LINC complex inhibitor polypeptide

Abstract

Nucleic acids encoding LINC complex inhibitory polypeptides are disclosed, as are LINC complex inhibitory polypeptides, compositions comprising such nucleic acids and polypeptides, and uses of such nucleic acids, polypeptides and compositions for LINC complex inhibition and for the treatment and prevention of diseases / conditions, particularly for the treatment of laminopathies and diseases characterized by hyperlipidemia.

Description

[Technical field]

[0001] This application claims priority to SG 10202113391P, filed December 1, 2021, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates to the fields of molecular and cell biology, and also to methods of medical treatment and prophylaxis. [Background technology]

[0003] Lamins A / C are components of the nuclear lamina, which lies inside the inner nuclear membrane, and are thought to provide mechanical support to the nucleus (Burke and Stewart, 2013). Mutations in LMNA, which encodes lamins A and C, result in a number of disorders known as laminopathies.

[0004] Disruption of the linker of nucleoskeletal and cytoskeleton (LINC) complex has been recently proposed as a therapeutic strategy for the treatment of laminopathies. The LINC complex consists of SUN domain proteins in the inner nuclear membrane and KASH domain proteins in the outer nuclear membrane (Lee and Burke, 2018). Paralogous Sun1 and Sun2 are the major SUN domain proteins and are widely expressed in mammalian tissues. Among the KASH domain proteins, Nesprin-1, Nesprin-2, and Nesprin-3 are the most widely expressed.

[0005] Crisp et al., 2006, describe LINC complex disruption via overexpression of a dominant-negative form of mouse Sun1 (DNSun1) protein. DNSun1 contains the entire luminal domain of Sun1, an N-terminal signal sequence, and a C-terminal KDEL Golgi-to-ER retrieval sequence. It functions by disrupting SUN-KASH interactions within the LINC complex, presumably by competing with endogenous SUN domain-containing proteins (e.g., Sun1 and Sun2) for binding to KASH domain-containing proteins. Unlike wild-type Sun1, DNSun1 is not anchored to the nuclear lamina, such that forces from the cytoskeleton transmitted to KASH domain proteins are not further transmitted by DNSun1 to the nuclear interior. A humanized version of DNSun1 is described in WO 2019 / 143300 A1.

[0006] The relatively large size of the coding sequence of DNSUN1 (about 1.5 kb) presents a challenge for its delivery, for example as a gene therapy. By way of illustration, in the self-complementary adeno-associated virus vector (scAAV) system, the entire transgene, including the viral inverted terminal repeats (ITRs), promoter and other regulatory sequences, as well as its own coding sequence, cannot exceed 2.3 kb.

[0007] The C-terminal SUN domain of SUN domain-containing proteins has been shown to be necessary but sufficient for interaction with KASH domain-containing proteins, and trimerization of the SUN domain mediated by the upstream helix and coil region is thought to be required for KASH interaction ( Sosa et al., 2012 ; Wang et al., 2012 ; Zhou et al., 2012 ; Jahed et al., 2018b ; Nie et al., 2016 ; Xu et al., 2018 ).

[0008] Zhou et al., 2012 and Sosa et al., 2012 demonstrated that luminal SUN constructs truncated before the start of the CC1 domain were able to associate with KASH domain proteins. Zhou et al., 2012, Wang et al., 2012, Jahed et al., 2018b and Nie et al., 2016 showed that constructs containing CC1-CC2-SUN could form trimers in gel filtration assays or interact with KASH domains in pull-down assays. Zhou et al., 2012 and Jahed et al., 2018b further showed that truncations starting in the middle of the CC1 domain and encompassing CC2 and SUN did not interact with KASH in pull-down assays and formed monomers in gel filtration assays. Wang et al., 2012 and Nie et al., 2016 showed that CC2-SUN constructs similarly did not interact with KASH in vitro and only formed monomers in gel filtration assays.

[0009] Although the majority of this work has focused on SUN2, there is a high degree of sequence conservation between SUN1 and SUN2, particularly in their luminal domains, and when Sun1 itself is examined, there is clear conservation at the structural level between Sun1 and Sun2 ( Xu et al., 2018 ; Gurusaran and Davies, 2021 ).

[0010] Importantly, there are conflicting reports on whether SUN protein variants containing certain truncations of the luminal domain can form trimers and interact with KASH domain proteins ( Jahed et al., 2018a ).

[0011] In studies by Zhou et al., 2012 and Wang et al., 2012, a Sun2 truncation variant consisting of G522 to H717 was able to interact with KASH domain-containing proteins (determined by co-immunoprecipitation analysis), and a Sun2 truncation variant consisting of V520 to H717 was able to form trimers and was used to resolve the SUN trimer structure by X-ray crystallography (the formation of SUN trimers is thought to be necessary for binding to KASH domain-containing proteins). Nie et al., 2016 determined that removal of the α1 (α2-α3-SUN) or both α1 and α2 (α3-SUN) helices from CC2-SUN abolished the autoinhibitory function of CC2, and the resulting Sun2 construct was able to interact with KASH domain proteins. Interestingly, α2-α3-SUN formed trimers in gel filtration assays, whereas α3-SUN was a mixture of monomers and trimers.

[0012] In contrast, Sosa et al., 2012 showed that a SUN2 truncation variant consisting of positions 507 to 717 (corresponding to α2-α3-SUN) was able to bind to the KASH domain, while further truncations resulted in weak (in the case of the SUN2(514–717) truncation variant) or negligible (in the case of the SUN2(521–717) truncation variant corresponding to α3-SUN) binding to the KASH domain.

[0013] The literature has addressed how truncation of the luminal domain of SUN proteins affects KASH binding, but it is unclear whether trimerization and KASH domain binding in vitro is predictive of their ability to disrupt LINC complexes at the cellular level.

[0014] Therefore, it remains to be seen whether truncation of the SUN luminal domain can be used as an agent for disrupting the LINC complex. Summary of the Invention

[0015] In a first aspect, the disclosure provides a nucleic acid encoding a LINC complex inhibitory polypeptide, the LINC complex inhibitory polypeptide comprising (i) an inhibitory region comprising an amino acid sequence corresponding to the α3 helix and a SUN domain of a CC2 region of a SUN domain-containing protein, and (ii) an endoplasmic reticulum retention motif; the LINC complex inhibitory polypeptide does not comprise the amino acid sequence of any one of SEQ ID NOs: 43, 45 or 58.

[0016] In some embodiments, the inhibitory region of a LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of SEQ ID NO: 44 or 46. In some embodiments, the inhibitory region of a LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of any one of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61 or 62.

[0017] In some embodiments, the inhibitory region of the LINC complex inhibitory polypeptide comprises: (i) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 66; (ii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 94; or (iii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 65; or (iv) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 63; or (v) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 64; or (vi) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 67 It essentially consists of:

[0018] In some embodiments, the LINC complex inhibitory polypeptide comprises: (i) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 72; (ii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 95; or (iii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 71; or (iv) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 69; or (v) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 70; or (vi) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 73 Containing or consisting essentially of.

[0019] In some embodiments, the LINC complex inhibitory polypeptide comprises a signal peptide.In some embodiments, the nucleic acid is a vector suitable for delivering the nucleic acid encoding the LINC complex inhibitory polypeptide as gene therapy.In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0020] The present disclosure provides a LINC complex inhibitory polypeptide comprising: (i) an inhibitory region comprising an amino acid sequence corresponding to the α3 helix and SUN domain of a CC2 region of a SUN domain-containing protein; and (ii) an endoplasmic reticulum retention motif, Also provided is a LINC complex inhibitory polypeptide that does not include the amino acid sequence of SEQ ID NO:43 or 45.

[0021] In some embodiments, the inhibitory region of the LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of SEQ ID NO: 44 or 46. In some embodiments, the inhibitory region of the LINC complex inhibitory polypeptide of claim 9 or claim 10, wherein the inhibitory region of the LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of any one of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62.

[0022] In some embodiments, the inhibitory region of the LINC complex inhibitory polypeptide comprises: (i) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 66; (ii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 94; or (iii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 65; or (iv) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 63; or (v) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 64; or (vi) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 67 It essentially consists of:

[0023] In some embodiments, the LINC complex inhibitory polypeptide comprises: (i) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 72; (ii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 95; or (iii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 71; or (iv) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 69; or (v) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 70; or (vi) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 73 Containing or consisting essentially of.

[0024] In some embodiments, the LINC complex inhibitory polypeptide comprises a signal peptide.

[0025] The present disclosure provides a method for identifying a LINC complex inhibitory polypeptide, comprising: introducing a nucleic acid encoding a candidate LINC complex inhibitory polypeptide, the candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to a SUN domain of a SUN domain-containing protein; and Then, the cells are analyzed to determine the subcellular localization of the interaction partner for the SUN domain-containing protein. wherein the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if a change in the subcellular localization of the interaction partner relative to the SUN domain-containing protein is detected.

[0026] The present disclosure provides a method for identifying a LINC complex inhibitory polypeptide, comprising: introducing a nucleic acid encoding a candidate LINC complex inhibitory polypeptide, the candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to a KASH domain of a KASH domain-containing protein; and Then, the cells are analyzed to determine the subcellular localization of the interacting partner for the KASH domain-containing protein. wherein the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if a change in the subcellular localization of the interaction partner relative to the KASH domain-containing protein is detected.

[0027] In some embodiments, a candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if an increase in the proportion of an interaction partner localized to the endoplasmic reticulum relative to the SUN / KASH domain-containing protein is detected.

[0028] The present disclosure also provides a LINC complex inhibitory polypeptide identified by a method according to the present disclosure.

[0029] The present disclosure also provides a nucleic acid encoding a LINC complex inhibitory polypeptide according to the present disclosure.

[0030] The present disclosure also provides a vector comprising a nucleic acid according to the present disclosure, the vector being suitable for delivering a nucleic acid encoding a LINC complex inhibitory polypeptide as a gene therapy.

[0031] In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0032] The present disclosure also provides a cell comprising a nucleic acid, a LINC complex inhibitory polypeptide, or a vector according to the present disclosure.

[0033] The present disclosure also provides a pharmaceutical composition comprising a nucleic acid, a LINC complex inhibitory polypeptide, a vector or a cell according to the present disclosure.

[0034] The present disclosure also provides a nucleic acid, a LINC complex inhibitory polypeptide, a vector, a cell or a pharmaceutical composition according to the present disclosure for use in a method of medical treatment or prophylaxis.

[0035] The present disclosure also provides a nucleic acid, a LINC complex inhibitory polypeptide, a vector, a cell or a pharmaceutical composition according to the present disclosure for use in a method of treating or preventing a laminopathies.

[0036] The disclosure also provides the use of a nucleic acid, a LINC complex inhibitory polypeptide, a vector, a cell or a pharmaceutical composition according to the disclosure in the manufacture of a medicament for treating or preventing a laminopathies.

[0037] The present disclosure also provides a method of treating or preventing a laminopathies, the method comprising administering to a subject a therapeutically or prophylactically effective amount of a nucleic acid, LINC complex inhibitory polypeptide, vector, cell or pharmaceutical composition according to the disclosure.

[0038] In some embodiments according to various aspects of the disclosure, the laminopathies are characterized by one or more of myopathy, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, myocardial dystrophy, skeletal muscular dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy, or dermatosis. In some embodiments, the laminopathies are associated with mutations to LMNA. In some embodiments, the laminopathies are characterized by one or more of Hutchinson-Gilford Progeria Syndrome; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-associated; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; muscular dystrophy; mandibular acrodysplasia with lipodystrophy type a; cardiomyopathy, dilated, 1a; Charcot-Marie-Tooth disease; limb-girdle muscular dystrophy; cardiomyopathy, dilated, with hypergonadotropic hypogonadism; Emery-Dreifuss muscular dystrophy, 1b; Fuss muscular dystrophy 3, autosomal recessive;lipodystrophy, familial partial, type 2;Emery-Dreifuss muscular dystrophy;Charcot-Marie-Tooth disease, axonal, type 2b1;heart-upper limb syndrome, Slovenian type;aging;familial partial lipodystrophy;restrictive cutis, fatal;arrhythmogenic right ventricular cardiomyopathy;dental disease;cardiac disease;Werner syndrome;hypertrophic cardiomyopathy;left ventricular noncompaction;atrioventricular block;calcification;acrosteolysis;autosomal dominant Limb-girdle muscular dystrophy;Diabetes mellitus, non-insulin dependent;Osteoporosis;Atrial fibrillation;Atrial asystole 1;Melanotic keratoderma;Cardiac conduction disorder;Catecholamine-induced polymorphic ventricular tachycardia;Micrognathia, deafness, progeria-like symptoms, and lipodystrophy syndrome;Sick sinus syndrome;Pelger-Houette nucleus anomaly;Charcot-Marie-Tooth disease, axonal, type 2e;Congenital generalized lipodystrophy;Restrictive cardiomyopathy;Congenital fiber type inequality;Lipodystrophy, congenital total body type, type 1;myofibrillar myopathy;lipodystrophy, familial partial, type 1;axonal neuropathy;atypical Werner syndrome;ovarian cystadenoma;Fanconi anemia, complementation group a;body mass index quantitative trait locus 11;skin diseases;ankylosing spinal muscular dystrophy 1;neuromuscular diseases;Harlermann-Streiff syndrome;Bethlem myopathy 1;acquired generalized lipodystrophy;cardiomyopathy, dilated, type 1e;lipodystrophy, congenital generalized, type 4;undifferentiated pleomorphic sarcoma;Lipodystrophy, familial partial, type 3;Muscular dystrophy, congenital merosin deficiency, type 1a;Proximal spinal muscular atrophy;Muscular dystrophy-dystroglycanopathy, type B, type 5;Muscular dystrophy, congenital, type 1b;Reynolds syndrome;Widermann-Rautenstrauch syndrome;Emery-Dreifuss muscular dystrophy 1, X-linked;Lipodystrophy, congenital generalized, type 2;Monogenic diabetes;Cardiomyopathy, dilated, type 1d;Myopathy, proximal, and ophthalmoplegia;Muscle tissue disease;Lipodystrophy, familial partial, type 4;Cardiomyopathy, dilated, type 1h;Second-degree atrioventricular block;Median neuropathy;Intrinsic cardiomyopathy;Prolapse of female genitalia;Complete generalized lipodystrophy;Ankylosing spinal muscular dystrophy; emelinopathy;ulnar neuropathy;limb-girdle muscular dystrophy type 1b;Lmna-associated dilated cardiomyopathy;pelvic muscle wasting;generalized lipodystrophy-associated progeria syndrome;muscle disease;cardiomyopathy, dilated, type 1b;autosomal inherited disease;familial isolated arrhythmogenic ventricular dysplasia, right dominant;familial isolated arrhythmogenic ventricular dysplasia, biventricular;familial isolated arrhythmogenic ventricular dysplasia, left dominant;Lmna-associated cardiocutaneous progeria syndrome;and autosomal semidominant severe lipodystrophy-laminopathies.;

[0039] The present disclosure also provides a nucleic acid, a LINC complex inhibitory polypeptide, a vector, a cell, or a pharmaceutical composition according to the present disclosure for use in a method of treating or preventing a disease characterized by hyperlipidemia.

[0040] The present disclosure also provides the use of a nucleic acid, a LINC complex inhibitory polypeptide, a vector, a cell or a pharmaceutical composition according to the present disclosure in the manufacture of a medicament for treating or preventing a disease characterized by hyperlipidemia.

[0041] The present disclosure also provides a method of treating or preventing a disease characterized by hyperlipidemia, the method comprising administering to a subject a therapeutically or prophylactically effective amount of a nucleic acid, LINC complex inhibitory polypeptide, vector, cell, or pharmaceutical composition according to the present disclosure.

[0042] In some embodiments according to various aspects of the disclosure, the disease characterized by hyperlipidemia is selected from atherosclerosis, cardiovascular disease, stroke, and familial hyperlipidemia. [Brief description of the drawings]

[0043] [Figure 1] A diagram showing alignment of the amino acid sequences of mouse Sun1, mouse Sun2, human SUN1 and human SUN2. [Diagram 2] Schematic diagram of human SUN1 protein and the various constructs characterized in Example 1 (i.e., constructs (A) to (H)). [Diagram 3] Schematic and micrographs showing the analysis of the ability of various putative dominant-negative SUN1 constructs to achieve LINC complex disruption, as determined by an increase in endoplasmic reticulum-localized Nesprin-2. [Figure 4] Schematic diagram of human SUN1 protein and the various constructs characterized in Example 5 (i.e., constructs A, D, D2, E and G). [Figure 5-1] Graph showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. [Figure 5-2] Graph showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. [Figure 5-3] Graph showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. [Figure 6-1] Graph showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. [Figure 6-2] Graph showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. [Figure 6-3]Graph showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] explanation The present disclosure is based on the inventors' unexpected discovery that, despite reports in the prior art that various truncation variants of the luminal domain of SUN domain-containing proteins are unable to associate with KASH domain-containing proteins and thus would be predicted not to function as LINC complex inhibitors, a wide range of such truncation variants do in fact function as LINC complex inhibitors.

[0045] Using a cell-based assay in which LINC complex disruption is determined through the observation of disruption of the normal subcellular localization of endogenous KASH domain-containing proteins (specifically, KASH domain-containing proteins localize to the endoplasmic reticulum rather than the nuclear envelope following LINC complex disruption), we demonstrate that polypeptides corresponding to a variety of different truncations of the luminal domain of SUN domain-containing proteins and containing a variety of their structural motifs (i.e., coiled-coil regions, their alpha helices) can affect LINC complex disruption. We further confirm that a variety of different dominant-negative LINC complex inhibitory polypeptides delivered as gene therapy via AAV reduce dilated cardiomyopathy (DCM) pathology in vivo in a mouse model of DCM established by cardiomyocyte-specific deletion of Lmna.

[0046] The present disclosure makes available a number of LINC complex inhibitory polypeptides suitable for applications related to existing and future gene therapy technologies. In particular, LINC complex inhibitory polypeptides smaller in size than the dominant-negative SUN domain-containing protein constructs described in WO 2019 / 143300 A1 are more suitable for use in vector systems with moderate packaging limits, such as scAAV vectors with a packaging limit of about 2.3 kb.

[0047] LINC complex structure and function The nucleoskeletal-cytoskeletal linker (LINC) complex is a polypeptide complex that contains SUN domain-containing proteins and KASH domain-containing proteins. The LINC complex structure is reviewed, 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.

[0048] The LINC complex connects the inner nuclear membrane (INM) and outer nuclear membrane (ONM) of the nuclear envelope. SUN domain-containing proteins span the INM and associate with nuclear lamins and chromatin-binding proteins on the nucleoplasmic side of the INM, as well as with 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 with SUN domain-containing proteins on the perinuclear side of the ONM. SUN domain proteins function as transtubular links for KASH domain proteins in the ONM.

[0049] As used herein, "SUN domain-containing protein" refers to any polypeptide that contains a SUN domain. SUN (Sad1 and UNC-84) domain proteins are important INM components that contain a conserved carboxy-terminal SUN domain that is localized to the nuclear cisternae. The SUN domain contains approximately 175 residues and is presented at the end of a helical stalk region. The nucleoplasmic domain of SUN proteins interacts with structural components of the nuclear scaffold.

[0050] The SUN domain can comprise or consist of an amino acid sequence set forth in SEQ ID NO:5, 14, 27, 28, 29 or 30, or an amino acid sequence having at least 60%, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:5, 14, 27, 28, 29 or 30.

[0051] 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.

[0052] In some embodiments, the SUN domain-containing protein can form a LINC complex. In some embodiments, the SUN domain-containing protein can interact with a KASH domain and / or a KASH domain-containing protein.

[0053] Human SUN1 is a polypeptide identified by UniProtKB O94901 and its amino acid sequence is set forth in SEQ ID NO: 1. Human SUN2 is a polypeptide identified by UniProtKB Q9UH99 and its amino acid sequence is set forth in SEQ ID NO: 13. Human SUN3 is a polypeptide identified by UniProtKB Q8TAQ9 and its amino acid sequence is set forth in SEQ ID NO: 23. Human SUN5 is a polypeptide identified by UniProtKB A9Z1W8 and its amino acid sequence is set forth in SEQ ID NO: 24. Human SPAG4 is a polypeptide identified by UniProtKB Q9NPE6 and its amino acid sequence is set forth in SEQ ID NO: 25. Human SUCO is a polypeptide identified by UniProtKB Q9UBS9 and its amino acid sequence is set forth in SEQ ID NO: 26.

[0054] As used herein, "SUN1," "SUN2," "SUN3," "SUN5," "SPAG4," and "SUCO" refer to SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO, respectively, from any species, including isoforms, fragments, variants, or homologs thereof.

[0055] As used herein, a "fragment," "variant," or "homolog" of a protein may optionally be characterized as having at least 60%, preferably one of 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 of a reference protein (e.g., a reference isoform of the reference protein). In some embodiments, a fragment / variant / isoform / homolog may be characterized by the ability to perform a function performed by the reference protein.

[0056] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions or other modifications compared to the amino acid sequence of the reference protein, but retains a significant degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the species of the reference protein. A "homolog" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0057] A "fragment" may be of any length (by 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 is derived), and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the reference protein.

[0058] The isoform, fragment, variant or homologue may optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of the reference protein, as determined by analysis with a suitable assay for that functional property / activity.

[0059] As used herein, reference to "SUN1" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 1, and fragments, variants or homologs thereof. In some embodiments, SUN1 comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1.

[0060] As used herein, reference to "SUN2" refers to a protein having an amino acid sequence as set forth in SEQ ID NO: 13, and fragments, variants or homologs thereof. In some embodiments, SUN2 comprises or consists of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 13.

[0061] As used herein, reference to "SUN3" refers to a protein having an amino acid sequence as set forth in SEQ ID NO: 23, and fragments, variants or homologs thereof. In some embodiments, SUN3 comprises or consists of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 23.

[0062] As used herein, reference to "SUN5" refers to a protein having an amino acid sequence as set forth in SEQ ID NO:24, and fragments, variants or homologs thereof. In some embodiments, SUN5 comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:24.

[0063] As used herein, reference to "SPAG4" refers to a protein having an amino acid sequence as set forth in SEQ ID NO: 25, and fragments, variants or homologs thereof. In some embodiments, SPAG4 comprises or consists of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 25.

[0064] As used herein, reference to "SUCO" refers to a protein having an amino acid sequence as set forth in SEQ ID NO: 26, and fragments, variants or homologs thereof. In some embodiments, a SUCO comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 26.

[0065] As used herein, "KASH domain-containing protein" refers to any polypeptide that contains a KASH domain. KASH (Klarsicht, ANC-1, Syne homology) domain proteins are carboxy-terminally tethered membrane proteins that are targeted to 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 transmembrane helix that spans the ONM and an approximately 30 amino acid region that extends into the nuclear cisternae.

[0066] The KASH domain may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 37, 38, 39, 40, 41 or 42, or an amino acid sequence having at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 37, 38, 39, 40, 41 or 42.

[0067] 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.

[0068] In some embodiments, the KASH domain containing protein is capable of forming a LINC complex, hi some embodiments, the KASH domain containing protein is capable of interacting with a SUN domain and / or a SUN domain containing protein.

[0069] Human Nesprin-1 is a polypeptide identified by UniProtKB Q8NF91, and its amino acid sequence is shown in SEQ ID NO: 31. Human Nesprin-2 is a polypeptide identified by UniProtKB Q8WXH0, and its amino acid sequence is shown in SEQ ID NO: 32. Human Nesprin-3 is a polypeptide identified by UniProtKB Q6ZMZ3, and its amino acid sequence is shown in SEQ ID NO: 33. Human Nesprin-4 is a polypeptide identified by UniProtKB Q8N205, and its amino acid sequence is shown in SEQ ID NO: 34. Human KASH5 is a polypeptide identified by UniProtKB Q8N6L0, and its amino acid sequence is shown in SEQ ID NO: 35. Human LRMP is a polypeptide identified by UniProtKB Q12912, and its amino acid sequence is shown in SEQ ID NO: 36.

[0070] As used herein, "Nesprin-1," "Nesprin-2," "Nesprin-3," "Nesprin-4," "KASH5," and "LRMP" refer to Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP, respectively, from any species, including isoforms, fragments, variants, or homologs thereof.

[0071] As used herein, reference to "Nesprin-1" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 31, and fragments, variants or homologs thereof. In some embodiments, Nesprin-1 comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 31.

[0072] As used herein, reference to "Nesprin-2" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 32, and fragments, variants or homologs thereof. In some embodiments, Nesprin-2 comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 32.

[0073] As used herein, reference to "Nesprin-3" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 33, and fragments, variants or homologs thereof. In some embodiments, Nesprin-3 comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 33.

[0074] As used herein, reference to "Nesprin-4" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 34, and fragments, variants or homologs thereof. In some embodiments, Nesprin-4 comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 34.

[0075] As used herein, reference to "KASH5" refers to a protein having an amino acid sequence as set forth in SEQ ID NO: 35, and fragments, variants or homologs thereof. In some embodiments, KASH5 comprises or consists of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 35.

[0076] As used herein, reference to "LRMP" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 36, and fragments, variants or homologs thereof. In some embodiments, the LMRP comprises or consists of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 36.

[0077] As used herein, "LINC complex" refers to a polypeptide complex comprising a SUN domain-containing protein and a KASH domain-containing protein.

[0078] LINC complex is formed by protein-protein interaction between SUN domain-containing protein and KASH domain-containing protein.LINC complex may include non-covalent and / or covalent interaction between SUN domain and KASH domain.Non-covalent interaction includes, for example, hydrogen bond, ionic interaction, van der Waals force and hydrophobic bond.Covalent interaction includes, for example, disulfide bond.

[0079] SUN domain proteins are believed to oligomerize and form trimers through interactions between their stalk regions to form coiled-coil triple helices (Zhou et al., J. Biol. Chem. (2012) 287:5317-5326). Deletion of the SUN domain protein stalk region has been shown to disrupt LINC complex formation. SUN domains assume a β-sandwich structure, with the SUN domains of the trimer interacting extensively with each other, in part through a protruding β-sheet known as the KASH lid; the KASH lid of one SUN domain overlaps in part with the β-sandwich of the adjacent SUN domain (Sosa et al., Cell (2012) 149:1035-1047).

[0080] KASH domain proteins can also oligomerize and may involve protein-protein interactions between transmembrane helices. A single KASH domain interacts with two adjacent SUN domains along a groove formed between the KASH lid of one SUN domain and the top region of the β-sandwich of the adjacent SUN domain. Thus, 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 important for SUN-KASH interactions; extension of the C-terminus by only one 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 transmission through the LINC complex (Jahed et al., Biophys. J. (2015) 109:501-509).

[0081] As explained herein above, the LINC complex is believed to form through the interaction of the SUN domains of three SUN domain-containing proteins with the KASH domains of three KASH domain-containing proteins. The interaction between SUN and KASH domain proteins appears to be promiscuous; SUN1 and SUN2 have been shown to interact with Nesprin-1, Nesprin-2 and Nesprin-3.

[0082] The LINC complex according to the present disclosure can include any SUN domain-containing protein and any KASH domain-containing protein. The SUN domain-containing protein of the LINC complex can be identical or not identical. The KASH domain-containing protein of the LINC complex can be identical or not identical.

[0083] LINC complex function is reviewed, for example, in Hieda, Cells (2017) 6(1):3, incorporated by reference herein above, and Stroud, Biophys Rev. (2018) 10(4):1033-1051, the entire contents of which are incorporated by reference herein.

[0084] LINC complexes perform diverse functions including providing structural support to the nucleus, shaping and positioning the nucleus, maintaining the connection between the centrosome and the nucleus and the spacing of the nuclear membrane, DNA repair, cell migration, and moving chromosomes within the nucleus during meiosis.

[0085] LINC complexes have a mechanosensing role, translating mechanical stimuli and changes in the extracellular matrix into signals that allow the cell to adapt to its environment by regulating cytoskeletal organization, gene expression, nuclear organization and structure.

[0086] Integrins mediate the transmission of forces from the external microenvironment to the intracellular cytoskeleton, and nuclear-cytoskeletal molecular connections transmit forces to the chromosomal organization in the nucleus. The nuclear lamina causes deformations in nuclear architecture and initiates changes in gene regulation.

[0087] The nuclear envelope is a key structure in such processes. On the nucleoplasmic side of the INM, the nuclear lamina (consisting of A- and B-type lamins) forms a lattice structure that contributes to the resistance of the nucleus to mechanical stress and is essential for the structural integrity of the nuclear envelope. Nuclear lamins are involved in processes that are crucial for cell function and survival, including the maintenance of nuclear integrity, cell cycle control, mechanical signaling, cell signaling and DNA repair.

[0088] Deviations from normal expression and / or function of nuclear envelope proteins, as well as deviations from normal expression and / or function of factors directly or indirectly associated with the nuclear envelope, are associated with a variety of diseases, including muscular dystrophies, cardiomyopathies, lipodystrophies, progeria, cancer and neurological diseases.

[0089] LINC complex inhibitor polypeptide The present disclosure relates to LINC complex inhibitory polypeptides. As used herein, "LINC complex inhibitory polypeptide" refers to a polypeptide that inhibits LINC complex formation and / or function.

