Disease treatment / prevention by LINC complex inhibition

Abstract

To provide pharmaceutical compositions for treating and preventing laminopathies and hyperlipidemia by inhibition of LINC complex.SOLUTION: Pharmaceutical compositions comprising LINC complex inhibitors for use in methods for treating or preventing laminopathies are provided. Also provided are pharmaceutical compositions comprising a LINC complex inhibitor for use in a method for treating or preventing a disease characterized by hyperlipidemia. The LINC complex inhibitors for the pharmaceutical compositions can bind to a LINC complex, a LINC complex protein or an interaction partner for a LINC complex protein.SELECTED DRAWING: Figure 19

Description

Technical Field

[0001] This application claims the priority of SG 10201906637U filed on July 17, 2019, and its content and elements are incorporated herein by reference for all purposes.

[0002] Field of the Invention The present invention relates to the treatment and prevention of diseases through LINC complex inhibition.

Background Art

[0003] Background of the Invention Dilated cardiomyopathy (DCM) is the most common disease affecting the heart muscle and accounts for approximately 60% of all cardiomyopathies. DCM is characterized by reduced systolic (contractile) function due to dilation and thinning of the left ventricular wall or, in some cases, both ventricles. DCM is associated with sudden heart failure and cardiac death, resulting in high hospitalization rates, the need for heart transplantation, and consequently high cost burdens (J.L. Jefferies and J.A. Towbin, Lancet 375:752 - 762 (2010); R.E. Hershberger et al., Nat Rev Cardiol 10:531 - 547 (2013)). The causes of DCM are diverse and include various extrinsic factors (viruses, autoimmune infiltrations, alcohol, and drugs). However, 30 - 40% of all cases have a basis related to a single gene, and mutations in approximately 40 genes have been associated with DCM. The gene most frequently mutated in DCM is TTN, which encodes titin, a giant sarcomere protein, and short - form variants of TTN account for approximately 15 - 25% of all congenital forms of DCM (D.S. Herman et al., N Engl J Med 366:619 - 628 (2012); U. Tayal, S. et al., Genome Med 9:20 (2017)). The second most frequently mutated gene is lamin A (LMNA), which accounts for 6 - 8% of congenital DCM patients (U. Tayal, S. et al., Genome Med 9:20 (2017)).

[0004] LMNA mutation-related DCM is characterized by a cardiac conduction disorder manifested by electrophysiological (ECG) abnormalities including atrioventricular block, ventricular arrhythmia, and fibrillation. The risk of sudden cardiac death is higher in patients with LMNA mutation-related cardiomyopathy compared to patients with other forms of DCM (J.H. Van Berlo et al., Hum Mol Genet 14:2839-2849 (2005)). Approximately 450 different mutations have been identified in the LMNA gene, most of which are missense, resulting in the majority of DCM cases having autosomal dominant inheritance and the diversity of LMNA mutations complicating genetic approaches to treating LMNA mutation-related DCM. Although limited, LMNA mutation-related DCM can be treated by pacemaker adjustment. However, ultimately, the effective treatment at present is achieved by heart transplantation (R.E. Hershberger and A. Morales, in GeneReviews, M.P. Adam et al. eds (Seattle (WA), 1993); G. Captur et al., Heart 104:468-479 (2018)).

[0005] Mouse lines carrying Lmna mutations typically die within a few weeks after birth (T. Sullivan et al., J Cell Biol 147:913-920 (1999); A.T. Bertrand et al., Hum Mol Genet 21:1037-1048 (2012); V. Nikolova et al., J Clin Invest 113:357-369 (2004); A.S. Wang et al., Differentiation; research in biological diversity, (2015)). The cause of early death in mice lacking Lmna is uncertain because multiple tissues are affected. Lmna mutant mice develop DCM with conduction abnormalities and focal myocyte degeneration (V. Nikolova et al., J Clin Invest 113:357-369 (2004); L.C. Mounkes et al., Hum Mol Genet 14:2167-2180 (2005)), and myocardial damage is considered a major factor, but the still unclear effects on other skeletal muscles may also contribute to early postnatal death.

[0006] Lamin is a nuclear intermediate filament protein and a major component of the nuclear lamina, a protein matrix located inside the inner nuclear membrane (INM). Lamin consists of A-type lamins, which are composed of the two major isoforms lamin A and lamin C, and A-type lamins are driven by alternative splicing of LMNA RNA, while the two B-type lamins (LMNB1 and 2) are encoded by two genes: LMNB1 and LMNB2, respectively (B. Burke and C.L. Stewart, Nat Rev Mol Cell Biol 14:13 - 24 (2013)). The lamina confers nuclear structural and mechanical integrity, maintains the shape and position of the nucleus within the cell, and is also a determinant of chromatin formation (T. Sullivan et al., J Cell Biol 147:913 - 920 (1999); I. Solovei et al., Cell 152:584 - 598 (2013)). Lamin interacts with a number of INM proteins including emerin, lamin - associated polypeptide (LAP), and SUN - domain - containing proteins (B. van Steensel and A. S. Belmont, Cell 169:780 - 791 (2017)), and many of these INM proteins are associated with heart disease when mutated or present as variants (H. J. Worman et al., Cold Spring Harbor perspectives in biology 2:a000760 (2010); C. L. Stewart et al., Exp Cell Res 313:2144 - 2156 (2007)). Furthermore, these proteins form an integrated protein network centered on lamin, where loss or mutation of lamin can lead to mislocalization or changes in the levels of expression of many lamin - associated proteins (e.g., emerin, SUN1, LBR, and Lap2α) (T. Sullivan et al., J Cell Biol 147:913 - 920 (1999); I. Solovei et al., Cell 152:584 - 598 (2013); C. Y. Chen et al., Cell 149:565 - 577 (2012); T. V. Cohen et al., Hum Mol Genet 22:2852 - 2869 (2013); F. Haque et al., J Biol Chem 285:3487 - 3498 (2010)). Among these proteins whose expression is affected by loss or mutation of Lmna are SUN1 and Lap2, and the levels of both increase.In the case of SUN1, the increase in levels is rather due to a decrease in metabolic turnover rather than an increase in expression, resulting in high levels accumulating in the Golgi and being considered cytotoxic, at least in the Lmna− / − and LmnaΔ9 mouse disease models (C.Y. Chen et al., Cell 149:565 - 577 (2012); C. Stewart and B. Burke, WO / 2013 / 158046). However, genetic reduction of SUN1 in mice with Lmna mutations triples lifespan and ameliorates most of the pathology (C.Y. Chen et al., Cell 149:565 - 577 (2012); C. Stewart and B. Burke, WO / 2013 / 158046). The median survival of wild-type or Sun1− / − exceeded 210 days, the tracking period of 7 months; Lmna− / − mice had a median survival of 41 days; Lmna− / −Sun1+ / - mice had a median survival of 54 days; Lmna− / −Sun1− / − mice had a median survival of 104 days (p < 0.01 comparing Lmna− / − and Lmna− / −Sun1− / −). Similarly, all LmnaΔ9 mice died by 30 days of age, but their LmnaΔ9Sun1− / − littermates survived beyond this period and most achieved a lifespan more than twice this survival period (C.Y. Chen et al., Cell 149:565 - 577 (2012)). At the cellular level, Hutchison. (Hutchison) - Gilford progeria syndrome - encompassing LMNA mutations Human fibroblasts also exhibited increased SUN1 levels. Greatly reducing SUN1 in these cells alleviated nuclear morphological abnormalities, further suggesting that excessive Sun1 due to LMNA mutations is cytotoxic (C.Y. Chen et al., Cell 149:565 - 577 (2012); C. Stewart and B. Burke, WO / 2013 / 158046).

[0007] SUN (Sad1p, UNC-84) domain proteins share a conserved C-terminal SUN domain and localize to the INM (C.J. Malone et al., Development 126:3171 - 3181 (1999)). In mammals, SUN1 and SUN2 are the two major SUN proteins that are widely expressed in substantially all tissues. In the nuclear envelope lumen between the INM and the outer nuclear membrane (ONM), the C-terminus of SUN1 and / or 2 binds to the C-terminus (KASH domain) of different Nesprin / SYNE / KASH proteins that traverse the ONM. Furthermore, these two protein families constitute the LINC complex that physically couples the interphase nucleus to the cytoskeleton (M. Crisp et al., J Cell Biol 172:41 - 53 (2006); E.C. Tapley and D.A. Starr, Curr Opin Cell Biol 25:57 - 62 (2013)). The N-terminus of SUN domain proteins protrudes into the nucleoplasm, and in SUN1, this region interacts with prelamin A and the nuclear pore complex. Whether the N-terminus of SUN2 interacts with any nucleoplasmic / NE proteins is not clear. In contrast, most of the Nesprin / KASH domain proteins extend into the cytoplasm adjacent to the ONM. Thus, depending on the specific Nesprin / KASH protein, they interact directly or indirectly with all three cytoskeletal protein networks (microtubules, actin microfilaments, and intermediate filaments) (H.F. Horn, Current topics in developmental biology 109:287 - 321 (2014)). Furthermore, the SUN and KASH / Nesprin proteins of the LINC complex establish a direct physical connection between the cytoplasmic cytoskeletal network (and its associated substances, such as cell adhesion complexes in the cell membrane) and the inside of the interphase nucleus or the nucleoplasm. The LINC complex is thought to mediate the transmission of forces between the nucleus and the cytoskeleton, and as a result, control changes in gene expression / chromatin formation in response to mechanical / physical stimuli (S.G. Alam et al., Scientific reports 6:38063 (2016)).Loss of only either SUN1 or SUN2 has no obvious effect on postnatal growth and survival, but SUN1 null mice are infertile and deaf (H.F. Horn et al., J Cell Biol. 2013 Sep 30;202(7):1023 - 39; H.F. Horn et al., J Clin Invest. 2013 Feb;123(2):740 - 50). Simultaneous loss of Sun1 and Sun2 results in perinatal lethality and shows some redundancy during embryogenesis (K. Lei et al., Proc Natl Acad Sci USA 106:10207 - 10212 (2009)).

[0008] Importantly, Chen et al., Cell (2012) 149, 565 - 577 disclosed that Sun1 knockout in mice reduces the pathology of Lmna in LmnaΔ9 mice and extends their lifespan, while this effect is due to preventing the over - accumulation of Sun1 in the Golgi apparatus. The cytotoxicity as a result of the accumulation of SUN proteins in the Golgi was one of several proposed mechanisms involved in laminopathy. Other proposed mechanisms included gain - of - function DNA damage after over - accumulation of SUN1 in the nuclear envelope and abnormal transcriptional activity driven by the SUN1 nucleoplasmic domain (see Starr, Curr Biol. (2012) 22(17):R678 - R680). - / - In recent years, Kim et al., Sci Transl Med (2018) 10 reported on the expression of a polypeptide containing the KASH domain of EGFP and Nesprin (KASH2) under the control of a smooth muscle cell - specific promoter - reduced aortic pathology in a mouse model of HGPS Lm

[0009] However, numerous studies have suggested that inhibition of the levels / functions of LINC complex proteins causes or exacerbates laminopathy - related pathologies. na G609G / G609G In a mouse model, the expression of a polypeptide containing the KASH domain of EGFP and Nesprin (KASH2) was reported under the control of a smooth muscle cell - specific promoter - reduced aortic pathology.

[0010] However, numerous studies have suggested that inhibition of the levels / functions of LINC complex proteins causes or exacerbates laminopathy - related pathologies.

[0011] Mutations in SYNE1, the gene encoding Nesprin-1, are involved in dilated cardiomyopathy (Zhou et al., Hum Mol Genet. (2017) 26(12):2258 - 2276; Haskell et al., Circ Cardiovasc Genet. (2017) 10(3) pii:e001443; Puckelwartz et al., J. Mol. Cell. (2010) Cardiol. 48, 600 - 60). Furthermore, the majority of cardiomyopathy - related mutations in SYNE1 and SYNE2 are located at the C - termini of Nesprin - 1 and - 2 (Stroud, Biophys Rev (2018) 10, 1033 - 1051), and are involved in disruption of KASH function in the disease state. Additionally, cardiac - specific ablation of Nesprin - 1 and Nesprin - 2 KASH domains has been reported to result in early - onset cardiac dysfunction, fibrosis, and fetal gene re - expression (Banjeree PLOS Genetics (2014) 10, e1004114), whereas global disruption of Nesprin - 1 and - 2 has been shown to result in perinatal lethality in mice (Zhang et al., Development (2007) 134, 901 - 908).

[0012] Similarly, SUN1 and SUN2 have been suggested to be modifier genes that exacerbate the effects of lamin A / C mutations - associated muscular dystrophy in humans (Meinke et al., PLoS Genet. (2014) 10, e1004605), and some EDMD patients have mutations in SUN1 that decrease the interaction between SUN1 and lamin A / C (Lie et al., Human Mutation (2014) 35, 452 - 461). Global disruption of SUN1 and SUN2 has been shown to result in perinatal lethality in mice and also results in increased genomic instability and DNA damage (Lei et al. Proc. Natl. Acad. Sci. U.S.A. (2009) 106, 10207 - 10212).

[0013] In addition to lamin A / C, considering the roles of Nesprin and SUN proteins in laminopathic EDMD, disruption of the LINC complex has been suggested to contribute to laminopathy (Chang et al., Nucleus (2015) 6, 77 - 88), and it has been proposed that disruption of the LINC complex results in defects in cellular structure and function that can contribute to the development of muscular dystrophy and cardiomyopathy (Lombardi et al. J Biol Chem (2011) 286, 26743 - 26753).

SUMMARY OF THE INVENTION

[0014] The present invention is based on the unexpected discovery by the inventors that inhibition of the LINC complex ameliorates the symptoms of a wide range of diseases caused by LMNA mutations with different functional consequences. The inventors have demonstrated in the experimental examples herein that disruption of the LINC complex (using a dominant - negative version of the LINC complex protein or through targeted disruption of the domains of the LINC complex proteins that interact to form the LINC complex) ameliorates laminopathy associated with reduced levels of lamin A / C as well as progerin - related laminopathy.

[0015] Thus, it has been revealed that disruption of the LINC complex is a viable strategy for the treatment and prevention of laminopathies arising from a wide range of LMNA mutations. The inventors have also unexpectedly discovered that inhibition of the LINC complex can ameliorate the symptoms of diseases characterized by hyperlipidemia. The inventors have demonstrated that disruption of the LINC complex reduces the symptoms of atherosclerosis.

[0016] In a first aspect, the present invention provides a LINC complex inhibitor for use in a method of treating or preventing laminopathy. The present invention also provides the use of a LINC complex inhibitor in the manufacture of a medicament for use in a method of treating or preventing laminopathy. ​

[0017] The present invention also provides a method of treating or preventing laminopathy, the method comprising administering to a subject a therapeutically or prophylactically effective amount of a LINC complex inhibitor. In some embodiments, the laminopathy is characterized by one or more of myopathy, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, cardiomyopathic muscular dystrophy, skeletal muscular dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy, or skin disease.

[0018] In some embodiments, the laminopathy is associated with a mutation in LMNA. In some embodiments, laminopathy includes Hutchinson-Gilford progeria syndrome; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-related; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; muscular dystrophy; mandibuloacral dysplasia with lipodystrophy; 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-hand syndrome, Slovenian type; aging; familial partial lipodystrophy; restrictive dermopathy, lethal; arrhythmogenic right ventricular cardiomyopathy; dental disease; heart disease; Werner syndrome; hypertrophic cardiomyopathy; left ventricular myocardial noncompaction; atrioventricular block; calcinosis; acro-osteolysis; autosomal dominant limb-girdle muscular dystrophy; true diabetes, insulin-independent; osteoporosis; atrial fibrillation; atrial standstill 1; cutaneous melanoma; cardiac conduction disorder; catecholamine-induced polymorphic ventricular tachycardia; micrognathia, deafness, progeroid features, and lipodystrophy syndrome; sinus node dysfunction syndrome; Pelger-Huet nuclear anomaly; Charcot-Marie-Tooth disease, axonal, type 2e; congenital generalized lipodystrophy; restrictive cardiomyopathy; congenital myofiber 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 disease; myotonic dystrophy 1; neuromuscular disease; Hallermann-Streiff 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-deficient, type 1a; proximal spinal muscular atrophy; muscular dystrophy-dystroglycanopathy, type B, 5; muscular dystrophy, congenital, type 1b; Reynolds Syndrome; Widemann-Rautenstrauch syndrome; Emery-Dreifuss muscular dyst Strophy 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; Limb-girdle muscular dystrophy type 1b; Lmna-associated dilated cardiomyopathy; Pelvic muscle wasting; Generalized lipodystrophy-associated progeria syndrome; Muscle disease; Cardiomyopathy, dilated, 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.

[0019] The present invention also provides a LINC complex inhibitor for use in a method of treating or preventing a disease characterized by hyperlipidemia. The invention also provides the use of a LINC complex inhibitor in the manufacture of a medicament for use in a method of treating or preventing a disease characterized by hyperlipidemia.

[0020] The present invention 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 LINC complex inhibitor.

[0021] In some embodiments, the disease characterized by hyperlipidemia is selected from atherosclerosis, cardiovascular disease, stroke, and familial hyperlipidemia. In some embodiments according to various aspects of the invention, the LINC complex inhibitor can bind to a LINC complex, a LINC complex protein, or an interaction partner for a LINC complex protein, or the LINC complex inhibitor can reduce expression of a LINC complex protein.

[0022] In some embodiments, the LINC complex inhibitor can inhibit the interaction between the LINC complex protein and its interaction partner. In some embodiments, the LINC complex inhibitor is a peptide / polypeptide, nucleic acid or small molecule.

[0023] In some embodiments, the LINC complex inhibitor can modify the gene encoding the LINC complex protein to reduce its expression. In some embodiments, the LINC complex inhibitor comprises a site-specific nuclease (SSN) that targets the gene encoding the LINC complex protein.

[0024] In some embodiments, the LINC complex inhibitor is an inhibitory nucleic acid capable of reducing the expression of the LINC complex protein by RNA interference (RNAi). In some embodiments, the method comprises administering to a subject a nucleic acid encoding the LINC complex inhibitor, or a nucleic acid encoding a factor necessary for the production of the LINC complex inhibitor.

Mode for Carrying Out the Invention

[0025] Description LINC Complex Structure and Function The linker of nucleoskeleton and cytoskeleton (LINC) complex is a polypeptide complex comprising SUN domain-containing proteins and KASH domain-containing proteins. The LINC complex structure is outlined, for example, in Sosa et al., Curr Opin Struct Biol. (2013) 23(2):285-91 and Hieda, Cells (2017) 6(1):3, both of which are incorporated herein by reference in their entirety.

[0026] The LINC complex connects the inner nuclear membrane (INM) to the outer nuclear membrane (ONM) or the nuclear envelope. SUN domain-containing proteins extend into 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 extend into the ONM and associate with cytoskeletal components on the cytoplasmic side of the ONM, such as actin filaments, microtubule motors, and intermediate filaments, as well as with SUN domain-containing proteins on the perinuclear side of the ONM. SUN domain proteins function as a trans-tubular linkage for KASH domain proteins in the ONM.

[0027] As used herein, "SUN domain-containing protein" refers to any polypeptide containing a SUN domain. SUN (Sad1 and UNC-84) domain proteins are important INM components containing a conserved carboxy-terminal SUN domain that localizes to the nuclear membrane cisternae. The SUN domain contains approximately 175 residues and is presented at the end of the helical stalk region. The nucleoplasmic domain of the SUN protein interacts with components of the nuclear skeleton.

[0028] The SUN domain can include, or consist of, an amino acid sequence set forth in SEQ ID NO: 82, 83, 84, 85, 86, or 87, or an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to an amino acid sequence set forth in SEQ ID NO: 82, 83, 84, 85, 86, or 87.

[0029] 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.

[0030] 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.

[0031] Human SUN1 is a polypeptide identified by UniProtKB O94901, and its amino acid sequence is shown in SEQ ID NO: 88. Human SUN2 is a polypeptide identified by UniProtKB Q9UH99, and its amino acid sequence is shown in SEQ ID NO: 89. Human SUN3 is a polypeptide identified by UniProtKB Q8TAQ9, and its amino acid sequence is shown in SEQ ID NO: 90. Human SUN5 is a polypeptide identified by UniProtKB A9Z1W8, and its amino acid sequence is shown in SEQ ID NO: 91. Human SPAG4 is a polypeptide identified by UniProtKB Q9NPE6, and its amino acid sequence is shown in SEQ ID NO: 92. Human SUCO is a polypeptide identified by UniProtKB Q9UBS9, and its amino acid sequence is shown in SEQ ID NO: 93.

[0032] As used herein, "SUN1", "SUN2", "SUN3", "SUN5", "SPAG4" and "SUCO" refer to SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO derived from any species, respectively, and include their isoforms, fragments, variants or homologs.

[0033] As used herein, a "fragment," "variant," or "homolog" of a protein may optionally be characterized as having an amino acid sequence identity of at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% 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 its ability to perform a function performed by the reference protein.

[0034] A "fragment" generally refers to a part of a reference protein. A "variant" includes one or more amino acid substitutions, insertions, deletions or other modifications compared to the amino acid sequence of a reference protein, but has a significant degree of sequence identity (e.g., at least 6 0%) with the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein. A "homolog" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0035] A "fragment" may be of any length (by number of amino acids), but optionally may 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.

[0036] An isoform, fragment, variant or homolog may optionally be a functional isoform, fragment, variant or homolog, such as having the functional characteristics / activities of the reference protein, as determined by analysis by a suitable assay for functional characteristics / activities.

[0037] As used herein, reference to "SUN1" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 88, and fragments, variants or homologs thereof. In some embodiments, SUN1 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 88.

[0038] As used herein, reference to "SUN2" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 89, and fragments, variants or homologs thereof. In some embodiments, SUN2 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 89.

[0039] As used herein, reference to "SUN3" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 90, and fragments, variants or homologs thereof. In some embodiments, SUN3 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 90.

[0040] As used herein, reference to "SUN5" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 91, and fragments, variants or homologs thereof. In some embodiments, SUN5 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 91.

[0041] As used herein, reference to "SPAG4" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 92, and fragments, variants or homologs thereof. In some embodiments, SPAG4 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 92.

[0042] As used herein, reference to "SUCO" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 93, and fragments, variants or homologs thereof. In some embodiments, SUCO comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 93.

[0043] 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-terminal anchored 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 extends into the ONM and an approximately 30 amino acid region that extends into the nuclear membrane cisternae.

[0044] The KASH domain may comprise, or consist of, an amino acid sequence having at least 70%, preferably 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 NOs: 97, 98, 99 or 100, or the amino acid sequence set forth in SEQ ID NOs: 97, 98, 99, 100 or 101.

[0045] 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.

[0046] In some embodiments, the KASH domain-containing protein can form a LINC complex. In some embodiments, the KASH domain-containing protein can interact with a SUN domain and / or a SUN domain-containing protein.

[0047] Human Nesprin-1 is a polypeptide identified by UniProtKB Q8NF91, and its amino acid sequence is shown in SEQ ID NO: 102. Human Nesprin-2 is a polypeptide identified by UniProtKB Q8WXH0, and its amino acid sequence is shown in SEQ ID NO: 103. Human Nesprin-3 is a polypeptide identified by UniProtKB Q6ZMZ3, and its amino acid sequence is shown in SEQ ID NO: 104. Human Nesprin-4 is a polypeptide identified by UniProtKB Q8N205, and its amino acid sequence is shown in SEQ ID NO: 105. Human KASH5 is a polypeptide identified by UniProtKB Q8N6L0, and its amino acid sequence is shown in SEQ ID NO: 106. Human LRMP is a polypeptide identified by UniProtKB Q12912, and its amino acid sequence is shown in SEQ ID NO: 113.

[0048] 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 from any species, respectively, and include their isoforms, fragments, variants, or homologs.

[0049] As used herein, reference to "Nesprin-1" refers to the polypeptide shown in SEQ ID NO: 102. It refers to a protein having an amino acid sequence, and fragments, variants or homologs thereof. In some embodiments, Nesprin-1 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 102.

[0050] As used herein, reference to "Nesprin-2" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 103, and fragments, variants or homologs thereof. In some embodiments, Nesprin-2 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 103.

[0051] As used herein, reference to "Nesprin-3" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 104, and fragments, variants or homologs thereof. In some embodiments, Nesprin-3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 104.

[0052] As used herein, reference to "Nesprin-4" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 105, and fragments, variants or homologs thereof. In some embodiments, Nesprin-4 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 105.

[0053] As used herein, reference to "KASH5" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 106, and fragments, variants or homologs thereof. In some embodiments, KASH5 comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 106.

[0054] As used herein, reference to "LRMP" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 113, and fragments, variants or homologs thereof. In some embodiments, LMRP comprises, or consists of, an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 113.

[0055] As used herein, "LINC complex" refers to a polypeptide complex comprising a SUN domain-containing protein and a KASH domain-containing protein. The LINC complex is formed by protein-protein interactions between SUN domain-containing proteins and KASH domain-containing proteins. The LINC complex may include non-covalent and / or covalent interactions between the SUN domain and the KASH domain. Examples of non-covalent interactions include hydrogen bonds, ionic interactions, van der Waals forces, and hydrophobic bonds. Examples of covalent interactions include disulfide bonds.

[0056] SUN domain proteins are thought 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. The SUN domain is assumed to have a β-sandwich structure, and the trimeric SUN domains interact extensively with each other through a protruding β-sheet known as the KASH lid; the KASH lid of one SUN domain partially overlaps with the β-sandwich of the adjacent SUN domain (Sosa et al., Cell (2012) 149:1035 - 1047).

[0057] KASH domain proteins can also form oligomers 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 upper region of the β-sandwich of the adjacent SUN domain. Thus, the SUN and KASH domains are thought to interact to form a 3:3 hexameric heterocomplex. Two to three proline residues immediately preceding the C-terminus of the KASH domain are thought to be accommodated in a deep pocket within the surface of the SUN domain. This region of KASH is important for SUN-KASH interaction; an extension of the C-terminus by just one amino acid disrupts LINC complex formation. The conserved cysteine residues of the SUN and KASH domains form disulfide bonds and further stabilize 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).

[0058] As described above herein, the LINC complex is thought to be formed through the interaction between the SUN domains of three SUN domain-containing proteins and the KASH domains of three KASH domain-containing proteins. The interaction between the SUN domain protein and the KASH domain protein is thought to be indiscriminate; SUN1 and SUN2 have been shown to interact with Nesprin-1, Nesprin-2, and Nesprin-3.

