Treatment of cardiomyopathy not associated with laminopathy

Abstract

This 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 diseases or conditions characterized by microtubule dysfunction and / or sarcomere deficiency or dysfunction.

Description

[Technical Field]

[0001] This application claims priority from GB2312938.0 filed on 24 August 2023 and GB2404416.6 filed on 27 March 2024, the contents and elements thereof being incorporated herein by reference for all purposes.

[0002] This disclosure relates to the treatment and prevention of diseases and conditions by LINC complex inhibition. [Background technology]

[0003] Mutations in LMNA, which encodes lamins A and C, result in a wide range of diseases known as laminopathy. Disruption of the linker of nucleoskeleton and cytoskeleton (LINC) complexes has recently been proposed as a therapeutic strategy for treating laminopathy (see, for example, Chai et al., Nature Communications (2021) 12:4722).

[0004] Desmoplakin is an essential component of desmosomes, which form intercellular junctions between adjacent cells. Desmosomes (also known as adhesion plaques) are specialized structures that mediate lateral intercellular adhesion between cells, particularly in tissues subjected to considerable mechanical stress, such as cardiomyocytes (and bladder tissue, gastrointestinal mucosa, and epithelium). The structure and function of desmosomes are outlined, 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, both of which are incorporated herein by reference in their entirety.

[0005] The N-terminal globular domain of human desmoplakin contains a 584-amino acid region necessary for interaction with placofilin and placoglobin (see, e.g., Stappenbeck et al., J Cell Biol. (1993) 123(3):691-705 and Kowalczyk et al., J Cell Biol. (1997) 139(3):773-84). Desmoplakin binds to the intermediate filament via its C-terminus (see, e.g., Stappenbeck and Green, J Cell Biol. (1992) 116(5):1197-209 and Stappenbeck et al., J Cell Biol. (1993) 123(3):691-705), which can be mediated by a glycine-serine-arginine repeat region at the C-terminus (see, e.g., Fogl et al., Nat Commun. (2016) 7:10827).

[0006] Mutations in desmoplakin (encoded by the DSP gene) cause cardiomyopathy, including dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). DSP mutations can also lead to skin diseases such as epidermolysis bullosa, as well as pleiotropic disorders such as dilated cardiomyopathy with trichorrhizophore and keratosis. These diseases are thought to result from a loss or reduction in desmoplakin expression levels, which reduces the number of desmosomes and / or inhibits their function.

[0007] Intercellular junctions (ICDs) between adjacent cardiomyocytes are fundamental to cell function and regulation, specifically facilitating the arrangement of cells into organized layers, thereby maintaining tissue integrity and efficient electromechanical coupling. The basic components of intercellular junctions can be broadly divided into those involved in mechanical coupling that maintains cell-cell adhesion and those involved in electrical coupling that facilitates rapid intercellular ion transfer, enabling rapid transmission of depolarization wave fronts (D. A. Brams, J. E. Faffitz, Cardioskeletal Myopathies in Children and Young Adults, 2017). Mechanical coupling and intercellular adhesion are related to the adhesion junction and Desmosome Provided by [unclear]. Electrical coupling between cardiomyocytes occurs primarily via gap junctions.

[0008] The desmosome of ICD is composed of desmosome cadherins that facilitate intercellular connections, and is intracellularly connected to desmoplakin via the adapter proteins placoglobin and placofilin 2, which then recruit desmoplakin to desmin intermediate filaments.

[0009] A sarcomere is a single contractile unit of striated muscle cell, composed of actin, myosin, and associated regulatory and scaffolding proteins. Repeating these sarcomeres forms myofibrils, the basic rod-shaped organelle units of cardiomyocytes (Warner E. et al. Circulation Research. 2022;130:1723-1741). Actin filaments are anchored to the Z disc. Z disc-associated proteins include α-actinin, titin, nebulin, and intermediate filaments such as desmin intermediate filaments.

[0010] Microtubules extend between Z-discs and appear to be stabilized at the Z-lines, thereby allowing their growth to pause. Intermediate filaments such as desmin anchor detyrosinated microtubules to the Z-discs, bridging the microtubules into the intermediate filament network, thereby increasing resistance and compressing the microtubules, resulting in buckling structures during cardiomyocyte contraction (Warner E. et al., 2022). Therefore, it has been proposed that desmin acts as a mechanical anchor for microtubules, restricting their distribution to specific spatial regions (Uchida K. et al., Annu. Rev. Physiol. 2022. 84:257-83). [Overview of the project]

[0011] In a first aspect, the disclosure provides a LINC complex inhibitor for use in methods of treating or preventing cardiomyopathy, wherein the cardiomyopathy is not laminopathy-related cardiomyopathy.

[0012] This disclosure also provides the use of LINC complex inhibitors in the manufacture of pharmaceuticals for use in the treatment or prevention of cardiomyopathy, wherein the cardiomyopathy is not laminopathy-related cardiomyopathy.

[0013] The disclosure also provides a method for treating or preventing cardiomyopathy, comprising administering a therapeutic or prophylactic effective dose of a LINC complex inhibitor to a target, wherein the cardiomyopathy is not laminopathy-related cardiomyopathy.

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

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

[0016] In some embodiments, the cardiomyopathy is associated with a mutation in a gene selected from the group consisting of DSP, DES, DSG2, DSC2, PKP2, and JUP.

[0017] In some embodiments, the cardiomyopathy is characterized by microtubule dysfunction.

[0018] In some embodiments, the cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy.

[0019] The present disclosure also provides a LINC complex inhibitor for use in a method of treating or preventing a disease or condition characterized by desmosome deficiency or dysfunction.

[0020] The present disclosure also provides use of a LINC complex inhibitor in the manufacture of a medicament for use in treating or preventing a disease or condition characterized by desmosome deficiency or dysfunction.

[0021] The present disclosure also provides a method of treating or preventing a disease or condition characterized by desmosome deficiency or dysfunction, comprising administering to a subject a therapeutically or prophylactically effective amount of a LINC complex inhibitor.

[0022] In some embodiments, according to various aspects of the present disclosure, the disease or condition characterized by desmosome deficiency or dysfunction is associated with a mutation in a gene encoding a desmosomal protein.

[0023] In some embodiments, the disease or condition characterized by desmosome deficiency or dysfunction is associated with a mutation in a gene selected from the group consisting of DSP, DES, DSG2, DSC2, PKP2, and JUP.

[0024] In some embodiments, the disease or condition characterized by desmosome deficiency or dysfunction is characterized by cardiomyopathy.

[0025] In some embodiments, a disease or condition characterized by desmosome deficiency or dysfunction is characterized by microtubule dysfunction.

[0026] This disclosure also provides LINC complex inhibitors for use in methods of treating or preventing diseases or conditions characterized by microtubule dysfunction.

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

[0028] This disclosure also provides a method for treating or preventing a disease or condition characterized by microtubule dysfunction, comprising administering a therapeutic or prophylactic effective dose of a LINC complex inhibitor to the target.

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

[0030] This disclosure also provides LINC complex inhibitors for use in methods of treating or preventing diseases or conditions characterized by sarcomere deficiency / incompleteness or dysfunction.

[0031] This disclosure also provides the use of LINC complex inhibitors in the manufacture of pharmaceuticals for use in the treatment or prevention of diseases or conditions characterized by sarcomere deficiency / incompleteness or dysfunction.

[0032] This disclosure also provides a method for treating or preventing a disease or condition characterized by sarcomere deficiency / incompleteness or dysfunction, comprising administering a therapeutic or prophylactic effective dose of a LINC complex inhibitor to the target.

[0033] In some embodiments, diseases or conditions characterized by sarcomere deficiency or dysfunction are associated with mutations in genes encoding sarcomere proteins.

[0034] In some embodiments, diseases or conditions characterized by sarcomere deficiency 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 embodiments, a disease or condition characterized by sarcomere deficiency / incompleteness or dysfunction is cardiomyopathy or is characterized by cardiomyopathy.

[0036] In some embodiments, a disease or condition characterized by sarcomere deficiency or dysfunction is characterized by microtubule dysfunction.

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

[0038] In some embodiments, according to various aspects of the present disclosure, treatment or prevention includes administering a LINC complex inhibitor or a nucleic acid encoding a LINC complex inhibitor to a target.

[0039] In some embodiments, treatment or prevention involves administering nucleic acids encoding LINC complex inhibitory polypeptides.

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

[0041] In some embodiments, the LINC complex inhibitory polypeptide comprises, or is essentially derived from, an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NOs. 99, 72, 95, 71, 69, 70, or 73.

[0042] In some embodiments, the nucleic acid encoding the LINC complex inhibitory polypeptide for the target is contained within an adeno-associated virus (AAV) vector.

[0043] explanation This disclosure states that LINC complex inhibition can treat laminopathy. other than This is based on the inventors' unexpected discovery that it is useful in treating / preventing the disease / condition, particularly cardiomyopathy with causes other than mutations in LMNA.

[0044] This disclosure is also based on the inventors' unexpected discovery that LINC complex inhibition is useful in treating / preventing diseases / conditions in which abnormal microtubule function pathologically affects the disease.

[0045] In this specification, the inventors demonstrate that LINC complex inhibition is useful for the treatment / prevention of diseases associated with desmosome deficiency / dysfunction, for example, resulting from mutations in desmosome proteins. In particular, the inventors demonstrate that the pathology of cardiomyopathy associated with DSP mutations can be ameliorated by LINC complex inhibition.

[0046] In this specification, the inventors demonstrate that LINC complex inhibition is useful for the treatment / prevention of diseases associated with sarcomere dysfunction, such as those resulting from mutations in sarcomere proteins. In particular, the inventors demonstrate that the pathology of cardiomyopathy associated with TTN mutations can be ameliorated by LINC complex inhibition.

[0047] The specific models of cardiomyopathy used in the experimental examples arise as a result of different factors (i.e., mutations in LMNA, DSP, or TTN). Mutation-associated cardiomyopathy, acquired heart disease (i.e., non-hereditary cardiomyopathy), and cardiomyopathy associated with other heart diseases are characterized by abnormal microtubule organization, which results in altered microtubule load-bearing capacity and force transmission. Thus, we unexpectedly demonstrate that inhibition of the LINC complex can reverse the dysfunctional microtubule organization. While we do not wish to be bound by any particular theory, we infer that discoupling the nuclear envelope from the microtubule network by disrupting the LINC complex reduces the anchoring sites of the perinuclear microtubule population, reversing the abnormal load-bearing capacity and force transmission from the microtubule network to the nuclear envelope, thereby improving the pathology of cardiomyopathy and other diseases.

[0048] This disclosure provides LINC complex inhibitors for use in methods of treating or preventing heart disease. This disclosure also provides the use of LINC complex inhibitors in the manufacture of pharmaceuticals for use in treating or preventing heart disease. This disclosure also provides methods of treating or preventing heart disease, comprising administering a therapeutic or prophylactic effective amount of a LINC complex inhibitor to a target. In some embodiments, the heart disease is not related to laminopathy.

[0049] Structure and function of LINC complex The linker (LINC) complex of the nuclear and cytoskeleton is a polypeptide complex containing SUN domain-containing proteins and KASH domain-containing proteins. The structure of the LINC complex is outlined, for example, in Sosa et al., Curr Opin Struct Biol. (2013) 23(2):285-91 and Hieda, Cells (2017) 6(1):3, both of 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. SUN domain-containing proteins span the INM and associate with nuclear lamin and chromatin-binding proteins on the nucleoplasmic side of the INM, as well as KASH domain-containing proteins on the perinuclear side of the INM. KASH domain-containing proteins span the ONM and associate with cytoskeletal structural components such as actin filaments, microtubule motors, and intermediate filaments on the cytoplasmic side of the ONM, as well as SUN domain-containing proteins on the perinuclear side of the ONM. SUN domain proteins function as transluminal tethers for KASH domain proteins in the ONM.

[0051] In this specification, "SUN domain-containing protein" refers to any polypeptide containing a SUN domain. S ad1 and UN C-84) domain proteins are important INM components containing a conserved carboxy-terminal SUN domain localized in the perinuclear space. The SUN domain consists of approximately 175 residues and is donated to the terminal of the helical stem region. The nucleoplasmic domain of SUN domain-containing proteins interacts with structural components of the nuclear scaffold.

[0052] The SUN domain may contain, or consist of, an amino acid sequence having at least 60% amino acid sequence identity with the amino acid sequence shown in SEQ ID NOs: 8, 18, 27, 28, 29, or 30, or at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identity with the amino acid sequence shown in SEQ ID NOs: 8, 18, 27, 28, 29, or 30.

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

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

[0055] Human SUN1 is a polypeptide identified by the amino acid sequence UniProtKB O94901 shown in SEQ ID NO: 1. Human SUN2 is a polypeptide identified by the amino acid sequence UniProtKB Q9UH99 shown in SEQ ID NO: 13. Human SUN3 is a polypeptide identified by the amino acid sequence UniProtKB Q8TAQ9 shown in SEQ ID NO: 23. Human SUN5 is a polypeptide identified by the amino acid sequence UniProtKB A9Z1W8 shown in SEQ ID NO: 24. Human SPAG4 is a polypeptide identified by the amino acid sequence UniProtKB Q9NPE6 shown in SEQ ID NO: 25. Human SUCO is a polypeptide identified by the amino acid sequence UniProtKB Q9UBS9 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 derived from any species, and include their isoforms, 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 may be characterized by its ability to perform a function performed by the reference protein.

[0058] A "fragment" generally refers to a fragment of a reference protein. A "variant" generally refers to a protein that has an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications to the amino acid sequence of a reference protein, but retains a considerable degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein. A "homolog" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologs include orthologues.

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

[0060] The isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs that possess the functional properties / activities of the reference protein, for example, as determined by analysis by a suitable assay for functional properties / activity.

[0061] In this specification, a reference to "SUN1" refers to the amino acid sequence shown in SEQ ID NO: 1, and proteins having fragments, variants, or homologs thereof. In some embodiments, SUN1 comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO: 1, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

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

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

[0064] In this specification, a reference to "SUN5" refers to the amino acid sequence shown in SEQ ID NO: 24, and proteins having fragments, variants, or homologs thereof. In some embodiments, SUN5 comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO: 24, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0065] In this specification, a reference to "SPAG4" refers to the amino acid sequence shown in SEQ ID NO: 25, and proteins having fragments, variants, or homologs thereof. In some embodiments, SPAG4 comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO: 25, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0066] In this specification, a reference to “SUCO” refers to the amino acid sequence shown in SEQ ID NO: 26, and proteins having fragments, variants, or homologs thereof. In some embodiments, SUCO comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO: 26, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

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

[0068] The KASH domain may contain, or consist of, 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: 37, 38, 39, 40, 41, or 42.

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

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

[0071] Human Nesprin-1 is a polypeptide identified by the amino acid sequence UniProtKB Q8NF91, shown in SEQ ID NO: 31. Human Nesprin-2 is a polypeptide identified by the amino acid sequence UniProtKB Q8WXH0, shown in SEQ ID NO: 32. Human Nesprin-3 is a polypeptide identified by the amino acid sequence UniProtKB Q6ZMZ3, shown in SEQ ID NO: 33. Human Nesprin-4 is a polypeptide identified by the amino acid sequence UniProtKB Q8N205, shown in SEQ ID NO: 34. Human KASH5 is a polypeptide identified by the amino acid sequence UniProtKB Q8N6L0, shown in SEQ ID NO: 35. Human LRMP is a polypeptide identified by the amino acid sequence UniProtKB Q12912, 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, respectively, and include their isoforms, fragments, variants, or homologs.

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

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

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

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

[0077] In this specification, a reference to "KASH5" refers to the amino acid sequence shown in SEQ ID NO: 35, and proteins having fragments, variants, or homologs thereof. In some embodiments, KASH5 comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO: 35, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

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

[0079] As used herein, “LINC complex” refers to a polypeptide complex comprising a SUN domain-containing protein and a KASH domain-containing protein.

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

[0081] The SUN domain protein is thought to oligomerize and trimerize via interactions between stem regions, forming a coiled triple helix (Zhou et al., J. Biol. Chem. (2012) 287: 5317-5326). Deletion of the stem region of the SUN domain protein has been shown to disrupt the formation of the LINC complex. The SUN domain is thought to have a β-sandwich structure, where the trimer SUN domains interact extensively with each other through a protruding β-sheet known as the KASH lid, and the KASH lid of one SUN domain partially overlaps with the β-sandwich of adjacent SUN domains (Sosa et al., Cell (2012) 149: 1035-1047).

[0082] KASH domain proteins can also oligomerize, which can involve protein-protein interactions between transmembrane helices. A single KASH domain interacts with two adjacent SUN domains along a groove formed between the KASH lid of one SUN domain and the upper region of the β-sandwich of the adjacent SUN domain. In this way, the SUN and KASH domains are thought to interact to form a 3:3 hexameric heterocomplex. Two to three proline residues immediately preceding the C-terminus of the KASH domain are thought to be accommodated in a deep pocket within the surface of the SUN domain. This region of KASH is crucial for the SUN-KASH interaction, and C-terminus elongation by only a single amino acid disrupts LINC complex formation. 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 via the LINC complex (Jahed et al., Biophys.J.(2015)109:501-509).

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

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

[0085] The functions of LINC complexes are outlined, for example, in Hieda, Cells (2017) 6(1):3 (incorporated herein by reference above) and Stroud, Biophys Rev. (2018) 10(4):1033-1051 (in their entirety incorporated herein by reference).

[0086] The LINC complex performs a variety of functions, including structural support for the nucleus, nuclear shaping and positioning, maintenance of the connection between the centrosome and the nucleus and the spacing of the nuclear membrane, DNA repair, cell migration, and chromosome movement within the nucleus during meiosis.

[0087] The LINC complex plays a mechanosensory role in converting mechanical stimuli and changes in the extracellular matrix into signals, enabling cells to adapt to their environment through cytoskeletal organization, gene expression, nuclear organization, and structural regulation. The microtubule cytoskeleton and LINC complex play crucial roles in nuclear protection and force transmission 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-cytoskeletal molecular connections transmit these forces to the chromosomal tissue within the nucleus. Nuclear laminas cause deformation of nuclear structure, initiating changes in gene regulation.

[0089] The nuclear envelope is a crucial structure in these processes. On the nucleoplasmic side of the INM, nuclear lamina (composed of type A and type B lamins) contribute to the mechanical stress resistance of the nucleus and form a lattice structure essential for the structural integrity of the nuclear envelope. Nuclear lamins are involved in processes critical to cellular function and viability, including maintaining nuclear integrity, regulating the cell cycle, mechanotransduction, cell signaling, and DNA repair.

[0090] Deviations from the normal expression and / or function of nuclear envelope proteins, as well as deviations from the normal expression and / or function of factors directly or indirectly related to the nuclear envelope, are involved in a variety of diseases, including muscular dystrophy, cardiomyopathy, lipodystrophy, progeria, cancer, and neurological disorders.

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

[0092] In light of the physiological requirements of rhythmic pumping, cardiomyocytes possess innate mechanical stretch-sensing capabilities. Microtubules, along with myofilaments and intermediate filaments, form a complex cytoskeleton that controls cell dynamics (Warner E. et al 2022). Crosslinking between microtubules and other cytoskeletal components (such as desmin and actin) can significantly amplify the mechanical forces transmitted within the cell (Warner E. et al 2022). The microtubule cytoskeleton has high inherent rigidity, can withstand compressive stress, and can exhibit resistance to movement.

[0093] Cardiomyocytes are equipped with mechanisms for rapidly generating force and movement, and microtubule buckling is estimated to provide approximately 20% of the contractile force of cardiomyocytes. Factors influencing mechanical force include microtubule growth rate, microtubule length, post-translational modification of microtubules, and microtubule network (MTN) density (Warner E. et al 2022). Microtubules link the extracellular space and contractile apparatus to the nucleus via the LINC complex, which enables the transmission of mechanical forces to regulate gene expression (Becker R. Cells 2020.9(6):1395).

[0094] Microtubules are constructed from α- and β-tubulin heterodimers. These tubulin subunits form higher-order assemblies containing protofilaments arranged in a hollow polymer. Microtubules are nucleated from microtubule organizing centers (MTOCs) containing γ-tubulin, which serve as structural templates for highly efficient nucleation (Uchida K. et al., 2022).

[0095] In mitotic cells, the centrosome MTOC continuously plays a role in microtubule nucleation throughout the cell's entire lifespan. However, when cardiomyocytes transform into postmitotic cells immediately after birth, the integrity of the centrosome is lost. Adult cardiomyocytes rely solely on the non-centrosome MTOC, which adorns the nuclear envelope and associated Golgi apparatus, for nucleation (Figure 5A, and Uchida K. et al., 2022). Thus, the entire cardiomyocyte nuclear envelope can be considered as the cellular MTOC.

[0096] Microtubule dysfunction is associated with various cardiac disorders, and variations in MTN density have been observed in post-translational microtubule modification (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) (Cheng G. et al., Am J Physiol Heart Circ Physiol. 2008;294:H2231-H2241).

[0097] The most well-studied post-translational modifications in cardiomyocytes are detyrosination and acetylation (Warner E. et al. 2022). Detyrosination enhances the biophysical rigidity of microtubules. Detyrosination by TCP (tubulin carboxypeptidase) preferentially occurs on microtubule polymers rather than tubulin dimers. Detyrosination removes the C-terminal tyrosine of α-tubulin.

[0098] Recent studies have demonstrated that enhanced microtubule detyrosination in myocyte cells, mediated by cross-linking of detyrosinated microtubules with desmin in the Z disc, is associated with increased mechanical resistance and reduced cell contraction under various pathophysiological conditions. Detyrosinated tubulin is significantly increased in patients with clinically diagnosed hypertrophic and dilated cardiomyopathy (HCM and DCM, respectively), along with a slight increase in total tubulin content (Robison P., et al. Science. 352, aaf0659 (2016)).

[0099] In cardiomyocytes, microtubules rich in acetylated α-tubulin increase cytoskeletal stiffness and viscoelastic resistance independently of changes in MTN density or detyrosinated microtubule levels (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 describes a decrease in α-tubulin acetylation levels in LMNA cardiomyopathy. The activity of HDAC6, a histone deacetylase that regulates tubulin deacetylation, increases under cardiac stress, and HDAC inhibitors have been shown to improve pressure overload hypertrophy and atrial fibrillation (Warner E. et al. 2022).

[0100] Microtubule-targeted therapies are currently available clinically for the treatment of cancer and neurodegenerative disorders. However, microtubule-targeted therapeutic interventions for the treatment of cardiovascular disease are limited to colchicine therapy, which is mainly due to the anti-inflammatory effects of colchicine at lower doses, such as in the treatment of gout (Warner E. et al. 2022).

[0101] LINC complex inhibition This disclosure relates to LINC complex inhibition. As used herein, “LINC complex inhibition” encompasses inhibition of LINC complex formation (i.e., inhibition of LINC complex assembly), disruption / degradation of LINC complex, and inhibition of LINC complex activity / function.

[0102] In some embodiments, LINC complex formation can be inhibited by inhibiting the gene and / or protein expression of the constituent proteins of the LINC complex. Examples of constituent proteins of the LINC complex include SUN domain-containing proteins and KASH domain-containing proteins. For brevity, in this disclosure, “constituent proteins of the LINC complex” may also be simply referred to as “LINC complex proteins.”

[0103] In some embodiments, inhibiting LINC complex formation 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 level of RNA encoding the LINC complex protein; inhibiting the transcription of nucleic acids encoding the LINC complex protein; increasing the degradation of RNA encoding the LINC complex protein; reducing the level of the LINC complex protein; disrupting normal post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding the LINC complex protein; and increasing the degradation of the LINC complex protein.

[0104] Gene expression can be determined by means well known to those skilled in the art. The levels of RNA encoding LINC complex constituent proteins can be determined by techniques such as RT-qPCR and Northern blotting. A decrease in the levels of RNA encoding LINC complex constituent proteins may result from, for example, a reduction in the transcription of nucleic acids encoding LINC complex proteins or an increase in the degradation of RNA encoding LINC complex proteins.

[0105] A reduction in the transcription of nucleic acids encoding LINC complex proteins may result from the inhibition of the assembly and / or activity of factors necessary for the transcription of DNA encoding LINC complex proteins. An increase in the degradation of RNA encoding LINC complex proteins may result from increased enzymatic degradation of RNA encoding LINC complex proteins, for example, as a result of RNA interference (RNAi), and / or a decrease in the stability of RNA encoding LINC complex proteins.

[0106] Protein expression can be determined by means well known to those skilled in the art. The levels of constituent proteins of the LINC complex can be determined, for example, by antibody-based methods including Western blotting, immunohistochemistry / cytochemistry, flow cytometry, and ELISA, or by reporter-based methods.

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

[0108] A decrease in LINC complex protein levels may result from, for example, a decrease in the level of RNA encoding LINC complex protein, reduced post-transcriptional processing of RNA encoding LINC complex protein, or increased degradation of LINC complex protein.

[0109] Disruption of normal post-transcriptional processing of LINC complex proteins may include, for example, reduced / altered splicing of mRNA precursors to mature mRNA encoding LINC complex proteins, reduced translation of mRNA encoding LINC complex proteins, or reduced / altered post-translational processing of LINC complex proteins.

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

[0111] In some embodiments, LINC complex formation can be inhibited by inhibiting the transport of LINC complex constituent proteins (e.g., SUN domain-containing proteins and / or KASH domain-containing proteins) and / or disrupting the normal subcellular localization of LINC complex constituent proteins. 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 inner 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 in the endoplasmic reticulum.

[0112] The intracellular localization of LINC complex constituent proteins can be analyzed using techniques known to those skilled in the art. Such techniques include, for example, immunocytochemistry and reporter-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 enable imaging of proteins in the ER, INM, and ONM. Using such methods, the levels / proportions of LINC complex constituent proteins can be analyzed in the nuclear envelope, inner nuclear membrane, and outer nuclear membrane.

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

[0114] In some embodiments, inhibiting the formation of the LINC complex includes one or more of the following: inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a KASH domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; and inhibiting the interaction between a KASH domain-containing protein and its interaction partner.

[0115] In some embodiments, LINC complex inhibition includes disruption / degradation of the LINC complex.

[0116] In some embodiments, disruption / degradation of the LINC complex includes one or more of the following: inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a KASH domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a KASH domain-containing protein and its interaction partner; and inhibiting the interaction between a KASH domain-containing protein and its interaction partner

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

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

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

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

[0121] LINC complex inhibition may be characterized by a decrease in the functional level of the LINC complex. In some embodiments, LINC complex inhibition may be determined by detecting a decrease in the correlation level of LINC complex function.