[0090] The LINC complex inhibitor polypeptide according to the present disclosure comprises: capable of binding to a component protein of the LINC complex (e.g., a KASH domain-containing protein, e.g., one or more of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP); It may inhibit interactions between component proteins of the LINC complex (e.g., interactions between SUN domain-containing proteins and KASH domain-containing proteins); may inhibit the formation of a LINC complex (i.e., a LINC complex; e.g., may inhibit the assembly of a LINC complex that includes a SUN domain-containing protein and a KASH domain-containing protein); may disrupt the LINC complex (e.g., cause or promote disassembly of the LINC complex; e.g., via replacement of one or more of the component proteins of the LINC complex, including SUN domain-containing proteins and KASH domain-containing proteins); may disrupt the normal subcellular localization of component proteins of the LINC complex (e.g., the LINC complex, which contains SUN domain-containing proteins and KASH domain-containing proteins); may increase the localization of component proteins of the LINC complex (e.g., the LINC complex that contains SUN domain-containing proteins and KASH domain-containing proteins) to the endoplasmic reticulum; It may reduce the levels of LINC complexes (e.g., LINC complexes that contain SUN domain-containing proteins and KASH domain-containing proteins); may inhibit the function / activity of a LINC complex (e.g., a LINC complex containing SUN domain-containing proteins and KASH domain-containing proteins); May increase survival of subjects with laminopathies; May increase survival time for subjects with laminopathies; may increase or inhibit the decline of cardiac function in subjects with laminopathies; may increase or inhibit the reduction of myocardial contractility in subjects with laminopathies; may increase or inhibit the reduction of ejection fraction and / or shortening fraction in subjects with laminopathies; and / or may reduce or inhibit the increase in left ventricular internal diameter in subjects with laminopathies; The left ventricular posterior wall thickness may be increased or its reduction inhibited in subjects with laminopathies.

[0091] It is recognized that the LINC complex inhibitor polypeptide according to the present disclosure may exhibit more than one of the above listed properties.A given polypeptide can be evaluated for the above listed properties using appropriate assays.The assay can be, for example, an in vitro assay, optionally a cell-based assay or a cell-free assay.The assay can be, for example, an in vivo assay, i.e., carried out in a non-human animal.

[0092] If the assay is a cell-based assay, the assay may include engineering cells (e.g., via transfection / transduction) to express a putative LINC complex inhibitory polypeptide to determine whether the polypeptide exhibits one or more of the listed properties. The assay may use molecular species labeled with a detectable entity to facilitate their detection. It is recognized that the cell is preferably a cell that expresses a component protein of the LINC complex.

[0093] Component proteins of the LINC complex include SUN domain-containing proteins and KASH domain-containing proteins. For simplicity, "component proteins of the LINC complex" may be referred to herein simply as "LINC complex proteins."

[0094] In a preferred embodiment, the LINC complex comprises a SUN domain-containing protein and a KASH domain-containing protein. In some embodiments, the SUN domain-containing protein can be selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO (more preferably selected from SUN1 and SUN2). In some embodiments, the KASH domain-containing protein is selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP (more preferably selected from Nesprin-1, Nesprin-2 and Nesprin-3).

[0095] The LINC complex inhibitory polypeptide according to the present disclosure preferably binds to a 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.

[0096] The present disclosure particularly relates to LINC complex inhibitory polypeptides that bind to LINC complex proteins that are interaction partners for SUN domain-containing proteins (e.g., SUN domain-containing proteins described herein). In some embodiments, the interaction partner for SUN domain-containing proteins is a KASH domain-containing protein (e.g., a KASH domain-containing protein described herein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP). In some embodiments, the interaction partner for SUN domain-containing proteins is a SUN domain-containing protein (e.g., a SUN domain-containing protein selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO); SUN domain-containing proteins are known to associate to form multimers (e.g., trimers).

[0097] The LINC complex inhibitory polypeptide according to the present disclosure may exhibit specific binding to an interaction partner for a SUN domain-containing protein (e.g., selected from KASH domain-containing proteins, e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP). As used herein, "specific binding" refers to binding that is selective and can be distinguished from non-specific binding to non-target molecules. A LINC complex inhibitory polypeptide that specifically binds to an interaction partner for a SUN domain-containing protein preferably binds to a factor associated with the associated protein with greater affinity and / or longer duration than the affinity / duration of binding to other non-target molecules.

[0098] The ability of a given polypeptide to specifically bind to a given target molecule can be determined by analysis according to methods known in the art, such as by 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.

[0099] In some embodiments, a LINC complex inhibitory polypeptide according to the present disclosure inhibits an interaction partner for a SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) in the micromolar range, i.e., K D =9.9×10 -4 From 1×10 -6 In some embodiments, the LINC complex inhibitory polypeptide binds to an interaction partner for a SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) with a submicromolar affinity, i.e., K D <1×10 -6 In some embodiments, the LINC complex inhibitory polypeptide binds to an interaction partner for a SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) with an affinity in the nanomolar range, i.e., K D =9.9×10 -7 From 1×10 -9In some embodiments, the LINC complex inhibitory polypeptide binds to an interaction partner for a SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) with subnanomolar affinity, i.e., K D <1×10 -9 In some embodiments, the LINC complex inhibitory polypeptide binds to an interaction partner for a SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) with an affinity in the picomolar range, i.e., K D =9.9×10 -10 From 1×10 -12 In some embodiments, the LINC complex inhibitory polypeptide binds to an interaction partner for a SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) with sub-picomolar affinity, i.e., K D <1×10 -12 Join with M.

[0100] A LINC complex inhibitory polypeptide according to the present disclosure may bind to an interaction partner for a SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) in the manner 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 a region of the interaction partner for a SUN domain-containing protein that is the same as, or overlaps with, a region bound by the SUN domain-containing protein.

[0101] 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 cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA and proteolytic-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is hereby incorporated by reference in its entirety.

[0102] In some embodiments, the LINC complex inhibitor polypeptide according to the present disclosure inhibits 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 for the SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP). Inhibition of the interaction between a SUN domain-containing protein and an interaction partner for the SUN domain-containing protein herein includes inhibition of binding between a SUN domain-containing protein and an interaction partner for the SUN domain-containing protein (thereby inhibiting the formation of a complex containing such proteins), as well as disruption of a complex containing a SUN domain-containing protein and an interaction partner for the SUN domain-containing protein (e.g., via replacement of component proteins of such complexes and resulting disassembly of such complexes).

[0103] 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 for the SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., 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 the SUN domain-containing protein to an interaction partner for the SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide blocks the SUN domain-containing protein from binding to an interaction partner for the SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide occupies the region of the interaction partner for 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, a LINC complex inhibitory polypeptide displaces a SUN domain-containing protein from a complex that includes a SUN domain-containing protein and an interaction partner for the SUN domain-containing protein.

[0104] The ability of a given polypeptide to inhibit the interaction between two factors can be determined, for example, by correlation analysis of such an interaction in the presence or after incubation of the polypeptide with one or both of the interaction partners.

[0105] The analysis may include detecting a complex containing the SUN domain-containing protein, an interaction partner for the SUN domain-containing protein, or a complex containing the SUN domain-containing protein and an interaction partner for the SUN domain-containing protein. Such techniques are well known to those of skill in the art and include, for example, antibody / reporter-based methods (Western blot, ELISA, immunohisto / cytochemistry, etc.).

[0106] A polypeptide that inhibits a given interaction (e.g., between a SUN domain-containing protein and its corresponding interaction partner, e.g., a KASH domain-containing protein) is identified by observing a decrease / reduction in the level of the correlate of the interaction between the interaction partners in the presence of the polypeptide or after incubation of one or both of the interaction partners with the polypeptide, 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 assays can be performed in vitro, for example, using recombinant interaction partners or using cells expressing the interaction partners. The cells expressing the interaction partners can be endogenous or can be derived from nucleic acids introduced into the cells. For the purposes of such assays, one or both of the interaction partners and / or the polypeptide can be labeled or used with a detectable construct for the purpose of detecting and / or measuring the level of the interaction.

[0107] A correlate of an interaction between two interaction partners can be, for example, a complex formed by the association between the interaction partners, a functional property of a complex formed by the association between the interaction partners, or a correlate of a downstream activity mediated by a complex formed by the association between the interaction partners.

[0108] A polypeptide that inhibits an 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 for the SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) - e.g., a polypeptide that inhibits the formation of a complex containing the SUN domain-containing protein and an interaction partner for the SUN domain-containing protein, and / or a polypeptide that disrupts a complex containing the SUN domain-containing protein and an interaction partner for the SUN domain-containing protein - can be expressed in the absence of the polypeptide (or in the presence of a suitable control polypeptide that is known not to inhibit the interaction between the interaction partners). (ii) a decrease in the level of a functional property of the SUN domain containing protein and a complex comprising an interaction partner for the SUN domain containing protein; (iii) a decrease in the level of a correlate of a downstream activity mediated by the SUN domain containing protein and a complex comprising an interaction partner for the SUN domain containing protein; (iv) an increase in the level of free (i.e., uncomplexed) SUN domain containing protein; and / or (v) an increase in the level of an interaction partner for free (i.e., uncomplexed) SUN domain containing protein, in the presence of the polypeptide or after incubation of the polypeptide with one or both of the interaction partners, compared to the levels observed in

[0109] A LINC complex inhibitor polypeptide according to the present disclosure may disrupt the normal subcellular localization of a LINC complex protein (eg, a SUN domain-containing protein or an interaction partner for a SUN domain-containing protein).

[0110] 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 interacting partners for LINC complex proteins 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.

[0111] In some embodiments, the LINC complex inhibitory polypeptide increases the level / proportion of LINC complex proteins that are not localized to the nuclear envelope. In some embodiments, the LINC complex inhibitory polypeptide increases the level / proportion of KASH domain-containing proteins that are not associated with the outer nuclear membrane (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP). 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).

[0112] In some embodiments, the LINC complex inhibitory polypeptide increases the level / proportion of LINC complex proteins localized to 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 to the endoplasmic reticulum. In some embodiments, the LINC complex inhibitory polypeptide increases the level / proportion of SUN domain-containing proteins (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO) that are not associated with the nuclear inner membrane.

[0113] The subcellular localization of the component proteins of the LINC complex in cells can be analyzed using techniques known to those skilled in the art. Such techniques include, for example, immunohistochemical staining and analysis by reporter-based methods. For example, Boni et al., J. Cell Biology (2015) 209(5):705-720 and Smoyer et al., J. Cell Biology (2016) 215(4):575-590, describe reporter systems that allow imaging of proteins in the ER, INM and ONM. Such methods can be used to analyze the levels / proportions of component proteins of the LINC complex in the nuclear envelope, inner nuclear membrane and outer nuclear membrane.

[0114] LINC complex inhibitory polypeptides that disrupt the normal subcellular localization of LINC complex proteins may be identified using assays that include detecting the presence or determining the proportion of the associated protein(s) at a given subcellular location, e.g., using antibody / reporter-based methods (Western blot, ELISA, immunohisto / cytochemistry, etc.). Subcellular localization may be analyzed, for example, by immunocytochemistry of extracts prepared from different cellular fractions, or Western blot, and may use organelle markers and / or labeled proteins of known subcellular localization.

[0115] The assay may involve expressing a putative LINC complex inhibitory polypeptide in a cell (e.g., from a nucleic acid encoding the polypeptide introduced (e.g., by transfection / transduction) into the cell), and subsequently comparing the subcellular localization of the relevant LINC complex protein(s) in such cells to that observed in cells under an appropriate control condition (e.g., untransfected cells, cells transfected / transduced with an empty vector, or cells transfected / transduced with a nucleic acid encoding a polypeptide that is known not to affect the subcellular localization of the relevant LINC complex protein(s).

[0116] In some embodiments, polypeptides may be assessed for their ability to behave as LINC complex inhibitory polypeptides essentially as described in Example 1 herein.

[0117] Nucleic acids for expressing putative LINC complex inhibitory polypeptides can be introduced into cells (e.g., by transfection), and the cells can then be evaluated to determine the subcellular localization of one or more LINC complex proteins (e.g., a SUN domain-containing protein (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO) and / or an interacting partner for the SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP)). A polypeptide may be identified as a LINC complex inhibitory polypeptide if its expression is determined to decrease the level / proportion of a LINC complex protein that is localized to the nuclear envelope or increase the level / proportion of a LINC complex protein that is not localized to the nuclear envelope in such an assay (e.g., its expression is determined to decrease the level / proportion of a KASH domain-containing protein that is associated with the nuclear envelope, or increase the level / proportion of a KASH domain-containing protein that is not associated with the nuclear envelope, or increase the level / proportion of a KASH domain-containing protein that is localized to the endoplasmic reticulum) compared to the level / proportion observed in cells of an appropriate control condition (e.g., untransfected cells, cells transfected / transduced with an empty vector, or cells transfected / transduced with a nucleic acid encoding a polypeptide that is known not to affect the subcellular localization of the relevant LINC complex protein(s)).

[0118] In some embodiments, expression in a cell of a LINC complex inhibitory polypeptide according to the disclosure is less than 1-fold / less than 100%, e.g., ≦0.99-fold / ≦99%, ≦0.95-fold / ≦95%, ≦0.9-fold / ≦90%, ≦0.85-fold / ≦85%, ≦0.8-fold / ≦80%, ≦0.75-fold / ≦75%, ≦0.7-fold / ≦70%, ≦0.65-fold, of the level observed in the absence of a LINC complex inhibitory polypeptide or in a suitable control condition in a given assay. / ≦65%, ≦0.6-fold / ≦60%, ≦0.55-fold / ≦55%, ≦0.5-fold / ≦50%, ≦0.45-fold / ≦45%, ≦0.4-fold / ≦40%, ≦0.35-fold / ≦35%, ≦0.3-fold / ≦30%, ≦0.25-fold / ≦25%, ≦0.2-fold / ≦20%, ≦0.15-fold / ≦15%, ≦0.1-fold / ≦10%, ≦0.05-fold / ≦5%, or ≦0.01-fold / ≦1%, (i) among component proteins of the LINC complex (e.g., the SUN domain); (ii) reduce the level of an interaction between a SUN domain-containing protein and a KASH domain-containing protein; (iii) reduce the level of a functional / activity correlate of a LINC complex (e.g., a LINC complex comprising a SUN domain-containing protein and a KASH domain-containing protein); (iv) reduce the level / proportion of a LINC complex protein (e.g., a SUN domain-containing protein or a KASH domain-containing protein) that is localized to the nuclear envelope; and / or (v) reduce the level / proportion of a KASH domain-containing protein (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) that is localized to the nuclear envelope / envelope.

[0119] A preferred level of reduction according to the previous paragraph is a reduction of less than or equal to 0.5-fold / ≦50%, for example, to one of ≦0.4-fold / ≦40%, ≦0.3-fold / ≦30%, ≦0.2-fold / ≦20%, ≦0.15-fold / ≦15%, or ≦0.1-fold / ≦10%.

[0120] In some embodiments, expression in a cell of a LINC complex inhibitory polypeptide according to the disclosure is greater than 1-fold the level observed in the absence of the LINC complex inhibitory polypeptide or in an appropriate control condition in a given assay, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, ≧10-fold, ≧11-fold, ≧12-fold, ≧13-fold, ≧14-fold, ≧15-fold, ≧16-fold, ≧17-fold, ≧18-fold, ≧19 ... fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, ≧10-fold, ≧50-fold, or ≧100-fold, (i) increasing the level / proportion of LINC complex proteins that are not localized to the nuclear envelope (e.g., SUN domain-containing proteins or KASH domain-containing proteins), and / or (ii) increasing the level / proportion of LINC complex proteins that are localized to the endoplasmic reticulum (e.g., SUN domain-containing proteins or KASH domain-containing proteins).

[0121] The polypeptides may be evaluated for certain functional properties in appropriate in vivo models. For example, the polypeptides may be evaluated for therapeutic / prophylactic effects in vivo in non-human animal models of the diseases / conditions described herein. In such assays, the polypeptides may be delivered in the form of nucleic acids encoding the proteins, for example, using viral vectors with appropriate tropism for cells / tissues where LINC complex inhibition confers therapeutic / prophylactic benefit. By way of illustration, in the experimental examples of the present disclosure, putative LINC complex inhibitory polypeptides are evaluated in a mouse model of dilated cardiomyopathy established by inducible cardiomyocyte-specific deletion of Lmna - see Example 5. The putative LINC complex inhibitory polypeptides are delivered as gene therapy in the form of AAV9 vectors expressing the polypeptides under the control of cardiomyocyte-specific promoters.

[0122] Subject's survival time and survival can be evaluated by monitoring survival over time in such models.Cardiac function and myocardial contractility can be evaluated by measuring their correlates, and ejection fraction, shortening fraction, left ventricular posterior wall thickness and / or left ventricular internal diameter can be measured by echocardiography / ultrasound.

[0123] It is understood that the subject is preferably evaluated for one or more of the characteristics listed above at a particular time point, for example, after a period sufficient for the effect of administration of the polypeptide to be observed in a relevant model. For example, the subject can be evaluated 7 days or more after administration of a viral vector encoding a putative LINC complex inhibitory polypeptide.

[0124] In some embodiments, administration of a LINC complex inhibitory polypeptide according to the present disclosure to a subject having a laminopathic disorder (e.g., a cardiomyopathy associated with a mutation to LMNA; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) results in greater than 1-fold the level observed in the absence of such treatment or following 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). For example, increasing survival or survival time of the subject by one of >1.01 fold, >1.02 fold, >1.03 fold, >1.04 fold, >1.05 fold, >1.1 fold, >1.2 fold, >1.3 fold, >1.4 fold, >1.5 fold, >1.6 fold, >1.7 fold, >1.8 fold, >1.9 fold, >2 fold, >3 fold, >4 fold, >5 fold, >6 fold, >7 fold, >8 fold, >9 fold, >10 fold, >50 fold or >100 fold, and / or increasing cardiac function, myocardial contractility, ejection fraction, fractional shortening and / or left ventricular posterior wall thickness in the subject.

[0125] In some embodiments, administration of a LINC complex inhibitory polypeptide according to the present disclosure to a subject having a laminopathic disorder (e.g., a cardiomyopathy associated with a mutation to LMNA; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) results in a reduction in IL-1 expression levels that are less than 1-fold / less than 100%, e.g., ≦0.99-fold / ≦99%, ≦0.95-fold / ≦100%, of levels observed in the absence of such treatment or following 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). and reducing left ventricular internal diameter in a subject by one of the following: 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% times, <0.05x / <5% or <0.01x / <1%.

[0126] In some embodiments, a LINC complex inhibitory polypeptide according to the present disclosure may have one or more new or improved functional properties compared to a LINC complex inhibitory polypeptide consisting of the amino acid sequence of SEQ ID NO:99.

[0127] In some embodiments, expression in a cell of a LINC complex inhibitory polypeptide according to the disclosure is at less than 1-fold / less than 100% of the level observed in expression in a cell (e.g., by a cell of the same type) of a polypeptide consisting of the amino acid sequence of SEQ ID NO:99, e.g., ≦0.99-fold / ≦99%, ≦0.95-fold / ≦95%, ≦0.9-fold / ≦90%, ≦0.85-fold / ≦85%, ≦0.8-fold / ≦80%, ≦0.75-fold / ≦75%, ≦0.7-fold / ≦70%, ≦0. 0.65-fold / ≦65%, ≦0.6-fold / ≦60%, ≦0.55-fold / ≦55%, ≦0.5-fold / ≦50%, ≦0.45-fold / ≦45%, ≦0.4-fold / ≦40%, ≦0.35-fold / ≦35%, ≦0.3-fold / ≦30%, ≦0.25-fold / ≦25%, ≦0.2-fold / ≦20%, ≦0.15-fold / ≦15%, ≦0.1-fold / ≦10%-fold, ≦0.05-fold / ≦5% or ≦0.01-fold / ≦1%; (i) between component proteins of the LINC complex (e.g., the SUN domain) (ii) reduce the level of an interaction between a SUN domain-containing protein and a KASH domain-containing protein; (iii) reduce the level of a functional / activity correlate of a LINC complex (e.g., a LINC complex comprising a SUN domain-containing protein and a KASH domain-containing protein); (iv) reduce the level / proportion of a LINC complex protein (e.g., a SUN domain-containing protein or a KASH domain-containing protein) that is localized to the nuclear envelope; and / or (v) reduce the level / proportion of a KASH domain-containing protein (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, and LRMP) that is localized to the nuclear envelope / envelope.

[0128] In some embodiments, expression in a cell of a LINC complex inhibitory polypeptide according to the disclosure is greater than 1-fold, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, or greater than the level observed in expression in a cell (e.g., by a cell of the same type) of a polypeptide consisting of the amino acid sequence of SEQ ID NO:99. , ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, ≧10-fold, ≧50-fold or ≧100-fold (i) increasing the level / proportion of LINC complex proteins that are not localized to the nuclear envelope (e.g., SUN domain-containing proteins or KASH domain-containing proteins), and / or (ii) increasing the level / proportion of LINC complex proteins that are localized to the endoplasmic reticulum (e.g., SUN domain-containing proteins or KASH domain-containing proteins).

[0129] In some embodiments, administration of a LINC complex inhibitory polypeptide according to the present disclosure to a subject having a laminopathic disorder (e.g., a cardiomyopathy associated with a mutation to LMNA; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) results in greater than 1-fold, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.06-fold, ≧1.07-fold, ≧1.09-fold, ≧1.10-fold, ≧1.20-fold, ≧1.21-fold, ≧1.30-fold, ≧1.40-fold, ≧1.50-fold, ≧1.60-fold, ≧1.70-fold, ≧1.80-fold, ≧1.90-fold, ≧1.80-fold, ≧1.90-fold, ≧1.90-fold, ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.06-fold, ≧1.07-fold, ≧1.08-fold, ≧1.10-fold, ≧1.25-fold, ≧1.30-fold, ≧1.40-fold, ≧1.50-fold, ≧1.60-fold, ≧1.70-fold, ≧1.80-fold, ≧1.90-fold, ≧1.90-fold, ≧1.05-fold, ≧1.06-fold, ≧1.07-fold, ≧1.08-fold, ≧1.09-fold, ≧1.10-fold, ≧1.25-fold, ≧1.30-fold, ≧1.40-fold increase the survival or survival time of the subject by one of the following: .03 fold, ≥ 1.04 fold, ≥ 1.05 fold, ≥ 1.1 fold, ≥ 1.2 fold, ≥ 1.3 fold, ≥ 1.4 fold, ≥ 1.5 fold, ≥ 1.6 fold, ≥ 1.7 fold, ≥ 1.8 fold, ≥ 1.9 fold, ≥ 2 fold, ≥ 3 fold, ≥ 4 fold, ≥ 5 fold, ≥ 6 fold, ≥ 7 fold, ≥ 8 fold, ≥ 9 fold, ≥ 10 fold, ≥ 50 fold or ≥ 100 fold; and / or increase cardiac function, myocardial contractility, ejection fraction, fractional shortening and / or left ventricular posterior wall thickness in the subject.

[0130] In some embodiments, administration of a LINC complex inhibitory polypeptide according to the present disclosure to a subject having a laminopathic disorder (e.g., a cardiomyopathy associated with a mutation to LMNA; e.g., via a viral vector encoding a LINC complex inhibitory polypeptide) results in less than 1-fold / less than 100%, e.g., ≦0.99-fold / ≦99%, ≦0.95-fold / ≦95%, ≦0.9-fold / ≦90%, ≦0.8-fold / ≦100%, of the level observed following administration of a polypeptide consisting of the amino acid sequence of SEQ ID NO:99 (e.g., delivery using the same viral vector). The method reduces left ventricular internal diameter in a subject by one of the following: 5-fold / ≦85%, ≦0.8-fold / ≦80%, ≦0.75-fold / ≦75%, ≦0.7-fold / ≦70%, ≦0.65-fold / ≦65%, ≦0.6-fold / ≦60%, ≦0.55-fold / ≦55%, ≦0.5-fold / ≦50%, ≦0.45-fold / ≦45%, ≦0.4-fold / ≦40%, ≦0.35-fold / ≦35%, ≦0.3-fold / ≦30%, ≦0.25-fold / ≦25%, ≦0.2-fold / ≦20%, ≦0.15-fold / ≦15%, ≦0.1-fold / ≦10%-fold, ≦0.05-fold / ≦5% or ≦0.01-fold / ≦1%.

[0131] A LINC complex inhibitory polypeptide according to the present disclosure may be based on a SUN domain-containing protein selected from, for example, SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO. In some embodiments, a LINC complex inhibitory polypeptide according to the present disclosure comprises an inhibitory region based on a SUN domain-containing protein selected from, for example, SUN1, SUN2, SUN3, SUN5, SPAG4, and SUCO.

[0132] As used herein, a peptide / polypeptide / amino acid sequence that is "based on" a reference protein comprises or consists of an amino acid sequence that has 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 a portion of the amino acids of the reference protein.

[0133] The "inhibitory region" of a LINC complex inhibitory polypeptide refers to a region of the polypeptide through which LINC complex inhibition is achieved. The inhibitory region according to the present disclosure typically has a high degree of 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 the present disclosure can be a region that the LINC complex inhibitory polypeptide uses to bind to an interaction partner for a SUN domain-containing protein (e.g., one or more of a KASH domain-containing protein, e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP). In aspects and embodiments of the present disclosure, the inhibitory region of a LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein.

[0134] A LINC complex inhibitory polypeptide according to the present disclosure can comprise or consist essentially of an amino acid sequence having a high degree of 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 the amino acid sequence of all or a portion of a SUN domain-containing protein selected from, for example, SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO. A LINC complex inhibitory polypeptide according to the present disclosure can include an inhibitory region that comprises, or consists of, an amino acid sequence having a high degree of 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 the amino acid sequence of all or a portion of a SUN domain-containing protein selected from, for example, SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO.

[0135] Such LINC complex inhibitory polypeptides preferably (i) retain the ability of the underlying SUN domain-containing protein to bind to a KASH domain-containing protein (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 underlying SUN domain-containing protein. In preferred embodiments, LINC complex inhibitory polypeptides lack or exhibit reduced ability to bind to nuclear lamins and / or chromatin-binding proteins, and / or lack or exhibit reduced ability to associate with (e.g., localize to) the inner nuclear membrane compared to their underlying SUN domain-containing protein.

[0136] For example, LINC complex inhibitory polypeptides / inhibitory regions thereof according to the present disclosure may contain the amino acid sequence(s) required 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) and may lack the amino acid sequence(s) required for one or more other properties of the SUN domain-containing protein on which they are based (e.g., the amino acid sequence(s) required for binding to nuclear lamins and / or chromatin-binding proteins and / or the amino acid sequence(s) required for association with the inner nuclear membrane).

[0137] It is understood that in some embodiments, a LINC complex inhibitory polypeptide according to the present disclosure consists essentially of a KASH domain-containing protein-binding fragment of a SUN domain-containing protein.Similarly, it is understood that in some embodiments, an inhibitory region of a LINC complex inhibitory polypeptide according to the present disclosure consists of a KASH domain-containing protein-binding fragment of a SUN domain-containing protein.

[0138] Such LINC complex inhibitory polypeptides may be referred to as "decoy," "dominant negative," or "mimetic" versions of the SUN domain-containing proteins from which they are based. That is, in aspects and embodiments of the present disclosure, the LINC complex inhibitory polypeptides may be dominant negative SUN domain-containing polypeptides.

[0139] The LINC complex inhibitory polypeptides according to the present disclosure preferably exhibit competitive inhibition of the interaction between a SUN domain-containing protein (e.g., selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO) and a KASH domain-containing protein (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP). The decoy / dominant negative / mimetic LINC complex inhibitory polypeptides preferably bind to a KASH domain-containing protein (e.g., selected from Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP) and occupy the site required for the interaction, thereby preventing these species from binding to endogenous SUN domain-containing proteins.

[0140] Such LINC complex inhibitory polypeptides may inhibit the formation of LINC complexes and / or disrupt existing LINC complexes via inhibition of assembly of endogenous interaction partners and / or via replacement of endogenous interaction partners. Decoy / dominant negative / mimetic LINC complex inhibitory polypeptides form non-functional LINC complexes or LINC complexes with reduced levels of function compared to LINC complexes formed by wild-type endogenous SUN and KASH domain-containing proteins.

[0141] As described herein above, dominant-negative versions of SUN domain-containing proteins have been previously described, for example, Crisp et al., J Cell Biol. (2006) 172(1):41-53, which describes a dominant-negative version of the mouse Sun1 protein, and WO2019 / 143300A1, which describes a humanized version thereof.

[0142] In aspects and embodiments according to the present disclosure, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein. It is understood that the amino acid sequence(s) of the LINC complex inhibitory polypeptide described herein that corresponds to one or more regions of a SUN domain-containing protein may be included in the inhibitory region of the LINC complex inhibitory polypeptide.

[0143] As used herein, an amino acid sequence that "corresponds" to a particular region of a reference polypeptide or amino acid sequence has 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 to the amino acid sequence of the polypeptide / amino acid sequence. An amino acid sequence that "corresponds" to a particular region of a reference polypeptide or amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, for example, using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960). As an illustration, the amino acid sequence from positions 522 to 717 of human SUN2 corresponds to the amino acid sequence from positions 616 to 812 of human SUN1.

[0144] In some embodiments, the LINC complex inhibitory polypeptide according to the present disclosure consists essentially of a human amino acid sequence. In some embodiments, the inhibitory region of the LINC complex inhibitory polypeptide according to the present disclosure consists essentially of a human amino acid sequence. As used herein, "human amino acid sequence" refers to the amino acid sequence encoded by the nucleic acid of the human genome. That is, in some embodiments, the LINC complex inhibitory polypeptide or the inhibitory region of the LINC complex inhibitory polypeptide consists essentially of an amino acid sequence having 100% amino acid sequence identity to the amino acid sequence encoded by the genome of a human subject. It is understood that in some embodiments, 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 an embodiment of the LINC complex inhibitory polypeptide is particularly contemplated when intended for administration to a human subject, for example, in the context of therapeutic / prophylactic intervention according to the present disclosure.

