Treatment / Prevention of Diseases by Inhibiting the LINC Complex

Abstract

A method for treating and preventing diseases characterized by laminopathies and hyperlipidemia by inhibiting the LINC complex is disclosed. Specifically, disrupting the LINC complex by expressing a dominant-negative LINC complex protein alleviates the pathophysiology of Lmna mutation-associated muscular dystrophy, progeria, and dilated cardiomyopathy. Furthermore, disrupting the LINC complex by expressing a dominant-negative LINC complex protein also alleviates the pathophysiology of atherosclerosis and familial hypercholesterolemia in mouse models.

Description

[Technical Field]

[0001] This application claims priority to SG 10201906637U, filed July 17, 2019, the contents and elements of which 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 the inhibition of LINC complexes. [Background technology]

[0003] Background of the Invention Dilated cardiomyopathy (DCM) is the most common disease affecting cardiac muscle, accounting for approximately 60% of all cardiomyopathies. DCM is characterized by reduced systolic (contraction) function due to enlargement and thinning of the left ventricular wall, or occasionally both ventricles. DCM is associated with sudden heart failure and cardiac death, leading to high rates of hospitalization, the need for heart transplantation, and consequently high costs (JL Jefferies and JA Towbin, Lancet 375:752-762 (2010); RE Hershberger et al., Nat Rev Cardiol 10:531-547 (2013)). The causes of DCM are diverse and include a variety of external factors (viruses, autoimmune infiltrates, alcohol, and drugs). However, 30-40% of all cases have a single-gene basis, with mutations in approximately 40 genes being associated with DCM. The most frequently mutated gene in DCM is TTN, which encodes the large sarcomeric protein titin, and truncating variants of TTN account for approximately 15–25% of all congenital forms of DCM (D.S. Sherman 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), accounting for 6–8% of congenital DCM cases (U. Tayal, S. et al., Genome Med 9:20 (2017)).

[0004] LMNA mutation-associated DCM is characterized by cardiac conduction disease manifested by electrophysiological (ECG) abnormalities, including atrioventricular block, ventricular arrhythmias, and fibrillation. The risk of sudden cardiac death is greater in patients with LMNA mutation-associated cardiomyopathy compared with patients with other forms of DCM (JH 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. As a result, the majority of DCM cases are inherited in an autosomal dominant manner. The diversity of LMNA mutations complicates genetic approaches to treat LMNA mutation-associated DCM. Although limited, LMNA mutation-associated DCM can be treated by pacemaker adjustment. However, currently, effective treatment is achieved through heart transplantation (RE Hershberger and A. Morales, in GeneReviews, edited by M.P. Adam et al., Seattle, WA, 1993; G. Captur et al., Heart 104:468-479 (2018)).

[0005] Mouse strains carrying Lmna mutations typically die within the first few weeks of life (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. Because 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)), myocardial damage is likely a major contributing factor, but other, as yet unclear, effects on skeletal muscle may also contribute to early postnatal death.

[0006] Lamins are nuclear intermediate filament proteins and are major components of the nuclear lamina, a protein matrix located inside the inner nuclear membrane (INM). The lamina consists of two major forms, A-type lamins, lamin A and lamin C. A-type lamins are driven by alternative splicing of the LMNA RNA, while two B-type lamins (LMNB1 and LMNB2) 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 structural and mechanical integrity to the nucleus, maintains its shape and position within the cell, and is also a determinant of chromatin organization (T. Sullivan et al., J Cell Biol 147:913-920 (1999); I. Solovei et al., Cell 152:584-598 (2013)). Lamins interact with numerous INM proteins, including emerin, lamina-associated polypeptide (LAP), and SUN domain-containing proteins (B. van Steensel and A.S. Belmont, Cell 169:780-791 (2017)), many of which, when mutated or present as variants, are associated with cardiac disease (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 constitute an integrated protein network centered around the lamina, where loss or mutation of lamina can result in mislocalization or altered expression levels of many lamina-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, both of which increase in expression level. In the case of SUN1, increased levels are due to reduced turnover rather than increased expression, resulting in high levels accumulating in the Golgi and appearing to be 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 carrying the Lmna mutation increases lifespan threefold 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- / - mice exceeded 210 days over a 7-month follow-up period; Lmna- / - mice had a median survival of 41 days; Lmna- / -Sun1+ / - mice had a median survival of 54 days; and Lmna- / -Sun1- / - mice had a median survival of 104 days (p<0.01 comparing Lmna- / - with Lmna- / -Sun1- / -). Similarly, while all LmnaΔ9 mice died by postnatal day 30, their LmnaΔ9Sun1- / - littermates survived beyond this time, with most achieving a survival period more than double this duration (Chen et al., Cell 149:565-577 (2012)).At the cellular level, human fibroblasts harboring LMNA mutations that cause Hutchison-Gilford progeria syndrome also exhibit increased SUN1 levels. Deep reduction of SUN1 in these cells alleviated nuclear morphological abnormalities, further suggesting that excess Sun1 caused by 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 widely expressed in virtually all tissues. In the nuclear cisternae between the INM and the outer nuclear membrane (ONM), the C-terminus of SUN1 and / or SUN2 binds to the C-termini (KASH domains) of different Nesprin / SYNE / KASH proteins that cross the ONM. Furthermore, these two protein families comprise the LINC complex, which 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; in SUN1, this region interacts with prelamin A and the nuclear pore complex. It is unclear whether the N-terminus of SUN2 interacts with any nucleoplasmic / NE proteins. In contrast, the majority of Nesprin / KASH domain proteins extend into the cytoplasm adjacent to the ONM. There, 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, SUN and KASH / Nesprin proteins of the LINC complex establish a direct physical connection between the cytoplasmic cytoskeletal network (and its connecting components, such as cell adhesion complexes at the plasma membrane) and the interphase interior of the nucleus or the nucleoplasm. The LINC complex is thought to mediate force transmission between the nucleus and the cytoskeleton, thereby controlling changes in gene expression and chromatin organization in response to mechanical / physical stimuli (SG Alam et al., Scientific Reports 6:38063 (2016)).Loss of either SUN1 or SUN2 alone has no apparent 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, indicating some degree of 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, showed that Sun1 knockout in mice resulted in the loss of Lmna in LmnaΔ9 mice. - / - While this effect was reported to reduce the pathology and extend the lifespan of laminopathies, this effect was attributed to the prevention of Sun1 overaccumulation in the Golgi apparatus. Cytotoxicity as a result of SUN protein accumulation in the Golgi was one of several proposed mechanisms involved in laminopathies. Other proposed mechanisms included hyperactive DNA damage following SUN1 overaccumulation at the nuclear envelope and aberrant transcriptional activity driven by the SUN1 nucleoplasmic domain (see Starr, Curr Biol. (2012) 22(17):R678-R680).

[0009] Recently, Kim et al., Sci Transl Med (2018) 10 reported that Lmna of HGPS G609G / G609G We report the expression of EGFP and a polypeptide containing the KASH domain of Nesprin (KASH2) under the control of a smooth muscle cell-specific promoter reducing aortic pathology in a mouse model.

[0010] However, numerous studies suggest that disruption of LINC complex protein levels / function may cause or exacerbate laminopathies-associated pathology.

[0011] Mutations in SYNE1, the gene encoding Nesprin-1, are implicated 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-associated mutations in SYNE1 and SYNE2 map to the C-terminus of Nesprin-1 and -2 (Stroud, Biophys Rev (2018) 10, 1033-1051), implicating disruption of KASH function in disease pathology. Furthermore, cardiac-specific elimination of the 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 comprehensive 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 as modifier genes that exacerbate the effects of muscular dystrophy associated with lamin A / C mutations in humans (Meinke et al., PLoS Genet. (2014) 10, e1004605), and some EDMD patients harbor mutations to SUN1 that reduce 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, as well as increased genomic instability and DNA damage (Lei et al., Proc. Natl. Acad. Sci. USA (2009) 106, 10207-10212).

[0013] In addition to lamin A / C, considering the role of Nesprin and SUN proteins in the laminopathic EDMD, disruption of the LINC complex has been suggested to contribute to laminopathies (Chang et al., Nucleus (2015) 6, 77-88), and LINC complex disruption has been proposed to result in defects in cellular structure and function that may contribute to the pathogenesis 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 inventors' unexpected discovery that inhibition of the LINC complex ameliorates symptoms of a wide range of diseases caused by LMNA mutations with different functional consequences. In the experimental examples herein, the inventors demonstrate that disruption of the LINC complex (using dominant-negative versions of LINC complex proteins or through targeted disruption of domains of LINC complex proteins that LINC complex proteins interact with to form the LINC complex) ameliorates laminopathies associated with reduced levels of lamin A / C as well as progerin-associated laminopathies.

[0015] Thus, LINC complex disruption is demonstrated to be a viable strategy for the treatment and prevention of laminopathies resulting from a wide range of LMNA mutations. The present inventors have also unexpectedly discovered that inhibition of the LINC complex can ameliorate the symptoms of diseases characterized by hyperlipidemia, and 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 a laminopathic disorder. 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 a laminopathic disorder.

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

[0018] In some embodiments, the laminopathies are associated with mutations to LMNA. In some embodiments, the laminopathies are selected from the group consisting of Hutchinson-Gilford progeria syndrome; dilated cardiomyopathy; muscular dystrophy, congenital, Lmna-related; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; muscular dystrophy; acromangial dysplasia with lipodystrophy type a; cardiomyopathy, dilated, 1a; Charcot-Marie-Tooth disease; limb-girdle muscular dystrophy; cardiomyopathy, dilated, with hypergonadotropic hypogonadism; Emery-Dreifuss muscular dystrophy 3, autosomal recessive; lipodystrophy Familial partial type, type 2; Emery-Dreifuss muscular dystrophy; Charcot-Marie-Tooth disease, axonal, type 2b1; heart-upper limb syndrome, Slovenian type; aging; familial partial lipodystrophy; restrictive skin disorder, fatal; arrhythmogenic right ventricular cardiomyopathy; dental disease; heart disease; Werner syndrome; hypertrophic cardiomyopathy; left ventricular noncompaction; atrioventricular block; calcification; acroosteolysis; autosomal dominant limb-girdle muscular dystrophy; diabetes mellitus, non-insulin dependent; osteoporosis; atrial fibrillation; atrial asystole 1; melanoma; cardiac conduction disorders; Catecholamine-induced polymorphic ventricular tachycardia; micrognathia, hearing loss, progeria-like symptoms, and lipodystrophy syndrome; sick sinus syndrome; Pelger-Houette anomaly; Charcot-Marie-Tooth disease, axonal, type 2e; congenital generalized lipodystrophy; restrictive cardiomyopathy; congenital fiber type disproportion; lipodystrophy, congenital generalized, type 1; myofibrillar myopathy; lipodystrophy, familial partial, type 1; axonal neuropathy; atypical Werner syndrome; ovarian cystadenoma; Fanconi anemia, complementation group A; body mass index Quantitative trait locus 11; skin diseases; ankylosing spinal muscular dystrophy 1; neuromuscular diseases; Hurlerman-Streiff syndrome; Bethlem myopathy 1; acquired generalized lipodystrophy; cardiomyopathy, dilated, type 1e; lipodystrophy, congenital generalized, type 4; undifferentiated pleomorphic sarcoma; lipodystrophy, familial partial, type 3; muscular dystrophy, congenital merosin deficiency, type 1a; proximal spinal muscular atrophy; muscular dystrophy-dystroglycanopathy, type B, type 5; muscular dystrophy, congenital, type 1b; Reynolds syndrome; Widamann-Rautenstrauch syndrome; Emery-Dreifuss muscular dystrophy 1, X-linked; lipodystrophy, congenital generalized, type 2;Selected from monogenic diabetes; cardiomyopathy, dilated, type 1d; myopathy, proximal, and ophthalmoplegia; muscle tissue disease; lipodystrophy, familial partial, type 4; cardiomyopathy, dilated, type 1h; second-degree atrioventricular block; median neuropathy; intrinsic cardiomyopathy; prolapse of female genitalia; complete generalized lipodystrophy; ankylosing spinal muscular dystrophy; emelinopathy; ulnar neuropathy; limb-girdle muscular dystrophy type 1b; Lmna-associated dilated cardiomyopathy; pelvic muscle wasting; generalized lipodystrophy-associated progeria syndrome; muscle disease; cardiomyopathy, dilated, type 1b; autosomal inherited disease; familial isolated arrhythmogenic ventricular dysplasia, right dominant; familial isolated arrhythmogenic ventricular dysplasia, biventricular; familial isolated arrhythmogenic ventricular dysplasia, left dominant; Lmna-associated cardiocutaneous progeria syndrome; and autosomal semi-dominant 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 for 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 present 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 the expression of a LINC complex protein.

[0022] In some embodiments, the LINC complex inhibitor can inhibit the interaction between a LINC complex protein and an interaction partner for the LINC complex protein. In some embodiments, the LINC complex inhibitor is a peptide / polypeptide, a nucleic acid, or a 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 a gene encoding a LINC complex protein.

[0024] In some embodiments, the LINC complex inhibitor is an inhibitory nucleic acid that can reduce the expression of a LINC complex protein by RNA interference (RNAi). In some embodiments, the method comprises administering to the subject a nucleic acid encoding a LINC complex inhibitor or a nucleic acid encoding a factor necessary for the production of a LINC complex inhibitor. DETAILED DESCRIPTION OF THE INVENTION

[0025] explanation LINC complex structure and function The nucleoskeletal-cytoskeletal linker (LINC) complex is a polypeptide complex containing 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) with the outer nuclear membrane (ONM) or nuclear envelope. SUN domain-containing proteins span the INM and associate with nuclear lamins and chromatin-binding proteins on the nucleoplasmic side of the INM, as well as with KASH domain-containing proteins on the perinuclear side of the INM. KASH domain-containing proteins span the ONM and associate with cytoskeletal structural components, such as actin filaments, microtubule motors, and intermediate filaments, on the cytoplasmic side of the ONM, as well as with SUN domain-containing proteins on the perinuclear side of the ONM. SUN domain proteins function as transductional links 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 that contain a conserved carboxy-terminal SUN domain localized in the nuclear cisternae. The SUN domain contains approximately 175 residues and is present at the end of a helical stalk region. The nucleoplasmic domain of SUN proteins interacts with structural components of the nucleoskeleton.

[0028] The SUN domain may comprise 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 the 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 set forth in SEQ ID NO: 88. Human SUN2 is a polypeptide identified by UniProtKB Q9UH99, and its amino acid sequence is set forth in SEQ ID NO: 89. Human SUN3 is a polypeptide identified by UniProtKB Q8TAQ9, and its amino acid sequence is set forth in SEQ ID NO: 90. Human SUN5 is a polypeptide identified by UniProtKB A9Z1W8, and its amino acid sequence is set forth in SEQ ID NO: 91. Human SPAG4 is a polypeptide identified by UniProtKB Q9NPE6, and its amino acid sequence is set forth in SEQ ID NO: 92. Human SUCO is a polypeptide identified by UniProtKB Q9UBS9, and its amino acid sequence is set forth 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, respectively, from any species, including isoforms, fragments, variants, or homologs thereof.

[0033] As used herein, a "fragment," "variant," or "homolog" of a protein may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with 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 portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of the reference protein, but retains a substantial degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the species of the reference protein. A "homolog" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0035] A "fragment" may be of any length (by number of amino acids), but may optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment is derived), and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the reference protein.

[0036] The isoform, fragment, variant or homologue may optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of the reference protein, as determined by analysis with a suitable assay for that functional property / activity.

[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, a 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 containing a KASH domain. KASH (Klarsicht, ANC-1, Syne homology) domain proteins are carboxy-terminally anchored membrane proteins targeted to the nuclear envelope. The 50-60 amino acid KASH domain is found at the C-terminus. The KASH domain is hydrophobic and contains a single transmembrane helix that spans the ONM and an approximately 30 amino acid region extending into the nuclear 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 NO: 97, 98, 99 or 100, or to the amino acid sequence set forth in SEQ ID NO: 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 is capable of forming a LINC complex. In some embodiments, the KASH domain-containing protein is capable of interacting 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 set forth in SEQ ID NO: 102. Human Nesprin-2 is a polypeptide identified by UniProtKB Q8WXH0, and its amino acid sequence is set forth in SEQ ID NO: 103. Human Nesprin-3 is a polypeptide identified by UniProtKB Q6ZMZ3, and its amino acid sequence is set forth in SEQ ID NO: 104. Human Nesprin-4 is a polypeptide identified by UniProtKB Q8N205, and its amino acid sequence is set forth in SEQ ID NO: 105. Human KASH5 is a polypeptide identified by UniProtKB Q8N6L0, and its amino acid sequence is set forth in SEQ ID NO: 106. Human LRMP is a polypeptide identified by UniProtKB Q12912, and its amino acid sequence is set forth 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, respectively, from any species, including isoforms, fragments, variants, or homologs thereof.

[0049] As used herein, reference to "Nesprin-1" refers to a protein having the amino acid sequence set forth in SEQ ID NO: 102, 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 to 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 an "LMRP" 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, an 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. LINC complexes are formed by protein-protein interactions between SUN domain-containing proteins and KASH domain-containing proteins. LINC complexes can include non-covalent and / or covalent interactions between SUN domains and KASH domains. Non-covalent interactions include, for example, hydrogen bonds, ionic interactions, van der Waals forces, and hydrophobic bonds. Covalent interactions include, for example, disulfide bonds.

[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 stalk region of SUN domain proteins has been shown to disrupt LINC complex formation. SUN domains assume a β-sandwich structure, and trimeric SUN domains interact extensively with each other, in part 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 oligomerize, potentially involving protein-protein interactions between transmembrane helices. A single KASH domain interacts with two adjacent SUN domains along a groove formed between the KASH lid of one SUN domain and the upper region of the β-sandwich of the adjacent SUN domain. In this way, 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 housed in a deep pocket within the surface of the SUN domain. This region of KASH is important for SUN-KASH interaction; extension of the C-terminus by only one amino acid disrupts LINC complex formation. Conserved cysteine ​​residues in the SUN and KASH domains form disulfide bonds, further stabilizing the SUN-KASH complex. Disulfide bonds may be important for force transmission through the LINC complex (Jahed et al., Biophys. J. (2015) 109:501-509).

[0058] As explained hereinabove, LINC complexes are thought to form through interactions between the SUN domains of three SUN domain-containing proteins and the KASH domains of three KASH domain-containing proteins. Interactions between SUN and KASH domain proteins appear to be promiscuous; SUN1 and SUN2 have been shown to interact with Nesprin-1, Nesprin-2, and Nesprin-3.

[0059] The LINC complex of the present invention can include 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 identical. The KASH domain-containing proteins of the LINC complex may or may not be identical.

[0060] LINC complex function is reviewed, for example, in Hieda, Cells (2017) 6(1):3 (incorporated herein by reference above) and Stroud, Biophys Rev. (2018) 10(4):1033-1051, which are incorporated herein by reference in their entireties.

[0061] LINC complexes perform diverse functions, including providing structural support to the nucleus, shaping and positioning the nucleus, maintaining the connection between the centrosome and the nucleus and the spacing of the nuclear membrane, DNA repair, cell migration, and moving chromosomes within the nucleus during meiosis.

[0062] LINC complexes have a mechanosensing role, translating mechanical stimuli and changes in the extracellular matrix into signals that allow cells to adapt to their environment by regulating cytoskeletal organization, gene expression, nuclear organization and structure.

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

[0064] The nuclear envelope is a key structure in these processes. On the nucleoplasmic side of the INM, the nuclear lamina (composed of A- and B-type lamins) forms a lattice structure that contributes to the resistance of the nucleus to mechanical stress and is essential for the structural integrity of the nuclear envelope. Nuclear lamins are involved in processes crucial for cellular function and survival, including maintaining nuclear integrity, cell cycle control, mechanical signaling, cell signaling, and DNA repair.

[0065] Deviations from normal expression and / or function of nuclear envelope proteins, as well as deviations from normal expression and / or function of factors directly or indirectly associated with the nuclear envelope, are associated with a variety of diseases, including muscular dystrophies, cardiomyopathies, lipodystrophies, progeria, cancer, and neurological disorders.

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

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

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

[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 RT-qPCR, Northern blot, etc. The 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] Decreased transcription of nucleic acids encoding LINC complex proteins can be the result of inhibition of the assembly and / or activity of factors required for transcription of DNA encoding LINC complex proteins. Increased degradation of RNA encoding LINC complex proteins can be the result of increased enzymatic degradation of RNA encoding LINC complex proteins, e.g., RNA interference (RNAi), and / or decreased stability of RNA encoding LINC complex proteins.

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

[0072] Protein degradation can be assessed, for example, by detecting or analyzing the level / rate of ubiquitinated proteins, association with ubiquitin ligase and / or proteosomal localization.

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

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

[0075] Decreased / altered splicing of pre-mRNA encoding a LINC complex protein into mature mRNA can be the result of inhibiting the assembly and / or activity of factors required for normal splicing. Decreased translation of mRNA encoding a LINC complex protein can be the result of inhibiting the assembly and / or activity of factors required for translation. Decreased / altered post-translational processing (e.g., enzymatic processing, folding) can be the result of inhibiting the assembly and / or activity of factors required for normal post-translational processing of a LINC complex protein. Increased degradation of a LINC complex protein can be the result of increased enzymatic (e.g., protease-mediated) degradation of the protein, which may be associated with, for example, misfolding.

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

[0077] The subcellular localization of LINC complex component proteins within cells can be analyzed using techniques known to those skilled in the art. Such techniques include immunohistochemistry and reporter-based analysis. 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 / proportions of LINC complex component proteins in the nuclear envelope, inner nuclear membrane, and outer nuclear membrane.

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

[0079] In some embodiments, inhibiting the formation of a LINC complex comprises one or more of: inhibiting the interaction between a SUN domain-containing protein and an interaction partner for the SUN domain-containing protein; inhibiting the interaction between a KASH domain-containing protein and an interaction partner for the KASH domain-containing protein; 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 a 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., a microfilament or component thereof (e.g., actin), a microtubule or component thereof (e.g., tubulin), or an intermediate filament or component thereof).

[0080] In some embodiments, LINC complex inhibition comprises disruption / disassembly of the LINC complex. In some embodiments, LINC complex disruption / disassembly includes one or more of: inhibiting the interaction between a SUN domain-containing protein and an interaction partner for the SUN domain-containing protein; inhibiting the interaction between a KASH domain-containing protein and an interaction partner for the KASH domain-containing protein; 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 a 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., microfilaments or components thereof (e.g., actin), microtubules or components thereof (e.g., tubulin), or intermediate filaments or components thereof); increasing LINC complex disassembly; increasing LINC complex degradation; displacing LINC complex proteins from LINC complexes; and decreasing LINC complexes.

[0081] As used herein, the 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. The interaction partner for a SUN domain-containing protein can be a protein that can form a complex with the SUN domain-containing protein through protein-protein interaction. In some embodiments, the 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, a 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 can be a SUN domain-containing protein (e.g., SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO), a KASH domain-containing protein (e.g., Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, or LRMP), a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, microtubule, tubulin, a microtubule motor, or an intermediate filament protein.

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

[0084] LINC complexes and their component proteins can be detected using, for example, methods well known to those skilled in the art such as Western blots, immunohisto / cytochemistry, flow cytometry, antibody-based methods including ELISA, or reporter-based methods.

[0085] LINC complex inhibition can be characterized by a decreased level of LINC complex function. In some embodiments, LINC complex inhibition can be determined by detecting a decreased level of a correlate of LINC complex function.

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

[0087] In some embodiments, modifying the gene encoding the KASH domain-containing protein or 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, inhibiting expression of the KASH domain-containing protein or 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 a SUN domain-containing protein and a KASH domain-containing protein is achieved using a dominant-negative SUN domain-containing protein, a dominant-negative KASH domain-containing protein, a small molecule inhibitor of the interaction between a SUN domain-containing protein and a KASH domain-containing protein, a KASH domain peptidomimetic, or a SUN domain peptidomimetic. It is understood that the interaction inhibition is between an endogenous SUN domain-containing protein and an endogenous KASH domain-containing protein.

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

[0090] Such agents may be effectors of LINC complex inhibition (i.e., may directly or indirectly cause LINC complex inhibition), as described above. LINC complex inhibitors are sometimes referred to herein as LINC complex antagonists.