[0122] In certain embodiments intended herein, LINC complex inhibition is achieved by one or more of the following: modifying a gene encoding a SUN domain-containing protein to reduce / prevent its expression; modifying a gene encoding a KASH domain-containing protein to reduce / prevent its expression; inhibiting the expression of a SUN domain-containing protein by RNAi; inhibiting the expression of a KASH domain-containing protein by RNAi; or inhibiting the interaction between a SUN domain-containing protein and a KASH domain-containing protein.

[0123] In some embodiments, modifying the gene encoding a KASH domain-containing protein or a SUN domain-containing protein to reduce or prevent its expression is achieved using a site-directed nuclease (SSN) system (e.g., a CRISPR-based system) that targets the relevant gene. In some embodiments, RNAi-mediated inhibition of the expression of a KASH domain-containing protein or a SUN domain-containing protein is achieved using siRNA, miRNA, or shRNA that targets the relevant protein.

[0124] In some embodiments, inhibiting the interaction between a SUN domain-containing protein and a KASH domain-containing protein is achieved using a dominant-negative SUN domain-containing protein, a dominant-negative KASH domain-containing protein, a small molecule inhibitor of the interaction between a SUN domain-containing protein and a KASH domain-containing protein, a peptide mimetic of the KASH domain, or a peptide mimetic of the SUN domain. It will be understood that the inhibition of the interaction is between an endogenous SUN domain-containing protein and an endogenous KASH domain-containing protein.

[0125] LINC complex inhibitors Aspects of this disclosure relate to LINC complex inhibition using LINC complex inhibitors. “LINC complex inhibitor” means any agent capable of achieving LINC complex inhibition. Examples of LINC complex inhibitors include agents that inhibit the formation of the LINC complex (i.e., inhibit LINC complex assembly), disrupt / degrade the LINC complex, or inhibit LINC complex function. Such agents may also be effectors of the above-described LINC complex inhibition (i.e., directly or indirectly). LINC complex inhibitors are also referred to herein as LINC complex antagonists.

[0126] LINC complex inhibitors are described, for example, in WO2019 / 143300A1, WO2021 / 010898A1, and WO2023 / 101607A2. In some embodiments, the LINC complex inhibitors according to this disclosure are the LINC complex inhibitors described in WO2019 / 143300A1, WO2021 / 010898A1, and WO2023 / 101607A2, which are incorporated herein by reference in their entirety.

[0127] In some embodiments, LINC complex inhibitors inhibit the formation of the LINC complex, disrupt / degrade the LINC complex, inhibit LINC complex activity, inhibit the expression of the LINC complex protein gene and / or protein, modify the gene encoding the LINC complex protein to reduce / prevent its expression, reduce the level of RNA encoding the LINC complex protein, inhibit the transcription of nucleic acids encoding the LINC complex protein, increase the degradation of RNA encoding the LINC complex protein, reduce the level of the LINC complex protein, disrupt normal post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding the LINC complex protein, increase the degradation of the LINC complex protein, inhibit the transport of the LINC complex protein, and / or disrupt its normal intracellular localization, reduce the level / proportion of LINC complex protein localized to the nuclear envelope, reduce the level / proportion of SUN domain-containing protein associated with the inner nuclear membrane, and reduce the level / proportion of KASH domain-containing protein associated with the outer nuclear membrane. This increases the retention of LINC complex proteins in the endoplasmic reticulum, increases the level / proportion of LINC complex proteins localized in the endoplasmic reticulum, inhibits interactions between LINC complex constituent proteins, inhibits interactions between LINC complex proteins and their interaction partners, inhibits interactions between SUN domain-containing proteins and their interaction partners, inhibits interactions between KASH domain-containing proteins and their interaction partners, inhibits interactions between SUN domain-containing proteins and KASH domain-containing proteins, inhibits interactions between SUN domain-containing proteins and Lamine, inhibits interactions between SUN domain-containing proteins and chromatin-binding proteins, inhibits interactions between KASH domain-containing proteins and SUN domain-containing proteins, and inhibits interactions between KASH domain-containing proteins and cytoskeletal components (e.g., microfilaments or their components (e.g., actin), microtubules or their components (e.g., tubulin)).This may inhibit interactions between the LINC complex (or intermediate filaments or their components), increase the dismantling of the LINC complex, increase the degradation of the LINC complex, replace LINC complex proteins from the LINC complex, and / or reduce the level of the LINC complex.

[0128] It will be understood that a given LINC complex inhibitor may exhibit two or more of the properties listed in the preceding paragraph. A given drug may be evaluated for the properties listed in the preceding paragraph using a suitable assay. The assay may be, for example, an in vitro assay, optionally a cell-based assay, or a cell-free assay. If the assay is cell-based, it may involve treating cells with the test agent to determine whether the drug exhibits one or more of the listed properties. The assay may use endogenously or recombinantly expressed proteins, and may use species labeled with detectable entities to facilitate their detection.

[0129] Drugs capable of reducing the gene expression of LINC complex proteins (e.g., by reducing the level of RNA encoding LINC complex proteins, reducing the transcription of nucleic acids encoding LINC complex proteins, and / or increasing the degradation of RNA encoding LINC complex proteins) may be identified using assays that include detecting the level of RNA encoding the relevant protein, e.g., RT-qPCR. Such assays may include treating cells / tissues with the drug and then comparing the level of RNA encoding the relevant protein in such cells / tissues with the level of RNA encoding the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues). Assays for detecting reduction / alteration of the splicing of mRNA precursors 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 those isoforms.

[0130] Drugs capable of reducing the protein expression of LINC complex proteins (e.g., lowering the level of LINC complex proteins, increasing the degradation of LINC complex proteins) can be identified using assays that involve detecting the level of the relevant proteins, for example, using antibody / reporter-based methods (Western blotting, ELISA, immunohistochemistry, etc.). Such assays may involve treating cells / tissues with the drug and then comparing the level of the relevant protein in such cells / tissues to the level of the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues). Protein degradation assays may include, for example, evaluating the ubiquitination or proteasome localization of the relevant proteins, and / or the proportion of the relevant proteins that are ubiquitinated or localized to the proteasome.

[0131] Drugs that can inhibit the transport of LINC complex proteins and / or disrupt their normal intracellular localization may be identified using assays that include detecting the presence of the relevant protein at a given intracellular location or determining its proportion, for example, using antibody / reporter-based methods (such as Western blotting, ELISA, or immunohistochemistry). Intracellular localization may be analyzed, for example, by immunocytochemistry or by Western blotting of extracts prepared from different cell fractions, or by using organelle markers and / or labeled proteins of known intracellular localization. The assay may include treating cells / tissues with the drug and then comparing the intracellular localization of the relevant protein in such cells with the intracellular localization of the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues).

[0132] Drugs that can inhibit the interaction between LINC complex proteins and their interaction partners can be identified using assays that involve detecting the level of interaction between LINC complex proteins and their interaction partners, for example, using antibody / reporter-based methods. The level of interaction between LINC complex proteins and their interaction partners can be analyzed using, for example, resonance energy transfer techniques (e.g., FRET, BRET), co-immunoprecipitation, or methods that analyze the correlation of the interaction (e.g., LINC complex function). The assay may involve treating cells / tissues with the drug and then comparing the level of interaction in such cells / tissues to the level of interaction in cells / tissues under appropriate control conditions (e.g., untreated / vehicle-treated cells / tissues). The interaction between LINC complex proteins and their interaction partners can also be analyzed using techniques such as ELISA, surface plasmon resonance (SPR, see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or by radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. The assay may involve comparing the level of interaction in the presence of a drug to the level of interaction under appropriate control conditions (e.g., in the absence of the drug).

[0133] Drugs that can inhibit the function of the LINC complex may be identified using an assay that includes detecting the correlation level of LINC complex function.

[0134] The LINC complex inhibitors according to this disclosure may be any / more agents that achieve the desired inhibitory activity. In some embodiments, the LINC complex inhibitors may be or include peptides / polypeptides, small molecules, nucleic acids, or biomolecules.

[0135] In some embodiments, the LINC complex inhibitor can bind to the LINC complex, the LINC complex protein, or an interaction partner of the LINC complex protein.

[0136] LINC complex inhibitors may exhibit specific binding to the relevant factor / complex (i.e., the LINC complex protein, or the interaction partner of the LINC complex protein). As used herein, “specific binding” refers to a binding that is selective and can be distinguished from nonspecific binding to non-target molecules. LINC complex inhibitors that specifically bind to the LINC complex protein or the interaction partner of the LINC complex protein preferably bind to the relevant factor with higher affinity and / or for a longer duration than other non-target molecules. Such LINC complex inhibitors may be described as “specific” to the relevant factor.

[0137] In some embodiments, LINC complex inhibitors can inhibit the interaction between the LINC complex protein and its interaction partner. In some embodiments, LINC complex inhibitors can inhibit LINC complex function. In some embodiments, LINC complex inhibitors act as competitive inhibitors of the interaction between the LINC complex protein and its interaction partner. LINC complex inhibitors may occupy or otherwise reduce access to the region of the LINC complex protein necessary for binding to its interaction partner, or occupy or otherwise reduce access to the region of the LINC complex protein's interaction partner necessary for binding to the LINC complex protein.

[0138] In some embodiments, LINC complex inhibitors mimic the interaction partners of LINC complex proteins.

[0139] In some embodiments, LINC complex inhibitors inhibit the interaction between the SUN domain and the KASH domain.

[0140] In some embodiments, the LINC complex inhibitor inhibits the association 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's access to the deep pocket of the SUN domain. In some embodiments, the LINC complex inhibitor binds to the KASH domain and inhibits the KASH domain's access to the deep pocket on the surface of the SUN domain.

[0141] In some embodiments, LINC complex inhibitors inhibit or disrupt the formation of disulfide bonds between the SUN domain and the KASH domain.

[0142] In some embodiments, LINC complex inhibitors target the C-terminal proline-rich region of KASH domain-containing proteins.

[0143] In some embodiments, LINC complex inhibitors inhibit the oligomerization of SUN domain-containing proteins. In some embodiments, LINC complex inhibitors target the stem region of SUN domain-containing proteins.

[0144] In some embodiments, the LINC complex inhibitor inhibits the protein-protein interaction between the SUN domain and the KASH domain. In some embodiments, the LINC complex inhibitor inhibits the protein-protein interaction 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 the protein-protein interaction 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 interaction partners can be evaluated, for example, by analyzing the interaction in the presence of one or both of the interaction partners with the candidate LINC complex inhibitor, or after incubation. A suitable assay for determining whether a given binder can inhibit the interaction between the LINC complex protein and its interaction partners is competitive ELISA.

[0146] In some embodiments, molecules that bind to the LINC complex protein or its interaction partners inhibit the LINC complex protein's ability to bind to its interaction partners.

[0147] In some embodiments, LINC complex inhibitors can bind to the LINC complex protein or its interaction partner, thereby inhibiting the interaction between the LINC complex protein and its interaction partner.

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

[0149] In some embodiments, LINC complex inhibitors that can bind to the LINC complex protein or its interaction partner and inhibit the interaction between the LINC complex protein and its interaction partner may be, for example, peptides / polypeptides.

[0150] LINC complex inhibitors may be based, for example, on the interaction partner of the relevant factor to which the inhibitor binds (i.e., the LINC complex protein, or the interaction partner of the LINC complex protein).

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

[0152] For example, a LINC complex inhibitor that binds to a LINC complex protein may contain / consist of a peptide / polypeptide fragment of the LINC complex protein's interaction partner. Similarly, a LINC complex inhibitor that binds to a LINC complex protein's interaction partner may contain / consist of a peptide / polypeptide fragment of the LINC complex protein.

[0153] Such inhibitors preferably have the ability to bind to the relevant factor, but lack or exhibit reduced levels 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(s) necessary for binding to the relevant factor, but may lack the amino acid sequence(s) necessary for one or more other properties of the protein on which it is based.

[0154] Such peptide / polypeptide LINC complex inhibitors may be called “decoy,” “dominant-negative,” or “mimicking” versions of the protein on which they are based, and preferably exhibit competitive inhibition of the interaction between the LINC complex protein and its interaction partner. In this way, such peptide / polypeptide LINC complex inhibitors inhibit the formation of the LINC complex and / or disrupt the existing LINC complex through the substitution of its endogenous interaction partner, forming a non-functional complex / complex with reduced levels of function.

[0155] Crisp et al. J Cell Biol. (2006) 172(1):41-53 describes a dominant-negative version of the mouse Sun1 protein, and WO2019 / 143300A1 describes its humanized version. A further dominant-negative version of SUN1 is described in WO2023 / 101607A2. Dominant-negative SUN2 is described, for example, in Stewart-Hutchinson et al. Exp Cell Res. (2008) 314(8):1892-905.

[0156] Dominant-negative versions of KASH1 are described, for example, by 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. Dominant-negative versions of KASH2 are described, for example, by 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. Dominant-negative versions of KASH3 are described, for example, by Stewart-Hutchinson et al. Exp Cell Res. (2008) 314(8):1892-905. Dominant-negative versions of KASH4 are described, for example, by Roux et al. Proc.Natl.Acad.Sci.USA (2009) 106:2194-2199. A dominant-negative version of KASH5 is described, for example, in Horn et al. J Cell Biol (2013) 202:1023-1039.

[0157] In some embodiments, the peptide / polypeptide LINC complex inhibitor is based on a SUN domain-containing protein (e.g., the SUN domain-containing protein described herein). In some embodiments, the peptide / polypeptide LINC complex inhibitor is a decoy / dominant-negative version of the SUN domain-containing protein.

[0158] LINC complex inhibitors based on SUN domain-containing proteins may exhibit binding to KASH domain-containing proteins (e.g., KASH domain-containing proteins described herein), but lack or exhibit reduced levels of one or more other properties of the SUN domain-containing protein on which they are based (e.g., binding to SUN domain-containing proteins, nucleoplasmic proteins, lamins, and / or chromatin-binding proteins).

[0159] LINC complex inhibitors based on SUN domain-containing proteins may exhibit binding to SUN domain-containing proteins (e.g., SUN domain-containing proteins described herein), but lack or exhibit reduced levels of one or more other properties of the SUN domain-containing protein on which they are based (e.g., binding to KASH domain-containing proteins, nucleoplasmic proteins, lamins, and / or chromatin-binding proteins).

[0160] LINC complex inhibitors based on SUN domain-containing proteins preferably contain a SUN domain. In some embodiments, the LINC complex consists of or is essentially composed of a SUN domain. The peptide / polypeptide may lack the amino acid sequence(s) of the SUN domain-containing protein that constitutes the protein domains other than the SUN domain. The peptide / polypeptide preferably lacks the properties of the endogenous SUN domain-containing protein other than those mediated by the SUN domain. The peptide / polypeptide may function as a dominant-negative peptide / polypeptide that can inhibit the interaction between the endogenous SUN domain-containing protein and its endogenous interaction partner.

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

[0162] LINC complex inhibitors based on KASH domain-containing proteins may exhibit binding to SUN domain-containing proteins (e.g., SUN domain-containing proteins described herein), but lack or exhibit 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 (e.g., KASH domain-containing proteins described herein), but lack or exhibit reduced levels of one or more other properties of the KASH domain-containing protein on which they are based (e.g., binding to SUN domain-containing proteins, 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 essentially composed of a KASH domain. The peptide / polypeptide may lack the amino acid sequence(s) of the KASH domain-containing protein that constitutes the protein domains other than the KASH domain. The peptide / polypeptide preferably lacks the properties of the endogenous KASH domain-containing protein other than those mediated by the KASH domain. The peptide / polypeptide may function as a dominant-negative peptide / polypeptide that can inhibit the interaction between the endogenous KASH domain-containing protein and its endogenous interaction partner.

[0165] In some embodiments, peptide / polypeptide LINC complex inhibitors that can bind to LINC complex / LINC complex protein / LINC complex protein interaction partners and inhibit the interaction between LINC complex protein and LINC complex protein interaction partners, and / or LINC complex function include, for example, peptide aptamers, thioredoxin, monobodies, antikalin, Knitz domains, avimers, Nottin, finomers, atrimers, DARPin, afibodies, nanobodies (i.e., single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRP), Obodies, and fibronectin, which are outlined, for example, in their entirety by reference in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101 (also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al. See also al.,Mabs (2011) 3:38-48). Further peptide / polypeptide LINC complex inhibitors intended in connection with this disclosure 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, diabody, triabody, scFv-Fc, minibody, single-domain antibodies (e.g., VhH), etc.).

[0166] Such peptide / polypeptide LINC complex inhibitors can be identified by screening a library of relevant peptides / polypeptides for LINC complex inhibition.

[0167] LINC complex inhibitory polypeptides are described in more detail below.

[0168] In some embodiments, the LINC complex inhibitor is a low-molecular-weight inhibitor of the LINC complex. As used herein, “low-molecular-weight” refers to an organic compound with a low molecular weight (less than 1000 daltons, typically about 300–700 daltons).

[0169] Small molecule LINC complex inhibitors can bind to the LINC complex, LINC complex proteins, or LINC complex protein interaction partners. Small molecule LINC complex inhibitors can inhibit the interaction between LINC complex proteins and their interaction partners. Small molecule LINC complex inhibitors can bind to the LINC complex and inhibit LINC complex function.

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

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

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

[0173] Nucleic acid aptamers are outlined, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202. They may be identified and / or produced by the phylogenetic evolution of ligands by exponential enrichment (SELEX) method or by developing SOMAmers (slow offrate modified aptamers) (Gold L et al. (2010) PLoS ONE 5(12):e15004). Aptamers and SELEX are described in Turek and Gold, Science (1990) 249(4968):505-10 and WO91 / 19813.

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

[0175] Nucleic acid aptamers can be synthesized chemically, for example, on a solid support. Solid-phase synthesis can utilize phosphoramidite chemistry. Briefly, a solid-supported nucleotide is detritylated and then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester bond. Next, capping occurs, followed by oxidation of the phosphite triester with an oxidizing agent, typically iodine. The cycle can then be repeated to assemble the aptamer (see, for example, Sinha, ND; Biernat, J.; McManus, J.; Koester, 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 reduce or prevent the expression of endogenous LINC complex proteins.

[0177] Inhibition of LINC complex protein expression results in a reduction in the amount of LINC complex protein and / or the LINC complex including its constituent proteins in cells / tissues / organs / organ systems / targets. For example, in a given cell, inhibition of LINC complex protein expression results in a decrease in the level of the LINC complex including LINC complex protein and / or its constituent proteins compared to untreated cells.

[0178] Inhibition may be partial. A preferred degree of inhibition is at least 50%, more preferably one of at least 60%, 70%, 80%, 85%, or 90%. Inhibition levels between 90% and 100% are considered "silencing" of expression or function. Gene and protein expression may be determined as described herein or by methods of the art that are well known to those skilled in the art.

[0179] In some embodiments, LINC complex inhibitors reduce or prevent the expression of SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO. In some embodiments, LINC complex inhibitors reduce or prevent the expression of SUN1 or SUN2.

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

[0181] In some embodiments, LINC complex inhibitors may target specific domains / regions of the LINC complex protein. In some embodiments, LINC complex inhibitors reduce or prevent the expression of isoforms of the LINC complex protein that contain one or more target domains / regions.

[0182] For example, the domain or region of interest may be, or may contain, a domain necessary for interaction with the SUN domain, the KASH domain, and / or interaction partners of the LINC complex protein. For example, the domain or region of interest may be necessary for interaction with KASH domain-containing proteins, SUN domain-containing proteins, nucleoplasmic proteins, lamins, chromatin-binding proteins, cytoplasmic proteins, cytoskeletal proteins, microfilaments, actin, microtubules, tubulin, microtubule motors, and / or intermediate filament proteins.

[0183] In some embodiments, LINC complex inhibitors may alter the splicing of mRNA precursors encoding the LINC complex protein to increase the proportion of mature mRNA encoding isoforms lacking the relevant domain / region and / or decrease the proportion of mature mRNA encoding isoforms containing the relevant domain / region.

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

[0185] In some embodiments, the LINC complex inhibitor is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an antisense nucleic acid. In some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide (ASO). The antisense oligonucleotide may be single-stranded or may be bound by a complementary sequence that binds to a target oligonucleotide, such as mRNA.

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

[0187] Oligonucleotides designed to inhibit / prevent the expression of LINC complex proteins or specific isoforms may have substantial sequence identity with respect to the LINC complex protein / related isoform, or a portion of the nucleic acid encoding a complementary sequence thereto.

[0188] In some embodiments, inhibitory nucleic acids reduce the expression of LINC complex proteins by RNA interference (RNAi). RNAi involves inhibiting gene expression and translation by targeting and inactivating mRNA molecules. In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA).

[0189] The role of RNAi mechanisms and small RNAs in the targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has been demonstrated. Double-stranded RNA (dsRNA)-dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific homologous genes for silencing in a short period of time. They act as signals that promote the degradation of mRNA with sequence identity. 20nt siRNAs are generally long enough to induce gene-specific silencing but short enough to avoid a host response. Reduction in the expression of the target gene product can be extensive, ranging from 90% silencing induced by several molecules of siRNA. RNAi-based therapies are progressing to phase I, II, and III clinical trials for numerous indications (Nature 2009 Jan 22;457(7228):426-433).

[0190] In the art, such RNA sequences are referred to as “short or small interfering RNA” (siRNA) or “microRNA” (miRNA), depending on their origin. Both types of sequences may be used to downregulate gene expression by binding to complementary RNA and causing mRNA removal (RNAi) or by halting the translation of mRNA into protein. siRNA is induced by the processing of long double-stranded RNA and, when found in nature, is typically of exogenous origin. Microinterfering RNA (microRNA, miRNA) is an endogenously encoded small non-coding RNA induced by the processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNA carrying a partially complementary target sequence without RNA cleavage and can degrade mRNA carrying a fully complementary sequence.

[0191] siRNAs are 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 so that it is short enough to reduce the host response, ensuring correct recognition of the siRNA by the RISC complex that mediates siRNA-mediated recognition of the mRNA target.

[0192] miRNAs are typically single-stranded and possess a partially complementary region that allows a ligand to form a hairpin. miRNAs are RNA genes that are transcribed from DNA but not translated into protein. The DNA sequence encoding a miRNA gene is longer than the miRNA itself. This DNA sequence contains the miRNA sequence and its approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complementary base pair pair up to form a partially double-stranded RNA segment. The design of microRNA sequences is discussed, for example, in John et al., PLoS Biology, 11(2), 1862-1879, 2004.

[0193] Typically, oligonucleotides intended to mimic the effects of siRNA or miRNA have 10 to 40 ribonucleotides (or synthetic analogs thereof), more preferably 17 to 30 ribonucleotides, more preferably 19 to 25 ribonucleotides, and most preferably 21 to 23 ribonucleotides. In some embodiments of this disclosure using double-stranded siRNA, the molecule may have a symmetrical 3' overhang, e.g., one or two (ribo)nucleotides, typically a UU or dTdT3' overhang. Based on the disclosures provided herein, those skilled in the art can easily design suitable siRNA and miRNA sequences using resources such as the Ambion siRNA finder, for example. siRNA and miRNA sequences can be produced synthetically, exogenously added to induce gene downregulation, or produced 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 the siRNAs used to achieve this are, for example, Oestlund 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 Biol Cell. (2017) 28(1):182-191, Espigat-Georger et al., J Cell Sci. (2016) 129(22):4227-4237, Yang et al., Int J Mol This is described in Med.(2013)32(4):805-12, Rajgor et al., PLoS One.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 RNA may be processed intracellularly to produce siRNA (see, for example, Myers (2003) Nature Biotechnology 21:324-328). Longer dsRNA molecules may have symmetric 3' or 5' overhangs, e.g., one or two (ribo)nucleotides, or they may have blunt ends. Longer dsRNA molecules may have 25 or more nucleotides. Preferably, longer dsRNA molecules are 25-30 nucleotides long. More preferably, longer dsRNA molecules are 25-27 nucleotides long. Most preferably, longer dsRNA molecules are 27 nucleotides long. dsRNAs longer than 30 nucleotides can be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

[0196] Another option is the expression of short hairpin RNA molecules (shRNA) in cells. shRNA is more stable than synthetic siRNA. shRNA consists of short reverse repeat sequences separated by small loop sequences. One reverse repeat sequence is complementary to the target gene. In cells, shRNA is processed into siRNA by DICER, which degrades the target gene mRNA and represses its expression. In some embodiments, shRNA is produced intracellularly by transcription from a vector. shRNA may be produced intracellularly by transfecting cells with a vector encoding an shRNA sequence under the control of an RNA polymerase III promoter, such as the human H1 or 7SK promoter or the RNA polymerase II promoter. Alternatively, shRNA may be synthesized exogenously (in vitro) by transcription from a vector. The shRNA may then be directly introduced into cells. Preferably, the shRNA molecule contains a partial sequence of a gene encoding a LINC complex protein. Preferably, the shRNA sequence is 40 to 100 nucleotides long, more preferably 40 to 70 nucleotides long. The stem portion of the hairpin is preferably 19 to 30 base pairs long. The stem portion may contain GU pairs to stabilize the hairpin structure.

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

[0198] In some embodiments, the inhibitory nucleic acid is a splice-switching oligonucleotide (SSO). Splice-switching oligonucleotides are outlined, 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 a target RNA transcript by blocking RNA-RNA base pairing and / or protein-RNA binding interactions that occur between the components of the splicing mechanism and the mRNA precursor. Using SSOs, the number / proportion of mature mRNA transcripts encoding the LINC complex protein or a particular isoform(s) may be altered. SSOs may be designed to target a specific region of the target transcript, for example, by skipping a desired exon(s), e.g., an exon encoding a desired domain / region.

[0199] SSO generally involves modifications to the oligonucleotide sugar-phosphate backbone to prevent RNAseH degradation, and may include, for example, phosphorodiamidate morpholino (PMO), peptide nucleic acid (PNA), roch nucleic acid (LNA), and / or 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications.

[0200] Inhibitory nucleic acids can be recombinantly produced, for example, by transcription of nucleic acid sequences contained within a vector. Transcription may be performed in a cell-free transcription reaction or within cells containing the nucleic acid encoding the inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is produced intracellularly, for example, by transcription from a vector. A vector encoding such a molecule may be introduced into cells by any method known in the art. Optionally, nucleic acid expression can be regulated using cell / tissue-specific promoters (e.g., heart, muscle).