[0145] In some embodiments, the LINC complex inhibitory polypeptide comprises 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 comprises an amino acid sequence corresponding to the α3 helix of the CC2 region of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the α3 helix of the CC2 region of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:22.

[0146] In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the SUN domain of a SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the SUN domain of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the SUN domain of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:18.

[0147] In some embodiments, the LINC complex inhibitory polypeptide comprises 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 comprises 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 comprises an inhibitory region comprising 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 comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:46.

[0148] In some embodiments, the LINC complex inhibitory polypeptide comprises 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 comprises an amino acid sequence corresponding to the α2 helix of the CC2 region of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the α2 helix of the CC2 region of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:21.

[0149] In some embodiments, the LINC complex inhibitory polypeptide comprises 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 comprises an amino acid sequence corresponding to the α1 helix of the CC2 region of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the α1 helix of the CC2 region of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0150] In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC1 region of a SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC1 region of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC1 region of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:19.

[0151] In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC1 and CC2 regions of a SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC1 and CC2 regions of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC1 and CC2 regions of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:88. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:91.

[0152] In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC2 region of a SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC2 region of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the CC2 region of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises 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 to the amino acid sequence of SEQ ID NO:89. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:92.

[0153] In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence 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 comprises an amino acid sequence corresponding to the α2 and α3 helices of the CC2 region of human SUN1. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the α2 and α3 helices of the CC2 region of human SUN2. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:93.

[0154] In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to a portion of the SMART coil-coiled 2 region, the CC1 region, and the CC2 region of a SUN domain-containing protein. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to positions 483 to 632 of human SUN1 (numbering according to SEQ ID NO: 1). In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to positions 388 to 538 of human SUN2 (numbering according to SEQ ID NO: 13). In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the LINC complex inhibitory polypeptide comprises an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:98.

[0155] In some embodiments, the LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of a dominant negative version of a SUN domain-containing protein disclosed in the prior art. In some embodiments, the LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of a truncated variant of a SUN domain-containing protein disclosed in the prior art. In some embodiments, the LINC complex inhibitory polypeptide does not comprise an inhibitory region consisting of the amino acid sequence of a dominant negative version of a SUN domain-containing protein disclosed in the prior art. In some embodiments, the LINC complex inhibitory polypeptide does not comprise an inhibitory region consisting of the amino acid sequence of a truncated variant of a SUN domain-containing protein disclosed in the prior art.

[0156] As used herein, a polypeptide or amino acid sequence "consisting essentially of" a reference amino acid sequence either (i) consists of the reference amino acid sequence or (ii) comprises the reference amino acid sequence, where the reference amino acid sequence constitutes at least 80% (e.g., one of ≧85% ≧86%, ≧87%, ≧88%, ≧89%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%) of the polypeptide / amino acid sequence. It is understood that a polypeptide or amino acid sequence "consisting essentially of" a reference amino acid sequence can include the reference amino acid sequence as well as additional amino acid(s) at either or both of the N-terminus and C-terminus of the reference amino acid sequence, provided that the additional amino acid(s) constitute ≦20% of the polypeptide / amino acid sequence.

[0157] As an illustration, a polypeptide consisting of the amino acid sequence of SEQ ID NO:74 consists essentially of the amino acid sequence of SEQ ID NO:44. SEQ ID NO:74 comprises the amino acid sequence of SEQ ID NO:44 (i.e., positions 1 to 197 of SEQ ID NO:74), with the amino acid sequence of SEQ ID NO:44 making up about 98% (i.e., residues 197 / 201) of the polypeptide. SEQ ID NO:74 includes a KDEL retention motif (SEQ ID NO:77) at the C-terminus of the amino acid sequence of SEQ ID NO:44.

[0158] In some embodiments, an amino acid sequence that consists essentially of a reference amino acid sequence consists of the reference amino acid sequence. That is, in some embodiments, an amino acid sequence that consists essentially of a reference amino acid sequence is identical to the reference amino acid sequence. In some embodiments, an amino acid sequence that consists essentially of a reference amino acid sequence comprises an amino acid sequence that is identical to the reference amino acid sequence and further comprises one or more additional amino acid(s) (e.g., 1-5, 1-10, or 1-20 amino acids) at one or both ends of the reference sequence.

[0159] In some embodiments, the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of SEQ ID NO: 43 or 45. In some embodiments, the LINC complex inhibitory polypeptide does not include an inhibitory region consisting of the amino acid sequence of any one of SEQ ID NO: 43 or 45.

[0160] In some embodiments, the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of SEQ ID NO: 44 or 46. In some embodiments, the LINC complex inhibitory polypeptide does not include an inhibitory region consisting of the amino acid sequence of any one of SEQ ID NO: 44 or 46.

[0161] In some embodiments, the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of SEQ ID NO: 43, 44, 45 or 46. In some embodiments, the LINC complex inhibitory polypeptide does not include an inhibitory region consisting of the amino acid sequence of any one of SEQ ID NO: 43, 44, 45 or 46.

[0162] In some embodiments, the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of any one of SEQ ID NOs: 43, 44, 45, 46, 47 or 48.

[0163] In some embodiments, the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of any one of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 74, or 75.

[0164] In some embodiments, the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of any one of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 58, 49, 50, 51, 52, 53, 54, 55, 56, 57, 74, or 75. In some embodiments, the LINC complex inhibitory polypeptide does not include an inhibitory region consisting of the amino acid sequence of any one of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 58, 49, 50, 51, 52, 53, 54, 55, 56, 57, 74, or 75.

[0165] In some embodiments, the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of any one of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 74, or 75. In some embodiments, the LINC complex inhibitory polypeptide does not include an inhibitory region consisting of the amino acid sequence of any one of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 74, or 75.

[0166] The present disclosure specifically contemplates disclaiming LINC complex inhibitory polypeptides consisting essentially of the amino acid sequence of any one of 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 or 95 individually or in combination with one or more other specified sequences. The present disclosure also specifically contemplates disclaiming LINC complex inhibitory polypeptides that contain an inhibitory region consisting of the amino acid sequence of any one of 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 or 95 individually or in combination with one or more other specified sequences.

[0167] However, in connection with certain aspects and embodiments of the disclosure, the LINC complex inhibitory polypeptide can comprise or consist essentially of the amino acid sequence of any one of 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 or 95. Similarly, in relation to certain aspects and embodiments of the present disclosure, a LINC complex inhibitory polypeptide can comprise an inhibitory region consisting of the amino acid sequence of any one of 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 or 95.

[0168] In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 64. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or essentially consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:67.In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 68. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 70. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or essentially consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:72.In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the LINC complex inhibitory polypeptide, or an inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the LINC complex inhibitory polypeptide, or an inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0169] In some embodiments, the LINC complex inhibitory polypeptide, or an inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the LINC complex inhibitory polypeptide, or an inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or essentially consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 103.

[0170] In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the LINC complex inhibitory polypeptide, or an inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the LINC complex inhibitory polypeptide, or an inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or essentially consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 108.In some embodiments, the LINC complex inhibitory polypeptide, or inhibitory region thereof, comprises or essentially consists of an amino acid sequence having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 109.

[0171] In some embodiments, a LINC complex inhibitory polypeptide, or inhibitory region thereof, according to the present disclosure is selected from the group consisting of positions 360-812 of SEQ ID NO:1; positions 361-812 of SEQ ID NO:1; positions 362-812 of SEQ ID NO:1; positions 363-812 of SEQ ID NO:1; positions 364-812 of SEQ ID NO:1; positions 365-812 of SEQ ID NO:1; positions 366-812 of SEQ ID NO:1; positions 367-812 of SEQ ID NO:1; positions 368-812 of SEQ ID NO:1; positions 369-812 of SEQ ID NO:1; positions 370-812 of SEQ ID NO:1; positions 371-812 of SEQ ID NO:1; positions 372-812 of SEQ ID NO:1; positions 373-812 of SEQ ID NO:1 Positions 3 to 812;Positions 374 to 812 of SEQ ID NO:1;Positions 375 to 812 of SEQ ID NO:1;Positions 376 to 812 of SEQ ID NO:1;Positions 377 to 812 of SEQ ID NO:1;Positions 378 to 812 of SEQ ID NO:1;Positions 379 to 812 of SEQ ID NO:1;Positions 380 to 812 of SEQ ID NO:1;Positions 381 to 812 of SEQ ID NO:1;Positions 382 to 812 of SEQ ID NO:1;Positions 383 to 812 of SEQ ID NO:1;Positions 384 to 812 of SEQ ID NO:1;Positions 385 to 812 of SEQ ID NO:1;Positions 386 to 812 of SEQ ID NO:1;Positions 387 to 812 of SEQ ID NO:1;Positions 388 to 812 of SEQ ID NO:1;Positions 389 to 812 of SEQ ID NO:1; Positions 390 to 812 of SEQ ID NO:1;Positions 391 to 812 of SEQ ID NO:1;Positions 392 to 812 of SEQ ID NO:1;Positions 393 to 812 of SEQ ID NO:1;Positions 394 to 812 of SEQ ID NO:1;Positions 395 to 812 of SEQ ID NO:1;Positions 396 to 812 of SEQ ID NO:1;Positions 397 to 812 of SEQ ID NO:1;Positions 398 to 812 of SEQ ID NO:1;Positions 399 to 812 of SEQ ID NO:1;Positions 400 to 812 of SEQ ID NO:1;Positions 401 to 812 of SEQ ID NO:1;Positions 402 to 812 of SEQ ID NO:1;Positions 403 to 812 of SEQ ID NO:1;Positions 404 to 812 of SEQ ID NO:1;Positions 405 to 812 of SEQ ID NO:1 06-812;positions 407-812 of SEQ ID NO:1;positions 408-812 of SEQ ID NO:1;positions 409-812 of SEQ ID NO:1;positions 410-812 of SEQ ID NO:1;positions 411-812 of SEQ ID NO:1;positions 412-812 of SEQ ID NO:1;positions 413-812 of SEQ ID NO:1;positions 414-812 of SEQ ID NO:1;positions 415-812 of SEQ ID NO:1;positions 416-812 of SEQ ID NO:1;positions 417-812 of SEQ ID NO:1;positions 418-812 of SEQ ID NO:1;positions 419-812 of SEQ ID NO:1;positions 420-812 of SEQ ID NO:1;positions 421-812 of SEQ ID NO:1;positions 422-812 of SEQ ID NO:1;Positions 423 to 812 of SEQ ID NO:1;Positions 424 to 812 of SEQ ID NO:1;Positions 425 to 812 of SEQ ID NO:1;Positions 426 to 812 of SEQ ID NO:1;Positions 427 to 812 of SEQ ID NO:1;Positions 428 to 812 of SEQ ID NO:1;Positions 429 to 812 of SEQ ID NO:1;Positions 430 to 812 of SEQ ID NO:1;Positions 431 to 812 of SEQ ID NO:1;Positions 432 to 812 of SEQ ID NO:1;Positions 433 to 812 of SEQ ID NO:1;Positions 434 to 812 of SEQ ID NO:1;Positions 435 to 812 of SEQ ID NO:1;Positions 436 to 812 of SEQ ID NO:1;Positions 437 to 812 of SEQ ID NO:1;Positions 438 to 812 of SEQ ID NO:1 Positions 39 to 812;Positions 440 to 812 of SEQ ID NO:1;Positions 441 to 812 of SEQ ID NO:1;Positions 442 to 812 of SEQ ID NO:1;Positions 443 to 812 of SEQ ID NO:1;Positions 444 to 812 of SEQ ID NO:1;Positions 445 to 812 of SEQ ID NO:1;Positions 446 to 812 of SEQ ID NO:1;Positions 447 to 812 of SEQ ID NO:1;Positions 448 to 812 of SEQ ID NO:1;Positions 449 to 812 of SEQ ID NO:1;Positions 450 to 812 of SEQ ID NO:1;Positions 451 to 812 of SEQ ID NO:1;Positions 452 to 812 of SEQ ID NO:1;Positions 453 to 812 of SEQ ID NO:1;Positions 454 to 812 of SEQ ID NO:1;Positions 455 to 812 of SEQ ID NO:1; Positions 456 to 812 of SEQ ID NO:1; Positions 457 to 812 of SEQ ID NO:1; Positions 458 to 812 of SEQ ID NO:1; Positions 459 to 812 of SEQ ID NO:1; Positions 460 to 812 of SEQ ID NO:1; Positions 461 to 812 of SEQ ID NO:1; Positions 462 to 812 of SEQ ID NO:1; Positions 463 to 812 of SEQ ID NO:1; Positions 464 to 812 of SEQ ID NO:1; Positions 465 to 812 of SEQ ID NO:1; Positions 466 to 812 of SEQ ID NO:1; Positions 467 to 812 of SEQ ID NO:1; Positions 468 to 812 of SEQ ID NO:1; Positions 469 to 812 of SEQ ID NO:1; Positions 470 to 812 of SEQ ID NO:1; Positions 471 to 812 of SEQ ID NO:1 Positions 72 to 812;Positions 473 to 812 of SEQ ID NO:1;Positions 474 to 812 of SEQ ID NO:1;Positions 475 to 812 of SEQ ID NO:1;Positions 476 to 812 of SEQ ID NO:1;Positions 477 to 812 of SEQ ID NO:1;Positions 478 to 812 of SEQ ID NO:1;Positions 479 to 812 of SEQ ID NO:1;Positions 480 to 812 of SEQ ID NO:1;Positions 481 to 812 of SEQ ID NO:1;Positions 482 to 812 of SEQ ID NO:1;Positions 483 to 812 of SEQ ID NO:1;Positions 484 to 812 of SEQ ID NO:1;Positions 485 to 812 of SEQ ID NO:1;Positions 486 to 812 of SEQ ID NO:1;Positions 487 to 812 of SEQ ID NO:1;Positions 488 to 812 of SEQ ID NO:1;Positions 489 to 812 of SEQ ID NO:1;Positions 490 to 812 of SEQ ID NO:1;Positions 491 to 812 of SEQ ID NO:1;Positions 492 to 812 of SEQ ID NO:1;Positions 493 to 812 of SEQ ID NO:1;Positions 494 to 812 of SEQ ID NO:1;Positions 495 to 812 of SEQ ID NO:1;Positions 496 to 812 of SEQ ID NO:1;Positions 497 to 812 of SEQ ID NO:1;Positions 498 to 812 of SEQ ID NO:1;Positions 499 to 812 of SEQ ID NO:1;Positions 500 to 812 of SEQ ID NO:1;Positions 501 to 812 of SEQ ID NO:1;Positions 502 to 812 of SEQ ID NO:1;Positions 503 to 812 of SEQ ID NO:1;Positions 504 to 812 of SEQ ID NO:1 05-812;positions 506-812 of SEQ ID NO:1;positions 507-812 of SEQ ID NO:1;positions 508-812 of SEQ ID NO:1;positions 509-812 of SEQ ID NO:1;positions 510-812 of SEQ ID NO:1;positions 511-812 of SEQ ID NO:1;positions 512-812 of SEQ ID NO:1;positions 513-812 of SEQ ID NO:1;positions 514-812 of SEQ ID NO:1;positions 515-812 of SEQ ID NO:1;positions 516-812 of SEQ ID NO:1;positions 517-812 of SEQ ID NO:1;positions 518-812 of SEQ ID NO:1;positions 519-812 of SEQ ID NO:1;positions 520-812 of SEQ ID NO:1;positions 521-812 of SEQ ID NO:1; Positions 522 to 812 of SEQ ID NO:1; Positions 523 to 812 of SEQ ID NO:1; Positions 524 to 812 of SEQ ID NO:1; Positions 525 to 812 of SEQ ID NO:1; Positions 526 to 812 of SEQ ID NO:1; Positions 527 to 812 of SEQ ID NO:1; Positions 528 to 812 of SEQ ID NO:1; Positions 529 to 812 of SEQ ID NO:1; Positions 530 to 812 of SEQ ID NO:1; Positions 531 to 812 of SEQ ID NO:1; Positions 532 to 812 of SEQ ID NO:1; Positions 533 to 812 of SEQ ID NO:1; Positions 534 to 812 of SEQ ID NO:1; Positions 535 to 812 of SEQ ID NO:1; Positions 536 to 812 of SEQ ID NO:1; Positions 537 to 812 of SEQ ID NO:1 Positions 38 to 812;Positions 539 to 812 of SEQ ID NO:1;Positions 540 to 812 of SEQ ID NO:1;Positions 541 to 812 of SEQ ID NO:1;Positions 542 to 812 of SEQ ID NO:1;Positions 543 to 812 of SEQ ID NO:1;Positions 544 to 812 of SEQ ID NO:1;Positions 545 to 812 of SEQ ID NO:1;Positions 546 to 812 of SEQ ID NO:1;Positions 547 to 812 of SEQ ID NO:1;Positions 548 to 812 of SEQ ID NO:1;Positions 549 to 812 of SEQ ID NO:1;Positions 550 to 812 of SEQ ID NO:1;Positions 551 to 812 of SEQ ID NO:1;Positions 552 to 812 of SEQ ID NO:1;Positions 553 to 812 of SEQ ID NO:1;Positions 554 to 812 of SEQ ID NO:1;Positions 555 to 812 of SEQ ID NO:1; Positions 556 to 812 of SEQ ID NO:1; Positions 557 to 812 of SEQ ID NO:1; Positions 558 to 812 of SEQ ID NO:1; Positions 559 to 812 of SEQ ID NO:1; Positions 560 to 812 of SEQ ID NO:1; Positions 561 to 812 of SEQ ID NO:1; Positions 562 to 812 of SEQ ID NO:1; Positions 563 to 812 of SEQ ID NO:1; Positions 564 to 812 of SEQ ID NO:1; Positions 565 to 812 of SEQ ID NO:1; Positions 566 to 812 of SEQ ID NO:1; Positions 567 to 812 of SEQ ID NO:1; Positions 568 to 812 of SEQ ID NO:1; Positions 569 to 812 of SEQ ID NO:1; Positions 570 to 812 of SEQ ID NO:1 Positions 571 to 812 of SEQ ID NO:1;Positions 572 to 812 of SEQ ID NO:1;Positions 573 to 812 of SEQ ID NO:1;Positions 574 to 812 of SEQ ID NO:1;Positions 575 to 812 of SEQ ID NO:1;Positions 576 to 812 of SEQ ID NO:1;Positions 577 to 812 of SEQ ID NO:1;Positions 578 to 812 of SEQ ID NO:1;Positions 579 to 812 of SEQ ID NO:1;Positions 580 to 812 of SEQ ID NO:1;Positions 581 to 812 of SEQ ID NO:1;Positions 582 to 812 of SEQ ID NO:1;Positions 583 to 812 of SEQ ID NO:1;Positions 584 to 812 of SEQ ID NO:1;Positions 585 to 812 of SEQ ID NO:1;Positions 586 to 812 of SEQ ID NO:1;SEQ ID NO: Positions 587 to 812 of SEQ ID NO:1;Positions 588 to 812 of SEQ ID NO:1;Positions 589 to 812 of SEQ ID NO:1;Positions 590 to 812 of SEQ ID NO:1;Positions 591 to 812 of SEQ ID NO:1;Positions 592 to 812 of SEQ ID NO:1;Positions 593 to 812 of SEQ ID NO:1;Positions 594 to 812 of SEQ ID NO:1;Positions 595 to 812 of SEQ ID NO:1;Positions 596 to 812 of SEQ ID NO:1;Positions 597 to 812 of SEQ ID NO:1;Positions 598 to 812 of SEQ ID NO:1;Positions 599 to 812 of SEQ ID NO:1;Positions 600 to 812 of SEQ ID NO:1;Positions 601 to 812 of SEQ ID NO:1;Positions 602 to 812 of SEQ ID NO:1;SEQ ID NO:1 1; positions 603 to 812 of SEQ ID NO:1; positions 604 to 812 of SEQ ID NO:1; positions 605 to 812 of SEQ ID NO:1; positions 606 to 812 of SEQ ID NO:1; positions 607 to 812 of SEQ ID NO:1; positions 608 to 812 of SEQ ID NO:1; positions 609 to 812 of SEQ ID NO:1; positions 610 to 812 of SEQ ID NO:1; positions 611 to 812 of SEQ ID NO:1; positions 612 to 812 of SEQ ID NO:1; positions 613 to 812 of SEQ ID NO:1; positions 614 to 812 of SEQ ID NO:1; positions 615 to 812 of SEQ ID NO:1; and positions 616 to 812 of SEQ ID NO:1;

[0172] In some embodiments, a LINC complex inhibitory polypeptide, or inhibitory region thereof, according to the present disclosure is selected from the group consisting of positions 258-717 of SEQ ID NO:13; positions 259-717 of SEQ ID NO:13; positions 260-717 of SEQ ID NO:13; positions 261-717 of SEQ ID NO:13; positions 262-717 of SEQ ID NO:13; positions 263-717 of SEQ ID NO:13; positions 264-717 of SEQ ID NO:13; positions 265-717 of SEQ ID NO:13; positions 266-717 of SEQ ID NO:13; positions 267-717 of SEQ ID NO:13; positions 268-717 of SEQ ID NO:13; positions 269-717 of SEQ ID NO:13; positions 270-717 of SEQ ID NO:13 717;positions 271 to 717 of SEQ ID NO:13;positions 272 to 717 of SEQ ID NO:13;positions 273 to 717 of SEQ ID NO:13;positions 274 to 717 of SEQ ID NO:13;positions 275 to 717 of SEQ ID NO:13;positions 276 to 717 of SEQ ID NO:13;positions 277 to 717 of SEQ ID NO:13;positions 278 to 717 of SEQ ID NO:13;positions 279 to 717 of SEQ ID NO:13;positions 280 to 717 of SEQ ID NO:13;positions 281 to 717 of SEQ ID NO:13;positions 282 to 717 of SEQ ID NO:13;positions 283 to 717 of SEQ ID NO:13;positions 284 to 717 of SEQ ID NO:13;positions 285 to 717 of SEQ ID NO:13;SEQ ID NO:13 Positions 286 to 717 of SEQ ID NO:13; Positions 287 to 717 of SEQ ID NO:13; Positions 288 to 717 of SEQ ID NO:13; Positions 289 to 717 of SEQ ID NO:13; Positions 290 to 717 of SEQ ID NO:13; Positions 291 to 717 of SEQ ID NO:13; Positions 292 to 717 of SEQ ID NO:13; Positions 293 to 717 of SEQ ID NO:13; Positions 294 to 717 of SEQ ID NO:13; Positions 295 to 717 of SEQ ID NO:13; Positions 296 to 717 of SEQ ID NO:13; Positions 297 to 717 of SEQ ID NO:13; Positions 298 to 717 of SEQ ID NO:13; Positions 299 to 717 of SEQ ID NO:13; Positions 300 to 717 of SEQ ID NO:13 Positions 1 to 717;Positions 302 to 717 of SEQ ID NO:13;Positions 303 to 717 of SEQ ID NO:13;Positions 304 to 717 of SEQ ID NO:13;Positions 305 to 717 of SEQ ID NO:13;Positions 306 to 717 of SEQ ID NO:13;Positions 307 to 717 of SEQ ID NO:13;Positions 308 to 717 of SEQ ID NO:13;Positions 309 to 717 of SEQ ID NO:13;Positions 310 to 717 of SEQ ID NO:13;Positions 311 to 717 of SEQ ID NO:13;Positions 312 to 717 of SEQ ID NO:13;Positions 313 to 717 of SEQ ID NO:13;Positions 314 to 717 of SEQ ID NO:13;Positions 315 to 717 of SEQ ID NO:13;Positions 316 to 717 of SEQ ID NO:13;Positions 317 to 717 of SEQ ID NO:13; Positions 318 to 717 of SEQ ID NO:13; Positions 319 to 717 of SEQ ID NO:13; Positions 320 to 717 of SEQ ID NO:13; Positions 321 to 717 of SEQ ID NO:13; Positions 322 to 717 of SEQ ID NO:13; Positions 323 to 717 of SEQ ID NO:13; Positions 324 to 717 of SEQ ID NO:13; Positions 325 to 717 of SEQ ID NO:13; Positions 326 to 717 of SEQ ID NO:13; Positions 327 to 717 of SEQ ID NO:13; Positions 328 to 717 of SEQ ID NO:13; Positions 329 to 717 of SEQ ID NO:13; Positions 330 to 717 of SEQ ID NO:13; Positions 331 to 717 of SEQ ID NO:13 Positions 32 to 717;Positions 333 to 717 of SEQ ID NO:13;Positions 334 to 717 of SEQ ID NO:13;Positions 335 to 717 of SEQ ID NO:13;Positions 336 to 717 of SEQ ID NO:13;Positions 337 to 717 of SEQ ID NO:13;Positions 338 to 717 of SEQ ID NO:13;Positions 339 to 717 of SEQ ID NO:13;Positions 340 to 717 of SEQ ID NO:13;Positions 341 to 717 of SEQ ID NO:13;Positions 342 to 717 of SEQ ID NO:13;Positions 343 to 717 of SEQ ID NO:13;Positions 344 to 717 of SEQ ID NO:13;Positions 345 to 717 of SEQ ID NO:13;Positions 346 to 717 of SEQ ID NO:13;Positions 347 to 717 of SEQ ID NO:13; Positions 348 to 717 of SEQ ID NO:13; Positions 349 to 717 of SEQ ID NO:13; Positions 350 to 717 of SEQ ID NO:13; Positions 351 to 717 of SEQ ID NO:13; Positions 352 to 717 of SEQ ID NO:13; Positions 353 to 717 of SEQ ID NO:13; Positions 354 to 717 of SEQ ID NO:13; Positions 355 to 717 of SEQ ID NO:13; Positions 356 to 717 of SEQ ID NO:13; Positions 357 to 717 of SEQ ID NO:13; Positions 358 to 717 of SEQ ID NO:13; Positions 359 to 717 of SEQ ID NO:13; Positions 360 to 717 of SEQ ID NO:13; Positions 361 to 717 of SEQ ID NO:13; Positions 362 to 717 of SEQ ID NO:13 Positions 63 to 717;Positions 364 to 717 of SEQ ID NO:13;Positions 365 to 717 of SEQ ID NO:13;Positions 366 to 717 of SEQ ID NO:13;Positions 367 to 717 of SEQ ID NO:13;Positions 368 to 717 of SEQ ID NO:13;Positions 369 to 717 of SEQ ID NO:13;Positions 370 to 717 of SEQ ID NO:13;Positions 371 to 717 of SEQ ID NO:13;Positions 372 to 717 of SEQ ID NO:13;Positions 373 to 717 of SEQ ID NO:13;Positions 374 to 717 of SEQ ID NO:13;Positions 375 to 717 of SEQ ID NO:13;Positions 376 to 717 of SEQ ID NO:13;Positions 377 to 717 of SEQ ID NO:13;Positions 378 to 717 of SEQ ID NO:13;Positions 379 to 717 of SEQ ID NO:13; Positions 380 to 717 of SEQ ID NO:13; Positions 381 to 717 of SEQ ID NO:13; Positions 382 to 717 of SEQ ID NO:13; Positions 383 to 717 of SEQ ID NO:13; Positions 384 to 717 of SEQ ID NO:13; Positions 385 to 717 of SEQ ID NO:13; Positions 386 to 717 of SEQ ID NO:13; Positions 387 to 717 of SEQ ID NO:13; Positions 388 to 717 of SEQ ID NO:13; Positions 389 to 717 of SEQ ID NO:13; Positions 390 to 717 of SEQ ID NO:13; Positions 391 to 717 of SEQ ID NO:13; Positions 392 to 717 of SEQ ID NO:13; Positions 393 to 717 of SEQ ID NO:13 94 to 717;395 to 717 of SEQ ID NO:13;396 to 717 of SEQ ID NO:13;397 to 717 of SEQ ID NO:13;398 to 717 of SEQ ID NO:13;399 to 717 of SEQ ID NO:13;400 to 717 of SEQ ID NO:13;401 to 717 of SEQ ID NO:13;402 to 717 of SEQ ID NO:13;403 to 717 of SEQ ID NO:13;404 to 717 of SEQ ID NO:13;405 to 717 of SEQ ID NO:13;406 to 717 of SEQ ID NO:13;407 to 717 of SEQ ID NO:13;408 to 717 of SEQ ID NO:13;409 to 717 of SEQ ID NO:13; Positions 410 to 717 of SEQ ID NO:13; Positions 411 to 717 of SEQ ID NO:13; Positions 412 to 717 of SEQ ID NO:13; Positions 413 to 717 of SEQ ID NO:13; Positions 414 to 717 of SEQ ID NO:13; Positions 415 to 717 of SEQ ID NO:13; Positions 416 to 717 of SEQ ID NO:13; Positions 417 to 717 of SEQ ID NO:13; Positions 418 to 717 of SEQ ID NO:13; Positions 419 to 717 of SEQ ID NO:13; Positions 420 to 717 of SEQ ID NO:13; Positions 421 to 717 of SEQ ID NO:13; Positions 422 to 717 of SEQ ID NO:13; Positions 423 to 717 of SEQ ID NO:13; Positions 424 to 717 of SEQ ID NO:13 Positions 25 to 717;Positions 426 to 717 of SEQ ID NO:13;Positions 427 to 717 of SEQ ID NO:13;Positions 428 to 717 of SEQ ID NO:13;Positions 429 to 717 of SEQ ID NO:13;Positions 430 to 717 of SEQ ID NO:13;Positions 431 to 717 of SEQ ID NO:13;Positions 432 to 717 of SEQ ID NO:13;Positions 433 to 717 of SEQ ID NO:13;Positions 434 to 717 of SEQ ID NO:13;Positions 435 to 717 of SEQ ID NO:13;Positions 436 to 717 of SEQ ID NO:13;Positions 437 to 717 of SEQ ID NO:13;Positions 438 to 717 of SEQ ID NO:13;Positions 439 to 717 of SEQ ID NO:13;Positions 440 to 717 of SEQ ID NO:13;Positions 441 to 717 of SEQ ID NO:13; Positions 442 to 717 of SEQ ID NO:13; Positions 443 to 717 of SEQ ID NO:13; Positions 444 to 717 of SEQ ID NO:13; Positions 445 to 717 of SEQ ID NO:13; Positions 446 to 717 of SEQ ID NO:13; Positions 447 to 717 of SEQ ID NO:13; Positions 448 to 717 of SEQ ID NO:13; Positions 449 to 717 of SEQ ID NO:13; Positions 450 to 717 of SEQ ID NO:13; Positions 451 to 717 of SEQ ID NO:13; Positions 452 to 717 of SEQ ID NO:13; Positions 453 to 717 of SEQ ID NO:13; Positions 454 to 717 of SEQ ID NO:13; Positions 455 to 717 of SEQ ID NO:13 Positions 56 to 717;Positions 457 to 717 of SEQ ID NO:13;Positions 458 to 717 of SEQ ID NO:13;Positions 459 to 717 of SEQ ID NO:13;Positions 460 to 717 of SEQ ID NO:13;Positions 461 to 717 of SEQ ID NO:13;Positions 462 to 717 of SEQ ID NO:13;Positions 463 to 717 of SEQ ID NO:13;Positions 464 to 717 of SEQ ID NO:13;Positions 465 to 717 of SEQ ID NO:13;Positions 466 to 717 of SEQ ID NO:13;Positions 467 to 717 of SEQ ID NO:13;Positions 468 to 717 of SEQ ID NO:13;Positions 469 to 717 of SEQ ID NO:13;Positions 470 to 717 of SEQ ID NO:13;Positions 471 to 717 of SEQ ID NO:13; Positions 472 to 717 of SEQ ID NO:13; Positions 473 to 717 of SEQ ID NO:13; Positions 474 to 717 of SEQ ID NO:13; Positions 475 to 717 of SEQ ID NO:13; Positions 476 to 717 of SEQ ID NO:13; Positions 477 to 717 of SEQ ID NO:13; Positions 478 to 717 of SEQ ID NO:13; Positions 479 to 717 of SEQ ID NO:13; Positions 480 to 717 of SEQ ID NO:13; Positions 481 to 717 of SEQ ID NO:13; Positions 482 to 717 of SEQ ID NO:13; Positions 483 to 717 of SEQ ID NO:13; Positions 484 to 717 of SEQ ID NO:13; Positions 485 to 717 of SEQ ID NO:13; Positions 486 to 717 of SEQ ID NO:13; Positions 87 to 717;Positions 488 to 717 of SEQ ID NO:13;Positions 489 to 717 of SEQ ID NO:13;Positions 490 to 717 of SEQ ID NO:13;Positions 491 to 717 of SEQ ID NO:13;Positions 492 to 717 of SEQ ID NO:13;Positions 493 to 717 of SEQ ID NO:13;Positions 494 to 717 of SEQ ID NO:13;Positions 495 to 717 of SEQ ID NO:13;Positions 496 to 717 of SEQ ID NO:13;Positions 497 to 717 of SEQ ID NO:13;Positions 498 to 717 of SEQ ID NO:13;Positions 499 to 717 of SEQ ID NO:13;Positions 500 to 717 of SEQ ID NO:13;Positions 501 to 717 of SEQ ID NO:13;Positions 502 to 717 of SEQ ID NO:13;Positions 503 to 717 of SEQ ID NO:13; Positions 504 to 717 of SEQ ID NO:13; Positions 505 to 717 of SEQ ID NO:13; Positions 506 to 717 of SEQ ID NO:13; Positions 507 to 717 of SEQ ID NO:13; Positions 508 to 717 of SEQ ID NO:13; Positions 509 to 717 of SEQ ID NO:13; Positions 510 to 717 of SEQ ID NO:13; Positions 511 to 717 of SEQ ID NO:13; Positions 512 to 717 of SEQ ID NO:13; Positions 513 to 717 of SEQ ID NO:13; Sequence 13; positions 514 to 717 of SEQ ID NO:13; positions 515 to 717 of SEQ ID NO:13; positions 516 to 717 of SEQ ID NO:13; positions 517 to 717 of SEQ ID NO:13; positions 518 to 717 of SEQ ID NO:13; positions 519 to 717 of SEQ ID NO:13; positions 520 to 717 of SEQ ID NO:13; positions 521 to 717 of SEQ ID NO:13; and positions 522 to 717 of SEQ ID NO:13;