[0091] In some embodiments, a LINC complex inhibitor inhibits the formation of a LINC complex; disrupts / disassembles a LINC complex; inhibits LINC complex activity; inhibits the gene and / or protein expression of a LINC complex protein; modifies a gene encoding a LINC complex protein to reduce / prevent its expression; reduces the level of an RNA encoding a LINC complex protein; inhibits the transcription of a nucleic acid encoding a LINC complex protein; increases the degradation of an RNA encoding a LINC complex protein; reduces the level of a LINC complex protein; disrupts normal post-transcriptional processing (e.g., splicing, translation, post-translational processing) of an RNA encoding a LINC complex protein; increases the degradation of a LINC complex protein; inhibits the transport and / or disrupts normal subcellular localization of a LINC complex protein; reduces the level / proportion of a LINC complex protein localized to the nuclear membrane; reduces the level / proportion of a SUN domain-containing protein associated with the nuclear membrane decrease the proportion of KASH domain-containing proteins associated with the outer nuclear membrane; decrease the level / proportion of KASH domain-containing proteins associated with the outer nuclear membrane; increase the retention of LINC complex proteins in the endoplasmic reticulum; increase the level / proportion of LINC complex proteins localized to the endoplasmic reticulum; inhibit interactions between LINC complex proteins; inhibit interactions between LINC complex proteins and interacting partners for LINC complex proteins; inhibit interactions between SUN domain-containing proteins and interacting partners for SUN domain-containing proteins; inhibit interactions between KASH domain-containing proteins and interacting partners for KASH domain-containing proteins; inhibit interactions between SUN domain-containing proteins and KASH domain-containing proteins; inhibit interactions between SUN domain-containing proteins and lamins; inhibit interactions between SUN domain-containing proteins and chromatin-binding proteins; inhibit interactions between KASH domain-containing proteins and SUN domain-containing proteins;inhibit the interaction between a KASH domain-containing protein and a cytoskeletal component (e.g., a microfilament or component thereof (e.g., actin), a microtubule or component thereof (e.g., tubulin), or an intermediate filament or component thereof); increase disassembly of the LINC complex; increase degradation of the LINC complex; displace a LINC complex protein from the LINC complex; and / or decrease the levels 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 an appropriate assay.The assay can be, for example, an in vitro assay, optionally a cell-based assay or a cell-free assay.

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

[0094] Agents capable of decreasing gene expression of a LINC complex protein (e.g., decreasing the level of RNA encoding the LINC complex protein, decreasing transcription of nucleic acids encoding the LINC complex protein, and / or increasing degradation of RNA encoding the LINC complex protein) may be identified using assays that include detecting the level of RNA encoding the relevant protein, e.g., by RT-qPCR. Such assays may 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). Assays for detecting reduced / altered splicing of pre-mRNA of a given protein may include detecting and / or quantifying one or more isoforms of the relevant protein or RNA encoding one or more of said isoforms.

[0095] Agents capable of decreasing protein expression of LINC complex proteins (e.g., decreasing the level of a LINC complex protein, increasing the degradation of a LINC complex protein) may be identified using assays that include detecting the level of the relevant protein, e.g., using antibody / reporter-based methods (Western blot, ELISA, immunohisto / cytochemistry, etc.). Such assays may include 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 of protein degradation may include, for example, assessing 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 capable of inhibiting the trafficking of LINC complex proteins and / or disrupting their normal subcellular localization can be identified using assays that detect the presence or proportion of the relevant protein at a given subcellular location, e.g., using antibody / reporter-based methods (Western blot, ELISA, immunohisto / cytochemistry, etc.). Subcellular localization can be analyzed, for example, by immunocytochemistry or Western blot of extracts prepared from different cellular fractions, and can use organelle markers and / or labeled proteins of known subcellular localization. Assays can include treating cells / tissues with an agent and subsequently comparing the subcellular localization of the relevant protein in such cells with that of cells / tissues in appropriate control conditions (e.g., untreated / solvent-treated cells / tissues).

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

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

[0099] In some embodiments, the LINC complex inhibitor is capable of binding to a LINC complex, a LINC complex protein, or an interaction partner for a LINC complex protein.

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

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

[0102] In some embodiments, the LINC complex inhibitor mimics an interaction partner for a LINC complex protein. In some embodiments, the 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 KASH domain from accessing the deep pocket of the SUN domain. In some embodiments, the LINC complex inhibitor binds to the KASH domain and inhibits the KASH domain from accessing the deep pocket on the surface of the SUN domain.

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

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

[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 assessed, for example, by analyzing the interaction in the presence of the candidate LINC complex inhibitor, or by incubating one or both of the interaction partners with the candidate LINC complex inhibitor. An example of a suitable assay for determining whether a given binding agent can inhibit the interaction between 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 a LINC complex protein inhibits the ability of the LINC complex protein to bind to an interaction partner for the LINC complex protein.

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

[0110] LINC complex inhibitors that can bind to a LINC complex protein or an interaction partner for a 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 a molecule to a related factor (i.e., a LINC complex protein or an interaction partner for a LINC complex protein) and the inhibition of the interaction between the LINC complex protein and the interaction partner for the LINC complex protein. Such assays may include, for example, detecting the formation of a complex between the related 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 can bind to a LINC complex protein or an interaction partner for a LINC complex protein and inhibit the interaction between the LINC complex protein and the interaction partner for the LINC complex protein may be, for example, a peptide / polypeptide.

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

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

[0114] For example, a LINC complex inhibitor that binds to a LINC complex protein may comprise / 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 associated factor but lack one or more other properties of the protein on which the inhibitor is based, or exhibit reduced levels of those properties. For example, a LINC complex inhibitor may contain the amino acid sequence(s) required for binding to the associated factor, but may lack the amino acid sequence(s) required for one or more other properties of the protein on which the inhibitor is based.

[0116] Such peptide / polypeptide LINC complex inhibitors may be referred to as "decoy," "dominant-negative," or "mimetic" versions of the proteins on which the inhibitors are based, and may preferably exhibit competitive inhibition of the interaction between a LINC complex protein and an interaction partner for the LINC complex protein. In this manner, such peptide / polypeptide LINC complex inhibitors inhibit the formation of LINC complexes and / or disrupt existing LINC complexes via displacement of endogenous interaction partners to form non-functional complexes / complexes with reduced levels of function.

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

[0118] 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. USA (2005) 102:4359-4364, and Libotte et al., MBoC (2005) 16:3411-3424. 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. Dominant-negative versions of KASH3 are described, for example, in Stewart-Hutchinson et al., Exp Cell Res. (2008) 314(8): 1892-905. Dominant-negative versions of KASH4 are described, for example, in Roux et al., Proc. Natl. Acad. Sci. USA (2009) 106: 2194-2199. A dominant-negative version of KASH5 is described, for example, in Horn et al., J Cell Biol (2013) 202:1023-1039.

[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 the 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 or exhibit reduced levels of 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).

[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 or exhibit reduced levels of 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).

[0122] LINC complex inhibitors based on SUN domain-containing proteins preferably contain a SUN domain. In some embodiments, the LINC complex consists of or essentially consists of a SUN domain. The peptide / polypeptide may lack the amino acid sequence(s) of the SUN domain-containing protein that constitute the protein domain other than the SUN domain. The peptide / polypeptide preferably lacks properties of the endogenous SUN domain-containing protein other than those mediated by the SUN domain. The peptide / polypeptide may behave as a dominant-negative peptide / polypeptide and may be able to inhibit the interaction between the endogenous SUN domain-containing protein and an 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 the SUN domain of SUN1, SUN2, SUN3, SUN5, SPAG4 or SUCO. In some embodiments, the LINC complex inhibitor comprises or consists 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 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 entire amino acid sequence of SUN1, SUN2, SUN3, SUN5, SPAG4, or SUCO. In some embodiments, the LINC complex inhibitor lacks an 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 for binding to a nucleocytoplasmic protein, a lamin, and / or a chromatin-binding protein. In some embodiments, the LINC complex inhibitor lacks the amino acid sequence of 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 the KASH domain-containing protein.

[0127] A LINC complex inhibitor based on a KASH domain-containing protein may exhibit binding to a SUN domain-containing protein (e.g., a SUN domain-containing protein described herein), but lacks or exhibits reduced levels of one or more other properties of the KASH domain-containing protein on which the inhibitor is based (e.g., binding to a KASH domain-containing protein, a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, microtubules, tubulin, a microtubule motor, or an intermediate filament protein).

[0128] A LINC complex inhibitor based on a KASH domain-containing protein may exhibit binding to a KASH domain-containing protein (e.g., a KASH domain-containing protein described herein), but lacks or exhibits reduced levels of one or more other properties of the KASH domain-containing protein on which the inhibitor is based (e.g., binding to a SUN domain-containing protein, a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, microtubules, tubulin, a microtubule motor, or an intermediate filament protein).

[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 constitutes the protein domain other than the KASH domain. The peptide / polypeptide preferably lacks properties of the endogenous KASH domain-containing protein other than those mediated by the KASH domain. The peptide / polypeptide may behave as a dominant-negative peptide / polypeptide and may be able to inhibit the interaction between the endogenous KASH domain-containing protein and an endogenous interaction partner for the KASH domain-containing protein.

[0130] In some embodiments, the LINC complex inhibitor comprises or consists of a KASH domain of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5, LRMP, or 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 or LRMP. In some embodiments, the LINC complex inhibitor comprises or consists of an amino acid sequence set forth in SEQ ID NO: 97, 98, 99, 100, 101 or 114, 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: 97, 98, 99, 100, 101 or 114.

[0131] In some embodiments, the LINC complex inhibitor lacks the entire 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 necessary for binding to a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, a microtubule, a tubulin, a microtubule motor, or an intermediate filament protein. In some embodiments, the LINC complex inhibitor lacks the amino acid sequence according to SEQ ID NO: 107 or 108.

[0133] In some embodiments, peptide / polypeptide LINC complex inhibitors that can bind to a LINC complex / LINC complex protein / interacting partner for a LINC complex protein and inhibit the interaction between the LINC complex protein and the interacting partner for the LINC complex protein and / or LINC complex function include, for example, peptide aptamers, thioredoxins, monobodies, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, nanobodies (i.e., single domain antibodies (sdAb)), affilins, armadillo repeat proteins (ArmRPs), OBody, and fibronectin, as reviewed, for example, in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, which is hereby incorporated by reference in its entirety (e.g., Boersma et al., J Biol (See also Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48.) Additionally, peptide / polypeptide LINC complex inhibitors contemplated in connection with the present invention include antibodies (immunoglobulins), such as monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), as well as fragments and derivatives thereof (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g., VhH), etc.).

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

[0135] Peptide / polypeptide LINC complex inhibitors may contain additional amino acids or amino acid sequences. For example, peptides / polypeptides may contain amino acid sequence(s) to facilitate expression, folding, transport, processing, purification, or detection. For example, peptides / polypeptides may contain sequences encoding His (e.g., 6xHis), Myc, GST, MBP, FLAG, HA, E, or biotin tags, as needed, at the N- or C-terminus of the antigen-binding molecule / polypeptide. In some embodiments, peptides / polypeptides contain a detectable moiety, such as a fluorescent, luminescent, immunodetectable, radioactive, chemical, nucleic acid, or enzymatic label.

[0136] In some embodiments, the peptide / polypeptide may comprise an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence. A peptide / polypeptide may further contain a signal peptide (also known as a leader sequence or signal sequence). A 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 in a newly synthesized peptide / polypeptide. The signal peptide is often removed by cleavage and therefore is not included in the mature peptide / polypeptide.

[0137] In some embodiments, the N-terminal signal sequence is derived from a secretory protein or a type I transmembrane protein. In some embodiments, the secretory protein or the type I transmembrane protein is selected from human serum albumin, proinsulin, transferrin receptor, EGF receptor, prepro-opiomelanocortin, carboxypeptidase, complement protein, fibrinogen, cytokine, chemokine, fibrinogen, pancreatic digestive enzymes (e.g., proteases, amylases, and lipases), 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 or is derived from one of the following: human serum albumin, proinsulin, transferrin receptor, EGF receptor, prepro-opiomelanocortin, pancreatic digestive enzymes (e.g., proteases, amylases, and lipases), 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 ER retention sequences are known in the art. In some embodiments, the ER retention motif is a KDEL sequence. The KDEL sequence, KDEL, or a variant thereof, serves to retain proteins in the endoplasmic reticulum. The KDEL variant can comprise or consist of the prosite motif [KRHQSA]-[DENQ]-EL (described in Hulo et al., 2006, Nucleic Acids Research 34:D227-D230), or a variant described by Raykhel et al., 2007 (J. Cell Biol. 179(6):1193-1204), who proposed an extended prosite motif definition. Raykhel demonstrated endoplasmic reticulum (ER) retention with variants including a range of residues at position 3 (i.e., position D) extending well beyond F, WY, and DENQ as alternatives to KRHQSA at position 4 (i.e., position K), F or M at position 1 (position L), and D at position 2 (i.e., position E). For example, the KDEL motif may be CDEL, KCEL, or HVEL, as proposed by Raykhel et al. Thus, in some embodiments disclosed herein, the endoplasmic reticulum (ER) retention motif is KDEL or a variant thereof that exhibits ER retention activity.

[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 structures are shown in Figures 11 and 12.

[0141] In some embodiments, the peptide / polypeptide comprises an epitope tag, which is either N-terminal or located in the peptide / polypeptide downstream (after) the C-terminal targeting peptide sequence (e.g., an endoplasmic reticulum retention motif such as a KDEL sequence), or located in the peptide / polypeptide except upstream (before) the N-terminal signal sequence. In some embodiments, the epitope tag is selected from 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 solved by Kutay and Schwartz labs [Sosa et al., Cell 149(5):1035-47 (2012)]) and an additional 20 amino acid residues upstream corresponding to the alpha-3 region of coiled-coil-2 are predicted to be sufficient to disrupt the SUN-KASH interaction, since they can bind to the KASH domain [Jahed et al., Biophysical Journal 114(5):1190-1203 (2018)]. This replaces the entire luminal domain (coiled-coil domain and SUN domain). The human SUN1 SUN domain nucleic acid sequence is shown in SEQ ID NO: 80. The presence of a signal sequence and KDEL sequence may be important for targeting to the perinuclear space.

[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, which contains the coiled-coil domain and the SUN domain but lacks the transmembrane domain. A schematic diagram of the structure of a 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, the KASH domain can be expressed to disrupt the LINC complex by competing with endogenous Nesprin (which contains 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-interacting peptide. In some embodiments, the peptide / polypeptide comprises a KASH domain that crosses the outer nuclear membrane, a SUN-interacting KASH peptide that extends into the perinuclear space at the C-terminus, and an N-terminal stabilizer polypeptide sequence in the cytoplasm.

[0148] It is understood that KASH domain constructs having extensions after the last C-terminal amino acid of a naturally occurring KASH domain are not expected to function, i.e., a C-terminal tag, or even an additional carboxy-terminal single amino acid, will disrupt 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; and 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 Figure 14.

[0151] In some embodiments, the KASH domain nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to 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 purposes of clinical use, the KASH domain is a human KASH1 domain of SYNE1 having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to 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 comprise any extension after the last C-terminal amino acid compared to a 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, including multiples and combinations thereof. In some embodiments, the N-terminal stabilizer polypeptide sequence forms a separately folded domain.

[0154] In some embodiments, the peptide / polypeptide comprises a KASH domain and an N-terminal stabilizer polypeptide sequence, wherein the KASH domain is selected from the group comprising 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, a "small molecule" refers to a low molecular weight (<1000 daltons, typically about 300-700 daltons) organic compound.

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

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

[0158] The nucleic acid may bind to a LINC complex, a LINC complex protein, or an interaction partner for a LINC complex protein. The nucleic acid may inhibit the interaction between a LINC complex protein and an interaction partner for a LINC complex protein. The nucleic acid may bind to a LINC complex and inhibit LINC complex function.

[0159] Nucleic acid aptamers are generally described, for example, in Zhou and Rossi, Nat Rev Drug Discov. 2017, 16(3):181-202. Nucleic acid aptamers can be identified and / or generated by the method of systematic evolution of ligands by exponential enrichment (SELEX) or by developing SOMAmers (slow off-rate modified aptamers) (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 International Publication No. 91 / 19813.

[0160] Nucleic acid aptamers can comprise DNA and / or RNA, and can be single-stranded or double-stranded.Nucleic acid aptamers can comprise, for example, chemically modified nucleic acids, in which sugars and / or phosphates and / or bases are chemically modified.Such modifications can improve the stability of aptamers or make aptamers more resistant to degradation, and can include modifications at the 2' position of ribose.

[0161] Nucleic acid aptamers can be chemically synthesized, for example, on 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. Capping can then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle can then be repeated to construct the aptamer (see, for example, Sinha, ND; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, SL; Lyer, RP (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 a LINC complex protein. In some embodiments, the LINC complex inhibitor reduces or prevents the expression of an endogenous LINC complex protein.

[0163] Inhibition of expression of a LINC complex protein reduces the amount of the LINC complex protein and / or the amount of the LINC complex, including its constituent proteins, in a cell / tissue / organ / organ system / subject. For example, inhibition of expression of a LINC complex protein in a given cell reduces the level of the INC complex protein and / or the LINC complex, including its constituent proteins, compared to an untreated cell.

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

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

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

[0167] In some embodiments, LINC complex inhibitors may target specific domains / regions of LINC complex proteins, hi some embodiments, LINC complex inhibitors reduce or prevent expression of isoforms of LINC complex proteins that contain one or more domains / regions of interest.

[0168] For example, the domain or region of interest can be or include a domain required for interaction with an interaction partner for a SUN domain, a KASH domain, and / or a LINC complex protein. For example, the domain or region of interest can be required for interaction with a KASH domain-containing protein, a SUN domain-containing protein, a nucleocytoplasmic protein, a lamin, a chromatin-binding protein, a cytoplasmic protein, a cytoskeletal protein, a microfilament, actin, microtubules, tubulin, a microtubule motor, and / or an intermediate filament protein.

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

[0170] In some embodiments, a LINC complex inhibitor may modify a nucleic acid encoding a LINC complex protein to increase expression of an isoform(s) that lack the relevant domain / region, and / or modify a nucleic acid encoding a LINC complex protein to decrease expression of an isoform(s) that include 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 may be single-stranded and may bind to a target oligonucleotide, for example, mRNA, by complementary sequence binding.

[0172] ASOs may be designed to inhibit / prevent the expression of LINC complex proteins or specific isoforms thereof. Oligonucleotides designed to inhibit / prevent expression of a LINC complex protein or a specific isoform thereof may have substantial sequence identity to a portion of the nucleic acid encoding the LINC complex protein / related 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 small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA).

[0174] The role of the RNAi machinery and small RNAs in targeting heterologous complexes and epigenetic gene silencing at specific chromosomal loci has been demonstrated. Double-stranded RNA (dsRNA)-dependent posttranscriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific homologous genes for silencing over a short period of time. This silencing serves as a signal to promote the degradation of mRNAs with sequence identity. 20-nt siRNAs are generally long enough to induce gene-specific silencing but short enough to avoid a host response. The reduction in expression of targeted gene products can be profound; 90% silencing can be induced with just a few siRNA molecules. RNAi-based therapeutics are currently in phase I, II, and III clinical trials for several indications (Nature 2009, January 22;457(7228):426-433).

[0175] In the art, such RNA sequences are termed "short or small interfering RNA" (siRNA) or "microRNA" (miRNA) depending on their origin. Both types of sequences can be used to downregulate gene expression by binding to complementary RNA and triggering mRNA elimination (RNAi) or halting mRNA translation into protein. siRNAs are derived by processing of long double-stranded RNAs and, when found in nature, are typically of exogenous origin. Microinterfering RNAs (miRNAs) are endogenously encoded small non-coding RNAs derived by processing of short hairpins.

[0176] Both siRNAs and miRNAs can inhibit the translation of mRNAs with partially complementary target sequences without RNA cleavage and degrade mRNAs with fully complementary sequences.

[0177] siRNAs are typically double-stranded, and to optimize the effectiveness of RNA-mediated downregulation of target gene function, the length of the siRNA molecule is preferably selected to be short enough to ensure correct recognition of the siRNA by the RISC complex, which mediates recognition of the mRNA target by the siRNA, and to reduce the host response.

[0178] miRNAs are typically single-stranded and have a partially complementary region that allows the ligand to form a hairpin. miRNAs are RNA genes that are 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 its approximate 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), pp. 1862-1879, 2004.

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

[0180] siRNA-mediated knockdown of LINC complex proteins and siRNAs for achieving this 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, all of which are hereby incorporated by reference in their entireties.

[0181] Relatively long double-stranded RNAs may be processed in cells to produce siRNAs (see, e.g., Myers (2003) Nature Biotechnology 21:324-328). Relatively long dsRNA molecules may have, for example, 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 to 30 nucleotides in length. More preferably, relatively long dsRNA molecules are 25 to 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 (shRNA) in cells. shRNAs are more stable than synthetic siRNAs. shRNAs consist of short inverted repeats separated by a small loop sequence. One inverted repeat is complementary to the gene target. In cells, shRNAs are processed by DICER into siRNAs that degrade the target gene mRNA and silence its expression. In some embodiments, shRNAs are produced intracellularly by transcription from a vector. shRNAs may be produced 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. shRNAs can then be directly introduced into cells. Preferably, shRNA molecules contain a partial sequence of a gene encoding a LINC complex protein. Preferably, the shRNA sequence is 40 to 100 bases long, more preferably 40 to 70 bases long. The base of the hairpin is preferably 19 to 30 base pairs long. The stem may contain GU pairings that stabilize the hairpin structure.

[0183] shRNA-mediated knockdown of LINC complex proteins and shRNAs 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 entireties.

[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. SSOs disrupt normal splicing of target RNA transcripts by blocking RNA-RNA base pairing and / or protein-RNA binding interactions between components of the splicing machinery and pre-mRNA. SSOs may be used to alter the number / proportion of mature mRNA transcripts encoding LINC complex proteins or specific isoform(s) thereof. SSOs may be designed to target specific regions of target transcripts, e.g., to cause skipping of exon(s) of interest, e.g., exons encoding domains / regions of interest.

[0185] SSOs generally involve modifications to the oligonucleotide sugar-phosphate backbone to prevent RNase H degradation and may include, for example, phosphorodiamidate morpholinos (PMOs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and / or 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications.

[0186] Inhibitory nucleic acids can be produced recombinantly, 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 that contains a 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, expression of the nucleic acid can be regulated using a cell / tissue (e.g., heart, muscle, etc.) specific promoter.

[0187] Inhibitory nucleic acids may 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 / molecules that can modify a nucleic acid encoding a LINC complex protein to reduce / prevent expression of the LINC complex protein or specific isoform(s) thereof.

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

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

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

[0191] In some embodiments, the modification may be to a region of a nucleic acid encoding a LINC complex protein that is involved in (e.g., required for) LINC complex formation. For example, in some embodiments, the modification may be targeted to a region encoding the SUN domain of a gene encoding a SUN domain-containing protein. In some embodiments, the modification may be targeted to a region encoding the KASH domain of a gene encoding a 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. The method for modifying the nucleic acid encoding the target protein and the agent for achieving this modification are well known in the art, and include, for example, the modification of target nucleic acid by homologous recombination and the targeted nucleic acid editing using site-specific nuclease (SSN).For example, the inventors have demonstrated the CRISPR / Cas9-mediated destruction of Sun1 and Nesprin-1 in the experimental examples herein.

[0193] Suitable methods may use targeting by homologous recombination, which is reviewed, 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. 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 SSNs. Gene editing using SSNs is reviewed, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10):e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into a target nucleic acid sequence(s) of interest. DSBs can be repaired by error-prone non-homologous end joining (NHEJ), in which the two ends of the break are rejoined, often with the insertion or deletion of nucleotides. Alternatively, DSBs can be repaired by advanced homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is provided and introduced into the DSB site.

[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 the clustered regularly interspaced palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) system.

[0196] The ZFN system is generally 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 contain 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 zinc finger arrays capable of binding to target nucleic acid sequences.

[0197] The TALEN system is generally 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 contain a programmable DNA-binding TALE domain and a DNA-cleavage domain (e.g., a FokI endonuclease domain). TALEs contain repeat domains consisting of 33-39 amino acid repeats, which are identical except for two residues at positions 12 and 13 of each repeat, which are repeat variable dinucleotides (RVDs). Each RVD determines the binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: "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. 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 nucleic acid sequence of interest.

[0199] In some embodiments, LINC complex inhibition utilizes a site-specific nuclease (SSN) system that targets LINC complex proteins. Thus, in some embodiments, LINC complex inhibitors comprise or consist of an SSN system that targets LINC complex proteins. In some embodiments, LINC complex inhibition utilizes a nucleic acid(s) encoding an SSN system that targets LINC complex proteins.

[0200] In some embodiments, the SSN system targets a region of a 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 expression of an exon encoding a SUN domain of a gene encoding a SUN domain-containing protein. In some embodiments, the SSN system can disrupt expression of an exon encoding a KASH domain of a gene encoding a KASH domain-containing protein.

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

[0202] In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a SUN domain-containing protein upstream of the sequence encoding tyrosine at position 154 of SEQ ID NO:82. In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a SUN domain-containing protein upstream of the sequence encoding tyrosine at position 153 of SEQ ID NO:83. In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a SUN domain-containing protein upstream of the sequence encoding tyrosine at position 153 of SEQ ID NO:84. In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a SUN domain-containing protein upstream of the sequence encoding tyrosine at position 151 of SEQ ID NO:85. In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a SUN domain-containing protein upstream of the sequence encoding tyrosine at position 152 of SEQ ID NO:86.