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

[0202] In some embodiments, LINC complex inhibitors are molecules / groups of molecules that can modify the nucleic acid encoding the LINC complex protein to reduce / prevent the expression of the LINC complex protein or a specific isoform(s) of that protein.

[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 an insertion, substitution, or deletion 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 shortened version and / or a non-functional version 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 version of the LINC complex protein.

[0205] The modification may be of a nucleic acid contained within the cell, such as an endogenous nucleic acid encoding the LINC complex protein. This modification results in a decrease in the level of gene and / or protein expression of the LINC complex protein or its specific isoform compared to an equivalent unmodified cell.

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

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

[0208] Methods for modifying nucleic acids encoding target proteins and agents for achieving this are well known in the art, and include, for example, modification of target nucleic acids by homologous recombination and targeted nucleic acid editing using site-directed nucleases (SSNs). For example, the inventors demonstrate CRISPR / Cas9-mediated disruption of SUN1 and Nesprin-1 in the experimental example WO2021 / 010898A1.

[0209] A preferred method may be homologous recombination targeting, as outlined, for example, 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 in their entirety). Homologous recombination targeting involves the exchange of nucleic acid sequences via a crossover event induced by homologous sequences.

[0210] In some embodiments, the method employs targeted nucleic acid editing using SSNs. Gene editing using SSNs is outlined, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct;48(10):e265, which is incorporated herein by reference in its entirety. An enzyme capable of producing site-directed double-strand breaks (DSBs) can be manipulated to introduce DSBs into the desired target nucleic acid sequence(s). The DSBs may be repaired by either error-prone non-homologous end joining (NHEJ), in which the two ends of the break are rejoined, often by nucleotide insertion or deletion. Alternatively, the DSBs may be repaired by homologous recombination repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced into the DSB site.

[0211] SSNs that can be manipulated to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered, regularly arranged palindromic repeat / CRISPR-related 9 (CRISPR / Cas9) systems.

[0212] The ZFN system is described, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is incorporated herein by reference in its entirety. A ZFN comprises a programmable zinc finger DNA-binding domain and a DNA-cleaving domain (e.g., a FokI endonuclease domain). The DNA-binding domain can be identified by screening Zinc Finger arrays capable of binding to target nucleic acid sequences.

[0213] The TALEN system is outlined, for example, in 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-cleaving domain (e.g., a FokI endonuclease domain). TALE contains a repeat domain consisting of 33-39 amino acid repeats, each repeat identical except for two residues at positions 12 and 13, which are repeat variable two residues (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the 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, are outlined, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, which is incorporated herein by reference in its entirety. These systems include an endonuclease (e.g., Cas9, Cpf1, etc.) and a single guide RNA (sgRNA) molecule. The sgRNA can be engineered so that its endonuclease activity targets a desired nucleic acid sequence.

[0215] In some embodiments, LINC complex inhibition utilizes a site-directed nuclease (SSN) system that targets the LINC complex protein. Therefore, in some embodiments, the LINC complex inhibitor comprises or consists of an SSN system that targets the LINC complex protein. In some embodiments, the LINC complex inhibitor utilizes a nucleic acid(s) encoding an SSN system that targets the LINC complex protein.

[0216] In some embodiments, the SSN system targets a region of nucleic acid encoding a LINC complex protein involved in (e.g., required for) LINC complex formation. For example, in some embodiments, the SSN system can disrupt the expression of an exon of a gene encoding a SUN domain-containing protein encoding a SUN domain. In some embodiments, the SSN system can disrupt the expression of an exon of a gene encoding a KASH domain-containing protein encoding a KASH domain.

[0217] In certain embodiments, the SSN system may introduce an insertion / deletion upstream of a sequence encoding a conserved tyrosine residue in the nucleic acid sequence of a gene encoding a SUN domain-containing protein.

[0218] In some embodiments, the SSN system may introduce an insertion / deletion upstream of the tyrosine-coding sequence at the position 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) in the nucleic acid sequence of the gene encoding the SUN domain-containing protein.

[0219] In certain embodiments, the SSN system may introduce an insertion / deletion upstream of a sequence encoding a conserved tyrosine residue in the nucleic acid sequence of a gene encoding a SUN domain-containing protein.

[0220] In some embodiments, the SSN system may introduce an insertion / deletion upstream of the sequence encoding the C-terminal proline-rich region in the nucleic acid sequence of the gene encoding the KASH domain-containing protein.

[0221] In some embodiments, the SSN system may introduce an insertion / deletion upstream of the proline-coding sequence at the position 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) in the nucleic acid sequence of the gene encoding the KASH domain-containing protein.

[0222] In some embodiments, 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 embodiments, the SSN system is a CRISPR / Cas9 system. In such embodiments, LINC complex inhibition may use nucleic acids (may include) encoding a CRISPR RNA (crRNA) that targets the nucleic acid encoding the LINC complex protein, and a transactivating crRNA (tracrRNA) for processing the crRNA into its mature form. CRISPR / Cas9 systems for targeted disruption of 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] Instead of disrupting the LINC complex by expressing components of the luminal domain or KASH domain of a SUN domain-containing protein and competing for binding with endogenous Nesprin (containing the KASH domain) or SUN1 and SUN2 (containing the SUN domain), another approach to disrupting the LINC complex is to modify the endogenous SUN domain or KASH domain so that it does not bind to its congeneral LINC complex-binding partner or has reduced binding ability.

[0225] Since both the SUN and KASH domains are located at the C-terminus of their respective proteins, one way to produce modified SUN or KASH domains is to modify the gene encoding the SUN or KASH domain protein using the CRISPR / Cas system, generating an early stop codon at the 3' end of the respective protein sequence following CRISPR-induced non-homologous end junctioning. This results in a truncated protein with a mutated SUN or KASH domain at the C-terminus. The truncated protein is expressed and membrane-localized, but cannot interact with its congeneral LINC complex partners.

[0226] Therefore, in some embodiments, the LINC complex inhibitor is a CRISPR-Cas or other synthetic nuclease system that can modify the nucleic acid encoding the SUN domain or KASH domain of the endogenous SUN or Nesprin protein, respectively.

[0227] In some embodiments, the CRISPR-Cas system disrupts the LINC complex by modifying the endogenous SUN domain or KASH domain of the SUN1 or Nesprin-1 protein, respectively. The nucleic acids are SUN1 and Syne1, respectively.

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

[0229] LINC complex inhibitory polypeptide In some embodiments, the LINC complex inhibitors according to this disclosure are LINC complex inhibitory polypeptides.

[0230] In this specification, “LINC complex inhibitory polypeptide” refers to a polypeptide that inhibits the formation and / or function of the LINC complex. LINC complex inhibitory polypeptides are described, for example, in WO2019 / 143300A1, WO2021 / 010898A1, and WO2023 / 101607A2. In some embodiments, the LINC complex inhibitors according to this disclosure are LINC complex inhibitory polypeptides according to any embodiment described in WO2019 / 143300A1, WO2021 / 010898A1, or WO2023 / 101607A2, all of which are incorporated herein by reference in their entirety.

[0231] The LINC complex inhibitory polypeptides according to this disclosure preferably bind to the LINC complex protein. The binding may be characterized by a non-covalent protein:protein interaction between the LINC complex inhibitory polypeptide and the LINC complex protein. The 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 polypeptide according to this disclosure has an affinity in the micromolar range, i.e., K D = 9.9 × 10 -4 ~1 × 10 -6 At M, the SUN domain-containing protein binds to an interaction partner (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In some embodiments, the LINC complex inhibitory polypeptide has submicromolar affinity, i.e., K D <1×10 -6 At M, the SUN domain-containing protein binds to an interaction partner (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In some embodiments, the LINC complex inhibitory polypeptide has an affinity in the nanomolar range, i.e., K D=9.9×10 -7 ~1×10 -9 M, and binds to an interaction partner of a SUN domain-containing protein (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In some embodiments, the LINC complex-inhibiting polypeptide binds with subnanomolar affinity, i.e., K D <1×10 -9 M, and binds to an interaction partner of a SUN domain-containing protein (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In some embodiments, the LINC complex-inhibiting polypeptide binds with affinity in the picomolar range, i.e., K D =9.9×10 -10 ~1×10 -12 M, and binds to an interaction partner of a SUN domain-containing protein (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In some embodiments, the LINC complex-inhibiting polypeptide binds with subpicomolar affinity, i.e., K D =<1×10 -12 M affinity to an interaction partner of a SUN domain-containing protein (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP).

[0233] The LINC complex inhibitory polypeptides according to this disclosure may bind to an interaction partner of a SUN domain-containing protein (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) in the form of a SUN domain-containing protein (e.g., a SUN domain-containing protein selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO). In some embodiments, the LINC complex inhibitory polypeptide may bind to the same or overlapping region of the interaction partner of the SUN domain-containing protein as the region bound by the SUN domain-containing protein.

[0234] The region of a given target molecule to which a polypeptide binds can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray cocrystallization of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolytic "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 polypeptides according to this disclosure inhibit the interaction between a SUN domain-containing protein (e.g., a SUN domain-containing protein selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) and an interaction partner of the SUN domain-containing protein (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). The inhibition of the interaction between a SUN domain-containing protein and an interaction partner of the SUN domain-containing protein as described herein includes inhibiting the binding of the SUN domain-containing protein to the interaction partner of the SUN domain-containing protein (thus inhibiting the formation of a complex containing such protein) and disrupting the complex containing the SUN domain-containing protein and the interaction partner of the SUN domain-containing protein (e.g., by substitution of the constituent proteins of such complex and the resulting disintegration of such complex).

[0236] In some embodiments, the LINC complex inhibitory polypeptide inhibits the binding of a SUN domain-containing protein (e.g., a SUN domain-containing protein selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) to an interaction partner of the SUN domain-containing protein (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In some embodiments, the LINC complex inhibitory polypeptide is a competitive inhibitor of the binding of a SUN domain-containing protein to an interaction partner of the SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide blocks the binding of a SUN domain-containing protein to an interaction partner of the SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide occupies the region of the interaction partner of the SUN domain-containing protein to which the SUN domain-containing protein binds, thereby inhibiting the interaction between the SUN domain-containing protein and its interaction partner. In some embodiments, the LINC complex inhibitory polypeptide replaces the SUN domain-containing protein in a complex that includes the SUN domain-containing protein and its interaction partner.

[0237] The ability of a given polypeptide to inhibit the interaction between two factors can be determined, for example, by analyzing the correlation of such interactions in the presence of one or both of its interaction partners, or after their incubation.

[0238] The analysis may involve detecting complexes containing SUN domain-containing proteins / interaction partners of SUN domain-containing proteins / SUN domain-containing proteins and their interaction partners. Such techniques are well known to those skilled in the art and include, for example, antibody / reporter-based methods (Western blotting, ELISA, immunohistochemistry, etc.).

[0239] Polypeptides that inhibit a given interaction (e.g., the interaction between a SUN domain-containing protein and its interaction partner, e.g., a KASH domain-containing protein) are identified by observing a decrease / reduction in the level of correlation of the interaction between the interaction partners in the presence of one or both of the polypeptide and its interaction partner, or after incubation of both, compared to the level observed in the absence of the polypeptide (or in the presence of a suitable control polypeptide known not to inhibit the interaction between the interaction partners). Suitable analyses can be performed in vitro, for example, using recombinant interaction partners or cells expressing the interaction partners. Cells expressing the interaction partners may be endogenous or derived from nucleic acids introduced into the cells. For the purposes of such assays, one or both of the interaction partners and / or polypeptides may be labeled or used in conjunction with a detectable entity for the purpose of detecting and / or measuring the level of interaction.

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

[0241] polypeptides that inhibit the interaction between a SUN domain-containing protein (e.g., a SUN domain-containing protein selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) and its interaction partner (e.g., a KASH domain-containing protein selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), for example polypeptides that inhibit the formation of a complex comprising a SUN domain-containing protein and its interaction partner, and / or polypeptides that disrupt a complex comprising a SUN domain-containing protein and its interaction partner, in the absence of the polypeptide (or in the presence of a suitable control polypeptide known not to inhibit the interaction between the interaction partners) Compared to the levels observed in ), in the presence of one or both of the polypeptide interaction partners, or after incubation thereof, one or more of the following observations may be identified: (i) a decrease in the level of the complex comprising the SUN domain-containing protein and the SUN domain-containing protein interaction partner; (ii) a decrease in the level of the functional properties of the complex comprising the SUN domain-containing protein and the SUN domain-containing protein interaction partner; (iii) a decrease in the level of the correlation of downstream activity mediated by the complex comprising the SUN domain-containing protein and the SUN domain-containing protein interaction partner; (iv) an increase in the level of free (i.e., uncomplexed) SUN domain-containing protein; and / or (v) an increase in the level of free (i.e., uncomplexed) interaction partner of the SUN domain-containing protein.

[0242] The LINC complex inhibitory polypeptides described herein can disrupt the normal intracellular localization of LINC complex proteins (e.g., SUN domain-containing proteins, or interaction partners of SUN domain-containing proteins).

[0243] In some embodiments, the LINC complex inhibitory polypeptide reduces the level / proportion of LINC complex proteins localized to the nuclear envelope. In some embodiments, the LINC complex inhibitory polypeptide reduces the level / proportion of interaction partners of the LINC complex inhibitory polypeptide localized to the nuclear envelope. In some embodiments, the LINC complex inhibitory polypeptide reduces the level / proportion of KASH domain-containing proteins associated with the outer nuclear membrane. In some embodiments, the LINC complex inhibitory polypeptide reduces the level / proportion of SUN domain-containing proteins associated with the inner nuclear membrane.

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

[0245] In some embodiments, the LINC complex inhibitory polypeptide increases the level / proportion of LINC complex proteins localized in the endoplasmic reticulum. In some embodiments, the LINC complex inhibitory polypeptide 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 in the endoplasmic reticulum. In some embodiments, the LINC complex inhibitory polypeptide increases the level / proportion of SUN domain-containing proteins that are not associated with the inner nuclear membrane (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO).

[0246] The intracellular localization of LINC complex constituent proteins can be analyzed using techniques known to those skilled in the art. Such techniques include, for example, immunocytochemistry and reporter-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 enable imaging of proteins in the ER, INM, and ONM. Using such methods, the levels / proportions of LINC complex constituent proteins can be analyzed in the nuclear envelope, inner nuclear membrane, and outer nuclear membrane.

[0247] LINC complex inhibitory polypeptides that disrupt the normal intracellular localization of LINC complex proteins can be identified using assays that involve detecting the presence or percentage of the relevant protein(s) at a given intracellular location, for example, using antibody / reporter-based methods (Western blotting, ELISA, immunohistochemistry, etc.). Intracellular localization may be analyzed, for example, by immunocytochemistry or by Western blotting of extracts prepared from different cell fractions, using organelle markers and / or labeled proteins of known intracellular localization.

[0248] The assay may include expressing a putative LINC complex inhibitory polypeptide intracellularly (e.g., from a nucleic acid encoding a polypeptide introduced into cells (e.g., by transfection / transduction)) and then comparing the intracellular localization of the associated LINC complex protein(s) in such cells with the intracellular 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 polypeptide known not to affect the intracellular localization of the associated LINC complex protein(s)).

[0249] In some embodiments, polypeptides may be evaluated for their ability to function as LINC complexes that essentially inhibit polypeptides, as described in Example 1 of WO2023 / 101607A2.

[0250] Nucleic acids for expressing a putative LINC complex inhibitory polypeptide may be introduced into cells (e.g., by transfection), and the cells may then be evaluated to determine the intracellular localization of one or more LINC complex proteins (e.g., SUN domain-containing proteins (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) and / or interaction partners of SUN domain-containing proteins (e.g., KASH domain-containing proteins selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP)). A polypeptide can be identified as a LINC complex inhibitory polypeptide if its expression is determined in such assay to decrease the level / percentage of LINC complex proteins localized to the nuclear envelope or increase the level / percentage of LINC complex proteins not localized to the nuclear envelope (for example, its expression decreases the level / percentage of KASH domain-containing proteins associated with the outer nuclear membrane, increases the level / percentage of KASH domain-containing proteins not associated with the outer nuclear membrane, or increases the level / percentage of KASH domain-containing proteins localized to the endoplasmic reticulum) compared to the level / percentage observed in cells under appropriate control conditions (e.g., untransfected cells, cells transfected with an empty vector, or cells transfected with a nucleic acid encoding a polypeptide known not to affect the intracellular localization of the relevant LINC complex proteins).

[0251] In some embodiments, cellular expression of LINC complex inhibitory polypeptides according to this disclosure (i) reduces the level of interaction between constituent proteins of the LINC complex (e.g., between SUN domain-containing proteins and KASH domain-containing proteins), (ii) reduces the level of the LINC complex (e.g., a LINC complex comprising SUN domain-containing proteins and KASH domain-containing proteins), (iii) reduces the level of function / activity correlation of the LINC complex (e.g., a LINC complex comprising SUN domain-containing proteins and KASH domain-containing proteins), (iv) reduces the level / proportion of LINC complex proteins localized to the nuclear envelope (e.g., SUN domain-containing proteins or KASH domain-containing proteins), and / or (v) reduces KASH domain-containing proteins localized to the nuclear envelope / outer nuclear membrane (e.g., Nes The level / percentage of a LINC complex inhibitory polypeptide (selected from prin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) is reduced to less than 1x / 100% of the level observed in a given assay in the absence of the LINC complex inhibitory polypeptide or under appropriate control conditions, e.g., ≤0.99x / ≤99%, ≤0.95x / ≤95%, ≤0.9x / ≤90%, ≤0.85x / ≤85%, ≤0.8x / ≤80%, ≤0.7x. Reduce the value to one of the following: 5x / ≦75%, ≦0.7x / ≦70%, ≦0.65x / ≦65%, ≦0.6x / ≦60%, ≦0.55x / ≦55%, ≦0.5x / ≦50%, ≦0.45x / ≦45%, ≦0.4x / ≦40%, ≦0.35x / ≦35%, ≦0.3x / ≦30%, ≦0.25x / ≦25%, ≦0.2x / ≦20%, ≦0.15x / ≦15%, ≦0.1x / ≦10%, ≦0.05x / ≦5%, or ≦0.01x / ≦1%.

[0252] The preferred reduction level according to the preceding paragraph is a reduction to less than 0.5 times / ≤50%, for example, to one of ≤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, cellular expression of LINC complex inhibitory polypeptides according to this disclosure (i) increases the level / proportion of LINC complex proteins not localized to the nuclear envelope (e.g., SUN domain-containing proteins or KASH domain-containing proteins), and / or (ii) increases the level / proportion of LINC complex proteins localized to the endoplasmic reticulum (e.g., SUN domain-containing proteins or KASH domain-containing proteins), thereby increasing the LINC complex level / proportion of LINC complex proteins. In the absence of a synthesis-inhibiting polypeptide, or under appropriate control conditions, the level is increased to more than 1x of the level observed in a given assay, for example, to one of the following: ≥1.01x, ≥1.02x, ≥1.03x, ≥1.04x, ≥1.05x, ≥1.1x, ≥1.2x, ≥1.3x, ≥1.4x, ≥1.5x, ≥1.6x, ≥1.7x, ≥1.8x, ≥1.9x, ≥2x, ≥3x, ≥4x, ≥5x, ≥6x, ≥7x, ≥8x, ≥9x, ≥10x, ≥50x, or ≥100x.

[0254] Polypeptides can be evaluated for specific functional properties in appropriate in vivo models. For example, polypeptides can be evaluated in vivo for therapeutic / preventive effects in non-human animal models of the diseases / conditions described herein. In such assays, polypeptides can be delivered in the form of protein-encoding nucleic acids using, for example, a viral vector having appropriate tropism to the cells / tissues to which LINC complex inhibition confers therapeutic / preventive benefits.

[0255] In such a model, the lifespan and survival of the subject can be evaluated by monitoring survival over time. Cardiac function and myocardial contractility can be evaluated by measuring their correlations, and ejection fraction, shortening ratio, left ventricular posterior wall thickness and / or left ventricular diameter can be measured by echocardiography / ultrasound.

[0256] It will be understood that subjects are preferably evaluated for one or more of the characteristics listed in the preceding paragraph at a specific point in time, for example, after a sufficient period has passed for the effects of polypeptide administration in the relevant model to be observed. For example, subjects may be evaluated at least 7 days after administration of a viral vector encoding a putative LINC complex inhibitory polypeptide.

[0257] In some embodiments, administration of LINC complex inhibitory polypeptides according to this disclosure to subjects having cardiomyopathy (e.g., cardiomyopathy associated with mutations to DSP; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) extends the survival or lifespan of the subjects and / or improves the subjects' cardiac function, myocardial contractility, ejection fraction, shortening rate, and / or left ventricular posterior wall thickness in the absence of such treatment or using an appropriate control polypeptide (e.g., using the same viral vector). To increase the level observed after administration of a polypeptide known not to inhibit the LINC complex delivered by means of (for example, ≥1.01, ≥1.02, ≥1.03, ≥1.04, ≥1.05, ≥1.1, ≥1.2, ≥1.3, ≥1.4, ≥1.5, ≥1.6, ≥1.7, ≥1.8, ≥1.9, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, ≥50, or ≥100.

[0258] In some embodiments, administration of the LINC complex inhibitory polypeptide according to this disclosure to a subject having cardiomyopathy (e.g., cardiomyopathy associated with mutations to DSP; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) reduces the subject's left ventricular diameter to less than 1x / 100% of the level observed in the absence of such treatment or after administration of a suitable control polypeptide (e.g., a polypeptide known not to inhibit the LINC complex, e.g., delivered using the same viral vector), e.g., ≤0.99x / ≤99%, ≤ Reduce to one of the following: 0.95x / ≦95%, ≦0.9x / ≦90%, ≦0.85x / ≦85%, ≦0.8x / ≦80%, ≦0.75x / ≦75%, ≦0.7x / ≦70%, ≦0.65x / ≦65%, ≦0.6x / ≦60%, ≦0.55x / ≦55%, ≦0.5x / ≦50%, ≦0.45x / ≦45%, ≦0.4x / ≦40%, ≦0.35x / ≦35%, ≦0.3x / ≦30%, ≦0.25x / ≦25%, ≦0.2x / ≦20%, ≦0.15x / ≦15%, ≦0.1x / ≦10%, ≦0.05x / ≦5%, or ≦0.01x / ≦1%.

[0259] In some embodiments, cellular expression of LINC complex inhibitory polypeptides according to this disclosure (i) reduces the level of interaction between constituent proteins of the LINC complex (e.g., between SUN domain-containing proteins and KASH domain-containing proteins), (ii) reduces the level of the LINC complex (e.g., the LINC complex comprising SUN domain-containing proteins and KASH domain-containing proteins), (iii) reduces the level of function / activity correlation of the LINC complex (e.g., the LINC complex comprising SUN domain-containing proteins and KASH domain-containing proteins), (iv) reduces the level / proportion of LINC complex proteins localized to the nuclear envelope (e.g., SUN domain-containing proteins or KASH domain-containing proteins), and / or (v) reduces the level of KASH domain-containing proteins localized to the nuclear envelope / outer nuclear membrane (e.g., Nes The level / percentage of LINC complex inhibitory polypeptides (selected from prin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) is defined as less than 1x / less than 100% of the level observed in the absence of cellular expression of LINC complex inhibitory polypeptides (e.g., by the same type of cells), e.g., ≤0.99x / ≤99%, ≤0.95x / ≤95%, ≤0.9x / ≤90%, ≤0.85x / ≤85%, ≤0.8x / ≤80%, ≤0.7x. Reduce the value to one of the following: 5x / ≦75%, ≦0.7x / ≦70%, ≦0.65x / ≦65%, ≦0.6x / ≦60%, ≦0.55x / ≦55%, ≦0.5x / ≦50%, ≦0.45x / ≦45%, ≦0.4x / ≦40%, ≦0.35x / ≦35%, ≦0.3x / ≦30%, ≦0.25x / ≦25%, ≦0.2x / ≦20%, ≦0.15x / ≦15%, ≦0.1x / ≦10%, ≦0.05x / ≦5%, or ≦0.01x / ≦1%.

[0260] In some embodiments, cellular expression of LINC complex inhibitory polypeptides according to this disclosure increases (i) the level / proportion of LINC complex proteins not localized to the nuclear envelope (e.g., SUN domain-containing proteins or KASH domain-containing proteins) and / or (ii) the level / proportion of LINC complex proteins localized to the endoplasmic reticulum (e.g., SUN domain-containing proteins or KASH domain-containing proteins), (e.g., It increases the level of LINC complex inhibitory polypeptides (by cells of the same type) to more than 1x the level observed in the absence of cellular expression of the LINC complex inhibitory polypeptide, for example, to one of the following: ≥1.01x, ≥1.02x, ≥1.03x, ≥1.04x, ≥1.05x, ≥1.1x, ≥1.2x, ≥1.3x, ≥1.4x, ≥1.5x, ≥1.6x, ≥1.7x, ≥1.8x, ≥1.9x, ≥2x, ≥3x, ≥4x, ≥5x, ≥6x, ≥7x, ≥8x, ≥9x, ≥10x, ≥50x, or ≥100x.

[0261] In some embodiments, administration of the LINC complex inhibitory polypeptide according to the present disclosure to a subject having cardiomyopathy (e.g., cardiomyopathy associated with mutations to DSP; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) extends the subject's survival or lifespan and / or increases the subject's cardiac function, myocardial contractility, ejection fraction, shortening ratio, and / or left ventricular posterior wall thickness to more than 1 times the level observed in the absence of such administration, for example, ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times, ≥10 times, ≥50 times, or ≥100 times.

[0262] In some embodiments, administration of the LINC complex inhibitory polypeptide according to this disclosure to a subject having cardiomyopathy (e.g., cardiomyopathy associated with mutations to DSP; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) reduces the subject's left ventricular diameter to less than 1 / 100% of the level observed in the absence of such administration, e.g., ≤0.99 / ≤99%, ≤0.95 / ≤95%, ≤0.9 / ≤90%, ≤0.85 / ≤85%, ≤0.8 Reduce to one of the following: ≤80%, ≤0.75x / ≤75%, ≤0.7x / ≤70%, ≤0.65x / ≤65%, ≤0.6x / ≤60%, ≤0.55x / ≤55%, ≤0.5x / ≤50%, ≤0.45x / ≤45%, ≤0.4x / ≤40%, ≤0.35x / ≤35%, ≤0.3x / ≤30%, ≤0.25x / ≤25%, ≤0.2x / ≤20%, ≤0.15x / ≤15%, ≤0.1x / ≤10%, ≤0.05x / ≤5%, or ≤0.01x / ≤1%.