[0173] In some embodiments, a LINC complex inhibitory polypeptide, or inhibitory region thereof, according to the present disclosure is selected from the group consisting of positions 360-812 of SEQ ID NO:1; positions 361-812 of SEQ ID NO:1; positions 362-812 of SEQ ID NO:1; positions 363-812 of SEQ ID NO:1; positions 364-812 of SEQ ID NO:1; positions 365-812 of SEQ ID NO:1; positions 366-812 of SEQ ID NO:1; positions 367-812 of SEQ ID NO:1; positions 368-812 of SEQ ID NO:1; positions 369-812 of SEQ ID NO:1; positions 370-812 of SEQ ID NO:1; positions 371-812 of SEQ ID NO:1; positions 372-812 of SEQ ID NO:1; positions 373-812 of SEQ ID NO:1 Positions 3 to 812;Positions 374 to 812 of SEQ ID NO:1;Positions 375 to 812 of SEQ ID NO:1;Positions 376 to 812 of SEQ ID NO:1;Positions 377 to 812 of SEQ ID NO:1;Positions 378 to 812 of SEQ ID NO:1;Positions 379 to 812 of SEQ ID NO:1;Positions 380 to 812 of SEQ ID NO:1;Positions 381 to 812 of SEQ ID NO:1;Positions 382 to 812 of SEQ ID NO:1;Positions 383 to 812 of SEQ ID NO:1;Positions 384 to 812 of SEQ ID NO:1;Positions 385 to 812 of SEQ ID NO:1;Positions 386 to 812 of SEQ ID NO:1;Positions 387 to 812 of SEQ ID NO:1;Positions 388 to 812 of SEQ ID NO:1;Positions 389 to 812 of SEQ ID NO:1; Positions 390 to 812 of SEQ ID NO:1;Positions 391 to 812 of SEQ ID NO:1;Positions 392 to 812 of SEQ ID NO:1;Positions 393 to 812 of SEQ ID NO:1;Positions 394 to 812 of SEQ ID NO:1;Positions 395 to 812 of SEQ ID NO:1;Positions 396 to 812 of SEQ ID NO:1;Positions 397 to 812 of SEQ ID NO:1;Positions 398 to 812 of SEQ ID NO:1;Positions 399 to 812 of SEQ ID NO:1;Positions 400 to 812 of SEQ ID NO:1;Positions 401 to 812 of SEQ ID NO:1;Positions 402 to 812 of SEQ ID NO:1;Positions 403 to 812 of SEQ ID NO:1;Positions 404 to 812 of SEQ ID NO:1;Positions 405 to 812 of SEQ ID NO:1 06-812;positions 407-812 of SEQ ID NO:1;positions 408-812 of SEQ ID NO:1;positions 409-812 of SEQ ID NO:1;positions 410-812 of SEQ ID NO:1;positions 411-812 of SEQ ID NO:1;positions 412-812 of SEQ ID NO:1;positions 413-812 of SEQ ID NO:1;positions 414-812 of SEQ ID NO:1;positions 415-812 of SEQ ID NO:1;positions 416-812 of SEQ ID NO:1;positions 417-812 of SEQ ID NO:1;positions 418-812 of SEQ ID NO:1;positions 419-812 of SEQ ID NO:1;positions 420-812 of SEQ ID NO:1;positions 421-812 of SEQ ID NO:1;positions 422-812 of SEQ ID NO:1;Positions 423 to 812 of SEQ ID NO:1;Positions 424 to 812 of SEQ ID NO:1;Positions 425 to 812 of SEQ ID NO:1;Positions 426 to 812 of SEQ ID NO:1;Positions 427 to 812 of SEQ ID NO:1;Positions 428 to 812 of SEQ ID NO:1;Positions 429 to 812 of SEQ ID NO:1;Positions 430 to 812 of SEQ ID NO:1;Positions 431 to 812 of SEQ ID NO:1;Positions 432 to 812 of SEQ ID NO:1;Positions 433 to 812 of SEQ ID NO:1;Positions 434 to 812 of SEQ ID NO:1;Positions 435 to 812 of SEQ ID NO:1;Positions 436 to 812 of SEQ ID NO:1;Positions 437 to 812 of SEQ ID NO:1;Positions 438 to 812 of SEQ ID NO:1 Positions 39 to 812;Positions 440 to 812 of SEQ ID NO:1;Positions 441 to 812 of SEQ ID NO:1;Positions 442 to 812 of SEQ ID NO:1;Positions 443 to 812 of SEQ ID NO:1;Positions 444 to 812 of SEQ ID NO:1;Positions 445 to 812 of SEQ ID NO:1;Positions 446 to 812 of SEQ ID NO:1;Positions 447 to 812 of SEQ ID NO:1;Positions 448 to 812 of SEQ ID NO:1;Positions 449 to 812 of SEQ ID NO:1;Positions 450 to 812 of SEQ ID NO:1;Positions 451 to 812 of SEQ ID NO:1;Positions 452 to 812 of SEQ ID NO:1;Positions 453 to 812 of SEQ ID NO:1;Positions 454 to 812 of SEQ ID NO:1;Positions 455 to 812 of SEQ ID NO:1; Positions 456 to 812 of SEQ ID NO:1; Positions 457 to 812 of SEQ ID NO:1; Positions 458 to 812 of SEQ ID NO:1; Positions 459 to 812 of SEQ ID NO:1; Positions 460 to 812 of SEQ ID NO:1; Positions 461 to 812 of SEQ ID NO:1; Positions 462 to 812 of SEQ ID NO:1; Positions 463 to 812 of SEQ ID NO:1; Positions 464 to 812 of SEQ ID NO:1; Positions 465 to 812 of SEQ ID NO:1; Positions 466 to 812 of SEQ ID NO:1; Positions 467 to 812 of SEQ ID NO:1; Positions 468 to 812 of SEQ ID NO:1; Positions 469 to 812 of SEQ ID NO:1; Positions 470 to 812 of SEQ ID NO:1; Positions 471 to 812 of SEQ ID NO:1 Positions 72 to 812;Positions 473 to 812 of SEQ ID NO:1;Positions 474 to 812 of SEQ ID NO:1;Positions 475 to 812 of SEQ ID NO:1;Positions 476 to 812 of SEQ ID NO:1;Positions 477 to 812 of SEQ ID NO:1;Positions 478 to 812 of SEQ ID NO:1;Positions 479 to 812 of SEQ ID NO:1;Positions 480 to 812 of SEQ ID NO:1;Positions 481 to 812 of SEQ ID NO:1;Positions 482 to 812 of SEQ ID NO:1;Positions 483 to 812 of SEQ ID NO:1;Positions 484 to 812 of SEQ ID NO:1;Positions 485 to 812 of SEQ ID NO:1;Positions 486 to 812 of SEQ ID NO:1;Positions 487 to 812 of SEQ ID NO:1;Positions 488 to 812 of SEQ ID NO:1;Positions 489 to 812 of SEQ ID NO:1;Positions 490 to 812 of SEQ ID NO:1;Positions 491 to 812 of SEQ ID NO:1;Positions 492 to 812 of SEQ ID NO:1;Positions 493 to 812 of SEQ ID NO:1;Positions 494 to 812 of SEQ ID NO:1;Positions 495 to 812 of SEQ ID NO:1;Positions 496 to 812 of SEQ ID NO:1;Positions 497 to 812 of SEQ ID NO:1;Positions 498 to 812 of SEQ ID NO:1;Positions 499 to 812 of SEQ ID NO:1;Positions 500 to 812 of SEQ ID NO:1;Positions 501 to 812 of SEQ ID NO:1;Positions 502 to 812 of SEQ ID NO:1;Positions 503 to 812 of SEQ ID NO:1;Positions 504 to 812 of SEQ ID NO:1 05-812;positions 506-812 of SEQ ID NO:1;positions 507-812 of SEQ ID NO:1;positions 508-812 of SEQ ID NO:1;positions 509-812 of SEQ ID NO:1;positions 510-812 of SEQ ID NO:1;positions 511-812 of SEQ ID NO:1;positions 512-812 of SEQ ID NO:1;positions 513-812 of SEQ ID NO:1;positions 514-812 of SEQ ID NO:1;positions 515-812 of SEQ ID NO:1;positions 516-812 of SEQ ID NO:1;positions 517-812 of SEQ ID NO:1;positions 518-812 of SEQ ID NO:1;positions 519-812 of SEQ ID NO:1;positions 520-812 of SEQ ID NO:1;positions 521-812 of SEQ ID NO:1; Positions 522 to 812 of SEQ ID NO:1; Positions 523 to 812 of SEQ ID NO:1; Positions 524 to 812 of SEQ ID NO:1; Positions 525 to 812 of SEQ ID NO:1; Positions 526 to 812 of SEQ ID NO:1; Positions 527 to 812 of SEQ ID NO:1; Positions 528 to 812 of SEQ ID NO:1; Positions 529 to 812 of SEQ ID NO:1; Positions 530 to 812 of SEQ ID NO:1; Positions 531 to 812 of SEQ ID NO:1; Positions 532 to 812 of SEQ ID NO:1; Positions 533 to 812 of SEQ ID NO:1; Positions 534 to 812 of SEQ ID NO:1; Positions 535 to 812 of SEQ ID NO:1; Positions 536 to 812 of SEQ ID NO:1; Positions 537 to 812 of SEQ ID NO:1 Positions 38 to 812;Positions 539 to 812 of SEQ ID NO:1;Positions 540 to 812 of SEQ ID NO:1;Positions 541 to 812 of SEQ ID NO:1;Positions 542 to 812 of SEQ ID NO:1;Positions 543 to 812 of SEQ ID NO:1;Positions 544 to 812 of SEQ ID NO:1;Positions 545 to 812 of SEQ ID NO:1;Positions 546 to 812 of SEQ ID NO:1;Positions 547 to 812 of SEQ ID NO:1;Positions 548 to 812 of SEQ ID NO:1;Positions 549 to 812 of SEQ ID NO:1;Positions 550 to 812 of SEQ ID NO:1;Positions 551 to 812 of SEQ ID NO:1;Positions 552 to 812 of SEQ ID NO:1;Positions 553 to 812 of SEQ ID NO:1;Positions 554 to 812 of SEQ ID NO:1;Positions 555 to 812 of SEQ ID NO:1;Positions 556 to 812 of SEQ ID NO:1;Positions 557 to 812 of SEQ ID NO:1;Positions 558 to 812 of SEQ ID NO:1;Positions 559 to 812 of SEQ ID NO:1;Positions 560 to 812 of SEQ ID NO:1;Positions 561 to 812 of SEQ ID NO:1;Positions 562 to 812 of SEQ ID NO:1;Positions 563 to 812 of SEQ ID NO:1;Positions 564 to 812 of SEQ ID NO:1;Positions 565 to 812 of SEQ ID NO:1;Positions 566 to 812 of SEQ ID NO:1;Positions 567 to 812 of SEQ ID NO:1;Positions 568 to 812 of SEQ ID NO:1;Positions 569 to 812 of SEQ ID NO:1;Positions 570 to 812 of SEQ ID NO:1;SEQ ID NO:1 Positions 571 to 812 of SEQ ID NO:1;Positions 572 to 812 of SEQ ID NO:1;Positions 573 to 812 of SEQ ID NO:1;Positions 574 to 812 of SEQ ID NO:1;Positions 575 to 812 of SEQ ID NO:1;Positions 576 to 812 of SEQ ID NO:1;Positions 577 to 812 of SEQ ID NO:1;Positions 578 to 812 of SEQ ID NO:1;Positions 579 to 812 of SEQ ID NO:1;Positions 580 to 812 of SEQ ID NO:1;Positions 581 to 812 of SEQ ID NO:1;Positions 582 to 812 of SEQ ID NO:1;Positions 583 to 812 of SEQ ID NO:1;Positions 584 to 812 of SEQ ID NO:1;Positions 585 to 812 of SEQ ID NO:1;Positions 586 to 812 of SEQ ID NO:1; Positions 587 to 812 of SEQ ID NO:1;Positions 588 to 812 of SEQ ID NO:1;Positions 589 to 812 of SEQ ID NO:1;Positions 590 to 812 of SEQ ID NO:1;Positions 591 to 812 of SEQ ID NO:1;Positions 592 to 812 of SEQ ID NO:1;Positions 593 to 812 of SEQ ID NO:1;Positions 594 to 812 of SEQ ID NO:1;Positions 595 to 812 of SEQ ID NO:1;Positions 596 to 812 of SEQ ID NO:1;Positions 597 to 812 of SEQ ID NO:1;Positions 598 to 812 of SEQ ID NO:1;Positions 599 to 812 of SEQ ID NO:1;Positions 600 to 812 of SEQ ID NO:1;Positions 601 to 812 of SEQ ID NO:1;Positions 602 to 812 of SEQ ID NO:1 does not contain, or does not essentially consist of, an amino acid sequence corresponding to one of positions 603 to 812; positions 604 to 812 of SEQ ID NO:1; positions 605 to 812 of SEQ ID NO:1; positions 606 to 812 of SEQ ID NO:1; positions 607 to 812 of SEQ ID NO:1; positions 608 to 812 of SEQ ID NO:1; positions 609 to 812 of SEQ ID NO:1; positions 610 to 812 of SEQ ID NO:1; positions 611 to 812 of SEQ ID NO:1; positions 612 to 812 of SEQ ID NO:1; positions 613 to 812 of SEQ ID NO:1; positions 614 to 812 of SEQ ID NO:1; positions 615 to 812 of SEQ ID NO:1; and positions 616 to 812 of SEQ ID NO:1;

[0174] In some embodiments, a LINC complex inhibitory polypeptide, or inhibitory region thereof, according to the present disclosure is selected from the group consisting of positions 258-717 of SEQ ID NO:13; positions 259-717 of SEQ ID NO:13; positions 260-717 of SEQ ID NO:13; positions 261-717 of SEQ ID NO:13; positions 262-717 of SEQ ID NO:13; positions 263-717 of SEQ ID NO:13; positions 264-717 of SEQ ID NO:13; positions 265-717 of SEQ ID NO:13; positions 266-717 of SEQ ID NO:13; positions 267-717 of SEQ ID NO:13; positions 268-717 of SEQ ID NO:13; positions 269-717 of SEQ ID NO:13; positions 270-717 of SEQ ID NO:13 717;positions 271 to 717 of SEQ ID NO:13;positions 272 to 717 of SEQ ID NO:13;positions 273 to 717 of SEQ ID NO:13;positions 274 to 717 of SEQ ID NO:13;positions 275 to 717 of SEQ ID NO:13;positions 276 to 717 of SEQ ID NO:13;positions 277 to 717 of SEQ ID NO:13;positions 278 to 717 of SEQ ID NO:13;positions 279 to 717 of SEQ ID NO:13;positions 280 to 717 of SEQ ID NO:13;positions 281 to 717 of SEQ ID NO:13;positions 282 to 717 of SEQ ID NO:13;positions 283 to 717 of SEQ ID NO:13;positions 284 to 717 of SEQ ID NO:13;positions 285 to 717 of SEQ ID NO:13;SEQ ID NO:13 Positions 286 to 717 of SEQ ID NO:13; Positions 287 to 717 of SEQ ID NO:13; Positions 288 to 717 of SEQ ID NO:13; Positions 289 to 717 of SEQ ID NO:13; Positions 290 to 717 of SEQ ID NO:13; Positions 291 to 717 of SEQ ID NO:13; Positions 292 to 717 of SEQ ID NO:13; Positions 293 to 717 of SEQ ID NO:13; Positions 294 to 717 of SEQ ID NO:13; Positions 295 to 717 of SEQ ID NO:13; Positions 296 to 717 of SEQ ID NO:13; Positions 297 to 717 of SEQ ID NO:13; Positions 298 to 717 of SEQ ID NO:13; Positions 299 to 717 of SEQ ID NO:13; Positions 300 to 717 of SEQ ID NO:13 Positions 1 to 717;Positions 302 to 717 of SEQ ID NO:13;Positions 303 to 717 of SEQ ID NO:13;Positions 304 to 717 of SEQ ID NO:13;Positions 305 to 717 of SEQ ID NO:13;Positions 306 to 717 of SEQ ID NO:13;Positions 307 to 717 of SEQ ID NO:13;Positions 308 to 717 of SEQ ID NO:13;Positions 309 to 717 of SEQ ID NO:13;Positions 310 to 717 of SEQ ID NO:13;Positions 311 to 717 of SEQ ID NO:13;Positions 312 to 717 of SEQ ID NO:13;Positions 313 to 717 of SEQ ID NO:13;Positions 314 to 717 of SEQ ID NO:13;Positions 315 to 717 of SEQ ID NO:13;Positions 316 to 717 of SEQ ID NO:13;Positions 317 to 717 of SEQ ID NO:13; Positions 318 to 717 of SEQ ID NO:13; Positions 319 to 717 of SEQ ID NO:13; Positions 320 to 717 of SEQ ID NO:13; Positions 321 to 717 of SEQ ID NO:13; Positions 322 to 717 of SEQ ID NO:13; Positions 323 to 717 of SEQ ID NO:13; Positions 324 to 717 of SEQ ID NO:13; Positions 325 to 717 of SEQ ID NO:13; Positions 326 to 717 of SEQ ID NO:13; Positions 327 to 717 of SEQ ID NO:13; Positions 328 to 717 of SEQ ID NO:13; Positions 329 to 717 of SEQ ID NO:13; Positions 330 to 717 of SEQ ID NO:13; Positions 331 to 717 of SEQ ID NO:13 Positions 32 to 717;Positions 333 to 717 of SEQ ID NO:13;Positions 334 to 717 of SEQ ID NO:13;Positions 335 to 717 of SEQ ID NO:13;Positions 336 to 717 of SEQ ID NO:13;Positions 337 to 717 of SEQ ID NO:13;Positions 338 to 717 of SEQ ID NO:13;Positions 339 to 717 of SEQ ID NO:13;Positions 340 to 717 of SEQ ID NO:13;Positions 341 to 717 of SEQ ID NO:13;Positions 342 to 717 of SEQ ID NO:13;Positions 343 to 717 of SEQ ID NO:13;Positions 344 to 717 of SEQ ID NO:13;Positions 345 to 717 of SEQ ID NO:13;Positions 346 to 717 of SEQ ID NO:13;Positions 347 to 717 of SEQ ID NO:13; Positions 348 to 717 of SEQ ID NO:13; Positions 349 to 717 of SEQ ID NO:13; Positions 350 to 717 of SEQ ID NO:13; Positions 351 to 717 of SEQ ID NO:13; Positions 352 to 717 of SEQ ID NO:13; Positions 353 to 717 of SEQ ID NO:13; Positions 354 to 717 of SEQ ID NO:13; Positions 355 to 717 of SEQ ID NO:13; Positions 356 to 717 of SEQ ID NO:13; Positions 357 to 717 of SEQ ID NO:13; Positions 358 to 717 of SEQ ID NO:13; Positions 359 to 717 of SEQ ID NO:13; Positions 360 to 717 of SEQ ID NO:13; Positions 361 to 717 of SEQ ID NO:13; Positions 362 to 717 of SEQ ID NO:13 Positions 63 to 717;Positions 364 to 717 of SEQ ID NO:13;Positions 365 to 717 of SEQ ID NO:13;Positions 366 to 717 of SEQ ID NO:13;Positions 367 to 717 of SEQ ID NO:13;Positions 368 to 717 of SEQ ID NO:13;Positions 369 to 717 of SEQ ID NO:13;Positions 370 to 717 of SEQ ID NO:13;Positions 371 to 717 of SEQ ID NO:13;Positions 372 to 717 of SEQ ID NO:13;Positions 373 to 717 of SEQ ID NO:13;Positions 374 to 717 of SEQ ID NO:13;Positions 375 to 717 of SEQ ID NO:13;Positions 376 to 717 of SEQ ID NO:13;Positions 377 to 717 of SEQ ID NO:13;Positions 378 to 717 of SEQ ID NO:13;Positions 379 to 717 of SEQ ID NO:13; Positions 380 to 717 of SEQ ID NO:13; Positions 381 to 717 of SEQ ID NO:13; Positions 382 to 717 of SEQ ID NO:13; Positions 383 to 717 of SEQ ID NO:13; Positions 384 to 717 of SEQ ID NO:13; Positions 385 to 717 of SEQ ID NO:13; Positions 386 to 717 of SEQ ID NO:13; Positions 387 to 717 of SEQ ID NO:13; Positions 388 to 717 of SEQ ID NO:13; Positions 389 to 717 of SEQ ID NO:13; Positions 390 to 717 of SEQ ID NO:13; Positions 391 to 717 of SEQ ID NO:13; Positions 392 to 717 of SEQ ID NO:13; Positions 393 to 717 of SEQ ID NO:13 94 to 717;395 to 717 of SEQ ID NO:13;396 to 717 of SEQ ID NO:13;397 to 717 of SEQ ID NO:13;398 to 717 of SEQ ID NO:13;399 to 717 of SEQ ID NO:13;400 to 717 of SEQ ID NO:13;401 to 717 of SEQ ID NO:13;402 to 717 of SEQ ID NO:13;403 to 717 of SEQ ID NO:13;404 to 717 of SEQ ID NO:13;405 to 717 of SEQ ID NO:13;406 to 717 of SEQ ID NO:13;407 to 717 of SEQ ID NO:13;408 to 717 of SEQ ID NO:13;409 to 717 of SEQ ID NO:13; Positions 410 to 717 of SEQ ID NO:13; Positions 411 to 717 of SEQ ID NO:13; Positions 412 to 717 of SEQ ID NO:13; Positions 413 to 717 of SEQ ID NO:13; Positions 414 to 717 of SEQ ID NO:13; Positions 415 to 717 of SEQ ID NO:13; Positions 416 to 717 of SEQ ID NO:13; Positions 417 to 717 of SEQ ID NO:13; Positions 418 to 717 of SEQ ID NO:13; Positions 419 to 717 of SEQ ID NO:13; Positions 420 to 717 of SEQ ID NO:13; Positions 421 to 717 of SEQ ID NO:13; Positions 422 to 717 of SEQ ID NO:13; Positions 423 to 717 of SEQ ID NO:13; Positions 424 to 717 of SEQ ID NO:13 Positions 25 to 717;Positions 426 to 717 of SEQ ID NO:13;Positions 427 to 717 of SEQ ID NO:13;Positions 428 to 717 of SEQ ID NO:13;Positions 429 to 717 of SEQ ID NO:13;Positions 430 to 717 of SEQ ID NO:13;Positions 431 to 717 of SEQ ID NO:13;Positions 432 to 717 of SEQ ID NO:13;Positions 433 to 717 of SEQ ID NO:13;Positions 434 to 717 of SEQ ID NO:13;Positions 435 to 717 of SEQ ID NO:13;Positions 436 to 717 of SEQ ID NO:13;Positions 437 to 717 of SEQ ID NO:13;Positions 438 to 717 of SEQ ID NO:13;Positions 439 to 717 of SEQ ID NO:13;Positions 440 to 717 of SEQ ID NO:13;Positions 441 to 717 of SEQ ID NO:13; Positions 442 to 717 of SEQ ID NO:13; Positions 443 to 717 of SEQ ID NO:13; Positions 444 to 717 of SEQ ID NO:13; Positions 445 to 717 of SEQ ID NO:13; Positions 446 to 717 of SEQ ID NO:13; Positions 447 to 717 of SEQ ID NO:13; Positions 448 to 717 of SEQ ID NO:13; Positions 449 to 717 of SEQ ID NO:13; Positions 450 to 717 of SEQ ID NO:13; Positions 451 to 717 of SEQ ID NO:13; Positions 452 to 717 of SEQ ID NO:13; Positions 453 to 717 of SEQ ID NO:13; Positions 454 to 717 of SEQ ID NO:13; Positions 455 to 717 of SEQ ID NO:13 Positions 56 to 717;Positions 457 to 717 of SEQ ID NO:13;Positions 458 to 717 of SEQ ID NO:13;Positions 459 to 717 of SEQ ID NO:13;Positions 460 to 717 of SEQ ID NO:13;Positions 461 to 717 of SEQ ID NO:13;Positions 462 to 717 of SEQ ID NO:13;Positions 463 to 717 of SEQ ID NO:13;Positions 464 to 717 of SEQ ID NO:13;Positions 465 to 717 of SEQ ID NO:13;Positions 466 to 717 of SEQ ID NO:13;Positions 467 to 717 of SEQ ID NO:13;Positions 468 to 717 of SEQ ID NO:13;Positions 469 to 717 of SEQ ID NO:13;Positions 470 to 717 of SEQ ID NO:13;Positions 471 to 717 of SEQ ID NO:13; Positions 472 to 717 of SEQ ID NO:13; Positions 473 to 717 of SEQ ID NO:13; Positions 474 to 717 of SEQ ID NO:13; Positions 475 to 717 of SEQ ID NO:13; Positions 476 to 717 of SEQ ID NO:13; Positions 477 to 717 of SEQ ID NO:13; Positions 478 to 717 of SEQ ID NO:13; Positions 479 to 717 of SEQ ID NO:13; Positions 480 to 717 of SEQ ID NO:13; Positions 481 to 717 of SEQ ID NO:13; Positions 482 to 717 of SEQ ID NO:13; Positions 483 to 717 of SEQ ID NO:13; Positions 484 to 717 of SEQ ID NO:13; Positions 485 to 717 of SEQ ID NO:13; Positions 486 to 717 of SEQ ID NO:13; Positions 87 to 717;Positions 488 to 717 of SEQ ID NO:13;Positions 489 to 717 of SEQ ID NO:13;Positions 490 to 717 of SEQ ID NO:13;Positions 491 to 717 of SEQ ID NO:13;Positions 492 to 717 of SEQ ID NO:13;Positions 493 to 717 of SEQ ID NO:13;Positions 494 to 717 of SEQ ID NO:13;Positions 495 to 717 of SEQ ID NO:13;Positions 496 to 717 of SEQ ID NO:13;Positions 497 to 717 of SEQ ID NO:13;Positions 498 to 717 of SEQ ID NO:13;Positions 499 to 717 of SEQ ID NO:13;Positions 500 to 717 of SEQ ID NO:13;Positions 501 to 717 of SEQ ID NO:13;Positions 502 to 717 of SEQ ID NO:13;Positions 503 to 717 of SEQ ID NO:13; Positions 504 to 717 of SEQ ID NO:13; Positions 505 to 717 of SEQ ID NO:13; Positions 506 to 717 of SEQ ID NO:13; Positions 507 to 717 of SEQ ID NO:13; Positions 508 to 717 of SEQ ID NO:13; Positions 509 to 717 of SEQ ID NO:13; Positions 510 to 717 of SEQ ID NO:13; Positions 511 to 717 of SEQ ID NO:13; Positions 512 to 717 of SEQ ID NO:13; Positions 513 to 717 of SEQ ID NO:13; 13; positions 514 to 717 of SEQ ID NO:13; positions 515 to 717 of SEQ ID NO:13; positions 516 to 717 of SEQ ID NO:13; positions 517 to 717 of SEQ ID NO:13; positions 518 to 717 of SEQ ID NO:13; positions 519 to 717 of SEQ ID NO:13; positions 520 to 717 of SEQ ID NO:13; positions 521 to 717 of SEQ ID NO:13; and positions 522 to 717 of SEQ ID NO:13;

[0175] A LINC complex inhibitory polypeptide according to the present disclosure may include one or more additional amino acids or sequences of amino acids, i.e., a LINC complex inhibitory polypeptide may include one or more amino acids or sequences of amino acids in addition to the inhibitory region of the polypeptide (i.e., the region including the amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein).