[0059] The LINC complex according to the present invention may comprise any SUN domain-containing protein and any KASH domain-containing protein. The SUN domain-containing proteins of the LINC complex may or may not be the same. The KASH domain-containing proteins of the LINC complex may or may not be the same.

[0060] The functions of the LINC complex are outlined, for example, in Hieda, Cells (2017) 6(1):3 (incorporated herein by reference above) and Stroud, Biophys Rev. (2018) 10(4):1033-1051, which is incorporated herein by reference in its entirety.

[0061] The LINC complex performs 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 nuclear envelope spacing, DNA repair, cell migration, and moving chromosomes into the nucleus during meiosis.

[0062] The LINC complex has a mechanosensory role in translating mechanical stimuli and changes in the extracellular matrix into signals that allow cells to adapt to their environment through the regulation of cytoskeletal organization, gene expression, nuclear composition, and structure.

[0063] Integrins mediate the transmission of forces from the external microenvironment to the intracellular cytoskeleton, and the nucleus-cytoskeleton molecular connection transmits forces to chromosome organization in the nucleus. The nuclear lamina causes deformation of the nuclear structure and initiates changes in gene regulation.

[0064] The nuclear envelope is an important structure in such processes. On the nucleoplasmic side of the INM, the nuclear lamina (consisting of type A and type B 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. The nuclear lamina is involved in processes that are extremely important for cell functions and survival, including maintenance of nuclear integrity, regulation of the cell cycle, mechanical signal transduction, cell signaling, and DNA repair.

[0065] Deviations from the normal expression and / or function of nuclear envelope proteins, as well as deviations from the normal expression and / or function of factors directly or indirectly associated with the nuclear envelope, are associated with various diseases including muscular dystrophy, cardiomyopathy, lipodystrophy, progeria, cancer, and neurological diseases.

[0066] Inhibition of the LINC complex The present invention relates to inhibition of the LINC complex. As used herein, "inhibition of the LINC complex" includes inhibition of the formation of the LINC complex (i.e., inhibition of LINC complex assembly), disruption / degradation of the LINC complex, and inhibition of LINC complex activity / function.

[0067] In some embodiments, formation of the LINC complex can be inhibited by inhibiting the gene and / or protein expression of the component proteins of the LINC complex. The component proteins of the LINC complex include SUN domain-containing proteins and KASH domain-containing proteins.

[0068] For brevity, herein "component proteins of the LINC complex" may sometimes simply be referred to as "LINC complex proteins". In some embodiments, inhibiting the formation of the LINC complex includes: inhibiting the gene or protein expression encoding the LINC complex protein; modifying the gene encoding the LINC complex protein to reduce / impede its expression; reducing the level of RNA encoding the LINC complex protein; inhibiting the transcription of the nucleic acid encoding the LINC complex protein; increasing the degradation of RNA encoding the LINC complex protein; reducing the level of the LINC complex protein; disrupting the normal post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding the LINC complex protein; and increasing the degradation of the LINC complex protein, including one or more of these.

[0069] Gene expression can be determined by means well known to those skilled in the art. The level of RNA encoding a component protein of the LINC complex can be determined by techniques such as, for example, RT-qPCR, Northern blot, etc. A decrease in the level of RNA encoding a component protein of the LINC complex can be the result of, for example, a decrease in the transcription of the nucleic acid encoding the LINC complex protein or an increase in the degradation of the RNA encoding the LINC complex protein.

[0070] A decrease in the transcription of the nucleic acid encoding the LINC complex protein can be the result of inhibition of the set and / or activity of factors necessary for the transcription of the DNA encoding the LINC complex protein. An increase in the degradation of the RNA encoding the LINC complex protein can be the result of an increase in the enzymatic degradation of the RNA encoding the LINC complex protein, for example, RNA interference (RNAi), and / or a decrease in the stability of the RNA encoding the LINC complex protein.

[0071] Protein expression can be determined by means well known to those skilled in the art. The level of a component protein of the LINC complex can be determined by, for example, antibody-based methods including Western blot, immunohistochemistry / cytochemistry, flow cytometry, ELISA, or by reporter-based methods.

[0072] Proteolysis can be evaluated, for example, by detection or analysis of the level / ratio of ubiquitinated proteins, association with ubiquitin ligase, and / or proteasome localization.

[0073] A decrease in the level of the LINC complex protein can be the result of, for example, a decrease in the level of the RNA encoding the LINC complex protein, a decrease in post-transcriptional processing of the RNA encoding the LINC complex protein, or an increase in the degradation of the LINC complex protein.

[0074] Disruption of normal post-transcriptional processing of the LINC complex protein can be, for example, a decrease / alteration in the splicing of pre-mRNA encoding the LINC complex protein into mature mRNA, a decrease in the translation of mRNA encoding the LINC complex protein, or a decrease / alteration in the post-translational processing of the LINC complex protein.

[0075] A decrease / alteration in the splicing of pre-mRNA encoding the LINC complex protein into mature mRNA can be the result of inhibition of the set and / or activity of factors necessary for normal splicing. A decrease in the translation of mRNA encoding the LINC complex protein can be the result of inhibition of the set and / or activity of factors necessary for translation. A decrease / alteration in post-translational processing (e.g., enzymatic processing, folding) can be the result of inhibition of the set and / or activity of factors necessary for normal post-translational processing of the LINC complex protein. An increase in the degradation of the LINC complex protein can be the result of an increase in the enzymatic (e.g., protease-mediated) degradation of proteins that may be associated with misfolding.

[0076] In some embodiments, the formation of the LINC complex can be inhibited by inhibiting the transport of the component proteins of the LINC complex (e.g., SUN domain-containing proteins and / or KASH domain-containing proteins) and / or disrupting normal intracellular localization. In some embodiments, inhibiting the formation of the LINC complex includes one or more of: decreasing the level / ratio of LINC complex proteins localized to the nuclear envelope; decreasing the level / ratio of SUN domain-containing proteins associated with the inner nuclear membrane; decreasing the level / ratio of KASH domain-containing proteins associated with the outer nuclear membrane; increasing the retention of LINC complex proteins in the endoplasmic reticulum; and increasing the level / ratio of LINC complex proteins localized to the endoplasmic reticulum.

[0077] The intracellular localization of the component proteins of the LINC complex within the cell can be analyzed using techniques known to those skilled in the art. Such techniques include, for example, analysis by immunohistochemical staining and 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 enable imaging of proteins in the ER, INM, and ONM. Such methods can be used to analyze the levels / ratio of the component proteins of the LINC complex in the nuclear envelope, inner nuclear membrane, and outer nuclear membrane.

[0078] In some embodiments, the formation of the LINC complex can be inhibited by inhibiting the interaction between the component proteins of the LINC complex (e.g., SUN domain-containing proteins and KASH domain-containing proteins). In some embodiments, the formation of the LINC complex can be inhibited by inhibiting the interaction between the component proteins of the LINC complex and the interaction partners for the component proteins of the LINC complex.

[0079] In some embodiments, inhibiting the formation of the LINC complex comprises inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a KASH domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and a KASH domain-containing protein; inhibiting the interaction between a SUN domain-containing protein and lamin; inhibiting the interaction between a SUN domain-containing protein and a chromatin-binding protein; inhibiting the interaction between a KASH domain-containing protein and a SUN domain-containing protein; and inhibiting the interaction between a KASH domain-containing protein and a cytoskeletal component (e.g., microfilament or its components (e.g., actin), microtubule or its components (e.g., tubulin) or intermediate filament or its components), including one or more of these.

[0080] In some embodiments, LINC complex inhibition comprises disruption / degradation of the LINC complex. In some embodiments, disrupting / degrading the LINC complex comprises inhibiting the interaction between a SUN domain-containing protein and its interaction partner; inhibiting the interaction between a KASH domain-containing protein and its interaction partner; inhibiting the interaction between a SUN domain-containing protein and a KASH domain-containing protein; inhibiting the interaction between a SUN domain-containing protein and lamin; inhibiting the interaction between a SUN domain-containing protein and a chromatin-binding protein; inhibiting the interaction between a KASH domain-containing protein and a SUN domain-containing protein; and inhibiting the interaction between a KASH domain-containing protein and a cytoskeletal component (e.g., microfilament or its component (e.g., actin), microtubule or its component (e.g., tubulin) or intermediate filament or its component); increasing LINC complex disassembly; increasing LINC complex degradation; moving LINC complex proteins from the LINC complex; and reducing the LINC complex, including one or more of the above.

[0081] As used herein, an interaction partner for a SUN domain-containing protein can be any molecule (e.g., protein / nucleic acid) with which the SUN domain-containing protein interacts. An interaction partner for a SUN domain-containing protein can be a protein that can form a complex with the SUN domain-containing protein through a protein-protein interaction. In some embodiments, an interaction partner for a SUN domain-containing protein can be a KASH domain-containing protein (e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4 or KASH5), a SUN domain-containing protein (e.g., SUN1, SUN2, SUN3, SUN5, SPAG4 or SUCO), a nucleoplasmic protein, lamin (e.g., lamin A, lamin C, lamin B1 or lamin B2) or a chromatin-binding protein.

[0082] As used herein, an interaction partner for a KASH domain-containing protein can be any molecule (e.g., protein / nucleic acid) with which the KASH domain-containing protein interacts. An interaction partner for a KASH domain-containing protein can be a protein that can form a complex with the KASH domain-containing protein through protein-protein interaction. In some embodiments, an interaction partner for a KASH domain-containing protein is a SUN domain-containing protein (e.g., SUN1, SUN2, SUN3, SUN5, SPAG4 or SUCO), a KASH domain-containing protein (e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 or LRMP), a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, a microtubule, tubulin, a microtubule motor or an intermediate filament protein. It can be a protein, a microfilament, actin, a microtubule, tubulin, a microtubule motor or an intermediate filament protein.

[0083] The interaction between the component proteins of the LINC complex and an interaction partner for such a protein can be analyzed using techniques well known to those skilled in the art, such as co-immunoprecipitation and resonance energy transfer (RET) assays using appropriate labeled molecular species. Inhibition of the interaction can be determined in such assays by detection of a decrease in the level of interaction compared to control, non-inhibited conditions.

[0084] The LINC complex and its component proteins can be detected using methods well known to those skilled in the art, such as Western blot, immunohistochemistry / cytochemistry, flow cytometry, antibody-based methods including ELISA or reporter-based methods.

[0085] LINC complex inhibition can be characterized by a decrease in the level of function of the LINC complex. In some embodiments, LINC complex inhibition can be determined by detection of a decrease in the level of a correlate of LINC complex function.

[0086] In the specific embodiments contemplated herein, LINC complex inhibition is achieved by one or more of: modifying the gene encoding the SUN domain-containing protein to reduce / prevent its expression; modifying the gene encoding the KASH domain-containing protein to reduce / prevent its expression; inhibiting the expression of the SUN domain-containing protein by RNAi; inhibiting the expression of the KASH domain-containing protein by RNAi; or inhibiting the interaction between the SUN domain-containing protein and the KASH domain-containing protein.

[0087] In some embodiments, modifying the gene encoding the KASH domain-containing protein or the SUN domain-containing protein to reduce / prevent its expression is achieved using a site-specific nuclease (SSN) system (e.g., a CRISPR-based system) that targets the relevant gene. In some embodiments, inhibition of the expression of the KASH domain-containing protein or the SUN domain-containing protein by RNAi is achieved using siRNA, miRNA, or shRNA that targets the relevant protein.

[0088] In some embodiments, inhibiting the interaction between the SUN domain-containing protein and the KASH domain-containing protein is achieved using a dominant-negative SUN domain-containing protein, a dominant-negative KASH domain-containing protein, a small molecule inhibitor of the interaction between the SUN domain-containing protein and the KASH domain-containing protein, a peptide mimetic of the KASH domain, or a peptide mimetic of the SUN domain. It is understood that the inhibition of the interaction is between the endogenous SUN domain-containing protein and the endogenous KASH domain-containing protein.

[0089] LINC complex inhibitor Aspects of the invention include LINC complex inhibition using an LINC complex inhibitor. The term "LINC complex inhibitor" refers to any agent capable of achieving LINC complex inhibition. LINC complex inhibitors include agents that can inhibit the formation of the LINC complex (i.e., inhibit LINC complex assembly), disrupt / decompose the LINC complex, or inhibit LINC complex function.

[0090] Such agents may also be effectors of LINC complex inhibition as described above (i.e., may cause LINC complex inhibition directly or indirectly). The LINC complex inhibitor may sometimes be referred to herein as a LINC complex antagonist.

[0091] In some embodiments, the LINC complex inhibitor inhibits the formation of the LINC complex; disrupts / degrades the LINC complex; inhibits the activity of the LINC complex; inhibits the gene and / or protein expression of the LINC complex protein; modifies the gene encoding the LINC complex protein to reduce / impede its expression; reduces the level of RNA encoding the LINC complex protein; inhibits the transcription of the nucleic acid encoding the LINC complex protein; increases the degradation of RNA encoding the LINC complex protein; reduces the level of the LINC complex protein; disrupts the normal post-transcriptional processing (e.g., splicing, translation, post-translational processing) of RNA encoding the LINC complex protein; increases the degradation of the LINC complex protein; inhibits the transport of the LINC complex protein and / or disrupts its normal intracellular localization; reduces the level / ratio of the LINC complex protein localized to the nuclear membrane; reduces the level / ratio of the SUN domain-containing protein associated with the inner nuclear membrane; reduces the level / ratio of the KASH domain-containing protein associated with the outer nuclear membrane; increases the retention of the LINC complex protein in the endoplasmic reticulum; increases the level / ratio of the LINC complex protein localized to the endoplasmic reticulum; inhibits the interaction between the constituent proteins of the LINC complex; inhibits the interaction between the LINC complex protein and its interaction partner for the LINC complex protein; inhibits the interaction between the SUN domain-containing protein and its interaction partner for the SUN domain-containing protein; inhibits the interaction between the KASH domain-containing protein and its interaction partner for the KASH domain-containing protein; inhibits the interaction between the SUN domain-containing protein and the KASH domain-containing protein; inhibits the interaction between the SUN domain-containing protein and lamin; inhibits the interaction between the SUN domain-containing protein and the chromatin-binding protein; inhibits the interaction between the KASH domain-containing protein and the SUN domain-containing protein;Inhibiting the interaction between KASH domain-containing proteins and cytoskeletal components (e.g., microfilaments or their constituents (e.g., actin), microtubules or their constituents (e.g., tubulin) or intermediate filaments or their constituents); increasing the disassembly of the LINC complex; increasing the degradation of the LINC complex; moving LINC complex proteins from the LINC complex; and / or reducing the level of the LINC complex.

[0092] It is recognized that a given LINC complex inhibitor may exhibit more than one of the properties listed above. A given agent can be evaluated for the properties listed above using appropriate assays. The assays can be, for example, in vitro assays, optionally cell-based assays or cell-free assays.

[0093] If the assay is a cell-based assay, the assay can include treating cells with a test agent to determine whether the agent exhibits one or more of the listed properties. The assay can use proteins expressed endogenously or recombinantly, and can use species labeled with a detectable entity to facilitate their detection.

[0094] Agents that can reduce the gene expression of LINC complex proteins (e.g., reducing the level of RNA encoding the LINC complex protein, reducing the transcription of nucleic acid encoding the LINC complex protein and / or increasing the degradation of RNA encoding the LINC complex protein) can be identified, for example, by RT-qPCR using an assay that includes detecting the level of RNA encoding the relevant protein. Such an assay can include treating cells / tissues with the agent and subsequently comparing the level of RNA encoding the relevant protein in such cells / tissues with the level of RNA encoding the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / solvent-treated cells / tissues). Decreased / altered pre-mRNA of a given protein Assays for detecting alternative splicing may include the step of detecting and / or quantifying one or more isoforms of a relevant protein or RNA encoding one or more of said isoforms.

[0095] Agents that can reduce the protein expression of LINC complex proteins (e.g., reduce the level of LINC complex proteins, increase the degradation of LINC complex proteins) can be identified using an assay that includes the step of detecting the level of a relevant protein, for example, using an antibody / reporter-based method (Western blot, ELISA, immunohistochemistry / cytochemistry, etc.). Such assays may include the step of treating cells / tissues with the agent and subsequently comparing the level of the relevant protein in such cells / tissues with the level of the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / solvent-treated cells / tissues). Assays for proteolysis may include, for example, the step of assessing the ubiquitination or proteosomal localization of the relevant protein, and / or the proportion of the relevant protein that is ubiquitinated or localized to the proteasome.

[0096] Agents that can inhibit the transport of LINC complex proteins and / or disrupt the normal intracellular localization of this protein can be identified using an assay that includes the step of detecting the presence or determining the proportion of the relevant protein at a given intracellular location, for example, using an antibody / reporter-based method (Western blot, ELISA, immunohistochemistry / cytochemistry, etc.). Intracellular localization can be analyzed, for example, by immunocytochemistry of extracts prepared from different cell fractions or by Western blot, and organelle markers and / or labeled proteins of known intracellular localization can be used. The assay may include the step of treating cells / tissues with the agent and subsequently comparing the intracellular localization of the relevant protein in such cells with the intracellular localization of the relevant protein in cells / tissues under appropriate control conditions (e.g., untreated / solvent-treated cells / tissues).

[0097] Agents that can inhibit the interaction between the LINC complex protein and its interaction partner can be identified using an assay that includes detecting the level of interaction between the LINC complex protein and its interaction partner, for example, using an antibody / reporter-based method. The level of interaction between the LINC complex protein and its interaction partner can be analyzed using, for example, resonance energy transfer techniques (e.g., FRET, BRET), co-immunoprecipitation, or methods for analyzing correlates of the interaction (e.g., the function of the LINC complex). The assay can include treating the cell / tissue with the agent and subsequently comparing the level of interaction in such cell / tissue with the level of interaction in a cell / tissue under appropriate control conditions (e.g., untreated / solvent-treated cell / tissue). The interaction between the LINC complex protein and its interaction partner can also be analyzed using techniques such as ELISA, surface plasmon resonance, or biolayer interferometry analysis. The assay can include comparing the level of interaction in the presence of the agent with the level of interaction under appropriate control conditions (e.g., in the absence of the agent).

[0098] Agents that can inhibit the function of the LINC complex can be identified using an assay that includes detecting the level of a correlate of the LINC complex function. The LINC complex inhibitor according to the present disclosure can be any agent(s) that achieve the desired inhibitory activity. In some embodiments, the LINC complex inhibitor can be or can include a peptide / polypeptide, small molecule, nucleic acid, or biomolecule.

[0099] In some embodiments, the LINC complex inhibitor can bind to the LINC complex, the LINC complex protein, or the interaction partner of the LINC complex protein.

[0100] A LINC complex inhibitor may exhibit specific binding to a related factor / complex (i.e., a LINC complex protein or an interaction partner for a LINC complex protein). As used herein, "specific binding" refers to a binding that is selective and distinguishable from non-specific binding to non-target molecules. A LINC complex inhibitor that specifically binds to a LINC complex protein or an interaction partner for a LINC complex protein preferably binds to the related factor with a greater affinity and / or for a longer duration than other non-target molecules; such a LINC complex inhibitor may be considered "specific for" the related factor.

[0101] In some embodiments, a LINC complex inhibitor can inhibit the interaction between a LINC complex protein and an interaction partner for the LINC complex protein. In some embodiments, a LINC complex inhibitor can inhibit LINC complex function. In some embodiments, a LINC complex inhibitor acts as a competitive inhibitor of the interaction between a LINC complex protein and an interaction partner for the LINC complex protein. A LINC complex inhibitor can occupy or otherwise reduce access to the region of the LINC complex protein required for binding to an interaction partner for the LINC complex protein, or can occupy or otherwise reduce access to the region of the interaction partner for the LINC complex protein required for binding to the LINC complex protein.

[0102] In some embodiments, a LINC complex inhibitor mimics an interaction partner for a LINC complex protein. In some embodiments, a LINC complex inhibitor inhibits the interaction between the SUN domain and the KASH domain.

[0103] In some embodiments, the LINC complex inhibitor inhibits the association between the C-terminal region of the KASH domain and the deep pocket on the surface of the SUN domain. In some embodiments, the LINC complex inhibitor binds to the SUN domain and inhibits the approach of the KASH domain to the deep pocket of the SUN domain. In some embodiments, the LINC complex inhibitor binds to the KASH domain and inhibits the approach of the KASH domain to the deep pocket on the surface of the SUN domain.

[0104] In some embodiments, the LINC complex inhibitor inhibits the formation of a disulfide bond between the SUN domain and the KASH domain or breaks this disulfide bond. In some embodiments, the LINC complex inhibitor targets the C-terminal proline-rich region of the KASH domain-containing protein.

[0105] In some embodiments, the LINC complex inhibitor inhibits the oligomerization of the SUN domain-containing protein. In some embodiments, the LINC complex inhibitor targets the stalk region of the SUN domain-containing protein.

[0106] In some embodiments, the LINC complex inhibitor inhibits the protein-protein interaction between the SUN domain and the KASH domain. In some embodiments, the LINC complex inhibitor inhibits the protein-protein interaction between SUN1 and Nesprin-1, SUN2 and Nesprin-1, SUN1 and Nesprin-2, SUN1 and Nesprin-3, SUN2 and Nesprin-2 or SUN2 and Nesprin-3. In some embodiments, the LINC complex inhibitor inhibits the protein-protein interaction between SUN1 and Nesprin-1.

[0107] The ability of a candidate LINC complex inhibitor to inhibit the interaction between a LINC complex protein and an interaction partner for the LINC complex protein can be evaluated, for example, by analysis of the interaction in the presence of the candidate LINC complex inhibitor or following incubation with one or both of the interaction partners together with the candidate LINC complex inhibitor. An example of an assay suitable for determining whether a given binder is capable of inhibiting the interaction between a LINC complex protein and an interaction partner for the LINC complex protein is a competitive ELISA.

[0108] In some embodiments, a molecule that binds to a LINC complex protein or an interaction partner for the LINC complex protein inhibits the ability of the LINC complex protein to bind to the interaction partner for the LINC complex protein.

[0109] In some embodiments, a LINC complex inhibitor can bind to a LINC complex protein or an interaction partner for the LINC complex protein and inhibit the interaction between the LINC complex protein and the interaction partner for the LINC complex protein.

[0110] A LINC complex inhibitor that can bind to a LINC complex protein or an interaction partner for the LINC complex protein and inhibit the interaction between the LINC complex protein and the interaction partner for the LINC complex protein can be identified using any assay suitable for detecting the binding of the molecule to the relevant factor (i.e., the LINC complex protein or the interaction partner for the LINC complex protein) and the inhibition of the interaction between the LINC complex protein and the interaction partner for the LINC complex protein. Such an assay can include, for example, steps of detecting the formation of a complex between the relevant factor and the candidate inhibitory molecule and / or detecting the formation of a complex between the LINC complex protein and the interaction partner for the LINC complex protein.

[0111] In some embodiments, a LINC complex inhibitor that binds to a LINC complex protein or an interaction partner for a LINC complex protein and inhibits the interaction between the LINC complex protein and the interaction partner for the LINC complex protein may be, for example, a peptide / polypeptide.

[0112] The LINC complex inhibitor may be based on, for example, an interaction partner for a relevant factor to which the inhibitor binds (i.e., a LINC complex protein, or an interaction partner for a LINC complex protein).

[0113] As used herein, a peptide / polypeptide "based on" a reference protein comprises or consists of an amino acid sequence having a high sequence identity (e.g., at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to all or a portion of the amino acids of the reference protein.

[0114] For example, a LINC complex inhibitor that binds to a LINC complex protein may comprise or consist of a peptide / polypeptide fragment of an interaction partner for the LINC complex protein. Similarly, a LINC complex inhibitor that binds to an interaction partner for a LINC complex protein may comprise / consist of a peptide / polypeptide fragment of the LINC complex protein.

[0115] Such inhibitors preferably have the ability to bind to the relevant factor, but lack one or more other properties of the protein that is the basis of the inhibitor or exhibit a reduced level of those properties. For example, a LINC complex inhibitor may contain the amino acid sequence(s) necessary for binding to the relevant factor and may lack the amino acid sequence(s) necessary for one or more other properties of the protein that is the basis of the inhibitor.

[0116] Such peptide / polypeptide LINC complex inhibitors are referred to as "decoy", "dominant negative" or "mimetic" versions of the protein on which the inhibitor is based and may preferably exhibit competitive inhibition of the interaction between the LINC complex protein and its interaction partner. In this way, such peptide / polypeptide LINC complex inhibitors inhibit the formation of the LINC complex and / or disrupt existing LINC complexes through substitution of the endogenous interaction partner to form non-functional complexes / complexes with reduced functional levels.

[0117] The dominant negative version of SUN1 is described, for example, in Crisp et al., J Cell Biol. (2006) 172(1): 41-53. The dominant negative SUN2 is described, for example, in Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8): 1892-905.

[0118] The dominant negative versions of KASH1 are described, for example, in Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8):1892-905, Grady et al., Proc. Natl. Acad. Sci. U.S.A. (2005) 102:4359-4364, and Libotte et al., MBoC (2005) 16:3411-3424. The dominant negative versions of KASH2 are described, for example, in Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8):1892-905, Libotte et al., MBoC (2005) 16:3411-3424, Zhen et al., J. Cell. Sci. (2002) 115:3207-3222, and Kim et al., Sci Transl Med (2018) 10. The dominant negative versions of KASH3 are described, for example, in Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8):1892-905. The dominant negative versions of KASH4 are described, for example, in Roux et al., Proc. Natl. Acad. Sci. U.S.A. (2009) 106:2194-2199. The dominant negative versions of KASH5 are described, for example, in Horn et al., J Cell Biol (2013) 202:1023-1039.

[0119] In some embodiments, the peptide / polypeptide LINC complex inhibitor is based on a SUN domain-containing protein (e.g., a SUN domain-containing protein described herein). In some embodiments, the peptide / polypeptide LINC complex inhibitor is a decoy / dominant negative version of a SUN domain-containing protein.

[0120] LINC complex inhibitors based on SUN domain-containing proteins may exhibit binding to KASH domain-containing proteins (e.g., KASH domain-containing proteins described herein), but lack one or more other properties of the SUN domain-containing protein on which the inhibitor is based (e.g., binding to SUN domain-containing proteins, nucleocytoplasmic proteins, lamins, and / or chromatin-binding proteins) or exhibit a decrease in the level of such properties.