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

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

[0205] In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a KASH domain-containing protein upstream of the sequence encoding proline at position 57 of SEQ ID NO:97. In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a KASH domain-containing protein upstream of the sequence encoding proline at position 57 of SEQ ID NO:98. In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a KASH domain-containing protein upstream of the sequence encoding proline at position 56 of SEQ ID NO:99. In some embodiments, the SSN system may introduce an insertion / deletion in the nucleic acid sequence of a gene encoding a 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, LINC complex inhibition may use a nucleic acid(s) encoding a CRISPR RNA (crRNA) that targets a nucleic acid encoding a LINC complex protein and a transactivating crRNA (tracrRNA) that processes the crRNA to its mature form. The CRISPR / Cas9 system for targeted destruction of 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.

[0208] Rather than disrupting the LINC complex by expressing luminal or KASH domain components of SUN domain-containing proteins to compete for binding with endogenous Nesprin (containing a KASH domain) or Sun1 and Sun2 (containing a SUN domain), an alternative approach to disrupting the LINC complex is to modify the endogenous SUN or KASH domain so that it is unable to bind to its cognate LINC complex binding partner or has a reduced ability to bind to this partner.

[0209] Because both the SUN and KASH domains are located at the C-terminus of their respective proteins, one method for generating modified SUN or KASH domains 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 create a premature stop codon at the 3' end of the respective protein sequence. This would result in a truncated protein with a mutated C-terminal SUN or KASH domain. The truncated protein would be expressed and localized to the membrane, but would be unable to interact with its cognate LINC complex partner.

[0210] Thus, in some embodiments, the LINC complex inhibitor is a CRISPR-Cas or other synthetic nuclease system capable of modifying nucleic acids encoding the SUN or KASH domains of endogenous SUN or Nesprin proteins, respectively.

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

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

[0213] In some embodiments, the CRISPR-Cas system comprises a gRNA nucleic acid that targets the human SUN1 domain set forth in SEQ ID NO: 80. In some embodiments, the gRNA nucleic acid sequence targets the end of exon 20, which comprises the nucleic acid sequence set forth 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, as set forth in Table 3.

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

[0215] In some embodiments, the CRISPR-Cas system comprises a gRNA nucleic acid sequence that targets the human KASH domain as set forth 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 as set forth 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 A LINC complex inhibitor according to the present invention is preferably administered to a subject in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to provide a therapeutic / prophylactic benefit to the subject.

[0217] The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated / prevented and the nature of the LINC complex inhibitor. Prescribing treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other medical professionals 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 medical practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0218] For therapeutic applications, the LINC complex inhibitors are preferably formulated as a medicament or pharmaceutical together with one or more other pharmaceutically acceptable ingredients 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, flavoring agents, and sweetening agents.

[0219] The term "pharmaceutically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, and the like that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject (e.g., human) in question without undue toxicity, irritation, allergic response, or other problem or difficulty, 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 ed., 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd ed., 1994.

[0221] The formulations can be prepared by any method well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a carrier (e.g., a liquid carrier, a finely divided solid carrier, etc.), and then, if necessary, shaping the product.

[0222] The formulations may be prepared for local, parenteral, systemic, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, subcutaneous, oral, or transdermal administration routes, which may include injection. Injectable formulations may contain the selected agent in a sterile or isotonic medium. The formulation and mode of administration may be selected depending on the agent to be administered and the disease to be treated / prevented.

[0223] Administration of a LINC complex inhibitor preferably alters the cell(s) that contain the LINC complex inhibitor described herein. LINC complex inhibitors can be formulated to facilitate delivery to and / or uptake by cells / tissues. LINC complex inhibitors can be linked to moieties to facilitate delivery to and / or uptake by cells / tissues. Strategies for facilitating intracellular delivery of molecular cargoes are reviewed, for example, in Li et al., Int. J. Mol. Sci. (2015) 16:19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9):1602-1608, which are hereby incorporated by reference in their entireties.

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

[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, polypeptide, poly(β-amino ester), DOPE, β-cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan, or polyphosphate ester nanoparticles.

[0226] In some embodiments, the LINC complex inhibitor is associated (covalently or non-covalently) with a cell-penetrating peptide (e.g., a protein transduction domain, a Trojan peptide, an arginine-rich peptide, a vectocell peptide), a cationic polymer, a cationic lipid, or a viral carrier. In some embodiments, the LINC complex inhibitor is associated 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 may be administered in the form of a nucleic acid encoding a peptide / polypeptide or 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" refers to a nucleic acid used as a vehicle for transferring an exogenous nucleic acid into a cell. The vector may be a vector for expressing a nucleic acid in a target cell. Such a vector may include a promoter sequence operably linked to the nucleic acid sequence to be expressed. The vector may also include a termination codon and an expression enhancer. As used herein, the term "operably linked" may include a situation in which a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked such that expression of the nucleotide sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of causing transcription of the nucleic acid sequence. If desired, the resulting transcript can then be translated into a desired polypeptide.

[0230] Any suitable vectors, promoters, enhancers and termination codons known in the art may 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. For example, in Example 5 herein, a dominant-negative version of SUN1 is administered using an adeno-associated viral vector.

[0231] In some embodiments, vectors are selected based on their tropism for the cell type / tissue / organ to which it is desired to deliver the nucleic acid, e.g., the cell type / tissue / organ affected by the disease being treated / prevented in accordance with the present invention (i.e., the cell / tissue / organ in which the symptoms of the disease are manifested).

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

[0233] AAV9 vectors were 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 viral vector of one of the following serotypes: AAV9, AAV1, AAV6, AAV8, AAV2i8, AAV9.45, AAV10, or AAVrh.74.

[0235] In some embodiments, the vector comprises a modification that increases binding and / or transduction into a cell type of interest (i.e., compared to the level of binding / transduction by the unmodified vector). In some embodiments, the modification is to a 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, e.g., a cell-targeting peptide shown in Table 1, 2, 3, or 4, which is hereby incorporated by reference in its entirety.

[0237] In some embodiments, the vector comprises a capsid protein comprising a mutation to one or more tyrosine residues, e.g., surface-exposed tyrosine residues. In some embodiments, the tyrosine residues are mutated to phenylalanine. In some embodiments, the vector comprises a capsid protein in which the tyrosine residues are mutated as described in Iida et al., Biomed Res Int. (2013) 2013:974-819, which is hereby incorporated by reference in its entirety.

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

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

[0240] The sequence for controlling the expression of a nucleic acid can provide expression of the nucleic acid by a specific type of cell or tissue. For example, expression can be under the control of a cell type- or tissue-specific promoter. For 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] Promoters for cell-type- or tissue-specific expression of the nucleic acids according to the invention can be selected according to the disease to be treated / prevented, for example, the promoter can drive expression in cells / tissues / organs affected by the disease (i.e., cells / tissues / organs in which symptoms of the disease are manifested).

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

[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 expression of the nucleic acid can, for example, provide expression of the nucleic acid in response to an agent / signal. For example, expression can be under the control of an inducible promoter. The agent can provide inducible expression of the nucleic acid in vivo by administration of the agent to a subject receiving cells modified according to the present disclosure, or ex vivo / in vitro by administration of the agent to cultured cells ex vivo or in vitro.

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

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

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

[0248] Embodiments of the present invention relate to the treatment / prevention of diseases in which LINC complex dysfunction is pathologically implicated. Such diseases include, for example, nuclear envelope diseases (e.g., laminopathies). The therapeutic utility of the agents and methods of the present invention extends to the treatment and / or prevention of any disease that is believed to derive therapeutic / prophylactic benefit from LINC complex inhibition.

[0249] The method may be directed to delaying / preventing the onset of disease symptoms; reducing the severity of disease symptoms (alleviating disease symptoms); reversing disease symptoms; reducing morbidity in a subject with the disease; reducing mortality in a subject with the disease; and / or delaying / preventing disease progression (e.g., to a later stage).

[0250] Aspects of the present 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 mutation, or a disease in which such mutation is a risk factor for the development or progression of the disease. In some embodiments, the mutation results in one or more of the following in cells containing one or more copies of a mutant allele of a gene, compared to cells containing two copies (i.e., homozygous) of a non-mutated (wild-type) reference allele of the gene: decreased levels of the gene product (e.g., RNA and / or protein (or a specific isoform thereof) of the wild-type allele; increased levels of the gene product of the non-wild-type allele; increased levels of the gene product of the wild-type allele.

[0251] An embodiment of the present invention relates 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 in or directly / indirectly associated with ONM, perinuclear space, or INM). 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. 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 present 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 entireties. Laminopathies are generally associated with tissue-specific defects in load-bearing at the nuclear level, which can reduce the cell's ability to withstand physical forces. In the experimental examples herein, the inventors demonstrate that inhibition of the LINC complex alleviates the symptoms of a range of laminopathies.

[0253] As used herein, a "laminopathy" is a disease / condition associated with mutations to genes encoding lamins. Genes encoding lamins include LMNA (encoding lamins A and C), and LMNB1, LMNB2, which encode lamins B1 and B2. Accordingly, embodiments 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 reduce levels of a lamin isoform encoded by a wild-type allele of a lamin-encoding gene (e.g., LMNA, LMNB1, or LMNB2). In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is known or predicted to generate a truncated version of a lamin encoded by a wild-type allele of a lamin-encoding gene. In some embodiments, the mutation is known or predicted to generate a misfolded and / or degraded lamin.

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

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

[0257] In some embodiments, the laminopathies are skeletal muscle laminopathies. In some embodiments, the laminopathies are myopathy. In some embodiments, the laminopathies are LMNA mutation-associated myopathy.

[0258] In some embodiments, the disease treated / prevented in accordance with the present invention is characterized by one or more of myopathy, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, myocardial dystrophy, skeletal muscular dystrophy, progeria, neuropathy, lipodystrophy, skeletal dysplasia, lipodystrophy, leukodystrophy, or dermatosis.

[0259] In some embodiments, the disease treated / prevented in accordance with the present invention is characterized by one or more of muscular dystrophy, cardiac dystrophy, or skeletal muscular dystrophy.

[0260] In some embodiments, the laminopathies are associated with mutations in LMNA, LMNB1, or LMNB2. In some embodiments, the laminopathies are selected from the group consisting of Hutchinson-Gilford progeria syndrome; Emery-Dreifuss muscular dystrophy; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; lipodystrophy, partial, acquired; epilepsy, progressive myoclonus, 9; Charcot-Marie-Tooth disease, axonal, type 2e; muscular dystrophy; lipodystrophy, familial partial, type 2; cardiomyopathy, dilated, 1h; Pelger-Huette anomaly; Reynolds syndrome; muscle diseases; leukodystrophy; dilated cardiomyopathy; muscular dystrophy. Strophy, congenital, Lmna-related; mandibular acrodysplasia with type a lipodystrophy; cardiomyopathy, dilated, 1a; restrictive dermatosis, fatal; familial partial lipodystrophy; epilepsy; lipodystrophy with diabetes, leukodystrophy, leukodystrophy, leukodystrophy, 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 type 1b; atrial asystole 1; limb-girdle muscular dystrophy; cardiomyopathy, dilated type, with hypergonadotropic hypogonadism; heart-upper limb syndrome, Slovenian type; monogenic diabetes; arrhythmogenic right ventricular cardiomyopathy; cardiomyopathy, dilated type 1e; aging; micrognathia, hearing loss, progeria-like symptoms, and lipodystrophy syndrome; adrenal muscular dystrophy; atypical Werner syndrome; endometriosis; spinocerebellar ataxia 31; progressive muscular atrophy; neurogenic bowel; autosomal dominant leukodystrophy with autonomic disease; Werner syndrome; myopathy; Lmna Related dilated cardiomyopathy; congenital muscular dystrophy, type 1b; hypertrophic cardiomyopathy; left ventricular noncompaction; diabetes mellitus, non-insulin-dependent; arrhythmogenic right ventricular dysplasia, familial, type 9; heart disease; atrial fibrillation; cardiac conduction disorders; myoclonus; progressive myoclonic epilepsy; myoclonic epilepsy; peripheral nervous system disease; dental disease; atrioventricular block; myofibrillar myopathy; autosomal dominant limb-girdle muscular dystrophy; Lmna-associated cardiocutaneous progeria syndrome; amyotrophic lateral sclerosis type 1; neural tube defects; cervical cancer; neural tube defects, folate-sensitive; brain degeneration; melanoma;3-Hydroxyacyl-CoA dehydrogenase deficiency; Congenital muscle fiber type inequality; Acroosteolysis; Wolff-Parkinson-White syndrome; Sick sinus syndrome; Calcification; Undifferentiated pleomorphic sarcoma; Ventricular tachycardia, catecholamine-induced polymorphic type 1, with or without atrial dysfunction and / or dilated cardiomyopathy; Lipodystrophy, familial partial type, type 1; Axonal neuropathy; Paroxysmal ventricular fibrillation; Brugada syndrome 5; Ankylosing spinal muscular dystrophy; Limb-girdle muscular dystrophy type 1b; Insulin-resistant acanthosis nigricans, type a; Generalized lipodystrophy-associated progeria syndrome; Osteoporosis; Ankylosing spinal muscular dystrophy 1; Neuropathy; Catecholamine-induced polymorphic ventricular tachycardia; Cataracts; Bethlem myopathy 1; Congenital Generalized lipodystrophy; Restrictive cardiomyopathy; Muscular dystrophy, congenital merosin deficiency type 1a; Proximal spinal muscular atrophy; Muscular dystrophy-dystroglycanopathy, type B, 5; Lipodystrophy, congenital generalized type 1; Emery-Dreifuss muscular dystrophy 1, X-linked; Cardiomyopathy, dilated type 1d; Myopathy, proximal, and ophthalmoplegia; Muscle tissue disease; Ovarian cystadenoma; Emelinopathy; Fanconi anemia, complementation group a; Body mass index quantitative trait locus 11; Myelodysplastic syndrome; Skin disease; Anorexia nervosa; Spinal muscular atrophy; Inclusion body myositis; Aniridia 1; Myositis; Trichohepatic-enteric syndrome 1; Neuromuscular disease; Nutritional deficiency; Thoracic outlet syndrome; Myopathy; Muscular atrophy; Harlerman-Streiff syndrome; Rere-related disorders Disorders); Miller-Dieker Lissencephaly Syndrome; Lipodystrophy, Congenital Generalized, Type 4; Lipodystrophy, Familial Partial, Type 3; Widamann-Rautenstrauch Syndrome; Lipodystrophy, Congenital Generalized, Type 2; Ataxia Neuropathy Spectrum; Hair Loss, Neurologic Deficit, and Endocrine Disorder Syndrome; Lipodystrophy, Familial Partial, Type 4; Second-Degree Atrioventricular Block; Acute Necrotizing Encephalitis; Median Neuropathy; Intrinsic Cardiomyopathy; Familial Isolated Arrhythmogenic Ventricular Dysplasia, Right Dominant; Prolapse of the Female Genitalia; Familial Isolated Arrhythmogenic Ventricular Dysplasia, Biventricular; Familial Isolated Arrhythmogenic Ventricular Dysplasia, Left Dominant; Complete Generalized Lipodystrophy;Blood type - Ahonen; autosomal semi-dominant severe lipodystrophy laminopathies; ulnar neuropathy; pelvic muscle wasting; Alzheimer's disease; stroke, ischemic; ataxia-telangiectasia; spondyloarthropathy 1; human immunodeficiency virus type 1; neuroblastoma; vascular disease; neurological disease; respiratory failure; Turner syndrome; carpal tunnel syndrome; Barrett's esophagus; sleep apnea; cerebrovascular disease; proteasome-associated autoinflammatory syndrome 1; Joubert syndrome 1; viral infections; dementia; personality disorders; neuropathy, hereditary sensory and autonomic, type III; Lowe oculocerebrorenal syndrome Syndrome); Diabetes Mellitus; Fatty Liver Disease; Leigh Syndrome; Duchenne Muscular Dystrophy; Hydrocephalus; Dermatomyositis; Hirschsprung Disease 1; Long Qt Syndrome; Angelman Syndrome; Central Nervous System Disease; Congenital Disorders of Glycosylation, Type 1; Alacrimation, Achalasia, and Mental Retardation Syndrome; Polycystic Ovary Syndrome; Hypoglycemia; Muscle Hypertrophy; Kearns-Sayre Syndrome; Cone-Rod Dystrophy 2; Aicardi-Goutieres Syndrome; Andersen Cardiodysrhythmic Periodic Paralysis Paralysis); Muscular dystrophy, Becker type; Legg-Calve-Perthes disease; Androgen insensitivity syndrome; Ehlers-Danlos syndrome; Axenfeld-Rieger syndrome; Muscular dystrophy-dystroglycanopathy, type C, 5; Glomerulonephritis; Seizure disorders; Chikungunya fever; West syndrome; Ullrich congenital muscular dystrophy 1; Focal segmental glomerulosclerosis; Walker-Warburg syndrome; Renal dysplasia / dysplasia 1 (Renal Hypodysplasia / aplasia 1); Popliteal fold syndrome; Microcephaly; Childhood dermatomyositis; Distal arthrogryposis; Myocarditis; Arterial tortuosity syndrome; Scoliosis; Membranous nephropathy; Microvascular complications of type 3 diabetes; Epidermolysis bullosa; Short's 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 arrhythmia; Muscular dystrophy-dystroglycanopathy, type a, 1; Ptosis; Laryngitis; Ablepharon-Macrostomia Syndrome; Supravalvular aortic stenosis; Myopathy, congenital;Metabolic encephalopathy crisis with relapses, rhabdomyolysis, cardiac arrhythmias, and neurodegeneration; lissencephaly; polycystic liver disease with or without renal cysts; idiopathic inflammatory myopathy; epidermolysis bullosa simplex; focal segmental glomerulosclerosis; genital dysgenesis; gyral chorioretinal atrophy; syringomyelia; ichthyosis vulgaris; arthrogryposis, peripheral, type 1a; acute insulin response; brachydactyly; cerebellar hypoplasia; craniometaphyseal dysplasia, autosomal dominant; Alport syndrome, X-linked; lissencephaly; muscular dystrophy-dystroglycanopathy, type B; diarrhea; tufting enteropathy Enteropathy), congenital; junctional epidermolysis bullosa; Aicardi-Goutieres syndrome 1; Miyoshi muscular dystrophy; retinitis; Marden-Walker syndrome; neuroretinopathy; polyglucosan body myopathy with or without immunodeficiency 1; epidermolysis bullosa, junctional, Herlitz type; macroglossia; Parkinson's disease 15, autosomal recessive, early-onset; myopathy, myofibrillar, 3; microvascular complications of type 7 diabetes; muscle-eye-encephalopathy; Melkersson-Rosenthal syndrome; myopathy, X-linked, with excessive autophagy; choroiditis; muscular dystrophy, limb-girdle type, autosomal recessive 8; Crouzon syndrome with acanthosis nigricans; muscular dystrophy, limb-girdle Type 6, autosomal recessive; polymicrogyria; dystrophinopathy; microvascular complications of type 6 diabetes; microvascular complications of type 4 diabetes; hypotonia; pontocerebellar hypoplasia; congenital fibrosarcoma; intrauterine growth retardation, metaphyseal dysplasia, congenital adrenal hypoplasia, and genital anomalies; muscular dystrophy, limb-girdle type, autosomal recessive 7; myopathy, congenital, with fiber type imbalance; hereditary amelogenesis imperfecta, type Ig; refractory anemia; fibrosis of extraocular muscles, congenital, 1; ataxia and polyneuropathy, adult-onset; Alrakad syndrome; senile cataract; muscular dystrophy-dystroglycanopathy, type C, 1; neuronal migration disorder; Ayme-Gripp syndrome Syndrome); Primary agammaglobulinemia; Autosomal recessive limb-girdle muscular dystrophy type 2a; Encephalitis; Muscular dystrophy, congenital, giant conoid type; Autosomal recessive limb-girdle muscular dystrophy; Alkuraya-Kucinskas syndrome; Muscular dystrophy-dystroglycanopathy, type C, 4; Congenital muscular dystrophy type 1a; Behr syndrome; Dandy-Walker complex;Muscular dystrophy / dystroglycanopathy, type C, 2; Emery-Dreifuss muscular dystrophy, X-linked; Muscular dystrophy, limb-girdle, autosomal recessive, 3; Autosomal recessive limb-girdle muscular dystrophy, type 2d; Cerebral small vessel disease with or without ocular abnormalities, 1; Familial sporadic dilated cardiomyopathy; Epithelial recurrent erosive dystrophy Dystrophy); Muscular dystrophy / dystroglycanopathy; Mycobacterium avium complex infection; Autosomal recessive limb-girdle muscular dystrophy type 2l; Visual epilepsy; Sinus of Valsalva aneurysm; Autosomal recessive limb-girdle muscular dystrophy type 2b; Creatine phosphokinase, elevated serum; Spastic paraplegia, ataxia, and mental retardation; Polynuclear neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia, and hydranencephaly; Patulous Eustachian tube; Autosomal inherited diseases; CK syndrome; Neuronitis; Hyperaknea 1; Reduced body myopathy; Polymicrogyria, bilateral temporooccipital; Isolated hyperckemia; Charcot-Marie-Tooth disease, axonal, type 2b2; Cardiac neuromuscular disease with hyaline masses and nemaline bodies; Congenital muscular dystrophy without intellectual disability; Blood type, I system (I System; Salih Myopathy; Adducted Thumbs Syndrome; Dural Sinus Malformation; Blood Group, Donbrock System; Blood Group, Colton System; Arthrochalasia Ehlers-Danlos Syndrome Syndrome); Lama2-related muscular dystrophy; Muscular dystrophy, congenital, with childhood cataracts and hypogonadism; Intrauterine infection; Muscular dystrophy, congenital, merosin-positive; Congenital muscular dystrophy with cerebellar involvement; Fukuyama muscular dystrophy; Chronic lymphoproliferative disorder of natural killer cells; Congenital muscular dystrophy with intellectual disability; Amelogenesis imperfecta hypoplasia type Ig; Androgen insensitivity syndrome, mild; Muscular dystrophy, congenital, resulting in arthrogryposis; Congenital alpha-dystroglycanopathy with cerebral and ocular abnormalities; Type VI collagen-related myopathy;Select from Emery-Dreifuss muscular dystrophy, dominant type; congenital muscular dystrophy due to dystroglycanopathy; proximal myopathy with focal mitochondrial deficiency; and childhood scoliosis.

[0261] In some embodiments, the laminopathies are laminopathies associated with mutations in LMNA. In some embodiments, the laminopathies are Hutchinson-Gilford progeria syndrome; dilated cardiomyopathy; congenital LMNA-associated muscular dystrophy; Emery-Dreifuss muscular dystrophy 2, autosomal dominant; muscular dystrophy; acromangial dysplasia with type a lipodystrophy; cardiomyopathy, dilated, type 1a; Charcot-Marie-Tooth disease; limb-girdle muscular dystrophy; cardiomyopathy, dilated, with hypergonadotropic hypogonadism; Emery-Dreifuss muscular dystrophy 3, autosomal recessive; lipodystrophy. , familial partial type, type 2; Emery-Dreifuss muscular dystrophy; Charcot-Marie-Tooth disease, axonal, type 2b1; heart-upper limb syndrome, Slovenian type; aging; familial partial lipodystrophy; restrictive dermatosis, fatal; arrhythmogenic right ventricular cardiomyopathy; dental disease; heart disease; Werner syndrome; hypertrophic cardiomyopathy; left ventricular noncompaction; atrioventricular block; calcification; acroosteolysis; autosomal dominant limb-girdle muscular dystrophy; diabetes mellitus, non-insulin dependent; osteoporosis; atrial fibrillation; atrial asystole 1; melanoma; cardiac conduction disorder; catechol Amine-induced polymorphic ventricular tachycardia; micrognathia, hearing loss, progeria-like symptoms, and lipodystrophy syndrome; sick sinus syndrome; Pelger-Houette anomaly; Charcot-Marie-Tooth disease, axonal, type 2e; congenital generalized lipodystrophy; restrictive cardiomyopathy; congenital fiber type disproportion; lipodystrophy, congenital generalized, type 1; myofibrillar myopathy; lipodystrophy, familial partial, type 1; axonal neuropathy; atypical Werner syndrome; ovarian cystadenoma; Fanconi anemia, complementation group A; body mass index quantitative trait loci 11; Skin diseases; Ankylosing spinal muscular dystrophy 1; Neuromuscular diseases; Hurlerman-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 deficiency, 1a; Proximal spinal muscular atrophy; Muscular dystrophy-dystroglycanopathy, type B, 5; Muscular dystrophy, congenital, 1b; Reynolds syndrome; Widamann-Rautenstrauch syndrome;Emery-Dreifuss muscular dystrophy 1, X-linked; Lipodystrophy, congenital generalized, type 2; Monogenic diabetes; Cardiomyopathy, dilated, type 1d; Myopathy, proximal, and ophthalmoplegia; Muscle tissue disease; Lipodystrophy, familial partial, type 4; Cardiomyopathy, dilated, type 1h; Second-degree atrioventricular block; Median neuropathy; Intrinsic cardiomyopathy; Prolapse of female genitalia; Complete generalized lipodystrophy; Ankylosing spinal muscular dystrophy; Emelinopathy; Ulnar neuropathy Selected from: limb-girdle muscular dystrophy type 1b; Lmna-associated dilated cardiomyopathy; pelvic muscle wasting; generalized lipodystrophy-associated progeria syndrome; muscle disease; cardiomyopathy, dilated, type 1b; autosomal inherited disease; familial isolated arrhythmogenic ventricular dysplasia, right dominant; familial isolated arrhythmogenic ventricular dysplasia, biventricular; familial isolated arrhythmogenic ventricular dysplasia, left dominant; Lmna-associated cardiocutaneous progeria syndrome; autosomal semi-dominant severe lipodystrophy-laminopathies;

[0262] In some embodiments, the disease to be treated / prevented in accordance with 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 in accordance with 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 regular font in Table 1. In some embodiments, the disease to be treated / prevented in accordance with the present invention is selected from the diseases shown in bold 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 laminopathies are not Hutchinson-Gilford Progeria Syndrome (HGPS). In some embodiments, when the mutation occurs in LMNA, the mutation is an LMNA mutation that does not result in increased levels of progerin. In some embodiments, when the mutation occurs in LMNA, the mutation is not an HGPS-associated mutation.