[0176] For example, a LINC complex inhibitory polypeptide can include an amino acid sequence(s) to facilitate expression, folding, transport, processing, purification, or detection.

[0177] According to various aspects and embodiments in accordance with the present disclosure, the LINC complex inhibitory polypeptide may include a sequence that prevents secretion of the polypeptide from a cell expressing the polypeptide. In some embodiments, the polypeptide includes an endoplasmic reticulum (ER) retention motif. Such a sequence may be provided at the C-terminus of the inhibitory region of the LINC complex inhibitory polypeptide in accordance with the present disclosure. In some embodiments, the sequence for preventing secretion of the polypeptide from a cell 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, the sequence for preventing secretion of the polypeptide from a cell expressing the polypeptide (e.g., an ER retention motif) is not followed by any other amino acids at the C-terminus of the polypeptide.

[0178] 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 that is effective for retaining a protein containing the motif at its C-terminus in the endoplasmic reticulum. The KDEL variant can comprise or consist of an amino acid sequence according to the prosite motif: [K / R / H / Q / S / A]-[D / E / N / Q]-EL (reported in Hulo et al., 2006, Nucleic acids research 34:D227-D230), or a variant reported by Raykhel et al., 2007 (J. Cell Biol. 179(6):1193-1204), who proposed an extended prosite motif definition. Raykhel et al. demonstrated endogenous conservation with variants including F or M at position 1 (L position), and D at position 2 (i.e., E position), a range of residues at position 3 (i.e., D position) extending well beyond F, WY, and DENQ as alternatives to KRHQSA at position 4 (i.e., K position).

[0179] For example, the variant of the KDEL motif may be one of, for example, CDEL, KCEL, or HVEL proposed by Raykhel et al. Thus, in some embodiments disclosed herein, the endoplasmic reticulum (ER) retention motif is KDEL or a variant thereof that exhibits ER retention activity.

[0180] In some embodiments, the ER retention motif comprises or consists of the amino acid sequence of SEQ ID NO:77, or a variant thereof comprising one or more (eg, one or two) substitutions in the amino acid sequence of SEQ ID NO:77.

[0181] In some embodiments, the LINC complex inhibitory polypeptide may include a signal peptide. The signal peptide is provided at the N-terminus of the inhibitory region of the LINC complex inhibitory polypeptide according to the present disclosure. In some embodiments, the signal peptide is provided at the N-terminus of the amino acid sequence of the polypeptide. In some embodiments, the signal peptide is not preceded by any other amino acids at the N-terminus of the polypeptide. A signal peptide usually consists of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often contain a signal peptide. The signal peptide may be present at the N-terminus of the peptide / polypeptide or may be present in a newly synthesized peptide / polypeptide. The signal peptide is often removed by cleavage and therefore is not included in the mature peptide / polypeptide.

[0182] 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, for example, 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 from a secreted protein or a type I transmembrane protein. In some embodiments, the secretory protein or type I transmembrane protein is selected from human serum albumin, proinsulin, transferrin receptor, EGF receptor, preproopiomelanocortin, 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.

[0183] In some embodiments, the signal peptide comprises a signal peptidase cleavage site. The signal peptidase cleavage site results in the removal of the signal peptide from the mature polypeptide. In some embodiments, the LINC complex inhibitor polypeptide according to the present disclosure comprises a signal peptidase cleavage site derived from a secreted protein or a type I transmembrane protein, such as a secreted protein or a type I transmembrane protein as described above.

[0184] In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 76. In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 60%, e.g., at least one of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100.

[0185] In some embodiments, the LINC complex inhibitory polypeptide may comprise a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radiolabel, a chemical, a nucleic acid or an enzymatic label. The LINC complex inhibitory polypeptide may be covalently or non-covalently labeled with a detectable moiety.

[0186] In some embodiments, the detectable moiety is provided at the N-terminus of the polypeptide (either before or after processing of the polypeptide with a signal peptidase to remove any signal peptide), In some embodiments, the detectable moiety is provided at the C-terminus of the polypeptide, e.g., downstream of a sequence to prevent secretion of the polypeptide from a cell expressing the polypeptide (e.g., downstream of an ER retention motif).

[0187] In some embodiments, the detectable moiety is or comprises an epitope tag. In some embodiments, the epitope tag is selected from hemagglutinin A (HA), ALFA, histidine (His; e.g., 6XHis), c-Myc, glutathione S-transferase (GST), green fluorescent protein (GFP), maltose binding protein (MBP), FLAG, E, biotin, protein A, protein G, streptavidin, T7, thioredoxin, V5 or vesicular stomatitis virus glycoprotein (VSV-G) tags. In some embodiments, the detectable moiety is or comprises a moiety that has detectable activity, e.g., enzymatic activity with a given substrate. Examples of such moieties include, e.g., horseradish peroxidase (HRP) and luciferase moieties.

[0188] In some embodiments, the detectable moiety is or comprises an HA tag. In some embodiments, the detectable moiety is or comprises an ALFA tag.

[0189] In some embodiments, the detectable moiety comprises or consists of the amino acid sequence of SEQ ID NO: 78, or a variant that contains one or more (e.g., 1, 2, 3 or 4) substitutions in the amino acid sequence of SEQ ID NO: 78. In some embodiments, the detectable moiety comprises or consists of the amino acid sequence of SEQ ID NO: 79, or a variant that contains one or more (e.g., 1, 2, 3 or 4) substitutions in the amino acid sequence of SEQ ID NO: 79.

[0190] In some embodiments, a LINC complex inhibitory polypeptide according to the present disclosure has the following structure: N-terminus-[signal peptide]-[detectable construct]-[inhibitory region comprising an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein]-[sequence that prevents secretion of the polypeptide from cells expressing the polypeptide]-C-terminus N-terminus-[detectable construct]-[signal peptide]-[inhibitory region comprising an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein]-[sequence that prevents secretion of the polypeptide from cells expressing the polypeptide]-C-terminus N-terminus-[detectable construct]-[inhibitory region comprising an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein]-[sequence that prevents secretion of the polypeptide from cells expressing the polypeptide]-C-terminus N-terminus - [an inhibitory region containing an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein] - [a sequence that prevents secretion of the polypeptide from cells expressing the polypeptide] - C-terminus It has one of the following:

[0191] In some embodiments, a LINC complex inhibitory polypeptide according to the present disclosure comprises one or more linker sequences between amino acid sequences. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, or 1-10 amino acids. In some embodiments, a linker sequence may be provided at one or both ends of one or more of: 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; a signal peptide; a sequence that prevents secretion of the polypeptide from a cell expressing the polypeptide; and / or a detectable component of the LINC complex inhibitory polypeptide.

[0192] 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 hereby incorporated by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences 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 percentage 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.

[0193] In a preferred embodiment, the LINC complex inhibitory polypeptide is of a size (i.e., in terms of the number of amino acids that make up 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.

[0194] In some embodiments, the LINC complex inhibitory polypeptide has a size such that the polynucleotide encoding the polypeptide has a size (i.e., in terms of the number of nucleotides that make up the polynucleotide) that is within the packaging limit of the vector for delivering the polynucleotide. In some embodiments, the LINC complex inhibitory polypeptide has a size such that the polynucleotide encoding the polypeptide has a size that is within the packaging limit 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 that is within the packaging limit of an adeno-associated virus (AAV) vector, such as 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 that is within the packaging limit of a scAAV vector.

[0195] In some embodiments, the LINC complex inhibitory polypeptide consists of an amino acid sequence comprising fewer than 510 amino acids. In some embodiments, the LINC complex inhibitory polypeptide consists of an amino acid sequence comprising fewer than 457 amino acids.

[0196] In some embodiments, the LINC complex inhibitory polypeptide consists of an amino acid sequence that contains fewer than 600 amino acids, e.g., one of < 550, < 500, < 450, < 400, < 350, < 340, < 330, < 320, < 310, < 300, < 290, < 280, < 270, < 260, < 250, < 240, < 230, < 220 or < 210 amino acids.

[0197] LINC complex inhibitory polypeptides according to the present disclosure can be prepared according to methods for the production of polypeptides known to those of skill in the art.

[0198] Polypeptides can be prepared by chemical synthesis, for example, liquid phase or solid phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described, for example, in Chandrudu et al., Molecules (2013), 18:4373-4388, which are hereby incorporated by reference in their entirety. Alternatively, antigen-binding molecules and polypeptides can be produced by recombinant expression. Suitable molecular biology techniques 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 ed.), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146, both of which are hereby incorporated by reference in their entirety. Methods for recombinant production of antigen-binding molecules have also been described in Frenzel et al., Front Immunol. (2013); 4:217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461, both of which are hereby incorporated by reference in their entireties.

[0199] Nucleic acids and vectors encoding LINC complex inhibitory polypeptides The present disclosure provides a nucleic acid that encodes a LINC complex inhibitory polypeptide according to the present disclosure.In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.In some embodiments, the nucleic acid is or comprises a polynucleotide.The nucleic acid can comprise or consist of a polynucleotide having a nucleotide sequence that encodes a polypeptide according to the present disclosure.

[0200] A LINC complex inhibitory polypeptide according to the present disclosure can be produced intracellularly by translation of an RNA encoding the polypeptide. A LINC complex inhibitory polypeptide according to the present disclosure can be produced intracellularly by transcription from a nucleic acid encoding the polypeptide followed by translation of the transcribed RNA.

[0201] In a preferred embodiment, the nucleic acid has a size that allows its delivery as a gene therapy, ie, in a suitable vector.

[0202] In some embodiments, the nucleic acid of the present disclosure comprises a nucleotide sequence that has a size within the packaging limit of the vector for delivering polynucleotide.In some embodiments, the nucleic acid comprises a nucleotide sequence that has a size within the packaging limit of the vector described herein.In some embodiments, the nucleic acid comprises a nucleotide sequence that has a size within the packaging limit of an adeno-associated virus (AAV) vector, such as the AAV vector described herein.In some embodiments, the nucleic acid comprises a nucleotide sequence that has a size within the packaging limit of a scAAV vector.

[0203] A nucleic acid according to the present disclosure may comprise a nucleotide sequence that encodes a polypeptide and may additionally comprise one or more non-polypeptide-encoding nucleotide sequence(s). The non-polypeptide-encoding nucleotide sequence(s) may be, for example, a 5' cap, a 5' UTR, a 3' UTR and / or a polyA tail sequence.

[0204] In some embodiments, the nucleotide sequence of the nucleic acid consists of fewer than 6,000 nucleotides, for example, one of ≦5,000, ≦4,500, ≦4,000, ≦3,500, ≦3,000, ≦2,500, ≦2,400 or ≦2,300 nucleotides.

[0205] In some embodiments, the protein-encoding nucleotide sequence of a nucleic acid of the disclosure consists of fewer than 1,530 nucleotides. In some embodiments, the protein-encoding nucleotide sequence of a nucleic acid of the disclosure consists of fewer than 1,371 nucleotides.

[0206] In some embodiments, the nucleotide sequence of the nucleic acid encoding the protein consists of fewer than 1,800 nucleotides, e.g., one of <1,650, <1,500, <1,350, <1,200, <1,050, <1,020, <990, <960, <930, <900, <870, <840, <810, <780, <750, <720, <690, <660 or <630 nucleotides.

[0207] In some embodiments, the nucleic acid is or is included in a vector. Thus, the present disclosure also provides a vector comprising a nucleic acid encoding a LINC complex inhibitory polypeptide according to the present disclosure.

[0208] The nucleic acids and vectors in accordance with this disclosure can be provided in purified or isolated form, ie, from other nucleic acids or naturally occurring biological materials.

[0209] As used herein, "vector" refers to a nucleic acid used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of a nucleic acid in a cell (i.e., the vector may be an expression vector). Such a vector may include a promoter sequence operably linked to a nucleotide sequence that codes for a sequence to be expressed. The vector may also include a termination codon and an expression enhancer. Any suitable vector, promoter, enhancer, and termination codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0210] The term "operably linked" may include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked to place expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of causing transcription of the nucleic acid sequence. The resulting transcript(s) can then be translated into a polypeptide, e.g., a LINC complex inhibitory polypeptide.

[0211] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors and herpes viral vectors), transposon-based vectors and artificial chromosomes (e.g., yeast artificial chromosomes), for example, as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4, 9, both of which are hereby incorporated by reference in their entireties. In a preferred embodiment, the vector is an adeno-associated viral vector or a lentiviral vector.

[0212] In some embodiments, the vector is selected based on its tropism for the cell type / tissue / organ to which it is desired to deliver the nucleic acid. In some embodiments, the vector is selected based on its tropism for the cell type / tissue / organ to which it is desired to express the LINC complex inhibitory polypeptide. For example, it may be desired to deliver the nucleic acid / express the LINC complex inhibitory polypeptide to a cell type / tissue / organ affected by the disease to be treated / prevented according to the present disclosure (e.g., a cell / tissue / organ in which the disease manifests symptoms).

[0213] In some embodiments, it is desirable to deliver a nucleic acid encoding a LINC complex inhibitory polypeptide to muscle cells / tissues (e.g., cardiac and / or skeletal muscle cells / tissues), and in such embodiments, a vector having tropism for such cells / tissues may be used. In some embodiments, the vector may be cardioactive. In some embodiments, the vector may be myotropic.

[0214] In a preferred embodiment, the vector is an adeno-associated virus vector. Adeno-associated virus vectors and their use to induce gene therapy are reviewed, for example, in Wang et al., Nat.Rev.Drug Discov. (2019) 18:358-378 and Li and Samulski, Nat.Rev.Genet. (2020) 12:255-272, both of which are incorporated herein by reference in their entirety. In some embodiments, the vector may be an adeno-associated virus vector as described in Wang et al., Nat.Rev.Drug Discov. (2019) 18:358-378. In some embodiments, the vector may be an adeno-associated virus vector as described in Li and Samulski, Nat.Rev.Genet. (2020) 12:255-272.

[0215] 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 hereby incorporated by reference in its entirety. Conventional AAV has a single-stranded DNA genome and depends on the DNA replication machinery of the transduced cell to synthesize a complementary strand, resulting in delayed transgene expression. In contrast, scAAV contains complementary sequences that spontaneously anneal upon infection, obviating the requirement for DNA synthesis in the transduced host cell. Compared to classical, single-stranded AAV vectors, scAAV vectors have been shown to result in accelerated onset of transgene expression and increased levels of transgene expression.

[0216] 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.

[0217] In some embodiments, the vector may be a cardioactive 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.

[0218] In some embodiments, the vector may be a skeletal muscle tropic adeno-associated viral vector. In some embodiments, the vector may be an adeno-associated viral vector of one of the following serotypes: AAV1, AAV6, AAV7, AAV8, AAV9, AAV9.45.

[0219] In some embodiments, the vector comprises a modification that increases binding and / or transduction into a cell type of interest (i.e., compared to the level of binding / transduction by an unmodified vector), hi some embodiments, the modification is to a capsid protein.

[0220] In some embodiments, the vector comprises a capsid protein comprising a cell targeting peptide. In some embodiments, the cell targeting peptide is a cell targeting peptide described in Buning and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248-265, such as a cell targeting peptide shown in Tables 1, 2, 3 or 4 thereof, which are hereby incorporated by reference in their entirety.

[0221] In some embodiments, the vector comprises a capsid protein comprising a substitution of one or more tyrosine residues, e.g., one or more surface-exposed tyrosine residues. In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments, the vector comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid as described in Iida et al., Biomed Res Int. (2013) 2013:974819, which is hereby incorporated by reference in its entirety.

[0222] In some embodiments, the vector may be an adeno-associated viral vector as described in Buning and Srivastava, supra. In some embodiments, the vector may be an adeno-associated viral vector as described in Iida et al., supra.

[0223] In some embodiments, the nucleic acid / vector comprises one or more sequences for controlling expression of the nucleic acid. Thus, in some embodiments, the nucleic acid / vector comprises a control element for inducible expression of the nucleic acid.

[0224] Sequences for controlling expression of a nucleic acid can provide for expression of the nucleic acid by a particular type of cell or tissue, for example, expression can be under the control of a cell type- or tissue-specific promoter.

[0225] Promoters for cell type- or tissue-specific expression of the nucleic acids according to the invention can be selected according to the disease to be treated / prevented. For example, the promoter can drive expression in cell types / tissues / organs affected by the disease (e.g., cell types / tissues / organs where symptoms of the disease are manifested).

[0226] 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 / striated muscle cell specific promoter (e.g., MCK, MHCK7 or desmin promoter). In some embodiments, the promoter is cTNT.

[0227] In some embodiments, the promoter may be a vascular endothelial cell specific promoter (e.g., Tie2 promoter). In some embodiments, the promoter may be a vascular smooth muscle cell specific promoter (e.g., SM22a promoter). In some embodiments, the promoter may be a monocyte / macrophage specific promoter (e.g., LysM promoter).

[0228] The sequence for controlling the expression of the nucleic acid can provide the expression of the nucleic acid, for example, in response to a given agent / signal. For example, the expression can be under the control of an inducible promoter. The agent can provide the inducible expression of the nucleic acid in vivo by administering the agent to a subject that has been administered the cells modified according to the present disclosure, or ex vivo / in vitro by administering the agent to cultured cells ex vivo or in vitro.

[0229] In some embodiments, the nucleic acid or vector according to the present disclosure may use a conditional expression system to control the expression of the nucleic acid encoding the LINC complex inhibitory polypeptide by cells containing the nucleic acid / vector. "Conditional expression", also referred to herein as "inducible expression", refers to expression that is conditional on a certain condition, for example, the presence of a certain drug. Conditional expression systems are well known in the art and are reviewed in Ryding et al., Journal of Endocrinology (2001) 171, pp. 1-14, which is hereby incorporated by reference in its entirety.

[0230] Cells encoding / containing / expressing LINC complex inhibitory polypeptides The present disclosure also provides a cell that contains or expresses a LINC complex inhibitory polypeptide according to the present disclosure. Cells that contain or express a nucleic acid or vector according to the present disclosure are also provided.

[0231] The cell may be a eukaryotic cell, for example a mammalian cell. The mammal may be a primate (rhesus monkey, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal of the order Rodentia), cat, dog, pig, sheep, goat, cow (including cattle, e.g. cow or any animal of the order Bos), horse (including any animal of the order Equidae), donkey and non-human primate). In a preferred embodiment, the cell may be a human cell.

[0232] The present disclosure also provides a method for producing a cell comprising a nucleic acid or vector according to the present disclosure or a cell comprising / expressing a LINC complex inhibitory polypeptide according to the present disclosure, comprising a step of introducing a nucleic acid or vector according to the present disclosure into a cell. In some embodiments, the step of introducing a nucleic acid / vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g., adeno-associated virus transduction). In some embodiments, the nucleic acid / vector is introduced into a cell in vivo, for example, after administration of a vector according to the present disclosure (e.g., a viral vector, e.g., an adeno-associated virus vector) to a subject. In some embodiments, the nucleic acid / vector is introduced into a cell in culture ex vivo or in vitro.

[0233] In some embodiments, the method further comprises culturing the cells under conditions suitable for expression of the nucleic acid or vector by the cells.

[0234] The present disclosure also provides cells obtained or obtainable by a method according to the present disclosure.

[0235] composition The present disclosure also provides compositions comprising the LINC complex inhibitory polypeptides, nucleic acids, vectors and cells described herein. In particular, the present disclosure provides pharmaceutical compositions and medicaments comprising the LINC complex inhibitory polypeptides, nucleic acids, vectors and cells of the present disclosure.

[0236] Such compositions may include the relevant articles (i.e., LINC complex inhibitory polypeptides / nucleic acids / vectors / cells) in a formulation suitable for clinical use. The present disclosure is particularly relevant to pharmaceutical compositions / medicines comprising the nucleic acids and vectors according to the present disclosure.

[0237] The compositions of the present disclosure may be formulated with one or more pharma- ceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), They may include antioxidants (e.g., 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, colorants (e.g., titanium dioxide).

[0238] The term "pharmacologically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, and the like that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject of interest (e.g., a human subject) without undue toxicity, irritation, allergic response, or other problem or difficulty, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring, or sweetener of a composition according to the present disclosure must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings, or sweetening agents can be found in standard pharmaceutical texts, such as Remington's 'The Science and Practice of Pharmacy' (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0239] A pharmaceutical composition / medicament according to the present disclosure may be formulated for administration to a subject, e.g., via a route of administration appropriate to the nature of the therapeutic agent and the disease to be treated / prevented. In some embodiments, the pharmaceutical composition / medicament may be formulated for parenteral, systemic, topical, intracavitary, intravascular, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral or transdermal administration. In some embodiments, the pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or by oral ingestion.

[0240] Medicaments and pharmaceutical compositions can be formulated for administration to a blood vessel or to a tissue / organ of interest (e.g., a tissue / organ affected by a disease / condition (e.g., a tissue / organ where symptoms of the disease / condition are manifested) that is affected by the condition.

[0241] The pharmaceutical composition / medicine may comprise a LINC complex inhibitory polypeptide / nucleic acid / vector / cell in a sterile or isotonic medium. The pharmaceutical composition / medicine may be provided in a liquid, including a gel form. The liquid formulation may be formulated for administration by injection or infusion (e.g., through a cannula) into a blood vessel or into a selected area of ​​the human or animal body. The pharmaceutical composition / medicine may be provided in a solid form, for example, a lyophilized form.

[0242] The present disclosure also provides a method for producing a pharmaceutical composition / medicine according to the present disclosure. Such a method may include mixing the LINC complex inhibitory polypeptide / nucleic acid / vector / cell described herein with a pharma- ceutically acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener. Such a method typically includes the step of bringing the LINC complex inhibitory polypeptide / nucleic acid / vector / cell into association with a carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a carrier (e.g., liquid carrier, finely divided solid carrier, etc.), and then, if necessary, shaping the product.

[0243] The LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions according to the present disclosure may be modified and / or formulated to facilitate delivery to and / or uptake by cells / tissues of interest (e.g., cardiac and / or skeletal muscle cells / tissues).

[0244] Strategies for targeted delivery of such species are reviewed, for example, in Li et al., Int. J. Mol. Sci. (2015) 16:19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9):1602-1608, which are hereby incorporated by reference in their entireties.

[0245] In some embodiments, the articles of the present disclosure may be encapsulated in nanoparticles or liposomes. In some embodiments, the articles of the present disclosure may be associated (covalently or non-covalently) with a cell penetrating peptide (e.g., a protein transduction domain, a trojan peptide, an arginine-rich peptide, a vectocell peptide), a cationic polymer, a cationic lipid, or a viral carrier.

[0246] The nanoparticles can be organic, such as micelles, liposomes, proteins, solid lipid particles, solid polymer particles, dendrimers, and polymeric therapeutic agents. The nanoparticles can be inorganic, such as nanotubes or metal particles, optionally with attached organic molecules. In some embodiments, the nanoparticles are those described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which is hereby incorporated by reference in its entirety. In some embodiments, the nanoparticles are PLGA, polypeptides, poly(β-amino esters), DOPE, β-cyclodextrin-containing polycations, linear PEI, PAMAM dendrimers, branched PEI, chitosan, or polyphosphate ester nanoparticles.

[0247] In some embodiments, LINC complex inhibitory polypeptides, nucleic acids, and vectors according to the present disclosure include modifications to incorporate one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest (e.g., cardiac and / or skeletal muscle cells / tissues). In some embodiments, LINC complex inhibitory polypeptides, nucleic acids, and vectors according to the present disclosure are linked (e.g., chemically conjugated) to one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest.

[0248] Moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest are described, for example, in Benizri et al., Bioconjug Chem. (2019) 30(2):366-383, which is hereby incorporated by reference in its entirety. Such moieties include, for example, N-acetylgalactosamine (GalNAc), α-tocopherol, cell penetrating peptides, nucleic acid aptamers, antibodies and antigen-binding fragments / derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g., palmitic acid), and nucleolipid moieties.

[0249] The articles of the present disclosure may be formulated in sustained release delivery systems to release the LINC complex inhibitory polypeptides, nucleic acids, vectors, cells or compositions at a predetermined rate. The sustained release delivery systems can maintain a constant drug / therapeutic / prophylactic concentration for a specified period of time. In some embodiments, the articles of the present disclosure are formulated in liposomes, gels, implants, devices or drug polymer conjugates, e.g., hydrogels.

[0250] Methods for identifying LINC complex inhibitor polypeptides The present disclosure also provides methods for identifying LINC complex inhibitory polypeptides.

[0251] The methods generally include the steps of introducing a nucleic acid encoding a candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to the SUN domain of a SUN domain-containing protein or corresponding to the KASH domain of a KASH domain-containing protein, and analyzing the intracellular localization of the interaction partner relative to the SUN domain-containing protein (if the candidate LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the SUN domain of a SUN domain-containing protein) or analyzing the intracellular localization of the interaction partner relative to the KASH domain-containing protein (if the candidate LINC complex inhibitory polypeptide comprises an amino acid sequence corresponding to the KASH domain of a KASH domain-containing protein) to determine whether the candidate LINC complex inhibitory polypeptide alters / disrupts the normal intracellular localization of an associated interaction partner.

[0252] More particularly, the present disclosure provides a method for identifying a LINC complex inhibitory polypeptide, comprising: introducing a nucleic acid encoding a candidate LINC complex inhibitory polypeptide, the candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to a SUN domain of a SUN domain-containing protein (e.g., SUN1, SUN2, SUN3, SUN5, SPAG4 or SUCO); and Then, the cells are analyzed to determine the subcellular localization of an interacting partner for the SUN domain-containing protein (e.g., a KASH domain-containing protein, e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, or LRMP). wherein the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if a change in the subcellular localization of the interaction partner relative to the SUN domain-containing protein is detected.

[0253] 1. A method for identifying a LINC complex inhibitor polypeptide, comprising: introducing a nucleic acid encoding a candidate LINC complex inhibitory polypeptide, the candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to a KASH domain of a KASH domain-containing protein (e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, or LRMP); and Then, the cells are analyzed to determine the subcellular localization of an interacting partner for the KASH domain-containing protein (e.g., a SUN domain-containing protein, such as SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO). Also provided is a method comprising:

[0254] It is understood that the changes in the subcellular localization of the relevant interaction partner referred to in the previous two paragraphs refer to changes compared to the subcellular localization of the interaction partner in control cells in which a nucleic acid encoding a candidate LINC complex inhibitory polypeptide has not been introduced, or in control cells in which a nucleic acid encoding a control polypeptide known to not affect the subcellular localization of the relevant interaction partner has been introduced.

[0255] Such methods are preferably performed using cells in in vitro culture.

[0256] In some embodiments, introducing the nucleic acid into a cell may include transformation, transfection, electroporation or transduction (eg, adeno-associated virus transduction).

[0257] In some embodiments, a nucleic acid encoding a candidate LINC complex inhibitory polypeptide may be provided in a vector, for example, in a vector according to an embodiment described herein.

[0258] The nucleic acid encoding the candidate LINC complex inhibitory polypeptide preferably comprises regulatory elements that provide for expression of the candidate LINC complex inhibitory polypeptide in a cell into which the nucleic acid has been introduced. Following introduction of the nucleic acid into the cell, the cell is preferably maintained under conditions suitable for expression of the candidate LINC complex inhibitory polypeptide from the nucleic acid in the cell.