[0121] LINC complex inhibitors based on SUN domain-containing proteins may exhibit binding to SUN domain-containing proteins (e.g., SUN domain-containing proteins described herein), but lack one or more other properties of the SUN domain-containing protein on which the inhibitor is based (e.g., binding to KASH domain-containing proteins, nucleocytoplasmic proteins, lamins, and / or chromatin-binding proteins) or exhibit a decrease in the level of such properties.

[0122] LINC complex inhibitors based on SUN domain-containing proteins preferably contain a SUN domain. In some embodiments, the LINC complex consists of or consists essentially of a SUN domain. The peptide / polypeptide may lack the amino acid sequence(s) of the SUN domain-containing protein that constitute protein domains other than the SUN domain. The peptide / polypeptide preferably lacks the properties of the endogenous SUN domain-containing protein other than the properties mediated by the SUN domain. The peptide / polypeptide may act as a dominant negative peptide / polypeptide and may be able to inhibit the interaction between the endogenous SUN domain-containing protein and the endogenous interaction partner for the SUN domain-containing protein.

[0123] In some embodiments, the LINC complex inhibitor comprises, or consists of, a SUN domain of SUN1, SUN2, SUN3, SUN5, SPAG4 or SUCO, or a SUN domain having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of the SUN domains of SUN1, SUN2, SUN3, SUN5, SPAG4 or SUCO. In some embodiments, the LINC complex inhibitor comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 82, 83, 84, 85, 86 or 87, or an amino acid sequence having at least 70%, preferably 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: 82, 83, 84, 85, 86 or 87.

[0124] In some embodiments, the LINC complex inhibitor lacks the complete amino acid sequence of SUN1, SUN2, SUN3, SUN5, SPAG4 or SUCO. In some embodiments, the LINC complex inhibitor lacks the amino acid sequence according to SEQ ID NO: 88, 89, 90, 91, 92 or 93.

[0125] In some embodiments, the LINC complex inhibitor lacks the amino acid sequence of a SUN domain-containing protein required to bind to a nucleocytoplasmic protein, lamins, and / or chromatin-binding proteins. In some embodiments, the LINC complex inhibitor lacks the amino acid sequence according to SEQ ID NO: 94, 95 or 96.

[0126] In some embodiments, the peptide / polypeptide LINC complex inhibitor is based on a KASH domain-containing protein (e.g., a KASH domain-containing protein described herein). In some embodiments, the peptide / polypeptide LINC complex inhibitor is a decoy / dominant negative version of a KASH domain-containing protein.

[0127] LINC complex inhibitors based on KASH domain-containing proteins may show binding to SUN domain-containing proteins (e.g., SUN domain-containing proteins described herein), but lack one or more other properties of the KASH domain-containing proteins on which these inhibitors are based (e.g., binding to KASH domain-containing proteins, cytoplasmic proteins, cytoskeletal proteins, microfilaments, actin, microtubules, tubulin, microtubule motors, or intermediate filament proteins) or show a decrease in the level of this property.

[0128] LINC complex inhibitors based on KASH domain-containing proteins may show binding to KASH domain-containing proteins (e.g., KASH domain-containing proteins described herein), but lack one or more other properties of the KASH domain-containing proteins on which these inhibitors are based (e.g., binding to SUN domain-containing proteins, cytoplasmic proteins, cytoskeletal proteins, microfilaments, actin, microtubules, tubulin, microtubule motors, or intermediate filament proteins) or show a decrease in the level of this property.

[0129] LINC complex inhibitors based on KASH domain-containing proteins preferably contain a KASH domain. In some embodiments, the LINC complex consists of or consists essentially of a KASH domain. The peptide / polypeptide may lack the amino acid sequence(s) of the KASH domain-containing protein that constitute protein domains other than the KASH domain. The peptide / polypeptide preferably lacks the properties of the endogenous KASH domain-containing protein other than those mediated by the KASH domain. The peptide / polypeptide may act as a dominant negative peptide / polypeptide and may be able to inhibit the interaction between the endogenous KASH domain-containing protein and the endogenous interaction partner for the KASH domain-containing protein.

[0130] In some embodiments, the LINC complex inhibitor comprises, or consists of, a KASH domain having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the KASH domain of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, LRMP, or a KASH domain of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 or LRMP. In some embodiments, the LINC complex inhibitor comprises, or consists of, an amino acid sequence having at least 70%, preferably 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: 97, 98, 99, 100, 101 or 114, or the amino acid sequence set forth in SEQ ID NO: 97, 98, 99, 100, 101 or 114.

[0131] In some embodiments, the LINC complex inhibitor lacks the complete amino acid sequence of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 or LRMP. In some embodiments, the LINC complex inhibitor lacks an amino acid sequence according to SEQ ID NO: 102, 103, 104, 105, 106 or 113.

[0132] In some embodiments, the LINC complex inhibitor lacks the amino acid sequence of a KASH domain-containing protein required to bind to a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, a microtubule, tubulin, a microtubule motor, or an intermediate filament protein. In some embodiments, the LINC complex inhibitor lacks an amino acid sequence according to SEQ ID NO: 107 or 108.

[0133] In some embodiments, a peptide / polypeptide LINC complex inhibitor that binds to a LINC complex / LINC complex protein / interaction partner for the LINC complex protein and inhibits the interaction between the LINC complex protein and the interaction partner for the LINC complex protein and / or LINC complex function can be, for example, a peptide aptamer, thioredoxin, monobody, anticalin, knotted domain, avimer, notchine, fynomers, atrimers, DARPin, affibodies, nanobodies (i.e., single domain antibodies (sdAb)) affilins, armadillo repeat proteins (ArmRP), OBody and fibronectin, for example, as reviewed in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, which is hereby incorporated by reference in its entirety herein (see also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48). Further, peptide / polypeptide LINC complex inhibitors contemplated in connection with the present invention include antibodies (immunoglobulins) such as monoclonal antibodies, polyclonal antibodies, single-specificity antibodies, multi-specificity antibodies (e.g., bispecific antibodies), and fragments and derivatives thereof (e.g., Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabody, tribody, scFv-Fc, minibody, single domain antibodies (e.g., VhH), etc.).

[0134] Such peptide / polypeptide LINC complex inhibitors can be identified by screening a library of peptides / polypeptides relevant for LINC complex inhibition.

[0135] The peptide / polypeptide LINC complex inhibitor may further contain additional amino acids or sequences of amino acids. For example, the peptide / polypeptide may contain amino acid sequence(s) for promoting expression, folding, transport, processing, purification, or detection. For example, the peptide / polypeptide may contain, at the N- or C-terminus of the antigen-binding molecule / polypeptide as necessary, a sequence encoding His (e.g., 6×His), Myc, GST, MBP, FLAG, HA, E, or a biotin tag. In some embodiments, the peptide / polypeptide contains a detectable moiety, such as a fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzymatic label.

[0136] In some embodiments, the peptide / polypeptide may contain an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence. The peptide / polypeptide may further contain a signal peptide (also known as a leader sequence or signal sequence). The signal peptide usually consists of a sequence of 5 to 30 hydrophobic amino acids that form a single alpha helix. Secreted proteins and proteins expressed on 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 the newly synthesized peptide / polypeptide. The signal peptide is often removed by cleavage and is thus not included in the mature peptide / polypeptide.

[0137] In some embodiments, the N-terminal signal sequence is derived from a secreted protein or a type I transmembrane protein. In some embodiments, the secreted protein or type I transmembrane protein is selected from human serum albumin, proinsulin, transferrin receptor, EGF receptor, preproopiomelanocortin, carboxypeptidase, complement protein, fibrinogen, cytokine, chemokine, fibrinogen, pancreatic digestive enzymes (e.g., protease, amylase, and lipase), endoplasmic reticulum lumen proteins such as protein disulfide isomerase and GRP94. In some embodiments, the N-terminal signal sequence is derived from human serum albumin. In some embodiments, the N-terminal signal sequence is not preceded by any other tag at its N-terminus.

[0138] In some embodiments, the peptide / polypeptide comprises a signal peptidase cleavage site. In some embodiments, the signal peptidase cleavage site is a signal peptidase cleavage site derived from or is one of human serum albumin, proinsulin, transferrin receptor, EGF receptor, preproopiomelanocortin, pancreatic digestive enzymes (e.g., protease, amylase, and lipase), endoplasmic reticulum lumen proteins , such as protein disulfide isomerase and GRP94. In some embodiments, the signal peptidase cleavage site is derived from human serum albumin.

[0139] In some embodiments, the peptide / polypeptide comprises a sequence for targeted intracellular transport of the peptide / polypeptide to the nuclear membrane. In some embodiments, the peptide / polypeptide comprises an endoplasmic reticulum (ER) retention motif. Suitable endoplasmic reticulum retention sequences are known in the art. In some embodiments, the ER retention motif is the KDEL sequence. The KDEL sequence, KDEL or a variant thereof, functions to retain a protein in the endoplasmic reticulum. KDEL variants can include or consist of the prosite motif [KRHQSA]-[DENQ]-E-L (described in Hulo et al., 2006, Nucleic acids research 34:D227-D230), or variants described by Raykhel et al., 2007 (J. Cell Biol. 179(6):1193-1204), who proposed an extended prosite motif definition. Raykhel demonstrated endoplasmic retention with variants that include F at the 4th position (i.e., the K position) as an alternative to KRHQSA, a range of residues at the 3rd position (i.e., the D position) extending well beyond DENQ, F or M at the 1st position (L position), and D at the 2nd position (i.e., the E position). For example, the KDEL motif can be CDEL, KCEL or HVEL proposed by Raykhel et al. Thus, in some aspects disclosed herein, the endoplasmic reticulum (ER) retention motif is KDEL or a variant thereof that exhibits ER retention activity.

[0140] In some embodiments, the peptide / polypeptide comprises a C-terminal targeting peptide sequence. In some embodiments, the C-terminal targeting peptide sequence prevents secretion of the peptide / polypeptide. In some embodiments, the C-terminal targeting peptide sequence is the KDEL tetrapeptide Golgi retrieval sequence. Examples of such constructs are shown in FIGS. 11 and 12.

[0141] In some embodiments, the peptide / polypeptide comprises an epitope tag. In some embodiments, the epitope tag is located either at the N-terminus or downstream (later) of the peptide / polypeptide of the C-terminal targeting peptide sequence [e.g., an endoplasmic reticulum retention motif such as the KDEL sequence], or at any of the peptide / polypeptide except upstream (before) of the N-terminal signal sequence. In some embodiments, the epitope tag is selected from a cellulose binding domain (CBD), chloramphenicol acetyltransferase (CAT), dihydrofolate reductase (DHFR), one or more FLAG tags, glutathione S-transferase (GST), green fluorescent protein (GFP), hemagglutinin A (HA), histidine (His), herpes simplex virus (HSV), luciferase, maltose binding protein (MBP), c-Myc, protein A, protein G, streptavidin, T7, thioredoxin, V5, vesicular stomatitis virus glycoprotein (VSV-G), and combinations thereof. In some embodiments, the epitope tag is hemagglutinin A (HA).

[0142] In some embodiments, the peptide / polypeptide comprises a signal sequence (i.e., prior to cleavage to remove the signal sequence), a humanized Sun1DN sequence and a KDEL sequence (e.g., encoded by SEQ ID NO: 4). In some embodiments, the peptide / polypeptide comprises a signal sequence, a humanized Sun2DN sequence and a KDEL sequence (e.g., encoded by SEQ ID NO: 5).

[0143] For the SUN domain construct, the SUN domain (crystal structure analyzed by the Kutay and Schwartz labs [Sosa et al., Cell 149(5):10 The additional 20 amino acid residues corresponding to the alpha-3 region of coil-coil-2 on pages 35-47 (2012) can bind to the KASH domain [Jahed et al., Biophysical Journal 114(5):1190-1203 (2018)] and are predicted to be sufficient to disrupt the SUN-KASH interaction. This is in place of the entire luminal domain (coil-coil domain and SUN domain). The nucleic acid sequence of the human SUN1 SUN domain is shown in SEQ ID NO: 80. The presence of the signal sequence and the KDEL sequence may be important for targeting to the perinuclear lumen.

[0144] In some embodiments, the LINC complex inhibitor comprises, or consists of, an amino acid sequence set forth in SEQ ID NO: 115, or an amino acid sequence having at least 70%, preferably 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: 115. In some embodiments, the LINC complex inhibitor comprises, or consists of, an amino acid sequence set forth in SEQ ID NO: 116, or an amino acid sequence having at least 70%, preferably 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: 116.

[0145] In some embodiments, the peptide / polypeptide comprises the luminal domain of a SUN domain-containing protein, or the SUN domain of a SUN domain-containing protein. In some embodiments, the luminal domain of Sun1 comprises amino acids 458-913 of full-length mouse Sun1 (UniProt: Q9D666) or its human equivalent that includes the coil-coil domain and the SUN domain and lacks the transmembrane domain. A schematic diagram of the dominant negative form of Sun1 is shown in Figure 7.

[0146] In some embodiments, the peptide / polypeptide comprises a humanized Sun1DN sequence or a humanized Sun2DN sequence. Rather than expressing components of the luminal domain of SUN domain-containing proteins, expression of the KASH domain can disrupt the LINC complex by competing with endogenous Nesprin (including the KASH domain) for binding to the SUN1 and SUN2 domains.

[0147] Thus, in some embodiments, the peptide / polypeptide comprises a KASH domain and an N-terminal stabilizer polypeptide sequence. In some embodiments, the peptide / polypeptide comprises a KASH domain that includes a transmembrane domain and a SUN interaction peptide. In some embodiments, the peptide / polypeptide comprises a KASH domain that traverses the outer nuclear membrane, a SUN interaction KASH peptide that extends into the perinuclear lumen at the C-terminus, and an N-terminal stabilizer polypeptide sequence in the cytoplasm.

[0148] It is not expected that a KASH domain construct having an extension after the last C-terminal amino acid of a naturally occurring KASH domain will function; that is, it is understood that even a C-terminal tag or an additional carboxy-terminal single amino acid will disrupt the KASH interaction with SUN.

[0149] In some embodiments, the KASH domain is selected from the group consisting of KASH1 (derived from Nesprin-1 (SYNE1 gene)), KASH2 (derived from Nesprin-2 (SYNE2 gene)), KASH3 (derived from Nesprin-3 (SYNE3 gene)), KASH4 (derived from Nesprin-4 (SYNE4 gene)), and KASH5 (derived from KASH5 / CCDC155 (KASH5 gene)).

[0150] In some embodiments, the KASH1 domain comprises the human amino acid sequence set forth in SEQ ID NO: 7; the KASH2 domain comprises the human amino acid sequence set forth in SEQ ID NO: 9; the KASH3 domain comprises the human amino acid sequence set forth in SEQ ID NO: 11; the KASH4 domain comprises the human amino acid sequence set forth in SEQ ID NO: 13; the KASH5 domain comprises the human amino acid sequence set forth in SEQ ID NO: 15. An alignment of the five KASH amino acid sequences is shown in FIG. 14.

[0151] In some embodiments, the KASH domain nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the nucleic acid sequence of the KASH1 domain set forth in SEQ ID NO: 6; the nucleic acid sequence of the KASH2 domain set forth in SEQ ID NO: 8; the nucleic acid sequence of the KASH3 domain set forth in SEQ ID NO: 10; the nucleic acid sequence of the KASH4 domain set forth in SEQ ID NO: 12; or the nucleic acid sequence of the KASH5 domain set forth in SEQ ID NO: 14.

[0152] In some embodiments, for example, for the purpose of clinical use, the KASH domain is the human KASH1 domain of SYNE1 having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the nucleic acid sequence of the human KASH1 domain set forth in SEQ ID NO: 6.

[0153] In some embodiments, the KASH domain does not include any extension after the last C-terminal amino acid as compared to the naturally occurring KASH domain. In some embodiments, the N-terminal stabilizer polypeptide sequence is selected from the group consisting of green fluorescent protein (GFP), cellulose binding domain (CBD), chloramphenicol acetyltransferase (CAT), dihydrofolate reductase (DHFR), glutathione S-transferase (GST), luciferase, maltose binding protein (MBP), protein A, protein G, streptavidin, thioredoxin, DHFR, multiples and combinations thereof. In some embodiments, the N-terminal stabilizer polypeptide sequence forms an individually folded domain.

[0154] In some embodiments, the peptide / polypeptide comprises a KASH domain and an N-terminal stabilizer polypeptide sequence, and the KASH domain is selected from the group consisting of KASH1, KASH2, KASH3, KASH4 and KASH5. In some embodiments, the N-terminal stabilizer polypeptide sequence is green fluorescent protein (GFP).

[0155] In some embodiments, the LINC complex inhibitor is a small molecule inhibitor of the LINC complex. As used herein, "small molecule" refers to an organic compound of low molecular weight (<1000 daltons, typically about 300-700 daltons).

[0156] The small molecule LINC complex inhibitor may bind to the LINC complex, a LINC complex protein, or an interaction partner for the LINC complex protein. The small molecule inhibitor LINC complex inhibitor may inhibit the interaction between the LINC complex protein and the interaction partner for the LINC complex protein. The small molecule inhibitor LINC complex inhibitor may bind to the LINC complex and inhibit LINC complex function.

[0157] Suitable small molecule LINC complex inhibitors can be identified, for example, by screening a small molecule library described in Example 7 herein. In some embodiments, the LINC complex inhibitor is or comprises a nucleic acid.

[0158] The nucleic acid can bind to a LINC complex, a LINC complex protein, or an interaction partner for the LINC complex protein. The nucleic acid can inhibit the interaction between the LINC complex protein and the interaction partner for the LINC complex protein. The nucleic acid can bind to the LINC complex and inhibit LINC complex function. The nucleic acid aptamer is outlined, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017, 16(3):181 - 202. The nucleic acid aptamer can be identified and / or produced by the systematic evolution of ligands by exponential enrichment (SELEX) method or by developing SOMAmer (slow off - rate modified aptamer) (Gold L et al., (2010) PLoS ONE 5(12):e15004). Aptamers and SELEX are described in Tuerk and Gold, Science (1990) 249(4968):505 - 10, and WO 91 / 19813.

[0159] The nucleic acid aptamer may contain DNA and / or RNA and may be single - stranded or double - stranded. The nucleic acid aptamer may contain, for example, chemically modified nucleic acids in which the sugar and / or phosphate and / or base are chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modifications at the 2'-position of ribose.

[0160]

[0161] ​Nucleic acid aptamers can be chemically synthesized, for example, on a solid support. Solid-phase synthesis can use phosphoramidite chemistry. Briefly, a solid-supported nucleotide is detritylated and then coupled with an appropriately activated nucleoside phosphoramidite to form a phosphite triester linkage. Next, capping may occur, followed by oxidation of the phosphite triester, typically with iodine. The cycle can then be repeated to construct the aptamer (see, for example, Sinha, N.D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, S.L.; Lyer, R.P. (1992). Tetrahedron 48(12):2223).

[0162] In some embodiments, the LINC complex inhibitor can reduce the expression (e.g., gene and / or protein expression) of the LINC complex protein. In some embodiments, the LINC complex inhibitor reduces or inhibits the expression of the endogenous LINC complex protein.

[0163] When the expression of the LINC complex protein is inhibited, the amount of the LINC complex protein and / or the amount of the LINC complex containing the constituent proteins in a cell / tissue / organ / organ system / subject decreases. For example, in a given cell, when the expression of the LINC complex protein is inhibited, the level of the INC complex protein and / or the LINC complex containing the constituent proteins decreases compared to untreated cells.

[0164] The inhibition may be partial. The preferred degree of inhibition is one of at least 50%, more preferably at least 60%, 70%, 80%, 85% or 90%. An inhibition level between 90% and 100% is considered "silencing" of expression or function. Gene and protein expression can be determined as described herein or by methods well known in the art to those of skill in the art.

[0165] In some embodiments, the LINC complex inhibitor reduces or prevents the expression of SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of SUN1 or SUN2. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of a polypeptide according to SEQ ID NO: 82, 83, 84, 85, 86 or 87. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of a polypeptide according to SEQ ID NO: 82 or 83.

[0166] In some embodiments, the LINC complex inhibitor reduces or prevents the expression of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, or LRMP. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of a polypeptide according to SEQ ID NO: 102, 103, 104, 105, 106, or 113. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of Nesprin-1, Nesprin-2, or Nesprin-3. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of a polypeptide according to SEQ ID NO: 102, 103, or 104.

[0167] In some embodiments, the LINC complex inhibitor can target specific domains / regions of the LINC complex protein. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of an isoform of the LINC complex protein that includes one or more domains / regions of interest.

[0168] For example, the domain or region of interest can be or can include the domain necessary for interaction with interaction partners for SUN domain, KASH domain, and / or LINC complex proteins. For example, the domain or region of interest can be necessary for interaction with KASH domain-containing proteins, SUN domain-containing proteins, nucleocytoplasmic proteins, lamins, chromatin-binding proteins, cytoplasmic proteins, cytoskeletal proteins, microfilaments, actin, microtubules, tubulin, microtubule motors, and / or intermediate filament proteins.

[0169] In some embodiments, the LINC complex inhibitor can alter the splicing of the pre-mRNA encoding the LINC complex protein to increase the proportion of mature mRNA encoding isoform(s) lacking the relevant domain / region and / or decrease the proportion of mature mRNA encoding isoform(s) containing the relevant domain / region.

[0170] In some embodiments, the LINC complex inhibitor can modify the nucleic acid encoding the LINC complex protein to increase the expression of isoform(s) lacking the relevant domain / region and / or modify the nucleic acid encoding the LINC complex protein to decrease the expression of isoform(s) containing the relevant domain / region of the protein.

[0171] In some embodiments, the LINC complex inhibitor is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an antisense nucleic acid. In some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide (ASO). The antisense oligonucleotide can be single-stranded and can bind to a target oligonucleotide, such as mRNA, by complementary sequence binding.

[0172] The ASO can be designed to inhibit / prevent the expression of the LINC complex protein or a specific isoform thereof. Oligonucleotides designed to inhibit or prevent the expression of LINC complex proteins or specific isoforms thereof may have substantial sequence identity to a portion of the nucleic acid encoding the LINC complex protein / associated isoform, or its complementary sequence.

[0173] In some embodiments, the inhibitory nucleic acid reduces the expression of LINC complex proteins by RNA interference (RNAi). RNAi involves the inhibition of gene expression and translation by targeted neutralization of mRNA molecules. In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). The targeting of heterochromatin complexes at specific chromosomal loci and the role of RNAi mechanisms and small RNAs in epigenetic gene silencing have been demonstrated. Double-stranded RNA (dsRNA)-dependent post-transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing for a short period of time. This silencing functions as a signal to promote the degradation of mRNA with sequence identity. 20-nt siRNAs are generally of sufficient length to induce gene-specific silencing but short enough to avoid the host response. The reduction in the expression of the targeted gene product can be substantial, with 90% silencing sometimes being induced by a small number of siRNA molecules. RNAi-based therapeutic agents are being advanced into Phase I, II, and III clinical trials for several indications (Nature, January 22, 2009; 457(7228):426-433).

[0174]

[0175] In the art, such RNA sequences are named "short or small interfering RNA" (siRNA) or "microRNA" (miRNA) depending on their origin. Both types of sequences can be used to down-regulate gene expression by binding to complementary RNA to initiate mRNA removal (RNAi) or by stopping mRNA translation into protein. siRNA is derived by processing of long double-stranded RNA and, when found in nature, is typically of exogenous origin. Micro interfering RNA (miRNA) is an endogenously encoded small non-coding RNA and is derived by processing of short hairpins.

[0176] Both siRNA and miRNA can inhibit the translation of mRNA having a partially complementary target sequence without RNA cleavage and can degrade mRNA having a fully complementary sequence.

[0177] SiRNA is typically double-stranded, and for optimizing the effectiveness of RNA-mediated down-regulation of the function of a target gene, the length of the siRNA molecule is preferably selected such that it ensures correct recognition of the siRNA by the RISC complex that mediates recognition of the mRNA target by the siRNA and is short enough for the siRNA to reduce the host response.

[0178] MiRNA is typically single-stranded and has a region that is partially complementary and allows the ligand to form a hairpin. miRNA is an RNA gene that is transcribed from DNA but not translated into protein. The DNA sequence encoding the miRNA gene is longer than the miRNA. This DNA sequence contains the miRNA sequence and approximately its reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complement base pair to form a partially double-stranded RNA segment. The design of microRNA sequences is discussed, for example, in John et al., PLoS Biology, 11(2), pages 1862-1879, 2004.

[0179] Typically, oligonucleotides intended to mimic the effects of siRNA or miRNA have 10 to 40 ribonucleotides (or synthetic analogs thereof), more preferably 17 to 30 ribonucleotides, still more preferably 19 to 25 ribonucleotides, and most preferably 21 to 23 ribonucleotides. In some embodiments of the invention using double-stranded siRNA, the molecule can have, for example, a symmetric 3' overhang of one or two (ribo)nucleotides, typically a dTdT 3' overhang of UU. Based on the disclosure provided herein, one of ordinary skill in the art can readily design appropriate siRNA and miRNA sequences using resources such as, for example, the Ambion siRNA Finder. siRNA and miRNA sequences can be made synthetically and added exogenously to cause gene downregulation or made using an expression system (e.g., a vector). In some embodiments, the siRNA is synthesized synthetically.

[0180] siRNA-mediated knockdown of LINC complex proteins and siRNAs for achieving such knockdown are described, for example, in Ostlund et al., Journal of Cell Science (2009) 122:4099-4108, Hatch and Hetzer, J Cell Biol. (2016) 215(1):27-36, Matsumoto et al., Nucleus. (2016) 7(1):68-83, Uzer et al., Stem Cells. (2015) 33(6):2063-76, Thakar et al., Mol Biol Cell. (2017) 28(1):182-191, Espigat-Georger et al., J Cell Sci. (2016) 129(22):4227-4237, Yang et al., Int J Mol Med. (2013) 32(4):805-12, Rajgor et al., PLoS One. 2012;7(7):e40098, Zhang et al., Exp Cell Res. (2016) 345(2):168-179, Warren et al., J Biol Chem. (2010) 285(2):1311-20 and King et al., Cytoskeleton (Hoboken) (2014) 71(7):423-34, the disclosures of which are hereby incorporated by reference in their entireties herein.