[0309] A further aspect of the present invention relates to the treatment / prevention of diseases characterized by hyperlipidemia. A further aspect of the present invention relates to the treatment / prevention of diseases associated with LDL receptor deficiency (ie, reduced levels 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 blood total cholesterol level of ≥ 5 mmol / L or a blood low-density lipoprotein (LDL) level of ≥ 3 mmol / L. The US NIH defines hypercholesterolemia as a blood total cholesterol level of ≥ 240 mg / dL. Hypertriglyceridemia is described, for example, in Berglund et al., J. Clin. Endocrinol. Metab. (2012) 97(9):2969-89, and is defined by a blood triglyceride level of ≥ 150 mg / dL (≥ 1.7 mmol / L).

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

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

[0314] In some embodiments, the disease treated / prevented in accordance with the present invention is characterized by one or more of hyperlipidemia, hypercholesterolemia, atherosclerosis, stenosis, or hypertension, hi some embodiments, the disease treated / prevented in accordance with the present invention is characterized by atherosclerosis.

[0315] In some embodiments, the disease being 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. 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, a subject may be suspected of having or suffering from a disease based, for example, on the presence of other symptoms indicative of the disease in the subject or in the subject's cells / tissues / organs. In some embodiments, a subject may be considered to be at risk for developing a disease due, for example, to a genetic predisposition or other risk factors for the disease.

[0317] In some embodiments, the method comprises determining whether the subject has a mutation in a gene described herein. In some embodiments, the method comprises detecting a mutation in a gene described herein.

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

[0319] Genetic factor can be assayed by the method known to those skilled in the art, including PCR-based and sequencing assay.For example, by determining the existence of genetic factor in the sample obtained from subject, diagnosis can be confirmed, and / or subject can be classified as having the risk of developing the disease described herein, and / or subject can be determined to be suitable for treatment with LINC complex inhibitor.

[0320] The assay may be performed in vitro on a sample obtained from a subject or following processing of the sample obtained from the subject. The sample obtained from the subject may be of any type. The 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, or a synovial fluid sample. The blood-derived sample may be a selected fraction of the patient's blood, for example, a selected cell-containing fraction or a plasma or serum fraction. The sample may include a tissue sample or biopsy; or cells isolated from the subject.

[0321] In some embodiments, the method includes determining whether a subject has a mutation in one or more alleles of LMNA, LMNBl, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62. In some embodiments, the method includes determining whether a subject has a mutation in one or more alleles of LMNA, LMNBl, and LMNB2. In some embodiments, the method includes determining whether a subject has a mutation in an allele of LMNA. In some embodiments, the method includes determining whether a subject has a mutation in an allele of LDLR.

[0322] In such embodiments, if a mutation is detected, the subject may be identified as a candidate for administration of a LINC complex inhibitor according to the present disclosure. Thus, in some embodiments, a method includes selecting a subject determined to contain a mutation in one or more of LMNA, LMNBl, LMNB2, EMD, LAP2, LBR, ZMPSTE24, SYNE-1, and NUP62 for administration of a LINC complex inhibitor. In some embodiments, a method includes selecting a subject determined to contain a mutation in one or more of LMNA, LMNBl, and LMNB2 for administration of a LINC complex inhibitor. In some embodiments, a method includes selecting a subject determined to contain a mutation in LMNA for administration of a LINC complex inhibitor. In some embodiments, a method includes selecting a subject determined to contain 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 the disease for the presence or absence of at least one LMNA mutation, wherein the presence of at least one LMNA mutation indicates that the subject should be administered a LINC complex inhibitor according to the present disclosure.

[0324] According to various aspects of the present invention, the method of treating and / or preventing a disease according to the present invention comprises administering to a subject a therapeutically effective amount of a compound selected from the group consisting of: Increasing survival of subjects with the disease; Increasing the lifespan of a subject with the disease; Steps to increase cardiac function; delaying the onset of cardiac decline; Steps to increase myocardial contractility; Increasing the ejection fraction and / or fractional shortening; Steps to reduce atherosclerosis; may include one or more of:

[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, e.g., a non-human mammal. The subject can be male or female.

[0326] The subject may be a patient. The patient may have a disease described herein. The subject may have been diagnosed with 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, subjects / patients may be selected for therapy / prevention by the methods described herein based on their characterization for markers of the diseases described herein.

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

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

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

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

[0332] 5. The nucleic acid molecule of paragraph 3 or 4, wherein the cardiomyocyte-specific promoter is a 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.

[0333] 7. The nucleic acid molecule of paragraph 6, wherein the viral expression vector is selected from the group comprising lentivirus, adenovirus, and adeno-associated virus (AAV). 8. The nucleic acid molecule of any one of the preceding paragraphs, wherein the adeno-associated viral expression vector (AAV) has / is cardiac 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. The nucleic acid molecule of any one of the preceding paragraphs, wherein the transgene comprises nucleic acid sequences for expressing the luminal 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 luminal 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. The nucleic acid molecule of any one of the preceding paragraphs, wherein the transgene further comprises nucleic acid sequences for expressing an N-terminal signal sequence, a signal peptidase cleavage site, and a C-terminal targeting peptide sequence.

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

[0339] 16. The nucleic acid molecule of any one of paragraphs 11 to 15, wherein the SUN domain protein is SUN1 or SUN2. 17. The nucleic acid molecule of 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., proteases, amylases, and lipases), endoplasmic reticulum luminal 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 of any one of paragraphs 11 to 19, wherein the N-terminal signal sequence is not preceded at its N-terminus by any other tag.

[0342] 21. The nucleic acid molecule of any one of paragraphs 11 to 20, wherein the signal peptidase cleavage site is, or is one of the group consisting of, a signal peptidase cleavage site derived from human serum albumin, proinsulin, transferrin receptor, EGF receptor, prepro-opiomelanocortin, carboxypeptidase, complement proteins, fibrinogen, cytokines, chemokines, fibrinogen, pancreatic digestive enzymes (e.g., proteases, amylases, and lipases), endoplasmic reticulum luminal proteins such as protein disulfide isomerase, GRP94, and combinations thereof.

[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 of any one of paragraphs 11 to 22, wherein the C-terminal targeting peptide sequence prevents secretion of the peptide expressed from the transgene of any one of paragraphs 1 to 19.

[0344] 24. The nucleic acid molecule of any one of paragraphs 11 to 23, wherein the C-terminal targeting peptide sequence is a KDEL sequence. 25. The nucleic acid molecule of 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 anywhere in the nucleic acid molecule except downstream of the C-terminal targeting peptide sequence [e.g., KDEL] (after), or anywhere in 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 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 viral vector (AAV) comprising a chicken cardiac troponin T promoter (cTnT) and the transgene of 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 a 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, e.g., Figure 10), The nucleic acid molecule of any one of the preceding paragraphs.

[0348] 30. The vector is an adeno-associated viral vector (AAV) comprising a chicken cardiac troponin T promoter (cTnT) and the transgene of 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 a 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: 5 (see, e.g., Figure 10), The nucleic acid molecule of any one of the preceding paragraphs.

[0349] 31. The nucleic acid molecule of any one of paragraphs 1 to 10, wherein the transgene comprises 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 comprises a transmembrane domain and a SUN-interacting peptide.

[0350] 33. The 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. The nucleic acid molecule of any one of paragraphs 1 to 10, wherein the transgene comprises 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 comprises a nucleic acid sequence for expressing CRISPR-Cas. 36. The nucleic acid molecule of any one of paragraphs 1 to 33, wherein the transgene is a dominant-negative construct.

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

[0354] 39. The nucleic acid molecule of paragraph 38, wherein the disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between a protein interaction 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. The nucleic acid molecule of paragraph 39, wherein disruption of the protein-protein interaction between SUN and KASH of the LINC complex occurs between Sun1 and Nesprin-1. 41. The nucleic acid molecule of any one of the preceding paragraphs, wherein the AAV vector is formulated for delivery to the myocardium of a subject.

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

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

[0358] 46. ​​The nucleic acid molecule of any one of paragraphs 44 or 45, wherein the cardiovascular disease is selected from the group consisting of laminopathies, cardiomyopathies, e.g., dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, isolated atrial fibrillation; muscular dystrophy (often associated with cardiomyopathy), e.g., 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 dystrophies; premature aging syndromes (which are primarily vascular but may also have cardiac involvement), e.g., cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome, and the like, and diseases represented in bold font in Table 1.

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

[0360] 48. A pharmaceutical composition comprising a nucleic acid molecule according to any one of paragraphs 1 to 41. 49. A method for treating a disease in a subject, the method comprising administering a pharmaceutically effective amount of a nucleic acid molecule described in any one of paragraphs 1 to 41 or a 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, or both lamin A / C isoforms, of the Lmna gene.

[0362] 52. The method of any one of paragraphs 49 to 51, wherein the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), acromandibular dysplasia with type A lipodystrophy, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and diseases represented in regular font in Table 1.

[0363] 53. The method of any one of paragraphs 49 to 51, wherein the disease is a cardiovascular disease selected from the group consisting of a laminopathy, a cardiomyopathic disorder, e.g., dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, isolated atrial fibrillation; a muscular dystrophy (often associated with a cardiomyopathy), e.g., 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 dystrophies; a premature aging syndrome (which is believed to be primarily vascular but may also have cardiac involvement), e.g., cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome; and a disease 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, mice were N195K mice (Lmna N195K / N195K ) or Lmna Conditional Knockout (Lmna flox / flox ) Any one of paragraphs 49 to 53.

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

[0366] 56. Use of paragraph 55, where the disease is selected from the group consisting of restrictive skin disorder, familial partial lipodystrophy (e.g., Dunnigan type), acromandibular dysplasia with lipodystrophy type A, metabolic syndrome, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease type 2B1, and diseases represented in regular font in Table 1.

[0367] 57. The use of paragraph 55, wherein the cardiovascular disease is selected from the group consisting of laminopathies, cardiomyopathies, such as dilated cardiomyopathy (DCM), dilated cardiomyopathy 1A, dilated cardiomyopathy with conduction system disorders, cardiomyopathy with high-degree AV block and arrhythmias, isolated atrial fibrillation; muscular dystrophies (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 dystrophies; premature aging syndromes (which are primarily vascular but may also have cardiac involvement), such as cardiomyopathy associated with atypical Werner syndrome, cardiomyopathy associated with Hutchinson-Gilford Progeria syndrome, etc., as well as diseases represented in bold font in Table 1.

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

[0369] 60. The method of paragraph 59, wherein the drug candidate disrupts a 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 Sun1 protein and Nesprin-1 protein.

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

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

[0372] 65. Recombinant SUN and KASH domains were used; Optionally, the recombinant SUN domain is immobilized on a solid surface and the recombinant KASH domain is labeled with an enzyme capable of producing a colorimetric or chemiluminescent readout (compounds that are unable to inhibit the SUN-KASH interaction will result in wells in the plate in which the recombinant SUN binds to the enzyme-linked KASH domain. After a washing step and incubation with a colorimetric or chemiluminescent enzyme substrate, the presence of the SUN-KASH interaction can be detected in a standard plate reader. If the compound is able to inhibit the SUN-KASH interaction, after a washing step the KASH domain will be removed and the enzymatic reaction in the well will be reduced or absent); Optionally, the KASH domain can be fluorescently labeled with a fluorescein moiety, and the fluorescence anisotropy of the KASH domain interacting with the SUN domain can be measured using standard equipment, such as a plate reader incorporating fluorescence spectrometer functionality; Optionally, if the amount of the first complex is less than the amount of the second complex, there will be a difference in the fluorescence anisotropy of the fluorescent KASH, and the drug is a drug candidate for inhibiting the protein interaction. Any one of paragraphs 59 to 65. Sequence identity Pairwise and multiple sequence alignment for determining the percent identity between two or more amino acid or nucleic acid sequences can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780 Software). When using such software, for example, default parameters 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] [Table 2-14]

[0387] [Table 2-15]

[0388] [Table 2-16]

[0389] [Table 2-17]

[0390] [Table 2-18]

[0391] [Table 2-19]

[0392] The present invention includes combinations of the described embodiments and preferred features except where such combinations are expressly disallowed or expressly avoided. The features disclosed in the foregoing description, or in the claims that follow, or in the accompanying drawings, expressed in particular forms or in terms of means for performing the disclosed functions, or, where appropriate, methods or processes for obtaining the disclosed results, may be utilized separately or in any combination of such features to realize the invention in diverse forms thereof.

[0393] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0394] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Throughout this specification, including the appended claims, unless the context requires otherwise, the words "comprise" and "comprises," and variations such as "comprise (singular)," "comprising," and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0395] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in connection with numerical values ​​is optional and means, for example, + / - 10%.

[0396] The methods disclosed herein may be performed, or products may exist, in vitro, ex vivo, or in vivo. The term "in vitro" is intended to encompass experiments with materials, biological substances, cells and / or tissues in laboratory conditions or in culture, while the term "in vivo" is intended to encompass experiments and procedures with intact multicellular organisms. "Ex vivo" refers to something that exists or occurs outside an organism, e.g., outside the human or animal body, which may be present on tissues (e.g., whole organs) or cells removed from an organism.

[0397] When a nucleic acid sequence is disclosed herein, its reverse complement is also expressly contemplated. For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3rd ed. 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0398] Aspects and embodiments of the present invention will now be 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 incorporated herein by reference. While the present invention has been described in conjunction with the exemplary embodiments set forth below, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the present invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments can be made without departing from the spirit and scope of the invention.

[0399] BRIEF DESCRIPTION OF THE DRAWINGS BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention are now discussed with reference to the accompanying figures. [Brief explanation of the drawings]