[0263] The LINC complex inhibitory polypeptides according to this disclosure may be based on a SUN domain-containing protein selected from, for example, SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO. In some embodiments, the LINC complex inhibitory polypeptides according to this disclosure include an inhibitory region based on a SUN domain-containing protein selected from, for example, SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO.

[0264] The “inhibitory region” of a LINC complex inhibitory polypeptide refers to the region of the polypeptide in which LINC complex inhibition is achieved. The inhibitory region according to this disclosure typically has high sequence identity with a portion of the amino acid sequence of a SUN domain-containing protein. The inhibitory region of a LINC complex inhibitory polypeptide according to this disclosure may be a region in which the LINC complex inhibitory polypeptide binds to an interaction partner of a SUN domain-containing protein (e.g., one or more KASH domain-containing proteins, e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). In aspects and embodiments of this disclosure, the inhibitory region of a LINC complex inhibitory polypeptide includes an amino acid sequence corresponding to the α3 helix of the CC2 region and the SUN domain of a SUN domain-containing protein.

[0265] The LINC complex inhibitory polypeptides according to this disclosure include, or may be essentially, amino acid sequences 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) to all or part of the amino acid sequences of SUN domain-containing proteins selected from, for example, SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO. The LINC complex inhibitory polypeptides according to this disclosure may include, for example, 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) to all or part of the amino acid sequence of a SUN domain-containing protein selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO, or an inhibitory region comprising such an amino acid sequence.

[0266] Such LINC complex inhibitory polypeptides preferably (i) retain the ability of their base SUN domain-containing protein to bind to KASH domain-containing proteins (e.g., one or more of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), but (ii) lack or exhibit reduced levels of one or more other properties of their base SUN domain-containing protein. In preferred embodiments, the LINC complex inhibitory polypeptide exhibits a lack of or reduced ability to bind to nuclear lamin and / or chromatin-binding proteins, and / or a lack of or reduced ability to associate with the inner nuclear membrane (e.g., localize to the inner nuclear membrane), compared to the base SUN domain-containing protein.

[0267] For example, the LINC complex inhibitory polypeptide / inhibitory region according to this disclosure may include an amino acid sequence(s) necessary for binding to a KASH domain-containing protein (e.g., one or more of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), but may lack an amino acid sequence(s) necessary for one or more other properties of the SUN domain-containing protein on which it is based (e.g., an amino acid sequence(s) necessary for binding to nuclear lamin and / or chromatin-binding proteins, and / or an amino acid sequence(s) necessary for association with the inner nuclear membrane).

[0268] As used herein, a polypeptide / amino acid sequence / domain "essentially derived from" a reference amino acid sequence is (i) consisting of the reference amino acid sequence or (ii) containing the reference amino acid sequence, the reference amino acid sequence constituting at least 80% of the polypeptide / amino acid sequence (e.g., one of ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%). It will be understood that a polypeptide or amino acid sequence "essentially derived from" a reference amino acid sequence may contain additional amino acids(s) at the reference amino acid sequence and either or both of the N-terminus and C-terminus of the reference amino acid sequence, provided that the additional amino acids(s) constitute no more than 20% of the polypeptide / amino acid sequence.

[0269] As an example, the 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 1-197 of SEQ ID NO: 74), and the amino acid sequence of SEQ ID NO: 44 constitutes approximately 98% (i.e., 197 / 201 residues) of the polypeptide. SEQ ID NO: 74 contains the KDEL-retaining motif (SEQ ID NO: 77) at the C-terminus of the amino acid sequence of SEQ ID NO: 44.

[0270] In some embodiments, it will be understood that the LINC complex inhibitory polypeptides according to this disclosure essentially consist of a KASH domain-containing protein-binding fragment of a SUN domain-containing protein. Similarly, in some embodiments, it will be understood that the inhibitory region of the LINC complex inhibitory polypeptides according to this disclosure consists of a KASH domain-containing protein-binding fragment of a SUN domain-containing protein. Such LINC complex inhibitory polypeptides may be referred to as “decoy,” “dominant-negative,” or “mimicking” versions of the SUN domain-containing protein on which they are based. That is, in aspects and embodiments of this disclosure, the LINC complex inhibitory polypeptide may be a dominant-negative SUN domain-containing polypeptide.

[0271] The LINC complex-inhibiting polypeptides according to this disclosure preferably exhibit competitive inhibition of the interaction between SUN domain-containing proteins (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO) and KASH domain-containing proteins (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP). The polypeptide-inhibiting decoy / dominant-negative / mimetic LINC complex preferably binds to KASH domain-containing proteins (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP), occupying the site necessary for interaction with endogenous SUN domain-containing proteins, thereby rendering these species unavailable for binding to endogenous SUN domain-containing proteins.

[0272] Such LINC complex inhibitory polypeptides may inhibit the formation of the LINC complex and / or disrupt existing LINC complexes by inhibiting the assembly of endogenous interaction partners and / or by substituting endogenous interaction partners. Decoy / dominant-negative / mimetic LINC complex inhibitory polypeptides form LINC complexes that are non-functional or have reduced functionality compared to LINC complexes formed by wild-type, endogenous SUN domain-containing proteins, and KASH domain-containing proteins.

[0273] In the embodiments and aspects of this disclosure, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the α3 helix of the CC2 region and the SUN domain of the SUN domain-containing protein. It will be understood that the inhibitory region of the LINC complex inhibitory polypeptide may include one or more amino acid sequences of the LINC complex inhibitory polypeptide described herein that correspond to one or more regions of the SUN domain-containing protein.

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

[0275] In some embodiments, the LINC complex inhibitory polypeptides according to this disclosure are essentially derived from a human amino acid sequence. In some embodiments, the inhibitory region of the LINC complex inhibitory polypeptides according to this disclosure is essentially derived from 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 polypeptide or the inhibitory region of the LINC complex inhibitory polypeptide is essentially derived from an amino acid sequence having 100% amino acid sequence identity with the amino acid sequence encoded by the genome of a human subject. In some embodiments, it will be understood that the amino acid sequence encoded by the genome of a human subject is the amino acid sequence of a human SUN domain-containing protein (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO; e.g., SUN1 or SUN2). Such embodiments of the LINC complex inhibitory polypeptides are intended, in particular, for example, in the context of therapeutic / preventive interventions according to this disclosure, when intended for administration to a human subject.

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

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

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

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

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

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

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

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

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

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

[0286] In some embodiments, the LINC complex inhibitory polypeptide or its inhibitory region is SEQ ID NOs: 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, It contains, or is essentially, an amino acid sequence having sequence identity with at least 60%, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence 104, 105, 106, 107, 108, or 109.

[0287] In some embodiments, the LINC complex-inhibiting polypeptide according to the present disclosure consists essentially of an inhibitory region consisting of an amino acid sequence having at least 60%, for example, 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: 43, 66, 94, 65, 63, 64, or 67.

[0288] In some embodiments, the LINC complex-inhibiting polypeptide according to the present disclosure comprises or consists essentially of an amino acid sequence having at least 60%, for example, 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: 99, 72, 95, 71, 69, 70, or 73.

[0289] The LINC complex-inhibiting polypeptide according to the present disclosure may comprise one or more additional amino acids or amino acid sequences. That is, in addition to the inhibitory region of the polypeptide (i.e., the region comprising the α3 helix of the CC2 region of a SUN domain-containing protein and the amino acid sequence corresponding to the SUN domain), the LINC complex-inhibiting polypeptide may further comprise one or more amino acids or an amino acid sequence.

[0290] According to various aspects and embodiments of the present disclosure, the LINC complex-inhibiting polypeptide may comprise a sequence that prevents secretion of the polypeptide from cells expressing the polypeptide. In some embodiments, the polypeptide comprises an endoplasmic reticulum (ER) retention motif. Such a sequence may be provided at the C-terminus of the inhibitory region of the LINC complex-inhibiting polypeptide according to the present disclosure. In some embodiments, a sequence for preventing secretion of the polypeptide from cells expressing the polypeptide (e.g., an ER retention motif) is provided at the C-terminus of the amino acid sequence of the polypeptide. In some embodiments, at the C-terminus of the polypeptide, the sequence for preventing secretion of the polypeptide from cells expressing the polypeptide (e.g., an ER retention motif) is not followed by any other amino acid.

[0291] Endoplasmic reticulum 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 is effective for retaining a protein comprising the motif at the C-terminus in the endoplasmic reticulum. A KDEL variant may comprise or consist of an amino acid sequence matching the PROSITE motif: [K / R / H / Q / S / A]-[D / E / N / Q]-E-L (described, for example, in Hulo et al., 2006, Nucleic Acids Research 34:D227-D230), or a variant described in Raykhel et al., 2007 (J. Cell Biol. 179(6):1193-1204) that proposed an expanded PROSITE motif definition. Raykhel et al. demonstrated endoplasmic reticulum retention for variants where position 4 (i.e., the K position) is F, W, Y as alternatives to KRHQSA, various position 3 (i.e., the D position) residues extending far beyond DENQ, F or M at position 1 (i.e., the L position), and D at position 2 (i.e., the E position).

[0292] For example, a variant of the KDEL motif may be one of CDEL, KCEL, or HVEL, as proposed, for example, by Raykhel et al. Thus, in some embodiments disclosed herein, the endoplasmic reticulum (ER)-retaining motif is KDEL or a variant thereof exhibiting ER-retaining activity.

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

[0294] In some embodiments, the LINC complex inhibitory polypeptide may include a signal peptide. The signal peptide may be provided at the N-terminus of the inhibitory region of the LINC complex inhibitory polypeptide, in accordance with this disclosure. In some embodiments, the signal peptide is provided at the N-terminus of the amino acid sequence of the polypeptide. In some embodiments, no other amino acids precede the signal peptide at the N-terminus of the polypeptide. The signal peptide typically consists of a sequence of 5 to 30 hydrophobic amino acids forming a single alpha-helix. Secretory proteins and proteins expressed on the cell surface often contain signal peptides. The signal peptide may be present at the N-terminus of the peptide / polypeptide or in a newly synthesized peptide / polypeptide. The signal peptide is often removed by cleavage and is therefore not present in mature peptides / polypeptides.

[0295] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, 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 a secreted protein or a type I transmembrane protein. In some embodiments, the secreted protein or type I transmembrane protein is selected from human serum albumin, proinsulin, transferrin receptor, EGF receptor, prepropiomelanocortin, carboxypeptidase, complement protein, fibrinogen, cytokine, chemokine, fibrinogen, pancreatic digestive enzyme (e.g., protease, amylase, or lipase), or endoplasmic reticulum lumen protein (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 inhibitory polypeptide according to this disclosure includes a signal peptidase cleavage site derived from a secretory protein or type I transmembrane protein, for example, the aforementioned secretory protein or type I transmembrane protein.

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

[0298] In some embodiments, the LINC complex inhibitory polypeptide may include a detectable moiety, such as a fluorescent label, phosphorescent label, luminescent label, immunodetectable label (e.g., an epitope tag), radioactive label, chemical label, nucleic acid label, or enzymatic label. The LINC complex inhibitory polypeptide may be labeled covalently or noncovalently at the detectable moiety.

[0299] In some embodiments, the detectable portion is provided at the N-terminus of the polypeptide (either before or after processing of the polypeptide by a signal peptidase to remove any signal peptide). In some embodiments, the detectable portion is provided at the C-terminus of the polypeptide, for example, downstream (after) a sequence to prevent secretion of the polypeptide from cells 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., 6X His), 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, for example, enzymatic activity on a given substrate. Examples of such portions include, for example, horseradish peroxidase (HRP) and luciferase portions.

[0301] In some embodiments, the LINC complex inhibitory polypeptide according to this disclosure comprises one of the following structures: N-term - [signal peptide] - [inhibitory region containing amino acid sequences corresponding to the α3 helix of the CC2 region of the SUN domain-containing protein and the SUN domain] - [sequence that prevents polypeptide secretion from cells expressing the polypeptide] - C-term N-term - [Inhibitory region containing amino acid sequences corresponding to the α3 helix and SUN domain of the CC2 region of the SUN domain-containing protein] - [Sequence that prevents polypeptide secretion from cells expressing the polypeptide] - C-term

[0302] In some embodiments, the LINC complex inhibitory polypeptide according to this disclosure comprises one or more linker sequences between amino acid sequences. In some embodiments, the linker sequences have a length of 1-2, 1-3, 1-4, 1-5, or 1-10 amino acids. In some embodiments, the linker sequences may be provided at one or both ends of an inhibitory region comprising an α3 helix of the CC2 region of a SUN domain-containing protein and an amino acid sequence corresponding to the SUN domain, a signal peptide, a sequence that prevents the secretion of the polypeptide from cells expressing the polypeptide, and / or a detectable entity of the LINC complex inhibitory polypeptide.

[0303] 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 for the relative movement of the amino acid sequence linked by the linker sequence. Flexible linkers are known to those skilled in the art and some are identified in Chen et al., Adv Drug Deliv Rev(2013) 65(10):1357-1369. Flexible linker sequences often 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.

[0304] In a preferred embodiment, the LINC complex inhibitory polypeptide is of a size (i.e., with respect to the number of amino acids constituting the LINC complex inhibitory polypeptide) that allows for delivery of the LINC complex inhibitory polypeptide as a gene therapy, i.e., in the form of a nucleic acid encoding the polypeptide.

[0305] In some embodiments, the LINC complex inhibitory polypeptide has a size such that the polynucleotide encoding the polypeptide has a size within the packaging limits of the vector for delivering the polynucleotide (i.e., with respect to the number of nucleotides constituting the polynucleotide). In some embodiments, the LINC complex inhibitory polypeptide has a size such that the polynucleotide encoding the polypeptide has a size within the packaging limits of the vector described herein. In some embodiments, the LINC complex inhibitory polypeptide has a size such that the polynucleotide encoding the polypeptide has a size within the packaging limits of an adeno-associated virus (AAV) vector, for example, the AAV vector described herein. In some embodiments, the LINC complex inhibitory polypeptide has a size such that the polynucleotide encoding the polypeptide has a size within the packaging limits of an scAAV vector.

[0306] In some embodiments, the LINC complex inhibitory polypeptide consists of an amino acid sequence containing fewer than 510 amino acids. In some embodiments, the LINC complex inhibitory polypeptide consists of an amino acid sequence containing fewer than 457 amino acids.

[0307] In some embodiments, the LINC complex inhibitory polypeptide consists of an amino acid sequence containing one amino acid from among less than 600 amino acids, for example, ≤550, ≤500, ≤450, ≤400, ≤350, ≤340, ≤330, ≤320, ≤310, ≤300, ≤290, ≤280, ≤270, ≤260, ≤250, ≤240, ≤230, ≤220, or ≤210.

[0308] The LINC complex-inhibiting polypeptide according to the present disclosure may be prepared according to methods for producing polypeptides known to those skilled in the art. The polypeptide may be prepared by chemical synthesis, for example, liquid phase or solid phase synthesis. For example, the peptide / polypeptide can be synthesized using the method described, for example, in Chandrudu et al., Molecules (2013), 18:4373-4388, which is incorporated herein by reference in its entirety. Alternatively, the antigen-binding molecule and the polypeptide can be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and Nat Methods. (2008); 5(2):135-146, all of which are incorporated herein by reference in their entirety. Methods for recombinant production of antigen-binding molecules are also disclosed in Front Immunol. (2013); 4:217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461, all of which are incorporated herein by reference in their entirety.

[0309] Cardiomyopathy Aspects and embodiments of the present disclosure relate to the treatment and prevention of cardiomyopathy. The experimental examples of the present disclosure demonstrate that LINC complex inhibition improves pathology in a mouse model of cardiomyopathy.

[0310] In this specification, “cardiomyopathy” refers to a pathology affecting the structure (e.g., size, shape) and / or function of myocardial tissue (i.e., myocardium), characterized by a decrease in cardiac function, i.e., a decrease in the heart’s ability to pump blood. Cardiomyopathy is outlined, for example, in 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 and include cardiovascular disease, genetic variations, infection, autoimmune diseases, and adverse effects of drug therapy.

[0311] Cardiomyopathy can be broadly divided into five main classes: 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).

[0312] Dilated cardiomyopathy (DCM) is characterized by enlargement (i.e., dilation) of one or both ventricles (typically the left ventricle), causing the ventricular walls to stretch and thin. The thinner ventricular walls are weaker and cannot contract with greater force, impairing cardiac function. Individuals with DCM may develop heart failure, with the most common primary symptoms being arrhythmias (ventricular / atrial) and sudden cardiac death. While the cause of dilated cardiomyopathy is most often hereditary, it is also associated with myocarditis (e.g., associated with viral infections), coronary artery disease, invasive disease, pregnancy (peripartum cardiomyopathy), hypertension, connective tissue disorders, drug abuse, and doxorubicin treatment.

[0313] Genetic variants of the TTN gene, which encodes the sarcomere protein titin (identified by UniProt Q8WZ42) that links actin and myosin, particularly variants resulting in titin shortening, which are thought to cause about 15-20% of dilated cardiomyopathy cases, account for about 25% of dilated cardiomyopathy cases (Schaefer et al., Nat Genet. (2017) 49(1):46-53). Mutations in LMNA, which encodes lamins A and C, account for about 5% of dilated cardiomyopathy cases. Mutations in PLN (which encodes phospholamban) and FLNC (which encodes filamin C) each account for about 1-5% of dilated cardiomyopathy cases. Other genes whose genetic variations are causally related to dilated cardiomyopathy include DSP (encodes desmoplakin), DMD (encodes dystrophin), ACTC (encodes cardiac actin), DES (encodes desmin), MYH7 (encodes the cardiac isoform of beta-myosin-heavy chain), TNNT2 (encodes troponin T), TNNI3 (encodes troponin I), SGCD (encodes delta-sarcoglycan), and SCN5A (encodes sodium channel, type V).

[0314] Ischemic cardiomyopathy is a common type of dilated cardiomyopathy and a common cause of systolic heart failure. Ischemic cardiomyopathy (ICM) is a term that refers to a decrease in the heart's ability to properly pump blood due to myocardial damage (reduced blood supply to the myocardium) caused by ischemia. Ischemic cardiomyopathy is associated with reduced blood supply to the myocardium caused by narrowing of the cardiac arteries, for example, due to coronary artery disease or a heart attack (known as myocardial infarction). In patients with ischemic cardiomyopathy, microtubule density and stability are significantly increased (see Chen C. et al. Nature Medicine (2018), 24, 1225-1233, which is incorporated herein in its entirety by reference).

[0315] Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the ventricular walls (15 mm or more in thickness), and most commonly, the left ventricular wall. The thickening of the ventricular walls reduces their size, decreasing the amount of blood they can hold, thus impairing cardiac function. Thicker walls are also often rigid and unable to fully relax, resulting in an inability to fill with large amounts of blood. The cause of hypertrophic cardiomyopathy is most often hereditary, frequently associated with mutations in genes encoding sarcomere proteins. However, there are HCM patients who do not have a genetic linkage and are sarcomere mutation-negative (Warner E. et al. 2022).

[0316] Mutations in MYBPC3, which encodes cardiac myosin-binding C in the intermediate filament protein, are thought to account for up to 40% of hypertrophic cardiomyopathy cases, while 15–25% of patients with hypertrophic cardiomyopathy also have mutations in MYH7. Mutations in TNNT2 account for 5–10% of cases, while TNNI3 mutations are present in 4–8% of cases. Other genes whose genetic variations are causally related to hypertrophic cardiomyopathy include MYL2 and MYL3 (encoding myosin light chain), TPM1 (encoding α-tropomyosin), and ACTC1 (encoding α-cardiac actin).

[0317] The hallmarks of hypertrophic cardiomyopathy are upregulated protein synthesis and the growth of the cytoskeletal network, 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-associated protein that promotes microtubule network stability and is upregulated in hypertrophic cardiomyocytes. A dense, stabilized microtubule network is formed, hindering cardiomyocyte contraction and microtubule-based transport.

[0318] Induction of microtubule depolymerization in hypertrophic cardiomyocytes under pressure overload, thereby reversing MTN density, is effective in reversing contractile dysfunction in animal models (Warner E. et al. 2022).

[0319] Restrictive cardiomyopathy is characterized by diastolic dysfunction, elevated end-diastolic pressure, and hardening of the ventricular walls, leading to atrial dilation. The stiffer ventricles cannot expand efficiently enough to fill with blood, impairing cardiac function. Restrictive cardiomyopathy is relatively rare and can be idiopathic or caused by a disease / condition affecting the heart.

[0320] Diseases / conditions that can cause restrictive cardiomyopathy include amyloidosis, sarcoidosis, primary hyperoxaluria, Fabry disease, Gaucher disease, hereditary hemochromatosis, glycogen storage disease, mucopolysaccharidosis type 1 (Harler syndrome), mucopolysaccharidosis type 2 (Hunter syndrome), Niemann-Pick disease, diabetes mellitus, scleroderma, myofibrillary myopathy, pseudoxanthoma elastica, sarcomere protein disorder, Werner syndrome, carcinoid heart disease, endocardial fibrosis, eosinophilic syndrome, cancer (e.g., metastatic cancer), chronic eosinophilic leukemia, and endocardial fibroelastosis. Restrictive cardiomyopathy can also occur as a side effect of treatment with certain drugs, including anthracyclines, serotonin, methysergide, ergotamine, mercury compounds, and busulfan, as well as as a side effect of radiotherapy.

[0321] Amyloidosis-associated mutations associated with restrictive cardiomyopathy include mutations to APOA1, as well as mutations to TTR such as V122I, I68L, L111M, T60A, S23N, P24S, W41L, V30M, and V20I. Fabry disease-associated mutations associated with restrictive cardiomyopathy include mutations to GLA, but mutations to GBA, associated with Gaucher disease, are also associated with restrictive cardiomyopathy. Hereditary hemochromatosis-associated mutations associated with restrictive cardiomyopathy include mutations to HAMP, HFE, HFE2, HJV, PNPLA3, SLC40A1, and TfR2. Mucopolysaccharidosis type I-associated mutations associated with restrictive cardiomyopathy include mutations to IDUA, but mutations to IDS, associated with mucopolysaccharidosis type II, are also associated with restrictive cardiomyopathy. Niemann-Pick disease-associated mutations associated with restrictive cardiomyopathy include mutations to NPC1, NPC2, and SMPD1. Myofibrillar myopathy-associated mutations associated with restrictive cardiomyopathy include mutations in BAG3, CRYAB, DES, DNAJB6, FHL1, FLNC, LDB3, and MYOT. Sarcomere protein disorder-associated mutations associated with restrictive cardiomyopathy include mutations in ACTC, β-MHC, TNNT2, TNNI3, TNNC1, DES, MYH, MYL3, and CRYAB. Pseudoxanthoma elastica-associated mutations associated with restrictive cardiomyopathy include mutations in ABCC6, but mutations in WRN, which are associated with Werner syndrome, are also associated with restrictive cardiomyopathy. Endocardial fibroelastosis-associated mutations associated with restrictive cardiomyopathy include mutations in BMP5, BMP7, and TAZ.

[0322] Arrhythmogenic cardiomyopathy (ACM) is another rare form of cardiomyopathy in which the muscular tissue of the ventricles (or ventricular dysplasia) is replaced by fatty and / or fibrous tissue, often leading to arrhythmias such as ventricular tachycardia. ACM was formerly thought to affect only the right ventricle and was therefore called arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic right ventricular dysplasia (ARVD), but further research has shown that the right ventricle, left ventricle, or both ventricles can be affected. The cause of arrhythmogenic cardiomyopathy is typically genetic.

[0323] Mutations in PKP2 (encoding placofilin 2) are thought to account for 34% to 74% of ACM cases, while mutations in DSG2 (encoding desmoglein 2) account for 5% to 26% of ACM cases, and mutations in DSP account for 2% to 39% of ACM cases. Other genes in which mutations that cause ACM exist include DSC2, JUP, TMEM43, DES, and PLN.

[0324] Diabetic cardiomyopathy is another form of cardiomyopathy, and diabetes mellitus leads to myocardial remodeling, loss of left ventricular (LV) pump function, and ultimately heart failure. Diabetic cardiomyopathy arises as a result of dysregulation of glucose and lipid metabolism associated with diabetes, which leads to increased oxidative stress, as well as activation of multiple inflammatory pathways that mediate 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, and hyperglycemia leads to the formation of advanced glycation end products (AGEs). AGEs promote microtubule stabilization by inhibiting the SIRT2 / acetylated α-tubulin signaling pathway (Warner E. et al. 2022).

[0325] The most common version of the nucleotide sequence of a given gene is sometimes called the wild-type allele of the gene. A version of the nucleotide sequence of a given gene that contains a “genetic variant” or “mutation” relative to the wild-type allele is sometimes called the “variant” or “mutant” allele of the gene. As used herein, a “genetic variant” of a given gene or a “mutation” to a given gene refers to an insertion, deletion, substitution, or larger translocation / rearrangement of the nucleotide sequence of a gene relative to the nucleotide sequence of a reference allele that does not contain a gene variant / mutation (e.g., the “non-mutant” or “wild-type” allele). It will be understood that the nucleotide sequence of a variant / mutant allele of a given gene will have a nucleotide sequence that is not identical to the nucleotide sequence of the wild-type allele.

[0326] Where a given disease / condition is described herein as “associated” with a given genetic variant / mutation or disease / condition, the genetic variant / mutation / disease / condition may be positively associated 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 genetic variant / mutation or disease / condition may be a risk factor for the onset or progression of the given disease / condition. In some embodiments, a disease / condition associated with a given genetic variant / mutation or disease / condition may be caused by or aggravated by the genetic variant / mutation / disease / condition. Where a given disease / condition is described herein as “caused” by a given genetic variant / mutation or disease / condition, the genetic variant / mutation / disease / condition may be directly or indirectly involved in the pathology of the given disease / condition.

[0327] Genetic variants / mutations may result in altered levels of expression (of genes and / or proteins) of the gene product (e.g., RNA and / or polypeptides) encoded by the wild-type allele of the relevant gene, and / or altered activity of the gene product (e.g., RNA and / or polypeptides). Genetic variants / mutations may result in decreased levels of the gene product encoded by the wild-type allele of the relevant gene. Genetic variants / mutations may result in increased levels of polypeptides encoded by the disease-associated allele of the relevant gene. Genetic variants / mutations may result in the production of shortened versions of polypeptides encoded by the wild-type allele of the relevant gene. Genetic variants / mutations may result in the production of incorrectly folded and / or degraded versions of polypeptides encoded by the wild-type allele of the relevant gene. Genetic variants / mutations may result in altered activity of polypeptides encoded by the wild-type allele of the relevant gene, e.g., decreased or increased levels of activity possessed by the polypeptide encoded by the wild-type allele, and / or the production of polypeptides with acquired activity not possessed by the polypeptide encoded by the wild-type allele.