[0181] Relatively long double-stranded RNAs may be processed in cells to generate siRNAs (see, for example, Myers (2003) Nature Biotechnology 21: 324-328). Relatively long dsRNA molecules may, for example, have symmetric 3' or 5' overhangs of one or two (ribo) nucleotides, or may have blunt ends. Relatively long dsRNA molecules may be 25 nucleotides or longer. Preferably, relatively long dsRNA molecules are 25-30 nucleotides in length. More preferably, relatively long dsRNA molecules are 25-27 nucleotides in length. Most preferably, relatively long dsRNA molecules are 27 nucleotides in length. dsRNAs that are 30 nucleotides or longer can be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

[0182] Another alternative is the expression of short hairpin RNA molecules (shRNAs) in cells. shRNAs are more stable than synthetic siRNAs. shRNAs consist of short inverted repeats separated by a small loop sequence. One of the inverted repeats is complementary to the gene target. In cells, shRNAs are processed by DICER into siRNAs that degrade the target gene mRNA and suppress expression. In some embodiments, shRNAs are generated intracellularly by transcription from a vector. shRNAs may be generated intracellularly by transfecting cells with a vector encoding the shRNA sequence under the control of an RNA polymerase III promoter such as the human H1 or 7SK promoter or an RNA polymerase II promoter. Alternatively, shRNAs may be synthesized exogenously (in vitro) by transcription from a vector. Next, the shRNAs can be introduced directly into cells. Preferably, the shRNA molecule comprises a partial sequence of the gene encoding the LINC complex protein. Preferably, the shRNA sequence is 40-100 bases in length, more preferably 40-70 bases in length. The base of the hairpin is preferably 19-30 base pairs in length. The base may contain G-U base pairs that stabilize the hairpin structure.

[0183] shRNA-mediated knockdown of the LINC complex protein and the shRNA for achieving this knockdown are described, for example, in Kelkar et al., Nucleus. (2015) 6(6):479-489, Mross et al., Nucleus. (2018) 9(1):503-515, Xing et al., International Journal of Cell Biology (2017) Article ID:8607532, Arsenovic et al., Biophys J. (2016) 110(1):34-43 and Li et al., Scientific Reports (2017) 7:Article number:9157, which are hereby incorporated by reference in their entirety herein.

[0184] In some embodiments, the inhibitory nucleic acid is a splice-switching oligonucleotide (SSO). Splice-switching oligonucleotides are reviewed, for example, in Haves and Hastings, Nucleic Acids Res. (2016) 44(14):6549-6563, which is hereby incorporated by reference in its entirety herein. SSOs disrupt the normal splicing of target RNA transcripts by blocking RNA-RNA base pairing and / or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. The number / ratio of mature mRNA transcripts encoding the LINC complex protein or its specific isoform(s) may be altered using SSOs. SSOs may be designed to target specific regions of the target transcript, for example, to cause skipping of an exon of interest, for example, an exon encoding a domain / region of interest.

[0185] ​SSOs generally include modifications to the oligonucleotide sugar-phosphate backbone to prevent ribonuclease H cleavage, and can include, for example, phosphorodiamidate morpholino (PMO), peptide nucleic acid (PNA), locked nucleic acid (LNA), and / or 2’O-methyl (2’OMe) and 2’-O-methoxyethyl (MOE) ribose modifications.

[0186] Inhibitory nucleic acids can be recombinantly produced, for example, by transcription of a nucleic acid sequence contained within a vector. Transcription can be performed in a cell-free transcription reaction or in a cell containing the nucleic acid encoding the inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is produced intracellularly, for example, by transcription from a vector. Vectors encoding such molecules can be introduced into cells by any method known in the art. Optionally, the expression of the nucleic acid can be regulated using a cell / tissue (e.g., heart, muscle, etc.)-specific promoter.

[0187] Inhibitory nucleic acids can be synthesized using standard solid-phase or solution-phase synthesis techniques known in the art. In some embodiments, a LINC complex inhibitor is a molecule or molecules that can modify a nucleic acid encoding a LINC complex protein to reduce / prevent the expression of the LINC complex protein or a specific isoform(s) thereof.

[0188] The step of modifying a nucleic acid encoding a LINC complex protein can include the step of modifying the gene encoding the LINC complex protein. In some embodiments, the step of modifying a nucleic acid encoding a LINC complex protein includes the step of introducing an insertion, substitution, or deletion into the nucleic acid sequence encoding the LINC complex protein.

[0189] In some embodiments, the step of modifying the nucleic acid encoding the LINC complex protein includes modifying the nucleic acid to introduce a premature stop codon in the sequence transcribed from the nucleic acid. In some embodiments, the step of modifying the nucleic acid encoding the LINC complex protein includes modifying the nucleic acid to encode a truncated and / or non-functional version of the LINC complex protein. In some embodiments, the step of modifying the nucleic acid encoding the LINC complex protein includes modifying the nucleic acid to encode a version of the LINC complex protein that is misfolded and / or degraded. and / or includes modifying the nucleic acid to encode a version of the LINC complex protein that is misfolded and / or degraded.

[0190] The modification may be of a nucleic acid contained in a cell, such as an endogenous nucleic acid encoding the LINC complex protein. Due to the modification, the cell has a reduced level of gene and / or protein expression of the LINC complex protein or a specific isoform(s) thereof compared to an equivalent unmodified cell.

[0191] In some embodiments, the modification may be to a region of the nucleic acid encoding the LINC complex protein that is involved in (e.g., required for) LINC complex formation. For example, in some embodiments, the modification may target the region encoding the SUN domain of the gene encoding the SUN domain-containing protein. In some embodiments, the modification may target the region encoding the KASH domain of the gene encoding the KASH domain-containing protein.

[0192] In some embodiments, the modification is performed in vitro or ex vivo. In some embodiments, the modification is performed in vivo. Methods for modifying nucleic acids encoding a protein of interest and agents for achieving such modification are well known in the art and include, for example, modification of target nucleic acids by homologous recombination and targeted nucleic acid editing using site-specific nucleases (SSNs). For example, the inventors have demonstrated CRISPR / Cas9-mediated disruption of Sun1 and Nesprin-1 in the experimental examples herein.

[0193] Suitable methods may use targeting by homologous recombination, which is outlined, for example, in Mortensen Curr Protoc Neurosci. (2007) Chapter 4: Unit 4.29 and Vasquez et al., PNAS 2001, 98(15): 8403-8410, both of which are hereby incorporated by reference in their entirety herein. Targeting by homologous recombination involves the exchange of nucleic acid sequences through crossover events guided by homologous sequences.

[0194] In some embodiments, the method uses targeted nucleic acid editing using an SSN. Gene editing using an SSN is outlined, for example, in Eid and Mahfouz, Exp Mol Med. October 2016; 48(10): e265, which is hereby incorporated by reference in its entirety herein. Enzymes capable of creating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into the target nucleic acid sequence(s) of interest. DSBs may be repaired by error-prone non-homologous end joining (NHEJ), where the two ends of the break are often rejoined with nucleotide insertions or deletions. Alternatively, DSBs may be repaired by high-fidelity homology-directed repair (HDR), in which case a DNA template with ends homologous to the site of the break is supplied and introduced at the site of the DSB.

[0195] SSNs that can be engineered to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) systems.

[0196] The ZFN system is described, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is hereby incorporated by reference in its entirety. ZFNs include a programmable zinc finger DNA-binding domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). The DNA-binding domain can be identified by screening a zinc finger array that can bind to a target nucleic acid sequence.

[0197] The TALEN system is described, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is hereby incorporated by reference in its entirety. TALENs include a programmable DNA-binding TALE domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). TALEs include a repeat domain consisting of 33-39 amino acid repeats, which domain is identical except for two residues at positions 12 and 13 of each repeat that are repeat variable diresidues (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the following relationships: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501).

[0198] CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3, are reviewed, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety herein. These systems include an endonuclease (e.g., Cas9, Cpf1, etc.) and a single-guide RNA (sgRNA) molecule. The sgRNA can be engineered to direct endonuclease activity to a target nucleic acid sequence.

[0199] In some embodiments, LINC complex inhibition uses a site-specific nuclease (SSN) system that targets a LINC complex protein. Thus, in some embodiments, a LINC complex inhibitor comprises or consists of an SSN system that targets a LINC complex protein. In some embodiments, LINC complex inhibition uses a nucleic acid(s) encoding an SSN system that targets a LINC complex protein.

[0200] In some embodiments, the SSN system targets a region of the nucleic acid encoding a LINC complex protein that is involved in (e.g., required for) LINC complex formation. For example, in some embodiments, the SSN system can disrupt the expression of an exon encoding the SUN domain of a gene encoding a SUN domain-containing protein. In some embodiments, the SSN system can disrupt the expression of an exon encoding the KASH domain of a gene encoding a KASH domain-containing protein.

[0201] In certain embodiments, the SSN system can introduce an insertion / deletion into the nucleic acid sequence of a gene encoding a SUN domain-containing protein upstream of the sequence encoding a conserved tyrosine residue.

[0202] In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the SUN domain-containing protein upstream of the sequence encoding tyrosine at position 154 of SEQ ID NO: 82. In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the SUN domain-containing protein upstream of the sequence encoding tyrosine at position 153 of SEQ ID NO: 83. In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the SUN domain-containing protein upstream of the sequence encoding tyrosine at position 153 of SEQ ID NO: 84. In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the SUN domain-containing protein upstream of the sequence encoding tyrosine at position 151 of SEQ ID NO: 85. In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the SUN domain-containing protein upstream of the sequence encoding tyrosine at position 152 of SEQ ID NO: 86. It can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the SUN domain-containing protein.

[0203] In certain embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the SUN domain-containing protein upstream of the sequence encoding the conserved tyrosine residue.

[0204] In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the KASH domain-containing protein upstream of the sequence encoding the C-terminal proline-rich region.

[0205] In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the KASH domain-containing protein upstream of the sequence encoding proline at position 57 of SEQ ID NO: 97. In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the KASH domain-containing protein upstream of the sequence encoding proline at position 57 of SEQ ID NO: 98. In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the KASH domain-containing protein upstream of the sequence encoding proline at position 56 of SEQ ID NO: 99. In some embodiments, the SSN system can introduce insertions / deletions into the nucleic acid sequence of the gene encoding the KASH domain-containing protein upstream of the sequence encoding proline at position 56 of SEQ ID NO: 100.

[0206] In some embodiments, the SSN system is a ZFN system, a TALEN system, a CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system, or a CRISPR / C2c3 system.

[0207] In some embodiments, the SSN system is a CRISPR / Cas9 system. In such embodiments, inhibition of the LINC complex may utilize a CRISPR RNA (crRNA) that targets the nucleic acid encoding the LINC complex protein and a nucleic acid(s) encoding a trans-activating crRNA (tracrRNA) for processing the crRNA to its mature form. The CRISPR / Cas9 system for targeted disruption of the LINC complex proteins SUN1 and SUN2 is described in Schaller et al., J Virol. (2017) 91(19):pii:e00463-17, which is hereby incorporated by reference in its entirety herein.

[0208] Rather than expressing components of the luminal domain or KASH domain of SUN domain-containing proteins and disrupting the LINC complex by competing for binding to endogenous Nesprin (including the KASH domain) or Sun1 and Sun2 (including the SUN domain), another approach for disrupting the LINC complex is to modify the endogenous SUN domain or KASH domain such that the endogenous SUN domain or KASH domain cannot bind to its cognate LINC complex binding partner, or such that its binding ability to this partner is reduced.

[0209] Since both the SUN domain and the KASH domain are located at the C-terminus of their respective proteins, one way to create a modified SUN or KASH domain is to use the CRISPR / Cas system to modify the gene encoding the SUN or KASH domain protein, followed by CRISPR-induced non-homologous end joining to generate a premature termination codon at the 3’ end of each protein sequence. This is thought to result in truncated proteins with a mutated C-terminal SUN or KASH domain. The truncated proteins are expressed and localized to the membrane, but are thought to be unable to interact with their cognate LINC complex partners.

[0210] Thus, in some embodiments, the LINC complex inhibitor is the CRISPR-Cas or another synthetic nuclease system capable of modifying the nucleic acid encoding the SUN domain or KASH domain of the endogenous SUN or Nesprin protein, respectively.

[0211] In some embodiments, the CRISPR-Cas system modifies the endogenous SUN domain or KASH domain of the Sun1 or Nesprin-1 protein to disrupt the LINC complex. The respective nucleic acids are Sun1 and Syne1.

[0212] In some embodiments, the CRISPR-Cas system includes a gRNA nucleic acid sequence comprising 5’-GCACAATAGCCTCGGATGTCG-3’ (SEQ ID NO: 66) and can modify the SUN domain of mouse Sun1.

[0213] In some embodiments, the CRISPR-Cas system includes a gRNA nucleic acid targeting the human SUN1 domain shown in SEQ ID NO: 80. In some embodiments, the gRNA nucleic acid sequence targets the end of exon 20 comprising the nucleic acid sequence shown in SEQ ID NO: 81. In some embodiments, the gRNA nucleic acid sequence targets a SUN1 nucleic acid sequence selected from the group consisting of SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; and SEQ ID NO: 65 shown in Table 3.

[0214] In some embodiments, the CRISPR-Cas system includes a gRNA nucleic acid sequence comprising 5’-CCGTTGGTATATCTGAGCAT-3’ (SEQ ID NO: 34) and can modify the KASH domain of mouse Syne-1.

[0215] In some embodiments, the CRISPR-Cas system includes a gRNA nucleic acid sequence targeting the human KASH domain shown in SEQ ID NO: 6. In some embodiments, the gRNA nucleic acid sequence targets a nucleic acid sequence selected from the group consisting of SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; and SEQ ID NO: 54 shown in Table 3. In some embodiments, the CRISPR-Cas system is a CRISPR-Cas9 system or a variant thereof.

[0216] Administration / delivery of LINC complex inhibitors Administration of the LINC complex inhibitor according to the present invention to a subject is preferably at a “therapeutically effective” amount or “prophylactically effective” amount, which is sufficient to exhibit a therapeutic / preventive effect on the subject.

[0217] The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the disease being treated / prevented, as well as the nature of the LINC complex inhibitor. The prescription of treatment, such as determination of dosage, etc., is within the responsibility of the general practitioner and other physicians, and typically takes into account the disease / condition being treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the practicing physician. Examples of the techniques and protocols described above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0218] In therapeutic applications, the LINC complex inhibitor is preferably formulated as a medicament or pharmaceutical composition together with one or more other pharmaceutically acceptable components well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, antioxidants, lubricants , stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavorants, and sweetening agents.

[0219] As used herein, the term “pharmaceutically acceptable” relates to compounds, ingredients, materials, compositions, dosage forms, etc. that are suitable for use in contact with the tissues of a subject (e.g., a human) within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problems or difficulties, and commensurate with a reasonable benefit / risk ratio. Each carrier, adjuvant, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

[0220] Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts such as Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994.

[0221] The formulations can be prepared by any method well known in the pharmaceutical art. Such methods include the step of associating the active compound with a carrier that constitutes one or more auxiliary components. Generally, the formulations are prepared by intimately and uniformly associating the active compound with a carrier (e.g., a liquid carrier, a micronized solid carrier, etc.) and then, if necessary, shaping the product.

[0222] The formulations can be prepared for local, parenteral, systemic, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intracorneal, subcutaneous, oral or transdermal administration routes, including injection. Injectable formulations may contain the agent selected in a sterile or isotonic medium. The formulations and the mode of administration may be selected according to the agent to be administered and the disease to be treated / prevented.

[0223] By administration of the LINC complex inhibitor, preferably, the cell(s) containing the LINC complex inhibitor described herein is / are modified. The LINC complex inhibitor can be formulated to facilitate delivery to and / or uptake by cells / tissues. The LINC complex inhibitor can be conjugated to a moiety to facilitate delivery to and / or uptake by cells / tissues. Strategies for facilitating intracellular delivery of molecular cargo 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 entirety herein.

[0224] In some embodiments, the LINC complex inhibitor is formulated with a cationic polymer. In some embodiments, the LINC complex inhibitor is encapsulated in nanoparticles or liposomes.

[0225] 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 nanoparticles.

[0226] In some embodiments, the LINC complex inhibitor is a cell penetrating peptide (e.g., a protein transduction domain, a trojan peptide, an arginine-rich peptide, a vector peptide, a vectocell ... In some embodiments, the LINC complex inhibitor is associated (covalently or non-covalently) with a peptide / polypeptide (e.g., an antibody, a peptide aptamer, a ligand for a cell surface molecule / fragment thereof) or a nucleic acid (e.g., a nucleic acid aptamer) capable of binding to a target cell of interest or an antigen thereof.

[0227] In some embodiments, the LINC complex inhibitor is administered in the form of a nucleic acid encoding the LINC complex inhibitor.For example, the LINC complex inhibitor can be administered in the form of a nucleic acid encoding a peptide / polypeptide or a nucleic acid LINC complex inhibitor or an SSN system targeting a LINC complex protein.

[0228] In some embodiments, the LINC complex inhibitor is administered in the form of a nucleic acid encoding factors necessary for the production of the LINC complex inhibitor (e.g., a nucleic acid encoding a precursor of the LINC complex inhibitor and / or a nucleic acid encoding factors necessary for the production / assembly of the LINC complex inhibitor). For example, the LINC complex inhibitor may be administered in the form of a nucleic acid encoding factors necessary for the production of a small molecule or biomolecule LINC complex inhibitor.

[0229] The nucleic acid may be a vector or may be contained in a vector. As used herein, a "vector" is a nucleic acid used as a vehicle to transfer foreign nucleic acid into a cell. The vector may be a vector for the expression of nucleic acid in a target cell. Such a vector may contain a promoter sequence operably linked to the nucleic acid sequence to be expressed. The vector may also contain a stop codon and an expression enhancer. As used herein, the term "operably linked" can include a situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or an enhancer) are covalently linked such that the nucleotide sequence expression is placed 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 can cause transcription of the nucleic acid sequence. Optionally, the resulting transcript can then be translated into the desired polypeptide.

[0230] Any suitable vectors, promoters, enhancers, and stop codons known in the art can be used. Suitable vectors include viral vectors such as retroviral 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), as described, for example, 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 entirety herein. As an example, in Example 5 herein, the dominant negative version of SUN1 is administered using an adeno-associated viral vector.

[0231] In some embodiments, the vector is selected based on tropism for the cell type / tissue / organ to which delivery of the nucleic acid is desired, e.g., the cell type / tissue / organ affected by the disease to be treated / prevented according to the present invention (i.e., the cell / tissue / organ in which symptoms of the disease appear).

[0232] For example, in some embodiments, it is desirable to deliver a nucleic acid encoding a LINC complex inhibitor to muscle cells / tissue (e.g., cardiac and / or skeletal muscle cells / tissue), and in such embodiments, a vector having tropism for such cells / tissue is used. is good. In some embodiments, the vector may be cardioactive. In some embodiments, the vector may be myotropic.

[0233] The AAV9 vector was recently used to deliver Lmna-targeted CRISPR / Cas9-mediated gene therapy for HGPS in the Lmna-G609G mouse model (Santiago-Fernandez et al., Nat Med. (2019) 25(3):423-426 and Beyret et al., Nat Med. (2019) 25(3):419-422).

[0234] In some embodiments, the vector may be an adeno-associated virus vector. In some embodiments, the vector may be an adeno-associated virus vector of one of the following serotypes: AAV9, AAV1, AAV6, AAV8, AAV2i8, AAV9.45, AAV10 or AAVrh.74.

[0235] In some embodiments, the vector includes modifications that increase binding and / or transduction into the cell type of interest (i.e., compared to the level of binding / transduction by the unmodified vector). In some embodiments, the modification is to the capsid protein.

[0236] 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 the cell targeting peptides shown in Tables 1, 2, 3, or 4, and the foregoing document is hereby incorporated by reference in its entirety herein.

[0237] In some embodiments, the vector comprises a capsid protein comprising a mutation to one or more tyrosine residues, such as surface-exposed tyrosine residues. In some embodiments, the tyrosine residue is mutated to phenylalanine. In some embodiments, the vector comprises a capsid protein in which tyrosine residues are mutated as described by Iida et al., Biomed Res Int. (2013) 2013:974819, and the foregoing document is hereby incorporated by reference in its entirety herein.

[0238] In some embodiments, the vector may be an adeno-associated virus vector as described in Buning and Srivastava, supra. In some embodiments, the vector may be an adeno-associated virus vector as described in Iida et al., supra.

[0239] In some embodiments, the nucleic acid / vector comprises one or more sequences for controlling the expression of the nucleic acid. Thus, in some embodiments, the nucleic acid / vector comprises control elements for the inducible expression of the nucleic acid.

[0240] Sequences for controlling the expression of the nucleic acid can provide for the expression of the nucleic acid by a particular type of cell or tissue. For example, expression may be under the control of a cell type- or tissue-specific promoter. As an example, in Example 5 herein, the expression of a construct encoding a dominant negative version of SUN1 is under the control of a cardiomyocyte-specific promoter.

[0241] The promoter for cell type- or tissue-specific expression of the nucleic acid according to the present invention can be selected according to the disease to be treated / prevented. For example, the promoter can drive expression in the cells / tissues / organs affected by the disease (i.e., the cells / tissues / organs in which the symptoms of the disease appear).

[0242] In some embodiments, the promoter can provide expression in muscle cells / tissues (e.g., cardiac and / or skeletal muscle cells / tissues). In some embodiments, the promoter may be a cardiac or cardiomyocyte-specific promoter (e.g., cTNT, α-MHC or MLC2v promoter). In some embodiments, the promoter may be a skeletal muscle / striated muscle cell-specific promoter (e.g., MCK, MHCK7 or desmin promoter).

[0243] 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).

[0244] The sequence for controlling the expression of the nucleic acid can, for example, provide the expression of the nucleic acid in response to a drug / signal. For example, the expression can be under the control of an inducible promoter. The drug can provide inducible expression of the nucleic acid in vivo by administration of the drug to the subject to whom the cells modified according to the present disclosure have been administered, or ex vivo / in vitro by administration of the drug to cultured cells ex vivo or in vitro.

[0245] In some embodiments, the nucleic acid(s) / vector(s) uses a conditional expression system to control the expression of a nucleic acid encoding a LINC complex inhibitor by a cell containing the nucleic acid(s) / vector(s). "Conditional expression" may also be referred to herein as "inducible expression" and refers to expression that is conditional on a particular condition, such as the presence of a particular agent. Conditional expression systems are well known in the art and are reviewed in Ryding et al., Journal of Endocrinology (2001) 171, pages 1-14, which is hereby incorporated by reference in its entirety.

[0246] Multiple doses of the LINC complex inhibitor may be provided. One or more of the doses, or each of them, may be accompanied by the simultaneous or sequential administration of another therapeutic agent. The multiple doses may be spaced apart at predetermined time intervals, which may be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days or one of 1, 2, 3, 4, 5, or 6 months. By way of example, the doses may be administered once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0247] Treatment / prevention of diseases through LINC complex inhibition The present invention provides methods and articles (drugs and compositions) for the treatment and / or prevention of diseases through LINC complex inhibition. Treatment / prevention of a disease is achieved, for example, by LINC complex inhibition in a cell, tissue / organ / organ system / subject.

[0248] Aspects of the present invention relate to the treatment / prevention of diseases in which LINC complex dysfunction is pathologically involved. Such diseases include, for example, nuclear envelope diseases (such as laminopathies). The therapeutic utility of the drugs and methods of the present invention extends to the treatment and / or prevention of any disease in which a therapeutic / preventive benefit can be derived from LINC complex inhibition.

[0249] The method delays / prevents the onset of symptoms of a disease; reduces the severity of symptoms of a disease (alleviates the symptoms of a disease); reverses the symptoms of a disease; reduces the pathological condition of a subject having the disease; reduces the mortality rate of a subject having the disease; and / or delays the progression of a disease / may be directed to preventing (e.g., until a later stage).

[0250] Aspects of the invention relate to the treatment of diseases associated with mutations 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 for 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 a cell containing one or more copies of the mutant allele of the gene as compared to a cell containing two copies (i.e., homozygous) of the non-mutated (wild-type) reference allele of the gene: a decreased level of the gene product (e.g., RNA and / or protein (or a particular 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.

[0251] Aspects of the invention relate to the treatment / prevention of nuclear envelope diseases. Nuclear envelope diseases are diseases / conditions associated with mutations in genes encoding nuclear envelope proteins (i.e., proteins contained within the ONM, perinuclear lumen or INM, or directly / indirectly related thereto). Nuclear envelope diseases are reviewed, for example, in Chi et al., Journal of Biomedical Science (2009) 16:96, which is hereby incorporated by reference in its entirety herein. Nuclear envelope diseases include diseases / conditions associated with mutations in LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1 and NUP62.

[0252] In particular, aspects of the invention relate to the treatment / prevention of laminopathies. 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 cell's resilience to physical forces. In the experimental examples herein, the inventors have demonstrated that LINC complex inhibition alleviates a range of laminopathy symptoms.

[0253] As used herein, "laminopathy" is a disease / condition associated with mutations in genes encoding lamins. Genes encoding lamins include LMNA (encoding lamin A and C), as well as LMNB1 and LMNB2 encoding lamin B1 and B2. Accordingly, aspects of the present invention relate to the treatment / prevention of diseases associated with mutations in LMNA, LMNB1, and / or LMNB2.

[0254] In some embodiments, the mutation is known or predicted to decrease the level of the lamin isoform encoded by the wild-type allele of the gene encoding lamin (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 the lamin encoded by the wild-type allele of the gene encoding lamin. In some embodiments, the mutation is known or predicted to generate a misfolded and / or degraded lamin.

[0255] In some embodiments, the mutation is known or predicted to increase the level of the lamin isoform encoded by the wild-type allele of the gene encoding lamin (e.g., LMNA, LMNB1, or LMNB2).

[0256] In some embodiments, the mutation is known or expected to increase the level of a disease-related lamin variant (e.g., progerin). In some embodiments, the mutation is known or expected to increase the level of a lamin encoded by a disease-related allele of the gene encoding lamin.

[0257] In some embodiments, the laminopathy is a skeletal muscle laminopathy. In some embodiments, the laminopathy is a myopathy. In some embodiments, the laminopathy is an LMNA mutation-related myopathy.

[0258] In some embodiments, the disease treated / prevented according to the present invention is characterized by one or more of myopathy, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, cardiomyopathic muscular dystrophy, skeletal muscle dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy or skin disease.