[0259] It will also be appreciated that following introduction of the nucleic acid into the cells, a period of time sufficient for a change in the subcellular localization of the relevant interaction partner preferably elapses before the cells are analyzed to assess the subcellular localization of the relevant interaction partner.

[0260] The subcellular localization of a given polypeptide (e.g., a given interaction partner for a SUN domain-containing protein or a KASH domain-containing protein) can be analyzed by methods well known to those of skill in the art. Such methods include antibody / reporter-based methods (Western blot, ELISA, immunohisto / cytochemistry, etc.). For example, subcellular localization can be analyzed by immunocytochemistry of extracts prepared from different cellular fractions, or by Western blot. Such analyses can use organelle markers and / or labeled proteins of known subcellular localization.

[0261] Such methods may use interaction partners for the SUN or KASH domain-containing proteins conjugated to a fluorescent label and may include analysis of the subcellular localization of such species by fluorescence microscopy. Such methods may use antibody-based detection of interaction partners for the SUN or KASH domain-containing proteins and may include analysis of the subcellular localization of such species by immunofluorescence microscopy.

[0262] In some embodiments, the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if a decrease in the proportion of relevant interaction partners localized to the nuclear envelope is detected (compared to the proportion localized to the nuclear envelope in control cells in which a nucleic acid encoding the candidate LINC complex inhibitory polypeptide has not been introduced, or in control cells in which a nucleic acid encoding a control polypeptide known to not affect the intracellular localization of the relevant interaction partner has been introduced).

[0263] In some embodiments, a candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if an increase in the proportion of relevant interaction partners not localized to the nuclear envelope or an increase in the proportion of relevant interaction partners localized to the endoplasmic reticulum is detected (compared to the proportion localized to the nuclear envelope in control cells in which a nucleic acid encoding the candidate LINC complex inhibitory polypeptide has not been introduced, or in control cells in which a nucleic acid encoding a control polypeptide known to not affect the intracellular localization of the relevant interaction partner has been introduced).

[0264] In some embodiments, methods for identifying a LINC complex inhibitory polypeptide may involve analysis of candidate LINC complex inhibitory polypeptides essentially as described in Example 1 herein.

[0265] In some embodiments, a method for identifying a LINC complex inhibitory polypeptide comprises: introducing into the cell a nucleic acid encoding a candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to the SUN domain of a SUN domain-containing protein; and Then, the cells are analyzed to determine the subcellular localization of Nesprin-2. and an increase in the proportion of Nesprin-2 localized to the endoplasmic reticulum is detected, the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide.

[0266] The present disclosure also provides a LINC complex inhibitory polypeptide identified by the methods of the present disclosure.

[0267] Therapeutic and preventive applications The LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions described herein are useful in therapeutic and prophylactic methods.

[0268] The present disclosure provides the LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions described herein for use in methods of medical treatment and prevention.Also provided is the use of the LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions described herein in the manufacture of a medicament for treating or preventing a disease / condition.Also provided is a method of treating or preventing a disease / condition, comprising administering to a subject a therapeutically or prophylactically effective amount of the LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions described herein.

[0269] The terms "disorder," "disease," and "condition" may be used interchangeably and refer to a pathological problem with a body part, organ, or system that can be characterized by an identifiable group of signs or symptoms.

[0270] Therapeutic or preventive interventions according to the present disclosure may be effective to reduce the onset or progression of a disease / condition, relieve symptoms of a disease / condition, or reduce the pathology of a disease / condition. Interventions may be effective to prevent the progression of a disease / condition, e.g., to prevent the disease / condition from worsening, or to slow the rate of onset. In some embodiments, interventions may result in an improvement of a disease / condition, e.g., a reduction in symptoms of a disease / condition, or a reduction in some other correlate of the severity / activity of a disease / condition. In some embodiments, interventions may prevent the onset of a disease / condition to a later stage (e.g., a more severe stage, or a chronic stage).

[0271] The terms, e.g., "develop," "developing," and "development" of a disorder, as used herein, refer to both the onset of a disease and the progression, worsening, or deterioration of the disease state / its correlates.

[0272] The present disclosure relates to the treatment / prevention of diseases in which LINC complex dysfunction is pathologically related. Such diseases include, for example, nuclear envelope diseases (e.g., laminopathies). The therapeutic and prophylactic usefulness of the agents and methods of the present disclosure extends to the treatment and / or prevention of any disease that is believed to derive therapeutic / prophylactic benefit from LINC complex inhibition.

[0273] Aspects of the present disclosure relate to the treatment of diseases associated with a mutation in a given gene or genes. As used herein, a disease "associated" with a mutation in a given gene(s) is a disease caused or exacerbated by such a mutation, or a disease in which such a mutation is a risk factor for the onset or progression of the disease. In some embodiments, the mutation results in one or more of the following in cells that contain one or more copies of a mutant allele of a gene, compared to cells that contain two copies (i.e., homozygous) of a non-mutated (wild-type) reference allele of the gene: a reduced level of the gene product (e.g., RNA and / or protein (or a specific isoform thereof) of the wild-type allele; an increased level of the gene product of the non-wild-type allele; an increased level of the gene product of the wild-type allele.

[0274] The present disclosure relates to the treatment / prevention of nuclear envelope disease.Nuclear envelope disease is a disease / condition associated with mutations in genes that code for nuclear envelope proteins (i.e., proteins that are contained in or directly / indirectly associated with ONM, perinuclear space or INM).Nuclear envelope disease is, for example, reviewed in Chi et al., Journal of Biomedical Science (2009) 16:96, which is hereby incorporated by reference in its entirety.Nuclear envelope disease includes diseases / conditions associated with mutations in LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1 and NUP62.

[0275] Thus, in some embodiments, the disease / condition treated / prevented by the present disclosure is characterized by mutations in one or more of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1 and NUP62.

[0276] In particular, aspects of the present disclosure relate to the treatment / prevention of laminopathies.

[0277] Laminopathies are reviewed, for example, in Burke and Stewart, Nat Rev Mol Cell Biol. (2013) 14(1): 13-24, and Hah and Kim, Cells (2019) 8(3): 231, both of which are hereby incorporated by reference in their entirety. Laminopathies are generally associated with tissue-specific defects in load-bearing at the nuclear level, which may reduce the cellular resistance to physical forces. In the experimental examples herein, the inventors demonstrate that LINC complex inhibition alleviates symptoms of a range of laminopathies.

[0278] As used herein, a "laminopathy" is a disease / condition associated with mutations to genes encoding lamins.

[0279] Genes encoding lamins include LMNA (encoding lamins A and C), and LMNB1, LMNB2, encoding lamins B1 and B2. Accordingly, embodiments of the present disclosure relate to the treatment / prevention of diseases associated with mutations to LMNA, LMNB1 and / or LMNB2.

[0280] In some embodiments, the mutation is known or predicted to reduce levels of a lamin isoform encoded by a wild-type allele of a lamin-encoding gene (e.g., LMNA, LMNB1 or LMNB2). In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is known or predicted to generate a truncated version of a lamin encoded by a wild-type allele of a lamin-encoding gene. In some embodiments, the mutation is known or predicted to generate a lamin that is misfolded and / or degraded.

[0281] In some embodiments, the mutation is known or predicted to increase levels of a lamin isoform encoded by a wild-type allele of a gene encoding a lamin (e.g., LMNA, LMNB1 or LMNB2).

[0282] In some embodiments, the mutation is known or predicted to increase levels of a disease-associated lamin variant (e.g., progerin), hi some embodiments, the mutation is known or predicted to increase levels of a lamin encoded by a disease-associated allele of a gene encoding a lamin.

[0283] In some embodiments, the laminopathies are skeletal muscle laminopathies. In some embodiments, the laminopathies are myopathy. In some embodiments, the laminopathies are LMNA mutation-associated myopathy.

[0284] In some embodiments, the disease treated / prevented according to the present disclosure is characterized by one or more of myopathy, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, myocardial dystrophy, skeletal muscular dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy, or dermatosis.

[0285] In some embodiments, the disease treated / prevented in accordance with the present disclosure is characterized by one or more of muscular dystrophy, cardiac dystrophy, or skeletal muscular dystrophy.

[0286] In some embodiments, the laminopathies are associated with mutations to LMNA, LMNB1 and / or LMNB2. In some embodiments, the laminopathies are Hutchinson-Gilford Progeria Syndrome;Emery-Dreifuss Muscular Dystrophy;Emery-Dreifuss Muscular Dystrophy 2, Autosomal Dominant;Lipodystrophy, Partial, Acquired;Epilepsy, Progressive Myoclonus, 9;Charcot-Marie-Tooth Disease, Axonal, Type 2e;Muscular Dystrophy;Lipodystrophy, Familial Partial, Type 2;Cardiomyopathy, Dilated, 1h;Pelger-Huette Anomaly;Reynolds Syndrome;Muscular Disease;Leukodystrophy;Dilated Cardiomyopathy;Muscular Dystrophy. Strophy, congenital, Lmna-related;mandibular acrodysplasia with lipodystrophy type a;cardiomyopathy, dilated, 1a;restrictive dermatosis, fatal;familial partial lipodystrophy;epilepsy;lipoatrophy with diabetes mellitus, vitiligo-melanotic papules, fatty liver, and hypertrophic cardiomyopathy;leukodystrophy, demyelinating, adult-onset, autosomal dominant;acquired generalized lipodystrophy;Emery-Dreifuss muscular dystrophy 3, autosomal recessive;Charcot-Marie-Tooth disease;Charcot-Marie-Tooth disease, axonal, type 2b1;cardiomyopathy, dilated, 1b;atrial stasis 1;limb-girdle muscular dystrophy;cardiomyopathy, dilated, with hypergonadotropic hypogonadism;heart-upper limb syndrome, Slovenian type;monogenic diabetes;arrhythmogenic right ventricular cardiomyopathy;cardiomyopathy, dilated, 1e;aging;micrognathia, deafness, progeria-like symptoms, and lipodystrophy syndrome;adrenal muscular dystrophy;atypical Werner syndrome;endometriosis;spinocerebellar ataxia 31;progressive muscular atrophy;neurogenic bowel;autosomal dominant leukodystrophy with autonomic disease;Werner syndrome;myopathy;Lmna Associated dilated cardiomyopathy;muscular dystrophy, congenital, 1b;hypertrophic cardiomyopathy;left ventricular noncompaction;diabetes mellitus, non-insulin dependent;arrhythmogenic right ventricular dysplasia, familial, 9;heart disease;atrial fibrillation;cardiac conduction disorder;myoclonus;progressive myoclonic epilepsy;myoclonic epilepsy;peripheral nervous system disease;dental disease;atrioventricular block;myofibrillary myopathy;autosomal dominant limb-girdle muscular dystrophy;Lmna-associated cardiocutaneous progeria syndrome;amyotrophic lateral sclerosis 1;neural tube defects;cervical cancer;neural tube defects, folate sensitive;cerebral degeneration;melanotic dermatosis;3-Hydroxyacyl-Coa dehydrogenase deficiency;congenital fiber type inequality;acrosteolysis;Wolf-Parkinson-White syndrome;sick sinus syndrome;calcification;undifferentiated pleomorphic sarcoma;ventricular tachycardia, catecholamine-induced polymorphic, 1, with or without atrial dysfunction and / or dilated cardiomyopathy;lipodystrophy, familial partial, type 1;axonal neuropathy;paroxysmal ventricular fibrillation;Brugada syndrome 5;ankylosing spinal muscular dystrophy;limb-girdle muscular dystrophy type 1b;insulin-resistant acanthosis nigricans, type a;generalized lipodystrophy-associated progeria syndrome;osteoporosis;ankylosing spinal muscular dystrophy 1;neuropathy;catecholamine-induced polymorphic ventricular tachycardia;cataracts;Bethlem myopathy 1;congenital Generalized lipodystrophy;Restrictive cardiomyopathy;Muscular dystrophy, congenital merosin deficiency, 1a;Proximal spinal muscular atrophy;Muscular dystrophy-dystroglycanopathy, type B, 5;Lipodystrophy, congenital generalized, type 1;Emery-Dreifuss muscular dystrophy 1, X-linked;Cardiomyopathy, dilated, 1d;Myopathy, proximal, and ophthalmoplegia;Muscle tissue disease;Ovarian cystadenoma;Emelinopathy;Fanconi anemia, complementation group a;Body mass index quantitative trait locus 11;Myelodysplastic syndrome;Skin disease;Anorexia nervosa;Spinal muscular atrophy;Inclusion body myositis;Aniridia 1;Myositis;Hair hepatic enteropathy syndrome 1;Neuromuscular disease;Nutritional deficiencies;Thoracic outlet syndrome;Myopathy;Muscular atrophy;Harlerman-Streiff syndrome;Rere-Related disorders Disorders);Miller-Dieker Lissencephaly Syndrome;Lipodystrophy, congenital generalized, type 4;Lipodystrophy, familial partial, type 3;Widermann-Rautenstrauch syndrome;Lipodystrophy, congenital generalized, type 2;Ataxia Neuropathy Spectrum;Hair loss, neurologic deficit, and endocrine disorder syndrome;Lipodystrophy, familial partial, type 4;Second-degree atrioventricular block;Acute necrotizing encephalitis;Median neuropathy;Intrinsic cardiomyopathy;Familial isolated arrhythmogenic ventricular dysplasia, right dominant type;Prolapse of female genitalia;Familial isolated arrhythmogenic ventricular dysplasia, biventricular type;Familial isolated arrhythmogenic ventricular dysplasia, left dominant type;Complete generalized lipodystrophy;Blood type - Ahonen;Autosomal semi-dominant severe lipodystrophy laminopathies;Ulnar neuropathy;Pelvic muscle wasting;Alzheimer's disease;Stroke, ischemic;Ataxia telangiectasia;Spondyloarthropathy 1;Human immunodeficiency virus type 1;Neuroblastoma;Vascular disease;Nervous system disease;Respiratory failure;Turner syndrome;Carpal tunnel syndrome;Barrett's esophagus;Sleep apnea;Cerebrovascular disease;Proteasome-associated autoinflammatory syndrome 1;Joubert syndrome 1;Viral infections;Dementia;Personality disorders;Neuropathy, hereditary sensory and autonomic, type III;Lowe oculocerebrorenal syndrome Syndrome);Diabetes Mellitus;Fatty Liver Disease;Leigh Syndrome;Duchenne Muscular Dystrophy;Hydrocephalus;Dermatomyositis;Hirschsprung Disease 1;Long Qt Syndrome;Angelman Syndrome;Central Nervous System Disease;Congenital Disorders of Glycosylation, Type I;Alacrimation, Achalasia, and Mental Retardation Syndrome;Polycystic Ovary Syndrome;Hypoglycemia;Muscular Hypertrophy;Kerns-Sayre Syndrome;Cone-Rod Dystrophy 2;Aicardi-Goutieres Syndrome;Andersen Cardiodysrhythmic Periodic Paralysis Paralysis);Muscular dystrophy, Becker type;Legg-Calve-Perthes disease;Androgen insensitivity syndrome;Ehlers-Danlos syndrome;Axenfeld-Rieger syndrome;Muscular dystrophy-dystroglycanopathy, type C, 5;Glomerulonephritis;Seizure disorders;Chikungunya fever;West syndrome;Ullrich congenital muscular dystrophy 1;Focal segmental glomerulosclerosis;Walker-Warburg syndrome;Renal dysplasia / dysplasia 1 (Renal Hypodysplasia / aplasia1);Popliteal fold syndrome;Microcephaly;Childhood-type dermatomyositis;Distal arthrogryposis;Myocarditis;Arterial tortuosity syndrome;Scoliosis;Membranous nephropathy;Microvascular complications of type 3 diabetes;Epidermolysis bullosa;Short's syndrome;Hyperakonia;Nonalcoholic fatty liver disease;Muscular dystrophy-dystroglycanopathy, type a, 4;Congenital hydrocephalus;Ataxia, combined cerebellar and peripheral, with hearing loss and diabetes;Cardiac arrhythmias;Muscular dystrophy-dystroglycanopathy, type a, 1;Ptosis;Laryngitis;Ablepharon-Macrostomia Syndrome;Supravalvular aortic stenosis;Myopathy, congenital;Metabolic encephalopathy crisis, with relapses, rhabdomyolysis, cardiac arrhythmias and neurodegeneration;lisencephaly1;polycystic liver disease, with or without renal cysts1;idiopathic inflammatory myopathy;epidermolysis bullosa simplex;focal segmental glomerulosclerosis1;genital hypoplasia;gyral chorioretinal atrophy;syringomyelia;ichthyosis vulgaris;arthrogryposis, peripheral, type 1a;acute insulin response;brachydactyly;cerebellar hypoplasia;craniometaphyseal dysplasia, autosomal dominant;Alport syndrome 1, X-linked;lisencephaly;muscular dystrophy-dystroglycanopathy, type B, 6;diarrhoea 5;tufting enteropathy (Tufting enteropathy), congenital;junctional epidermolysis bullosa;Aicardi-Goutieres syndrome 1;Miyoshi muscular dystrophy;retinitis;Marden-Walker syndrome;neuroretinopathy;Polyglucosan body myopathy with or without immunodeficiency 1;Epidermolysis bullosa, junctional, Herlitz type;Macroglossia;Parkinson's disease 15, autosomal recessive early-onset;Myopathy, myofibrillar, 3;Microvascular complications of type 7 diabetes mellitus;Muscle-eye-brain disease;Melkersson-Rosenthal syndrome;Myopathy, X-linked, with excessive autophagy;Chrioretinitis;Muscular dystrophy, limb-girdle type, autosomal recessive 8;Crouzon syndrome with acanthosis nigricans;Muscular dystrophy, limb-girdle type 6, autosomal recessive;polymicrogyria;dystrophinopathy;microvascular complications of diabetes mellitus type 6;microvascular complications of diabetes mellitus type 4;hypotonia;pontocerebellar hypoplasia;congenital fibrosarcoma;intrauterine growth retardation, metaphyseal dysplasia, congenital adrenal hypoplasia, and genital anomalies;muscular dystrophy, limb-girdle type, autosomal recessive 7;myopathy, congenital, with fiber-type imbalance;hereditary amelogenesis imperfecta, type Ig;refractory anemia;fibrosis of extraocular muscles, congenital, 1;ataxia and polyneuropathy, adult-onset;Arlacado syndrome;senile cataract;muscular dystrophy-dystroglycanopathy, type C, 1;neuronal migration disorder;Ayme-Gripp syndrome Syndrome);Primary agammaglobulinemia;Autosomal recessive limb-girdle muscular dystrophy type 2a;Encephalitis;Muscular dystrophy, congenital, giant conoid;Autosomal recessive limb-girdle muscular dystrophy;Alkuraya-Kucinskas syndrome;Muscular dystrophy-dystroglycanopathy, type C, 4;Congenital muscular dystrophy type 1a;Behr syndrome;Dandee-Walker complex;Muscular dystrophy-dystroglycanopathy, type C; Emery-Dreifuss muscular dystrophy, X-linked; Muscular dystrophy, limb-girdle, autosomal recessive; Autosomal recessive limb-girdle muscular dystrophy type 2d; Cerebral small vessel disease with or without ocular abnormalities; Familial isolated dilated cardiomyopathy; Epithelial recurrent erosive dystrophy Dystrophy);Muscular dystrophy / dystroglycanopathy;Mycobacterium avium complex infection;Autosomal recessive limb-girdle muscular dystrophy type 2l;Visual epilepsy;Sinus of Valsalva aneurysm;Autosomal recessive limb-girdle muscular dystrophy type 2b;Creatine phosphokinase, elevated serum;Spastic paraplegia, ataxia, and mental retardation;Multinucleated neuron, anhydramnios, renal dysplasia, cerebellar hypoplasia, and hydranencephaly;Eustachian tube dysfunction;Autosomal inherited diseases;CK syndrome;Neuronitis;Hyperakicola 1;Reduced body myopathy;Polymicrogyria, bilateral temporooccipital;Isolated hyperckemia;Charcot-Marie-Tooth disease, axonal, type 2b2;Cardiac neuromuscular disease with hyaline masses and nemaline bodies;Congenital muscular dystrophy without intellectual disability;Blood type, I system (I System);Salih Myopathy;Adducted Thumbs Syndrome;Dural Sinus Malformation;Blood Type, Donbrock System;Blood Type, Colton System;Arthrochalasia Ehlers-Danlos Syndrome Syndrome);Lama2-related muscular dystrophy;Muscular dystrophy, congenital, with infantile cataracts and hypogonadism;Intrauterine infection;Muscular dystrophy, congenital, merosin positive;Congenital muscular dystrophy with cerebellar involvement;Fukuyama muscular dystrophy;Chronic lymphoproliferative disorder of natural killer cells;Congenital muscular dystrophy with intellectual disability;Amelogenesis imperfecta hypoplasia, Ig;Androgen insensitivity syndrome, mild type;Muscular dystrophy, congenital, resulting in arthrogryposis;Congenital muscular alpha-dystroglycanopathy with brain and eye abnormalities;Type VI collagen-related myopathy;Selected from: Emery-Dreifuss muscular dystrophy, dominant type; congenital muscular dystrophy with dystroglycanopathy; proximal myopathy with focal mitochondrial deficiency; infantile scoliosis;

[0287] In some embodiments, the laminopathies are laminopathies associated with mutations to LMNA. In some embodiments, the laminopathies are Hutchinson-Gilford progeria syndrome; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-associated; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; muscular dystrophy; mandibular acrodysplasia with lipodystrophy type a; cardiomyopathy, dilated, 1a; Charcot-Marie-Tooth disease; limb-girdle muscular dystrophy; cardiomyopathy, dilated, with hypergonadotropic hypogonadism; Emery-Dreifuss muscular dystrophy 3, autosomal recessive; lipodystrophy , familial partial, type 2;Emery-Dreifuss muscular dystrophy;Charcot-Marie-Tooth disease, axonal, type 2b1;heart-upper limb syndrome, Slovenian type;aging;familial partial lipodystrophy;restrictive cutis, fatal;arrhythmogenic right ventricular cardiomyopathy;dental disease;cardiac disease;Werner syndrome;hypertrophic cardiomyopathy;left ventricular noncompaction;atrioventricular block;calcification;acral osteolysis;autosomal dominant limb-girdle muscular dystrophy;diabetes mellitus, non-insulin dependent;osteoporosis;atrial fibrillation;atrial asystole 1;melanotic keratoderma;cardiac conduction disorder;catechol Amine-induced polymorphic ventricular tachycardia;Micrognathia, deafness, progeria-like symptoms, and lipodystrophy syndrome;Sick sinus syndrome;Pelger-Houette anomaly;Charcot-Marie-Tooth disease, axonal, type 2e;Congenital generalized lipodystrophy;Restrictive cardiomyopathy;Congenital fiber type disproportion;Lipodystrophy, congenital generalized, type 1;Myofibrillary myopathy;Lipodystrophy, familial partial, type 1;Axonal neuropathy;Atypical Werner syndrome;Ovarian cystadenoma;Fanconi anemia, complementation group a;Body mass index quantitative trait locus 11;Skin diseases;Ankylosing spinal muscular dystrophy 1;Neuromuscular diseases;Harlerman-Strife syndrome;Bethlem myopathy 1;Acquired generalized lipodystrophy;Cardiomyopathy, dilated, 1e;Lipodystrophy, congenital generalized, type 4;Undifferentiated pleomorphic sarcoma;Lipodystrophy, familial partial, type 3;Muscular dystrophy, congenital merosin deficiency, 1a;Proximal spinal muscular atrophy;Muscular dystrophy-dystroglycanopathy, type B, 5;Muscular dystrophy, congenital, 1b;Reynolds syndrome;Widamann-Rautenstrauch syndrome;Emery-Dreifuss muscular dystrophy 1, X-linked;Lipodystrophy, congenital generalized, type 2;Monogenic diabetes;Cardiomyopathy, dilated, 1d;Myopathy, proximal, and ophthalmoplegia;Muscle tissue disease;Lipodystrophy, familial partial, type 4;Cardiomyopathy, dilated, 1h;Second-degree atrioventricular block;Median neuropathy;Intrinsic cardiomyopathy;Prolapse of female genitalia;Complete generalized lipodystrophy;Ankylosing spinal muscular dystrophy;Emelinopathy;Ulnar neuropathy selected from;limb-girdle muscular dystrophy type 1b;Lmna-associated dilated cardiomyopathy;pelvic muscle wasting;generalized lipodystrophy-associated progeria syndrome;muscle disease;cardiomyopathy, dilated, 1b;autosomal genetic disease;familial isolated arrhythmogenic ventricular dysplasia, right dominant;familial isolated arrhythmogenic ventricular dysplasia, biventricular;familial isolated arrhythmogenic ventricular dysplasia, left dominant;Lmna-associated cardiocutaneous progeria syndrome;autosomal semidominant severe lipodystrophy laminopathies;

[0288] In some embodiments, the disease to be treated / prevented in accordance with the present disclosure is selected from diseases associated with the cDNA or protein variants shown in Table 1 (listed on next page).

[0289] In some embodiments, the disease to be treated / prevented in accordance with the present disclosure is selected from the diseases shown in Table 1.

[0290] In some embodiments, the disease to be treated / prevented in accordance with the present disclosure is selected from those diseases shown in regular font in Table 1.

[0291] In some embodiments, the disease to be treated / prevented in accordance with the present disclosure is selected from the diseases shown in bold font in Table 1.

[0292] [Table 1-1]

[0293] [Table 1-2]

[0294]

Table 1-3

[0295]

Table 1-4

[0296]

Table 1-5

[0297]

Table 1-6

[0298]

Table 1-7

[0299]

Table 1-8

[0300]

Table 1-9

[0301]

Table 1-10

[0302]

Table 1-11

[0303]

Table 1-12

[0304]

Table 1-13

[0305]

Table 1-14

[0306]

Table 1-15

[0307]

Table 1-16

[0308]

Table 1-17

[0309]

Table 1-18

[0310]

Table 1-19

[0311]

Table 1-20

[0312]

Table 1-21

[0313]

Table 1-22

[0314]

Table 1-23

[0315]

Table 1-24

[0316]

Table 1-25

[0317]

Table 1-26

[0318]

Table 1-27

[0319]

Table 1-28

[0320]

Table 1-29

[0321]

Table 1-30

[0322]

Table 1-31

[0323]

Table 1-32

[0324]

Table 1-33

[0325]

Table 1-34

[0326]

Table 1-35

[0327]

Table 1-36

[0328]

Table 1-37

[0329]

Table 1-38

[0330]

Table 1-39

[0331]

Table 1-40

[0332]

Table 1-41

[0333]

Table 1-42

[0334]

Table 1-43

[0335] [Table 1-44]

[0336] A further aspect of the present disclosure relates to the treatment / prevention of diseases characterized by dyslipidemia, such as diseases characterized by hyperlipidemia. LINC complex inhibition has recently been shown to be a suitable therapeutic / preventive intervention for dyslipidemia, particularly hyperlipidemia, such as hypercholesterolemia, see, for example, WO 2021 / 010898 A1, for example, Example 18.

[0337] A further aspect of the present disclosure relates to the treatment / prevention of diseases associated with LDL receptor deficiency (ie, reduced levels of LDL receptor protein and / or function).

[0338] In some embodiments, the disease / condition treated / prevented by the present disclosure is dyslipidemia and / or hypercholesterolemia. In some embodiments, the disease / condition is a disease / condition characterized by dyslipidemia and / or hypercholesterolemia. In some embodiments, the disease / condition is a disease / condition associated with dyslipidemia and / or hypercholesterolemia (e.g., a disease / condition in which dyslipidemia and / or hypercholesterolemia is a risk factor for the onset, development or progression of the disease / condition).

[0339] Dyslipidemia is defined as having too high or too low blood lipid levels. The present disclosure is particularly relevant to the treatment of hyperlipidemia when the levels of lipids or lipoproteins in the blood are elevated. Hyperlipidemia includes hypertriglyceridemia, hypercholesterolemia and combined hyperlipidemia (combination of hypertriglyceridemia and hypercholesterolemia). Hyperlipidemia is associated with, for example, atherosclerosis, hypertension and cardiovascular disease.

[0340] Hyperlipidemia refers to an elevated level of lipids or lipoproteins in the blood. Hyperlipidemia includes hypertriglyceridemia, hypercholesterolemia, and combined hyperlipidemia (combination of hypertriglyceridemia and hypercholesterolemia). Hyperlipidemia is associated with, for example, atherosclerosis, hypertension, and cardiovascular disease.

[0341] Hypercholesterolemia refers to high cholesterol levels in blood.In many cases, hypercholesterolemia is the result of high-fat diet and sedentary lifestyle in combination with genetic risk factors.Hypercholesterolemia can also occur as a result of genetic mutation (e.g., in the case of familial hypercholesterolemia), type 2 diabetes, hypothyroidism, kidney disease, or as a side effect of certain drugs, such as corticosteroid treatment.

[0342] Hypercholesterolemia is described, for example, in Bhatnagar et al., BMJ (2008) 337:a993. The UK NHS defines hypercholesterolemia as a blood total cholesterol level of ≧5 mmol / L or a blood low-density lipoprotein (LDL) level of ≧3 mmol / L. The US NIH defines hypercholesterolemia as a blood total cholesterol level of ≧240 mg / dL. Hypertriglyceridemia is described, for example, in Berglund et al., J. Clin. Endocrinol. Metab. (2012) 97(9):2969-89, and is defined by a blood triglyceride level of ≧150 mg / dL (≧1.7 mmol / L). Hypercholesterolemia is a well-recognized risk factor for cardiovascular disease, particularly cardiovascular disease resulting from atherosclerosis - see, e.g., Nelson Prim Care. (2013) Mar;40(1):195-211. Hypercholesterolemia has also been reported to result in steatosis and non-alcoholic liver disease - see, e.g., Arguello et al., Biochim Biophys Acta (2015) 1852(9):1765-78.