[0328] In some embodiments, the cardiomyopathy described herein is dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, or an unclassified cardiomyopathy.

[0329] In some embodiments, the cardiomyopathy described herein is dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic cardiomyopathy, diabetic cardiomyopathy, ischemic cardiomyopathy, or an unclassified cardiomyopathy.

[0330] In some embodiments, the cardiomyopathy according to this disclosure is dilated cardiomyopathy. In some embodiments, the cardiomyopathy is cardiomyopathy (e.g., dilated cardiomyopathy) associated with a genetic variant, infection, viral infection, myocarditis, coronary artery disease, invasive disease, pregnancy, hypertension, connective tissue disease, drug abuse, or doxorubicin treatment. In some embodiments, the cardiomyopathy is cardiomyopathy (e.g., dilated cardiomyopathy) associated with a mutation in a gene selected from TTN, LMNA, PLN, FLNC, DSP, DMD, ACTC, DES, MYH7, TNNT2, TNNI3, SGCD, and SCN5A. In some embodiments, the cardiomyopathy is cardiomyopathy (e.g., dilated cardiomyopathy) associated with a mutation in a gene selected from TTN, PLN, FLNC, DSP, DMD, ACTC, DES, MYH7, TNNT2, TNNI3, SGCD, and SCN5A. In some embodiments, the cardiomyopathy is cardiomyopathy (e.g., dilated cardiomyopathy) associated with a mutation in DSP or DES. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation to DSP (e.g., dilated cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation to DMD (e.g., dilated cardiomyopathy).

[0331] In some embodiments, cardiomyopathy is a type of cardiomyopathy associated with mutations in genes encoding sarcomere proteins (e.g., dilated cardiomyopathy). As used herein, “sarcomere protein” or “sarcomere protein” refers to the constituent polypeptides of sarcomeres. Examples of sarcomere constituent polypeptides, i.e., sarcomere proteins, include titin, troponin T, troponin I, troponin C1, cardiac myosin-binding C, α-tropomyosin, α-cardiac actin, myosin, and filamin C. In some embodiments, cardiomyopathy is a type of cardiomyopathy associated with mutations in genes selected from TTN, TNNT2, TNNI3, TNNC1, MYBPC3, MYH7, TPM1, ACTC1, MYL2, MYL3, and FLNC. In some embodiments, cardiomyopathy is a cardiomyopathy associated with mutations in genes selected from TTN, TNNT2, TNNI3, TNNC1, MYBPC3, MYH7, TPM1, ACTC1, MYL2, and MYL3. In some embodiments, cardiomyopathy is a cardiomyopathy associated with mutations in TTN (e.g., dilated cardiomyopathy).

[0332] In some embodiments, the cardiomyopathy according to this disclosure is hypertrophic cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a genetic variant (e.g., hypertrophic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation in a gene selected from MYBPC3, MYH7, TNNT2, TNNI3, MYL2, MYL3, TPM1, and ACTC1 (e.g., hypertrophic cardiomyopathy).

[0333] In some embodiments, the cardiomyopathy according to this disclosure is restrictive cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a genetic variant, amyloidosis, sarcoidosis, primary hyperoxaluria, Fabry disease, Gaucher disease, hereditary hemochromatosis, glycogen storage disease, mucopolysaccharidosis type I (Harler syndrome), mucopolysaccharidosis type II (Hunter syndrome), Niemann-Pick disease, diabetes mellitus, scleroderma, myofibrilary myopathy, pseudoxanthoma elastica, sarcomere protein disorder, Werner syndrome, carcinoid heart disease, endocardial fibrosis, eosinophilic syndrome, cancer (e.g., metastatic cancer), chronic eosinophilic leukemia, endocardial fibroelastosis, chemotherapy, radiotherapy, or treatment with anthracyclines, serotonin, methysergide, ergotamine, mercury agents, or busulfan (e.g., restrictive cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with mutations in genes selected from APOA1, TTR (e.g., V122I, I68L, L111M, T60A, S23N, P24S, W41L, V30M, or V20I), GLA, GBA HAMP, 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 (e.g., restrictive cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with mutations in DES (e.g., restrictive cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation to FLNC (e.g., restrictive cardiomyopathy). do not It is cardiomyopathy (for example, restrictive cardiomyopathy).

[0334] In some embodiments, the cardiomyopathy according to this disclosure is arrhythmogenic cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a genetic variant (e.g., arrhythmogenic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation in a gene selected from PKP2, DSG2, DSP, DSC2, JUP, TMEM43, DES, and PLN (e.g., arrhythmogenic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation in a gene selected from PKP2, DSG2, DES, DSC2, JUP, and DES (e.g., arrhythmogenic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation in DSP (e.g., arrhythmogenic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation in PKP2 (e.g., arrhythmogenic cardiomyopathy).

[0335] In some embodiments, the cardiomyopathy is a cardiomyopathy associated with mutations in genes selected from DSP, TTN, FLNC, and PKP2 (e.g., arrhythmogenic cardiomyopathy). In some embodiments, the cardiomyopathy is a cardiomyopathy associated with mutations in genes selected from DSP, TTN, and PKP2 (e.g., dilated cardiomyopathy).

[0336] In some embodiments, cardiomyopathy is a type of cardiomyopathy associated with laminopathy. do not have Cardiomyopathy associated with laminopathy may be a type of cardiomyopathy caused by mutations in the gene encoding lamin. In some embodiments, cardiomyopathy is a type of cardiomyopathy associated with laminopathy. other thanIt is a type of cardiomyopathy. As used herein, “laminopathy” is a disease / pathological condition associated with mutations in the genes encoding lamin. Laminopathy is outlined, for example, by Burke and Stewart, Nat Rev Mol Cell Biol. (2013) 14(1):13-24 and Hah and Kim, Cells (2019) 8(3):231, both of which are incorporated herein by reference in their entirety. Laminopathy is generally associated with tissue-specific defects in load-bearing capacity at the nuclear level, which can reduce the cell’s resistance to physical forces.

[0337] Genes encoding lamins include LMNA (encoding lamins A and C), and LMNB1 and LMNB2 (encoding lamins B1 and B2). In some embodiments, cardiomyopathy is associated with mutations in LMNA. do not have In some embodiments, cardiomyopathy is cardiomyopathy associated with mutations in LMNA. other than This is a cardiomyopathy. In some embodiments, the cardiomyopathy is a cardiomyopathy associated with a mutation in any of LMNA, LMNB1, and LMNB2. do not have (In other words, cardiomyopathy that causes mutations in LMNA) no In cardiomyopathy associated with mutations in LMNB1 no (not cardiomyopathy associated with mutations to LMNB2). In some embodiments, cardiomyopathy associated with mutations to any of LMNA, LMNB1, and LMNB2. other than Cardiomyopathy.

[0338] In some embodiments, cardiomyopathy is a type of cardiomyopathy associated with nuclear envelopeopathy. do not haveNuclear envelope cardiopathy is a disease / pathological condition associated with mutations in genes encoding nuclear envelope proteins (i.e., proteins contained in or directly / indirectly related to ONM, perinuclear space, or INM). Nuclear envelope cardiopathy is outlined, for example, in Chi et al., Journal of Biomedical Science (2009) 16:96, which is incorporated herein by reference in its entirety. Nuclear envelope cardiopathy includes disease / pathological conditions associated with genetic variations of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62. In some embodiments, cardiomyopathy is associated with mutations in any of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62. do not have .

[0339] In some embodiments, the cardiomyopathy described herein is diabetic cardiomyopathy. In some embodiments, the cardiomyopathy is cardiomyopathy associated with one or more of the following: type 1 diabetes, type 2 diabetes, prediabetes, metabolic syndrome, pregnancy-related hyperglycemia (i.e., gestational diabetes), obesity, and hyperglycemia (e.g., diabetic cardiomyopathy).

[0340] In some embodiments, the cardiomyopathy according to this disclosure is ischemic cardiomyopathy. In some embodiments, the cardiomyopathy is cardiomyopathy associated with one or more of the following conditions: coronary artery disease, myocardial infarction, diabetes mellitus, prediabetes, metabolic syndrome, pregnancy-related hyperglycemia (i.e., gestational diabetes mellitus), hyperglycemia, amyloidosis, hypertension, obesity, and hypercholesterolemia (e.g., ischemic cardiomyopathy).

[0341] In some embodiments, cardiomyopathy is associated with a genetic variant, for example, the genetic variant described herein. do not have It is cardiomyopathy (for example, dilated cardiomyopathy).

[0342] In some embodiments, the cardiomyopathy described herein is cardiomyopathy associated with desmosome deficiency / deficiency or dysfunction.

[0343] In some embodiments, the cardiomyopathy described herein is a cardiomyopathy associated with mutations in genes encoding desmosome proteins. As used herein, “desmosome proteins” refers to the constituent polypeptides of desmosomes. Examples of desmosome constituent polypeptides include desmoplakin, desmin, desmoglein (e.g., desmoglein 1, 2, 3, 4), desmocolin (e.g., desmocolin 1, 2, 3), placofilin (e.g., placofilin 1, 2, 3), and placoglobin. In some embodiments, the genes encoding desmosome proteins are selected from DSP, DES, DSG1, DSG2, DSG3, DSG4, DSC1, DSC2, DSC3, PKP1, PKP2, PKP3, and JUP.

[0344] In some embodiments, the cardiomyopathy described herein is a cardiomyopathy associated with mutations in genes encoding constituent polypeptides of the desmosome intermediate filament complex (DIFC).

[0345] Desmosomes (also known as adhesion plaques) are specialized structures that mediate lateral intercellular adhesion between cells, particularly in tissues subjected to considerable mechanical stress, such as cardiomyocytes, bladder tissue, gastrointestinal mucosa, and epithelium. The structure and function of desmosomes are outlined, 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, both of which are incorporated herein by reference in their entirety. Desmosomes consist of a desmosome intermediate filament complex (DIFC) formed from a network of cadherins, linker proteins, and intermediate filaments. Desmosomes have an extracellular core region known as the desmoglare, which contains cadherins (e.g., desmogleins and desmocolins, which associate via heteroaffinic interactions between their N-terminuses). At their C-terminuses, cadherins associate with placofilin 1 and 2 and placoglobin in the outer dense plaque (ODP). Placofilin 1 and 2 and placoglobin are then linked by desmoplakin to the inner dense plaque (IDP), which includes intermediate filaments.

[0346] In some embodiments, the cardiomyopathy described herein is a cardiomyopathy associated with mutations in genes selected from DSP, DES, DSG2, DSC2, PKP2, and JUP.

[0347] In some embodiments, the cardiomyopathy described herein is cardiomyopathy associated with desmoplakin deficiency / insufficiency or dysfunction.

[0348] In some embodiments, the cardiomyopathy described herein is cardiomyopathy associated with mutations in DSP. It has even been suggested that cardiomyopathy associated with mutations in DSP forms another class of cardiomyopathy, so-called “desmoplakin cardiomyopathy”—see Smith et al., Circulation (2020) 141(23):1872-1884.

[0349] Desmoplakin (also known as DSP) is a protein identified by UniProt P15924. The structure and function of desmoplakin are described, for example, Yuan et al., Chin Med J(Engl).(2021)134(15):1771-1779, Mueller 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.

[0350] Desmoplakins are essential components of desmosomes, which form intercellular junctions between adjacent cells. Desmoplakins bind via their C-terminus to intermediate filaments of the cytoskeleton, which vary depending on the cell type (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). In cardiomyocytes, desmoplakins bind to intermediate filaments desmin; in endothelial and epithelial cells, desmoplakins bind to cytokeratin-type intermediate filaments; and in dendritic cells, desmoplakins bind to vimentin-type intermediate filaments. At their N-terminus, desmoplakins bind to armadillo family proteins such as placofilin 1 and 2 and placoglobin, which then bind to desmosome cadherins, particularly desmocolin and desmoglein. Therefore, desmoplakin functions as an adapter protein, ligating the intermediate filament to the cadherin of the desmosome.

[0351] Mutations in the human DSP gene cause a variety of cardiomyopathy, including dilated cardiomyopathy and arrhythmogenic cardiomyopathy. Such mutations in DSP are described, for example, in Mohammed and Chidgey, Journal of Structural Biology (2021) 213(3):107749 (the entire text is incorporated herein by reference; see Figure 4e in particular), and include G2056R, D2757H, K2689T, R2834H, T2595I, R2541K, R2541S, R2639Q, R2366H, R2366C, D230N, S299R, N375I, S442F, I445V, N458Y, K470E, S507F, N287D, N287S, A206T, W207C, E422K, I5 33T, A556T, N661I, Y787C, R808C, R808H, R1255K, R1255T, L1348R, T1373A, L1654P, R1775I, R1838H, Q198P, D203N, R222L, D241N, E290 K, I305F, A306T, R315C, R315P, M316V, A349P, Y350H, T356K, I368T, E384Q, K401N, N408K, R451G, R451H, I461V, C482Y, Y494F, P515L, L5 40P, T587N, N593S, M599R, R606W, E721K, Y895H, I870M, I870T, R907H, R908H, L933F, G939S, S987P, T1960P, Y1981C, L1995S, A2019S, A 2019V, P2061S, H2062R, D2070N, V2107L, R2083C, K2103E, A2148T, T2177N, R2189Q, T2196I, I2263T, T2267S, A2294G, Q2295H, G2338R, E 2343K, 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, K1581 E, K1592R, M1601I, S1623C, V1639M, S1658F, R1666Q, R1666W, H1684R, E1721D, R173 This includes 8Q, E1740K, E1833V, R1852H, R2366H, T2770C, G2832V, S2616L, G2647D, R1838H, T13I, E21K, V30M, R44Q, C81Y, Q90R, R105Q, E109K, M118K, I125F, C174F, and A566T. Mutations to DSPs associated with erythematous keratoderma-cardiamyocardial syndrome include L622P, Q616P, and H618P. Mutations in DSPs associated with cardiocutaneous syndromes such as Naxos disease and Carvajal syndrome (characterized by a combination of dilated cardiomyopathy, keratoderma, and trichomer) include I560F, T564I, L583P, S597L, E2193K, L2329P, Q2371K, G2375R, and A2655D.

[0352] In some embodiments, the cardiomyopathy according to this disclosure is cardiomyopathy associated with one or more of the following mutations in DSP: 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, R1255 T, L1348R, T1373A, L1654P, R1775I, R1838H, Q198P, D203N, R222L, D241N, E 290K, I305F, A306T, R315C, R315P, M316V, A349P, Y350H, T356K, I368T, E384 Q, K401N, N408K, R451G, R451H, I461V, C482Y, Y494F, P515L, L540P, T587N, N593S, M599R, R606W, E721K, Y895H, I870M, I870T, R907H, R908H, L933F, G93 9S, S987P, T1960P, Y1981C, L1995S, A2019S, A2019V, P2061S, H2062R, D207 0N, V2107L, R2083C, K2103E, A2148T, T2177N, R2189Q, T2196I, I2263T, T22 67S, A2294G, Q2295H, G2338R, E2343K, I2347V, D2579H, I2593S, T2664N, G2 666D, A2674T, M2684V, M2707T, A2709T, R2759S, P2777H, I2797T, M2819L, G2 844V, I2869V, K1094N, Y1169N, L1178R, L1178V, Y1188H, R1207K, T1217M, D 1251N, D1258E, A1274P, K1288Q, T1319I, R1341H, E1357D, R1392Q, R1392W, E 1407D, 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, A566T, L622P, Q616P, H618P, I560F, T564I, L583P, S597L, E2193K, L2329P, Q2371K, G2375R, or A2655D.

[0353] In some embodiments of various aspects of the present disclosure, the cardiomyopathy according to the present disclosure is a cardiomyopathy characterized by microtubule dysfunction.

[0354] Diseases / conditions characterized by microtubule dysfunction The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and conditions including / characterized by microtubule dysfunction. Experimental examples of this disclosure demonstrate that LINC complex inhibition improves pathology in a mouse model of a disease characterized by microtubule network dysfunction.

[0355] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure may be a disease / condition in which microtubule dysfunction or deficiency / impairment is pathologically involved, for example, a disease / condition in which microtubule dysfunction or deficiency / impairment is positively associated 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 / impairment may be a risk factor for the onset, development, or progression of the disease / condition.

[0356] As described above, microtubule dysfunction is associated with heart disease (Warner E. et al., 2022, Table A below). In some embodiments, the disease / condition characterized by microtubule dysfunction is heart disease.

[0357] Microtubule dysfunction is often associated with cardiomyopathy. In some embodiments, the disease / condition characterized by microtubule dysfunction is cardiomyopathy.

[0358] In some embodiments, a disease or condition characterized by microtubule dysfunction is characterized by cardiomyopathy. That is, in some embodiments, cardiomyopathy is a symptom of a disease or condition characterized by microtubule dysfunction.

[0359] In some embodiments, cardiomyopathy is the cardiomyopathy described herein. In some embodiments, cardiomyopathy is a non-hereditary cardiomyopathy.

[0360] Various conditions characterized by microtubule dysfunction are outlined in Warner E. et al. 2022, Circulation Research. 2022;130:1723-1741, which is incorporated herein by reference in its entirety. For example, in patients with cardiomyopathy such as ischemic cardiomyopathy, hypertrophic cardiomyopathy, diabetic cardiomyopathy, and dilated cardiomyopathy, microtubule density and stability are dramatically increased. Dystrophic cardiomyopathy, a type of cardiomyopathy resulting from mutations in DMD (which encodes dystrophin), is also associated with microtubule dysfunction (see Table A below).

[0361] In some embodiments, the disease / condition characterized by microtubule dysfunction is or features ischemic cardiomyopathy, hypertrophic cardiomyopathy, diabetic cardiomyopathy, and dilated cardiomyopathy. In some embodiments, the disease / condition characterized by microtubule dysfunction is or features dystrophic cardiomyopathy.

[0362] Heart failure can occur when the myocardium is stiff and unable to relax properly (diastolic dysfunction). In dysfunctional cardiomyocytes, increased stiffness of the microtubule network (MTN) leads to contractile dysfunction. This increase in MTN stiffness is often induced by increased interaction between detyrosinized microtubules and intermediate filaments desmin in the sarcomere Z disc (Warner E. et al., 2022). That is, detyrosinized microtubules provide mechanical resistance that can hinder the movement of contracting cardiomyocytes (Chen C. et al., Nature Medicine (2018) volume 24, 1225-1233).

[0363] Failure of cardiomyocytes is also often characterized by increased microtubule network density, which is associated with increased myocyte stiffness and decreased contractility (Chen C. et al., Nature Medicine (2018) volume 24, 1225-1233). Increased MTN density is accompanied by a marked upregulation of total tubulin in hearts affected by ischemic cardiomyopathy, hypertrophic cardiomyopathy, and dilated cardiomyopathy.

[0364] Levels of detyrosinated α-tubulin were significantly higher in cardiomyocytes isolated from ischemic hearts compared to cardiomyocytes isolated from mice that underwent sham surgery, in contrast to the rest of the cell pool tested, e.g., immune cells, fibroblasts, and endothelial cells (Yu X. et al. 2021 Nature:594,560-565, the whole of which is incorporated herein by reference).

[0365] In some embodiments, the disease / condition characterized by microtubule dysfunction is heart failure. In some embodiments, heart failure is associated with ischemic injury; that is, in some embodiments, heart failure is ischemic heart failure.

[0366] As summarized in Table A below, various models of heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) have been reported to be characterized by microtubule dysfunction.

[0367] heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome in which a patient has signs and symptoms of heart failure as a result of high left ventricular (LV) filling pressure, despite having a normal or near-normal LV ejection fraction (LVEF; ≥50%) (Harper A. et al. Clin Med (Lond). 2018;18(Suppl2):s24-s29). Heart failure with reduced ejection fraction (HFrEF) is heart failure with reduced left ventricular ejection fraction (LVEF is <40%). In some cases, hypertrophic cardiomyopathy can lead to heart failure with preserved ejection fraction (HFpEF) or heart failure with reduced ejection fraction (HFrEF).

[0368] In some embodiments, the disease / condition characterized by microtubule dysfunction is heart failure with preserved ejection fraction (HFpEF). In some embodiments, the disease / condition characterized by microtubule dysfunction is heart failure with reduced ejection fraction (HFrEF). In some embodiments, HFpEF and / or HFrEF are associated with hypertrophic cardiomyopathy.

[0369] 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 embodiments, aortic stenosis is a disease / condition characterized by microtubule dysfunction.

[0370] Stones R. et al. J Mol Cell Cardiol. 2013;56:91-96 describes microtubule proliferation as a common response to pulmonary hypertension in right ventricular dysfunction (see Table A below). Prins K. et al. J Am Heart Assoc. 2017;6(6):e006195 describes pulmonary arterial hypertension (PAH) as being characterized by abnormal microtubule structure in right ventricular (RV) cardiomyocytes that impair RV function (see Table A below). Drug-induced pulmonary arterial hypertension (D-PAH) is a form of pulmonary hypertension (PH) defined by severe small vessel loss and occlusive vascular damage leading to progressive right heart failure and death (Orcholski M et al. Am J Physiol Lung Cell Mol Physiol. 2018;314(6):L967-L983).

[0371] In some embodiments, the disease / condition characterized by microtubule dysfunction is pulmonary arterial hypertension (PAH). In some embodiments, the disease / condition characterized by microtubule dysfunction is pulmonary hypertension (PH). In some embodiments, the disease / condition characterized by microtubule dysfunction is drug-induced pulmonary hypertension or hypoxia-induced pulmonary hypertension (e.g., hypoxia-induced neonatal pulmonary hypertension).

[0372] Microtubule dysfunction has also been described as characterizing right ventricular pressure overload and left ventricular pressure overload (summarized in Table A below). In some embodiments, the disease / condition characterized by microtubule dysfunction is right ventricular (RV) pressure overload. In some embodiments, the disease / condition characterized by microtubule dysfunction is left ventricular (LV) pressure overload.

[0373] Myocardial infarction (also known as heart attack) is associated with increased phosphorylation of microtubule-associated protein 4 (MAP4) (see Table A below). In some embodiments, diseases / conditions characterized by microtubule dysfunction include myocardial infarction, such as acute myocardial infarction.

[0374] As used herein, “microtubule dysfunction” or “microtubule network dysfunction” refers to a condition in which the normal function of the microtubule cytoskeleton is impaired / abnormal / insufficient, resulting in microtubule dysfunction in a particular tissue (e.g., cardiac tissue) and inappropriate function of the associated tissue. The normal function of the microtubule network and microtubule dysfunction are outlined, for example, in Warner E. et al. (2022).

[0375] In some embodiments, the disease / condition to be treated / prevented in accordance with this disclosure is a disease / condition characterized by abnormal organization of microtubule networks (e.g., intracellular) compared to the organization observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-disease cells).

[0376] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a disease / condition characterized by an increased density compared to the density of the microtubule network (e.g., within cells such as cardiomyocytes) observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-disease cells). In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a disease / condition characterized by an increased density compared to the density of the perinuclear microtubule network (e.g., within cells such as cardiomyocytes) observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-disease cells).

[0377] 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 may be determined using immunohistochemistry as described herein and, for example, Chen C. et al., Nature Medicine (2018), volume 24, 1225-1233, which is incorporated herein in its entirety by reference.

[0378] In some embodiments, the disease / condition to be treated / prevented in accordance with this disclosure is a disease / condition characterized by increased mechanical resistance compared to the mechanical resistance of the microtubule network (e.g., within cells such as cardiomyocytes) observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-disease cells).

[0379] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is, for example, a disease / condition characterized by an increase in mechanical force compared to the mechanical force transmitted to the cell nucleus / nuclear envelope (e.g., within cells such as cardiomyocytes) observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-disease cells).

[0380] In some embodiments, the diseases / conditions treated / prevented in accordance with this disclosure are, for example, diseases / conditions characterized by reduced contractility of cardiac tissue or cardiomyocytes compared to the contractility of cardiac tissue or cardiomyocytes in the absence of the disease / condition (e.g., in healthy subjects). Chen C. et al., Nature Medicine (2018) volume 24, 1225-1233, describes a method for measuring cell contractility.

[0381] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a disease / condition characterized by an increased level / expression of tubulin (e.g., intracellularly, such as cardiomyocytes) compared to the level / expression of tubulin (e.g., in healthy subjects or in equivalent non-disease cells) in the absence of the disease / condition. In some embodiments, the disease / condition is characterized by an increased level / expression of α-tubulin. In some embodiments, the disease / condition is characterized by an increased level / expression of β-tubulin.

[0382] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a disease / condition characterized by an elevated level / proportion of detyrosinated tubulin (e.g., intracellular, such as cardiomyocytes) compared to the level / expression (e.g., in healthy subjects or equivalent non-disease cells) in the absence of the disease / condition. The proportion of detyrosinated tubulin refers to the ratio of detyrosinated α-tubulin to total tubulin (e.g., intracellular). "Total tubulin" may refer to the protein levels of α-tubulin and / or β-tubulin, which are the main components of microtubules.

[0383] In some embodiments, the disease / condition treated / prevented according to this disclosure is a disease / condition characterized by an elevated level / expression compared to the level / percentage of acetylated tubulin in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-disease cells). In some embodiments, the disease / condition treated / prevented according to this disclosure is a disease / condition characterized by a decreased level / expression compared to the level / percentage of acetylated tubulin in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-disease cells). The percentage of acetylated tubulin refers to the ratio of acetylated α-tubulin to total tubulin (e.g., intracellular). "Total tubulin" may refer to the protein levels of α-tubulin and / or β-tubulin, which are the main components of microtubules.

[0384] In some embodiments, the disease / condition to be treated / prevented in accordance with this disclosure is a disease / condition characterized by an increased density of detyrosinated microtubules (e.g., intracellular, such as cardiomyocytes) compared to the percentage of detyrosinated microtubules (e.g., in healthy subjects or equivalent non-disease cells) in the absence of the disease / condition.

[0385] In some embodiments, microtubule dysfunction refers to perinuclear microtubule dysfunction. That is, the dysfunction relates to the population / density / function / detyrosine levels of perinuclear microtubules, as described above.