[0259] In some embodiments, the disease treated / prevented according to the present invention is characterized by one or more of muscular dystrophy, cardiomyopathic muscular dystrophy or skeletal muscle dystrophy.

[0260] In some embodiments, laminopathy is associated with mutations in LMNA, LMNB1, or LMNB2. In some embodiments, laminopathy is Hutchinson-Gilford progeria syndrome; Emery-Dreifuss muscular dystrophy; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; lipodystrophy, partial, acquired; epilepsy, progressive myoclonic, 9; Charcot-Marie-Tooth disease, axonal, type 2e; muscular dystrophy; lipodystrophy, familial partial, type 2; cardiomyopathy, dilated, 1h; Pelger-Huet nuclear anomaly; Reynolds syndrome; muscle disease; leukodystrophy; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-related; mandibuloacral dysplasia with a-type lipodystrophy; cardiomyopathy, dilated, 1a; restrictive dermopathy, lethal; familial partial lipodystrophy; epilepsy; lipoatrophy with diabetes, vitiligo, black skin 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 standstill 1; limb-girdle muscular dystrophy; cardiomyopathy, dilated, with hypergonadotropic hypogonadism; heart-hand syndrome, Slovenian type; monogenic diabetes; arrhythmogenic right ventricular cardiomyopathy; cardiomyopathy, dilated, 1e; aging; micrognathia, hearing loss, progeria-like symptoms, and lipodystrophy syndrome; adrenoleukodystrophy; atypical Werner syndrome; endometriosis; spinocerebellar ataxia 31; progressive muscular atrophy; neurogenic bowel; autosomal dominant leukodystrophy with autonomic disease; Werner syndrome; myopathy; Lmna-related dilated cardiomyopathy; muscular dystrophy, congenital, 1b; hypertrophic cardiomyopathy; left ventricular noncompaction; true diabetes, insulin-independent; arrhythmogenic right ventricular dysplasia, familial, 9; heart disease; atrial fibrillation; heart conduction disorder; myoclonus; progressive myoclonic epilepsy; myoclonic epilepsy; peripheral nervous system disease; dental disease; atrioventricular block; myofibrillar myopathy; autosomal dominant limb-girdle muscular dystrophy; Lmna-related cardio-dermatological progeria syndrome; amyotrophic lateral sclerosis 1; neural tube defect; cervical cancer; neural tube defect, folate-sensitive; brain degeneration; melanoma;3-Hydroxyacyl-CoA dehydrogenase deficiency; congenital fiber type disproportion myopathy; acro-osteolysis; Wolff-Parkinson-White syndrome; sinoatrial node dysfunction syndrome; calcinosis; 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; myotonic dystrophy; limb-girdle muscular dystrophy type 1B; insulin-resistant acanthosis nigricans, type A; systemic lipodystrophy-related premature aging; syndrome; osteoporosis; myotonic dystrophy 1; neuropathy; catecholamine-induced polymorphic ventricular tachycardia; cataract; Bethlem myopathy 1; congenital generalized lipodystrophy; restrictive cardiomyopathy; muscular dystrophy, congenital merosin-deficient type, 1A; proximal spinal muscular atrophy; muscular dystrophy-dystroglycanopathy, type B, 5; lipodystrophy, congenital generalized type, 1; Emery-Dreifuss muscular dystrophy type 1, X-linked; cardiomyopathy, dilated, 1D; myopathy, proximal, and ophthalmoplegia; myopathy; ovarian cystadenoma; emerinopathy; Fanconi anemia, complementation group A; obesity index quantitative trait locus 11; myelodysplastic syndrome; skin disease; anorexia nervosa; spinal muscular atrophy; inclusion body myositis; aniridia 1; myositis; trichothiodystrophy 1; neuromuscular disease; nutritional deficiency; thoracic outlet syndrome; muscle disorder; muscular atrophy; Hurler-Scheie syndrome; Rere-Related Disorders; Miller-Dieker Lissencephaly Syndrome; lipodystrophy, congenital generalized type, 4; lipodystrophy, familial partial type, 3; Wiedemann-Rautenstrauch syndrome; lipodystrophy, congenital generalized type, 2; ataxia neuropathy spectrum (Ataxia Neuropathy Spectrum); Hair loss, Neurological deficits, and Endocrine disease syndrome; Lipodystrophy, Familial partial type, type 4; Second-degree atrioventricular block; Acute necrotizing encephalitis; Median nerve disorder; Dilated cardiomyopathy; Familial isolated arrhythmogenic ventricular dysplasia, right-dominant type; Female genital prolapse; Familial isolated arrhythmogenic ventricular dysplasia, biventricular type; Familial isolated arrhythmogenic ventricular dysplasia, left-dominant type; Complete generalized lipodystrophy; Blood type - Ahoonen; Autosomal semi-dominant severe lipodystrophy laminopathy; Ulnar nerve disorder; Pelvic muscle wasting; Alzheimer's disease; Stroke, ischemic; Ataxia-telangiectasia; Spondylosis 1; Human immunodeficiency virus type 1; Neuroblastoma; Vascular disease; Nervous system disease; Respiratory failure; Turner syndrome; Carpal tunnel syndrome; Barrett esophagus; Sleep apnea; Cerebrovascular disease; Proteasome-related autoinflammatory syndrome 1; Joubert syndrome 1; Viral infection; Dementia; Personality disorder; Neuropathy, hereditary sensory and autonomic, type III; Lowe Oculocerebrorenal Syndrome; True diabetes; Fatty liver disease; Leigh syndrome; Muscular dystrophy, Duchenne type; Hydrocephalus; Dermatomyositis; Hirschsprung disease 1; QT prolongation syndrome; Angelman syndrome; Central nervous system disease; Congenital disorder of glycosylation, type in; Anhidrosis, achalasia, and mental retardation syndrome; Polycystic ovary syndrome; Hypoglycemia; Muscle hypertrophy; Kearns-Sayre syndrome; Cone-rod dystrophy 2; Aicardi-Goutières syndrome; Andersen Cardiodysrhythmic Periodic Paralysis; Muscular dystrophy, Becker type; Legg-Calvé-Perthes disease; Androgen insensitivity; Ehlers-Danlos syndrome; Axenfeld-Rieger syndrome; Muscular dystrophy - dystroglycanopathy, type C, 5; Glomerulonephritis; Episodic disease; Chikungunya fever; West syndrome; Ullrich congenital muscular dystrophy type 1; Focal segmental glomerulosclerosis; Walker-Warburg syndrome; Renal hypodysplasia / aplasia 1; Popliteal pterygium syndrome; Microcephaly; Juvenile dermatomyositis; Distal arthrogryposis; Myocarditis; Arterial tortuosity syndrome; Scoliosis; Membranous nephropathy; Microvascular complications of type 3 diabetes; Epidermolysis bullosa; Short syndrome;Hyperalgesia; 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 Syndrome); Supravalvular aortic stenosis; Myopathy, congenital; Metabolic encephalopathy crisis, with recurrence, rhabdomyolysis, cardiac arrhythmias and neurodegeneration; Lissencephaly1; Polycystic liver disease, with or without renal cysts1; Idiopathic inflammatory myopathy; Epidermolysis bullosa simplex; Focal segmental glomerulosclerosis1; Genital hypoplasia; Gyrate chorioretinal atrophy; Syringomyelia; Ichthyosis vulgaris; Arthrogryposis, peripheral, type 1a; Acute insulin response; Brachydactyly; Cerebellar hypoplasia; Craniometaphyseal dysplasia, autosomal dominant; Alport syndrome 1, X-linked; Lissencephaly; Muscular dystrophy-dystroglycanopathy, type B, 6; Diarrhea 5, Tufting Enteropathy; With; Congenital; Junctional Epidermolysis Bullosa; Aicardi-Goutieres Syndrome 1; Miyoshi Muscular Dystrophy; Retinitis; Marden-Walker Syndrome; Optic Retinopathy; Polyglucosan Body Myopathy 1 with or without Immunodeficiency; Epidermolysis Bullosa, Junctional, Herlitz Type; Macroglossia; Parkinson Disease 15, Autosomal Recessive Early-Onset Type; Myopathy, Myofibrillar, 3; Microvascular Complications of Type 7 Diabetes; Muscle-Eye-Brain Disease; Melkersson-Rosenthal Syndrome; Myopathy, X-Linked, with Excessive Autophagy; Choroiditis; Muscular Dystrophy, Limb-Girdle, Autosomal Recessive 8; Crouzon Syndrome with Melanosis; Muscular Dystrophy, Limb-Girdle, Autosomal Recessive 6; Polymicrogyria; Dystrophinopathy; Microvascular Complications of Type 6 Diabetes; Microvascular Complications of Type 4 Diabetes; Hypotonia; Pontocerebellar Hypoplasia; Congenital Fibrosarcoma; Intrauterine Fetal Growth Retardation, Metaphyseal Dysplasia, Congenital Adrenal Hypoplasia, and Genital Anomalies; Muscular Dystrophy, Limb-Girdle, Autosomal Recessive 7; Myopathy, Congenital, with Fibrous Type Imbalance; Hereditary Enamel Hypoplasia, Ig Type; Refractory Anemia; Fibrosis of Extraocular Muscles, Congenital, 1; Ataxia and Multiple Neuropathy, Adult-Onset; Allgrove Syndrome; Senile Cataract; Muscular Dystrophy-Dystroglycanopathy, Type C, 1; Neuronal Migration Disorder; Ayme-Gripp Syndrome; Primary Agammaglobulinemia; Autosomal Recessive Limb-Girdle Muscular Dystrophy Type 2a; Encephalitis; Muscular Dystrophy, Congenital, Large Conical Type; Autosomal Recessive Limb-Girdle Muscular Dystrophy; Alkuraya-Kucinskas Syndrome; Muscular Dystrophy-Dystroglycanopathy, Type C, 4; Congenital Muscular Dystrophy Type 1a; Baraitser-Winter Syndrome; Dandy-Walker Complex; Muscular Dystrophy-Dystroglycanopathy, Type C, 2; Emery-Dreifuss Muscular Dystrophy, X-Linked Type; Muscular Dystrophy, Limb-Girdle, Autosomal Recessive 3; Autosomal Recessive Limb-Girdle Muscular Dystrophy Type 2d; Cerebral Small Vessel Disease 1 with or without Ocular Abnormalities; Familial Isolated Dilated Cardiomyopathy; Epithelial Recurrent Erosion Dystrophy; Muscular Dystrophy-Dystroglycanopathy; Mycobacterium Avium Complex Infection; Autosomal Recessive Limb-Girdle Muscular Dystrophy Type 2l; Visual Epilepsy;Barlow's aneurysm; autosomal recessive limb-girdle muscular dystrophy type 2B; creatine phosphokinase, elevated serum; spastic paraplegia, ataxia, and mental retardation; multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia, and anencephaly; patulous eustachian tubes; autosomal genetic disease; Ck syndrome; neuronitis; hyperekplexia 1; reducing 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 group, I system; Salih myopathy; adducted thumbs syndrome; dural sinus malformation; blood group, Donath-Landsteiner system; blood group, Colton system; arthrochalasia Ehlers-Danlos syndrome; Lama2-related muscular dystrophy; muscular dystrophy, congenital, with pediatric cataract and hypogonadism; intrauterine infection; muscular dystrophy, congenital, merosin positive; congenital muscular dystrophy with cerebellar disorder; Fukuyama type muscular dystrophy; chronic lymphoproliferative disorder of natural killer cells; congenital muscular dystrophy with intellectual disability; enamel hypoplasia, hypomorphic, Ig; androgen insensitivity syndrome, mild form; muscular dystrophy, congenital, resulting in joint contractures; congenital muscular alpha-dystroglycanopathy with brain and eye abnormalities; type VI collagen-related myopathy; Emery-Dreifuss muscular dystrophy, dominant form; congenital muscular dystrophy due to dystroglycanopathy; proximal myopathy with ragged red fibers; selected from pediatric idiopathic scoliosis.;

[0261] In some embodiments, the laminopathy is a laminopathy associated with a mutation in LMNA. In some embodiments, the laminopathy is Hutchinson-Gilford progeria syndrome; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-related; Emery-Dre Fusiform muscular dystrophy 2, autosomal dominant; Muscular dystrophy; Mandibuloacral dysplasia with lipodystrophy; 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-hand syndrome, Slovenian type; Aging; Familial partial lipodystrophy; Restrictive cutaneous disorder, lethal; Arrhythmogenic right ventricular cardiomyopathy; Dental disease; Heart disease; Werner syndrome; Hypertrophic cardiomyopathy; Left ventricular noncompaction; Atrioventricular block; Calcinosis; Acro-osteolysis; Autosomal dominant limb-girdle muscular dystrophy; True diabetes mellitus, insulin-independent; Osteoporosis; Atrial fibrillation; Atrial standstill 1; Melanotic macule; Cardiac conduction disorder; Catecholamine-induced polymorphic ventricular tachycardia; Micrognathia, deafness, progeroid features, and lipodystrophy syndrome; Sinus node dysfunction syndrome; Pelger-Huet nuclear anomaly; Charcot-Marie-Tooth disease, axonal, type 2e; Congenital generalized lipodystrophy; Restrictive cardiomyopathy; Congenital myofiber 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; Obesity index quantitative trait locus 11; Skin disease; Myotonic dystrophy 1; Neuromuscular disease; Hallermann-Streiff 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-deficient, type 1a; Proximal spinal muscular atrophy; Muscular dystrophy-dystroglycanopathy, type B, 5; Muscular dystrophy, congenital, type 1b; Reynolds syndrome; Wiedemann-Rautenstrauch syndrome; Emery-Dreifuss muscular dystrophy 1, X-linked; Lipodystrophy, congenital generalized, type 2; Monogenic diabetes; Cardiomyopathy, dilated, 1d; Myopathy, proximal, and ophthalmoplegia; Myopathic disease; Lipodystrophy, familial partial, type 4; Cardiomyopathy, dilated, 1h; Second-degree atrioventricular block; Median nerve disorder;Intrinsic cardiomyopathy;Prolapse of female genital organs;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 disease;Cardiomyopathy, dilated, type 1b;Autosomal inherited disease;Familial isolated arrhythmogenic ventricular dysplasia, right dominant type;Familial isolated arrhythmogenic ventricular dysplasia, biventricular type;Familial isolated arrhythmogenic ventricular dysplasia, left dominant type;Lmna-associated cardiocutaneous progeria syndrome;Autosomal semidominant severe lipodystrophy-laminopathies;

[0262] In some embodiments, the disease to be treated / prevented according to the present invention is selected from diseases associated with the cDNA or protein variants shown in Table 1. In some embodiments, the disease to be treated / prevented according to the present invention is selected from the diseases shown in Table 1.

[0263] In some embodiments, the disease to be treated / prevented in accordance with the present invention is selected from the diseases shown in Table 1 in regular font. In some embodiments, the disease to be treated / prevented in accordance with the present invention is selected from the diseases shown in bold font in Table 1.

[0264] [Table 1-1]

[0265] [Table 1-2]

[0266] [Table 1-3]

[0267] [Table 1-4]

[0268]

Table 1-5

[0269]

Table 1-6

[0270]

Table 1-7

[0271]

Table 1-8

[0272]

Table 1-9

[0273]

Table 1-10

[0274]

Table 1-11

[0275]

Table 1-12

[0276]

Table 1-13

[0277]

Table 1-14

[0278]

Table 1-15

[0279]

Table 1-16

[0280]

Table 1-17

[0281]

Table 1-18

[0282]

Table 1-19

[0283]

Table 1-20

[0284]

Table 1-21

[0285]

Table 1-22

[0286]

Table 1-23

[0287]

Table 1-24

[0288]

Table 1-25

[0289]

Table 1-26

[0290]

Table 1-27

[0291]

Table 1-28

[0292]

Table 1-29

[0293]

Table 1-30

[0294]

Table 1-31

[0295]

Table 1-32

[0296]

Table 1-33

[0297]

Table 1-34

[0298]

Table 1-35

[0299]

Table 1-36

[0300]

Table 1-37

[0301]

Table 1-38

[0302]

Table 1-39

[0303]

Table 1-40

[0304]

Table 1-41

[0305]

Table 1-42

[0306]

Table 1-43

[0307]

Table 1-44

[0308] In some embodiments, the laminopathy is not Hutchinson-Gilford progeria syndrome (HGPS). In some embodiments, when a mutation occurs in LMNA, the mutation is an LMNA mutation that does not result in an increase in the level of progerin. In some embodiments, when a mutation occurs in LMNA, the mutation is not an HGPS-related mutation.

[0309] A further aspect of the invention relates to the treatment / prevention of a disease characterized by hyperlipidemia. A further aspect of the invention relates to the treatment / prevention of a disease associated with LDL receptor deficiency (i.e., a reduced level of LDL receptor protein and / or function).

[0310] Hyperlipidemia refers to elevated levels of lipids or lipoproteins in the blood. Hyperlipidemia includes hypertriglyceridemia, hypercholesterolemia, and combined hyperlipidemia (a combination of hypertriglyceridemia and hypercholesterolemia). Hyperlipidemia is associated with, for example, atherosclerosis, hypertension, and cardiovascular disease.

[0311] Hypercholesterolemia is described, for example, in Bhatnagar et al., BMJ (2008) 337: a993. The UK NHS defines hypercholesterolemia as a total blood 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 total blood cholesterol level of ≥ 240 mg / dL. Hypertriglyceridemia is described, for example, in Berglund et al., J. Clin. Endocrinol. Metab. (2012) 97(9): 2969-2989, and is defined by a blood triglyceride level of ≥ 150 mg / dL (≥ 1.7 mmol / L).

[0312] In some embodiments, the disease characterized by hyperlipidemia may be familial hyperlipidemia or acquired (secondary) hyperlipidemia. In some embodiments, familial hyperlipidemia is selected from Burger-Grütz syndrome, familial apoprotein CII deficiency, type Ic hyperlipoproteinemia, familial hypercholesterolemia, familial combined hyperlipidemia, familial dysbetalipoproteinemia, familial hypertriglyceridemia, and type V hyperlipoproteinemia. In some embodiments, familial hyperlipidemia is familial hypercholesterolemia.

[0313] LDL receptor deficiency can occur, for example, as a result of mutations in the LDLR. Thus, aspects of the invention relate to the treatment / prevention of diseases associated with mutations in the LDLR. In some embodiments, the mutation is known or predicted to decrease the level of one or more LDL receptor isoforms encoded by the wild-type LDLR allele and / or increase the level of one or more disease-associated LDL receptor variants. In some embodiments, the disease associated with the mutation in the LDLR is familial hypercholesterolemia.

[0314] In some embodiments, the disease treated / prevented according to the invention is characterized by one or more of hyperlipidemia, hypercholesterolemia, atherosclerosis, stenosis, or hypertension. In some embodiments, the disease treated / prevented according to the invention is characterized by atherosclerosis.

[0315] In some embodiments, the disease treated / prevented is selected from atherosclerosis, cardiovascular disease, stroke, and familial hyperlipidemia. In some embodiments, the method includes determining whether the subject has a disease described herein. In some embodiments, the method includes diagnosing a disease described herein. The step of determining whether the subject has a disease described herein may include analyzing the subject for one or more symptoms / correlates of the disease.

[0316] In some embodiments, for example, based on the presence of other symptoms indicative of a disease in a subject or in the cells / tissues / organs of a subject, the subject may be suspected of having or being afflicted with a disease. In some embodiments, for example, due to a genetic predisposition or other risk factors for a disease, the subject may be considered at risk of developing the disease.

[0317] 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.

[0318] Determination of a mutation in a gene described herein may confirm a diagnosis or a suspected diagnosis, or may confirm that the subject is at risk of developing a disease. The determination may diagnose a disease or a predisposition to a disease for treatment / prevention with a LINC complex inhibitor.

[0319] Genetic factors may be assayed by methods known to those of skill in the art, including PCR-based and sequencing assays. For example, in a sample obtained from a subject, diagnosis may be confirmed by determining the presence of a genetic factor, and / or the subject may be classified as being at risk of developing a disease described herein, and / or the subject may be determined to be suitable for treatment with a LINC complex inhibitor.

[0320] The assay may be performed in vitro on a sample obtained from a subject or following processing of a sample obtained from a subject. The sample obtained from the subject may be of any type. A biological sample may be taken from any tissue or body fluid, for example, a blood sample, a blood-derived sample, a serum sample, a lymph sample, a semen sample, a saliva sample, a bone fluid sample. A blood-derived sample may be a selected fraction of patient blood, for example, a selected cell-containing fraction or a plasma or serum fraction. The sample may be an isolated tissue sample or biopsy from the subject; or may contain cells.

[0321] In some embodiments, the method includes 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 includes determining whether the subject comprises a mutation in one or more alleles of LMNA, LMNB1, and LMNB2. In some embodiments, the method includes determining whether the subject comprises a mutation in an allele of LMNA. In some embodiments, the method includes determining whether the subject comprises a mutation in an allele of LDLR.

[0322] In such embodiments, if a mutation is detected, the subject can be identified as a subject to be administered a LINC complex inhibitor according to the present disclosure. Thus, in some embodiments, the method includes selecting a subject determined to comprise a mutation in one or more of LMNA, LMNB1, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62 for administration of a LINC complex inhibitor. In some embodiments, the method includes selecting a subject determined to comprise a mutation in one or more of LMNA, LMNB1, and LMNB2 for administration of a LINC complex inhibitor. In some embodiments, the method includes selecting a subject determined to comprise a mutation in LMNA for administration of a LINC complex inhibitor. In some embodiments, the method includes selecting a subject determined to comprise a mutation in LDLR for administration of a LINC complex inhibitor.

[0323] In some embodiments, the method includes testing a sample obtained from a subject suspected of having a disease for the presence or absence of at least one LMNA mutation, the presence of the at least one LMNA mutation indicating that the subject should be administered a LINC complex inhibitor according to the present disclosure.

[0324] According to various aspects of the present invention, methods of treating and / or preventing a disease according to the present invention are as follows: increasing the survival of a subject having the disease; increasing the lifespan of a subject having the disease; increasing cardiac function; delaying the onset of decline of cardiac function; increasing myocardial contractility; increasing ejection fraction and / or shortening fraction; decreasing atherosclerosis; may include one or more of the above.

[0325] subject A subject according to the present disclosure can be any animal. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some embodiments, the subject can be a non-human animal, such as a non-human mammal. The subject can be male or female.

[0326] The subject can be a patient. The patient may have a disease described herein. The subject may be diagnosed as having a disease described herein, may be suspected of having a disease described herein, or may be at risk of developing a disease described herein.

[0327] In embodiments according to the present invention, the subject / patient can be selected for therapy / prevention by the methods described herein based on the characterization of markers of the disease described herein.

[0328] numbered paragraphs The following numbered paragraphs (paras) provide further description of the properties and combinations of properties considered in connection with the present invention.

[0329] In some aspects and embodiments of the present invention, the subject matter according to the following numbered paragraphs may not be specifically claimed. 1. An isolated nucleic acid molecule, wherein the nucleic acid molecule comprises an expression vector and a transgene, the transgene is operably linked to the expression vector, and expression of the transgene in a transfected cell results in disruption of the nuclear skeleton-cytoskeleton linker (LINC) complex in the transfected cell.

[0330] 2. The nucleic acid molecule of paragraph 1, wherein the expression vector is a heart or cardiomyocyte-specific expression vector. 3. The nucleic acid molecule of paragraph 1 or paragraph 2, wherein the expression vector comprises a heart or cardiomyocyte-specific promoter.

[0331] 4. The nucleic acid molecule of paragraph 3, wherein the expression vector comprises a heart or cardiomyocyte-specific promoter selected from the group consisting of the cardiac troponin T promoter (cTnT), the α-myosin heavy chain (α-MHC) promoter, and the myosin light chain (MLC2v) promoter.

[0332] 5. The nucleic acid molecule of paragraph 3 or paragraph 4, wherein the cardiomyocyte-specific promoter is the chicken cardiac troponin T (cTnT) promoter. 6. The nucleic acid molecule of any one of the preceding paragraphs, wherein the expression vector is a viral expression vector. molecule.

[0333] 7. The nucleic acid molecule of paragraph 6, wherein the viral expression vector is selected from the group consisting of lentivirus, adenovirus, and adeno-associated virus (AAV). 8. The nucleic acid molecule of any one of the preceding paragraphs, wherein the adeno-associated virus expression vector (AAV) is heart tropic.

[0334] 9. The nucleic acid molecule of any one of the preceding paragraphs, wherein the AAV vector is selected from the group consisting of AAV9 (serotype 9), AAV1 (serotype 1), AAV6 (serotype 6), AAV8 (serotype 8), AAV2i8, and AAV9.45.

[0335] 10. The nucleic acid molecule of any one of the preceding paragraphs, wherein the AAV vector is AAV9 (serotype 9). 11. A nucleic acid molecule according to any one of the preceding paragraphs, wherein the transgene comprises a nucleic acid sequence for expressing an intraluminal domain of a SUN domain-containing protein, an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence.

[0336] 12. The nucleic acid molecule of paragraph 11, wherein the intraluminal domain of the SUN domain-containing protein comprises a coiled-coil domain and a SUN domain. 13. The nucleic acid molecule of paragraph 11, wherein the coiled-coil domain is upstream of the SUN domain.

[0337] 14. A nucleic acid molecule according to any one of the preceding paragraphs, wherein the transgene further comprises a nucleic acid sequence for expressing an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence.

[0338] 15. A nucleic acid molecule according to any one of the preceding paragraphs, wherein the transgene comprises a nucleic acid sequence for expressing an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence, and a nucleic acid sequence for expressing either an intraluminal domain of a SUN domain-containing protein or a SUN domain.

[0339] 16. A nucleic acid molecule according to any one of paragraphs 11 to 15, wherein the SUN domain protein is SUN1 or SUN2. 17. A nucleic acid molecule according to any one of paragraphs 11 to 16, wherein the N-terminal signal sequence is derived from a secreted protein or a type I transmembrane protein.

[0340] 18. The nucleic acid molecule of paragraph 17, wherein the secreted protein or type I transmembrane protein is selected from the group consisting of human serum albumin, proinsulin, transferrin receptor, EGF receptor, preproopiomelanocortin, pancreatic digestive enzymes (e.g., protease, amylase, and lipase), endoplasmic reticulum lumen proteins such as protein disulfide isomerase, GRP94, and combinations thereof.