[0400] [Figure 1] FIG. 1 is a schematic diagram showing mutations in the lamin A / C gene LMNA and the laminopathies resulting from these mutations. [Figure 2] FIG. 1 is a schematic diagram showing the arrangement of components of the nuclear envelope membrane and lamina. [Figure 3]This is a schematic diagram illustrating the connection between the nucleus and the extracellular matrix via the LINC complex and how mutations in lamin A / C can result in DCM. The plasma membrane, cytoskeleton, and nucleus form a mechanically and physically linked entity. In Lmna mutants, the nucleus is structurally weaker. It is much more susceptible to mechanical stress from cytoskeletal forces. This leads to severe damage to the myocyte nucleus, which in turn leads to a cascade of events, including apoptosis and fibrosis, that ultimately result in DCM. [Figure 4] Figure 1 shows the effect of microinjection of dextran under low pressure into the nuclei of Lmna+ / + and Lmna- / - mice. In wild-type cells, dextran remains in the nucleus, whereas in Lmna mutant cells, dextran effluxes from the nucleus into the cytoplasm. [Figure 5] 5A-5B are schematic diagrams showing the LINC complex (FIG. 5A) and the interaction between KASH and SUN (FIG. 5B). [Figure 6]This figure shows that the weight and lifespan defects in Lmna- / - and LmnaΔ9 mice are ameliorated in homozygous Sun1 knockout Lmna- / -Sun1- / - and LmnaΔ9Sun1- / - animals. (A) Weights are averaged for mice with the indicated genotypes. The number of animals (n) used is indicated. (B) Kaplan-Meier plots showing the increased survival of Lmna- / -Sun1- / - mice compared with Lmna- / - mice. The median survival of wild-type or Sun1- / - mice was over 210 days over a 7-month follow-up period; Lmna- / - mice had a median survival of 41 days; Lmna- / -Sun1+ / - mice had a median survival of 54 days; and Lmna- / -Sun1- / - mice had a median survival of 104 days (p<0.01 comparing Lmna- / - with Lmna- / -Sun1- / -). (C) Body weight of LmnaΔ9 mice that are wild-type, heterozygous, or homozygous for Sun1 deficiency. Wild-type and Sun1- / - cohorts are graphed for comparison. Values ​​are the mean ± SEM of animals in each cohort. The number of animals (n) is indicated (p<0.0001 comparing LmnaΔ9Sun1+ / + with LmnaΔ9Sun1- / -). (D) Kaplan-Meier graph showing increased survival of LmnaΔ9Sun1- / - mice compared to LmnaΔ9Sun1+ / + mice. LmnaΔ9Sun1+ / - mice are also graphed (p<0.0001 comparing LmnaΔ9Sun1+ / + with LmnaΔ9Sun1- / -). (E) Cell proliferation of the indicated MEFs. Curves are the mean ± SD and represent four or more independent isolates from embryos of the indicated genotype. (F) Growth curves of MAFs (mouse adult fibroblasts) from WT, Sun1- / -, LmnaΔ9Sun1+ / +, and LmnaΔ9Sun1- / - mice. MAFs were seeded at a density of 1,000 cells per well. Growth was measured, and the normalized cell index (mean ± SD) is shown. [Figure 7] FIG. 1 is a schematic diagram showing the characteristics of the Sun1 protein and the components used to generate dominant-negative SUN1 proteins, including the signal sequence, coiled-coil sequence, SUN domain sequence, and KDEL sequence. [Figure 8] FIG. 1 is a schematic diagram of the plasmid (SEQ ID NO: 1) used for AAV production. [Figure 9] FIG. 1 is a schematic diagram showing a plasmid (SEQ ID NO: 2) for AAV production, containing sequences from AAV2 and AAV9. [Figure 10] FIG. 1 is a schematic diagram showing an AAV expression construct (SEQ ID NO: 3) containing a cardiac-specific promoter and a Sun1 dominant-negative sequence. [Figure 11] FIG. 1 is a schematic diagram showing the characteristics of a dominant-negative Sun1 protein, including a signal sequence, a coiled-coil sequence, a SUN domain sequence, and a KDEL sequence (SEQ ID NO: 4). [Figure 12] FIG. 1 is a schematic diagram showing the characteristics of a dominant-negative Sun2 protein containing a signal sequence, a luminal domain sequence, and a KDEL sequence (SEQ ID NO: 5). [Figure 13] FIG. 1 is a schematic diagram showing the regions of the Sun1 protein used in dominant-negative constructs. [Figure 14] FIG. 1 shows an alignment of KASH1 to KASH5 domain amino acid sequences (SEQ ID NOs: 7, 9, 11, 13, and 15, respectively) with conserved residues. [Figure 15] Schematic diagrams showing LINC complexes in wild-type mice, Sun1 KO mice, AAV dominant-negative SUN mice, and mice with altered KASH domains. The schematic diagram of wild-type mice is taken from Brian Burke, 2012. The schematic diagram of Sun1 KO mice represents the results of Chen et al., 2012. The schematic diagrams of AAV dominant-negative SUN domains and altered KASH domains represent the inventors' submission at the priority date regarding methods for disrupting LINC complexes to ameliorate laminopathies and are based on data obtained at that time. [Figure 16] Figure 1 shows Kaplan-Meier curves for Lmna KO mice surviving an average of 28 days, Sun1 KO mice living for more than 300 days, and cardiac Lmna KO / Sun1 KO mice living for more than 300 days. [Figure 17] FIG. 1 shows H&E-stained sections of hearts from Sun1 KO mice, cardiac Lmna KO mice, and cardiac Lmna KO / Sun1 KO mice, with LmnaKO / Sun1WT hearts showing left ventricular dilation (DCM) compared to WT and LmnaKo / Sun1KO hearts. [Figure 18] Figure 1 shows a schematic diagram showing disruption of the LINC complex in Nesprin-1 ΔKASH mice. LmnaKO Nesprin-1WT mice have a survival time of approximately 20 days. LmnaKO Nesprin-1-ΔKASH mice survive for approximately 40 days, comparable to LmnaKOSun1KO mice. [Figure 19] Figures 19A-19B show a schematic diagram (Figure 19A) showing the predicted AAV-cTNT-DN-SUN expression and competition between exogenous DN-SUN and native Sun1 for binding to the KASH domain, DN-SUN is shown in 19B (top panel), and the effect of transfected DN-SUN on the location of native Nesprin2G in cells (Figure 19B), where two nuclei in the middle panel express DN-SUN and both in the merge panel show loss of Nesprin2 from the nuclear membrane. [Figure 20] Kaplan-Meier curves showing that in vivo disruption of the SUN-KASH interaction using AAV9-cTNT dominant-negative Sun1 (DNSun1) extends the lifespan of heart-specific Lmna KO in male and female mice. [Figure 21] Figure 1 shows the C-terminal amino acids of the KASH domain of Nesprin-2 (KASH2). A sequence of 14 or 18 amino acids from the KASH2 C-terminus can physically interact with the SUN domain of SUN2. Loss of the last four amino acids from KASH2 or addition of a single alanine amino acid at the C-terminus of KASH2 is sufficient to disrupt the interaction between the KASH2 domain and the SUN domain. [Figure 22] FIG. 1 is a schematic diagram showing a screening method for detecting agents that disrupt LINC complexes. [Figure 23]1 is a flow chart showing a more detailed screening method for identifying small molecules that disrupt LINC complexes. [Figure 24] Kaplan-Meier curves showing that wild-type (C57 / Bl6) mice with or without the Nesprin-1 KASH-disrupting (C'TΔ8) mutation have a normal survival time. Mice wild-type (Nesp1+ / +) or heterozygous for Nesp1-C'TΔ8 (Nesp1+ / C'TΔ8) with the Lmna null / KO mutation (LA-ZP3creΔ / Δ) have a median survival time of 15 or 18 days, while median survival increases to 38 days in Lmna KO / Nesp1 homozygous (LA-ZP3creΔ / Δ; Nesp1C'TΔ8 / C'TΔ8) mice. [Figure 25] Kaplan-Meier curves show that mice with wild-type Lmna (N1CTΔ8 / CTΔ8LA+ / +MCre+ / -) or mice with a loxP-transduced Lmna allele but lacking a cardiac-specific Cre driver (N1CTΔ8 / CTΔ8LAf / fMCre+ / + and N1WT / WTLAf / fMCre+ / +) survive for the duration of the experiment (approximately 80 days at the time of priority filing, extending to 120 days without change). Mice with cardiomyocyte-specific deletion of Lmna (N1WT / WTLAf / fMCre+ / -) survive 22-24 days after induction of Cre / loxP-mediated deletion by tamoxifen (TMX) delivery, and survival increases for mice with TMX-induced cardiomyocyte-specific deletion of Lmna and a homozygous mutation for Nesprin-1 (N1CTΔ8 / CTΔ8LAf / fMCre+ / -). [Figure 26A]Figures 26A-26D are graphs showing Kaplan-Meier curves demonstrating that Sun1 loss extends the lifespan of Lmna mutant mice. (Figure 26A) Wild-type (C57 / Bl6) mice with or without Sun1 had a normal lifespan, whereas LmnaFlx / Flx:Zp3 mice, in which lamin A was deleted in all tissues, had a mean postnatal survival of 17.5 days (***P ≤ 0.0001, log-rank test). On the Sun1- / - background, lifespan increased to 32.5 days. (Figure 26B) When LmnaFlx / Flx mice were specifically and constitutively deleted in the heart by crossing them with the CreαMyHC line, LmnaFlx / Flx:αMyHC mice lived an average of 26.5 days. On the Sun1- / - background, these mice lived for more than 6 months. (Figure 26C) When 3- to 5-month-old LmnaFlx / Flx mice were crossed with Tmx-inducible cardiomyocyte-specific Cre Tg{Myh6-cre / Esr1) (abbreviated as mcm), the mice died within 3-4 weeks after a single injection of Tmx. On the Sun1- / - background, these mice lived for over 1 year. (Figure 26D) Compared with LmnaN195K / N195KSun1- / - mice, which had a mean survival time of 111 days, LmnaN195K / N195K mice lived an average of 78 days (***P ≤ 0.0001, **P = 0.0073, log-rank test). [Figure 26B]Figures 26A-26D are graphs showing Kaplan-Meier curves demonstrating that Sun1 loss extends the lifespan of Lmna mutant mice. (Figure 26A) Wild-type (C57 / Bl6) mice with or without Sun1 had a normal lifespan, whereas LmnaFlx / Flx:Zp3 mice, in which lamin A was deleted in all tissues, had a mean postnatal survival of 17.5 days (***P ≤ 0.0001, log-rank test). On the Sun1- / - background, lifespan increased to 32.5 days. (Figure 26B) When LmnaFlx / Flx mice were specifically and constitutively deleted in the heart by crossing them with the CreαMyHC line, LmnaFlx / Flx:αMyHC mice lived an average of 26.5 days. On the Sun1- / - background, these mice lived for more than 6 months. (Figure 26C) When 3- to 5-month-old LmnaFlx / Flx mice were crossed with Tmx-inducible cardiomyocyte-specific Cre Tg{Myh6-cre / Esr1) (abbreviated as mcm), the mice died within 3-4 weeks after a single injection of Tmx. On the Sun1- / - background, these mice lived for over 1 year. (Figure 26D) Compared with LmnaN195K / N195KSun1- / - mice, which had a mean survival time of 111 days, LmnaN195K / N195K mice lived an average of 78 days (***P ≤ 0.0001, **P = 0.0073, log-rank test). [Figure 26C]Figures 26A-26D are graphs showing Kaplan-Meier curves demonstrating that Sun1 loss extends the lifespan of Lmna mutant mice. (Figure 26A) Wild-type (C57 / Bl6) mice with or without Sun1 had a normal lifespan, whereas LmnaFlx / Flx:Zp3 mice, in which lamin A was deleted in all tissues, had a mean postnatal survival of 17.5 days (***P ≤ 0.0001, log-rank test). On the Sun1- / - background, lifespan increased to 32.5 days. (Figure 26B) When LmnaFlx / Flx mice were specifically and constitutively deleted in the heart by crossing them with the CreαMyHC line, LmnaFlx / Flx:αMyHC mice lived an average of 26.5 days. On the Sun1- / - background, these mice lived for more than 6 months. (Figure 26C) When 3- to 5-month-old LmnaFlx / Flx mice were crossed with Tmx-inducible cardiomyocyte-specific Cre Tg{Myh6-cre / Esr1) (abbreviated as mcm), the mice died within 3-4 weeks after a single injection of Tmx. On the Sun1- / - background, these mice lived for over 1 year. (Figure 26D) Compared with LmnaN195K / N195KSun1- / - mice, which had a mean survival time of 111 days, LmnaN195K / N195K mice lived an average of 78 days (***P ≤ 0.0001, **P = 0.0073, log-rank test). [Figure 26D]Figures 26A-26D are graphs showing Kaplan-Meier curves demonstrating that Sun1 loss extends the lifespan of Lmna mutant mice. (Figure 26A) Wild-type (C57 / Bl6) mice with or without Sun1 had a normal lifespan, whereas LmnaFlx / Flx:Zp3 mice, in which lamin A was deleted in all tissues, had a mean postnatal survival of 17.5 days (***P ≤ 0.0001, log-rank test). On the Sun1- / - background, lifespan increased to 32.5 days. (Figure 26B) When LmnaFlx / Flx mice were specifically and constitutively deleted in the heart by crossing them with the CreαMyHC line, LmnaFlx / Flx:αMyHC mice lived an average of 26.5 days. On the Sun1- / - background, these mice lived for more than 6 months. (Figure 26C) When 3- to 5-month-old LmnaFlx / Flx mice were crossed with Tmx-inducible cardiomyocyte-specific Cre Tg{Myh6-cre / Esr1) (abbreviated as mcm), the mice died within 3-4 weeks after a single injection of Tmx. On the Sun1- / - background, these mice lived for over 1 year. (Figure 26D) Compared with LmnaN195K / N195KSun1- / - mice, which had a mean survival time of 111 days, LmnaN195K / N195K mice lived an average of 78 days (***P ≤ 0.0001, **P = 0.0073, log-rank test). [Figure 27A]Figures 27A-27E show the survival and phenotype of LmnaFlx / Flx:mcm+Tmx mice. (Figure 27A) The mean survival time of LmnaFlx / Flx:mcm mice was 27 days after a single Tmx injection (***P≦0.0001; log-rank test). (Figure 27B) PCR detected the loxP-transduced (deleted) Lmna gene (arrowhead) only in cardiac tissue after Tmx injection, but not in other tissues, even when Tmx was not injected. (Figure 27C) LmnaFlx / Flx:mcm+Tmx mice developed kyphosis (arrowhead) by 21 days after injection. (Figure 27D) 21 days after Tmx, cardiomyocyte (CM) nuclei were detected by PCM-1 staining. Lamin A / C protein, detected by immunofluorescence, was present in controls (i, iii) but was reduced / absent (white arrowheads) in CM nuclei of both isolated CMs (second panel of ii) and heart sections (iv). (Figure 27E) 21 days after Tmx, lamin A / C levels were quantified by Western analysis of whole heart lysates. A significant decrease in A-type lamin protein (***P ≤ 0.0001; T-test) was detected, but lamin C levels were not similarly reduced in LmnaFlx / Flx:mcm+Tmx mice compared with LmnaFlx / Flx:mcm+CTL. (Figure 27F) Quantitative analysis was performed 21 days after Tmx. The presence of LoxP sites in the WT-Lmna gene (LmnaFlx / Flx) resulted in reduced Lmna transcript levels compared to LmnaWt / Wt levels, but this had no apparent effect on lifespan or growth / survival. [Figure 27B]Figures 27A-27E show the survival and phenotype of LmnaFlx / Flx:mcm+Tmx mice. (Figure 27A) The mean survival time of LmnaFlx / Flx:mcm mice was 27 days after a single Tmx injection (***P≦0.0001; log-rank test). (Figure 27B) PCR detected the loxP-transduced (deleted) Lmna gene (arrowhead) only in cardiac tissue after Tmx injection, but not in other tissues, even when Tmx was not injected. (Figure 27C) LmnaFlx / Flx:mcm+Tmx mice developed kyphosis (arrowhead) by 21 days after injection. (Figure 27D) 21 days after Tmx, cardiomyocyte (CM) nuclei were detected by PCM-1 staining. Lamin A / C protein, detected by immunofluorescence, was present in controls (i, iii) but was reduced / absent (white arrowheads) in CM nuclei of both isolated CMs (second panel of ii) and heart sections (iv). (Figure 27E) 21 days after Tmx, lamin A / C levels were quantified by Western analysis of whole heart lysates. A significant decrease in A-type lamin protein (***P ≤ 0.0001; T-test) was detected, but lamin C levels were not similarly reduced in LmnaFlx / Flx:mcm+Tmx mice compared with LmnaFlx / Flx:mcm+CTL. (Figure 27F) Quantitative analysis was performed 21 days after Tmx. The presence of LoxP sites in the WT-Lmna gene (LmnaFlx / Flx) resulted in reduced Lmna transcript levels compared to LmnaWt / Wt levels, but this had no apparent effect on lifespan or growth / survival. [Figure 27C]Figures 27A-27E show the survival and phenotype of LmnaFlx / Flx:mcm+Tmx mice. (Figure 27A) The mean survival time of LmnaFlx / Flx:mcm mice was 27 days after a single Tmx injection (***P≦0.0001; log-rank test). (Figure 27B) PCR detected the loxP-transduced (deleted) Lmna gene (arrowhead) only in cardiac tissue after Tmx injection, but not in other tissues, even when Tmx was not injected. (Figure 27C) LmnaFlx / Flx:mcm+Tmx mice developed kyphosis (arrowhead) by 21 days after injection. (Figure 27D) 21 days after Tmx, cardiomyocyte (CM) nuclei were detected by PCM-1 staining. Lamin A / C protein, detected by immunofluorescence, was present in controls (i, iii) but was reduced / absent (white arrowheads) in CM nuclei of both isolated CMs (second panel of ii) and heart sections (iv). (Figure 27E) 21 days after Tmx, lamin A / C levels were quantified by Western analysis of whole heart lysates. A significant decrease in A-type lamin protein (***P ≤ 0.0001; T-test) was detected, but lamin C levels were not similarly reduced in LmnaFlx / Flx:mcm+Tmx mice compared with LmnaFlx / Flx:mcm+CTL. (Figure 27F) Quantitative analysis was performed 21 days after Tmx. The presence of LoxP sites in the WT-Lmna gene (LmnaFlx / Flx) resulted in reduced Lmna transcript levels compared to LmnaWt / Wt levels, but this had no apparent effect on lifespan or growth / survival. [Figure 27D]Figures 27A-27E show the survival and phenotype of LmnaFlx / Flx:mcm+Tmx mice. (Figure 27A) The mean survival time of LmnaFlx / Flx:mcm mice was 27 days after a single Tmx injection (***P≦0.0001; log-rank test). (Figure 27B) PCR detected the loxP-transduced (deleted) Lmna gene (arrowhead) only in cardiac tissue after Tmx injection, but not in other tissues, even when Tmx was not injected. (Figure 27C) LmnaFlx / Flx:mcm+Tmx mice developed kyphosis (arrowhead) by 21 days after injection. (Figure 27D) 21 days after Tmx, cardiomyocyte (CM) nuclei were detected by PCM-1 staining. Lamin A / C protein, detected by immunofluorescence, was present in controls (i, iii) but was reduced / absent (white arrowheads) in CM nuclei of both isolated CMs (second panel of ii) and heart sections (iv). (Figure 27E) 21 days after Tmx, lamin A / C levels were quantified by Western analysis of whole heart lysates. A significant decrease in A-type lamin protein (***P ≤ 0.0001; T-test) was detected, but lamin C levels were not similarly reduced in LmnaFlx / Flx:mcm+Tmx mice compared with LmnaFlx / Flx:mcm+CTL. (Figure 27F) Quantitative analysis was performed 21 days after Tmx. The presence of LoxP sites in the WT-Lmna gene (LmnaFlx / Flx) resulted in reduced Lmna transcript levels compared to LmnaWt / Wt levels, but this had no apparent effect on lifespan or growth / survival. [Figure 27E]Figures 27A-27E show the survival and phenotype of LmnaFlx / Flx:mcm+Tmx mice. (Figure 27A) The mean survival time of LmnaFlx / Flx:mcm mice was 27 days after a single Tmx injection (***P≦0.0001; log-rank test). (Figure 27B) PCR detected the loxP-transduced (deleted) Lmna gene (arrowhead) only in cardiac tissue after Tmx injection, but not in other tissues, even when Tmx was not injected. (Figure 27C) LmnaFlx / Flx:mcm+Tmx mice developed kyphosis (arrowhead) by 21 days after injection. (Figure 27D) 21 days after Tmx, cardiomyocyte (CM) nuclei were detected by PCM-1 staining. Lamin A / C protein, detected by immunofluorescence, was present in controls (i, iii) but was reduced / absent (white arrowheads) in CM nuclei of both isolated CMs (second panel of ii) and heart sections (iv). (Figure 27E) 21 days after Tmx, lamin A / C levels were quantified by Western analysis of whole heart lysates. A significant decrease in A-type lamin protein (***P ≤ 0.0001; T-test) was detected, but lamin C levels were not similarly reduced in LmnaFlx / Flx:mcm+Tmx mice compared with LmnaFlx / Flx:mcm+CTL. (Figure 27F) Quantitative analysis was performed 21 days after Tmx. The presence of LoxP sites in the WT-Lmna gene (LmnaFlx / Flx) resulted in reduced Lmna transcript levels compared to LmnaWt / Wt levels, but this had no apparent effect on lifespan or growth / survival. [Figure 27F]Figures 27A-27E show the survival and phenotype of LmnaFlx / Flx:mcm+Tmx mice. (Figure 27A) The mean survival time of LmnaFlx / Flx:mcm mice was 27 days after a single Tmx injection (***P≦0.0001; log-rank test). (Figure 27B) PCR detected the loxP-transduced (deleted) Lmna gene (arrowhead) only in cardiac tissue after Tmx injection, but not in other tissues, even when Tmx was not injected. (Figure 27C) LmnaFlx / Flx:mcm+Tmx mice developed kyphosis (arrowhead) by 21 days after injection. (Figure 27D) 21 days after Tmx, cardiomyocyte (CM) nuclei were detected by PCM-1 staining. Lamin A / C protein, detected by immunofluorescence, was present in controls (i, iii) but was reduced / absent (white arrowheads) in CM nuclei of both isolated CMs (second panel of ii) and heart sections (iv). (Figure 27E) 21 days after Tmx, lamin A / C levels were quantified by Western analysis of whole heart lysates. A significant decrease in A-type lamin protein (***P ≤ 0.0001; T-test) was detected, but lamin C levels were not similarly reduced in LmnaFlx / Flx:mcm+Tmx mice compared with LmnaFlx / Flx:mcm+CTL. (Figure 27F) Quantitative analysis was performed 21 days after Tmx. The presence of LoxP sites in the WT-Lmna gene (LmnaFlx / Flx) resulted in reduced Lmna transcript levels compared to LmnaWt / Wt levels, but this had no apparent effect on lifespan or growth / survival. [Figure 28A]Figures 28A-28D show echocardiograms, cardiac function, and histology of LmnaFlx / Flx:mcm+Tmx mice. (Figure 28A) LmnaFlx / Flx:mcm+Tmx mice exhibited impaired cardiac contractile function. (Figure 28B) LmnaFlx / Flx:mcm hearts exhibited decreased EF% and FS% and increased LVID (***P≦0.0001, **P=0.0010; two-way ANOVA). (Figure 28C) Histological analysis of the hearts revealed increased infiltration of nucleated cells and intercellular spaces in LmnaFlx / Flx:mcm hearts (i and ii). Significantly fewer viable (brick-like) CMs were isolated from LmnaFlx / Flx:mcm hearts compared with LmnaFlx / Flx:mcm controls (iii). Further magnification revealed that cardiomyocytes isolated from LmnaFlx / Flx:mcm hearts contained large intracellular vacuoles (arrowhead, iv). (Fig. 28D) The left ventricular lumen in LmnaFlx / Flx:mcm hearts was enlarged (i), accompanied by increased fibrosis (ii) (**P = 0.0007, seen as the light gray area in the middle panel ii of 28D and the left panel of iv) and an increase in apoptotic nuclei as revealed by TUNEL staining (*P = 0.0220; one-way ANOVA) (right panels of iii and iv). All sampling and analysis was performed on hearts 21 days after Tmx injection. [Figure 28B]Figures 28A-28D show echocardiograms, cardiac function, and histology of LmnaFlx / Flx:mcm+Tmx mice. (Figure 28A) LmnaFlx / Flx:mcm+Tmx mice exhibited impaired cardiac contractile function. (Figure 28B) LmnaFlx / Flx:mcm hearts exhibited decreased EF% and FS% and increased LVID (***P≦0.0001, **P=0.0010; two-way ANOVA). (Figure 28C) Histological analysis of the hearts revealed increased infiltration of nucleated cells and intercellular spaces in LmnaFlx / Flx:mcm hearts (i and ii). Significantly fewer viable (brick-like) CMs were isolated from LmnaFlx / Flx:mcm hearts compared with LmnaFlx / Flx:mcm controls (iii). Further magnification revealed that cardiomyocytes isolated from LmnaFlx / Flx:mcm hearts contained large intracellular vacuoles (arrowhead, iv). (Fig. 28D) The left ventricular lumen in LmnaFlx / Flx:mcm hearts was enlarged (i), accompanied by increased fibrosis (ii) (**P = 0.0007, seen as the light gray area in the middle panel ii of 28D and the left panel of iv) and an increase in apoptotic nuclei as revealed by TUNEL staining (*P = 0.0220; one-way ANOVA) (right panels of iii and iv). All sampling and analysis was performed on hearts 21 days after Tmx injection. [Figure 28C]Figures 28A-28D show echocardiograms, cardiac function, and histology of LmnaFlx / Flx:mcm+Tmx mice. (Figure 28A) LmnaFlx / Flx:mcm+Tmx mice exhibited impaired cardiac contractile function. (Figure 28B) LmnaFlx / Flx:mcm hearts exhibited decreased EF% and FS% and increased LVID (***P≦0.0001, **P=0.0010; two-way ANOVA). (Figure 28C) Histological analysis of the hearts revealed increased infiltration of nucleated cells and intercellular spaces in LmnaFlx / Flx:mcm hearts (i and ii). Significantly fewer viable (brick-like) CMs were isolated from LmnaFlx / Flx:mcm hearts compared with LmnaFlx / Flx:mcm controls (iii). Further magnification revealed that cardiomyocytes isolated from LmnaFlx / Flx:mcm hearts contained large intracellular vacuoles (arrowhead, iv). (Fig. 28D) The left ventricular lumen in LmnaFlx / Flx:mcm hearts was enlarged (i), accompanied by increased fibrosis (ii) (**P = 0.0007, seen as the light gray area in the middle panel ii of 28D and the left panel of iv) and an increase in apoptotic nuclei as revealed by TUNEL staining (*P = 0.0220; one-way ANOVA) (right panels of iii and iv). All sampling and analysis was performed on hearts 21 days after Tmx injection. [Figure 28D]Figures 28A-28D show echocardiograms, cardiac function, and histology of LmnaFlx / Flx:mcm+Tmx mice. (Figure 28A) LmnaFlx / Flx:mcm+Tmx mice exhibited impaired cardiac contractile function. (Figure 28B) LmnaFlx / Flx:mcm hearts exhibited decreased EF% and FS% and increased LVID (***P≦0.0001, **P=0.0010; two-way ANOVA). (Figure 28C) Histological analysis of the hearts revealed increased infiltration of nucleated cells and intercellular spaces in LmnaFlx / Flx:mcm hearts (i and ii). Significantly fewer viable (brick-like) CMs were isolated from LmnaFlx / Flx:mcm hearts compared with LmnaFlx / Flx:mcm controls (iii). Further magnification revealed that cardiomyocytes isolated from LmnaFlx / Flx:mcm hearts contained large intracellular vacuoles (arrowhead, iv). (Fig. 28D) The left ventricular lumen in LmnaFlx / Flx:mcm hearts was enlarged (i), accompanied by increased fibrosis (ii) (**P = 0.0007, seen as the light gray area in the middle panel ii of 28D and the left panel of iv) and an increase in apoptotic nuclei as revealed by TUNEL staining (*P = 0.0220; one-way ANOVA) (right panels of iii and iv). All sampling and analysis was performed on hearts 21 days after Tmx injection. [Figure 29A-1] Figures 29A-29D show changes in nuclear morphology and cardiac structure in LmnaFlx / Flx:mcm mice with or without Sun1 after Tmx injection. (Figure 29A) CM nuclei with reduced or absent lamin A / C expression are indicated by white arrowheads (1, 3). CM nuclei with normal lamin A / C levels (2, 4) are indicated by gray arrowheads. LMNA protein levels, measured by both fluorescence intensity (5) and Western blot (6), were significantly reduced in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice (***P=0.0009; t-test) and LmnaFlx / Flx:mcmSun1- / -+Tmx mice (*P=0.0359; t-test) compared with LmnaFlx / FlxmcmSun1+ / + controls (lower graph, 6). [Figure 29A-2]Figures 29A-29D show changes in nuclear morphology and cardiac structure in LmnaFlx / Flx:mcm mice with or without Sun1 after Tmx injection. (Figure 29A) CM nuclei with reduced or absent lamin A / C expression are indicated by white arrowheads (1, 3). CM nuclei with normal lamin A / C levels (2, 4) are indicated by gray arrowheads. LMNA protein levels, measured by both fluorescence intensity (5) and Western blot (6), were significantly reduced in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice (***P=0.0009; t-test) and LmnaFlx / Flx:mcmSun1- / -+Tmx mice (*P=0.0359; t-test) compared with LmnaFlx / FlxmcmSun1+ / + controls (lower graph, 6). [Figure 29B] (FIG. 29B) Left ventricular (LV) enlargement was evident in LmnaFlx / Flx:mcmSun1+ / ++Tmx hearts (panel 1), but not in the LV of LmnaFlx / Flx:mcmSun1- / -+Tmx hearts (panel 2). LmnaFlx / Flx:mcmSun1+ / ++Tmx mice had significantly increased fibrosis compared with controls (panel 3, fibrosis is gray), but there was no significant increase in fibrosis in LmnaFlx / FlxmcmSun1- / -+Tmx hearts (panel 3) compared with controls (panels 4, quantified in panel 5; ***P=0.0001, one-way ANOVA). Cardiac papillary muscle force measurements from LmnaFlx / Flx:mcmSun1+ / ++Tmx mice were significantly reduced compared with LmnaFlx / Flx:mcmSun1+ / + controls (**P=0.0047; T-test) and LmnaFlx / Flx:mcmSun1- / -+Tmx (*P=0.0113; T-test) (Panel 6). [Figure 29C](Figure 29C) CM nuclear morphology was significantly altered in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice (panel 1, filled arrowheads). In the absence of TMX, control heart sections (CTL, panel 2) display a few nuclear abnormalities. In the absence of Sun1, LmnaFlx / Flx:mcmSun1- / -+Tmx cardiomyocytes showed no nuclear abnormalities (panels 3 and 4). Panel 5 of Figure 29C reveals that, in summary, 70% of CMs in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice had ruptured / distorted NEs or malformed nuclei, compared with less than 1% of CM nuclei in LmnaFlx / Flx:mcmSun1- / -+Tmx mice. [Figure 29D] (Figure 29D) Echocardiogram analysis was performed on TMX-treated and control mice after Tmx induction. Echocardiograms (ECGs) performed 28 days after Tmx injection on 3- to 5-month-old mice (Panel 1). ECGs performed before and after Cre induction revealed a progressive deterioration of cardiac contractility in LmnaFlx / Flx:mcmSun1+ / ++Tmx mice (solid black line) compared with LmnaFlx / Flx:mcmSun1- / -+Tmx mice (Panels 2-4). Loss of SUN1 preserved EF (Panel 2), FS (Panel 3), and longitudinal global strain (GLS, Panel 4) in LmnaFlx / Flx:mcmSun1- / -+Tmx mice compared with LmnaFlx / Flx:mcmSun1+ / -+Tmx mice. [Figure 30]Figure 30A is a Kaplan-Meier graph showing the effect of SUN1 deletion on cardiac pathology induced by a missense mutation (N195K) in the Lmna gene. Absence of Sun1 significantly increased the survival time of LmnaN195K / Flx:mcmSun1- / -+Tmx mice compared with LmnaN195K / Flx:mcmSun1+ / ++Tmx mice (*P=0.0101; log-rank test). Mice with only one copy of the N195K mutation (LmnaN195K / -:mcmSun1+ / ++Tmx) had a median survival time of 47 days, approximately half the survival time of mice homozygous for two copies of the N195K allele. Figure 30B shows the effect of SUN1 deletion on cardiac function induced by a missense mutation (N195K) in the Lmna gene. Echocardiograms (ECGs) performed before and after Cre induction revealed a progressive deterioration of cardiac contractility in LmnaN195K / -:mcmSun1+ / ++Tmx mice over time compared with LmnaN195K / -:mcmSun1- / -+Tmx mice. ECG images were recorded 28 days after Tmx injection (left-hand panel). Loss of SUN1 preserved EF, FS, and GLS in LmnaN195K / Flx:mcmSun1- / -+Tmx mice compared with LmnaN195K / Flx:mcmSun1+ / ++Tmx mice (lower three right-hand panel). [Figure 31A] Figures 31A-G show that AAV-transduced LmnaFlxx / Flx:mcm+Tmx mice expressing DNSun1 exhibit improved cardiac function and increased lifespan. (Figure 31A) Protocol for AAV-mediated transduction of DN-Sun1 miniprotein into LmnaFlxx / Flx:mcm+Tmx mice. A single Tmx (IP) injection is performed on postnatal day 14 to induce Lmna deletion. Then, AAV9-DNSun1 or AAV9-GFP viral particles are injected into the thoracic cavity on postnatal day 15. The experimental endpoint was set at 100 days after Tmx. [Figure 31B](Figure 31B) The DNSun1 miniprotein competes with endogenous Sun1 for binding to the KASH domain of Nesprin (which is Nesprin1 in CM). The miniprotein competes with endogenous SUN1 for binding to the KASH domain of Nesprin. Because the DNSun1 miniprotein is not anchored in the INM, it effectively displaces endogenous SUN proteins from binding to the KASH domain, thus disrupting the LINC. [Figure 31C] (Figure 31C) The presence of the recombinant Lmna gene after Tmx injection was confirmed by PCR in heart tissue (upper panel). Strong expression of both AAV9-DNSun1 and AAV9-GFP proteins (dosage: 5x10^10 vg / g mouse) was detected in extracts from whole hearts 99 days after AAV injection (lower panel). [Figure 31D] (Fig. 31D) CM derived from human iPS cells were transduced with DNSun1 using AVV-DJ as a vector. In CM expressing high levels of DNSun1, as indicated by gray arrows, Nesprin1 localization to the NE is reduced or absent. Nesprin1 localization to the NE is maintained in CM that do not express or express low levels of AVV-DJ-DNSun1 (white arrowhead). [Figure 31E] (Figure 31E) LmnaFlx / Flx:mcm+Tmx+AAV9-GFP mice lived an average of 34.5 days after Tmx induction, whereas LmnaFlx / Flx:mcm+Tmx mice injected with AA9-DNSun1 (5 x 10^10 vg / g / mouse) lived significantly longer (**P = 0.0038, log-rank test) until at least 100 days after Tmx, at which point they were sacrificed for analysis. This data set was derived from that shown in Figure 20 and adjusted by excluding female mice and mice treated with different doses of virus. Figure E(i) represents male mice, and Figure E(ii) represents female mice. [Figure 31F](FIG. 31F) At 35 days after Tmx, extensive fibrosis (blue in the original image, gray here) and ventricular enlargement were detected in LmnaFlx / Flx:mcm+Tmx+AAV9-GFP hearts compared with LmnaFlx / Flx:mcm+Tmx+AAV9-DNSun1 hearts. [Figure 31G] (Figure 31G) ECG analysis confirmed that LmnaFlx / Flx:mcm+Tmx+AAV9-DNSun1 hearts had better cardiac function compared with LmnaFlx / Flx:mcm+Tmx+AAV9-GFP hearts 35 days after Tmx injection. [Figure 32] Figure 32A shows a model of how disrupting LINCs by disrupting Sun1 protects cardiomyocytes from contraction-induced stress. Cardiomyocyte nuclei expressing LmnaA / C can withstand mechanical and tensile stress transmitted from the cytoplasm to the NE via the LINC complex. Figure 32B shows a model of how disrupting LINCs by disrupting Sun1 protects cardiomyocytes from contraction-induced stress. Loss of the Lmna gene or introduction of a mutation within the Lmna gene results in the loss and / or incorrect assembly of the nuclear lamina, weakening the lamina / NE. Weakened nuclei are damaged by tension / stress exerted by the contracting sarcomeres of cardiomyocytes via the LINC complex. Figure 32C shows a model of how disrupting LINCs by disrupting Sun1 protects cardiomyocytes from contraction-induced stress. In the absence of SUN1, untethered LINC complexes exert less tension on cardiomyocyte nuclei, allowing Lmna mutant cardiomyocytes to survive. Figure 32D shows a model for how disrupting LINCs by disrupting Sun1 protects cardiomyocytes from contraction-induced stress. By disrupting the binding of SUN1 to the KASH domain by expressing DNSun1, the untethered LINC complex exerts less tension on the cardiomyocyte nucleus, allowing Lmna mutant cardiomyocytes to survive. [Figure 33]Figure 1 shows the structure of the LmnaFlx / Flx conditional allele. Primer positions for identifying the genotype of the Lmna gene both before and after Cre recombination are shown for the LmnaFlx allele (Flox), the Lmna deletion allele (Δ), and the wild-type allele [AS Wang et al., Differentiation 89:11-21 (2015)]. [Figure 34] Figure 1 shows the recombinant AAV9-DNSun1 and AAV9-GFP miniproteins. DN-Sun1 contains the SUN domain, HA tag, signal sequence (SS, for targeting the protein to the ER), and KDEL (an ER retention signal) [M. Crisp et al., J Cell Biol. 172:41-53 (2006)]. AAV9-GFP contains the SS and KDEL sequences. GFP was used instead of Sun1L-KDEL as a control. [Figure 35] This is a photomicrograph showing cardiomyocyte-specific expression of Cre recombinase after Tmx injection. LmnaFlx / Flx:mcm mice were crossed with mT / mG (JAX:Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J) reporter mice. In the absence of Cre, RFP is expressed. When Cre is induced, GFP is expressed. Only CMs of LmnaFlx / Flx:mcm mice express GFP upon TMX injection. Heart tissue was analyzed 7 days after Tmx injection. [Figure 36] Figure 1 shows that loss of Sun2 does not rescue loss of Lmna. Loss of Sun2 does not extend the survival of LmnaΔ / ΔSun2− / − mice. [Figure 37A] Figures 37A-37E show the phenotypes of LmnaFlx / FlxmcmSun1+ / + and LmnaFlx / FlxmcmSun1- / - hearts 12-14 months after Tmx injection. (Figure 37A) Histological analysis of senescent LmnaFlx / Flx / :mcmSun1+ / + hearts 12-14 months after Tmx injection reveals no significant morphological changes, e.g., LV enlargement, compared to controls. [Figure 37B](Figure 37B) Histological analysis of senescent LmnaFlx / Flx / :mcmSun1+ / + hearts 12-14 months after Tmx injection revealing no significant morphological changes, e.g., fibrotic changes, compared to controls. [Figure 37C] (Figure 37C) PCR analysis confirmed the sustained deletion of the Lmna gene. [Figure 37D] (FIG. 37D) Protein quantification revealed a significant decrease in LMNA levels in LmnaFlx / Flx:mcmSun1− / −+Tmx hearts 14 months after TMX. [Figure 37E] (FIG. 37E) Echocardiograms from aged mice (left-hand panel) showed decreased EF and FS (right-hand panel) in both LmnaFlx / Flx:mcmSun1+ / ++CTL aged mice and LmnaFlx / Flx:mcmSun1− / −+Tmx aged mice. [Figure 38] (A) AAV9-DNSun1 rescue is dependent on the dosage of injected viral particles. Survival of LmnaFlx / Flx:mcm+TMX mice is dependent on the AAV9-DNSun1 dosage, with lower concentrations resulting in shorter survival. Each point represents a mouse, and the horizontal line indicates the mean. [Figure 39A] Figures 39A-39C show the levels of lamin A / C and the expression of AAV-expressed proteins after Tmx induction. (Figure 39A) Lamin A / C levels were significantly reduced after Tmx induction, and the presence of either AAV9-DNSun1 or AAV9-GFP proteins did not alter LMNA protein levels (quantification of lamin A / C immunofluorescence intensity). The amounts of lamin A / C, DNSun1, and GFP proteins in whole hearts were also quantified by Western analysis (bottom three graphs). (Analysis performed 35 days after Tmx.) [Figure 39B] (Figure 39B) The expression of both DNSun1 and GFP proteins depended on the concentration of injected viral particles. [Figure 39C](Figure 39C) Immunofluorescence revealed that the majority of CMs were successfully infected and expressed GFP with 5x10^10 vg / g AAV9-GFP (left image) compared with infection with a ten-fold lower concentration of viral particles (5x10^9 AAV9-GFP, right image). [Figure 40A] Figures 40A-40C show that CRISPR targeting of the Sun1 SUN domain results in loss of Sun1 protein. Clustal alignment of the Sun1 DNA sequence (Figure 40A) (SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, respectively) and amino acid sequence (Figure 40B) (SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, respectively) flanking the CRISPR-induced mutations in wild-type Sun1, Sun1 with a 4-bp insertion (Sun1_plus4), and Sun1 with a 7-bp deletion (Sun1_del7). Numbering is for the Sun1 coding sequence (A) and the Sun1 protein sequence (B). Bold text in (B) indicates the SUN domain. (Figure 40C) Immunofluorescence staining of adult mouse fibroblasts derived from wild-type and Sun1 mutant mice. Sun1 expression is lost in mutant mice, while Sun2 and Nesprin-1 expression is similar in all three genotypes. Scale bar = 10 μm. [Figure 40B]Figures 40A-40C show that CRISPR targeting of the Sun1 SUN domain results in loss of Sun1 protein. Clustal alignment of the Sun1 DNA sequence (Figure 40A) (SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, respectively) and amino acid sequence (Figure 40B) (SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, respectively) flanking the CRISPR-induced mutations in wild-type Sun1, Sun1 with a 4-bp insertion (Sun1_plus4), and Sun1 with a 7-bp deletion (Sun1_del7). Numbering is for the Sun1 coding sequence (A) and the Sun1 protein sequence (B). Bold text in (B) indicates the SUN domain. (Figure 40C) Immunofluorescence staining of adult mouse fibroblasts derived from wild-type and Sun1 mutant mice. Sun1 expression is lost in mutant mice, while Sun2 and Nesprin-1 expression is similar in all three genotypes. Scale bar = 10 μm. [Figure 40C] Figures 40A-40C show that CRISPR targeting of the Sun1 SUN domain results in loss of Sun1 protein. Clustal alignment of the Sun1 DNA sequence (Figure 40A) (SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, respectively) and amino acid sequence (Figure 40B) (SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, respectively) flanking the CRISPR-induced mutations in wild-type Sun1, Sun1 with a 4-bp insertion (Sun1_plus4), and Sun1 with a 7-bp deletion (Sun1_del7). Numbering is for the Sun1 coding sequence (A) and the Sun1 protein sequence (B). Bold text in (B) indicates the SUN domain. (Figure 40C) Immunofluorescence staining of adult mouse fibroblasts derived from wild-type and Sun1 mutant mice. Sun1 expression is lost in mutant mice, while Sun2 and Nesprin-1 expression is similar in all three genotypes. Scale bar = 10 μm. [Figure 41-1]Figures 41A-41D show that targeting the Syne1 C-terminus with CRISPR results in the expression of mutant Nesprin-1 protein. (Figures 41A, 41B) Clustal alignment of wild-type Nesprin-1 DNA (SEQ ID NO: 75) and Nesprin-1C'TΔ8 (Nesprin1_CTdel8) (SEQ ID NO: 76) (Figure 41A) and the amino acid sequences flanking the CRISPR-induced mutations in wild-type Nesprin-1 and Nesprin-1C'TΔ8 (Nesprin1_CTdel8) (SEQ ID NO: 77, SEQ ID NO: 78, respectively) (Figure 41B). Bold TGA indicates the stop codon of the Syne1 / Nesprin-1 gene. (Figures 41C, 41D) Immunoblots of Nesprin-1 from Syne1 / Nesprin-1 wild-type and Syne1 / Nesprin-1C'TΔ8 mutant cardiac tissue. [Figure 41-2] Figures 41A-41D show that targeting the Syne1 C-terminus with CRISPR results in the expression of mutant Nesprin-1 protein. (Figures 41A, 41B) Clustal alignment of wild-type Nesprin-1 DNA (SEQ ID NO: 75) and Nesprin-1C'TΔ8 (Nesprin1_CTdel8) (SEQ ID NO: 76) (Figure 41A) and the amino acid sequences flanking the CRISPR-induced mutations in wild-type Nesprin-1 and Nesprin-1C'TΔ8 (Nesprin1_CTdel8) (SEQ ID NO: 77, SEQ ID NO: 78, respectively) (Figure 41B). Bold TGA indicates the stop codon of the Syne1 / Nesprin-1 gene. (Figures 41C, 41D) Immunoblots of Nesprin-1 from Syne1 / Nesprin-1 wild-type and Syne1 / Nesprin-1C'TΔ8 mutant cardiac tissue. [Figure 42]Figures 42A-42B are photomicrographs showing that CRISPR-induced Syne1 mutation results in mislocalized "KASH-free" Nesprin-1 protein. Immunofluorescence staining of mouse adult fibroblasts (Figure 42A) and primary myotubes (Figure 42B) derived from wild-type (WT) and Syne1C'TΔ8 mutant mice. Nesprin-1 is mislocalized from the nuclear envelope in mutant samples. Merged images show Nesprin-1 and DNA staining. Scale bar = 10 μm. [Figure 43] Figures 43A-43C are photomicrographs showing that Syne1 mutations do not disrupt the localization of certain nuclear envelope proteins. Immunofluorescence staining of mouse primary myotubes from wild-type (WT) and Syne1C'TΔ8 mutant mice shows that Sun1 (Figure 43A), Sun2 and emerin (Figure 43B), and lamin A / C (Figure 43A) normally localize to the nuclear envelope. Merged images show protein and DNA staining. Arrows indicate examples of normally localized nuclear envelope proteins. Scale bar = 10 μm. [Figure 44] Figures 44A-44C are photomicrographs showing that Syne1 mutations disrupt the localization of nuclear envelope-localized centrosomal proteins. (Figures 44A-44C) Immunofluorescence staining of mouse primary myotubes derived from wild-type (WT) and Syne1C'TΔ8 mutant mice. Pcm1, which normally localizes to the nuclear envelope in myotubes, is shown to be displaced from the nuclear envelope in Syne1C'TΔ8 mutant myotubes. MF20 is an antibody directed against myosin heavy chain, a myotube marker. Merged images show protein and DNA staining. Arrows indicate typical nuclear envelope staining for these centrosomal proteins. Scale bar = 10 μm. [Figure 45] Figures 45A-C show that Syne1 mutations do not affect mouse phenotype. (AB) Representative images of 12-week-old male (A) and female (B) mice. (C) Body weights of male and female wild-type (WT) and Syne1C'TΔ8 mutant mice over a 6-week period. [Figure 46]Figures 46A-46C show that Syne2 constructs and Syne1 / Syne2 double mutant mice experience perinatal lethality. (Figure 46A) Design of the IRES-βgal PGK-Neo targeting construct to generate Syne2 mutations. (Figure 46B) Immunofluorescence staining of adult mouse fibroblasts from wild-type (WT) and Syne2 mutant mice showing loss of Nesprin-2. (Figure 46C) Images of newborn pups. The top row shows healthy, pink-appearing mice with at least one wild-type Syne1 or Syne2 allele. The bottom row shows cyanotic, double-mutant Syne1C'TΔ8 / C'TΔ8:syne2- / - pups that appear blue and die at birth. [Figure 47] Figure 1. Kaplan-Meier graph showing that Syne2 mutation does not ameliorate Lmna pathology. Figure 2. Kaplan-Meier survival curve showing that LmnaΔ / Δ mice die within 3 weeks of birth, regardless of Syne2 mutation status (wild-type, heterozygous, or mutant). [Figure 48] Figures 48A and 48B. Schematic of the AAV viral capsid and its genomic payload (derived from Figures 1B and 1C in Lipinski et al., Prog Retin Eye Res. (2013) 32:22-47). (Figure 48A) The 20 nm icosahedral capsid of the AAV virion containing the single-stranded DNA AAV genome. (Figure 48B) The genome can be engineered to contain an expression cassette with a maximum size of 4.7 kb bounded by inverted terminal repeats (ITRs). The transgene minimally includes a promoter, transgene, and poly(A) tail. [Figure 49]Figures 49A and 49B. Loss of Sun1 rescues the lethality of a skeletal muscle-specific knockout of Lmna. (Figure 49A) Mice carrying a loxP-sequenced allele of Lmna and a transgene containing myosin light chain regulatory sequences driving Cre recombinase (LmnaFlx / Flx;MLC_Cre, N=21) survive an average of 18 days after birth. However, (Figure 49B) the absence of one copy of Sun1 in these mice extends their survival from 18 days to approximately 24 days (LmnaFlx / Flx;MLC_Cre;Sun1+ / -, N=10), and loss of both copies of Sun1 (LmnaFlx / Flx;MLC_Cre;Sun1- / -, N=8) doubles their survival from 18 days to 35 days. [Figure 50] Figure 50. Mortality of progerin-expressing mice is rescued by loss of Sun1. Mice homozygous for the Lmna-G609G progerin splice variant allele (LmnaG609G / G609G) have a median survival of 116 days, which increases to 152 days after loss of one copy of Sun1 (LmnaG609G-CR / G609G-CR; Sun1+ / -) and to 174 days after loss of both copies of Sun1 (LmnaG609G-CR / G609G-CR; Sun1- / -). Mice heterozygous for the Lmna-G609G progerin splice variant allele (LmnaG609G-CR / +) live for a median of 290 days, and loss of Sun1 (LmnaG609G-CR / +; Sun1- / -) extends survival to more than one year. The total number of mice (N) and the number of mice of each sex (M-male, F-female) for the four genotypes are shown below the figure. [Figure 51A] Figures 51A to 51C. Loss of Sun1 reduces atherosclerotic lesion area in the aortic arch. To assess atherosclerosis in mice with Sun1 deletion, wild-type (Sun1+ / +) and Sun1- / - mice were generated on an atherogenic Ldlr- / - background. Mice were fed a Western diet for 15 weeks. (Figure 51A) Representative images of Oil Red O-stained atherosclerotic lesions in the aortic arch of Sun1+ / +;Ldlr- / - and Sun1- / -;Ldlr- / - mice. [Figure 51B] (Figure 51B) A significant reduction in lesion area was observed in quantification of the area occupied by atherosclerotic lesions, shown as a percentage of total surface area, and (Figure 51C) as actual lesion area in square microns. N=8-9 each. [Figure 51C] (Figure 51B) A significant reduction in lesion area was observed in quantification of the area occupied by atherosclerotic lesions, shown as a percentage of total surface area, and (Figure 51C) as actual lesion area in square microns. N=8-9 each. [Figure 52A] Figures 52A to 52E. Loss of Sun1 significantly reduces atherosclerotic lesions in the aortic sinus. Either wild-type (Sun1+ / +; Ldlr- / -) or atherogenic Ldlr- / - mice mutant for Sun1 (Sun1+ / +; Ldlr- / -) were fed a Western diet for 15 weeks. (Figure 52A) Representative image of hematoxylin, phloxine, and saffron (HPS) staining of the aortic sinus. [Figure 52B] (Figure 52B) Significantly reduced atherosclerotic lesion area and (Figure 52C) unchanged total lesion numbers, (Figure 52D) a significant decrease in the number of severe lesions as classified by the American Heart Association criteria, and (Figure 52E) a significant increase in the number of mild lesions were observed in the absence of Sun1 in Sun1- / -;Ldlr- / - mice. [Figure 52C] (Figure 52B) Significantly reduced atherosclerotic lesion area and (Figure 52C) unchanged total lesion numbers, (Figure 52D) a significant decrease in the number of severe lesions as classified by the American Heart Association criteria, and (Figure 52E) a significant increase in the number of mild lesions were observed in the absence of Sun1 in Sun1- / -;Ldlr- / - mice. [Figure 52D](Figure 52B) Significantly reduced atherosclerotic lesion area and (Figure 52C) unchanged total lesion numbers, (Figure 52D) a significant decrease in the number of severe lesions as classified by the American Heart Association criteria, and (Figure 52E) a significant increase in the number of mild lesions were observed in the absence of Sun1 in Sun1- / -;Ldlr- / - mice. [Figure 52E] (Figure 52B) Significantly reduced atherosclerotic lesion area and (Figure 52C) unchanged total lesion numbers, (Figure 52D) a significant decrease in the number of severe lesions as classified by the American Heart Association criteria, and (Figure 52E) a significant increase in the number of mild lesions were observed in the absence of Sun1 in Sun1- / -;Ldlr- / - mice. [Figure 53] Figures 53A and 53B. Loss of Sun1 significantly reduces the lesional macrophage area in the aortic sinus. Mice mutant for the low-density lipoprotein receptor gene and either wild-type (Sun1+ / +;Ldlr- / -) or mutant for Sun1 (Sun1+ / +;Ldlr- / -) were fed a Western diet for 15 weeks. (Figure 53A) Representative image of aortic sinus stained with an antibody to the macrophage marker Mac-3, and (Figure 53B) the significantly reduced macrophage-positive area in Sun1- / -;Ldlr- / - mice. [Figure 54A] Figures 54A to 54D. Loss of Sun1 in a model of rapidly progressing atherosclerosis does not affect body weight or lipid levels. Mice mutant for the low-density lipoprotein receptor gene, and either wild-type (Sun1+ / +; Ldlr- / -) or mutant for Sun1 (Sun1- / -; Ldlr- / -), were fed a Western diet for 15 weeks. (Figure 54A) No change in mouse body weight was observed. [Figure 54B] (FIG. 54B) Plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol (FIG. 54C), and non-HDL cholesterol (FIG. 54D) were unchanged in Sun1 − / − ;Ldlr − / − mice. [Figure 54C](FIG. 54B) Plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol (FIG. 54C), and non-HDL cholesterol (FIG. 54D) were unchanged in Sun1 − / − ;Ldlr − / − mice. [Figure 54D] (FIG. 54B) Plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol (FIG. 54C), and non-HDL cholesterol (FIG. 54D) were unchanged in Sun1 − / − ;Ldlr − / − mice. [Figure 55A] Figures 55A to 55F. AAV9-mediated delivery of a human Sun1 dominant-negative construct improves cardiac function and survival in the LmnaFlx / FlxMcm mouse model of DCM. (Figure 55A) Schematic diagram of an AAV9 construct (AAV9-huSUN1DN) encoding dominant-negative human Sun1. The dominant-negative SUN1 sequence encompasses a portion of the luminal domain, including the Sun domain. A MYC-tag sequence is fused to the NH2-terminus. ITR = AAV2 inverted terminal repeat; cTNT = chicken cardiac troponin promoter; intron = β-globin / IgG chimeric intron; signal sequence = 1-25 aa of human serum albumin (Uniprot P02768 signal peptide + propeptide); Myc = Myc epitope tag; SUN1DN = 1046-2404 nt of NM 001130965; KDEL = Golgi-to-ER retrieval sequence; RGB pA = rabbit globin poly(A) tail. [Figure 55B] (Figure 55B) Schematic of the experimental setup. Tamoxifen (TMX) was injected intraperitoneally on postnatal day 14, followed by retro-orbital injection of AAV9 huSUN1DN or AAV9 GFP (AAV) as a control on postnatal day 15. The predicted survival time of LmnaFlx / FlxMcm + TMX injected with AAV9 GFP is 33 days after TMX injection. The endpoint of this study is the day of death (DOD) of LmnaFlx / FlxMcm animals. [Figure 55C](Figure 55C) AAV9 huSUN1DN (S1DN) transduction extends the survival of LmnaFlx / FlxMcm+TMX animals. LmnaFlx / FlxMcm+TMX+huSUN1DN mice survive an average of 66 days after TMX injection, whereas LmnaFlx / FlxMcm+TMX+GFP mice have a shorter survival time (36 days) (P<0.0001). [Figure 55D] (Figures 55D to 55F) Cardiac function after transduction with AAV9 huSUN1DN is improved in LmnaFlx / FlxMcm+TMX animals. Echocardiographic analysis showed improved fractional shortening (FS; Figure 55D), longitudinal global strain (GLS; Figure 55E), and ejection fraction (EF; Figure 55F) in LmnaFlx / FlxMcm+TMX+huSUN1DN animals compared with LmnaFlx / FlxMcm+TMX+GFP control animals (day 28: FS P<0.002; GLS P<0.0006; EF P<0.0001). [Figure 55E] (Figures 55D to 55F) Cardiac function after transduction with AAV9 huSUN1DN is improved in LmnaFlx / FlxMcm+TMX animals. Echocardiographic analysis showed improved fractional shortening (FS; Figure 55D), longitudinal global strain (GLS; Figure 55E), and ejection fraction (EF; Figure 55F) in LmnaFlx / FlxMcm+TMX+huSUN1DN animals compared with LmnaFlx / FlxMcm+TMX+GFP control animals (day 28: FS P<0.002; GLS P<0.0006; EF P<0.0001). [Figure 55F] (Figures 55D to 55F) Cardiac function after transduction with AAV9 huSUN1DN is improved in LmnaFlx / FlxMcm+TMX animals. Echocardiographic analysis showed improved fractional shortening (FS; Figure 55D), longitudinal global strain (GLS; Figure 55E), and ejection fraction (EF; Figure 55F) in LmnaFlx / FlxMcm+TMX+huSUN1DN animals compared with LmnaFlx / FlxMcm+TMX+GFP control animals (day 28: FS P<0.002; GLS P<0.0006; EF P<0.0001). [Figure 56A]Figures 56A to 56D. AAV9-mediated delivery of a human Sun1 dominant-negative construct improves cardiac function and survival in a dose-dependent manner in the LmnaFlx / FlxMcm mouse model of DCM. (Figure 56A) Transduction of a high dose (5x10^10 viral genomes / g body weight) of AAV9 huSUN1DN further extends the survival of LmnaFlx / FlxMcm+TMX animals. LmnaFlx / flxMcm+TMX mice injected with a high dose (5x10^1 viral genomes / g body weight) of huSUN1DN survived an average of 205 days after TMX injection, whereas LmnaFlx / FlxMcm+TMX mice injected with a standard dose (2x10^1 viral genomes / g body weight) of huSUN1DN had a shorter survival time (66 days) (P<0.0001). [Figure 56B] (Figures 56B to 56D) Cardiac function is improved in a dose-dependent manner. Echocardiographic analysis showed improved fractional shortening (FS; Figure 56B), longitudinal global strain (GLS; Figure 56C), and ejection fraction (EF; Figure 56D) in LmnaFlx / FlxMcm + TMX + high-dose huSUN1 animals compared with standard-dose transduced animals. [Figure 56C] (Figures 56B to 56D) Cardiac function is improved in a dose-dependent manner. Echocardiographic analysis showed improved fractional shortening (FS; Figure 56B), longitudinal global strain (GLS; Figure 56C), and ejection fraction (EF; Figure 56D) in LmnaFlx / FlxMcm + TMX + high-dose huSUN1 animals compared with standard-dose transduced animals. [Figure 56D] (Figures 56B to 56D) Cardiac function is improved in a dose-dependent manner. Echocardiographic analysis showed improved fractional shortening (FS; Figure 56B), longitudinal global strain (GLS; Figure 56C), and ejection fraction (EF; Figure 56D) in LmnaFlx / FlxMcm + TMX + high-dose huSUN1 animals compared with standard-dose transduced animals. [Example]