[0386] Diseases / conditions in which microtubule dysfunction is pathologically complex may be characterized by one or more of the following: For example, decreased contractility compared to the contractility of cells (e.g., cardiomyocytes) observed in the absence of disease / condition; For example, abnormal organization compared to microtubule network organization (e.g., intracellular) observed in the absence of disease / condition; For example, an increase in mechanical force compared to the level of mechanical force transmitted to the cell nucleus / nuclear envelope in the absence of disease / condition; For example, abnormal organization compared to sarcomere organization (e.g., intracellular) observed in the absence of disease / condition; For example, an increase in the mechanical resistance of the microtubule network compared to levels in the absence of disease / condition; For example, a decrease in cardiomyocyte contraction compared to the contraction of cardiomyocytes in the absence of disease / condition; For example, increased density of microtubule networks, e.g., perinuclear, interfibrous, and / or cortical microtubules, compared to the absence of disease / condition; For example, an increase in the level / expression of tubulin (e.g., α-tubulin and / or β-tubulin) compared to the level / expression in the absence of disease / condition; For example, an increase in the level / percentage of detyrosinated tubulin compared to the level / percentage in the absence of the disease / condition; For example, an increase in the level / percentage of intermediate filaments, e.g., microtubules interacting with desmin, compared to the level / percentage in the absence of disease / condition; and For example, an increase in the level / percentage of acetylated tubulin compared to the level / percentage in the absence of the disease / condition; For example, a decrease in the level / percentage of acetylated tubulin compared to the level / percentage in the absence of the disease / condition; For example, abnormal organization compared to the organization of peripheral microtubule networks (e.g., intracellular) observed in the absence of disease / condition; For example, an increase in mechanical force compared to the level of mechanical force transmitted to the cell nucleus / nuclear envelope by the perinuclear microtubule network in the absence of disease / condition; For example, increased mechanical resistance of the perinuclear microtubule network compared to levels in the absence of disease / condition; For example, increased density compared to the density of the perinuclear microtubule network in the absence of disease / condition; For example, increased levels / expression of perinuclear tubulins (e.g., α-tubulin and / or β-tubulin) compared to levels / expression in the absence of disease / condition; For example, an increase in the level / percentage compared to the level / percentage of detyrosinized perinuclear tubulin in the absence of disease / condition; and For example, an increase in the level / percentage of intermediate filaments, e.g., perinuclear microtubules interacting with desmin, compared to the level / percentage in the absence of disease / condition.

[0387] Disease models in which microtubule dysfunction is observed Chen C and colleagues performed functional tests on human muscle cells to support the role of microtubules in regulating proliferation and dysfunction to treat various heart diseases. Chen C et al. noted that previous studies had demonstrated 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, dystrophic cardiomyopathy, and diabetic cardiomyopathy, as summarized in Table A below.

[0388] Multiple other pieces of evidence suggest microtubule dysfunction in cardiac disease. HDAC6 inhibition, which has been shown to increase acetylated tubulin protein levels, has been proposed as a treatment for HFpEF; deficiency of microtubule affinity regulatory kinase 4 (MARK4) reduces left ventricular ejection fraction after acute myocardial infarction in mice; and microtubule-associated protein 4 (MAP4) phosphorylation has been found to be increased in mouse models of myocardial infarction (MI) and transverse aortic stenosis (TAC). Furthermore, tubulin and detyrosinated tubulin levels were increased in ZSF1 obese rats, an HFpEF model. [Table 1-1] [Table 1-2] [Table 1-3]

[0389] Diseases / conditions characterized by desmosome / desmoplakin deficiency / dysfunction The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and conditions including / characterized by desmosome deficiency / failure or dysfunction. Experimental examples of this disclosure demonstrate that LINC complex inhibition improves pathology in a mouse model of a disease characterized by desmosome deficiency / failure.

[0390] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure may be a disease / condition in which desmosome dysfunction or deficiency / impairment is pathologically involved, for example, a disease / condition in which desmosome dysfunction or deficiency / impairment is positively associated 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, desmosome dysfunction or deficiency / impairment may be a risk factor for the onset, development, or progression of the disease / condition.

[0391] In some embodiments, the disease / condition to be treated / prevented in accordance with this disclosure is a disease / condition characterized by a reduction in the number of desmosomes (e.g., formed between adjacent cells of a given tissue) compared to the number observed in the absence of the disease / condition (e.g., in a healthy subject or in equivalent non-infected tissue).

[0392] Diseases / conditions characterized by desmosome deficiency / deficiency or dysfunction include diseases / conditions characterized by reduced expression / activity of desmosome proteins (for example, diseases / conditions characterized by reduced expression / activity of constituent polypeptides of the desmosome intermediate filament complex (DIFC)).

[0393] Diseases / conditions characterized by desmosome deficiency / deficiency or dysfunction include diseases / conditions associated with mutations in genes encoding desmosome proteins. In some embodiments, the disease / condition treated / prevented according to this disclosure is a disease / condition associated with mutations in genes encoding constituent polypeptides of the desmosome intermediate filament complex (DIFC). In some embodiments, the disease / condition is associated with mutations in genes selected from DSP, DES, DSG1, DSG2, DSG3, DSG4, DSC1, DSC2, DSC3, PKP1, PKP2, PKP3, and JUP. In some embodiments, the disease / condition is associated with mutations in genes selected from DSP, DES, DSG2, DSC2, PKP2, and JUP.

[0394] The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and conditions including / characterized by desmoplakin deficiency / insufficiency or dysfunction. Experimental examples of this disclosure demonstrate that LINC complex inhibition improves pathology in a mouse model of a disease caused by desmoplakin deficiency / insufficiency.

[0395] In some embodiments, a disease or condition characterized by desmosome / desmoplakin deficiency / deficiency or dysfunction is characterized by cardiomyopathy. That is, in some embodiments, cardiomyopathy is a symptom of a disease or condition characterized by desmosome / desmoplakin deficiency / deficiency or dysfunction.

[0396] As explained above, mutations in genes encoding desmosomal proteins cause cardiomyopathy. Mutations in DSP are associated with dilated cardiomyopathy and arrhythmogenic cardiomyopathy. Mutations in DES are associated with dilated cardiomyopathy, arrhythmogenic cardiomyopathy, and restrictive cardiomyopathy. Mutations in DSG2, PKP2, and JUP are all associated with arrhythmogenic cardiomyopathy.

[0397] Mutations in DSP cause cardiomyopathy (particularly dilated cardiomyopathy and arrhythmogenic cardiomyopathy), as well as cardiocutaneous syndromes such as erythematous keratoderma-cardiamiocarditis and Naxos disease and Carvajal syndrome. Mutations in DSP are also associated with a variety of other diseases / conditions, including cutaneous fragility (epidermolysis bullosa) and palmoplantar keratosis. Autoantibodies against desmoplakin are characteristic of paraneoplastic pemphigus, an autoimmune disease. Reduced desmoplakin expression has also been observed in patients with oropharyngeal cancer and breast cancer, and it is thought that this reduced expression may alter cell-cell adhesion properties and enhance metastasis.

[0398] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure may be a disease / condition in which desmoplakin deficiency / insufficiency or dysfunction has a pathological effect, for example, a disease / condition in which desmoplakin deficiency / insufficiency or dysfunction is positively associated 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, desmoplakin deficiency / insufficiency or dysfunction may be a risk factor for the onset, development, or progression of the disease / condition.

[0399] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a disease / condition characterized by reduced expression / activity compared to, for example, the level of desmoplakin expression / activity in the absence of the disease / condition (e.g., in a healthy subject or in equivalent non-infected tissue). In some embodiments, the disease / condition is characterized by a decrease in number compared to, for example, the number of desmosomes observed (e.g., formed between adjacent cells of a given tissue) in the absence of the disease / condition (e.g., in a healthy subject or in equivalent non-infected tissue).

[0400] Diseases / conditions characterized by desmosome / desmoplakin deficiency / deficiency or dysfunction include diseases / conditions associated with mutations in DSPs.

[0401] In some embodiments, mutations to DSP are known or predicted to reduce the level of desmoplakin isoforms encoded by the wild-type allele (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 shortened version of the desmoplakin isoform encoded by the wild-type allele. In some embodiments, the mutation is known or predicted to result in the production of a desmoplakin isoform that is dysfunctional and / or nonfunctional. In some embodiments, the mutation is known or predicted to result in the production of a desmoplakin isoform that exhibits a reduced level of desmoplakin-mediated function compared to the level of the associated function indicated by the desmoplakin isoform encoded by the wild-type allele (e.g., DPI, DPII, and / or DSPIa). In some embodiments, the mutation is known or predicted to result in the production of a misfolded and / or degraded desmoplakin isoform. In some embodiments, the mutation is known or predicted to increase the level of disease-associated desmoplakin variants. In some embodiments, the mutation is known or predicted to increase the level of desmoplakin isoforms encoded by disease-associated alleles of DSP.

[0402] Diseases / conditions associated with mutations in DSPs are described, for example, in 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 of which are incorporated herein by reference in their entirety. Disease-related missense mutations in DSPs 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, and Y78. 7C, R808C, R808H, R1255K, R1255T, L1348R, T1373A, L1654P, R1775I, R1838H, Q198P, D203N, R222L, D241N, E290K, I 305F, A306T, R315C, R315P, M316V, A349P, Y350H, T356K, I368T, E384Q, K401N, N408K, R451G, R451H, I461V, C482Y, Y 494F, 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, R2189 Q, T2196I, I2263T, T2267S, A2294G, Q2295H, G2338R, E2343K, I2347V, D2579H, I2593S, T2664N, G2666D, A2674T, M2 684V, M2707T, A2709T, R2759S, P2777H, I2797T, M2819L, G2844V, I2869V, K1094N, Y1169N, L1178R, L1178V, Y1188H,R1207K, T1217M, D1251N, D1258E, A1274P, K1288Q, T1319I, R1341H, E135 7D, R1392Q, R1392W, E1407D, A1435P, R1458G, Y1512C, L1514P, L1535P, K 1537C, K1537H, T1557M, K1581E, K1592R, M1601I, S1623C, V1639M, S1658 F, R1666Q, R1666W, H1684R, E1721D, R1738Q, E1740K, E1833V, R1852H, R23 Examples include 66H, 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.

[0403] In some embodiments, the DSP mutation is a mutation associated with heart disease, such as cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic cardiomyopathy. Mutations affecting DSP 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, R808H, R1255K, R1255T, L1348R, T1373A, L1654P, R1775I, R1838H, Q198P, D203N, R222L, D241N, E290K, I305F, A306T, R3 15C, R315P, M316V, A349P, Y350H, T356K, I368T, E384Q, K401N, N408K, R 451G, 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, V 2107L, R2083C, K2103E, A2148T, T2177N, R2189Q, T2196I, I2263T, T226 7S, A2294G, Q2295H, G2338R, E2343K, I2347V, D2579H, I2593S, T2664N, G2666D, A2674T, M2684V, M2707T, A2709T, R2759S, P2777H, I2797T, M28 19L, G2844V, I2869V, K1094N, Y1169N, L1178R, L1178V, Y1188H, R1207 K, T1217M, D1251N, D1258E, A1274P, K1288Q, T1319I, R1341H, E1357D, R 1392Q, R1392W, E1407D, A1435P, R1458G, Y1512C, L1514P, L1535P, K153 7C, K1537H, T1557M, K1581E, K1592R, M1601I, S1623C, V1639M, S1658F,Examples include 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.

[0404] In some embodiments, DSP mutations are associated with cardiocutaneous syndromes (CCS). For example, the DSP deletion variant c.825_827del has been reported in subjects with cardiocutaneous syndromes characterized by palmoplantar keratoderma and arrhythmogenic cardiomyopathy (Cimen et al., J Clin Med. (2023) 12(3):913). Examples of cardiocutaneous syndromes include Naxos disease and Carvajal syndrome.

[0405] In some embodiments, the DSP mutation is a mutation associated with Naxos disease or Carvajal syndrome. Examples of such mutations for DSP include I560F, T564I, L583P, S597L, E2193K, L2329P, Q2371K, G2375R, and A2655D.

[0406] In some embodiments, the DSP mutation is a mutation associated with skin fragility-trichome syndrome. Examples of such mutations in DSP include N287K, R2366H, and R2366C.

[0407] In some embodiments, DSP mutations are mutations associated with erythematous keratoderma-cardiamyocardial syndrome. Examples of such DSP mutations include L622P, Q616P, and H618P.

[0408] In some embodiments, DSP mutations are mutations associated with asymptomatic alopecia. An example of such a DSP mutation is P498L.

[0409] In some embodiments, the DSP mutation is a mutation associated with palmo-sacral keratoderma. An example of such a DSP mutation is R1184W.

[0410] In some embodiments, DSP mutations are those associated with severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. Examples of such DSP mutations include S610P, H586P, and H586Y.

[0411] In aspects and embodiments of this disclosure, the diseases / conditions to be treated / prevented include cardiomyopathy, dilated cardiomyopathy, arrhythmogenic cardiomyopathy, diseases / conditions in which desmosome deficiency / failure or dysfunction has a pathological impact, diseases / conditions characterized by a reduction in the number of desmosomes, diseases / conditions associated with mutations in genes encoding desmosome proteins (e.g., selected from DSP, DES, DSG2, DSC2, PKP2, and JUP), and desmosome proteins The following are selected from diseases / conditions characterized by reduced expression / activity of desmoplakin, diseases / conditions in which desmoplakin deficiency / insufficiency or dysfunction has pathological effects, diseases / conditions characterized by reduced expression / activity of desmoplakin, diseases / conditions associated with mutations to DSP, skin diseases, cardiocutaneous syndromes (CCS), Naxos disease, Carvajal syndrome, keratosis, palmoplantar keratosis, epidermolysis bullosa, cutaneous fragility, trichorrheal syndrome, erythematous keratosis-cardiomyopathy syndrome, asymptomatic alopecia, palmoplantar keratosis and severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndromes.

[0412] Diseases / conditions characterized by sarcomere / titin deficiency / dysfunction The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and conditions including / characterized by sarcomere / titin deficiency / deficiency or dysfunction. Experimental examples of this disclosure demonstrate that LINC complex inhibition improves pathology in a mouse model of a disease characterized by sarcomere / titin deficiency / deficiency.

[0413] In some embodiments, the diseases / conditions treated / prevented in accordance with this disclosure are diseases / conditions in which sarcomere / titin dysfunction or deficiency / deficiency pathologically affects, for example, diseases / conditions in which sarcomere / titin dysfunction or deficiency / deficiency is positively associated 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, sarcomere / titin dysfunction or deficiency / deficiency may be a risk factor for the onset, development, or progression of the disease / condition.

[0414] In some embodiments, the diseases / conditions treated / prevented in accordance with this disclosure are, for example, diseases / conditions characterized by sarcomere disorganization resulting in reduced contractility compared to sarcomere organization observed in the absence of the disease / condition (e.g., in healthy subjects or in equivalent non-infected tissue).

[0415] In some embodiments, the disease / condition to be treated / prevented in accordance with this disclosure is, for example, a disease / condition characterized by increased metabolic stress compared to cardiac metabolism observed in the absence of the disease / condition (e.g., in a healthy subject or in equivalent non-infected tissue).

[0416] Diseases / conditions characterized by titin deficiency / deficiency or dysfunction include diseases / conditions characterized by reduced titin protein expression / activity (for example, diseases / conditions characterized by reduced titin polypeptide expression / activity).

[0417] Diseases / conditions characterized by sarcomere deficiency / deficiency or dysfunction include diseases / conditions associated with mutations in genes encoding sarcomere proteins. In some embodiments, the disease / condition treated / prevented according to this disclosure is a disease / condition associated with mutations in genes encoding sarcomere constituent polypeptides. In some embodiments, the disease / condition is associated with mutations in genes selected from TTN, MYH7, TNNT2, TNNI3, TNNC1, TPM1, MYBPC3, MYL2, MYL3, CSRP3, RBM20, ACTN2, TCAP, and ACTC1. In some embodiments, the disease / condition is associated with mutations in genes selected from TTN, TNNT2, TPM1, MYH7, and MYBPC3.

[0418] The aspects and embodiments of this disclosure relate to the treatment and prevention of diseases and conditions including / characterized by titin deficiency / deficiency or dysfunction. Experimental examples of this disclosure demonstrate that LINC complex inhibition improves pathology in mouse models of diseases caused by titin deficiency / deficiency.

[0419] In some embodiments, a disease or condition characterized by sarcomere / titin deficiency / deficiency or dysfunction is characterized by cardiomyopathy. That is, in some embodiments, cardiomyopathy is a symptom of a disease or condition characterized by sarcomere / titin deficiency / deficiency or dysfunction.

[0420] As explained above, mutations in genes encoding sarcomere proteins cause cardiomyopathy. Mutations in TTN are associated with dilated cardiomyopathy, arrhythmogenic cardiomyopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy. Mutations in TNNT2 are associated with dilated cardiomyopathy and hypertrophic cardiomyopathy. Mutations in TPM1 are associated with dilated cardiomyopathy. Mutations in MYH7 are associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, and left ventricular noncompaction cardiomyopathy. All mutations in MYBPC3 are associated with hypertrophic cardiomyopathy and left ventricular noncompaction cardiomyopathy.

[0421] Mutations in TTN cause cardiomyopathy (particularly dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic cardiomyopathy), as well as diseases / conditions characterized by cardiomyopathy such as early adult-onset degenerative distal titinopathy, early-onset myopathy with fatal cardiomyopathy, multiminicore disease with heart disease, and Emery-Dreyfus muscular dystrophy (EDMD). Titin mutations are also associated with a variety of other diseases / conditions, including pure skeletal muscle myopathy: limb cingulate 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).

[0422] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure may be a disease / condition in which titin deficiency / impairment or dysfunction pathologically affects, for example, a disease / condition in which titin deficiency / impairment or dysfunction is positively associated 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, titin deficiency / impairment or dysfunction may be a risk factor for the onset, development, or progression of the disease / condition.

[0423] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is, for example, a disease / condition characterized by reduced titin expression / activity compared to the level of expression / activity in the absence of the disease / condition (e.g., in a healthy subject or in equivalent unaffected tissue). In some embodiments, the disease / condition is characterized by sarcomere breakdown resulting in reduced contractility compared to sarcomere organization observed in the absence of the disease / condition (e.g., in a healthy subject or in equivalent unaffected tissue). In some embodiments, the disease / condition is characterized by increased metabolic stress compared to cardiac metabolism observed in the absence of the disease / condition (e.g., in a healthy subject or in equivalent unaffected tissue).

[0424] Diseases / conditions characterized by sarcomere / titin deficiency / deficiency or dysfunction include diseases / conditions associated with mutations to TTN.

[0425] In some embodiments, mutations to TTN are known or predicted to reduce the levels of titin isoforms encoded by the wild-type allele (e.g., the putative full isoforms (ICs) N2BA-G, N2BA-N2, N2BA-A1, N2BA-A2, N2B, Novex-1, Novex-2, Novex-3, N2A-soleus muscle, and N2A-psoas muscle). 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 shortened version of the titin isoform encoded by the wild-type allele. In some embodiments, the mutation is known or predicted to result in the production of a titin isoform that is dysfunctional and / or nonfunctional. 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 level of associated function indicated by the titin isoform encoded by the wild-type allele (e.g., the putative complete isoforms (ICs) N2BA-G, N2BA-N2, N2BA-A1, N2BA-A2, N2B, Novex-1, Novex-2, Novex-3, N2A-soleus muscle, and N2A-psoas muscle). 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 level of a disease-associated titin variant. In some embodiments, this mutation is known or predicted to increase the level of a titin isoform encoded by the disease-associated allele of TTN.

[0426] Diseases / conditions associated with mutations to TTN are described, for example, 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-related missense mutations in TTN (UniProt:Q8WZ42-1) include Val54Met, Arg740Leu, Ala743Val, Trp976Arg, Val1034Met, Arg2083fs, Thr2896lle, Glu2989GlufsX4, Ser3799Tyr, Gln4053X, Gln4249X, Ser4465Asn, Leu5782Phe, Ser6395fs, Glu7004Lys, Gly7933fs, Asp7971_Ile7976del, Tyr8958Cys, Arg9427His, Arg9531Gln, His9775Tyr, Gly10159, Val11396, 11477InsPro, Val11879, Val11879, Val11879Leu, Ala12675, Ala12873fs, Pro13298_Thr17642dup, C ys13771X, Glu14779fs, Asp14909, Tyr15045Cys, Phe15108fs, Asp16122, Gly16189X, Trp16359X, Lys16782, Arg 17295X, Ala17342Thr, Arg17470X, Glu17715fs, Glu17783X, Ile17876Thr, Lys17898fs, Glu17978fs, Glu18141, Asp18235, His18335, Cys18789X, Arg18858X, Arg18985X, Ala19506Thr, Arg19560X, Pro20049fs, Ala20236Ser, Val20591Glu, Gln20809, Arg20858X, Thr21135, Tyr21301, Gly21497, Lys21640fs, Ile21923 and subsequent abbreviations (Truncation afterIle21923), Glu21956fs, Ala22353fs, Pro22582fs, Asn23824fs, Gln24059fs, Ser24241fs, Gln24281X, Gln2 5689X, Trp26632X, Arg26949X, Lys27016X, Ser27052IfsX1, Trp27147X, Ser27179fs, Glu27300, Tyr27567X, T yr28326fs, Glu28386LysfsX10, Trp29318X, Arg29415X, Glu29510X, Thr29725fs, Pro30068Arg, Cys30071Ar g, Gln30081X, Trp30088Arg, Trp30088Cys, Trp30088Leu, Pro30091Leu, Asn30145Lys, Gly30150Asp, Thr3016 5, Asn30348fs, Thr30513fs, Arg30857, Arg31126fs, Arg31195X, Lys31371X, Pro31774Leu, Ser31841X, Asn3 2379ThrfsX9, Trp32431Arg, Arg32450Trp, Arg32534X, Pro32976fs, Arg33041Gly, His33534fs, Gln33637X, G Examples include ly33698fs, Ser33828SerfsX11, Lys33915fs, Met34218Thr, Ser34242GlnfsX10, Glu34286_Trp34289delinsValLysGluLys, His34305Pro, Iso34306Asp, Leu34315Pro, Lys34322AsnfsX9, Lys34322fs, and Gln34323X.

[0427] In some embodiments, TTN mutations are mutations associated with heart disease, 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, Gly7933 fs, Asp7971_Ile7976del, Tyr8958Cys, Arg9427His, Arg9531Gln, His9775Tyr, Gly10159, Val11396, 11477I nsPro, Val11879, Val11879, Val11879Leu, Ala12675, Ala12873fs, Pro13298_Thr17642dup, Cys13771X, Glu 14779fs, Asp14909, Tyr15045Cys, Phe15108fs, Asp16122, Gly16189X, Trp16359X, Lys16782, Arg17295X, Al a17342Thr, Arg17470X, Glu17715fs, Glu17783X, Ile17876Thr, Lys17898fs, Glu17978fs, Glu18141, Asp182 35, His18335, Cys18789X, Arg18858X, Arg18985X, Ala19506Thr, Arg19560X, Pro20049fs, Ala20236Ser, Val20591Glu, Gln20809, Arg20858X, Thr21135, Tyr21301, Gly21497, Lys21640fs, Ile21923 and subsequent abbreviations (Truncation afterIle21923), Glu21956fs, Ala22353fs, Pro22582fs, Asn23824fs, Gln24059fs, Ser24241fs, Gln24281X, Gln2 5689X, Trp26632X, Arg26949X, Lys27016X, Ser27052IfsX1, Trp27147X, Ser27179fs, Glu27300, Tyr27567X, T yr28326fs, Glu28386LysfsX10, Trp29318X, Arg29415X, Glu29510X, Thr29725fs, Pro30068Arg, Cys30071Ar g, Gln30081X, Trp30088Arg, Trp30088Cys, Trp30088Leu, Pro30091Leu, Asn30145Lys, Gly30150Asp, Thr3016 5, Asn30348fs, Thr30513fs, Arg30857, Arg31126fs, Arg31195X, Lys31371X, Pro31774Leu, Ser31841X, Asn3 2379ThrfsX9, Trp32431Arg, Arg32450Trp, Arg32534X, Pro32976fs, Arg33041Gly, His33534fs, Gln33637X, G Examples include ly33698fs, Ser33828SerfsX11, Lys33915fs, Met34218Thr, Ser34242GlnfsX10, Glu34286_Trp34289delinsValLysGluLys, His34305Pro, Iso34306Asp, Leu34315Pro, Lys34322AsnfsX9, Lys34322fs, and Gln34323X.

[0428] In some embodiments, TTN mutations are mutations associated with disease / conditions characterized by cardiomyopathy, such as early-onset myopathy with fatal cardiomyopathy (EOMFC), characterized by dilated cardiomyopathy, delayed motor development with generalized muscle weakness primarily affecting the proximal and distal lower extremities. Examples of titin-deficient 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. Examples include P6613fs, Y13414fs, P10340fs, P11641fs, P11267fs, E16226Ter, E17867Ter, E8802Ter, E15299Ter, E8927Ter, E8994Ter, T21129fs, T13897fs, T20202fs, T22770fs, T13705fs, T13830fs, R17915Ter, R15347Ter, R9042Ter, R8975Ter, R16274Ter, and R8850Ter.

[0429] In some embodiments, TTN mutations are mutations associated with 2J-type autosomal recessive limb-girdle muscular dystrophy. Examples of 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, E11681Ter, E11307Ter, D2143fs, D2189fs, K25643fs, K32015fs, K25518fs, K25710fs, K32942fs, K34583fs, M25640fs, M25707fs, M34580fs, M 32939fs, M25515fs, M32012fs, E19273fs, E19398fs, E19465fs, E25770 fs, E26697fs, E28338fs, E18442fs, E27315fs, E18375fs, E25674fs, E1 8250fs, E24747fs, I16818fs, I16943fs, I17010fs, I23315fs, I24242 fs, I25883fs, P15942fs, P24815fs, P15875fs, P23174fs, P15750fs, P2 2247fs, D888fs, D842fs, T25284fs, T25476fs, T34349fs, T31781fs, T3 2708fs, T25409fs, C31712R, C30071R, C29144R, C22647R, C22772R, C22 839R, Q13385Ter, Q13510Ter, Q13577Ter, Q19882Ter, Q20809Ter, Q22450Ter, C10433fs, C10500fs, C10308fs, C16805fs, C17732fs, C19373fs , Q24894Ter, Q26535Ter, 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、Examples include R26109fs, R26301fs, R32606fs, E12913Ter, Q26794Ter, Q17921Ter, Q24226Ter, Q17854Ter, Q17729Ter, and Q25153Ter.