[0341] 19. The nucleic acid molecule of paragraph 18, wherein the N-terminal signal sequence is derived from human serum albumin. 20. The nucleic acid molecule according to any one of paragraphs 11 to 19, wherein the N-terminal signal sequence is not preceded by any other tag at its N-terminus.

[0342] 21. The signal peptidase cleavage site is a signal peptidase cleavage site derived from human serum albumin, proinsulin, transferrin receptor, EGF receptor, preproopiomelanocortin, carboxypeptidase, complement protein, fibrinogen, cytokine, chemokine, fibrinogen, pancreatic digestive enzyme (e.g., protease, amylase, and lipase), endoplasmic reticulum lumen protein, such as protein disulfide isomerase, GRP94, and combinations thereof, or is one of the group consisting of them, and the nucleic acid molecule according to any one of paragraphs 11 to 20. 9. The nucleic acid molecule according to any one of paragraphs 11 to 20, wherein the signal peptidase cleavage site is a signal peptidase cleavage site derived from human serum albumin.

[0343] 22. The nucleic acid molecule of paragraph 21, wherein the signal peptidase cleavage site is a signal peptidase cleavage site derived from human serum albumin. 23. The nucleic acid molecule according to any one of paragraphs 11 to 22, wherein the C-terminal targeting peptide sequence inhibits the secretion of the peptide expressed from the transgene described in any one of paragraphs 1 to 19.

[0344] 24. The nucleic acid molecule according to any one of paragraphs 11 to 23, wherein the C-terminal targeting peptide sequence is a KDEL sequence. 25. The nucleic acid molecule according to any one of paragraphs 1 to 24, wherein the transgene further comprises an epitope tag.

[0345] 26. The nucleic acid molecule of paragraph 25, wherein the optional epitope tag is located at the N-terminus, or at any location of the nucleic acid molecule except downstream of the C-terminal targeting peptide sequence [e.g., KDEL] (after), or at any location of the nucleic acid molecule except upstream of the N-terminal signal sequence (before).

[0346] 27. The nucleic acid molecule of paragraph 26, wherein the optional epitope tag is selected from the group consisting of a cellulose binding domain (CBD), chloramphenicol acetyltransferase (CAT), dihydrofolate reductase (DHFR), one or more FLAG tags, glutathione S-transferase (GST), green fluorescent protein (GFP), hemagglutinin A (HA), histidine (His), herpes simplex virus (HSV), luciferase, maltose binding protein (MBP), c-Myc, protein A, protein G, streptavidin, T7, thioredoxin, V5, vesicular stomatitis virus glycoprotein (VSV-G), and combinations thereof.

[0347] 28. The nucleic acid molecule of paragraph 27, wherein the epitope tag is hemagglutinin A (HA). 29. The vector is an adeno-associated virus vector (AAV) comprising a chicken cardiac troponin T promoter (cTnT) and a transgene as described in any one of paragraphs 1 to 28, wherein the luminal domain of the SUN domain-containing protein is derived from SUN1, the N-terminal signal sequence and signal peptidase cleavage site are each derived from human serum albumin, the C-terminal targeting peptide sequence is the KDEL sequence, and the transgene further comprises hemagglutinin (HA) as an N-terminal epitope tag; Optionally, the vector comprises the nucleic acid sequence shown in SEQ ID NO: 3 (see, for example, FIG. 10). The nucleic acid molecule of any one of the preceding paragraphs.

[0348] 30. The vector is an adeno-associated virus vector (AAV) comprising a chicken cardiac troponin T promoter (cTnT) and a transgene as described in any one of paragraphs 1 to 28, wherein the luminal domain of the SUN domain-containing protein is derived from SUN2, the N-terminal signal sequence and signal peptidase cleavage site are each derived from human serum albumin, the C-terminal targeting peptide sequence is the KDEL sequence, and the transgene further comprises hemagglutinin (HA) as an N-terminal epitope tag; Optionally containing a nucleic acid sequence in which the vector is shown in SEQ ID NO: 5 (see, for example, FIG. 10). Any one nucleic acid molecule of the preceding paragraphs.

[0349] 31. A nucleic acid molecule of any one of paragraphs 1 to 10, wherein the transgene contains a nucleic acid sequence for expressing a KASH domain and an N-terminal stabilizing polypeptide sequence. 32. The nucleic acid molecule of paragraph 31, wherein the KASH domain contains a transmembrane domain and a SUN interaction peptide.

[0350] 33. A nucleic acid molecule of any one of paragraphs 31 or 32, wherein the KASH domain is selected from the group consisting of KASH1 (derived from Nesprin-1 (SYNE1 gene)), KASH2 (derived from Nesprin-2 (SYNE2 gene)), KASH3 (derived from Nesprin-3 (SYNE3 gene)), KASH4 (derived from Nesprin-4 (SYNE4 gene)), and KASH5 (derived from KASH5 / CCDC155 (KASH5 gene)).

[0351] 34. A nucleic acid molecule of any one of paragraphs 1 to 10, wherein the transgene contains a nucleic acid sequence for expressing a CRISPR-Cas or other synthetic nuclease system that modifies a nucleic acid encoding the SUN domain of an endogenous Sun protein or the KASH domain of an endogenous Nesprin protein.

[0352] 35. The nucleic acid molecule of paragraph 34, wherein the transgene contains a nucleic acid sequence for expressing CRISPR-Cas. 36. A nucleic acid molecule of any one of paragraphs 1 to 33, wherein the transgene is a dominant negative construct.

[0353] 37. A nucleic acid molecule of any one of the preceding paragraphs, wherein the transgene is a humanized or human transgene. 38. A nucleic acid molecule of any one of the preceding paragraphs, wherein the expression of the transgene results in disruption of the protein-protein interaction between SUN and KASH of the LINC complex.​

[0354] 39. A nucleic acid molecule according to paragraph 38, wherein disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between protein-protein interactions selected from the group consisting of Sun1+Nesprin-1, Sun2+Nesprin-1, Sun1+Nesprin-2, Sun1+Nesprin-3, Sun2+Nesprin-2, and Sun2+Nesprin-3.

[0355] 40. A nucleic acid molecule according to paragraph 39, wherein disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between Sun1 and Nesprin-1. 41. A nucleic acid molecule according to any one of the preceding paragraphs, wherein the AAV vector is formulated for delivery to the myocardium of a subject.

[0356] 42. A nucleic acid molecule according to any one of the preceding paragraphs for use in treating a disease caused by one or more Lmna mutations in a subject. 43. A nucleic acid molecule according to paragraph 42, wherein the disease is selected from the group consisting of restrictive dermopathy, familial partial lipodystrophy (e.g., Dunnigan type), mandibuloacral dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and the diseases represented in normal font in Table 1.

[0357] 44. A nucleic acid molecule according to any one of the preceding paragraphs for use in treating cardiovascular disease in a subject. 45. A nucleic acid molecule according to paragraph 42 or 43, wherein the disease or cardiovascular disease is characterized by the presence of at least one Lmna mutation.

[0358] 46. The cardiovascular disease is laminopathy, cardiomyopathy, such as dilated cardiomyopathy (DCM), dilated Cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmia, isolated atrial fibrillation; muscular dystrophy (often associated with cardiomyopathy), for example, cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, cardiomyopathy associated with congenital muscular dystrophy; premature aging syndromes (mainly vascular, but may also have heart lesions), for example, cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford progeria syndrome, etc., and a nucleic acid molecule selected from the group consisting of the diseases represented in bold font in Table 1, any one of paragraphs 44 or 45.

[0359] 47. An adeno-associated virus vector (AAV) comprising the cardiac troponin T promoter (cTnT) and a transgene as described in any one of paragraphs 11 to 30 or paragraphs 34 to 38.

[0360] 48. A pharmaceutical composition comprising the nucleic acid molecule as described in any one of paragraphs 1 to 41. 49. A method for treating a disease in a subject, comprising administering a pharmaceutically effective amount of the nucleic acid molecule as described in any one of paragraphs 1 to 41 or the pharmaceutical composition of paragraph 48.

[0361] 50. The method of paragraph 49, wherein the disease is characterized by the presence of at least one Lmna mutation. 51. The method of any one of paragraphs 49 or 50, wherein the Lmna mutation affects the lamin A isoform or the lamin C isoform of the Lmna gene, or both the lamin A / C isoforms.

[0362] 52. A method according to any one of paragraphs 49 to 51, wherein the disease is selected from the group consisting of restrictive skin disorders, familial partial lipodystrophy (such as the Dunnigan type), mandibular terminal dysplasia with type A lipodystrophy, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and the diseases represented in normal font in Table 1.

[0363] 53. A method according to any one of paragraphs 49 to 51, wherein the disease is laminopathy, cardiomyopathy such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorder, cardiomyopathy with high-degree AV block and arrhythmia, isolated atrial fibrillation; muscular dystrophy (often associated with cardiomyopathy), such as cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, cardiomyopathy associated with congenital muscular dystrophy; premature aging syndrome (mainly vascular but may have heart lesions), such as cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford progeria syndrome; and a cardiovascular disease selected from the group consisting of the diseases represented in bold font in Table 1.

[0364] 54. The subject is a non-human mammal or a human; Optionally, the non-human mammal is a mouse; Optionally, the mouse is an N195K mouse (Lmna N195K / N195K ) or an Lmna conditional knockout (Lmna flox / flox ), A method according to any one of paragraphs 49 to 53.

[0365] 55. Use of the pharmaceutical composition according to paragraph 48 or the nucleic acid molecule according to any one of paragraphs 1 to 41 in the manufacture of a medicament for treating a disease or cardiovascular disease caused by one or more Lmna mutations.

[0366] 56. Use according to paragraph 55, wherein the disease is selected from the group consisting of constrictive skin disorders, familial partial lipodystrophy (such as the Dunnigan type), mandibular terminal dysplasia with type A lipodystrophy, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and the diseases represented in normal font in Table 1.

[0367] 57. Use according to paragraph 55, wherein the cardiovascular disease is selected from the group consisting of laminopathy, cardiomyopathy such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorder, cardiomyopathy with high-degree AV block and arrhythmia, isolated atrial fibrillation; muscular dystrophy (often associated with cardiomyopathy), such as cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal dominant), cardiomyopathy associated with Emery-Dreifuss muscular dystrophy (autosomal recessive), cardiomyopathy associated with limb-girdle muscular dystrophy type 1B, cardiomyopathy associated with congenital muscular dystrophy; premature aging syndromes (mainly vascular but may also have heart lesions), such as cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford progeria syndrome, etc., and the diseases represented in bold font in Table 1.

[0368] 58. The pharmaceutical composition according to paragraph 48 for use in therapy. 59. A method for screening drug candidates capable of inhibiting the protein interaction of the LINC complex in cells, (a) combining the proteins of the LINC complex in the presence of a drug to form a first complex; (b) combining the proteins in the absence of a drug to form a second complex; (c) measuring the amounts of the first complex and the second complex; and (d) comparing the amount of the first complex with the amount of the second complex, comprising A method wherein when the amount of the first complex is less than the amount of the second complex, the drug is a drug candidate for inhibiting the protein interaction of the LINC complex in cells.

[0369] 60. The method of paragraph 59, wherein the drug candidate disrupts the protein-protein interaction between SUN and KASH of the LINC complex. 61. The method of paragraph 60, wherein the drug candidate disrupts the interaction between the Sun1 protein and the Nesprin-1 protein.

[0370] 62. The method according to any one of paragraphs 59 to 61, wherein the screening is an in vitro screening. 63. The method according to any one of paragraphs 59 to 62, wherein the complex is measured by an ELISA method or a fluorescence anisotropy measurement method.

[0371] 64. The method according to any one of paragraphs 59 to 63, wherein when the amount of the first complex is less than the amount of the second complex, the drug is a drug candidate for inhibiting protein interaction. 65. The method according to any one of paragraphs 59 to 64, wherein recombinant SUN and KASH domains are used.

[0372] 65. Recombinant SUN and KASH domains are used; Optionally, the recombinant SUN domain is immobilized on a solid surface, and the recombinant KASH domain is labeled with an enzyme capable of generating a colorimetric or chemiluminescent readout (a compound that cannot inhibit the SUN-KASH interaction results in a well in the plate where the recombinant SUN binds to the enzyme-linked KASH domain. After the washing step and incubation with the colorimetric or chemiluminescent enzyme substrate, the presence of the SUN-KASH interaction can be detected in a standard plate -reader. If the compound can inhibit the SUN-KASH interaction, after the washing step, the KASH domain is removed, and the enzyme reaction in the well is considered to be reduced or absent). Optionally, the KASH domain is fluorescently labeled using a fluorescein moiety, and the fluorescence anisotropy of the KASH domain that interacts with the SUN domain can be measured using standard equipment such as a plate reader incorporating a fluorescence spectrometer function; Optionally, if the amount of the first complex is less than the amount of the second complex, a difference is thought to exist in the fluorescence anisotropy of the fluorescent KASH, and it becomes a drug candidate for inhibiting the interaction of the protein, Any one of the methods from paragraphs 59 to 65. Sequence identity Pairwise and multiple sequence alignments for the purpose of determining the percent identity between two or more amino acid or nucleic acid sequences can be achieved by various methods 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, the default parameters for gap penalties and extension penalties are preferably used.

[0373] [Table 2-1]

[0374] [Table 2-2]

[0375] [Table 2-3]

[0376]

Table 2-4

[0377]

Table 2-5

[0378]

Table 2-6

[0379]

Table 2-7

[0380]

Table 2-8

[0381]

Table 2-9

[0382]

Table 2-10

[0383]

Table 2-11

[0384]

Table 2-12

[0385]

Table 2-13

[0386]

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[0387]

Table 2-15

[0388]

Table 2-16

[0389]

Table 2-17

[0390]

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[0391]

Table 2-19

[0392] The present invention encompasses the described aspects and combinations of preferred features, except where such combinations are clearly not admitted or are clearly avoided. In the foregoing description, or in the following claims, or in the accompanying drawings, features disclosed in a particular form or from the point of view of means for performing the disclosed functions, or, if necessary, methods or processes for obtaining the disclosed results, may be used separately or in any combination of such features to implement the present invention in its various forms.

[0393] To avoid any doubt, any theoretical explanations provided in this specification are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0394] The title of any item used in this specification is for purposes of organization only and should not be construed as limiting the subject matter described. Throughout this specification, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" shall 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.

[0395] 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. It should be noted that ranges may be expressed in this specification as from "about" a particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from a particular value and / or to another 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. The term "about" associated with a numerical value is optional and means, for example, + / - 10%. The methods disclosed herein may be performed, or the products may exist, in vitro, ex vivo, or in vivo. The term "in vitro" is intended to include experiments using materials, biological substances, cells and / or tissues under laboratory conditions or in culture, whereas the term "in vivo" is intended to include experiments and procedures using intact multicellular organisms. "Ex vivo" refers to something that exists or is done outside of a living organism, e.g., outside of a human or animal body, that may be present on tissue (e.g., an organ) or cells taken from a living organism.

[0396]

[0397] ​ When a nucleic acid sequence is disclosed herein, the reverse complement thereof is also clearly intended. Regarding standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0398] As such, aspects and embodiments of the present invention are discussed 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 hereby incorporated by reference into this specification. While the present invention is described in conjunction with the exemplary embodiments described below, many equivalent modifications and variations will be apparent to those skilled in the art upon the disclosure of this application. Accordingly, the exemplary embodiments of the present invention described above are to be considered illustrative and not restrictive. Various changes to the described embodiments can be made without departing from the spirit and scope of the present invention.

[0399] Brief Description of the Drawings Embodiments and experiments illustrating the principles of the present invention are considered herein with reference to the accompanying figures.

Brief Description of the Drawings

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Example

[0401] In the following examples, the inventors have demonstrated that disruption of the LINC complex improves laminopathy, which is associated with mutations in the gene encoding lamin A / C, including knockout mutations, missense mutations, and progerin-related mutations. The inventors have also shown that disruption of the LINC complex can improve the symptoms of diseases characterized by hyperlipidemia. The inventors have shown that disruption of the LINC complex reduces atherosclerosis.

[0402] Example 1: Materials and Methods Mice were maintained at the STAR Biological Resource Centre facility and the NUS animal facility according to the guidelines of the animal ethics committees of each facility. Lmna * mice were generated and characterized as previously described [A.S. Wang et al., Differentiation; research in biological diversity, (2015); I. Solovei et al., Cell 152:584 - 598 (2013)] (Figure 33). To obtain mice with a complete deletion of Lmna (Lmna Flx / Flx ), an allele with loxP sequences introduced (Lmna Δ / Δ ) was introduced by mating into mice in which Cre recombinase is driven by the regulatory sequences of the mouse zona pellucida 3 gene (Zp3; Tg(Zp3-cre)93Knw, JAX stock 003651) [W.N. de Vries et al., Genesis 26:110 - 112 (2000)]. To obtain a cardiomyocyte-specific deletion of Lmna (Lmna Flx / Flx ), first, Lmna Flx / Flx / NIMhc mice were mated with mice in which Cre expression is driven by the cardiac-specific mouse alpha myosin heavy chain (Myh6, myosin, heavy polypeptide 6, cardiac muscle, alpha) promoter (MyHC; Tg(Myhca-cre)2182Mds, JAX stock 011038). To obtain a tamoxifen-inducible cardiomyocyte-specific deletion of Lmna (LmnaFlx / Flx:mcm), Lmna Flx / Flx mice were first mated with mice in which Cre expression is driven by the cardiac-specific mouse alpha myosin heavy chain (Myh6, myosin, heavy polypeptide 6, cardiac muscle, alpha) promoter (MyHC; Tg(Myhca-cre)2182Mds, JAX stock 011038). To obtain a tamoxifen-inducible cardiomyocyte-specific deletion of Lmna (LmnaFlx / Flx:mcm), Lmna Flx / Flxwas crossed with a mouse (mcm; Tg(Myh6-cre / Esr1 * )1Jmk, JAX stock 005657) in which Cre expression is driven by the murine cardiac-specific alpha-myosin heavy chain promoter (αMHC or alpha-MHC; Myh6), which specifically expresses tamoxifen-inducible Cre recombinase (MerCreMer) in young and adult cardiac myocytes. The specificity of mcm Cre expression for cardiomyocytes was confirmed by crossing the Cre line to mT / mG reporter mice [M.D. Muzumdar et al., Genesis 45:593-605 (2007)] (Figure 35). Sun1 - / - The generation of mice has been described previously [Y.H. Chi et al., Development 136:965-973 (2009)], and Lmna N195K / N195K mice were similarly described [L.C. Mounkes et al., Hum Mol Genet 14:2167-2180 (2005)]. Sun1 - / - Since Lmna Δ / Δ :Sun1 - / - and Lmna Flx / Flxmcm:Sun1 - / - mice were obtained by crossing each lamin-Cre mouse strain to Sun1 + / - mice.

[0403] To test for the insertion of loxP sites and conditional deletion alleles, genotyping was performed by a duplex PCR protocol using the following primers. FLX / FLX-F1: 5’-CCAGCTTACAGAGCACCGAGCT-3’ (SEQ ID NO: 16) FLX / FLX-F2: 5’-TCCTTGCAGTCCCTCTTGCATC-3’ (SEQ ID NO: 17) FLX / FLX-R1: 5’-AGGCACCATTGTCACAGGGTC-3’ (SEQ ID NO: 18) To test for Sun1 deletion, the following primers were used. ​

[0404] Sun1-F: 5'-GGC AAG TGG ATC TCT TGT GAA TTC TTG AC-3' (SEQ ID NO: 19) Sun1-R: 5'-GTA GCA CCC ACC TTG GTG AGC TGG TAC-3' (SEQ ID NO: 20) Sun1-E8: 5'-AGC CAC ATA ACC ACC TGG AG-3' (SEQ ID NO: 21) To test the MyHC transgene, the following primers were used.

[0405] MyHC-tF: 5'-ATG ACA GAC AGA TCC CTC CTA TCT CC-3' (SEQ ID NO: 22) MyHC-tR: 5'-CTC ATC ACT CGT TGC ATC ATC GAC-3' (SEQ ID NO: 23) MyHC-F: 5'-CAA ATG TTG CTT GTC TGG TG-3' (SEQ ID NO: 24) MyHC-R: 5'-GTC AGT CGA GTG CAC AGT TT-3' (SEQ ID NO: 25) To test the presence of the mcm transgene, the following primers were used.

[0406] mcm-3798t: 5'-AGG TGG ACC TGA TCA TGG AG-3' (SEQ ID NO: 26) mcm-8346t: 5'-ATA CCG GAG ATC ATG CAA GC-3' (SEQ ID NO: 27) mcm-7338: 5'-CTA GGC CAC AGA ATT GAA AGA TCT-3' (SEQ ID NO: 28) mcm-7339: 5'-GTA GGT GGA AAT TCT AGC ATC ATC C-3' (SEQ ID NO: 29) 1.1 Tamoxifen injection and tissue collection Newborn mice (14 days old) and adult mice (3 - 5 months old) were injected once with 40 mg / kg of tamoxifen (Sigma) dissolved in corn oil (Sigma). At various time points after tamoxifen injection, the mice were sacrificed by CO2 euthanasia or anesthetized with a gas mixture of 1.5% isoflurane (BioMac) and 1.5 L O2. Cardiac arrest was induced by injection of 15% KCl, followed by washing with PBS to remove blood. Hearts for paraffin embedding were further washed with 4% paraformaldehyde (PFA), left overnight in 4% paraformaldehyde (PFA), dehydrated in 70% ethanol for at least 24 hours, and embedded in paraffin. Hearts for cryosectioning were embedded in tragacanth gum (Sigma), frozen in liquid N2-cooled isopentane (BDH-AnalaR), sectioned into 9-μm sections using a cryostat (Leica CM3050), collected on charged slides, and stored at -20 °C for histological and immunofluorescence staining. Hearts for protein and RNA extraction were snap-frozen in liquid N2 and stored for further processing.

[0407] 1.2 Isolation of cardiomyocytes Cardiomyocyte isolation was performed as per the standard protocol [M. Ackers-Johnson et al., Circulation Research 119:909 (2016)]. Briefly, mice were anesthetized using isoflurane (100% O2 at 0.5 L / min, 4% isoflurane nebulizer dial). The mouse heart was arrested using 15% KCl, the descending aorta was dissected, and 7 mL of EDTA buffer was injected into the right ventricle to wash the heart. The ascending aorta was occluded using Reynolds forceps, and the entire heart was removed and placed in a 60 mm dish containing fresh EDTA buffer. The heart was digested by sequential injection of 10 mL of EDTA buffer, 3 mL of perfusion buffer, and 30 - 50 mL of collagenase buffer into the left ventricle. Using forceps, the digested heart was gently torn into smaller pieces of approximately 1 mm and subjected to gentle trituration. Enzymatic activity was inhibited by adding 5 ml of stop buffer. The cell suspension was passed through a 100 um filter, and cardiomyocytes were concentrated by four consecutive rounds of gravity sedimentation, finally obtaining a highly pure cardiomyocyte fraction. The cardiomyocyte pellet was snap-frozen in liquid N2 and stored at -80 °C for further processing.

[0408] 1.3 Histological and Immunofluorescence Microscopy For histological studies, sections (9 μm) were stained using standard hematoxylin and eosin for cell morphology, Masson's trichrome staining to detect collagen, and TUNEL assay to detect apoptotic nuclei. Images were acquired with a Zeiss Axio Imager microscope. For immunofluorescence on frozen heart sections, sections were warmed to room temperature, rehydrated with PBS, blocked with M.O.M block (Vector Shields) and donkey serum (Sigma-Aldrich), and incubated overnight at 4 °C with primary antibodies. The slides were then washed in PBS, incubated for 60 min with secondary antibodies and Hoechst dye (Sigma-Aldrich), washed with PBS, and mounted with Prolong-Gold antifade reagent (Invitrogen). Primary antibodies: LMNA / C N-18 (goat, 1:50, Santa Cruz), Sun1 monoclonal (mouse, undiluted, from B. Burke), PCM-1 (rabbit, 1:200, Sigma), and sarcomeric α-actinin (mouse, 1:100, abcam); secondary antibodies were Alexa Fluor488, 568, and 647 (1:250, Invitrogen). For immunofluorescence of isolated cardiomyocytes, myocytes were stained in suspension, gently spun down with each solution change, and then placed on glass slides for imaging using a Zeiss LSM510 inverted confocal microscope.

[0409] 1.4 Western analysis of LMNA, SUN1, Ha-tag, and GFP The whole heart and quadriceps were homogenized in RIPA lysis buffer and centrifuged at 13,200 g for 10 minutes at 4 °C. The total cell lysate was electrophoresed, transferred to a PVDF membrane, and blocked with Odyssey blocking buffer (Li-Cor Biosciences). The membrane was incubated with the primary antibody at room temperature for 2 hours. Subsequently, the membrane was washed in TBST wash buffer and incubated in Odyssey IRDye secondary antibody for 1 hour, and then visualized with an Odyssey infrared imaging system (Li-Cor Biosciences). Primary antibodies used for the detection of LMNA / C (rabbit, Cell Signalling), Sun1 monoclonal (mouse, 1:500, Burke), and control beta-tubulin (rabbit, 1:1000, Abcam), which are specific for the epitope in the first 50 amino acids of LMNA.

[0410] 1.5 Measurement of the contractile force of cardiac papillary muscles Mouse papillary muscles from the left ventricle were prepared according to a previously described method [C.N. Toepfer et al., J Physiol 594:5237 - 5254 (2016)]. Briefly, the explanted mouse heart was immediately rinsed with oxygenated ice-cold Krebs-Henseleit solution containing 12 units / mL sodium heparin (EDQM ) and 30 mM 2,3-butanedione monoxime (BDM, Sigma) to remove excess blood. Subsequently, the heart was transferred to ice-cold Krebs-Henseleit solution in a glass Petri dish under a dissecting microscope equipped with a cooling stage. Cylindrical papillae (diameter 200 - 300 μm and length 1.5 - 2 mm) were excised from the left ventricle. A perforated T-shaped aluminum clip was crimped onto both ends of the papillary preparation, and the prepared papillary mass was fixed onto a glass Petri dish containing a layer of PDMS sylgard184 (Dow Corning) using pins. The papillary preparation was immersed in a 2% Triton X-100 solution at 4 °C overnight.

