3, TNNC1, TPM1, MYBPC3, MYL2, MYL3, CSRP3, RBM20, ACTN2, TCAP and ACTC1 .

30. The LINC complex inhibitor used according to any one of claims 25, 28 or 29, the use according to any one of claims 26, 28 or 29, or the method according to any one of claims 27 to 29, wherein the disease or disorder characterized by sarcomere defects or dysfunction is characterized by cardiomyopathy.

31. The LINC complex inhibitor used according to any one of claims 25 and 28 to 30, the use according to any one of claims 26 and 28 to 30, or the method according to any one of claims 27 to 30, wherein the disease or disorder characterized by sarcomere defects or dysfunction is characterized by microtubule dysfunction.

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