[0430] In some embodiments, TTN mutations are mutations associated with tibial anterior muscular dystrophy. Such TTN mutations 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, K26898fs, K35963fs, K33395fs, K34322fs, R33703Ter, R32062Ter, R24638Ter, R31135Ter, R24763Ter, R24830Ter, P3173 2L, P29164L, P22792L, P22859L, P30091L, P22667L, R22499Ter, R20858Ter, R19931Ter, R13626Ter, R13434Ter, R13559Ter, R23868Ter, R21300 Ter, R22227Ter, R14803Ter, R14995Ter, R14928Ter, L29811fs, L23439fs, L30738fs, L23314fs, L23506fs, L32379fs, R18878Ter, R20519Ter, R 11579Ter, R11646Ter, R17951Ter, R11454Ter, P22521fs, P28826fs, P29753fs, P31394fs, P22329fs, P22454fs, Y24539Ter, Y27107Ter, Y25466 Ter, 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, R221 83Ter, R20626Ter, R18058Ter, R18985Ter, R11561Ter, R11686Ter, R11753Ter, R32102Ter, R33743Te r, R24678Ter, R31175Ter, R24803Ter, R24870Ter, R22975Ter, R24616Ter, R15676Ter, R15743Ter, R1 5551Ter, R22048Ter, R20026Ter, R21667Ter, R12727Ter, R19099Ter, R12602Ter, R12794Ter, R25552 Ter, R22984Ter, R16487Ter, R16679Ter, R16612Ter, R23911Ter, G25912Ter, G27553Ter, G24985Ter, G18488Ter, G18613Ter, G18680Ter, R16290Ter, R9918Ter, R17217Ter, R18858Ter, R9793Ter, R9985T Examples include er, R19856fs, R19923fs, R26228fs, R19731fs, R28796fs, A27689fs, A18624fs, A25121fs, A26048fs, A18749fs, A18816fs, W23158Ter, W23283Ter, W23350Ter, W29655Ter, W30582Ter, and W32223Ter.

[0431] In some embodiments, TTN mutations are mutations associated with myopathy, myofibrillar, 9, with early respiratory failure. Examples of such TTN mutations include C31712R, C30071R, C29144R, C22647R, C22772R, C22839R, I21018fs, I12145fs, I19377fs, I11953fs, I12078fs, I18450fs, Y32573Ter, Y35141Ter, Y26268Ter, Y33500Ter, Y26076Ter, Y26201Ter, Q26794Ter, Q17921Ter, Q24226Ter, Q17854Ter, and Q1772 9Ter, Q25153Ter, K27023fs, K27090fs, K26898fs, K35963fs, K33395fs, K34322fs, R33703Ter, R32062Ter, R24638Ter, R31135Ter, R2 4763Ter, R24830Ter, P31732L, P29164L, P22792L, P22859L, P30091L, P22667L, R22499Ter, R20858Ter, R19931Ter, R13626Ter, R13434 Ter, R13559Ter, R23868Ter, R21300Ter, R22227Ter, R14803Ter, R14995Ter, R14928Ter, L29811fs, L23439fs, L30738fs, L23314fs, L 23506fs, L32379fs, R18878Ter, R20519Ter, R11579Ter, R11646Ter, R17951Ter, R11454Ter, P22521fs, P28826fs, P29753fs, P31394fs , P22329fs, P22454fs, Y24539Ter, Y27107Ter, Y25466Ter, Y18042Ter, Y18167Ter, Y18234Ter, N20902fs, N21094fs, N21027fs, N2832 6fs, N27399fs, N29967fs, R23469Ter, R25110Ter, R16237Ter, R16045Ter, R16170Ter, R22542Ter, R28364Ter, R19299Ter, R25796Ter,R19424Ter, R26723Ter, R19491Ter, E24419Ter, E26060Ter, E23492Ter, E16995Ter, E17120Ter, E17187Ter, R 31056Ter, R29415Ter, R21991Ter, R28488Ter, R22116Ter, R22183Ter, R20626Ter, R18058Ter, R18985Ter, R11 561Ter, R11686Ter, R11753Ter, R32102Ter, R33743Ter, R24678Ter, R31175Ter, R24803Ter, R24870Ter, R229 75Ter, R24616Ter, R15676Ter, R15743Ter, R15551Ter, R22048Ter, R20026Ter, R21667Ter, R12727Ter, R19099 Ter, R12602Ter, R12794Ter, R25552Ter, R22984Ter, R16487Ter, R16679Ter, R16612Ter, R23911Ter, G25912T er, G27553Ter, G24985Ter, G18488Ter, G18613Ter, G18680Ter, R16290Ter, R9918Ter, R17217Ter, R18858Ter, Examples include R9793Ter, R9985Ter, R19856fs, R19923fs, R26228fs, R19731fs, R28796fs, A27689fs, A18624fs, A25121fs, A26048fs, A18749fs, A18816fs, W23158Ter, W23283Ter, W23350Ter, W29655Ter, W30582Ter, and W32223Ter.

[0432] In aspects and embodiments of this disclosure, the disease / condition to be treated / prevented is selected from: cardiomyopathy, dilated cardiomyopathy, arrhythmogenic cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, disease / condition in which sarcomere deficiency / failure or dysfunction has a pathological effect, disease / condition in which sarcomere deficiency / failure or dysfunction has a pathological effect, disease / condition characterized by sarcomere breakdown and the resulting decrease in contractility, gene encoding sarcomere proteins (e.g., TTN, TNNT2, TPM1, MYH) Diseases / conditions associated with mutations to (selected from 7 and MYBPC3), diseases / conditions characterized by reduced expression / activity of sarcomere proteins, diseases / conditions in which titin deficiency / impairment or dysfunction has pathological effects, diseases / conditions characterized by reduced expression / activity of titin, diseases / conditions associated with mutations to TTN, myopathy, autosomal recessive limb-girdle muscular dystrophy type 2J, myofibrils myopathy with early respiratory failure, early-onset myopathy with fatal cardiomyopathy, and tibialis anterior muscular dystrophy.

[0433] Treatment / prevention of diseases / conditions through LINC complex inhibition This disclosure provides methods and articles (pharmaceuticals and compositions) for the treatment and / or prevention of diseases / conditions by LINC complex inhibition. The treatment / prevention of diseases is achieved, for example, by LINC complex inhibition in cells, tissues / organs / organ systems / subjects.

[0434] Aspects of this disclosure relate to the treatment / prevention of diseases / conditions described herein by LINC complex inhibition.

[0435] The methods may aim to delay / prevent the onset of disease / condition symptoms, reduce (i.e., alleviate) the severity of disease / condition symptoms, reduce the level of correlation of disease / condition pathology, reverse disease / condition symptoms, reduce morbidity in individuals with disease / condition, increase survival rates in individuals with disease / condition, extend lifespan in individuals with disease / condition, reduce mortality in individuals with disease / condition, and / or delay / prevent the progression of disease / condition (e.g., to later stages).

[0436] In some embodiments, the disease / condition treated / prevented according to this disclosure is laminopathy. do not have In some embodiments, the disease / condition is associated with mutations in LMNA. do not have In some embodiments, the disease / condition is associated with a mutation in one of LMNA, LMNB1, and LMNB2. do not have In some embodiments, the disease / condition is associated with a mutation in one of LMNA, LMNB1, and LMNB2. do not have .

[0437] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is nuclear envelopedism. do not have In some embodiments, the disease / condition is associated with a mutation in one of the following: LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62. do not have .

[0438] The LINC complex inhibitors described herein may be provided in the form of nucleic acids comprising or encoding LINC complex inhibitors. Such nucleic acids may comprise or consist of DNA and / or RNA, and may be polynucleotides or comprise polynucleotides.

[0439] If the LINC complex inhibitor is a nucleic acid, the LINC complex inhibitor may be provided in the form of a larger nucleic acid containing the LINC complex inhibitor. If the LINC complex inhibitor is a peptide / polypeptide or SSN system that targets the LINC complex protein, the LINC complex inhibitor may be provided in the form of a nucleic acid encoding the LINC complex inhibitor.

[0440] In some embodiments, LINC complex inhibitors are administered in the form of nucleic acids encoding factors necessary for the production of LINC complex inhibitors (e.g., nucleic acids encoding precursors of LINC complex inhibitors and / or nucleic acids encoding factors necessary for the production / assembly of LINC complex inhibitors). For example, LINC complex inhibitors may be administered in the form of nucleic acids encoding factors necessary for the production of small molecule or biomolecule LINC complex inhibitors.

[0441] The nucleic acid may contain a LINC complex that inhibits polypeptide-coding nucleotide sequences, and may additionally contain one or more non-polypeptide-coding nucleotide sequences. The non-polypeptide-coding nucleotide sequence(s) may be, for example, a 5' cap, 5' UTR, 3' UTR, and / or poly(A) tail sequence.

[0442] The nucleic acid may be a vector or may be contained within a vector. As used herein, “vector” refers to a nucleic acid used as a vehicle for transferring an exogenous nucleic acid to a cell. The vector may be a vector for the expression of nucleic acid in a cell (i.e., the vector may be an expression vector). Such a vector may include a promoter sequence operably ligated to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a termination codon and an expression enhancer. A peptide or polypeptide can be expressed from the vector according to this disclosure using any suitable vector, promoter, enhancer, and termination codon known in the art.

[0443] The term “operatably linked” may include situations in which a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in a manner such as the expression of the nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, if the regulatory sequence can result in the transcription of the nucleic acid sequence, then the regulatory sequence is operatably linked to the selected nucleic acid sequence. The resulting transcript(s) may then be translated into polypeptides, such as LINC complex inhibitory polypeptides.

[0444] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus vectors (e.g., vectors derived from mouse 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 Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4,9 (both incorporated herein by reference in their entirety).

[0445] In some embodiments, the vector is selected based on the tropism of the cell types / tissues / organs to which it is desirable to deliver the nucleic acid containing / encoding the LINC complex inhibitor. In some embodiments, the vector is selected based on the tropism of the cell types / tissues / organs to which it is desirable to express the LINC complex inhibitor. For example, it may be desirable to deliver the nucleic acid / LINC complex inhibitor to cell types / tissues / organs affected by the disease to be treated / prevented according to this disclosure (e.g., cell types / tissues / organs to which symptoms of the disease appear).

[0446] In some embodiments, it is desirable to deliver the nucleic acid encoding the LINC complex inhibitor to muscle cells / tissues (e.g., cardiac and / or skeletal muscle cells / tissues), and in such embodiments, a vector having tropism to such cells / tissues may be used. In some embodiments, the vector may be cardiotropic. In some embodiments, the vector may be myotropic.

[0447] In preferred embodiments, the vector is an adeno-associated virus vector. Adeno-associated virus vectors and their use in vector gene therapy are outlined, 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, both of which are incorporated herein by reference in their entirety. In some embodiments, the vector may be the adeno-associated virus vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18:358-378. In some embodiments, the vector may be the adeno-associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12:255-272.

[0448] In some embodiments, 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 in its entirety. Conventional AAVs have a single-stranded DNA genome and synthesize complementary strands depending on the DNA replication mechanism of the transduced cell, delaying the expression of the transduced gene. In contrast, scAAVs contain complementary sequences that spontaneously anneal upon infection, eliminating the need for DNA synthesis in the transduced host cell. Compared to classical single-stranded AAV vectors, scAAV vectors have been shown to accelerate the initiation of transgene expression and increase the level of transgene expression.

[0449] 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.

[0450] 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, AAV9.45.

[0451] In some embodiments, the vector may be a skeletal muscle-targeting 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.

[0452] In some embodiments, the vector includes modifications to increase binding to and / or transduction to the target cell type (i.e., compared to the level of binding / transduction by an unmodified vector). In some embodiments, the modifications are to the capsid protein.

[0453] In some embodiments, the vector comprises a capsid protein containing a cell-targeting peptide. In some embodiments, the cell-targeting peptide is a cell-targeting peptide described in Buening and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12:248-265, which is incorporated herein in its entirety by reference, for example, the cell-targeting peptides shown in Tables 1, 2, 3, or 4.

[0454] In some embodiments, the vector comprises a capsid protein whose one or more tyrosine residues are substituted, for example, 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 vector comprises a capsid protein in which one or more tyrosine residues are substituted with other amino acids, such as those described in Iida et al., Biomed Res Int. (2013) 2013:974819, which is incorporated herein in its entirety by reference.

[0455] In some embodiments, the vector may be the adeno-associated virus vector described above by Buening and Srivastava. In some embodiments, the vector may be the adeno-associated virus vector described above by Iida et al.

[0456] In some embodiments, the nucleic acid / vector includes one or more sequences for controlling nucleic acid expression. Therefore, in some embodiments, the nucleic acid / vector includes a control element for inducible expression of the nucleic acid. Sequences for controlling nucleic acid expression may provide nucleic acid expression by cells of a particular type or tissue. For example, expression may be under the control of a cell-type or tissue-specific promoter.

[0457] The promoters for cell-type or tissue-specific expression of nucleic acids provided in this disclosure can be selected depending on the disease being treated / prevented. For example, the promoter may drive expression in disease-affected cell types / tissues / organs (e.g., cell types / tissues / organs in which disease symptoms appear).

[0458] In some embodiments, the promoter may provide expression in muscle cells / tissues (e.g., cardiac and / or skeletal muscle cells / tissues). In some embodiments, the promoter may be a cardiac or cardiomyocyte-specific promoter (e.g., cTNT, α-MHC, or MLC2v promoter). In some embodiments, the promoter may be a skeletal muscle / striated muscle cell-specific promoter (e.g., MCK, MHCK7, or desmin promoter). In some embodiments, the promoter is cTNT.

[0459] 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).

[0460] Sequences for controlling nucleic acid expression may, for example, provide nucleic acid expression in response to a given drug / signal. For example, expression may be under the control of an inducible promoter. The drug may provide inducible nucleic acid expression in vivo by administering the drug to a target administered with the modified cells according to this disclosure, or ex vivo / in vitro by administering the drug to cells in cultures ex vivo or in vitro.

[0461] In some embodiments, the nucleic acids or vectors according to this disclosure may use a conditional expression system to control the expression of the nucleic acid containing / encoding a LINC complex inhibitor by a cell containing the nucleic acid / vector. "Conditional expression" may also be referred to herein as "inducible expression" and refers to expression that depends on specific conditions, such as the presence of a specific drug. Conditional expression systems are well known in the art and are outlined, for example, in Ryding et al. Journal of Endocrinology (2001) 171, 1-14, which are incorporated herein by reference in their entirety.

[0462] In a preferred embodiment, the nucleic acid has a size that allows for its delivery as a gene therapy, i.e., in a suitable vector.

[0463] In some embodiments, the nucleic acid comprising / encoding the LINC complex inhibitor consists of a nucleotide sequence having a size within the packaging limits of a vector for delivering polynucleotides. In some embodiments, the nucleic acid consists of a nucleotide sequence having a size within the packaging limits of a vector described herein. In some embodiments, the nucleic acid consists of a nucleotide sequence having a size within the packaging limits of an adeno-associated virus (AAV) vector, e.g., an AAV vector described herein. In some embodiments, the nucleic acid consists of a nucleotide sequence having a size within the packaging limits of an scAAV vector. In some embodiments, the nucleotide sequence of the nucleic acid consists of one nucleotide with fewer than 6,000 nucleotides, e.g., ≤5,000, ≤4,500, ≤4,000, ≤3,500, ≤3,000, ≤2,500, ≤2,400, or ≤2,300.

[0464] The LINC complex inhibitors according to this disclosure may be produced intracellularly. For example, if the LINC complex inhibitor is a LINC complex inhibitory polypeptide, the LINC complex inhibitor may be produced intracellularly by transcription from the nucleic acid encoding the polypeptide and subsequent translation of the transcribed RNA.

[0465] The LINC complex inhibitors described herein may be provided in the form of compositions comprising a LINC complex inhibitor and / or compositions comprising a nucleic acid comprising / encoding a LINC complex inhibitor. Such compositions may also include relevant articles (i.e., LINC complex inhibitory polypeptides / nucleic acids / vectors) in formulations suitable for clinical use.

[0466] The composition contains 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), auxiliaries, 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. It may also contain vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, lubricants (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), 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 (e.g., titanium dioxide).

[0467] As used herein, the term “pharmaceutically acceptable” means, within reasonable medical judgment, a compound, component, material, composition, dosage form, etc., that is suitable for use in contact with the tissue of the subject of concern (e.g., a human subject) without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, auxiliary, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener of a composition according to this disclosure must also be “acceptable” in the sense that it is compatible with the other components of the formulation. Suitable carriers, diluents, excipients, auxiliaries, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners can be found in standard pharmaceutical literature, for example, Remington's "The Science and Practice of Pharmacy" (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0468] The articles of this disclosure may be formulated for administration to a subject, for example, via a route of administration suitable for the nature of the therapeutic agent and the disease being treated / prevented. Administration of the articles of this disclosure may be parenteral, systemic, topical, intracavitary, intravascular, intravenous, intraarterial, intramuscular, subarachnoid, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral, or percutaneous. In some embodiments, administration may be by injection or infusion, or by ingestion.

[0469] In some embodiments and settings, the articles of the Disclosure may be administered to a target tissue / organ (e.g., a tissue / organ affected by a disease / condition (e.g., a tissue / organ on which symptoms of the disease / condition appear)). In some embodiments and settings, the articles of the Disclosure may be administered into the blood by injection or infusion (e.g., via a cannula) (i.e., intravenous / intra-arterial administration), or subcutaneously or orally. The specific mode and / or site of administration may be selected according to the location where the therapeutic / preventive effect is desired (e.g., cardiomyocytes / tissues).

[0470] The pharmaceutical composition / pharmaceutical may contain a LINC complex inhibitor / nucleic acid / vector in a sterile or isotonic medium. The pharmaceutical composition / pharmaceutical may be provided in a fluid form, including a gel. The fluid formulation may be formulated for administration by injection or infusion (e.g., via a cannula) into a blood vessel or a selected area of ​​a human or animal body. The pharmaceutical composition / pharmaceutical may be provided in a solid form, for example, in a lyophilized form.

[0471] The LINC complex inhibitors, nucleic acids, vectors, cells, and compositions relating to this disclosure may be modified and / or formulated to facilitate delivery to and / or uptake of target cells / tissues (e.g., cardiomyocytes / tissues). Strategies for such targeted delivery are outlined, 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.

[0472] In some embodiments, the articles of the Disclosure may be encapsulated in nanoparticles or liposomes. In some embodiments, the articles of the Disclosure may be associated (covalently or noncovalently) with cell-permeable peptides (e.g., protein transduction domains, Trojan horse peptides, arginine-rich peptides, vectocell peptides), cationic polymers, cationic lipids, or viral carriers.

[0473] The nanoparticles may be organic, such as micelles, liposomes, proteins, solid lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. The nanoparticles may also be inorganic, such as nanotubes or metal particles, and optionally, organic molecules may be added. In some embodiments, the nanoparticles are those described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which are incorporated herein by reference in their entirety. In some embodiments, the nanoparticles are PLGA, polypeptides, poly(β-aminoester), DOPE, β-cyclodextrin-containing polycations, linear PEI, PAMAM dendrimers, branched PEI, chitosan, or polyphosphoester nanoparticles.

[0474] In some embodiments, the LINC complex inhibitors, nucleic acids, and vectors according to this disclosure include modifications to incorporate one or more moieties that facilitate delivery to and / or uptake of a target cell type or tissue (e.g., cardiac and / or skeletal muscle cells / tissues). In some embodiments, the LINC complex inhibitors, nucleic acids, and vectors according to this disclosure are ligated (e.g., chemically conjugated) to one or more moieties that facilitate delivery to and / or uptake of a target cell type or tissue.

[0475] The portions that facilitate delivery to and / or uptake of target cell types or tissues are described, for example, in their entirety by reference in Benizri et al., Bioconjug Chem. (2019) 30(2):366-383. Such portions include, for example, N-acetylgalactosamine (GalNAc), α-tocopherol, cell-permeable peptides, their nucleic acid aptamers, antibodies and antigen-binding fragments / derivatives, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g., palmitic acid), and nucleolid portions.

[0476] The articles of this disclosure may be formulated in a sustained-release delivery system to release LINC complex inhibitors, nucleic acids, vectors, cells, or compositions at a predetermined rate. The sustained-release delivery system can maintain a constant drug / therapeutic / prophylactic concentration for a specified period. In some embodiments, the articles of this disclosure are formulated in liposomes, gels, implants, devices, or drug-polymer conjugates, such as hydrogels.

[0477] The administration of LINC complex inhibitors to subjects according to this disclosure is preferably a “therapeutic effective” or “preventive effective” dose, which is sufficient to demonstrate a therapeutic / preventive benefit to the subjects. The administration of LINC complex inhibitors preferably results in modification of one or more cells containing the LINC complex inhibitors described herein.

[0478] The actual amount administered, as well as the rate and duration of administration, depends on the nature and severity of the disease being treated / prevented, and the properties of the LINC complex inhibitor. The determination of treatment prescriptions, such as dosage, is the responsibility of the general practitioner and other physicians, typically taking into account the disease / condition being treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's 'The Science and Practice of Pharmacy' (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0479] In a preferred embodiment, administration is the administration of a nucleic acid / vector or a composition comprising a nucleic acid / vector according to the Disclosure. In a preferred embodiment, administration results in the modification of one or more cells to contain / express a nucleic acid / vector and / or a polypeptide according to the Disclosure. That is, the nucleic acid / vector / composition is used as gene therapy.

[0480] In some embodiments, the therapeutic or prophylactic interventions described herein may further include the administration of another agent for the treatment / prevention of the associated disease / condition. The administration of LINC complex inhibitors / nucleic acids / vectors / compositions described herein may be administered simultaneously or sequentially, alone or in combination with other treatments, depending on the condition being treated. Simultaneous administration means, for example, administration as a pharmaceutical composition (combination preparation) containing both agents, or immediately after each other, and optionally via the same route of administration (e.g., the same tissue, artery, vein, or other vessel) together with another therapeutic agent. Sequential administration means administering one agent, followed by the separate administration of another agent after a given time interval. The two agents do not need to be administered via the same route, but may be in some embodiments. The time interval may be any time interval.

[0481] Multiple doses of a LINC complex inhibitor / nucleic acid / vector / composition may be provided. One or more of these doses, or each of them, may be accompanied by the simultaneous or sequential administration of another therapeutic / prophylactic agent. The multiple doses may be separated by a predetermined time interval, which may be selected from 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, doses may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0482] According to various aspects of this disclosure, methods for treating and / or preventing a disease / condition (e.g., cardiomyopathy) may include one or more of the following: promoting normal contractility of cells (e.g., cardiomyocytes); promoting normal organization of microtubule networks (e.g., intracellular); promoting normal organization of sarcomeres (e.g., intracellular); reducing the mechanical forces transmitted to the cell nucleus / nuclear envelope; reducing the mechanical resistance of the microtubule network; increasing the contractility of cardiomyocytes; reducing the density of the microtubule network; for example, lowering the level of intracellular tubulin (e.g., α-tubulin and / or β-tubulin); lowering the level / proportion of detyrosinated tubulin; and increasing the level / proportion of acetylated tubulin. To increase, decrease the level / proportion of acetylated tubulin, decrease the level / proportion of intermediate filaments, e.g., microtubules that interact with desmin, promote normal organization of the perinuclear microtubule network (e.g., intracellular), reduce the mechanical forces transmitted to the cell nucleus / nuclear envelope by the perinuclear microtubule network, reduce the mechanical resistance of the perinuclear microtubule network, reduce the density of the perinuclear microtubule network, e.g., decrease the level of intracellular perinuclear tubulin (e.g., α-tubulin and / or β-tubulin), decrease the level / proportion of detyrosinated perinuclear tubulin, and decrease the level / proportion of intermediate filaments, e.g., microtubules that interact with desmin.

[0483] This disclosure further promotes normal contraction / contractility of cells (e.g., cardiomyocytes), promotes normal organization of microtubule networks (e.g., intracellular), promotes normal organization of sarcomeres (e.g., intracellular), reduces mechanical forces transmitted to the cell nucleus / nuclear envelope, reduces mechanical resistance of microtubule networks, increases the amount of contraction of cardiomyocytes, reduces the density of microtubule networks, for example, lowers the level of intracellular tubulin (e.g., α-tubulin and / or β-tubulin), lowers the level / percentage of detyrosinated tubulin, increases the level / percentage of acetylated tubulin, lowers the level / percentage of acetylated tubulin, intermediate filaments, e.g. The present disclosure provides for the use of LINC complex inhibitors to reduce the level / proportion of microtubules interacting with desmin, promote normal organization of the perinuclear microtubule network (e.g., intracellular), reduce the mechanical forces transmitted to the cell nucleus / nuclear envelope by the perinuclear microtubule network, reduce the mechanical resistance of the perinuclear microtubule network, reduce the density of the perinuclear microtubule network, for example, reduce the level of intracellular perinuclear tubulin (e.g., α-tubulin and / or β-tubulin), reduce the level / proportion of detyrosinated perinuclear tubulin, and / or reduce the level / proportion of intermediate filaments, for example, microtubules interacting with desmin.

[0484] This disclosure further describes how LINC complex inhibitors according to this disclosure can be used to promote normal contractility / contractility of cells (e.g., cardiomyocytes), promote normal organization of microtubule networks (e.g., intracellular), promote normal organization of sarcomeres (e.g., intracellular), reduce mechanical forces transmitted to the cell nucleus / nuclear envelope, reduce mechanical resistance of microtubule networks, increase the contractility of cardiomyocytes, reduce the density of microtubule networks, for example, decrease the level of intracellular tubulin (e.g., α-tubulin and / or β-tubulin), decrease the level / percentage of detyrosinated tubulin, increase the level / percentage of acetylated tubulin, and increase the level / percentage of acetylated tubulin The present invention provides methods for reducing the proportion of intermediate filaments, e.g., microtubules interacting with desmin; promoting normal organization of the perinuclear microtubule network (e.g., intracellular); reducing the mechanical forces transmitted to the cell nucleus / nuclear envelope by the perinuclear microtubule network; reducing the mechanical resistance of the perinuclear microtubule network; reducing the density of the perinuclear microtubule network, e.g., reducing the level of intracellular perinuclear tubulin (e.g., α-tubulin and / or β-tubulin); reducing the level / proportion of detyrosinated perinuclear tubulin; and / or reducing the level / proportion of intermediate filaments, e.g., microtubules interacting with desmin.