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

[0402] Example 1: Materials and Methods Mouse, A * They were maintained at the STAR Biological Resource Centre facility and the NUS Animal Facility in accordance with the guidelines of the Animal Care and Use Committees of each institution. Flx / Flx 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). Δ / Δ In order to obtain mice carrying the loxP sequence, a loxP sequence was introduced into the allele (Lmna Flx / Flx ) was introduced by crossing into mice carrying the Cre recombinase driven by the regulatory sequences of the mouse zona pellucida 3 gene (Zp3; Tg(Zp3-cre)93Knw, JAX stock 003651) [W. de Vries et al., Genesis 26:110-112 (2000)]. Flx / Flx / NIMhc ) to get Lmna Flx / Flx Mice were crossed to mice in which Cre expression was 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 tamoxifen-inducible cardiomyocyte-specific deletion of Lmna (LmnaFlx / Flx:mcm). Flx / FlxWe developed mice (mcm;Tg(Myh6-cre / Esr1)) in which Cre expression is driven by the mouse cardiac-specific alpha-myosin heavy chain promoter (αMHC or alpha-MHC;Myh6) that specifically express tamoxifen-inducible Cre recombinase (MerCreMer) in immature and adult cardiac myocytes. * )1Jmk, JAX stock 005657). The specificity of mcm Cre expression to cardiomyocytes was confirmed by crossing the Cre line to mT / mG reporter mice [MD Muzumdar et al., Genesis 45:593-605 (2007)] (Figure 35). Sun1 - / - The generation of Lmna mice has been previously described [YHChi et al., Development 136:965-973 (2009)]. N195K / N195K Mice have also been described [LC Mounkes et al., Hum Mol Genet 14:2167-2180 (2005)]. - / - Because mice are sterile, Lmna Δ / Δ :Sun1 - / - and Lmna Flx / Flxmcm:Sun1 - / - The mice were the Sun1 Lamin-Cre mouse strains. + / - It was obtained by mating with mice.