[0411] Force measurements were performed as previously described [C. Toepfer et al., J Biol Chem 288:13446~13454(2013). T-shaped aluminum clips at both ends of the papilla preparation were attached to the hooks of a force transducer (AE801, HJK Sensoren+Systeme), and the servomotor of the experimental rig was adhered using shellac in ethanol (Sigma) to minimize movement during the experiment. The papillary contractile force was measured at 20 °C. The maximum contractile force was measured in an activating solution (100 mM TES, 6.5 mM MgCl2, 25 mM Ca-EGTA, 5.7 mM Na2ATP, 20 mM glutathione, 21.5 mM sodium creatine phosphate, pH = 7.1, ionic strength is 150 mmol / L) containing 32 μmol / L free Ca2+. Data were collected and processed from a DAQ data acquisition device (National Instrument) using a force transducer and customized software programmed by LabVIEW 2013 (National instrument). At least 5 fibers were tested in each mouse, and at least 3 mice were tested for each experimental group.

[0412] 1.6 AAV9-DN-Sun1 and AAV9-GFP viruses The DN-Sun1 (SS-HA-Sun1L-KDEL) and GFP (SS-GFP-KDEL) vectors were as described [M. Crisp et al., J Cell Biol. 172:41-53 (2006)]. Briefly, HA was tagged at the NH2 terminus of almost the entire luminal domain of Sun1 (HA-Sun1L). To introduce HA-Sun1L into the lumen of the ER and PNS in a soluble form, the signal sequence and signal peptidase cleavage site of human serum albumin were fused to the NH2 terminus of HA-Sun1L to obtain SS-HA-Sun1L. To prevent its secretion, the KDEL tetrapeptide was fused to the COOH terminus of SS-HA-Sun1L to form the final SS-HA-Sun1L-KDEL. The HA-Sun1L region was replaced with the GFP sequence to generate SS-GFP-KDEL. The DN-Sun1 and GFP fragments were amplified using the primers listed below (the same forward primer was used for both fragments) and ligated into the pENN-AAV-cTnT-PI-eGFP plasmid (gift from Dr. J. Jian), digested with Ncol and Kpnl to generate Penn-AAV-cTnT-Sun1DN (Figure 10, SEQ ID NO: 3).

[0413] aav Sun1 F:5’-CgagaattcacgcgggccgccATGAAGTGGGTAACCTTTATTTC-3’ (SEQ ID NO: 30) aav Sun1 R:5’-CgggtcgactctagaggtaccttaCTACAACTCATCTTTCTGGATG-3’ (SEQ ID NO: 31) aav GFP Sun R:5’-CgggtcgactctagaggtacttaCTACAACTCATCTTTGGATCC-3’ (SEQ ID NO: 32) All restriction enzymes were purchased from NEB. PCR reactions were performed using Q5® Hot Start High-Fidelity 2X Master Mix (NEB, M0494L). Ligation was performed using NEBuilder® HiFi Performed using DNA Assembly Master Mix (NEB, E2621L). Isothermal assembly was used. Primers used to construct the plasmids were ordered from IDT.

[0414] AAV viruses were produced according to the standard protocol [H. Wakimoto et al., in Current Protocols in Molecular Biology. (John Wiley & Sons, Inc., 2001)]. Materials supplied by R. Foo: transplasmid encoding pAAV2 / 9-AAV replicase and capsid genes (SEQ ID NO: 2, available from the University of Pennsylvania Penn Vector Core); pAdDeltaF6-adenovirus helper plasmid (SEQ ID NO: 1) (available from the University of Pennsylvania Penn Vector Core); QIAGEN Plasmid Maxi Kit; HEK293T cells (ATCC); transfection reagent (polyethyleneimine, e.g., Polysciences). AAV-DJ capsid was obtained from Cell Biolabs, Inc. pAAV2 / 9, AAV-DJ, pAdDeltaF6, DN-Sun1, and GFP plasmids were purified using the QIAGEN Plasmid Maxi Kit. HEK293T cells were transfected with a viral combination of the pAAV2 / 9 plasmid, the pAdDeltaF6 plasmid, and either the DN-Sun1 plasmid or the GFP plasmid. Cells were collected and the virus was purified by iodixanol gradient ultracentrifugation.

[0415] The following schedule was used for mouse heart infection. Mouse genotypes were identified at 10 days after birth. Mice were then subjected to a single IP injection of Tmx (40 mg / kg mouse body weight) at 14 days after birth, followed by an intrathoracic injection of AAV9-DN-Sun1 or AAV9-GFP virus at a concentration of 5×10^10 vg / g at 15 days after birth. Adult mice (3 - 5 months old) were given a single dose of Tmx (40 mg / kg mouse body weight) by IP injection, followed by an intrathoracic injection of AAV at a concentration of 5×10^10 vg / g of AAV9-DN-Sun1 or AAV9-GFP virus. Young and adult mice were anesthetized using a gas mixture of 1.5% isoflurane (BioMac) and 1.5 L O2 before virus injection was performed.

[0416] 1.7 Plasmid construction and generation of Cas9 mRNA and sgRNA pX330 was obtained from Addgene (#42230, Cambridge, MA, USA). 20-nt Sun1 and Syne1 single-guide RNA (sgRNA) sequences were designed using the CRISPR Design Tool (crispr.genome-engineering.org). The region of the gene of interest was given to the tool to identify suitable target sites. Since off-target mutations can occur in CRISPR / Cas9-mediated targeted mutagenesis in mice, the CRISPR Design Tool experimentally evaluates off-target genome modifications for each gRNA target site and provides computationally predicted off-target sites for each desired target, allowing the target sequences to be ranked according to a quantitative specificity analysis regarding the identification, position, and distribution effects of base pair mismatches. Complementary oligonucleotides containing the gRNA target sequences were annealed and cloned into the Bbsl site of pX330. The guide RNA sequences were as follows.

[0417] For Sun1ΔSUN, 5’-GCACAATAGCCTCGGATGTCG-3’ (SEQ ID NO: 33) For Syne1-stop, 5’-CCGTTGGTATATCTGAGCAT-3’ (SEQ ID NO: 34) For tyrosinase 4a, 5’-GGTTATGGCCGATAGGTGCAT-3’ (SEQ ID NO: 35) These plasmids (pSun1ΔSUN, pSyne1-stop, and p tyrosinase 4a) were then sequenced to verify the exact insertion of the target sequence. For in vitro transcription, PCR was performed using a common reverse primer (AAAAGCACCGACTCGGTGCC-3’, SEQ ID NO: 36), and a gRNA-specific forward primer encoding the following T7 promoter sequence to generate an appropriate transcription template.

[0418] Sun1ΔSUN: 5’-TTAATACGACTCACTATAGCACAATAGCCTCGGATGTCG-3’ (SEQ ID NO: 37); Syne1-stop: 5’-TTAATACGACTCACTATAGCCGTTGGTATATCTGAGCAT-3’ (SEQ ID NO: 38); tyrosinase 4a: 5’-TTAATACGACTCACTATAGGTTATGGCCGATAGGTGCAT-3’ (SEQ ID NO: 39) The gRNA PCR products were then subjected to agarose gel electrophoresis (1.5% agarose) to confirm successful PCR, gel purified, and used as templates for in vitro transcription using the MEGAshortscript T7 kit (Life Technologies). The gRNA was purified using the MEGAclear kit (Life Technologies) and eluted in RNase-free water. Samples of the purified gRNA were then subjected to agarose gel electrophoresis for quality confirmation and then injected into the zygotes.

[0419] 1.8 Generation of mutant mice using CRISPR / Cas9 C57BL / 6N female mice at 3 - 4 weeks of age were superovulated using pregnant mare serum gonadotropin (Calbiochem, 36722, 5 IU / ml). After 48 hours, the females were injected with human chorionic gonadotropin (Sigma, CG10, 5 IU / ml) and mated with C57BL6 males. The next day, fertilized 0.5 dpc embryos were collected from the oviducts. Cas9 mRNA (Sigma, CAS9MRNA, 100 ng / ul), tyrosinase 4a gRNA (50 ng / ul), and gene - specific gRNA (50 ng / ul) were co - injected into the cytoplasm of embryos in M2 medium (EmbryoMax® Sigma) using a micro - injection system (Nikon). Syne1 - stop sgRNA was used to obtain Syne1 C’T mutant mice, and Sun1ΔSUN sgRNA was used to obtain Sun1ΔSUN mutant mice. The injected zygotes were cultured in KSOM (EmbryoMax® Sigma) containing amino acids for 2 hours in an incubator maintained at 37 °C, 5% CO2, and 5% O2, and then transplanted into 0.5 dpc pseudopregnant C3H - ICR females.

[0420] 1.9 DNA Extraction for Genotyping of CRISPR / Cas9 Mice Mouse tails were cut and placed into separate 1.5 ml Eppendorf tubes. 80 μl of lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 12) was dispensed into the tubes and heated at 95 °C for 60 minutes. After heating, the buffer was neutralized with an equal volume of 40 mM Tris - HCl, pH 5. For certain applications, DNA was extracted and purified from mouse tails using the DNeasy Blood and Tissue Kit (QIAGEN).

[0421] 1.10 Genotyping of CRISPR / Cas9 Mice The genotypes of CRISPR - modified mutant mice were identified by PCR followed by gel electrophoresis using high - resolution agarose (2% MetaPhor agarose, Lonza).

[0422] The primers for Syne1CT’Δ8 mice were Forward: 5'-TGCTCCTGCTGCTGCTTATT-3' (SEQ ID NO: 40) Reverse: 5'-ACATGGTGGAGCATTTGTCTCC-3' (SEQ ID NO: 41 ) It was.

[0423] The primers for the Sun1 CRISPR mouse were Forward: 5'-TGACCTTGAGCTGAAACTGC-3' (SEQ ID NO: 42) Reverse: 5'-TCAGAACACTGGCACACACA-3' (SEQ ID NO: 43) It was.

[0424] The genotypes of the Lmna mutant mice were identified as described in Example 1. To determine the sequences of the CRISPR-induced mutations, PCR products from mouse tail DNA were subjected to TOPO cloning (Zero Blunt™ TOPO™ PCR Cloning Kit, 450245, Thermo Fisher Scientific). Plasmid DNA from at least 10 bacterial colonies was isolated using a mini-prep kit (QIAGEN, QIAprepSpin Miniprep Kit) and subjected to Sanger sequencing.

[0425] 1.11 Acquisition of Myoblasts, Fibroblasts, and Cell Cultures for CRISPR / Cas9 Research To isolate myoblasts, limbs were obtained from euthanized mice and the muscle was dissected from the bone. Tissue digestion was performed by incubating muscle tissue in an enzyme solution consisting of dispase II (Roche, cat. 04942078001) at a concentration of 2.4 U / ml and 1% collagenase II (GIBCO® Invitrogen, cat 17101-015) in a 37°C water bath for 30 minutes with intermittent mixing every 10 minutes. After 30 minutes, the enzyme solution was neutralized in D10 medium (Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum). The mixture was then filtered through a 70 μm sterile filter (BD Falcon™, cat 352350) and a 40 μm sterile filter (BD Falcon™, cat 352340). The suspension was then centrifuged, the supernatant removed, and then resuspended in F10 medium (GIBCO® Invitrogen, cat.11550043) supplemented with 10 μg / ml bFGF (GIBCO®, cat PHG0264) and plated onto 100 mm plates. After the mouse adult fibroblasts were allowed to settle for 2 - 3 hours, the supernatant (containing floating myoblasts) was collected and replated onto 60 mm plates coated with 0.15% gelatin (Sigma, cat G1393). D10 medium was added to the 100 mm plates with MAF. To finally differentiate the myoblasts into myotubes, the medium was changed to DMEM supplemented with 2% horse serum (Thermo Fisher Scientific GIBCO®, cat 16050122).

[0426] 1.12 Immunoblotting for CRISPR / Cas9 studies Whole cell lysates were generated using the Lysis-M kit solution (cOmplete, Roche). Cells were washed in ice-cold PBS, lysed using Roche Lysis M buffer, and centrifuged at 14,000 g for 10 minutes to remove cell debris. To extract proteins from tissue samples, small slices of tissue were quickly placed into Lysing Matrix D tubes (MP Biomedicals) and snap-frozen in liquid nitrogen. After snap-freezing, the tubes were stored at -80 °C or used immediately for protein analysis. Protein extraction buffer (in distilled water, 50 mM Tris (pH 7.4), 500 mM NaCl, 0.4% SDS, 5 mM EDTA (pH 7.4), 1× protease inhibitor (cOmplete™ EDTA-free protease inhibitor cocktail, catalog number 04693159001, Roche), 2% Triton, 1 mM dithiothreitol) was added to the tissue, and then homogenized using a FastPrep™-24 instrument (MP Biomedicals). The samples were then centrifuged at 14,000 g for 10 minutes to remove cell debris After quantifying the protein concentration using a bicinchoninic acid (BCA) protein kit (Bio-Rad), the protein samples were loaded onto polyacrylamide gels to ensure equal amounts were analyzed. All protein samples were separated by SDS-PAGE gel analysis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore) at 20 V and 4 °C for 48 h by wet transfer. The membrane was blocked for 1 h at room temperature in TBS (TBST) containing 0.1% Tween 20 supplemented with 5% powdered milk (Anlene). Western blot analysis was performed using a primary antibody diluted in 5% powdered milk (diluted in TBST). The membrane was incubated for 2 h at room temperature or overnight at 4 °C. For the secondary antibody, an antibody conjugated to horseradish peroxidase (HRP) (Invitrogen) was used for chemiluminescent imaging. The membrane was incubated with the secondary antibody for 1 h at room temperature. For immunoblots visualized by chemiluminescence, the membrane was incubated in ECL substrate (Pierce) for 1 min, then exposed to chemiluminescent-sensitive film (Thermo Scientific) and then processed.

[0427] 1.13 Immunofluorescence for CRISPR / Cas9 research Cells were grown in 8-well slides (Ibidi) and fixed in ice-cold methanol at -20 °C for 15 minutes. The cells were then washed twice in PBS and permeabilized and blocked at room temperature for 15 minutes using 0.1% Triton X and 3% BSA in PBS. The fixed and permeabilized cells were then washed three times in PBS. The samples were then incubated with the primary antibody (Table 2) for 2 hours at room temperature or overnight at 4 °C. The samples were then washed three times with PBS, followed by incubation with the secondary antibody (Life Technologies) and DAPI (Life Technologies) for 1 hour at room temperature. After three washes in PBS, the cells were mounted with an anti-fading agent (1% DABCO, 90% glycerol, 10% PBS) and examined using a Zeiss 510 Meta confocal microscope or an Axiovert 200 inverted epifluorescence microscope (Zeiss). Images were recorded and analyzed using Zeiss ZEN, Metamorph, or Image J (NIH) software.

[0428]

Table 3

[0429] 1.14 Mouse genetics Lmna mice and tamoxifen injection were performed as described in Example 1. Lmna Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 and Lmna Flx / Flxmcm :Syne1 C’ TΔ8 / C’TΔ8 To obtain double mutant mice, Lmna Δ / + or Lmna Flx / Flxmcm mice were crossed with Syne1 C’TΔ8 / C’TΔ8 mice. In the Syne2 mouse model, an IRES-β-gal neomycin selection cassette (PgkNeo) flanked by loxP sites was inserted into the Syne2 gene, resulting in the deletion of a part of exon 102 and all of exons 103 - 104. The neomycin cassette was then removed by mating with Cre recombinase mice. Syne1 C’TΔ8 / +or Syne1 C’TΔ8 / C’TΔ8 Mice were crossed with Syne2 + / - or Syne2 - / - mice to obtain mice with mutant Syne1 and Syne2 alleles, and these were intercrossed to obtain double mutant mice. Kaplan-Meier method was used to depict the survival curves.

[0430] 1.15 Human guide RNA sequences Potential guide RNA sequences that disrupt the human SYNE1 KASH domain or the SUN1 SUN domain were determined using the CRISPR tool in Benchling software (Benchling Inc., USA) and are shown in Table 3.

[0431]

Table 4

[0432] 1.16 Statistical analysis All statistical analyses were performed using Graphpad Prism software. Example 2: Cardiomyocyte-specific loss of Lmna leads to rapid onset of heart failure To further define the interaction between Sun1 and Lmna in the postnatal pathology of mice, conditional Lmna Flx / Flx strains of mice that result in complete loss of lamin A / C protein when recombined by Cre activation (Figure 33) were used to specifically remove the Lmna gene in different tissues [A.S. Wang et al., Differentiation; research in biological diversity, (2015); I. Solovei et al., Cell 152:584~598 (2013)]. Lmna Flx / Flx mice were crossed with Zp3-Cre mice to obtain Lmna Flx / FlxWhen deleted constitutively in all tissues [W.N. de Vries et al., Genesis 26:110 - 112 (2000)], the mean postnatal survival period was 17.5 days (Figure 26A). When the same deletion was induced in the absence of Sun1, Lmna Δ / Δ :Sun1 - / - mice lived on average up to 32.5 days and the lifespan was almost doubled (Figure 26A). Performing the same Lmna deletion with respect to the Sun2 null background did not extend the lifespan of Lmna Δ / Δ mice, demonstrating that the lifespan extension is specific to the loss of Sun1 (Figure 36). Since A-type lamins are widely expressed in almost all adult tissues, we then determined to what extent Lmna deletion, specifically Lmna deletion in cardiomyocytes, contributes to the early postnatal death of Lmna Δ / Δ mice. Furthermore, we sought to determine whether the loss of Sun1 could increase the lifespan of these mice including Lmna-deficient cardiomyocytes. First, Lmna Flx / Flx was crossed with a constitutive myh6 Cre [R.Agah et al., J Clin Invest 100:169 - 179 (1997)] in which Cre expression is constitutive but restricted to cardiomyocytes and starts during embryogenesis. These mice lived slightly longer than Lmna Δ / Δ and survived up to an average of 26.5 days postnatally (Figure 26C). When the same cardiomyocyte-specific deletion was performed with respect to the Sun1 - / - background, this resulted in a significant increase in lifespan up to at least 6 months postnatally and beyond (Figure 26C). To further define the loss of Lmna and its effects in postnatal / adult cardiomyocytes, we obtained homozygous mice for the Lmna Flx / Flx allele carrying an inducible cardiomyocyte-specific Cre Tg(Myh6-cre / Esr1) (hereafter abbreviated as mcm) in which Cre is induced by a single injection of tamoxifen (Tmx) [D.S. Sohal et al., Circ Res 89:20 - 25 (2001)]. Lmna Flx / Flx:mcmThe mean survival time after Cre induction in mice was 27 days (Figure 27A). The control was not affected by Tmx injection. PCR and immunofluorescence analysis confirmed that Lmna deletion was specific to cardiomyocytes and that detectable recombination did not occur in the brain, diaphragm, lung, liver, and skeletal muscle, nor in wild-type control animals (Figure 27B). By 21 days after injection, Lmna Flx / Flx:mcm mice showed labored breathing, a disheveled and unkempt appearance, increased lethargy, and kyphosis (Figure 27C). Immunofluorescence analysis of isolated cardiomyocytes (CMs) and sections of the Lmna Flx / Flx:mcm heart showed reduced levels of lamin A protein and cardiomyocyte nuclei without lamin A expression (Figure 27D). Lamin A protein levels were reduced to one-third in the Lmna Flx / Flx:mcm heart after Cre induction compared to the non-induced Lmna Flx / Flx:mcm and Lmna Flx / Flx:mcm hearts (Figure 27E). Sampling of Lmna + / + / mcm mice at certain time points after Tmx injection estimated that it took 7 - 14 days for the LMNA protein level to drop by 50% after Cre induction (data not shown), which is consistent with the rate shown by studies using siRNA LMNA knockdown in human fibroblasts that further decreased to one-third after 48 hours and to one-fourth after 10.5 days [A. Buchwalter and M. W. Hetzer, Nature communications 8:328 (2017); T. Sieprath et al., Nucleus 6:236 - 246 (2015)]. Echocardiograms (ECGs) performed 21 days after Cre induction revealed insufficient cardiac contractility in Lmna Flx / Flx:mcm mice compared to controls (Figure 28A). There were significant decreases in ejection fraction (EF%) and fractional shortening (FS%) (P < 0.0001) (Figure 28B). Left ventricular end-systolic and end-diastolic diameters (LVID) were enlarged in Lmna Flx / Flx:mcm mice compared to controls (Figure 28B). Significantly fewer viable (brick-like) cardiomyocytes were present in Lmna Flx / Flx:mcm compared to controls (Figure 28C). Flx / Flx:mcm compared to controls (Figure 28B). Lmna Flx / Flx:mcm compared to controls (Figure 28C). Significantly fewer viable (brick-like) cardiomyocytes were present in Lmna Flx / Flx:mcm+Tmx isolated from the heart (Figure 28C). Visual analysis showed that Lmna Fl x / Flx:mcm +Tmx cardiomyocytes isolated from the heart contained large intracellular vacuoles (Figure 28C). Lmna Flx / Flx:mcm +Tmx histological analysis of the heart revealed infiltration of nucleated cells and an increased intercellular space between cardiomyocytes compared to control hearts (Figure 28D). Lmna Flx / Flx:mcm +Tmx left ventricular cavity in the heart was significantly enlarged, and at the same time, a significantly increased level of fibrosis (P = 0.0098) compared to the control was observed in Lmna Flx / Flx:mcm +Tmx heart (Figure 28D). An increased number of apoptotic cells compared to control hearts was also identified in Lmna Flx / Flx:mcm +Tmx heart (Figure 28D). However, as evaluated by Rad51, MRE11, H2AX phosphor-Ser, and 53BP1 immunostaining, there was no evidence of extensive DNA damage detectable in cardiomyocytes (data not shown). Flx / Flx:mcm Flx / Flx:mcm Flx / Flx:mcm +Tmx heart (Figure 28D). However, as evaluated by Rad51, MRE11, H2AX phosphor-Ser, and 53BP1 immunostaining, there was no evidence of extensive DNA damage detectable in cardiomyocytes (data not shown).

[0433] Example 3: Deletion of Sun1 ameliorates heart pathologies induced by Lmna loss Mice with Lmna mutations show a significant increase in lifespan and a significant improvement in health in the absence of Sun1 [C.Y. Chen et al., Cell 149:565 - 577 (2012)]. As described, inducible deletion of Lmna (Lmna Flx / Flx:mcm +Tmx) in cardiomyocytes results in death within one month after Cre induction (Figure 26C). Surprisingly, when the same deletion was induced in a Sun1 null background, the mice survived for more than one year after Cre induction (Figure 26C). Lmna Flx / Flx:mcm Sun1 - / - Hearts from +Tmx mice were collected three weeks after induction, Lmna Flx / Flx:mcm Sun1 + / +We determined the extent to which loss of SUN1 improved the pathological changes induced by loss of Lmna in cardiomyocytes compared to the heart from +Tmx. Immunofluorescence imaging for lamin A / C identified many elongated and distorted nuclei. In some of these, residual lamin A / C had migrated to one pole of the nucleus in the +Tmx heart (panel 1 and inset in Figure 29A). In contrast, in the Lmna Flx / Flx:mcm Sun1 + / + +Tmx heart, many elongated nuclei were present even in the absence of lamin A / C staining, and these showed little distortion if any (yellow arrowheads in panel 3 of Figure 29A). Western analysis of whole hearts revealed a significant decrease in lamin A / C compared to controls (P = 0.0359) in the Lmna Flx / Flx:mcm Sun1 - / - +Tmx heart lysate (lower panel in Figure 29A). The Lmna Flx / Flx:mcm Sun1 - / - +Tmx cardiomyocyte nuclei presented with an increased longitudinal length and a segmented appearance, with the segments connected by narrow bridges (arrows in panels 1 of Figure 29A and C). However, in the absence of Sun1, the Lmna Flx / Flx:mcm Sun1 + / + cardiomyocyte nuclei showed no abnormalities or segmentation (panels 3 and 4 in Figure 29C). Overall, for less than 1% of cardiomyocytes from Lmna Flx / Flx:mcm Sun1 + / + 70% of the cardiomyocytes in the mouse had ruptured or malformed nuclei (panel 5 in Figure 29C). A clear enlargement of the left ventricle (LV) was evident in the Lmna Flx / Flxmcm Sun1 - / - mouse but not in the LV of the Lmna Flx / Flx:mcm Sun1 - / - +Tmx heart (panels 1 and 2 in Figure 29B). The Lmna Flx / Flx:mcm Sun1 + / + mouse, but not in the LV of the Lmna Flx / Flx:mcm Sun1 + / + mouse was evident, but not in the LV of the Lmna Flx / Flx:mcm Sun1 - / - +Tmx heart (panels 1 and 2 in Figure 29B). The Lmna Flx / Flx:mcm Sun1 + / +The heart exhibited a significantly increased level of fibrosis compared to the control (P<0.0001), Lmna Flx / Flx:mcm Sun1 - / - There was no significant fibrosis in the heart (Figure 29B, panels 3 - 5).

[0434] As a model for the mechanism of action of the left ventricular muscle, the force exerted on the cardiac papillary muscle was measured. The force exerted was significantly, 66% decreased (P = 0.0028) in Lmna Flx / Flx:mcm :Sun1 + / + +Tmx papillary muscles compared to Lmna Flx / Flx:mcm Sun1 + / + +CTL. In the absence of SUN1, the Lmna Flx / Flx:mcm Sun1 - / - +Tmx cardiac papillary force was maintained at a level not significantly different from the control force (panel 6 of Figure 29B). It was maintained (panel 6 of Figure 29B).

[0435] Echocardiograms performed before and after Cre induction revealed a progressive deterioration of cardiac contractility in Lmna Flx / Flx:mcm Sun1 + / + +Tmx mice compared to Lmna Flx / Fl:mcm Sun1 - / - +Tmx mice (Figure 29D). Loss of SUN1 preserved EF, FS, and longitudinal global strain (GLS) (GLS is an independent parameter used to evaluate myocardial contractility and a better predictor of heart failure) in Lmna Flx / Flx:mcm Sun1 - / - +Tmx mice compared to all of Lmna Flx / Flx:mcm Sun1 + / + +Tmx mice.

