Transcriptional regulatory elements for cardiac-specific gene expression

Abstract

The present disclosure relates to transcriptional regulatory elements comprising an enhancer and a promoter, and polynucleotides, vectors and cells comprising the same. Also provided are uses of the transcriptional regulatory elements for inducing cardiac-specific gene expression, and methods of treating or preventing a cardiomyopathy comprising administering a vector comprising the transcriptional regulatory elements.

Description

[0001] Transcriptional Regulatory Elements for Cardiac-Specific Gene Expression

[0002] This application claims priority from GB2415588.9 filed 23 October 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the field of molecular and cellular biology, and also to methods of medical treatment and prophylaxis using gene therapy.

[0005] Background

[0006] Gene therapies targeting cardiac dysfunction, such as dilated cardiomyopathy, require delivery of vectors that drive high and specific expression of gene(s) of interest in cardiomyocytes. High expression in cardiomyocytes permits the use of lower vector doses to achieve sufficient efficacy. Specific expression in cardiomyocytes minimises any potential off-target expression of the gene of interest, particularly in organs such as the liver that are known to receive high levels of gene therapy products. Collectively, these serve to reduce the potential risks of toxicity due to the delivery of high vector doses as well as high expression in off-target tissues.

[0007] Design of such highly expressing, tissue-specific promoters is challenging, especially as gene delivery vectors, such as AAV9, often have size limitations in the cargo that they can carry, and the promoters also have to be short, so that the promoter, gene of interest, and other regulatory elements can fit into the delivery vector. It can be appreciated that the shorter the designed promoter, the delivery vector will be able to accommodate genes of interest of longer lengths and this is a highly desired property in addition to its tissue specificity and high expression.

[0008] One way to increase specificity and expression of promoters is to combine regulatory elements together into one promoter, such as combining enhancers and promoters together. Prior studies have identified regulatory sequences, including enhancers and promoters, that drive organ-specific expression of genes, including cardiomyocytes (Rincon et al., 2015) and skeletal muscle (Sarcar et al., 2019). Sometimes specific combinations of enhancer and promoter sequences have been demonstrated to drive synergistically higher gene expression than would be predicted by additive effect alone. However, such potential synergistic effects cannot be predicted based on the strengths of the individual enhancer and promoter sequences, but instead require experimental validation of various enhancer-promoter combinations in suitable in vivo model systems. An example would be Sarcar et al., 2019, where the enhancer SK-CRM4 had dramatic synergistic effects with the promoter Desmin but not SPc5-12.

[0009] Furthermore, very often highly expressing promoters (or enhancer-promoter combinations) are less tissue-specific, with there being an inverse relationship between expression and tissue specificity. Often enhancer-promoter combinations stated to be highly tissue specific have their expression tested in just a few off-target organs and a comprehensive biodistribution profile is lacking. Dilated Cardiomyopathy (DCM) is the most common disease affecting heart muscle, accounting for approximately 60% of all cardiomyopathies. It is characterised by reduced systolic (contractile) function due to enlargement and thinning of the left ventricular wall or in some cases both ventricles. DCM is associated with sudden heart failure and cardiac death, resulting in high rates of hospital admission, the need for heart transplantation and consequently a high-cost burden (Jefferies and Towbin, 2010;

[0010] Hershberger et al., 2013). The causes of DCM are varied, but include a variety of extrinsic factors, (viral, autoimmune infiltration, alcohol, and drugs). However 30-40% of all cases have a monogenic basis, with mutations in some 40 genes being linked to DCM. The most frequently mutated gene in DCM is TTN, that encodes the giant sarcomeric protein titin, with truncating variants in TTN accounting for almost 15-25% of all congenital forms of DCM (Herman et al., 2012; Tayal et al, 2017). The second most frequently mutated gene is Lamin A (LMNA) accounts for as many as 6-8% of congenital DCM patients (Tayal et al., 2017).

[0011] There exists a need in the art for methods of increasing cardiac-specific gene expression, for use in gene therapy approaches that aim to treat and prevent diseases that effect the heart, such as DCM.

[0012] Summary

[0013] The invention is as defined in the claims.

[0014] In a first aspect, the disclosure provides a transcriptional regulatory element comprising an enhancer and a promoter, wherein the enhancer is selected from CS-CRM4 and SK-CRM3; and the promoter is selected from Synthetic hTNNT2, hTNNT2(-502 to +42), cTnT-129 (269-201)neg, Original cTnT (442), TNNT2, CMV IE (608) and cTnT (-555 to +38). The transcriptional regulatory element may be a polynucleotide. Thus, the first aspect may be a polynucleotide comprising an enhancer and a promoter, wherein the enhancer is selected from CS-CRM4 and SK-CRM3; and the promoter is selected from Synthetic hTNNT2, hTNNT2(-502 to +42), cTnT-129 (269-201)neg, Original cTnT (442), TNNT2, CMV IE (608) and cTnT (-555 to +38).

[0015] In some aspects where the enhancer is CS-CRM4 the promoter may be selected from Synthetic hTNNT2, Original cTnT (442), cTnT-129 (269-201)neg, hTNNT2(-502 to +42) and TNNT2. In other aspects where the enhancer is SK-CRM3 the promotor is selected from cTnT-129 (269-201)neg, hTNNT2(-502 to +42), synthetic hTNNT2, and TNNT2. In some aspects, the enhancer is CS-CRM4 and the promotor is Synthetic hTNNT2.