[0403] To test for the insertion of the loxP site and the conditional deletion allele, 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 for 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 for 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 Young (14 days old) and adult mice (3-5 months old) received a single injection of 40 mg / kg tamoxifen (Sigma) dissolved in corn oil (Sigma). At various time points after tamoxifen injection, mice were either sacrificed by CO2 euthanasia or anesthetized using a gas mixture of 1.5% isoflurane (BioMac) and 1.5 L O2. Cardiac arrest was induced by injection of 15% KCl, followed by a PBS wash to remove blood. Hearts for paraffin embedding were further washed with 4% paraformaldehyde (PFA), left overnight in 4% PFA, dehydrated in 70% ethanol for at least 24 hours, and embedded in paraffin. Hearts for cryosectioning were embedded in gum tragacanth (Sigma), frozen in liquid N2-cooled isopentane (BDH-AnalaR), cut into 9 μm sections using a cryostat (Leica CM3050), collected onto charged slides, and stored at -20°C for histology and immunofluorescence staining. Hearts for protein and RNA extraction were snap-frozen in liquid N2 and stored for further processing.

[0407] 1.2 Cardiomyocyte isolation Cardiomyocyte isolation was performed according to standard protocols [M. Ackers-Johnson et al., Circulation Research 119:909 (2016)]. Briefly, mice were anesthetized with isoflurane (0.5 L / min 100% O2, 4% isoflurane nebulizer dial). The mouse heart was arrested with 15% KCl, the descending aorta was isolated, and 7 mL of EDTA buffer was injected into the right ventricle to flush the heart. The ascending aorta was clamped using Reynolds forceps, and the whole 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, approximately 1 mm pieces and subjected to gentle trituration. Enzyme activity was inhibited by adding 5 mL of stop buffer. The cell suspension was passed through a 100 μm filter and concentrated by four successive rounds of gravity sedimentation to finally obtain a highly pure myocyte fraction. The myocyte 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 with standard hematoxylin and eosin for cell morphology, Masson's trichrome stain 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 MOM block (Vector Shields) and donkey serum (Sigma-Aldrich), and incubated with primary antibodies overnight at 4°C. Slides were then washed in PBS, incubated with secondary antibodies and Hoechst dye (Sigma-Aldrich) for 60 minutes, washed with PBS, and mounted with Prolong-Gold antifade reagent (Invitrogen). Primary antibodies were: 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 Fluor 488, 568, and 647 (1:250, Invitrogen). For immunofluorescence of isolated cardiomyocytes, myocytes were stained in suspension, gently spun down after 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 Whole hearts and quadriceps muscles were homogenized in RIPA lysis buffer and centrifuged at 13,200 g for 10 minutes at 4°C. Total cell lysates were electrophoresed, transferred to PVDF membranes, and blocked with Odyssey blocking buffer (Li-Cor Biosciences). The membranes were incubated with primary antibodies for 2 hours at room temperature. The membranes were then washed in TBST wash buffer and incubated in Odyssey IRDye secondary antibodies for 1 hour before visualization with an Odyssey infrared imaging system (Li-Cor Biosciences). Primary antibodies used for detection of LMNA / C were rabbit (Cell Signaling), specific for an epitope in the first 50 amino acids of LMNA, Sun1 monoclonal (mouse, 1:500, Burke), and control beta-tubulin (rabbit, 1:1000, Abcam).

[0410] 1.5 Measurement of cardiac papillary muscle force 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, explanted mouse hearts were immediately rinsed with oxygenated ice-cold Krebs-Henseleit solution containing 12 units / mL heparin sodium (EDQM) and 30 mM 2,3-butanedione monoxime (BDM, Sigma) to remove excess blood. The hearts were then transferred to ice-cold Krebs-Henseleit solution in a glass Petri dish under a dissecting microscope equipped with a cooling stage. Cylindrical papillae (200–300 μm in diameter and 1.5–2 mm in length) were excised from the left ventricle. T-shaped aluminum clips with holes were crimped onto both ends of the nipple preparation, and the prepared nipple mass was fixed with pins onto a glass Petri dish containing a layer of PDMS sylgard 184 (Dow Corning). The nipple preparation was immersed overnight in a 2% Triton X-100 solution at 4°C.

[0411] Force measurements were performed as previously described [C. Toepfer et al., J Biol Chem 288:13446–13454 (2013)]. T-shaped aluminum clips on both ends of the nipple preparation were attached to the hooks of a force transducer (AE801, HJK Sensoren+Systeme), and the servomotor of the experimental rig was glued using shellac in ethanol (Sigma) to minimize movement during the experiment. Nipple contraction force was measured at 20°C. Maximum contraction force was measured in an activating solution containing 32 μmol / L free Ca2+ (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 = 150 mmol / L). Data were collected and processed from the force transducer and a DAQ data acquisition device (National Instrument) using customized software programmed with LabVIEW 2013. At least five fibers were tested in each mouse, and at least three mice were tested per 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 prepared as previously described [M. Crisp et al., J Cell Biol. 172:41-53 (2006)]. Briefly, almost the entire luminal domain of Sun1 was tagged with HA at its NH2-terminus (HA-Sun1L). To deliver HA-Sun1L to the ER lumen and PNS as a soluble form, the human serum albumin signal sequence and signal peptidase cleavage site were fused to the NH2-terminus of HA-Sun1L, yielding SS-HA-Sun1L. To prevent its secretion, the KDEL tetrapeptide was fused to the COOH-terminus of SS-HA-Sun1L, forming the final SS-HA-Sun1L-KDEL vector. The HA-Sun1L region was replaced with a 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), ligated into the pENN-AAV-cTnT-PI-eGFP plasmid (a gift from Dr. J. Jian), and digested with Ncol and Kpnl to generate Penn-AAV-cTnT-Sun1DN (Figure 10, sequence number 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). Ligations were performed using isothermal assembly with NEBuilder® HiFi DNA Assembly Master Mix (NEB, E2621L). Primers used to construct the plasmids were ordered from IDT.

[0414] AAV virus was produced according to standard protocols [H. Wakimoto et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc., 2001)]. Materials provided by R. Foo: pAAV2 / 9-transformer plasmid encoding the 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 a QIAGEN Plasmid Maxi Kit. HEK293T cells were transfected with a viral combination of pAAV2 / 9, pAdDeltaF6, and either DN-Sun1 or GFP plasmids. Cells were harvested and the virus was purified by iodixanol gradient ultracentrifugation.

[0415] The following schedule was used for infection of mouse hearts. Mice were genotyped on postnatal day 10. Then, on postnatal day 14, mice received a single IP injection of Tmx (40 mg / kg mouse body weight), followed by intrathoracic injection of AAV9-DN-Sun1 or AAV9-GFP virus at a concentration of 5 x 10^10 vg / g on postnatal day 15. Adult mice (3-5 months old) received an IP injection of a single dose of Tmx (40 mg / kg mouse body weight), followed by intrathoracic injection of AAV at a concentration of 5 x 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.

[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 provided to the tool to identify suitable target sites. Because off-target mutations can occur during CRISPR / Cas9-mediated targeted mutagenesis in mice, the CRISPR Design Tool experimentally evaluates off-target genome modifications for each gRNA target site, provides computationally predicted off-target sites for each intended target, and allows target sequences to be ranked according to quantitative specificity analysis of the effects of base pair mismatch identity, location, and distribution. 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] 5'-GCACAATAGCCTCGGATGTCG-3' (SEQ ID NO: 33) for Sun1ΔSUN 5'-CCGTTGGTATATCTGAGCAT-3' (SEQ ID NO: 34) for Syne1-stop 5'-GGTTATGGCCGATAGGTGCAT-3' (SEQ ID NO: 35) for tyrosinase 4a These plasmids (pSun1ΔSUN, pSyne1-stop, and pTyrosinase4a) were then sequenced to verify correct insertion of the target sequence. For in vitro transcription, PCR was performed using a general reverse primer (AAAAGCACCGACTCGGTGCC-3', SEQ ID NO: 36) and a gRNA-specific forward primer encoding the T7 promoter sequence as follows to generate appropriate transcription templates:

[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 product was then subjected to agarose gel electrophoresis (1.5% agarose) to confirm successful PCR, gel purified, and used as a template 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. A sample of the purified gRNA was then subjected to agarose gel electrophoresis to confirm quality before injection into zygotes.

[0419] 1.8 Generating mutant mice using CRISPR / Cas9 Three- to four-week-old C57BL / 6N females were superovulated with pregnant mare serum gonadotropin (Calbiochem, 36722, 5 IU / ml). 48 hours later, the females were injected with human chorionic gonadotropin (Sigma, CG10, 5 IU / ml) and mated with C57BL6 males. The following day, fertilized 0.5-dpc embryos were collected from the oviduct. Cas9 mRNA (Sigma, CAS9 mRNA, 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 microinjection system (Nikon). Syne1-stop sgRNA was used to generate Syne1 C'T mutant mice, and Sun1ΔSUN sgRNA was used to generate Sun1ΔSUN mutant mice. 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 implanted 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 in 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 the 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 Sun1 CRISPR mouse are: Forward: 5'-TGACCTTGAGCTGAAACTGC-3' (SEQ ID NO: 42) Reverse: 5'-TCAGAACACTGGCACACACA-3' (SEQ ID NO: 43) It was.

[0424] The genotype of Lmna mutant mice was identified as described in Example 1. To determine the sequence of the CRISPR-induced mutation, 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 Obtaining myoblasts, fibroblasts, and cell cultures for CRISPR / Cas9 studies To isolate myoblasts, limbs were obtained from euthanized mice, and the muscles were dissected from the bone. Tissue digestion was performed by incubating muscle tissue in an enzyme solution consisting of equal volumes of 2.4 U / ml Dispase II (Roche, cat. 04942078001) and 1% Collagenase II (GIBCO® Invitrogen, cat. 17101-015) in a 37°C water bath for 30 minutes, with occasional 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 the cells were resuspended in F10 medium (GIBCO® Invitrogen, cat. 11550043) supplemented with 10 μg / ml bFGF (GIBCO®, cat. PHG0264) and plated onto 100 mm plates. After allowing the mouse adult fibroblasts 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 containing MAFs. To 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 research Whole cell lysates were generated using Lysis-M kit solution (cOmplete, Roche). Cells were washed in ice-cold PBS, lysed with 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 in Lysing Matrix D tubes (MP Biomedicals) and flash-frozen in liquid nitrogen. After flash-freezing, the tubes were either stored at -80°C or used immediately for protein analysis. Protein extraction buffer (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 in distilled water) was added to the tissue, which was then homogenized using a FastPrep™-24 instrument (MP Biomedicals). Samples were then centrifuged at 14,000 g for 10 minutes to remove cellular debris. Protein concentrations were quantified using a bicinchoninic acid (BCA) protein kit (Bio-Rad), after which 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 polyvinylidene fluoride (PVDF) membranes (Millipore) by wet transfer at 20V for 48 hours at 4°C. The membranes were blocked for 1 hour at room temperature in TBS containing 0.1% Tween 20 (TBST) supplemented with 5% dry milk (Anlene). Western blot analysis was performed using primary antibodies diluted in 5% dry milk (diluted in TBST). The membranes were incubated for 2 hours at room temperature or overnight at 4°C. For secondary antibodies, antibodies conjugated to horseradish peroxidase (HRP) (Invitrogen) were used for chemiluminescence imaging. The membranes were incubated with the secondary antibodies for 1 hour at room temperature. For immunoblots visualized by chemiluminescence, the membranes were incubated in ECL substrate (Pierce) for 1 minute, then exposed to chemiluminescence-sensitive film (Thermo Scientific) and subsequently 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. Cells were then rinsed twice in PBS and permeabilized and blocked with 0.1% Triton X and 3% BSA in PBS for 15 minutes at room temperature. Fixed and permeabilized cells were then rinsed three times in PBS. Samples were then incubated with primary antibodies (Table 2) for 2 hours at room temperature or overnight at 4°C. Samples were then washed three times with PBS and subsequently incubated with secondary antibodies (Life Technologies) and DAPI (Life Technologies) for 1 hour at room temperature. After three washes in PBS, cells were mounted with antifade 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 injections were described in Example 1. 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 Syne1 mice C’TΔ8 / C’TΔ8In 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 part of exon 102 and all of exons 103–104. The neomycin cassette was then removed by breeding with Cre recombinase-expressing mice. C’TΔ8 / + or Syne1 C’TΔ8 / C’TΔ8 Mouse Syne2 + / - or Syne2 - / - Mice carrying mutant Syne1 and Syne2 alleles were crossed with mice carrying mutant Syne1 and Syne2 alleles to obtain double mutant mice. Survival curves were plotted using the Kaplan-Meier method.

[0430] 1.15 Human guide RNA sequences Potential guide RNA sequences that disrupt the human SYNE1 KASH domain or 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 postnatal pathology in mice, we developed a conditional Lmna gene that, when recombined by Cre activation, leads to the complete loss of lamin A / C protein. Flx / FlxBy using a mouse strain (Figure 33), the Lmna gene was specifically deleted in different tissues [A.S. Wang et al., Differentiation; research in biological diversity, (2015); I. Solovei et al., Cell 152:584-598 (2013)]. Flx / Flx Lmna mice by crossing them with Zp3-Cre mice Flx / Flx When Lmna was constitutively deleted in all tissues [W. de Vries et al., Genesis 26:110-112 (2000)], the mean postnatal survival time was 17.5 days (Fig. 26A). When the same deletion was induced in the absence of Sun1, Lmna Δ / Δ :Sun1 - / - Mice lived an average of 32.5 days, nearly doubling lifespan (Fig. 26A). Performing the same Lmna deletion on a Sun2 null background resulted in Lmna Δ / Δ We found that loss of Sun1 did not extend the lifespan of mice, and that the lifespan extension was specific to loss of Sun1 (Figure 36). Because A-type lamins are widely expressed in almost all adult tissues, we next hypothesized that loss of Lmna, specifically in cardiomyocytes, would result in loss of Lmna. Δ / Δ We determined the extent to which Sun1 contributes to early postnatal death in mice. Furthermore, we wanted to determine whether loss of Sun1 could increase the lifespan of these mice, which contain Lmna-deficient cardiomyocytes. Flx / Flx These mice were crossed with a constitutive myh6Cre gene [R. Agah et al., J Clin Invest 100:169-179 (1997)] in which Cre expression is constitutive but restricted to cardiomyocytes and initiated during embryogenesis. Δ / Δ The survival time was slightly longer than that of Sun1 mice, averaging 26.5 days after birth (Fig. 26C). - / -When performed against a background mouse model, this resulted in a significant increase in lifespan up to and beyond 6 months of age (Figure 26C). To further define the loss of Lmna and its effects in postnatal / adult cardiomyocytes, Cre was induced by a single injection of tamoxifen (Tmx) [DS Sohal et al., Circ Res 89:20-25 (2001)]. Lmna carrying an inducible cardiomyocyte-specific Cre Tg(Myh6-cre / Esr1) (abbreviated here as mcm) was then cultured. Flx / Flx Mice homozygous for the allele were obtained from this mating. Flx / Flx:mcm The mean survival time of mice after Cre induction was 27 days (Figure 27A). Controls were unaffected by Tmx injection. PCR and immunofluorescence analysis demonstrated that Lmna deletion resulted in the loss of Lmna Flx / Flx:mcm The results confirmed that the recombination was specific to cardiomyocytes, with no detectable recombination occurring in the brain, diaphragm, lung, liver, or skeletal muscle, nor in wild-type control animals (Figure 27B). Flx / Flx:mcm Mice exhibited labored breathing, a ruffled and ungroomed appearance of the fur, increased lethargy, and kyphosis (Figure 27C). Flx / Flx:mcm Immunofluorescence analysis of cardiac sections showed reduced levels of lamin A protein and cardiomyocyte nuclei lacking lamin A expression (Figure 27D). Lamin A protein levels were significantly elevated after Cre induction of Lmna Flx / Flx:mcm In the heart, Lmna was not induced Flx / Flx:mcm and Lmna + / + / mcm The Lmna expression level was 3.5-fold lower than that of the heart (Figure 27E). Flx / Flx:mcmBy sampling mice, we estimated that it took 7–14 days for LMNA protein levels to fall by 50% after Cre induction (data not shown), a rate consistent with studies using siRNA LMNA knockdown in human fibroblasts [A. Buchwalter and M.W. Hetzer, Nature Communications 8:328 (2017); T. Sieprath et al., Nucleus 6:236–246 (2015)], which showed a further decline of 1.3-fold after 48 hours and 4-fold after 10.5 days. Echocardiograms (ECGs) performed 21 days after Cre induction showed that Lmna Flx / Flx:mcm In mice, Lmna Flx / Flx:mcm Compared to controls, the subjects revealed insufficient cardiac contractility (Fig. 28A). There was a significant decrease in ejection fraction (EF%) and fractional shortening (FS%) (P<0.0001) (Fig. 28B). Left ventricular systolic and diastolic internal diameters (LVID) were significantly decreased (Lmna Flx / Flx:mcm expanded compared to the control (Figure 28B). Flx / Flx:mcm Significantly fewer viable (brick-like) cardiomyocytes were detected in Lmna compared to controls. Flx / Flx:mcm +Tmx hearts (Figure 28C). Flx / Flx:mcm We found that cardiomyocytes isolated from +Tmx hearts contained large intracellular vacuoles (Figure 28C). Flx / Flx:mcm Histological analysis of +Tmx hearts revealed infiltration of nucleated cells, and Lmna Flx / Flx:mcm Increased intercellular spaces between cardiomyocytes were evident compared to control hearts (Figure 28D). Flx / Flx:mcm The left ventricular cavity in the +Tmx heart was significantly enlarged, and simultaneously, Lmna Flx / Flx:mcm There was a significantly increased level of fibrosis in Lmna compared with controls (P = 0.0098). Flx / Flx:mcm +Tmx hearts (Figure 28D). Increased numbers of apoptotic cells compared to control hearts were also observed in Lmna Flx / Flx:mcm+Tmx hearts (Figure 28D). However, there was no evidence of detectable widespread DNA damage in cardiomyocytes, as assessed by Rad51, MRE11, H2AX phosphor-Ser, and 53BP1 immunostaining (data not shown).

[0433] Example 3: Deletion of Sun1 ameliorates cardiac pathology induced by Lmna loss Mice carrying the Lmna mutation exhibit a significant increase in lifespan and health in the absence of Sun1 [Chen et al., Cell 149:565-577 (2012)]. As described, inducible deletion of Lmna in cardiomyocytes (Lmna Flx / Flx:mcm +Tmx) resulted in death within one month after Cre induction (Figure 26C). Surprisingly, when the same deletion was induced on a Sun1 null background, mice survived for more than one year after Cre induction (Figure 26C). Flx / Flx:mcm Sun1 - / - Hearts from +Tmx mice were cultured at 3 weeks post-induction in Lmna Flx / Flx:mcm Sun1 + / + We determined the extent to which SUN1 loss ameliorated the pathological changes induced by Lmna loss in cardiomyocytes compared with hearts from +Tmx. Immunofluorescence imaging for lamin A / C identified many elongated and distorted nuclei. In some of these, residual lamin A / C was associated with Lmna loss. Flx / Flx:mcm Sun1 + / + +Tmx hearts migrated to one pole of the nucleus (Panel 1 and inset in Figure 29A). Flx / Flx:mcm Sun1 - / - In +Tmx hearts, even in the absence of lamin A / C staining, many elongated nuclei were present, but these showed little, if any, distortion (yellow arrowhead in panel 3 of Figure 29A). Western analysis of whole hearts revealed that Lmna Flx / Flx:mcm Sun1 - / - + In Tmx cardiac lysate, Lmna Flx / Flx:mcm Sun1 + / + A significant decrease in lamin A / C was observed compared to the control (P=0.0359) (Figure 29A, bottom panel). Flx / Flx:mcmSun1 + / + +Tmx cardiomyocyte nuclei exhibited increased longitudinal length with a segmented appearance, with segments connected by narrow bridges (Figure 29A and C, panel 1, arrowheads). However, in the absence of Sun1, Lmna Flx / Flxmcm Sun1 - / - Cardiomyocyte nuclei showed no abnormalities or compartmentalization (Figure 29C, panels 3 and 4). Flx / Flx:mcm Sun1 - / - For less than 1% of cardiomyocytes from Flx / Flx:mcm Sun1 + / + 70% of the cardiomyocytes in the mice had ruptured or malformed nuclei (Panel 5 of Figure 29C). Flx / Flx:mcm Sun1 + / + Although it was clear in mice, Lmna Flx / Flx:mcm Sun1 - / - +Tmx hearts (Fig. 29B, panels 1 and 2). Flx / Flx:mcm Sun1 + / + The hearts exhibited significantly increased levels of fibrosis compared with controls (P<0.0001), whereas Lmna Flx / Flx:mcm Sun1 - / - There was no significant fibrosis in the heart (FIG. 29B, panels 3-5).

[0434] As a model of the left ventricular muscle's operating mechanism, we measured the force acting on the cardiac papillary muscle. Flx / Flx:mcm :Sun1 + / + + In Tmx papillary muscles, Lmna Flx / Flx:mcm Sun1 + / + The expression of Lmna was significantly reduced by 66% compared to that of Lmna + CTL (P = 0.0028). Flx / Flx:mcm Sun1 - / - +Tmx cardiac papillary force was maintained at a level not significantly different from that of the control (Figure 29B, panel 6).

[0435] Echocardiograms performed before and after Cre induction were performed using Lmna Flx / Flx:mcm Sun1 + / + Lmna caused a progressive deterioration of cardiac contractility in +Tmx mice.Flx / Fl:mcm Sun1 - / - +Tmx mice (Fig. 29D). Flx / Flx:mcm Sun1 - / - In +Tmx mice, EF, FS, and longitudinal global strain (GLS) (GLS is an independent parameter used to assess myocardial contractility and is a better predictor of heart failure) were all significantly reduced. Flx / Flx:mcm Sun1 + / + +Tmx mice.