[0343] In some embodiments, the disease characterized by hyperlipidemia can be familial hyperlipidemia or acquired (secondary) hyperlipidemia.

[0344] In some embodiments, the familial hyperlipidemia is selected from Burger-Glitz syndrome, familial apoprotein CII deficiency, hyperlipoproteinemia type Ic, familial hypercholesterolemia, familial combined hyperlipidemia, familial dysbetalipoproteinemia, familial hypertriglyceridemia, and hyperlipoproteinemia type V. In some embodiments, the familial hyperlipidemia is familial hypercholesterolemia.

[0345] LDL receptor deficiency may result from, for example, mutation to LDLR.Accordingly, aspects of the present disclosure relate to the treatment / prevention of disease associated with mutation to LDLR.In some embodiments, the mutation is known or predicted to reduce the level of one or more LDL receptor isoforms encoded by wild-type LDLR alleles and / or increase the level of one or more disease-related LDL receptor variants.In some embodiments, the disease associated with mutation to LDLR is familial hypercholesterolemia.

[0346] In some embodiments, the disease treated / prevented according to the present disclosure is characterized by one or more of hyperlipidemia, hypercholesterolemia, atherosclerosis, stenosis, or hypertension. In some embodiments, the disease treated / prevented according to the present disclosure is characterized by atherosclerosis.

[0347] In some embodiments, the disease being treated / prevented is selected from atherosclerosis, cardiovascular disease, stroke and familial hyperlipidemia.

[0348] According to various aspects of the present disclosure, the method of treating and / or preventing a disease / condition according to the present disclosure comprises: Increasing survival of a subject with the disease; Increasing survival time in subjects with the disease; Increasing or inhibiting the decline of cardiac function; delaying the onset of cardiac decline; Increasing or inhibiting the decrease in myocardial contractility; Increasing or inhibiting the decline of ejection fraction and / or shortening fraction; Reducing or inhibiting an increase in left ventricular internal diameter; Increasing or inhibiting a decrease in left ventricular posterior wall thickness; and / or Reducing or Inhibiting the Development of Atherosclerosis may include one or more of:

[0349] According to various aspects of the present disclosure, methods are provided for or including (e.g., in the context of treating / preventing diseases / conditions described herein) one or more of: inhibiting the interaction between SUN domain-containing protein and KASH domain-containing protein; inhibiting the formation of LINC complexes comprising SUN domain-containing protein and KASH domain-containing protein; disrupting LINC complexes comprising SUN domain-containing protein and KASH domain-containing protein; disrupting the normal intracellular localization of SUN domain-containing protein or KASH domain-containing protein; increasing the localization of LINC complex component proteins (e.g., KASH domain-containing protein) to the endoplasmic reticulum; decreasing the level of LINC complexes comprising SUN domain-containing protein and KASH domain-containing protein; and / or inhibiting the function / activity of LINC complexes comprising SUN domain-containing protein and KASH domain-containing protein. Also provided are agents according to the present disclosure for use in such methods, and the use of agents according to the present disclosure in the manufacture of pharmaceutical compositions or medicaments for use in such methods. It is understood that the methods typically include administering to a subject a LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein.

[0350] Similarly, following therapeutic or prophylactic intervention according to the present disclosure (e.g., compared to pre-intervention levels), one or more of the following are observed in the subject: inhibition of interaction between SUN domain-containing proteins and KASH domain-containing proteins; inhibition of formation of LINC complexes comprising SUN domain-containing proteins and KASH domain-containing proteins; disruption of LINC complexes comprising SUN domain-containing proteins and KASH domain-containing proteins; disruption of normal intracellular localization of SUN domain-containing proteins or KASH domain-containing proteins; increased localization of component proteins of LINC complexes (e.g., KASH domain-containing proteins) to the endoplasmic reticulum; reduced levels of LINC complexes comprising SUN domain-containing proteins and KASH domain-containing proteins; and / or inhibition of function / activity of LINC complexes comprising SUN domain-containing proteins and ASH domain-containing proteins.

[0351] In some embodiments, therapeutic / prophylactic interventions according to the present disclosure may be described as "associated with" one or more of the effects described in the preceding paragraph. Those skilled in the art can readily assess such characteristics using techniques routinely practiced in the art.

[0352] Administration of the LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions of the present disclosure is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to exhibit a therapeutic or prophylactic effect on the subject. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease / condition, and the specific item administered. Prescription of treatment, such as determining dosage, is within the responsibility of general practitioners and other physicians, and typically takes into account the disease / disorder 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 techniques and protocols mentioned above can be found in Remington's 'The Science and Practice of Pharmacy' (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0353] In a preferred embodiment, the administration is of a nucleic acid / vector according to the present disclosure or of a composition comprising a nucleic acid / vector. In a preferred embodiment, the administration results in the modification of one or more cells that contain / express a nucleic acid / vector according to the present disclosure and / or that contain / express a LINC complex inhibitory polypeptide.

[0354] Administration of the articles of the present disclosure can be, for example, parenteral, systemic, topical, intracavitary, intravascular, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral or transdermal. Administration can be by injection, infusion or oral ingestion.

[0355] In some aspects and embodiments, the articles of the present disclosure may be administered to a tissue / organ of interest (e.g., a tissue / organ affected by a disease / condition that is affected by the condition (e.g., a tissue / organ where symptoms of the disease / condition are manifested). In some aspects and embodiments, the articles of the present disclosure may be administered into the blood (i.e., intravenously / intraarterially) by injection or infusion (e.g., through a cannula), or may be administered subcutaneously or orally.

[0356] In some aspects and embodiments according to the present disclosure, there may be targeted delivery of the article of the present disclosure, i.e., the concentration of the relevant agent in the subject is increased in a given tissue(s) / organ(s) compared to other parts of the body. In some embodiments, the method includes intravascular (e.g., intravenous or intraarterial), intramuscular or subcutaneous administration, and the relevant article is formulated into a targeted drug delivery system (e.g., as described above).

[0357] The particular mode and / or site of administration may be selected according to where LINC complex inhibition is required, for example, cardiac and / or skeletal muscle cells / tissues.

[0358] In some embodiments, therapeutic or prophylactic intervention according to the present disclosure may further comprise administering another agent for the treatment / prevention of an associated disease / condition.

[0359] The administration of the LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions described herein can be either simultaneously or sequentially, alone or in combination with other treatments, depending on the condition to be treated. Simultaneous administration refers to administration in conjunction with another therapeutic agent, for example as a pharmaceutical composition (combined preparation) containing both agents, or immediately after each other and optionally through the same route of administration (e.g., into the same tissue, artery, vein or other blood vessel). Sequential administration refers to the administration of one agent followed by another administration of another agent after a given time interval. Although in some embodiments this is the case, it is not necessary that the two agents are administered by the same route. The time interval can be any time interval.

[0360] Multiple doses of the LINC complex inhibitory polypeptides, nucleic acids, vectors, cells and compositions of the disclosure may be provided. One or more of the doses, or each, may be accompanied by simultaneous or sequential administration of another therapeutic agent.

[0361] The multiple doses may be separated by a predetermined time interval, which may be selected to be one of 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. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 day).

[0362] In some embodiments, the method according to the present disclosure may include determining whether the subject has a disease as described herein. In some embodiments, the method includes diagnosing the disease as described herein. Determining whether the subject has a disease as described herein may include analyzing the subject for one or more symptoms / correlates of the disease.

[0363] In some embodiments, a subject may be suspected of having or suffering from a disease, for example, based on the presence of other symptoms indicative of the disease in the subject or in the subject's cells / tissues / organs. In some embodiments, a subject may be considered to be at risk for developing a disease, for example, due to a genetic predisposition or other risk factors for the disease.

[0364] In some embodiments, the method includes determining whether the subject has a mutation in a gene described herein. In some embodiments, the method includes detecting a mutation in a gene described herein.

[0365] Determining a mutation in a gene described herein may confirm a diagnosis or suspected diagnosis, or may confirm that a subject is at risk for developing a disease. The determination may diagnose a disease, or a predisposition to a disease, for treatment / prevention with a LINC complex inhibitor polypeptide, nucleic acid, vector, cell, or composition described herein.

[0366] Genetic factors can be assayed by methods known to those skilled in the art, including PCR-based and sequencing assays.For example, by determining the presence of genetic factors in the sample obtained from the subject, diagnosis can be confirmed, and / or the subject can be classified as being at risk of developing the disease described herein, and / or the subject can be determined to be suitable for treatment with the LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein.

[0367] The assay may be performed in vitro on a sample obtained from a subject or following processing of the sample obtained from a subject. The sample obtained from a subject may be of any type. The biological sample may be taken from any tissue or bodily fluid, for example, a blood sample, a blood-derived sample, a serum sample, a lymph sample, a semen sample, a saliva sample, a synovial fluid sample. The blood-derived sample may be a selected fraction of a patient's blood, for example, a selected cell-containing fraction or a plasma or serum fraction. The sample may include a tissue sample or biopsy; or cells isolated from a subject.

[0368] In some embodiments, the method comprises determining whether the subject comprises a mutation in one or more alleles of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1 and NUP62. In some embodiments, the method comprises determining whether the subject comprises a mutation in one or more alleles of LMNA, LMNB1 and LMNB2. In some embodiments, the method comprises determining whether the subject comprises a mutation in an allele of LMNA. In some embodiments, the method comprises determining whether the subject comprises a mutation in an allele of LDLR.

[0369] In such an embodiment, if a mutation is detected, the subject may be identified as a subject to be administered with a LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein. Thus, in some embodiments, the method comprises selecting a subject determined to contain a mutation in one or more of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1 and NUP62 for administration of a LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein. In some embodiments, the method comprises selecting a subject determined to contain a mutation in one or more of LMNA, LMNB1 and LMNB2 for administration of a LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein. In some embodiments, the method comprises selecting a subject determined to contain a mutation in LMNA for administration of a LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein. In some embodiments, the method comprises selecting a subject determined to contain a mutation in LDLR for administration of a LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein.

[0370] In some embodiments, the methods include testing a sample obtained from a subject suspected of having the disease for the presence or absence of at least one LMNA mutation, where the presence of at least one LMNA mutation indicates that the subject should be administered a LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein.

[0371] subject The subject according to various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in humans or animals (veterinary use). The subject treated with the LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein may be a subject in need thereof. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient.

[0372] A subject may have been diagnosed with a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / suffering such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to a method based on a signature for certain markers of such a disease / condition.

[0373] In some embodiments, the subject comprises a mutation according to any of the preceding embodiments. In some embodiments, the subject comprises a mutation that results in a disease / condition described herein (e.g., a laminopathic disorder).

[0374] kit In some aspects of the present disclosure, a kit of parts is provided. In some embodiments, the kit may comprise at least one container comprising a predetermined amount of the LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition described herein.

[0375] In some embodiments, the kits may include materials for producing a LINC complex inhibitory polypeptide, nucleic acid, vector, cell, or composition described herein.

[0376] The kit may be provided with instructions for administering the LINC complex inhibitory polypeptide, nucleic acid, vector, cell or composition to a patient to treat a particular disease / condition.

[0377] In some embodiments, the kit may further comprise at least one container containing a predetermined amount of another therapeutic agent. In such embodiments, the kit may also comprise a second medicament or pharmaceutical composition, so that the two medicaments or pharmaceutical compositions can be administered simultaneously or separately to provide a combination treatment for a particular disease or condition.

[0378] Kits according to the present disclosure may include instructions for use, for example in the form of instructions or pamphlets. The instructions may include protocols for carrying out any one or more of the methods described herein.

[0379] Sequence identity Pairwise and multiple sequence alignment for the purpose of determining the percent identity between two or more amino acid or nucleic acid sequences can be accomplished in a variety of ways known to those skilled in the art, for example using publicly available computer software such as ClustalOmega (Soding, 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) software. When using such software, for example, default parameters of gap penalties and extension penalties are preferably used.

[0380] [Table 2-1]

[0381] [Table 2-2]

[0382]

Table 2-3

[0383]

Table 2-4

[0384]

Table 2-5

[0385]

Table 2-6

[0386]

Table 2-7

[0387]

Table 2-8

[0388]

Table 2-9

[0389]

Table 2-10

[0390]

Table 2-11

[0391]

Table 2-12

[0392] [Table 2-13]

[0393] [Table 2-14]

[0394] Numbered paragraphs The following numbered paragraphs (para) provide further description of the properties and property combinations contemplated in connection with the present invention: 1. A nucleic acid encoding a LINC complex inhibitory polypeptide, the LINC complex inhibitory polypeptide comprising (i) an inhibitory region comprising an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein, and (ii) an endoplasmic reticulum retention motif; A LINC complex inhibitory polypeptide is a nucleic acid that does not include the amino acid sequence of any one of SEQ ID NOs: 43, 45 or 58.

[0395] 2. The nucleic acid of paragraph 1, wherein the inhibitory region of the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of SEQ ID NO: 44 or 46.

[0396] 3. The nucleic acid of paragraph 1 or paragraph 2, wherein the inhibitory region of the LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of any one of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61 or 62.

[0397] 4. The nucleic acid of any one of paragraphs 1 to 3, wherein the inhibitory region of the LINC complex inhibitory polypeptide consists essentially of an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of any one of SEQ ID NOs: 63, 64, 65, 66, 67, 68 or 94.

[0398] 5. The nucleic acid of any one of paragraphs 1 to 4, wherein the LINC complex inhibitory polypeptide comprises or consists essentially of an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence of any one of SEQ ID NOs: 69, 70, 71, 72, 73, 74 or 95.

[0399] 6. The nucleic acid of any one of paragraphs 1 to 5, wherein the LINC complex inhibitory polypeptide comprises a signal peptide.

[0400] 7. The nucleic acid of any one of paragraphs 1 to 6, wherein the nucleic acid is a vector suitable for delivering the nucleic acid encoding the LINC complex inhibitory polypeptide as gene therapy.

[0401] 8. The nucleic acid of paragraph 7, wherein the vector is an adeno-associated virus (AAV) vector.

[0402] 9. A LINC complex inhibitory polypeptide comprising: (i) an inhibitory region comprising an amino acid sequence corresponding to the α3 helix and SUN domain of the CC2 region of a SUN domain-containing protein; and (ii) an endoplasmic reticulum retention motif, A LINC complex inhibitory polypeptide that does not comprise the amino acid sequence of SEQ ID NO: 43 or 45.

[0403] 10. The LINC complex inhibitory polypeptide of paragraph 9, wherein the inhibitory region of the LINC complex inhibitory polypeptide does not consist essentially of the amino acid sequence of SEQ ID NO: 44 or 46.

[0404] 11. The LINC complex inhibitory polypeptide of paragraph 9 or paragraph 10, wherein the inhibitory region of the LINC complex inhibitory polypeptide does not essentially consist of the amino acid sequence of any one of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62.

[0405] 12. The LINC complex inhibitory polypeptide of any one of paragraphs 9 to 11, wherein the inhibitory region of the LINC complex inhibitory polypeptide consists essentially of an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of any one of SEQ ID NOs: 63, 64, 65, 66, 67, 68 or 94.

[0406] 13. The LINC complex inhibitory polypeptide of any one of paragraphs 9 to 12, wherein the LINC complex inhibitory polypeptide comprises, or essentially consists of, an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of any one of SEQ ID NOs: 69, 70, 71, 72, 73, 74 or 95.

[0407] 14. The LINC complex inhibitory polypeptide of any one of paragraphs 9 to 13, wherein the LINC complex inhibitory polypeptide comprises a signal peptide.

[0408] 15. A method for identifying a LINC complex inhibitory polypeptide, comprising: introducing a nucleic acid encoding a candidate LINC complex inhibitory polypeptide, the candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to a SUN domain of a SUN domain-containing protein; and Then, the cells are analyzed to determine the subcellular localization of the interaction partner for the SUN domain-containing protein. wherein the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if a change in the subcellular localization of the interaction partner relative to the SUN domain-containing protein is detected.

[0409] 16. A method for identifying a LINC complex inhibitory polypeptide, comprising: introducing a nucleic acid encoding a candidate LINC complex inhibitory polypeptide, the candidate LINC complex inhibitory polypeptide comprising an amino acid sequence corresponding to a KASH domain of a KASH domain-containing protein; and Then, the cells are analyzed to determine the subcellular localization of the interacting partner for the KASH domain-containing protein. wherein the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if a change in the subcellular localization of the interaction partner relative to the KASH domain-containing protein is detected.

[0410] 17. The method of paragraph 15 or paragraph 16, wherein the candidate LINC complex inhibitory polypeptide is determined to be a LINC complex inhibitory polypeptide if an increase in the proportion of the interaction partner for the SUN / KASH domain-containing protein localized to the endoplasmic reticulum is detected.

[0411] 18. A LINC complex inhibitory polypeptide identified by the method of any one of paragraphs 15 to 17.

[0412] 19. A nucleic acid encoding a LINC complex inhibitory polypeptide of any one of paragraphs 9 to 14 or paragraph 18.

[0413] 20. A vector comprising the nucleic acid of paragraph 19, which is a vector suitable for delivering a nucleic acid encoding a LINC complex inhibitory polypeptide as a gene therapy.

[0414] 21. The vector of paragraph 20, which is an adeno-associated virus (AAV) vector.

[0415] 22. A cell comprising a nucleic acid according to any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide according to any one of paragraphs 9 to 14 or paragraph 18, or a vector according to paragraph 20 or paragraph 21.

[0416] 23. A pharmaceutical composition comprising a nucleic acid according to any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide according to any one of paragraphs 9 to 14 or paragraph 18, a vector according to paragraph 20 or paragraph 21, or a cell according to paragraph 22.

[0417] 24. A nucleic acid according to any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide according to any one of paragraphs 9 to 14 or paragraph 18, a vector according to paragraph 20 or paragraph 21, a cell according to paragraph 22 or a pharmaceutical composition according to paragraph 23 for use in a method of medical treatment and prophylaxis.

[0418] 25. A nucleic acid according to any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide according to any one of paragraphs 9 to 14 or paragraph 18, a vector according to paragraph 20 or paragraph 21, a cell according to paragraph 22 or a pharmaceutical composition according to paragraph 23 for use in a method for treating or preventing a laminopathies.

[0419] 26. Use of a nucleic acid according to any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide according to any one of paragraphs 9 to 14 or paragraph 18, a vector according to paragraph 20 or paragraph 21, a cell according to paragraph 22 or a pharmaceutical composition according to paragraph 23 in the manufacture of a medicament for treating or preventing a laminopathies.

[0420] 27. A method for treating or preventing a laminopathies, comprising administering to a subject a therapeutically or prophylactically effective amount of the nucleic acid of any one of paragraphs 1 to 8 or paragraph 19, the LINC complex inhibitory polypeptide of any one of paragraphs 9 to 14 or paragraph 18, the vector of paragraph 20 or paragraph 21, the cell of paragraph 22 or the pharmaceutical composition of paragraph 23.

[0421] 28. The nucleic acid, the LINC complex inhibitory polypeptide, the vector, the cell or the pharmaceutical composition for use of paragraph 25, the use of paragraph 26 or the method of paragraph 27, wherein the laminopathies are characterized by one or more of myopathies, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, myocardial dystrophy, skeletal muscular dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy or dermatosis.

[0422] 29. The nucleic acid for use, LINC complex inhibitory polypeptide, vector, cell or pharmaceutical composition, use or method according to any one of paragraphs 25 to 28, wherein the laminopathies are associated with mutations to LMNA.

[0423] 30. Laminopathies include: Hutchinson-Gilford progeria syndrome; Dilated cardiomyopathy; Muscular dystrophy, congenital, Lmna-related; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; Muscular dystrophy; Mandibular acrodysplasia with lipodystrophy type a; Cardiomyopathy, dilated, 1a; Charcot-Marie-Tooth disease; Limb-girdle muscular dystrophy; Cardiomyopathy, dilated, with hypergonadotropic hypogonadism; Emery-Dreifuss muscular dystrophy 3, autosomal recessive; Lipodystrophy, familial partial, type 2; Emery-Dreifuss Muscular dystrophy;Charcot-Marie-Tooth disease, axonal, type 2b1;Heart-upper limb syndrome, Slovenian type;Aging;Familial partial lipodystrophy;Restrictive cutis, fatal;Arrhythmogenic right ventricular cardiomyopathy;Dental disease;Heart disease;Werner syndrome;Hypertrophic cardiomyopathy;Left ventricular noncompaction;Atrioventricular block;Calcification;Acroosteolysis;Autosomal dominant limb-girdle muscular dystrophy;Diabetes mellitus, non-insulin dependent;Osteoporosis;Atrial fibrillation;Atrial asystole 1;Melanotic dermatosis;Cardiac conduction disorder;Catecholamine-induced polymorphic ventricular tachycardia;Micrognathia, hearing loss, progeria-like symptoms, and lipo Dystrophic syndromes;Sick sinus syndrome;Pelger-Huette anomaly;Charcot-Marie-Tooth disease, axonal, type 2e;Congenital generalized lipodystrophy;Restrictive cardiomyopathy;Congenital fiber type disproportion;Lipodystrophy, congenital generalized, type 1;Myofibrillary myopathy;Lipodystrophy, familial partial, type 1;Axonal neuropathy;Atypical Werner syndrome;Ovarian cystadenoma;Fanconi anemia, complementation group a;Body mass index quantitative trait locus 11;Skin disorders;Ankylosing spinal dystrophy 1;Neuromuscular disorders;Harlerman-Strife syndrome;Bethlem myopathy muscular dystrophy 1;acquired generalized lipodystrophy;cardiomyopathy, dilated, 1e;lipodystrophy, congenital generalized, type 4;undifferentiated pleomorphic sarcoma;lipodystrophy, familial partial, type 3;muscular dystrophy, congenital merosin deficiency, 1a;proximal spinal muscular atrophy;muscular dystrophy-dystroglycanopathy, type B, 5;muscular dystrophy, congenital, 1b;Reynolds syndrome;Widermann-Rautenstrauch syndrome;Emery-Dreifuss muscular dystrophy 1, X-linked;lipodystrophy, congenital generalized, type 2;monogenic diabetes mellitus;cardiomyopathy, dilated, 1d;Myopathy, proximal, and ophthalmoplegia;Muscle tissue diseases;Lipodystrophy, familial partial, type 4;Cardiomyopathy, dilated, 1h;Second-degree atrioventricular block;Median neuropathy;Intrinsic cardiomyopathy;Prolapse of female genitalia;Complete generalized lipodystrophy;Ankylosing spinal muscular dystrophy;Emelinopathy;Ulnar neuropathy;Limb-girdle muscular dystrophy type 1b;Lmna-associated dilated cardiomyopathy;Pelvic muscle wasting;Generalized lipodystrophy-associated progeria syndrome;Muscular diseases;Cardiomyopathy, dilated, 1 b; the nucleic acid, LINC complex inhibitory polypeptide, vector, cell or pharmaceutical composition, use or method for use according to any one of paragraphs 25 to 29, selected from autosomal genetic diseases; familial isolated arrhythmogenic ventricular dysplasia, right dominant; familial isolated arrhythmogenic ventricular dysplasia, biventricular; familial isolated arrhythmogenic ventricular dysplasia, left dominant; Lmna-associated cardiocutaneous progeria syndrome; and autosomal semi-dominant severe lipodystrophy laminopathies;

[0424] 31. A nucleic acid according to any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide according to any one of paragraphs 9 to 14 or paragraph 18, a vector according to paragraph 20 or paragraph 21, a cell according to paragraph 22 or a pharmaceutical composition according to paragraph 23 for use in a method for treating or preventing a disease characterized by hyperlipidemia.

[0425] 32. Use of a nucleic acid according to any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide according to any one of paragraphs 9 to 14 or paragraph 18, a vector according to paragraph 20 or paragraph 21, a cell according to paragraph 22 or a pharmaceutical composition according to paragraph 23 in the manufacture of a medicament for treating or preventing a disease characterized by hyperlipidemia.

[0426] 33. A method for treating or preventing a disease characterized by hyperlipidemia, comprising administering to a subject a therapeutically or prophylactically effective amount of a nucleic acid of any one of paragraphs 1 to 8 or paragraph 19, a LINC complex inhibitory polypeptide of any one of paragraphs 9 to 14 or paragraph 18, a vector of paragraph 20 or paragraph 21, a cell of paragraph 22 or a pharmaceutical composition of paragraph 23.

[0427] 34. The nucleic acid, LINC complex inhibitory polypeptide, vector, cell or pharmaceutical composition for use according to paragraph 31, the use according to paragraph 32 or the method according to paragraph 33, wherein the disease characterized by hyperlipidemia is selected from atherosclerosis, cardiovascular disease, stroke and familial hyperlipidemia.

[0428] The present invention includes combinations of the described embodiments and preferred features except where such combinations are expressly not permitted or expressly avoided.

[0429] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0430] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0431] Throughout this specification, including the appended claims, unless the context requires otherwise, the word "comprises", as well as variations such as "comprises (singular)", "comprising", etc., will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0432] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0433] When a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0434] The methods described herein can be carried out in vitro or in vivo. In some embodiments, the methods described herein can be carried out in vitro. The term "in vitro" is intended to include experiments using cells in culture, whereas the term "in vivo" is intended to include experiments using intact multicellular organisms.

[0435] BRIEF DESCRIPTION OF THE DRAWINGS BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention are now discussed with reference to the accompanying figures.

[0436] Figure 1. Alignment of the amino acid sequences of mouse Sun1, mouse Sun2, human SUN1 and human SUN2. The amino acid sequences starting six amino acids upstream of the start of the luminal domain were aligned using ClustalOmega software. Residues and domains of interest are indicated.

[0437] Figure 2. Schematic diagram of human SUN1 protein and various constructs (i.e., constructs (A) through (H)) characterized in Example 1. The domains of the luminal domain of human SUN1 included in the constructs are indicated.

[0438] Figure 3. Schematic and micrographs showing the results of an analysis of the ability of various putative dominant-negative SUN1 constructs to achieve LINC complex disruption as determined by increased Nesprin-2 localization to the endoplasmic reticulum. Arrows in the micrographs indicate cells expressing high levels of the relevant construct. It is indicated whether the construct was determined to result in Nesprin-2 localization.

[0439] Figure 4. Schematic diagram of human SUN1 protein and various constructs characterized in Example 5 (i.e., constructs A, D, D2, E and G). The domains of the luminal domain of human SUN1 included in the constructs are indicated. The length of the SUN1-derived sequence + KDEL is indicated.

[0440] 5A to 5D. Graphs showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. Briefly, Lmna FF:mcm Mice were injected with tamoxifen (TMX) to induce deletion of Lmna. Seventeen days after treatment with TMX, mice were injected with a single dose (100 ng / mL) of AAV9 encoding DNhSUN1_201aa ("201aa"; construct G in FIG. 4), DNhSUN1_255aa ("255aa"; construct E in FIG. 4), DNhSUN1_334aa ("334aa"; construct D2 in FIG. 4) or DNhSUN1_457aa ("457aa"; construct A in FIG. 4) under the control of the cTNT promoter. * 10 14 As a control condition, wild-type mice (WT) or Lmna mice were administered TMX. FF:mcm Mice (DCM) were injected with a single dose (1 * 10 14 Vaccination was performed at 10-20°C for 12-24 h. Survival was monitored and cardiac function was assessed by echocardiography, and left ventricular internal diameter (LVID) and left ventricular posterior wall (LVPW) thickness in diastole (d) and systole (s) were assessed by ultrasound. (5A) Percent survival over time for mice in the various treatment groups. N=6 for each group. *** Long-rank (Mantel-Cox) P=0.0005, pairwise comparison versus DCM controls. (5B) Fractional shortening (FS) and ejection fraction (EF) over time are shown for mice in different treatment groups. **Mann-Whitney P<0.0087, pairwise comparison vs. DCM controls. Data are presented as mean ± SD. (5C and 5D) Left ventricular internal diameter (LVID; 5C) and left ventricular posterior wall (LVPW) thickness (5D) over time in diastole (d) and systole (s), respectively. Data are presented as mean ± SD.

[0441] 6A to 6D. Graphs showing the results of an analysis of the therapeutic efficacy of various putative dominant-negative SUN1 constructs in a mouse model of dilated cardiomyopathy. Briefly, the Lmna construct harboring a cardiac-specific Cre FF:mcm Mice were injected with tamoxifen (TMX) to induce deletion of Lmna. 17 days after treatment with TMX, mice were injected with a single dose (1 s) of AAV9 encoding DNhSUN1_322aa ("322aa"; construct D in Figure 4) or DNhSUN1_457aa ("457aa"; construct A in Figure 4) under the control of the cTNT promoter. * 10 14 As a control condition, wild-type mice (WT) or Lmna mice were administered TMX. FF:mcm Mice (DCM) were injected with a single dose (1 * 10 14 vg / kg). Survival was monitored and cardiac function was assessed by echocardiography, with left ventricular internal diameter (LVID) and left ventricular posterior wall (LVPW) thickness in diastole (d) and systole (s) assessed by ultrasound. (6A) Percentage survival over time for mice in different treatment groups. 322aa group N=6, 457aa group N=6, DCM group N=5, WT group N=7. Log-rank (Mantel-Cox) P<0.0007, pairwise comparison against DCM control. Data are presented as mean ± SD. (6B) Fractional shortening (FS) and ejection fraction (EF) over time for mice in different treatment groups. **Mann-Whitney P<0.0095, pairwise comparison vs. DCM controls. Data are presented as mean ± SD. (6C and 6D) Left ventricular internal diameter (LVID; 6C) and left ventricular posterior wall (LVPW) thickness (6D) over time in diastole (d) and systole (s), respectively. Data are presented as mean ± SD. EXAMPLES

[0442] Example 1: In vitro analysis of LINC complex inhibition by various dominant-negative SUN1 constructs 1.1 Materials and Methods 1.1.1 Bioinformatics The amino acid sequences of SUN domain-containing proteins and their orthologues were obtained from NCBI and Uniprot. Sequence alignment was performed using ClustalOmega (https: / / www.ebi.ac.uk / Tools / msa / clustalo). Domain analysis was performed using SMART (http: / / smart.embl-heidelberg.de / ).