[0436] PCR analysis of the aged Lmna Flx / Flx:mcm Sun1 - / - +Tmx heart 12 - 14 months after Tmx injection confirmed a persistent deletion of the Lmna gene (Figure 37C), and protein quantification showed a significant decrease in Lmna 12 - 14 months after TMX in Lmna Flx / Flx:mcm Sun1 - / -+Tmx revealed a significant decrease in LMNA levels in the heart (Figure 37D). At 12 - 14 months of Lmna Flx / Flx:mcm Sun1 - / - +Tmx histological analysis of the heart revealed no significant increase in fibrosis compared to controls (Figures 37A and B). However, echocardiograms of these aging mice showed decreased EF and FS in both Lmna Flx / Flx:mcm Sun1 + / + +CTL mice and Lmna Flx / Flxmcm Sun1 - / - +Tmx mice (Figure 37E). The mean survival of Lmna Flx / Flx mice was 13 - 14 months (Figure 26C), so the decreased systolic function was considered to be due to aging. Furthermore, these findings demonstrate that loss of Lmna in adult (2 - 3 months) cardiomyocytes is sufficient to cause heart failure within 3 - 4 weeks after Cre activation, but this pathology is surprisingly alleviated by deleting Sun1, and this alleviation persists for one year.

[0437] Example 4: Loss of SUN1 extends the lifespan of Lmna missense mutants Since the majority of cases of LMNA - induced DCM are caused by missense mutations, we determined the effect of loss of SUN1 on the lifespan and cardiac function of a previously described Lmna mutant mouse line [L.C. Mounkes et al., Hum Mol Genet 14:2167 - 2180 (2005)] that carries the N195K missense mutation identified in two unrelated patients diagnosed with AD - EDMD [D. Fatkin et al., N Engl J Med 341:1715 - 1724 (1999); J.P. van Tintelen et al., Am Heart J 154:1130 - 1139 (2007)]. Here too, we found that the absence of SUN1 significantly extended the lifespan of this mutant mouse line, along with improved cardiac function (Figure 26D). These findings were obtained by introducing loxP sequences into the WT - Lmna allele to obtain mice that are heterozygous for the N195K mutation, i.e., Lmna N195K / Flx ×Sun1+ / + were extended by. Inducing the Tmx-inducible cardiomyocyte Cre allele in these mice (Lmna N195K / Flx:mcm +Tmx) resulted in deletion of the WT flox Lmna allele, making the cardiomyocytes hemizygous for the Lmna N195K / - mutation. These mice had an average lifespan of less than 50 days, which was half the lifespan of the original Lmna N195K / N195K homozygous mice (Figure 30A). When the Lmna N195K / Flx:mcm +Tmx mutation was induced with respect to the Sun1 null background, the lifespan significantly extended from less than 50 days to over 200 days (Figure 30A), revealing that loss of Sun1 was also effective in preventing DCM caused by the Lmna missense mutation, specifically the Lmna missense mutation in cardiomyocytes.

[0438] Echocardiograms performed before and after Cre induction revealed progressive deterioration of cardiac contractility in Lmna N195K / Flx:mcm Sun1 + / + mice compared to Lmna N195K / Flx:mcm Sun1 - / - mice (Figure 30B). Loss of SUN1 preserved EF, FS, and global longitudinal strain (GLS) in the long-axis direction in Lmna N195K / -:mcm Sun1 - / - mice compared to Lmna N195K / -:mcm Sun1 + / + mice (Figure 30B).

[0439] Example 5: AAV9-mediated transduction and expression of DNSun1 extend the lifespan of Lmna Flx / Flx:mcm +Tmx mice The above results demonstrated that genetically removing the function of SUN1 or genetically reducing the level of SUN1 may have therapeutic value in treating DCM. The inventors then examined whether this is due to complete elimination of the function of SUN1, which overcomes the toxicity of excessive SUN1 by specifically disrupting the role of SUN1 in the LINC complex that connects the nucleus to the cytoskeletal components by connecting the KASH domain protein in the ONM without changing the level of SUN1. To distinguish between these two possibilities, adeno-associated virus (AAV) was used to specifically transduce and express in cardiomyocytes a dominant-negative SUN1 minigene [M. Crisp et al., J Cell Biol 172:41-53 (2006)] whose protein product can compete with both SUN1-KASH binding and SUN2-KASH binding in the nuclear envelope cisternae of cardiomyocytes. Schematic diagrams of the AAV viral capsid and its genomic payload are shown in FIGS. 48A and 49B (obtained from FIGS. 1B and 1C of Lipinski et al., Prog Retin Eye Res. (2013) 32:22-47).

[0440] In the region corresponding to the entire luminal domain of the Sun1 gene, the HA (HA-Sun1L) epitope was tagged at its N-terminus. To localize the resulting protein product to the endoplasmic reticulum (ER) and the perinuclear space (between the INM and ONM - PNS), the signal sequence of human serum albumin and the signal peptidase cleavage site were fused to the N-terminus of HA-Sun1L to obtain SS-HA-Sun1L. To prevent the secretion of the mini-protein, the KDEL tetrapeptide was ligated to the C-terminus of SS-HA-Sun1L to form SS-HA-Sun1L-KDEL (Figure 34). The signal sequence can ensure that HA-Sun1KDEL accumulates in the continuous perinuclear ER and PNS lumen intracellularly. The cDNA sequence encoding the mini-gene was fused to the chicken cardiac troponin promoter (cTnT) to ensure that the mini-gene was transcribed only in cardiomyocytes [K.M. Prasad et al., Gene Ther 18:43 - 52 (2011)]. Figures 15 (the third panel) and 31B show diagrams of how SS-HA-Sun1L (DN-Sun1) moves the KASH domain protein from the LINC complex in the PNS to the ER.

[0441] To verify that DN-Sun1 functioned in cardiomyocytes (CM), first, the AAV-DJ system [D. Grimm et al., J Virol. 82(12):5887 - 911 (2008)], which provides a higher infection rate in cultured cells than the AAV9 serotype, was used under the transcriptional control of the cTnT promoter in the mouse heart to transduce human CM derived from iPS stem cells. As shown in Figure 31D, DN-Sun1 was effective in moving Nesprin-1 from the nuclear envelope in CM expressing DN-Sun1. Cells expressing high and low levels of DN-Sun1 are indicated by gray and white arrowheads, respectively. High-level DN-Sun1 expression resulted in the movement of Nesprin-1 from the nuclear envelope. This confirmed that DN-Sun1 was effective in disrupting the LINC complex in CM.

[0442] Using AAV (serotype 9), the DN-Sun1 minigene was transduced and expressed in the hearts of postnatal mice by intrathoracic injection. The procedure is summarized in Fig. 31A, and all mice were T sacrificed for analysis 100 days after Tmx injection. Detection of Lmna deletion in the heart by PCR confirmed Cre induction by Tmx injection (Fig. 31C). To determine the localization and expression level of the DN-Sun1 minigene, total protein was extracted from half of the heart. Western analysis revealed strong expression of both AAV9-DNSun1 protein and AAV9-GFP control protein (injection dose: 5×10^10 vg / g mouse) 99 days after AAV injection (Fig. 31C), and the expression levels of both proteins were dependent on the dose of injected virus particles (Fig. 38). Expression of either AAV9-DNSun1 protein or AAV9-GFP protein did not affect the LMNA protein level (Fig. 39A).

[0443] Immunofluorescence analysis revealed that a larger percentage of cardiomyocytes expressed GFP with 5×10^10 vg / g of AAV9-GFP compared to the levels resulting from one-tenth the dose of virus particles (5×10^9 AAV9-GFP) (Figs. 39B and 39C).

[0444] Lmna injected with AAV9-GFP control Flx / Flx:mcm +Tmx mice survived for an average of 34.5 days after Tmx, but Lmna injected with AA9-DNSun1 (5×10^10 vg / g mouse) Flx / Flx:mcm +Tmx mice survived significantly longer, and the majority survived until at least 100 days after Tmx and were then sacrificed for analysis (P = 0.0002) (Fig. 20 shows the results for the initial time period for male and female mice, and Fig. 31E shows the results at 100 days, presented in a separate graph for male and female mice with mice having different virus injection titers removed). Echocardiogram analysis showed that the Lmna Flx / Flx:mcm +Tmx+AAV9-DNSun1 heart at 35 days after Tmx had Lmna Flx / Flx:mcmIt was confirmed that +Tmx+AAV9-GFP functioned better than the heart (Figure 31G). Lmna Flx / Flx:mcm The +Tmx+AAV9-DNSun1 mice survived at 100 days after induction, but both EF% and FS% were significantly lower compared to the control Lmna Flx / FlxWT +Tmx mice (Figure 31G). At 35 days after Tmx, increased fibrosis was detected in both the Lmna Flx / Flx:mcm +Tmx+AAV9-DNSun1 heart and the Lmna Flxx / Flxxmcm +Tmx+AAV9-GFP heart (Figure 31F), but the fibrosis in the Lmna Flx / Flx:mcm +Tmx+AAV9-DNSun1 heart was significantly less than that in the Lmna Flxx / Flxxmcm +Tmx+AAV9-GFP heart (lower panel of Figure 31F).

[0445] Example 6: Disruption of the LINC complex in mice using CRISPR / Cas9 Whether systemic or heart-specific mutations, mice containing various Lmna mutations show a significant increase in lifespan and a significant improvement in health in the absence of Sun1 [(Chen et al., Cell 149:565 - 577 (2012), and Examples 2 - 4]. Prior to the findings described in Examples 2 - 5, the mechanism of this rescue was not clear but was hypothesized to be due to the toxic effect of excessive Sun1 in Lmna mutants [Chen et al., Cell 149:565 - 577 (2012)]. AAV-mediated expression of a dominant-negative LINC complex-disrupting transgene improves the pathology associated with Lmna mutations [Example 5]. The findings in Examples 2 - 5 are consistent with the idea that LINC complex function rather than excessive Sun1 is the molecular driver of Lmna pathology. This is demonstrated by the genetic disruption of the LINC complex through the loss of Sun1 and Sun2 in mice [K. Lei et al., Proc It was surprising that the cardiac-specific disruption of Nesprin-1 and Nesprin-2 [Banerjee et al., PLOS Genet 10(2):e1004114 (2014)] or Natl Acad Sci USA 106:10207~10212 (2009) led to various pathologies.

[0446] To develop alternative means of disrupting the LINC complex in vivo, we examined whether CRISPR / Cas9 genome editing could be used to disrupt the SUN and KASH domains of the proteins that make up the LINC complex. Since both the SUN domain and the KASH domain are located at the C-terminus of their respective proteins, we hypothesized that a CRISPR guide RNA targeting the 3’ end of the gene encoding the SUN or KASH domain protein could generate premature stop codons after CRISPR-induced non-homologous end joining. This could result in truncated proteins with mutated C-terminal SUN or KASH domains. The truncated proteins could be expressed and membrane-localized, but unable to interact with their cognate LINC complex partners. In Example 2, we found that loss of Sun2 did not improve Lmna-related pathologies. Therefore, since Sun1 is thought to be the dominant SUN domain protein mediating Lmna pathologies, we chose to use CRISPR to target the Sun1 SUN domain. Among the KASH domain proteins, only Nesprin-1, Nesprin-2, and Nesprin-3 are widely expressed [H.F. Horn, Current topics in developmental biology 109:287~321 (2014)]. Nesprin-1 and Nesprin-2 are closely related paralogs with functionally overlapping roles. They interact with the actin and microtubule cytoskeletons, while Nesprin-3 is thought to interact specifically with intermediate filaments [Kim et al., Biol. Chem. 396:295~310 (2015)]. Since we already had Nesprin-2 and Nesprin-3 mutant mouse lines obtained by conventional gene targeting available in the laboratory, we chose to use CRISPR to target the KASH domain of Nesprin-1 and verify whether CRISPR / Cas9 could be used in vivo for the treatment of laminopathy.C57 / Bl6 mice were microinjected with Cas9 mRNA and gRNA targeting the SUN1 domain (5’-GCACAATAGCCTCGGATGTCG-3’, SEQ ID NO: 66) or gRNA targeting the KASH1 domain (5’-CCGTTGGTATATCTGAGCAT-3’, SEQ ID NO: 67), and then transplanted into surrogate mothers, directly targeting the Sun1 gene and the Syne1 gene encoding the Nesprin-1 protein in vivo. Note that the SUN1 gRNA targets Sun1 upstream of the SUN domain to remove the SUN domain. gRNA targeting the tyrosinase gene (5’-GGTTATGGCCGATAGGTGCAT-3’, SEQ ID NO: 68) was co-injected - Offspring that received CRISPR genome editing may have a white or mosaic coat color due to tyrosinase disruption. The genotypes of these pups were identified to confirm successful gene disruption, and these pups were used as founder animals to establish Sun1 or Nesprin-1 mutant colonies.

[0447] 6.1 Characterization of mutant mice After Sanger sequencing of founder animals and F1 offspring, we focused on characterizing the Sun1 mutant allele with a 7-bp deletion (Sun1_del7 or Sun1Δ7, SEQ ID NO: 71) and the Sun1 mutant allele with a 4-bp insertion (Sun1_plus4, SEQ ID NO: 70) (Figure 40A), as well as the Syne1 (Nesprin-1) mutant allele with an 8-bp deletion (Syne1_CTdel8 or Syne1C’TΔ8, SEQ ID NO: 76) (Figure 41A). The Sun1 mutant allele was predicted to produce an mRNA with a premature stop codon, resulting in a truncated Sun1 protein lacking the SUN domain (Figure 40B). Tail tip fibroblasts were isolated from Sun1 homozygous mutant animals.

[0448] Immunofluorescence staining revealed the loss of the Sun1 protein (Figure 40C), suggesting that the insertions and deletions generated by CRISPR caused a nonsense mutation-dependent degradation mechanism of Sun1 mRNA. It was not clear whether the site of the mutation, which was outside rather than inside the SUN domain, had an effect on the expression of the mutant gene. SU Since we were unable to obtain Sun1 mutant alleles that produce Sun1 proteins lacking the SU

[0449] The Syne1 C’TΔ8 allele is predicted to produce a protein in which the final 11 amino acids in the wild-type sequence (SEQ ID NO: 77) are mutated, followed by an additional 50 amino acids encoded by the alternative reading frame (Figure 41B, SEQ ID NO: 78). Immunoblotting performed on Syne1WT and Syne1C’TΔ8 heart and muscle tissues revealed a band of approximately 120 kDa corresponding to the Nesprin-1α isoform of the Syne1 gene, which is abundant in striated muscle in WT (Figure 41C, D). In C’TΔ8 heart and muscle tissues, the putative Nesprin-1α polypeptide was thought to be less abundant and have a lower electrophoretic mobility than in the wild type (Figure 41C, D). This is consistent with the 8 bp deletion in the Syne1C’TΔ8 allele introducing a new stop codon downstream, resulting in a protein of higher molecular weight. In addition, a band of approximately 1 MDa, which is likely to correspond to Nesprin-1 Giant, was observed in heart tissues from both Syne1WT and Syne1C’TΔ8 mice.

[0450] Immunofluorescence analysis of mouse adult fibroblasts (MAF) derived from 12-week-old mice revealed that Nesprin-1 was mislocalized from the nuclear envelope to the cytoplasm in Syne1C’TΔ8 MAF (Figure 42A). Similarly, in myotubes, Nesprin-1 redistributes to the cytoplasm in Syne1C’TΔ8 myotubes compared to Syne1WT myotubes (Figure 42B). Other LINC complex and NE proteins, such as SUN1, SUN2, emerin, and lamin A, remained localized to the NE (Figures 43A–C). Consistent with previous reports [Gimpel et al., Curr. Biol. 27:2999–3009.e9. (2017)], disruption of Nesprin-1 in myotubes resulted in mislocalization of centrosomal proteins PCM1, Pcnt, and Akap450 from the myotube nuclear envelope (Figures 44A–C). The mislocalization of Nesprin-1 from the nuclear envelope coincides with disruption of the Nesprin-1 KASH domain, preventing the Nesprin-1C’TΔ8 mutant protein from interacting with the SUN domains of Sun1 and Sun2, which normally can restrict Nesprin-1 to the nuclear envelope. Since the transmembrane region is not disrupted, Nesprin-1 is likely to be mislocalized to the endoplasmic reticulum (ER) in the C’TΔ8 mutant. This is because the ER and the nuclear membrane cisternae form a continuous membrane system.

[0451] Similar to one Nesprin-1 mouse model reported previously [Zhang et al., Development 134(5):901-8(2007)], and in contrast to two other models [Puckelwartz et al., Hum Mol Genet 18:607-620(2009); Zhang et al., Hum Mol Genet 19:329-341(2010)], disruption of the KASH domain of Nesprin-1 does not result in an obvious phenotypic difference between Syne1 wild type (WT) and the Syne1C’TΔ8 mutation (Figs. 45A-B). Both male and female homozygous mutants are fertile, and there is no significant difference in body weight between Syne1WT mice and Syne1C’TΔ8 mice (Fig. 45C). Syne1C’TΔ8 mice also do not exhibit any growth retardation or obvious muscular dystrophy, and show no difficulty in movement or grooming that could be an indicator of muscle decline.

[0452] To explore the role of other KASH domain proteins in the Lmna pathology, mouse mutations regarding Syne2 that encodes Nesprin-2 were generated by conventional gene targeting (Fig. 46A). To characterize the mutations, immunofluorescence microscopy of tail tip fibroblasts was performed. Syne2 - / - homozygous mutant fibroblasts hardly or did not express Nesprin-2 (Fig. 46B). Consistent with previous findings [Zhang et al., Development 134(5):901-8(2007)], Sy ne2 - / - mice were clearly normal and had no growth retardation or infertility, but Nesprin-1 / 2 double mutant mice (Syne1 C’TΔ8 / C’TΔ8 :Syne2 - / - ) were perinatal lethal (Fig. 46C).

[0453] 6.2 Disruption of the Nesprin-1 KASH domain improves the Lmna pathology Even if Nesprin-1 is still expressed, the Nesprin-1-containing LINC complex is Syne1 C’TΔ8 / C’TΔ8It was thought not to be formed in cells and animals. Since in vivo AAV-mediated disruption of the LINC complex using dominant-negative SUN1 rescues the Lmna pathology (Example 5), the inventors inferred that the “KASH-less” Nesprin-1 mutant alleles generated by the inventors might also rescue the Lmna pathology. To test this hypothesis, mice heterozygous for the Lmna null (Lmna Δ / Δ ) allele (Example 1) were crossed with Syne1C’TΔ8 mice to obtain Lmna Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 double mutant mice. Lmna Δ / Δ mice survived for 15 - 17 days, whereas Lmna Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 double mutant mice survived up to 42 days (Figure 24). Lmna null mice heterozygous for the Syne1 C’TΔ8 allele did not experience any extension of their lifespan. Lmna - / - mice on a Syne2 Δ / Δ homozygous mutant background also did not experience an extension of their lifespan (Figure 47), indicating that the Lmna pathology is mainly mediated by the Nesprin-1 / Sun1 LINC complex. To examine the effect of the Syne1 C’TΔ8 / C’TΔ8 allele in mice with cardiac-specific loss of Lmna, conditional Lmna Flx / Flx mice homozygous for the allele, carrying an inducible cardiomyocyte-specific Cre Tg(Myh6-cre / Esr1) (abbreviated here as mcm) in which Cre is induced by a single injection of tamoxifen (Tmx), were used as described in Examples 1 - 2. Cardiac-specific deletion of Lmna led to death within 1 month, whereas mice with the same deletion induced on a homozygous Syne1 C’TΔ8 / C’TΔ8 background survived for at least 120 days after Tmx induction (Figure 25, no change from days 80 - 120).

[0454] Example 7: Method for screening small molecules that block SUN-KASH interaction Crystallographic studies of human SUN2 reveal that the SUN domains assemble as a cloverleaf-like trimeric structure [Sosa et al., Cell 149(5):1035-47 (2012)]. Trimerization is mediated by a triple-helical coiled coil with an estimated length of 40-45 nm. This is sufficient to crosslink the nuclear envelope cisternae (PNS) and allows the SUN and KASH domains to interact directly [Sosa et al., Cell 149(5):1035-47 (2012)]. The KASH-binding site is mainly formed within a groove formed at the boundary between adjacent SUN domains (Figure 5B, left panel of Figure 21). This groove accommodates a portion of the ~18-residue KASH domain in an extended conformation. However, that portion is the C-terminal tetrapeptide of the KASH domain, characterized by three proline residues followed by a terminal aliphatic residue, Leu or Thr (in the cases of Nesp1 and Nesp2, respectively), and is extremely important for the SUN-KASH interaction (right panel of Figure 21, cited from Figure 1 of Sosa et al., Cell 149(5):1035-47 (2012)). The importance of this tetrapeptide lies in its positioning within a well-defined pocket formed within a single SUN monomer. Modification of this peptide by any means, including the addition of a single residue (Ala) at the C-terminus, completely abrogates SUN-KASH association throughout the SUN-KASH contact region (Sosa et al., Cell 149(5):1035-47 (2012) and left panel of Figure 22). In conclusion, stable binding of the KASH domain requires 18-20 residues, but it is the C-term...

Claims

1. A LIN Complex inhibitor for use in a method of treating or preventing laminopathy.

2. Use of a LIN Complex inhibitor in the manufacture of a medicament for use in a method of treating or preventing laminopathy.

3. A method of treating or preventing laminopathy, the method comprising administering to a subject a therapeutically or prophylactically effective amount of a LIN Complex inhibitor.

4. The LIN Complex inhibitor for use according to claim 1, the use according to claim 2, or the method according to claim 3, wherein the laminopathy is characterized by one or more of myopathy, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, cardiomyopathic muscular dystrophy, skeletal muscular dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy or skin disease.

5. The LIN Complex inhibitor, use or method for use according to any one of claims 1 to 4, wherein the laminopathy is associated with a mutation in LMNA.

6. Laminopathy is Hutchinson-Gilford progeria syndrome; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-related; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; muscular dystrophy; mandibuloacral dysplasia with lipodystrophy, a type; 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-hand syndrome, Slovenian type; aging; familial partial lipodystrophy; restrictive dermopathy, lethal; arrhythmogenic right ventricular cardiomyopathy; dental disease; Heart disease; Werner syndrome; Hypertrophic cardiomyopathy; Left ventricular noncompaction; Atrioventricular block; Calcification; Acro-osteolysis; Autosomal dominant limb-girdle muscular dystrophy; True diabetes, insulin-independent; Osteoporosis; Atrial fibrillation; Atrial standstill 1; Melanoma; Cardiac conduction disorder; Catecholamine-induced polymorphic ventricular tachycardia; Micrognathia, deafness, progeroid features, and lipodystrophy syndrome; Sinus node dysfunction syndrome; Pelger-Huet nuclear anomaly; Charcot-Marie-Tooth disease, axonal, type 2E; Congenital generalized lipodystrophy; Restrictive cardiomyopathy; Congenital myofibrillar 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; Obesity index quantitative trait locus 11; Skin disease; Myotonic dystrophy 1; Neuromuscular disease; Hurler-Scheie 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-deficient, type 1A; Proximal spinal muscular atrophy; Muscular dystrophy-dystroglycanopathy, type B, 5; Muscular dystrophy, congenital, type 1B; Reynolds syndrome; Wiedemann-Rautenstrauch syndrome; Emery-Dreifuss muscular dystrophy 1, X-linked type; Lipodystrophy, congenital generalized, type 2; Monogenic diabetes; Cardiomyopathy, dilated, type 1D; Myopathy, proximal, and ophthalmoplegia; Myopathy; Lipodystrophy, familial partial, type 4; Cardiomyopathy, dilated, type 1H; Second-degree atrioventricular block; Median nerve disorder; Endogenous cardiomyopathy; Prolapse of female genital organs; Complete generalized lipodystrophy; Myotonic dystrophy; Emerinopathy; Ulnar nerve disorder; Limb-girdle muscular dystrophy type 1B; Lmna-related dilated cardiomyopathy; Pelvic muscle wasting; Generalized lipodystrophy-related progeroid syndrome; Muscle disease; Cardiomyopathy, dilated, type 1B; Autosomal genetic disease; Family Familial isolated arrhythmogenic ventricular dysplasia, right-dominant type; Familial isolated arrhythmogenic ventricular dysplasia, biventricular type; A LIN Complex inhibitor, use or method for use according to any one of claims 1 to 5, selected from familial isolated arrhythmogenic ventricular dysplasia, left-dominant type; Lmna-related cardio-cutaneous progeria syndrome; and autosomal semi-dominant severe lipodystrophy laminopathy.

7. A LIN Complex inhibitor for use in a method of treating or preventing a disease characterized by hyperlipidemia.

8. Use of a LIN Complex inhibitor in the manufacture of a medicament for use in a method of treating or preventing a disease characterized by hyperlipidemia.

9. A method of treating or preventing a disease characterized by hyperlipidemia, the method comprising the step of administering a therapeutically or prophylactically effective amount of a LIN Complex inhibitor to a subject.

10. A LIN Complex inhibitor for use according to claim 7, use according to claim 8, or method according to claim 9, wherein the disease characterized by hyperlipidemia is selected from atherosclerosis, cardiovascular disease, stroke, and familial hyperlipidemia.

11. A LIN Complex inhibitor, use or method for use according to any one of claims 1 to 10, wherein the LIN Complex inhibitor is capable of binding to a LIN Complex, a LIN Complex protein, or an interaction partner for a LIN Complex protein, or wherein the LIN Complex inhibitor is capable of reducing the expression of a LIN Complex protein.

12. A LIN Complex inhibitor, use or method for use according to claim 11, wherein the LIN Complex inhibitor is capable of inhibiting the interaction between a LIN Complex protein and an interaction partner for a LIN Complex protein.

13. A LIN Complex inhibitor, use or method for use according to claim 11 or claim 12, wherein the LIN Complex inhibitor is a peptide / polypeptide, nucleic acid, or small molecule.

14. A LIN Complex inhibitor, use or method for use according to claim 11, wherein the LIN Complex inhibitor is capable of modifying a gene encoding a LIN Complex protein to reduce its expression.

15. A LIN Complex inhibitor, use or method for use according to claim 14, wherein the LIN Complex inhibitor comprises a site-specific nuclease (SSN) targeting a gene encoding a LIN Complex protein.

16. The LIN Complex inhibitor, use or method for use according to claim 11, wherein the LIN Complex inhibitor is an inhibitory nucleic acid capable of reducing the expression of LIN Complex protein by RNA interference (RNAi).

17. The LIN Complex inhibitor, use or method for use according to any one of claims 11 to 16, wherein the method comprises administering to a subject a nucleic acid encoding a LIN Complex inhibitor or a nucleic acid encoding a factor necessary for the production of a LIN Complex inhibitor.

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