[0436] Aging Lmna 12-14 months after Tmx injection Flx / Flx:mcm Sun1 - / - PCR analysis of +Tmx hearts confirmed the persistent deletion of the Lmna gene (Figure 37C), and protein quantification demonstrated the presence of Lmna at 12–14 months after TMX. Flx / Flx:mcm Sun1 - / - + revealed a significant decrease in LMNA levels in Tmx hearts (Figure 37D). Flx / Flx:mcm Sun1 - / - Histological analysis of +Tmx hearts revealed no significant increase in fibrosis compared to controls (Figures 37A and B). However, echocardiograms of these aged mice showed no significant increase in fibrosis. Flx / Flx:mcm Sun1 + / + +CTL mice and Lmna Flx / Flxmcm Sun1 - / - +Tmx mice showed decreased EF and FS (Fig. 37E), whereas Lmna Flx / Flx The median survival time of the mice was 13-14 months (Figure 26C; therefore, the reduced contractile function was likely due to aging). Furthermore, these findings demonstrate that loss of Lmna in adult (2-3 month old) cardiomyocytes is sufficient to result in cardiac failure within 3-4 weeks after Cre activation, but that this pathology is remarkably alleviated by deletion of Sun1, and this alleviation persists for 1 year.

[0437] Example 4: Loss of SUN1 extends the lifespan of Lmna missense mutants Because most 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 harbors the N195K missense mutation, which has been 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)). Again, we found that the absence of SUN1 significantly extended the survival time of this mutant mouse line, along with improved cardiac function (Figure 26D). These findings were confirmed by obtaining mice heterozygous for the N195K mutation by introducing a loxP sequence into the WT-Lmna allele, i.e., Lmna N195K / Flx ×Sun1 + / + In these mice, the Tmx-inducible cardiomyocyte Cre allele (Lmna) was introduced. N195K / Flx:mcm +Tmx) results in the deletion of the WT flox Lmna allele, causing cardiomyocytes to become Lmna N195K / - These mice had a mean survival time of less than 50 days and were hemizygous for the Lmna mutation. N195K / N195K The lifespan of Lmna mice was half that of homozygous mice (Figure 30A). N195K / Flx:mcm When the +Tmx mutation was induced on a Sun1-null background, lifespan was significantly extended from less than 50 days to more than 200 days (Figure 30A), revealing that loss of Sun1 was also effective in preventing DCM caused by Lmna missense mutations, specifically in cardiomyocytes.

[0438] Echocardiograms performed before and after Cre induction were performed using Lmna N195K / Flx:mcm Sun1 + / + Lmna causes progressive deterioration of cardiac contractility in mice N195K / Flx:mcm Sun1 - / - This was revealed by comparison with mice (Figure 30B). Loss of SUN1 significantly reduced the expression of LmnaN195K / -:mcm Sun1 - / - In mice, EF, FS, and longitudinal global strain (GLS) were all significantly reduced by Lmna. N195K / -:mcm Sun1 + / + Conserved compared to mice (Figure 30B).

[0439] Example 5: AAV9-mediated transduction and expression of DNSun1 Flx / Flx:mcm Extends survival of +Tmx mice These results demonstrate that genetically ablating SUN1 function or genetically reducing SUN1 levels may have therapeutic value in treating DCM. We next examined whether this was due to complete elimination of SUN1 function, overcoming toxic SUN1 overload without altering SUN1 levels and by specifically disrupting SUN1's LINC complex-associated role in linking the nucleus to cytoskeletal components by tethering KASH domain proteins in the ONM. To distinguish between these two possibilities, we used adenovirus-associated virus (AAV) to specifically transduce and express a dominant-negative SUN1 minigene [M. Crisp et al., J Cell Biol 172:41-53 (2006)] in cardiomyocytes, whose protein product can compete for both SUN1-KASH and SUN2-KASH binding in cardiomyocyte nuclear cisternae. A schematic diagram of the AAV viral capsid and its genomic payload is shown in Figure 48A and Figure 49B (taken from Figure 1B and Figure 1C in Lipinski et al., Prog Retin Eye Res. (2013) 32:22-47).

[0440] The Sun1 gene was tagged at its N-terminus with an HA (HA-Sun1L) epitope in a region corresponding to the entire luminal domain. To localize the resulting protein product to the endoplasmic reticulum (ER) and nuclear cisternae (between the INM and ONM—PNS), the signal sequence and signal peptidase cleavage site of human serum albumin were fused to the N-terminus of HA-Sun1L to obtain SS-HA-Sun1L. To prevent secretion of the miniprotein, the KDEL tetrapeptide was linked to the C-terminus of SS-HA-Sun1L to form SS-HA-Sun1L-KDEL (Figure 34). The signal sequence may ensure that HA-Sun1KDEL accumulates within the continuous perinuclear ER and PNS lumen within the cell. The cDNA sequence encoding the minigene was fused to the chicken cardiac troponin promoter (cTnT) to ensure that the minigene was transcribed exclusively in cardiomyocytes [KM Prasad et al., Gene Ther 18:43-52 (2011)]. A diagram of how SS-HA-Sun1L (DN-Sun1) translocates KASH domain proteins from the LINC complex in the PNS to the ER is shown in Figure 15 (third panel) and Figure 31B.

[0441] To verify that DN-Sun1 functioned in cardiomyocytes (CMs), we first transduced human CMs derived from iPS cells using 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 and is used to transduce DN-Sun1 under the transcriptional control of the cTnT promoter in mouse hearts. DN-Sun1 was effective in displacing Nesprin-1 from the nuclear envelope in CMs expressing DN-Sun1, as shown in Figure 31D. Cells expressing high and low levels of DN-Sun1 are indicated by gray and white arrowheads, respectively. High levels of DN-Sun1 expression resulted in the dislocation of Nesprin-1 from the nuclear envelope. This confirmed that DN-Sun1 was effective in disrupting the LINC complex in CMs.

[0442] Using AAV (serotype 9), the DN-Sun1 minigene was transduced and expressed in the hearts of postnatal mice via intrathoracic injection. The procedure is summarized in Figure 31A. All mice were sacrificed for analysis 100 days after Tmx injection. PCR detection of Lmna deletion in the heart confirmed Cre induction by Tmx injection (Figure 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 the AAV9-DNSun1 protein and the AAV9-GFP control protein (injection dose: 5 x 10^10 vg / g mouse) 99 days after AAV injection (Figure 31C). The expression levels of both proteins depended on the dose of injected viral particles (Figure 38). Expression of either the AAV9-DNSun1 protein or the AAV9-GFP protein did not affect LMNA protein levels (Figure 39A).

[0443] Immunofluorescence analysis revealed that at 5x10^10 vg / g AAV9-GFP, a greater percentage of cardiomyocytes expressed GFP compared with the levels resulting from a ten-fold lower dose of viral particles (5x10^9 AAV9-GFP) (Figures 39B and 39C).

[0444] Lmna injected with AAV9-GFP control Flx / Flx:mcm +Tmx mice lived an average of 34.5 days after Tmx, whereas Lmna mice injected with AA9-DNSun1 (5×10^10vg / g mouse) Flx / Flx:mcm +Tmx mice lived significantly longer, with the majority surviving at least 100 days post-Tmx before being sacrificed for analysis (P=0.0002). (Figure 20 shows the results for the early time period for male and female mice, and Figure 31E shows the results at 100 days with separate graphs for male and female mice, where mice with different virus injection titers were removed.) Echocardiography analysis showed that Lmna Flx / Flx:mcm +Tmx+AAV9-DNSun1 hearts were Lmna at 35 days after Tmx Flx / Flx:mcmThe Lmna+Tmx+AAV9-GFP hearts functioned better than the Lmna+Tmx+AAV9-GFP hearts (Figure 31G). Flx / Flx:mcm +Tmx+AAV9-DNSun1 mice were alive at 100 days post-induction, but both EF% and FS% were significantly higher than those of the control Lmna Flx / FlxWT +Tmx mice (Fig. 31G). At 35 days after Tmx, increased fibrosis was observed in Lmna mice. Flx / Flx:mcm +Tmx+AAV9-DNSun1 Heart and Lmna Flxx / Flxxmcm +Tmx+AAV9-GFP were detected in both hearts (Fig. 31F), but Lmna Flx / Flx:mcm Fibrosis in the heart with +Tmx+AAV9-DNSun1 was associated with Lmna Flxx / Flxxmcm +Tmx+AAV9-GFP hearts (lower panel in Figure 31F).

[0445] Example 6: Disruption of the LINC complex in mice using CRISPR / Cas9 Mice harboring multiple Lmna mutations, both systemic and cardiac-specific, exhibit significantly increased lifespan and 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 unclear but was speculated to be due to a toxic effect of excess Sun1 in Lmna mutants [Chen et al., Cell 149:565-577 (2012)]. AAV-mediated expression of a dominant-negative LINC complex-disrupting transgene ameliorates 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 excess Sun1, is the molecular driver of Lmna pathology. This is supported by the finding that genetic disruption of the LINC complex through loss of Sun1 and Sun2 in mice [K. Lei et al., Proc Natl Acad Sci USA 106:10207-10212 (2009)] or cardiac-specific disruption of Nesprin-1 and Nesprin-2 [Banerjee et al., PLOS Genet 10(2):e1004114 (2014)] was surprising because it resulted in a variety of pathologies.

[0446] To develop an alternative method for disrupting the LINC complex in vivo, we investigated whether CRISPR / Cas9 genome editing could be used to disrupt the SUN and KASH domains of proteins that make up the LINC complex. Because both the SUN and KASH domains are located at the C-terminus of the respective proteins, we hypothesized that CRISPR guide RNAs targeting the 3' end of genes encoding SUN or KASH domain proteins might result in premature stop codons after CRISPR-induced non-homologous end joining. This could result in truncated proteins with mutated C-terminal SUN or KASH domains. While the truncated proteins could be expressed and membrane-localized, they would be unable to interact with their cognate LINC complex partners. In Example 2, we found that loss of Sun2 did not ameliorate Lmna-associated pathology. Therefore, we chose to target the Sun1 SUN domain using CRISPR because Sun1 is believed to be the dominant SUN domain protein mediating Lmna pathology. Among KASH domain proteins, only Nesprin-1, Nesprin-2, and Nesprin-3 are ubiquitously expressed [HF Horn, Current Topics in Developmental Biology 109:287-321 (2014)]. Nesprin-1 and Nesprin-2 are closely related paralogs with overlapping functions. They interact with the actin and microtubule cytoskeleton, whereas Nesprin-3 appears to interact specifically with intermediate filaments [Kim et al., Biol. Chem. 396:295-310 (2015)]. Because we already had Nesprin-2 and Nesprin-3 mutant mouse lines obtained by conventional gene targeting available in our laboratory, we chose to target the KASH domain of Nesprin-1 using CRISPR to test whether CRISPR / Cas9 could be used in vivo to treat laminopathies.The Sun1 gene and the Syne1 gene, encoding the Nesprin-1 protein, were directly targeted in vivo by microinjecting Cas9 mRNA into C57 / B16 mouse zygotes with either a gRNA targeting the SUN1 domain (5'-GCACAATAGCCTCGGATGTCG-3', SEQ ID NO: 66) or a gRNA targeting the KASH1 domain (5'-CCGTTGGTATATCTGAGCAT-3', SEQ ID NO: 67), followed by transplantation into surrogate mothers. Note that the SUN1 gRNA targets Sun1, which is upstream of the SUN domain, removing it. Co-injection with a gRNA targeting the tyrosinase gene (5'-GGTTATGGCCGATAGGTGCAT-3', SEQ ID NO: 68) resulted in CRISPR-edited offspring with white or mosaic coat color due to tyrosinase disruption. These pups were genotyped to confirm successful gene disruption and used as founder animals to establish Sun1 or Nesprin-1 mutant colonies.

[0447] 6.1 Characterization of mutant mice After Sanger sequencing of the founder animals and F1 progeny, we focused on characterizing the Sun1 mutant alleles with a 7-bp deletion (Sun1_del7 or Sun1Δ7, SEQ ID NO: 71) and 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 alleles were predicted to produce 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 loss of Sun1 protein (Figure 40C), suggesting that the CRISPR-generated indel triggered nonsense-mediated decay of Sun1 mRNA. It is unclear whether the location of the mutation, which was outside the SUN domain rather than inside it, had an effect on mutant gene expression. Because we were unable to obtain Sun1 mutant alleles that produced Sun1 protein lacking the SUN domain and instead essentially obtained Sun1-null animals, we did not further characterize these mutant lines.

[0449] The Syne1 C'TΔ8 allele is predicted to produce a protein (Figure 41B, SEQ ID NO: 78) in which the last 11 amino acids in the wild-type sequence (SEQ ID NO: 77) are mutated, followed by an additional 50 amino acids encoded by an alternative reading frame. 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 appeared less abundant and had 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 novel stop codon downstream, resulting in a higher molecular weight protein. In addition, a band of approximately 1 MDa, likely corresponding to Nesprin-1Giant, was observed in cardiac tissue from both Syne1WT and Syne1C'TΔ8 mice.

[0450] Immunofluorescence analysis of mouse adult fibroblasts (MAFs) derived from 12-week-old mice revealed that Nesprin-1 was mislocalized from the nuclear envelope to the cytoplasm in Syne1C'TΔ8 MAFs (Figure 4A). Similarly, in myotubes, Nesprin-1 redistributed to the cytoplasm in Syne1C'TΔ8 myotubes compared to Syne1WT myotubes (Figure 4B). Other LINC complex and NE proteins, such as SUN1, SUN2, emerin, and lamin A, remained localized to the NE (Figure 4C). Consistent with previous reports [Gimpel et al., Curr. Biol. 27:2999-3009.e9.(2017)], disruption of Nesprin-1 in myotubes resulted in the mislocalization of the centrosomal proteins PCM1, Pcnt, and Akap450 from the myotube nuclear envelope (Figure 4D). Mislocalization of Nesprin-1 from the nuclear envelope is consistent 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 would normally restrict Nesprin-1 to the nuclear envelope. Because the transmembrane domain is not disrupted, Nesprin-1 is likely mislocalized to the endoplasmic reticulum (ER) in the C'TΔ8 mutant because the ER and nuclear cisternae form a continuous membrane system.

[0451] Similar to one previously reported Nesprin-1 mouse model [Zhang et al., Development 134(5):901-908 (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)], the disrupted KASH domain of Nesprin-1 did not result in any obvious phenotypic differences between Syne1 wild-type (WT) and Syne1C'TΔ8 mutants (Figure 45A-B). Both male and female homozygous mutants were fertile, and there was no significant difference in body weight between Syne1WT and Syne1C'TΔ8 mice (Figure 45C). Syne1C'TΔ8 mice also did not exhibit any growth retardation or overt muscular dystrophy, and did not show any difficulties with movement or grooming, which could be indicators of muscle loss.

[0452] To explore the role of other KASH domain proteins in Lmna pathology, we generated mouse mutants for Syne2, which encodes Nesprin-2, by conventional gene targeting (Figure 46A). To characterize the mutations, we performed immunofluorescence microscopy of tail tip fibroblasts. Syne2 - / - Homozygous mutant fibroblasts expressed little or no Nesprin-2 (Fig. 46B). Consistent with previous findings [Zhang et al., Development 134(5):901-8(2007)], Syne2 - / - The mice were apparently normal and had neither growth retardation nor infertility, but the Nesprin-1 / 2 double mutant mice (Syne1 C’TΔ8 / C’TΔ8 :Syne2 - / - ) was perinatally lethal (Figure 46C).

[0453] 6.2 Disruption of the Nesprin-1 KASH domain ameliorates Lmna pathology Even if Nesprin-1 is still expressed, the Nesprin-1-containing LINC complex is not involved in the expression of Syne1. C’TΔ8 / C’TΔ8Because AAV-mediated disruption of the LINC complex in vivo using dominant-negative SUN1 rescues Lmna pathology (Example 5), we reasoned that the "KASH-free" Nesprin-1 mutant allele we generated might also rescue Lmna pathology. To test this hypothesis, we created an Lmna null (Lmna Δ / Δ Mice heterozygous for the Lmna allele (Example 1) were crossed with Syne1C'TΔ8 mice to produce Lmna Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 We obtained double mutant mice. Δ / Δ The mice lived for 15–17 days, but Lmna Δ / Δ :Syne1 C’TΔ8 / C’TΔ8 The double mutant mice lived for up to 42 days (Figure 24). C’TΔ8 Lmna null mice heterozygous for the allele did not experience any survival benefit. - / - Lmna on a homozygous mutant background Δ / Δ Mice also did not experience an increased survival time (Figure 47), indicating that Lmna pathology is primarily mediated by the Nesprin-1 / Sun1 LINC complex. Syne1 in mice with cardiac-specific loss of Lmna C’TΔ8 / C’TΔ8 To examine the effect of the allele, we transduced conditional Lmna mice carrying an inducible cardiomyocyte-specific Cre Tg(Myh6-cre / Esr1) (abbreviated as mcm here) in which Cre expression was induced by a single injection of tamoxifen (Tmx). Flx / Flx Mice homozygous for the allele were used as described in Examples 1 and 2. Cardiac-specific deletion of Lmna resulted in death within one month, whereas homozygous Syne1 C’TΔ8 / C’TΔ8 Mice with the same deletion induced on the background survived for at least 120 days after Tmx induction (FIG. 25, no change from day 80 to day 120).

[0454] Example 7: Methods for screening small molecules that block SUN-KASH interaction Crystallographic studies of human SUN2 reveal that the SUN domain assembles into 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 bridge the nuclear cisternae (PNS), allowing the SUN and KASH domains to directly interact [Sosa et al., Cell 149(5):1035-47(2012)]. The KASH-binding site is primarily formed within a groove formed at the interface between adjacent SUN domains (Figure 5B, left panel of Figure 21). This groove accommodates a portion of the approximately 18-residue KASH domain in an extended conformation. However, the C-terminal tetrapeptide of the KASH domain, characterized by three proline residues followed by a terminal aliphatic residue, Leu or Thr (for Nesp1 and Nesp2, respectively), is crucial for the SUN-KASH interaction (right panel of Figure 21, adapted from Figure 1 in Sosa et al., Cell 149(5):1035-47 (2012)). The importance of this tetrapeptide is that it is located in 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 abolishes SUN-KASH association across the entire SUN-KASH contact region (Sosa et al., Cell 149(5):1035-47 (2012) and left panel of Figure 22). Consequently, stable binding of the KASH domain requires 18–20 residues, but it is the C-terminal tetrapeptide that actually initiates binding. Therefore, blocking the tetrapeptide-binding pocket within the SUN monomer can disrupt the SUN-KASH association. In this disclosure, we describe an AAV-based gene therapy strategy that disrupts the endogenous SUN-KASH interaction as a treatment for laminopathies, including dilated cardiomyopathy. Alternatively, small molecules that block the SUN-KASH interaction at the SUN-binding pocket can disrupt the LINC complex and similarly treat laminopathies. There are a variety of standard methods for screening small molecule drugs in vitro.

[0455] In vitro screens can be constructed using recombinant SUN and KASH domains, or KASH peptides, whose production methods have been published [Sosa et al., Cell 149(5):1035-47 (2012)]. One such screen involves an assay technique similar to an enzyme-linked immunosorbent assay (right panel of Figure 22, equivalent to Lepourcelet et al., Cancer Cell. 5(1):91-102 (2004)). Recombinant SUN domains are immobilized on a solid surface, typically a 96-well plate, and then complexed with recombinant KASH domains linked to an enzyme capable of generating a colorimetric or chemiluminescent readout. One method for enabling this linkage is to synthesize biotinylated KASH peptides, which can then be linked to commercially available streptavidin-horseradish peroxidase (HRP) conjugates. Candidate compounds are obtained from appropriate suppliers and screened in vitro for their ability to inhibit KASH-SUN association. Compounds that cannot inhibit the SUN-KASH interaction will result in wells in which recombinant SUN binds to the enzyme-linked KASH domain. After a washing step and incubation with a colorimetric or chemiluminescent HRP substrate, the presence of the SUN-KASH interaction is detected in a standard plate reader. If the compound can inhibit the SUN-KASH interaction, the KASH domain will be removed after the washing step, and the enzyme reaction in the well will be reduced or absent.

[0456] Alternatively, fluorescence anisotropy or polarization can be used to screen for small molecule inhibitors of the SUN-KASH interaction in vitro [Lea, WA and Simeonov, A. Expert Opin Drug Discov 6:17-32 (2011)]. This assay also uses recombinant SUN and KASH domains. The KASH domains are fluorescently labeled; for example, chemically synthesized KASH peptides can be easily functionalized with fluorescein moieties. The fluorescence anisotropy of interacting KASH domains that interact with the SUN domain can be measured using standard instruments such as a plate reader. Because fluorescent KASH changes fluorescence anisotropy when not bound to SUN, small molecule inhibitors that disrupt the SUN-KASH interaction can be easily detected.

[0457] As is typical in drug screening processes, compounds that successfully pass the in vitro primary screen can then be subjected to a cell-based secondary screen (Figure 23). In this case, immunofluorescence microscopy can be used to identify compounds that can dissociate the LINC complex. This is manifested as dispersion of KASH components to the perinuclear endoplasmic reticulum, while the cognate SUN protein is retained at the inner nuclear membrane. This microscopy-based assay can be initially performed on HeLa cells. Active compounds are then evaluated in cultured cells from disease-related tissue, such as cardiac cells. Additional secondary screens may include whether identified compounds are able to rescue the growth defect in Lmna knockout cells. After hit-to-lead optimization of identified compounds using standard methods, compounds can be tested for Lmna dilated cardiomyopathy in a mouse model of laminopathies, such as those described herein. Lead efficacy can be assessed using survival time and echocardiograms of mutant mice to assess cardiac function, as described herein.

[0458] Example 8: Discussion DCM caused by LMNA is considered progressive and often leads to premature death or heart transplantation [M. Pasotti et al., J Am Coll Cardiol 52:1250-1260 (2008); MR Taylor et al., J Am Coll Cardiol 41:771-780 (2003)]. By age 60, 55% of LMNA mutation carriers will die of cardiovascular failure or undergo heart transplantation, compared with 11% of patients with idiopathic cardiomyopathy. Attempts to ameliorate DCM by adjusting pacemakers are, at best, only temporarily beneficial. Therefore, the development of novel therapeutic approaches to treat DCM caused by LMNA mutations is necessary.

[0459] The majority of LMNA mutations that cause DCM are dominant-negative missense. Conventional gene therapy to...

Claims

1. 1. A pharmaceutical composition for use in a method for treating or preventing a laminopathies associated with mutations to LMNA, comprising a nucleic acid encoding a LINC complex inhibitor, wherein the LINC complex inhibitor is a dominant-negative SUN domain-containing protein that inhibits the interaction between (i) SUN1 or SUN2 and (ii) Nesprin-1, Nesprin-2, or Nesprin-3; A pharmaceutical composition, wherein: (a) the dominant-negative SUN domain-containing protein comprises the SUN domain of SUN1 and does not comprise the amino acid sequence of SEQ ID NO: 94 and does not comprise the amino acid sequence of SEQ ID NO: 95; or (b) the dominant-negative SUN domain-containing protein comprises the SUN domain of SUN2 and does not comprise the amino acid sequence of SEQ ID NO:

96.

2. The pharmaceutical composition described in claim 1, wherein the laminopathies associated with mutations in LMNA are characterized by one or more of myopathy, cardiomyopathy, dilated cardiomyopathy, muscular dystrophy, myocardial dystrophy, and skeletal muscular dystrophy.

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

4. The pharmaceutical composition of claim 3 , wherein the nucleic acid is contained in a vector.

5. The pharmaceutical composition according to claim 4, wherein the vector is an adeno-associated virus vector.

6. 6. The pharmaceutical composition of claim 4 or claim 5, wherein the vector is an adeno-associated viral vector selected from one of the following serotypes: AAV9, AAV1, AAV6, AAV8, AAV2i8, AAV9.45, AAV10, and AAVrh.

74.

7. The pharmaceutical composition of any one of claims 4 to 6, wherein the vector comprises a promoter that provides for expression of the nucleic acid in cardiac and / or skeletal muscle cells or tissues.

8. The pharmaceutical composition of claim 7 , wherein the promoter is a cardiac or cardiomyocyte-specific promoter.

9. The pharmaceutical composition of claim 8, wherein the heart or cardiomyocyte-specific promoter is selected from the group consisting of cTNT, α-MHC, and MLC2v promoters.

10. The pharmaceutical composition described in claim 7, wherein the promoter is a skeletal muscle or striated muscle cell-specific promoter.

11. The pharmaceutical composition described in claim 10, wherein the skeletal muscle or striated muscle cell-specific promoter is selected from the MCK, MHCK7 or desmin promoter.

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