[0485] Therefore, this disclosure promotes normal contraction / contractility of cells (e.g., cardiomyocytes), promotes normal organization of microtubule networks (e.g., intracellular), promotes normal organization of sarcomeres (e.g., intracellular), reduces mechanical forces transmitted to the cell nucleus / nuclear envelope, reduces mechanical resistance of microtubule networks, increases the amount of contraction of cardiomyocytes, reduces the density of microtubule networks, for example, reduces the level of intracellular tubulin (e.g., α-tubulin and / or β-tubulin), reduces the level / percentage of detyrosinated tubulin, reduces the level / percentage of intermediate filaments, for example, microtubules interacting with desmin. The present disclosure provides a method for promoting normal organization of the perinuclear microtubule network (e.g., intracellular), reducing the mechanical forces transmitted to the cell nucleus / nuclear envelope by the perinuclear microtubule network, reducing the mechanical resistance of the perinuclear microtubule network, reducing the density of the perinuclear microtubule network, for example, lowering the level of intracellular perinuclear tubulin (e.g., α-tubulin and / or β-tubulin), lowering the level / percentage of detyrosinated perinuclear tubulin, and / or lowering the level / percentage of intermediate filaments, for example, microtubules that interact with desmin, comprising administering a LINC complex inhibitor according to the present disclosure to a target.

[0486] subject The subjects of various aspects of this disclosure may be any animal or human. The therapeutic and preventive uses may be in humans or animals (veterinary use).

[0487] Subjects administered with the Articles of this Disclosure (for example, in accordance with therapeutic or prophylactic interventions) may be subjects requiring such interventions. Subjects are preferably mammals, more preferably humans. Subjects may be non-human mammals, but more preferably humans. Subjects may be male or female. Subjects may be patients.

[0488] Subjects may have (e.g., be diagnosed with) any of the diseases or conditions described herein (e.g., cardiomyopathy, or any disease or condition characterized by desmoplakin dysfunction / deficiency / impairment, any disease or condition characterized by titin dysfunction / deficiency / impairment, or any disease or condition characterized by microtubule dysfunction), be suspected of having such a disease / condition, or be at risk of developing such a disease / condition. In embodiments of this disclosure, subjects may be selected for treatment according to the method, for example, as described above, based on the characterization of one or more markers of such disease / condition. Subjects may have or be suspected of having any of the diseases / conditions described herein based on the presence of other symptoms indicating the disease / condition in the subject or the cells / tissues / organs of the subject. Subjects may be considered at risk of developing a disease / condition due to genetic predisposition or other risk factors for the disease.

[0489] In some embodiments, the subject includes mutations according to any embodiment described herein. In some embodiments, the subject includes mutations that cause the disease / condition described herein.

[0490] In some embodiments, the subject includes laminopathy. do not have In some embodiments, the subjects include disease-associated mutations in LMNA. do not have In some embodiments, the subjects do not include mutations in LMNA (i.e., are homozygous for the wild-type allele of LMNA). In some embodiments, the subjects include disease-associated mutations in any of LMNA, LMNB1, and LMNB2. do not have In some embodiments, the subjects do not have mutations in any of LMNA, LMNB1, and LMNB2 (i.e., they are homozygous for the wild-type alleles of LMNA, LMNB1, and LMNB2). In some embodiments, the subjects do not have nuclear envelopepathies. do not have In some embodiments, the target teethThe subjects do not contain disease-related mutations in any of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62. In some embodiments, the subjects do not contain mutations in any of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62 (i.e., they are homozygous for the wild-type alleles of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62).

[0491] In some embodiments, the methods according to this disclosure may include determining whether a subject has a disease / condition described herein. In some embodiments, the methods include diagnosing a disease / condition described herein. Determining whether a subject has a disease / condition described herein may include analyzing the subject for one or more symptoms / correlations of the disease / condition. Genetic factors may be assayed by methods known to those skilled in the art, including PCR-based assays and sequencing assays. For example, by determining the presence of genetic factors in a sample obtained from a subject, the diagnosis may be confirmed, and / or the subject may be classified as being at risk of developing a disease / condition described herein, and / or the subject may be identified as suitable for treatment with a LINC complex inhibitor / nucleic acid / vector / composition described herein.

[0492] The assay may be performed in vitro on a sample obtained from a subject, or after processing of the sample obtained from the subject. The sample obtained from the subject may be of any type. Biological samples may be collected 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 may be selected fractions of the patient's blood, such as selected cell-containing fractions, or plasma or serum fractions. Samples may include tissue samples or biopsies, or cells isolated from the subject.

[0493] Sequence identity As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in a target sequence that are identical to those in a reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage of sequence identity between the sequences. Pairwise and multiple sequence alignments for the purpose of determining the percentage of identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to those skilled in the art using publicly available computer software such as ClustalOmega (Soeding, 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 such software, it is preferable to use default parameters such as gap penalties and extension penalties. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 [Table 2-25] [Table 2-26] [Table 2-27] [Table 2-28] [Table 2-29] [Table 2-30] [Table 2-31] ***

[0494] The present invention includes combinations of the described embodiments and preferred features, except where such combinations are clearly unacceptable or expressly avoided.

[0495] The section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described.

[0496] Hereafter, aspects and embodiments of this disclosure will 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 referenced herein are incorporated herein by reference.

[0497] Throughout this Specified Specification, including the following claims, unless the context specifically requires otherwise, the word “comprise,” and its variations such as “comprises” and “comprising,” are understood to imply that they encompass an explicitly stated integer value or step, or group of integer values ​​or steps, but do not exclude any other integer value or step, or group of integer values ​​or steps.

[0498] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. Ranges may be expressed herein as “about” one particular value to and / or “about” another particular value. Where such ranges are expressed, another embodiment includes one particular value to and / or another particular value. Similarly, where a value is expressed as an approximation by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0499] The methods described herein may preferably be performed in vitro. The term “in vitro” is intended to encompass procedures performed using cells in culture, while the term “in vivo” is intended to encompass procedures using / for intact multicellular organisms.

[0500] Embodiments and experiments illustrating the principles of this disclosure are described herein with reference to the accompanying drawings. [Brief explanation of the drawing]

[0501] [Figure 1]Functional rescue of LMNA N195K iPSC-CM by DNSUN1. (A) Schematic diagram of the experimental setup for the functional assay performed using the CardioExcyte system. (B) Exemplary snapshots of EFP in control and LMNA N195K iPSC-CM, as well as in LMNA N195K iPSC-CM transduced with DNSUN1. (C) Figure showing the phenotype observed in LMNA N195K iPSC-CM during EFP measurement, namely an increase in variation coefficient indicating arrhythmia, as well as an increase in amplitude and beat rate. Induction of DNSUN1 results in rescue or mitigation of these phenotypes. (D) Figure showing the decrease in amplitude observed in LMNA N195K iPSC-CM during contractility measurement, with the introduction of DNSUN1 resulting in rescue of this phenotype. Each dot corresponds to a well. [Figure 2] DNSUN1 reduces the microtubule network in cardiomyocytes. (A) Immunostaining using a specific anti-tubulin antibody and nuclear stain DAPI in untransduced and DNSUN1-transduced cardiomyocytes. The top panel includes a magnified view of the Tyr-α-Tub inset. Treated cells show weaker, less uniformly distributed perinuclear microtubule signals. (B) Quantification of microtubule intensity normalized to DAPI, where each dot represents a different imaging field. Statistical significance was determined by the Mann-Whitney U test. [Figure 3] A diagram showing the experimental setup of in vivo proximity markers (BioIDs). [Figure 4] This figure shows the desmin proximity network in healthy cardiomyocytes and DSP-associated diseased cardiomyocytes. The proximity score, represented by a gray gradient, reflects the proximity between proteins belonging to different functional units (square boxes) and the desmin bait. The bait (DES) is clearly outlined with a solid black border. Among the acquired interactions, the most notable finding is the association between desmin and the disease state's MTOC (microtubule organizing center). Proteins particularly affected by the disease state are marked with a black dashed line. IF: Intermediate filament. AJ: Adhesion junction. [Figure 5] Schematic diagram of microtubules and MTOCs in adult cardiomyocytes. (A) Immediately after birth, cardiomyocytes transition from mitotic (i) to postmitotic (ii), changing the localization of microtubule nucleation factors such as microtubule organizing centers (MTOCs), and thus the polarity of the network. Mature cardiomyocytes consist of three microtubule populations: interfibrillary, cortical, and perinuclear. (B) Enlarged view of the Z disc, the interaction site between microtubules and desmin. Each myofibrils within a cardiomyocyte contain many individual units called sarcomeres. Sarcomeres are mainly composed of actin and myosin. α-actinin plays a role in anchoring actin to the Z disc. Desmin, cross-linked by plectin, is also present in the Z disc and can reinforce microtubules at this site. (C) Enlarged view of the interaction site between microtubules, desmin, and the nuclear envelope. The rectangular box highlights the enhanced interaction between desmin and the subdistal appendage of MTOCs in disease states. Furthermore, the schematic diagram shows the site of action of DNSUN1 (SUN1-2) that dismantles the connection between the nuclear envelope and the cytoskeleton. Figures 5A and 5B are adapted from Uchida, K., Scarborough, EA, & Prosser, BL (2021). [Figure 6] Elevated total microtubule and detyrosinated microtubule levels in shDSP-affected hearts. (A) Western blot comparing total microtubule and detyrosinated microtubule levels in healthy controls and shDSP-affected hearts, with beta-actin serving as a loading control. (B) Quantitative analysis of fold changes in total tubulin and detyrosinated tubulin, normalized to control (shLacZ). Bar heights indicate the mean, and error bars indicate the standard deviation. Statistical analysis performed using two-way ANOVA. *P≦0.05, **P≦0.01 [Figure 7A]Cardiac function in mice after Dsp knockdown (KD) and during rescue by DNSUN1. (A) Diagram 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 from the cardiac troponin promoter. shDSP - AAV expressing shRNA targeting Dsp. cTnT-DNSUN1 - AAV expressing dominant-negative SUN1 from the cardiac troponin promoter. [Figure 7B] Cardiac function in mice after Dsp knockdown (KD) and during rescue with DNSUN1. (B) In mice with DSP KD (knockdown), ejection fraction (EF) and fetal shortening (FS) were reduced, but the rescue group mice had EF and FS similar to the control group. (****P<0.0001, ***P<0.001; one-way ANOVA, mean ± SD). From day 84, group 4 consisted of 3 mice because one mouse died due to excessive exposure to anesthesia during preparation for echocardiography. From day 133, group 2 consisted of 4 mice because one mouse died due to accidental hypoxia and asphyxiation in a parallel experiment with an electrocardiogram tunnel that was too small for the size of the mouse. [Figure 8] Cardiac function in a second batch of 20 mice after Dsp knockdown (KD) and rescue with DNSUN1. (A) Diagram 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 from the cardiac troponin promoter. shDSP-AAV expressing shRNA targeting Dsp. cTnT-DNSUN1-AAV expressing dominant-negative SUN1 from the cardiac troponin promoter. (B) Mice with Dsp KD showed decreased EF% and FS%. As shown in (A), data were analyzed from the total number of animals per treatment group (N). (****P<0.0001, one-way ANOVA, mean ± SD). [Figure 9]Cardiac function in mice after TTN knockdown (KD) and rescue with DNSUN1. Mice with TTN KD had reduced ejection fraction (EF) (A) and fasting rate (FS) (B) compared to the control group. 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 ± SD). AAVs expressing shRNA targeting Ctrl-LacZ. AAVs expressing EGFP-EGFP. AAVs expressing shRNA targeting shTTN-Citin. AAVs expressing DNSUN1-dominant negative SUN1. [Figure 10A] DNSun1 treatment reduces tubulin levels and improves cardiac function in an LMNA-DCM mouse model. This Western blot compares tubulin levels in healthy controls (WT), LMNA-DCM-affected hearts, and DNSUN1-treated LMNA-DCM mice, with GAPDH serving as the load control. [Figure 10B] DNSun1 treatment reduces tubulin levels and improves cardiac function in LMNA-DCM mouse models. The quantification of the tubulin level change, normalized to the control (WT), is shown. Bar heights represent the mean, and error bars represent the standard deviation. Statistical analysis was performed using standard one-way ANOVA. **P≦0.01, ****P≦0.0001. [Figure 10C] DNSun1 treatment reduces tubulin levels and improves cardiac function in LMNA-DCM mouse models. A negative correlation was observed between LVEF (EF%) and normalized tubulin expression in control mice (WT), affected mice (LMNA-DCM), and treated mice (LMNA-DCM + DNSUN1). [Figure 11]Elevated microtubule levels in shTTN-affected hearts. (A) Western blot comparing tubulin levels in healthy controls and shTTN-affected hearts, with histone H2B serving as the loading control. Western blotting was performed using antibodies specific to α-tubulin and histone H2B, following the standard protocol available on the Bio-Rad website. (B) Quantitative change in tubulin folds normalized to control (shLacZ), bar heights indicate the mean and error bars indicate the standard deviation. Statistical analysis was performed using an unpaired t-test. *P ≤ 0.05. [Examples]

[0502] Example 1: Decoupling of the LINC complex using DNSUN1 reversed pathological microtubule organization in human iPSC-derived cardiomyocytes. DNSUN1 competes with the endogenous SUN protein for binding to the KASH protein in the perinuclear space of cardiomyocytes, thereby decoupling the nucleus from the cytoskeleton (Crisp, M. et al. J. Cell Biol. 172, 41-53 (2006)).

[0503] Based on the above findings and the fact that various cardiac conditions (e.g., cardiomyopathy, whether of genetic or non-genetic origin, as described above) share a common feature: a substantial and maladaptive increase in microtubule density within cardiomyocytes, we investigated whether DNSUN1 could potentially function as an effective treatment for multiple conditions by reversing the formation of pathological microtubule networks.

[0504] The inventors initially tested their hypothesis using an in vitro model of LMNA cardiomyopathy. This decision was based on substantial evidence demonstrating the efficacy of DNSUN1 in treating LMNA cardiomyopathy in a mouse model (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, WO2019 / 143300A1, and WO2021 / 010898A1).

[0505] The in vitro model specifically refers to LMNA N195K cardiomyopathy. The substitution of asparagine (N) to lysine (K) at amino acid 195 in variants of type A lamin causes dilated cardiomyopathy (DCM) in humans (Fatkin, et al. (1999) N.Engl.J.Med., 341, 1715-1724).

[0506] Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA-N195K mutation patients exhibit both electrophysiological abnormalities and nuclear morphological irregularities. Therefore, they serve as a suitable model for rescue experiments. (Shemer Y et al. (2021) Int J Mol Sci. 23;22(15):7874).

[0507] 1.1 Decoupling of the LINC complex by DNSUN1 reverses the electrophysiological disorder of LMNA N195K iPSC-CM. The CardioExcyte system was used for electrophysiological studies of the LMNA N195K iPSC-CM.

[0508] The CardioExcyte system allows for the simultaneous measurement of both impedance and extracellular potential (EFP) from a single well. An electrode positioned at the bottom of the well measures the impedance (resistance) introduced by a monolayer located above the electrode. The composition of the cells forming the monolayer affects the impedance; flattened cells (e.g., relaxed CMs) result in high impedance, while round cells (e.g., contracted CMs) result in low impedance. Capturing impedance changes with high temporal resolution makes it possible to assess cell contractility. Furthermore, variations in the membrane potential of a pulsating CM affect the ion concentration trapped between the monolayer and the detection electrode. Therefore, measuring ion concentration (i.e., EFP) with high temporal resolution serves as a proxy for changes in the CM membrane potential.

[0509] EFP analysis of LMNA N195K iPSC-CM revealed arrhythmia phenotypes, along with increased pulsity and amplitude compared to normal isogenic CM (control) (Figures 1B and 1C). Contractility measurements showed reduced amplitude compared to normal isogenic CM (Figure 1D). In particular, treatment of LMNA N195K iPSC-CM with AAV6-DNSUN1 resulted in the rescue or mitigation of all these disease phenotypes (Figures 1B, 1C, and 1D), namely, the reversal of arrhythmia phenotypes indicated by a reduction in the variation coefficient, decreased membrane potential amplitude and pulsity (Figure 1C), and the reversal of decreased contractility indicated by an increase in impedance amplitude.

[0510] The observed rescue of electrophysiological irregularities in LMNA N195K iPSC-CM by DNSUN1 makes this in vitro system a suitable tool for deciphering the mechanism of action of DNSUN1.

[0511] 1.2 DNSUN1 can reverse pathological microtubule tissue formation in iPSC-CM. To investigate the effect of DNSUN1 on the microtubule network in iPSC-CMs, immunostaining was performed using antibodies targeting tyrosinated and detyrosinated microtubules. In untreated LMNA N195K iPSC-CMs, a distinct microtubule network was observed at the nuclear periphery. Conversely, DNSUN1-treated cardiomyocytes showed a more uniformly distributed pattern throughout the cytoplasm and reduced cytoplasmic microtubule density compared to untreated LMNA N195K iPSC-CMs (Figure 2). These findings suggest that DNSUN1 may be able to reverse the pathological microtubule organization found in disease states.

[0512] Based on the findings described in this embodiment, the inventors propose a novel mechanism by which inhibition of the LINC complex (e.g., by DNSUN1) rescues cardiomyopathy, such as LMNA-associated cardiomyopathy. This mechanism involves mitigating the pathological effects of dysfunctional microtubules. Two potentially additional protective effects are anticipated. 1. It protects the nucleus from excessive forces caused by disordered microtubule structures, thereby reducing the risk of DNA damage. 2. Protection against reduced contractile properties due to changes in microtubule load-bearing capacity and force transmission with sarcomeres. Decoupling of the nuclear envelope from the cytoskeleton mediated by disruption of the LINC complex by DNSUN1 may reduce anchoring sites in the perinuclear microtubule population, leading to reduced stability.

[0513] These data suggest that inhibition of the LINC complex (e.g., by DNSUN1) may be a promising therapeutic agent for several conditions associated with abnormal microtubule density or microtubule tissue within cardiomyocytes.

[0514] 1.3 Materials and Methods AAV6 production AAV6 was produced by triple transfection and purified using a modified method from Arden et al., J Biol Methods. 2016;3(2):e38 or Strobel et al., Hum Gene Ther Methods. 2015;26(4):147-57.

[0515] iPSC-CM experiment Human iPSC lines derived from PGP1 donors (Personal Genome Project) were engineered to carry a homozygous junction mutation in LMNA (N195K) using the CRISPR / Cas9 system. The iPSC lines were then instructed to differentiate into beating cardiomyocytes (CMs) and cultured according to a published protocol (Lian, X., et al. PNAS. (2012) 109(27), E1848-57).

[0516] For the functional assay (Figure 1A), cells were plated into each CardioExcyte well (Nanion Technologies). One day after seeding, LMNA-N195K iPSC-CM and control (WT) were treated with AAV6-DNSUN1 (encoding DNSUN1, SEQ ID NO: 112) or AAV6-GFP. Seed cells were stimulated with endothelin 1. EFP and contractility were then evaluated according to the manufacturer's protocol. Statistical analysis was performed using one-way ANOVA with GraphPad Prism. The experiment was repeated three times. Each dot in Figures 1C and 1D represents a well.

[0517] For immunofluorescence experiments, CM cells were plated in 96-well plastic phenoplates coated with Matrigel. One day after seeding, treated cells were transduced with AAV6 carrying DNSUN1. Seven days after transduction, cells were fixed with 4% paraformaldehyde and subjected to immunostaining using a standard protocol with microtubule-specific antibodies. Images were captured three times each from treated and untreated wells using a Perkin Elmer Opera Phenix (High-Content) microscope system. The mean fluorescence intensity of microtubules in the area of ​​interest was analyzed using IMARIS 8.2 software (Bitplane) and normalized to the mean nuclear intensity. For statistical analysis, the Mann-Whitney U test was performed using Prism software.

[0518] Example 2: In vivo proximity labeling (BioID) identifies abnormal microtubule organization in DSP-associated cardiomyopathy. 2.1 Identify changes in the desmin proximity network between healthy hearts and DSP-related affected hearts. To identify potential cytoskeletal rearrangements associated with desmoplakin cardiomyopathy, we performed in vivo comparisons of the desmin interactome between healthy hearts and shDSP-induced affected hearts (Figure 3).

[0519] The method used to identify proteins that interact with desmin was 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 in its entirety. This method screens for physiologically relevant protein interactions occurring in living cells. This technique utilizes a non-discriminatory biotin ligase on biotinylated proteins based on proximity. The ligase is fused to the target protein and expressed in the cell, where the proximal endogenous protein is biotinylated. Because this protein modification is rare in nature, biotinylation of these endogenous proteins by BioID-fused proteins allows for selective isolation and identification by standard biotin affinity capture.

[0520] Desmin is a component of the intermediate filament and functions as a key protein that cross-links the nuclear intercalation plate to the envelope (through interaction with the LINC complex via plectin) (via direct binding to desmoplakin).

[0521] The inventors identified 18 proteins that showed differential proximity to desmin in DSP-associated pathological samples compared to healthy cardiac tissue samples. Of these, nine showed increased proximity and nine showed decreased proximity. Among the p...

Claims

1. A LINC complex inhibitor for use in methods of treating or preventing cardiomyopathy, wherein the cardiomyopathy is not laminopathy-related cardiomyopathy.

2. The use of a LINC complex inhibitor in the manufacture of a pharmaceutical product for use in the treatment or prevention of cardiomyopathy, wherein the cardiomyopathy is not laminopathy-related cardiomyopathy.

3. A method for treating or preventing cardiomyopathy, comprising administering a therapeutic or prophylactic effective amount of a LINC complex inhibitor to a target, wherein the cardiomyopathy is not laminopathy-related cardiomyopathy.

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

6. A LINC complex inhibitor for use according to any one of claims 1, 4, or 5, wherein the cardiomyopathy is associated with a mutation in a gene selected from DSP, DES, DSG2, DSC2, PKP2, and JUP; the use according to any one of claims 2, 4, or 5; or the method according to any one of claims 3 to 5.

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

8. LINC complex inhibitors for use in methods of treating or preventing diseases or conditions characterized by desmosome deficiency or dysfunction.

9. Use of LINC complex inhibitors in the manufacture of pharmaceuticals for use in the treatment or prevention of diseases or conditions characterized by desmosome deficiency or dysfunction.

10. A method for treating or preventing a disease or condition characterized by desmosome deficiency or dysfunction, comprising administering a therapeutic or prophylactic effective dose of a LINC complex inhibitor to the target.

11. A LINC complex inhibitor for use according to claim 8, for use according to claim 9, or by method according to claim 10, wherein the disease or condition characterized by desmosome deficiency or dysfunction is associated with a mutation in a gene encoding a desmosome protein.

12. A LINC complex inhibitor for use according to claim 8 or 11, the use according to claim 9 or 11, or the method according to claim 10 or 11, wherein the disease or condition characterized by desmosome deficiency or dysfunction is associated with a mutation in a gene selected from DSP, DES, DSG2, DSC2, PKP2, and JUP.

13. A LINC complex inhibitor for use according to any one of claims 8, 11, or 12, wherein the disease or condition characterized by desmosome deficiency or dysfunction is characterized by cardiomyopathy, the use according to any one of claims 9, 11, or 12, or the method according to any one of claims 10 to 12.

14. A LINC complex inhibitor for use according to any one of claims 8 and 11-13, wherein the disease or condition characterized by desmosome deficiency or dysfunction is characterized by microtubule dysfunction, the use according to any one of claims 9 and 11-13, or the method according to any one of claims 10-13.

15. LINC complex inhibitors for use in methods of treating or preventing diseases or conditions characterized by microtubule dysfunction.

16. Use of LINC complex inhibitors in the manufacture of pharmaceuticals for use in the treatment or prevention of diseases or conditions characterized by microtubule dysfunction.

17. A method for treating or preventing a disease or condition characterized by microtubule dysfunction, comprising administering a therapeutic or prophylactic effective dose of a LINC complex inhibitor to the target.

18. The LINC complex inhibitor for use according to claim 15, the use according to claim 16, or the method according to claim 17, wherein the disease or condition characterized by microtubule dysfunction is cardiomyopathy, or characterized by cardiomyopathy.

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

20. A LINC complex inhibitor for use according to any one of claims 1, 4, 5, 7, 8, 11, 12, 14, 15, 18, or 19, wherein the treatment or prevention comprises administering a LINC complex inhibitor to a nucleic acid encoding it; a 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 3-5, 7, 10-12, 14, or 17-19.

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

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

23. A LINC complex inhibitor for use according to claim 21 or 22, or the method according to claim 21 or 22, wherein the LINC complex inhibitory polypeptide comprises or is essentially therefrom 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.

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

25. LINC complex inhibitors for use in methods of treating or preventing diseases or conditions characterized by sarcomere deficiency or dysfunction.

26. Use of LINC complex inhibitors in the manufacture of pharmaceuticals for use in the treatment or prevention of diseases or conditions characterized by sarcomere deficiency or dysfunction.

27. A method for treating or preventing a disease or condition characterized by sarcomere deficiency or dysfunction, comprising administering a therapeutic or prophylactic effective dose of a LINC complex inhibitor to the target.

28. A LINC complex inhibitor for use according to claim 25, for use according to claim 26, or by method according to claim 27, wherein the disease or condition characterized by sarcomere deficiency or dysfunction is associated with a mutation in a gene encoding a sarcomere protein.

29. A LINC complex inhibitor for use according to claim 25 or 28, the use according to claim 26 or 28, or the method according to claim 27 or 28, wherein the disease or condition characterized by sarcomere deficiency or dysfunction is associated with a mutation in a gene selected from TTN, MYH7, TNNT2, TNNI3, TNNC1, TPM1, MYBPC3, MYL2, MYL3, CSRP3, RBM20, ACTN2, TCAP, and ACTC1.

30. A LINC complex inhibitor for use according to any one of claims 25, 28, or 29, wherein the disease or condition characterized by sarcomere deficiency or dysfunction is characterized by cardiomyopathy, the use according to any one of claims 26, 28, or 29, or the method according to any one of claims 27 to 29.

31. A LINC complex inhibitor for use according to any one of claims 25 and 28-30, wherein the disease or condition characterized by sarcomere deficiency or dysfunction is characterized by microtubule dysfunction, the use according to any one of claims 26 and 28-30, or the method according to any one of claims 27-30.

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