[0443] 1.1.2 Molecular Biology DNA sequences encoding the full-length DNSUN1 coding sequence and its truncated variants were synthesized and cloned into pTwist EF1 alpha plasmid. The nucleic acid encoding the DNSUN1 construct was composed of an N-terminal human serum albumin signal sequence followed by an HA tag and an ALFA tag, followed by one of a variety of different truncated luminal SUN1 domain sequences followed by a C-terminal KDEL sequence. The amino acid sequences of the dominant negative human SUN1 (DNhSUN1) constructs characterized in the examples of the present disclosure are shown in SEQ ID NOs: 80 to 87 and 96, and their sequence characteristics are shown diagrammatically in Figures 1, 2 and 4.

[0444] [Table 3]

[0445] 1.1.3 Cell culture and transfection HeLa cells were cultured at 37°C, 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / L D-glucose, 4 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, and 100 U / ml penicillin; 100 mg / ml streptomycin. For immunofluorescence studies, cells were seeded onto coverslips or 8-well slides (ibidi, catalog no. 80826). Each well of an 8-well slide was transfected with 200 ng of plasmid DNA encoding a putative dominant-negative SUN1 construct by addition of preformed transfection complexes containing 0.3 ml Lipofectamine 3000 (Invitrogen) and 0.2 ml P3000 reagent (Invitrogen) per 100 ng of plasmid DNA in Opti-MEM medium (Invitrogen) for 15 min.

[0446] 1.1.4 Immunofluorescence microscopy The day after transfection, HeLa cells were fixed in cold methanol at −20°C for 10 min, permeabilized, and blocked for 30 min in blocking buffer containing 3% bovine serum albumin and 0.5% Trixon X-100 in phosphate-buffered saline (PBS).

[0447] To detect related DNSUN1 constructs, cells were stained with anti-Nesprin-2 mouse monoclonal antibody, anti-ALFA antibody (FluoTag®-X2 anti-ALFA conjugated to Abberior Star580, NanoTag Biotechnologies N1502-Ab580-L), and for nuclear envelope with mouse monoclonal anti-lamin B2 antibody. Cells were incubated with primary antibodies in blocking buffer for 1-2 hours at room temperature. Following primary antibody incubation, cells were washed three times with 0.5% Triton X-100 in PBS and then incubated for 30 min to 1 h at room temperature with fluorophore-conjugated mouse antibody isotype-specific secondary antibodies (Thermo Fisher Scientific A-21121, goat anti-mouse IgG1 conjugated to Alexa Fluor 488 and A-21242, goat anti-mouse IgG2b conjugated to Alexa Fluor 647) and Hoechst to stain DNA. Cells were washed again in 10% PBS / 90% glycerin before mounting in Prolong Diamond or 1% DABCO (1,4-diazabicyclo[2.2.2]octane). Wide-field epifluorescence microscopy imaging was performed on an Olympus IX-83 with a motorized stage equipped with a 20x0.45 NA LUCPlan FL N or 40x0.6 NA Ph2 LUCPlan FL N objective. Digital images were captured using CellSens software and processed using Fiji software.

[0448] 1.2 Results To determine which regions of SUN1 are necessary for the protein to function as a dominant-negative SUN1 protein to achieve LINC complex inhibition, we assessed the ability of various truncated variants of SUN1 containing the C-terminal KDEL sequence. Specifically, we investigated the contribution of two coiled-coil regions identified by SMART domain analysis, such as the CC1 and CC2 domains described in Xu et al., 2018, as well as the involvement of the three alpha helices of the CC2 domain.

[0449] An alignment of the luminal regions of human SUN1, human SUN1, mouse Sun1 and mouse Sun2 is shown in FIG.

[0450] The various truncated variants investigated in this example are shown diagrammatically in FIG.

[0451] The ability of various truncated SUN1 constructs to function as dominant-negative LINC complex inhibitors was evaluated by transiently expressing them in cells and then assessing the subcellular localization of the KASH domain-containing protein Nesprin-2. At low levels of expression, DNSUN1 localizes to the nuclear envelope at the nuclear cisternae. When expressed at high levels, DNSUN1 localizes throughout the endoplasmic reticulum lumen, the same membrane compartment as the nuclear cisternae. See Crisp et al., 2006.

[0452] In cells expressing high levels of dominant-negative SUN domain-containing LINC complex inhibitory proteins, the normal nuclear envelope localization of KASH domain-containing proteins is disrupted, and the KASH domain-containing proteins localized to the endoplasmic reticulum as a large excess of dominant-negative proteins outcompete endogenous SUN domain-containing proteins for interactions with KASH domain-containing proteins, which can be readily visualized using immunofluorescence microscopy with antibodies specific for the KASH domain-containing proteins.

[0453] In this study, plasmids encoding various truncated SUN1 constructs were expressed in HeLa cells by transient transfection, and the cells were subsequently analyzed by immunofluorescence staining for DNSUN1 and Nesprin-2, a KASH domain-containing protein highly expressed in HeLa cells.

[0454] The results are shown in Figure 3. As expected, full-length DNSUN1 (i.e., a protein having the amino acid sequence of SEQ ID NO: 80; DNhSUN1_457aa) - which has been demonstrated to disrupt the LINC complex in HeLa cells and suppress the pathology of LMNA-associated mutations in a mouse model of laminopathies - was found to displace Nesprin-2 from the nuclear envelope. Also as expected, a DNSUN1 construct containing only the SUN domain (i.e., encoding a protein having the amino acid sequence of SEQ ID NO: 87; DNhSUN1_181aa) and lacking all preceding coiled-coil domains of its alpha helices that promote multimerization, failed to displace Nesprin-2.

[0455] Unexpectedly, all other DNhSUN1 constructs tested (DNhSUN1_413aa, DNhSUN1_362aa, DNhSUN1_322aa, DNhSUN1_255aa, DNhSUN1_216aa and DNhSUN1_201aa) were able to displace Nesprin-2 from the nuclear envelope to the ER, even though previous studies predicted that certain constructs would not be able to interact with KASH domain-containing proteins.

[0456] Nie et al., 2016 and Jahed et al., 2018b disclosed that a truncated variant of Sun2 containing the complete CC2 and SUN domains was inhibited by the CC2 domain from forming a trimeric complex required for interaction with the KASH domain of Nesprin-2. Similarly, Sosa et al., 2012 reported that a truncated variant of Sun2 containing the α3 helix of the CC2 domain and the SUN domain was KASH-binding defective.

[0457] However, in this study, we found that paralogous constructs based on SUN1 disrupted the normal localization of Nesprin-2, thereby behaving as LINC complex inhibitors.

[0458] 1.3 Discussion In this example, we demonstrated that a series of SUN1 luminal domain sequences of various lengths (shown in SEQ ID NOs: 43, 44, 63-67 and 94) can affect LINC complex disruption. Surprisingly, previous structural and biochemical studies of the luminal domain of SUN proteins did not predict the effect of truncating the luminal domain of SUN1 in the present DNSUN1 construct.

[0459] The characterized dominant-negative SUN1 proteins can achieve LINC complex disruption through the formation of multimers (e.g., trimers) that behave as competitive inhibitors of endogenous SUN domain-containing proteins for binding to KASH domain-containing proteins. The fact that only low levels of dominant-negative SUN1 proteins localize to the nuclear envelope suggests that dominant-negative SUN1 proteins may also achieve LINC complex disruption by forming hetero-oligomers with endogenous SUN domain-containing proteins, thereby weakening the link between the cytoskeleton and the nuclear lamina mediated by intact LINC complexes.

[0460] Even though the dominant-negative SUN1 construct cannot homotrimerize, it is still able to form hetero-oligomers with endogenous SUN proteins. This may explain why truncated luminal SUN proteins, which are reported to be monomeric and cannot bind to KASH in vitro, can still have the ability to disrupt the LINC complex as demonstrated in this example. Alternatively, the in vitro situation in which the binding and gel filtration experiments were performed is simply biochemically different from the cellular environment and therefore does not predict how truncated luminal SUN proteins function in cells.

[0461] Example 2: Further in vitro analysis of LINC complex inhibition of various dominant-negative SUN constructs In further experiments, we characterize all possible truncation mutants of human SUN1 and human SUN2 luminal domains for their ability to function as LINC complex inhibitors.

[0462] Briefly, constructs are prepared as described in Example 1.1.2 (i.e., containing the N-terminal signal peptide, HA / ALFA tag and region derived from a SUN domain-containing protein and the C-terminal KDEL motif), except that all possible truncation mutants of the SUN1 luminal domain from SUN1(361-812) to SUN1(636-812) are prepared (i.e., SUN1(361-812), SUN1(363-812), SUN1(364-812), see below).

[0463] Similarly, prepare all possible truncation mutants of the SUN2 luminal domain derived from SUN2(258-717) through SUN2(522-717) (i.e., SUN2(259-717), SUN2(260-717), SUN2(261-717), SUN2(262-717), see below).

[0464] As described in Example 1.1, various constructs are evaluated for their ability to disrupt the normal subcellular localization of Nesprin-2 in HeLa cells.

[0465] All constructs evaluated that contain at least the α3 helix and SUN domain of the CC2 region (i.e., constructs containing at least positions 616-812 of human SUN1 and constructs containing at least positions 522-717 of human SUN2) are determined to displace Nesprin-2 from the nuclear envelope to the endoplasmic reticulum and therefore behave as LINC complex inhibitors.

[0466] Example 3: In vivo validation of the utility of the LINC complex inhibitor polypeptides characterized in Examples 1 and 2 for the treatment of laminopathies associated with mutations to Lmna Mice that are mouse models of Lmna mutation-associated cardiomyopathy, muscular dystrophy and progeria are administered an AAV or lentiviral vector encoding a nucleic acid encoding a LINC complex inhibitory polypeptide as described in Examples 1 and 2, or nanoparticles containing such a nucleic acid.

[0467] Mouse models of Lmna mutation-associated muscular dystrophy include the conditional Lmna knockout mouse. Flx / Flx The generation of Lmna mice and MLC-Cre mice is described in Wang et al., Differentiation. (2015) 89(1-2):11-21 and Mourkioti et al., Genesis. (2008) 46(8):424-30, respectively. Flx / + To obtain MLC-Cre mice, Flx / Flx The mice were crossed with MLC-Cre mice and then Lmna Flx / Flx ;Lmna to generate MLC-Cre Flx / + mice, resulting in a conditional knockout of Lmna in muscle cells.

[0468] Mouse models of Lmna mutation-associated cardiomyopathies and muscular dystrophies include, for example, the Lmna-H222P mouse described in Arimura et al., Hum Mol Genet. (2005) 14(1): 155-69, the Lmna-N195K mouse described in Mounkes et al., Hum Mol Genet. (2005) 14(15): 2167-80, and the conditional Lmna deletion-associated mouse model of dilated cardiomyopathy described in, for example, Chai et al., Nat Commun (2021) 12(1): 4722 (see Example 5.1 below).

[0469] Mouse models of Lmna mutation-associated progeria include the Lmna-G609G mouse, described in Osori et al., Sci Transl Med. (2011) 3(106):106ra107.

[0470] Mice with Lmna mutation-associated cardiomyopathy administered a nucleic acid encoding a LINC complex inhibitory polypeptide described in Examples 1 or 2 had improved cardiac function (e.g., increased myocardial contractility, increased ejection fraction and / or increased fractional shortening) compared to untreated mice or mice treated with vehicle only (e.g., empty vector).

[0471] Mice with Lmna mutation-associated muscular dystrophy or Lmna mutation-associated progeria administered a nucleic acid encoding a LINC complex inhibitory polypeptide described in Examples 1 or 2 had extended survival times compared to untreated mice or mice treated with vehicle only (e.g., an empty vector).

[0472] Example 4: In vivo confirmation of the utility of the LINC complex inhibitor polypeptides characterized in Examples 1 and 2 for the treatment of hyperlipidemia Mice, which are mouse models of atherosclerosis and familial hypercholesterolemia, are administered an AAV or lentiviral vector encoding a nucleic acid encoding a LINC complex inhibitory polypeptide as described in Examples 1 and 2, or nanoparticles containing such a nucleic acid.

[0473] A mouse model of atherosclerosis and familial hypercholesterolemia is established by feeding Ldlr knockout mice (C57BL / 6JInv, Jackson Laboratory) a Western-type diet (WTD; D12079B, Research Diets, NJ) for 15 weeks.

[0474] A subject with atherosclerosis and familial hypercholesterolemia who was administered a nucleic acid encoding a LINC complex inhibitor polypeptide described in Example 1 or 2, LDLR - / - The mice have smaller, less advanced, and less complex atherosclerotic lesions compared to untreated mice or mice treated with vehicle only (eg, empty vector).

[0475] Example 5: In vivo evaluation of the therapeutic efficacy of various different LINC complex inhibitory polypeptides We next investigated the ability of various DNhSUN1 LINC complex inhibitory polypeptides to rescue the deleterious effects of LNMA mutation-associated dilated cardiomyopathy in vivo in a mouse model.

[0476] 5.1 Materials and Methods Preparation of AAV encoding LINC complex inhibitory polypeptide The coding sequence for the DNhSUN1 construct was PCR amplified from a synthetic gene, restriction digested, and ligated into an AAV transfer plasmid containing a cTnT cardiac promoter, a chimeric intron, a WPRE (marmot hepatitis virus post-transcriptional regulatory element) and a bovine growth hormone polyA tail. For the preparation of AAV-vectored GFP for use as an experimental subject, an AAV transfer plasmid was also prepared that instead contained the coding sequence of GFP. The LINC complex inhibitory polypeptide used in the experiments of this example differs from that in Example 1, which uses a minimal human serum albumin signal peptide (SEQ ID NO: 100) and lacks the HA and ALFA tags:

[0477]

Table 4

[0478] Adeno-associated virus was produced by transient triple transfection of HEK293T cells with an adenovirus helper plasmid, a rep / cap plasmid encoding AAV2 Rep and AAV9 cap, and a transgene-specific plasmid. Four days after transfection, cell pellets and supernatants were collected. The supernatant was clarified by filtration and applied to POROS™ CaptureSelect™ AAV9 Affinity Resin (Thermo Fisher Scientific, Waltham, MA, USA) by gravity flow. Cells were resuspended in lysis buffer (phosphate-buffered saline, 200 mM NaCl, 0.001% Pluronic F-68), lysed by freeze / thawing 4-5 times, sonicated, and treated with benzonase to shear and digest DNA. Cell debris was pelleted by centrifugation, and cell lysate was collected and filtered through a 0.45 μm syringe filter. The filtered lysate was then applied to the AAV9 affinity resin by gravity flow. After washing in wash buffer (phosphate-buffered saline, 500 mM NaCl), AAV9 virions were eluted using 100 mM glycine, pH 2.5, and collected into microtubes containing 1 / 10 volume of 1 M Tris, pH 8. After two buffer exchanges into PBS containing 0.01% Pluronic F-68 and concentration via an Amicon® Ultra 100 kDA concentrator (Merck KGaA, Darmstadt, Germany), the solution containing AAV virions was filtered through a 0.22 μm 4 mm Millex syringe filter unit (Merck KGaA, Darmstadt, Germany) and stored at 4° C. or −80° C. Viral titers were determined using alkaline gel electrophoresis (Kohlbrenner and Weber, Methods Mol. Biol. (2017) 1521:91-107) with plasmid DNA standards, gel imaging on a ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA), and image quantification using Image Lab software (Bio-Rad Laboratories).

[0479] Lmna-deficient dilated cardiomyopathy (DCM) mouse model Mouse strains were maintained in a ventilated animal barrier facility with a 12-h light / dark cycle at a temperature of 21 ± 1 °C, humidity of 55-70%, and food and water provided ad libitum, in accordance with all ethical regulations.

[0480] To generate Lmna deletion-associated mouse models of dilated cardiomyopathy, mice were generated that were either conditionally "knocked out" or "loxP-transduced" Lmna as described in Wang et al., Differentiation. (2015) 89(1-2):11-21. Flx / Flx The mouse strains were crossed with mice harboring a tamoxifen-inducible Cre recombinase under the control of the cardiomyocyte-specific alpha myosin heavy chain promoter (αMHC-MerCreMer, abbreviated as mcm) purchased from the Jackson Laboratory (JAX stock number 005657; see also Chai et al., Nat Commun (2021) 12(1):4722). FF:mcm To induce dilated cardiomyopathy in mice, 40 mg / kg tamoxifen was injected intraperitoneally on postnatal day 14 (P14) to produce cardiomyocyte-specific deletion of Lmna. Tamoxifen (Merck) solution (16 g / L) was prepared in corn oil (Sigma).

[0481] In vivo delivery of adeno-associated virus Seventeen days after tamoxifen treatment, mice were inoculated with various AAV9s at 1 × 10 14 vg / kg. Animals were randomly selected. TMX and AAV9 injections were performed under anesthesia with 1.5% isoflurane mixed with oxygen. AAV9 dilutions were freshly prepared prior to administration using PBS containing 0.001% Pluronic F-68.

[0482] Echocardiogram Cardiac function was assessed by ultrasound recordings using a Prospect-T1 (Sharp-S). Mice were shaved 1 day before ultrasound examination. Animals were anesthetized with 1.5% isoflurane mixed with oxygen. B-mode and M-mode readings were obtained at heart rates between 450 bpm and 350 bpm. Fractional shortening (FS) and ejection fraction (EF) were calculated from the parasternal long axis using the instrument's software analysis tools. Cardiac measurements of left ventricular internal diameter (LVID) and left ventricular posterior wall (LVPW) were obtained from the parasternal short axis for diastolic (d) and systolic (s) states.

[0483] 5.2 Results and Discussion In the first experiment, P14 Lmna harboring the cardiac-specific Cre recombinase was FF:mcm Mice were injected with tamoxifen (TMX) to induce deletion of the Lmn gene. Seventeen days after treatment with TMX, mice were injected with a single dose (1 × 10 ) of AAV9 encoding DNhSUN1_201aa (construct G in FIG. 4), DNhSUN1_255aa (construct E in FIG. 4), DNhSUN1_334aa (construct D2 in FIG. 4) or DNhSUN1_457aa (construct A in FIG. 4) under the control of the cTNT promoter. 14 As a control condition, wild-type mice (WT) or Lmna mice administered TMX from day 17 were used. FF:mcm Mice (DCM) were administered a single dose (1 × 10 14 Survival was monitored, cardiac function was assessed by echocardiography, and left ventricular internal diameter (LVID) and left ventricular posterior wall (LVPW) thickness in diastole (d) and systole (s) were assessed by ultrasound.

[0484] The results are shown in Figures 5A to 5D.

[0485] FIG. 5A shows that treatment with all DNhSUN1 variants resulted in an extension of median survival compared to DCM control animals (39 days). The shortest construct, DNhSUN1_201aa (construct G), resulted in the shortest extension of survival at 66.5 days. Mice treated with DNhSUN1_334aa (construct D2) survived a median of 214.5 days, surpassing the full-length, DNhSUN1_457aa (construct A), which survived 162.5 days. Animals treated with DNhSUN1_255aa (construct E) showed the longest extension of survival; only two animals died, with four mice still alive after 275 days.

[0486] FIG. 5B shows that treatment with AAVs encoding DNhSUN1_201aa, DNhSUN1_255aa, DNhSUN1_334aa, or DNhSUN1_457aa improved cardiac function in Lmna DCM mice. Echocardiographic analysis showed that all AAV9-cTnT-DNhSUN1 truncated variants treated Lmna DCM mice improved cardiac function. FF:mcm revealed a significant improvement in fractional shortening (FS) and ejection fraction (EF) 17 days after administration of AAV9 (i.e., day 31 after administration of TMX) compared with AAV9-cTNT-GFP-treated mice (DCM).

[0487] Figures 5C and 5D show that LVID is increased and LVPW is decreased in Lmna DCM mice; this is more evident in systole. Left ventricular dilation and thinning of the heart wall occurs earlier in mice treated with DNhSUN1_201aa (construct G) than in other DNhSUN1 variants (DNhSUN1_255aa / construct E, DNhSUN1_334aa / construct D2, DNhSUN1_457aa / construct A), which correlates with the decline in cardiac function and the early onset of DCM in mice treated with AAV delivering DNhSUN1_201aa.

[0488] Thus, AAV encoding DNhSUN1_201aa, DNhSUN1_255aa, DNhSUN1_334aa, or DNhSUN1_457aa was found to treat Lmna deletion-associated DCM. DNhSUN1_201aa was found to extend survival to a lesser extent than DNhSUN1_457aa, while DNhSUN1_334aa and DNhSUN1_255aa extended survival to a greater extent than DNhSUN1_457aa.

[0489] In the second experiment, the efficacy of DNhSUN1_322aa was compared with that of DNhSUN1_457aa. FF:mcm Mice were injected with tamoxifen (TMX) to induce deletion of the Lmn gene. Seventeen days after treatment with TMX, mice were injected with a single dose (1 × 10 14 As a control condition, wild-type mice (WT) or Lmna mice administered TMX from day 17 were used. FF:mcm Mice (DCM) were administered a single dose (1 × 10 14 Survival was monitored, cardiac function was assessed by echocardiography, and left ventricular internal diameter (LVID) and left ventricular posterior wall (LVPW) thickness in diastole (d) and systole (s) were assessed by ultrasound.

[0490] The results are shown in Figures 6A to 6D.

[0491] Figure 6A shows that treatment with AAV encoding DNhSUN1_322aa or DNhSUN1_457aa extended the median survival time compared to DCM control animals, with a median survival of 39 days. All mice in the groups administered AAV encoding DNhSUN1_322aa or DNhSUN1_457aa were still alive at day 170.

[0492] FIG. 6B shows that treatment with AAV encoding DNhSUN1_322aa or DNhSUN1_457aa improved cardiac function in Lmna DC mice. Echocardiographic analysis showed that Lmna DC mice treated with AAV9 delivering the 322aa or DNhSUN1_457aa variants improved cardiac function. FF:mcm revealed a significant improvement in fractional shortening (FS) and ejection fraction (EF) 17 days after administration of AAV (i.e., 31 days after administration of TMX) compared with AAV9-cTNT-GFP-treated mice (DCM).

[0493] Figures 6C and 6D show that LVID increases and LVPW decreases in Lmna DCM mice; this is even more evident in the systole phase. For AAV9-cTnT-DNhSUN1-treated animals, which survive longer than DCM control animals, LVID increases while LVPW remains stable and does not decrease.

[0494] It was thus found that AAV encoding DNhSUN1_322aa or DNhSUN1_457aa treats Lmna deletion-associated DCM.

[0495] References

[0496] [Table 5-1]

[0497] [Table 5-2]

Claims

1. (i) (a) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 65, and (b) an endoplasmic reticulum retention motif; or (ii) (a) a signal peptide, (b) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 65, and (c) an endoplasmic reticulum retention motif A LINC complex inhibitory polypeptide comprising:

2. A LINC complex inhibitory polypeptide as described in claim 1, wherein the endoplasmic reticulum retention motif is a KDEL motif or a variant thereof selected from CDEL, KCEL or HVEL.

3. A LINC complex inhibitory polypeptide described in claim 1 or 2, wherein the signal peptide consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

100.

4. A LINC complex inhibitory polypeptide described in claim 1 or 2, consisting of an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 71 or 104.

5. A LINC complex inhibitory polypeptide described in claim 1 or 2, consisting of the amino acid sequence of SEQ ID NO: 71 or 104.

6. A polynucleotide encoding the LINC complex inhibitory polypeptide of claim 1.

7. A polynucleotide as described in claim 6, further comprising a promoter for providing expression of the polynucleotide in cardiac and / or skeletal muscle cells or tissues.

8. The polynucleotide of claim 7, wherein the promoter is a cardiac or cardiac muscle cell-specific promoter, optionally a cTNT, α-MHC or MLC2v promoter.

9. The polynucleotide described in claim 7, wherein the promoter is a skeletal muscle / striated muscle cell-specific promoter, optionally an MCK, MHCK7 or desmin promoter.

10. A vector comprising the polynucleotide of claim 6, which is suitable for delivering a polynucleotide encoding a LINC complex inhibitory polypeptide as a gene therapy.

11. The vector of claim 10, which is an adeno-associated virus (AAV) vector.

12. The vector of claim 10 or 11, which is an AAV vector selected from one of the following serotypes: AAV2, AAV1, AAV2i8, AAV5, AAV6, AAV8, AAV9, AAV9.45, AAV10 or AAVrh74.

13. A pharmaceutical composition comprising the LINC complex inhibitory polypeptide described in claim 1, the polynucleotide described in claim 6, or the vector described in claim 10.

14. A medicament for treating or preventing laminopathies or diseases characterized by hyperlipidemia, comprising:

2. The LINC complex inhibitor polypeptide of claim 1 . The polynucleotide of claim 6 . A vector according to claim 10, or A pharmaceutical composition comprising the LINC complex inhibitor polypeptide of claim 1, the polynucleotide of claim 6, or the vector of claim 10. Pharmaceuticals, including

15. 15. The pharmaceutical of claim 14, wherein the laminopathies are characterized by one or more of myopathies, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, myocardial dystrophy, skeletal muscular dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy, or dermatosis.

16. The pharmaceutical of claim 14, wherein the laminopathies are associated with mutations in LMNA.

17. Laminopathies include Hutchinson-Gilford progeria syndrome; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-related; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; muscular dystrophy; mandibular acrodysplasia with lipodystrophy type a; cardiomyopathy, dilated, type 1a; Charcot-Marie-Tooth disease; limb-girdle muscular dystrophy; cardiomyopathy, dilated, with hypergonadotropic hypogonadism; Emery-Dreifuss muscular dystrophy 3, autosomal recessive; lipodystrophy, familial partial, type 2; Emery-Dreifuss muscular dystrophy; Charcot-Marie-Tooth disease, axonal, type 2b1; heart-upper limb syndrome, Slovenian type; aging; familial partial lipodystrophy; restrictive skin disorder, fatal; arrhythmogenic right ventricular cardiomyopathy; dental disease; Heart disease; Werner syndrome; hypertrophic cardiomyopathy; left ventricular noncompaction; atrioventricular block; calcification; acroosteolysis; autosomal dominant limb-girdle muscular dystrophy; diabetes mellitus, non-insulin dependent; osteoporosis; atrial fibrillation; atrial asystole 1; melanoma; cardiac conduction disorders; catecholamine-induced polymorphic ventricular tachycardia; micrognathia, hearing loss, progeria-like symptoms, and lipodystrophy syndrome; sick sinus syndrome; Pelger-Houette anomaly; Charcot-Marie-Tooth disease, axonal, type 2e; congenital generalized lipodystrophy; restriction Cardiomyopathy; Congenital fiber type disproportion; Lipodystrophy, congenital generalized, type 1; Myofibrillar myopathy; Lipodystrophy, familial partial, type 1; Axonal neuropathy; Atypical Werner syndrome; Ovarian cystadenoma; Fanconi anemia, complementation group a; Body mass index quantitative trait locus 11; Skin diseases; Ankylosing spinal muscular dystrophy 1; Neuromuscular diseases; Harlermann-Stryff syndrome; Bethlem myopathy 1; Acquired generalized lipodystrophy; Cardiomyopathy, dilated, type 1e; Lipodystrophy, congenital generalized, Type 4; undifferentiated pleomorphic sarcoma; lipodystrophy, familial partial type; type 3; muscular dystrophy, congenital merosin deficiency type; 1a; proximal spinal muscular atrophy; muscular dystrophy-dystroglycanopathy, type B; 5; muscular dystrophy, congenital; 1b; Reynolds syndrome; Widermann-Rautenstrauch syndrome; Emery-Dreifuss muscular dystrophy 1, X-linked; lipodystrophy, congenital generalized type; monogenic diabetes; cardiomyopathy, dilated type; 1d; myopathy, proximal and ophthalmoplegia; muscle tissue disease; lipodystrophy Strophy, familial partial, type 4; Cardiomyopathy, dilated, type 1h; Second-degree atrioventricular block; Median neuropathy; Intrinsic cardiomyopathy; Prolapse of female genitalia; Complete generalized lipodystrophy; Ankylosing spinal muscular dystrophy; Emelinopathy; Ulnar neuropathy; Limb-girdle muscular dystrophy type 1b; Lmna-associated dilated cardiomyopathy; Pelvic muscle wasting; Generalized lipodystrophy-associated progeria syndrome; Muscle disease; Cardiomyopathy, dilated, type 1b; Autosomal inherited disease; Familial isolated arrhythmogenic ventricular dysplasia, right dominant; Familial isolated arrhythmogenic ventricular dysplasia, biventricular; The pharmaceutical of claim 14, which is selected from familial isolated arrhythmogenic ventricular dysplasia, left dominant; Lmna-associated cardiocutaneous progeria syndrome; and autosomal semi-dominant severe lipodystrophy laminopathies.

18. The method according to claim 14, wherein the disease characterized by hyperlipidemia is selected from atherosclerosis, cardiovascular disease, stroke, and familial hyperlipidemia.

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