[0016] In some aspects CS-CRM4 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 1 ; and / or SK-CRM3 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 2; and / or Synthetic hTNNT2 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 6; and / or hTNNT2(-502 to +42) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 4; and / or cTnT-129 (269-201)neg is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 3; and / or Original cTnT (442) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 5; and / or TNNT2 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 7; and / or CMV IE (608) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 8; and / or cTnT (-555 to +38) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO:9. In some transcriptional regulatory elements described herein, a restriction enzyme recognition site is arranged between the enhancer and the promoter.

[0017] The transcriptional regulatory element may have a sequence selected from any one of SEQ ID NOs: 11 to 21.

[0018] In a second aspect, the disclosure provides a vector comprising the transcriptional regulatory element according to any one of the preceding claims and a gene of interest. The gene of interest may encode a therapeutic protein. The gene of interest may encode a LINC complex inhibiting polypeptide or a focal adhesion protein. The LINC complex inhibiting polypeptide may comprises or consist essentially of any one of SEQ ID NOs: 22 to 86. The focal adhesion protein may comprise or consist essentially of any one of SEQ ID NOs: 90 to 94. The vector may comprise at least one, several, or all elements selected from an intron, a Kozak sequence, a signal peptide and a polyA sequence. The vector may be an adeno-associated virus (AAV) vector. The vector may comprise or consist or consist of a sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10. The vector may comprise, or consist of, a sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 96.

[0019] In a third aspect, there is provided the use of a transcriptional regulatory element described herein to induce cardiac-specific expression of a GOL

[0020] In a fourth aspect, there is provided a method for inducing cardiac-specific expression of a gene of interest.

[0021] In a fifth aspect, the disclosure provides a cell comprising a vector as described herein.

[0022] In a sixth aspect, the disclosure provides a method of treatment or prophylaxis comprising administering the a vector described herein to a subject in need thereof. The transcriptional regulatory element or vector may be administered in a therapeutically useful amount. The method of treatment or prophylaxis may be a method of gene therapy. Transcriptional regulatory elements, or vectors, for use in such methods are also described, as well as the use of transcriptional regulatory elements or vectors in the manufacture of a medicament for use in a method of treatment or prevention. The treatment or prevention may be the treatment or prevention of a cardiomyopathy.

[0023] Description

[0024] Gene therapies targeting cardiac dysfunction, such as dilated cardiomyopathy, require delivery of vectors that drive high and specific expression of gene(s) of interest in the heart. The present invention is based on the inventors’ unexpected finding that transcriptional regulatory elements comprising certain enhancer and promoter combinations, including a novel combination of the CS-CRM4 enhancer and a synthetic hTNNT2 promoter, that drive high and specific gene expression in cardiac tissue upon systemic delivery in a gene therapy vector.

[0025] In particular, the enhancer / promoter combinations drive increased gene expression in various tissues of the heart without resulting in significant gene expression in the liver. This is particularly advantageous, as some viral vectors such as AAV have a strong tropism for the liver, so this tissue receives a high copy number of the viral genome. The inventors demonstrate in the experimental examples herein that transcriptional regulatory elements comprising certain enhancer / promoter combinations can be used to drive high and specific gene expression of various genes of interest (GOIs) such as therapeutic genes or reporter genes in cardiomyocytes, showing that the transcriptional regulatory elements are suitable for gene therapy targeting the heart.

[0026] Transcriptional regulatory elements

[0027] The present invention relates to regulatory sequences, and specifically to DNA regulatory sequences referred to herein as transcriptional regulatory elements. As used herein, the term “transcriptional regulatory element” refers to a nucleic acid that, when operably linked to a gene of interest (GOI), controls expression of that GOI. Transcriptional regulatory elements described herein increase expression of the GOI in cardiac tissue. The transcriptional regulatory elements described herein may increase cardiacspecific expression of the GOI. Transcriptional regulatory elements disclosed herein comprise an enhancer and a promoter. In some embodiments, the transcriptional regulatory element is a polynucleotide. As such, where the present disclosure refers to a transcriptional regulatory element, it may be interpreted as referring to a polynucleotide.

[0028] In some embodiments, the transcriptional regulatory element comprises an enhancer that is contiguous with the promoter. In other words, the nucleic acid of the enhancer is connected directly to the nucleic acid of the promoter, with no additional nucleic acid between them. This is advantageous as the total length of the transcriptional regulatory element is reduced, meaning that the transcriptional regulatory element takes up less packaging space in a viral vector, allowing for a larger GOI may be incorporated in the vector. In other embodiments the enhancer and promoter are not contiguous. For example, in some embodiments, the transcriptional regulatory element may comprise additional nucleic acids between the enhancer and promoter. For example, the transcriptional regulatory element may comprise an intervening stretch of nucleotides between the enhancer and the promoter, such as an intervening stretch of 1-10, 2-9, 3-8 or 4-7 nucleotides, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0029] In some embodiments, the transcriptional regulatory elements comprise a nucleic acid sequence encoding restriction enzyme recognition site positioned between the enhancer and promoter sequences. In other words, described herein is a polynucleotide comprising or consisting of an enhancer and a promoter, the enhancer and promoter being linked by a nuclei acid sequence encoding a restriction enzyme recognition site. Numerous restriction enzymes and their recognition sites are known in the art. In some embodiments, the nucleic acid sequence encodes a BsrGI recognition site. The BsrGI recognition site may comprise or consist of the nucleic acid sequence TGTACA.

[0030] In some embodiments, the enhancer is located upstream of the promoter. “Upstream” refers to a first sequence that is, when reading a nucleic acid sequence in the 5’ to 3’ direction, located before a second sequence ( / .e. the first sequence is located 5’ with respect to the second sequence). For example, the enhancer may be located before the promoter, when reading the DNA in the 5’ to 3’ direction. In other words, the enhancer may be positioned 5’ of the promoter. In other words, the promoter may be positioned 3’ of the enhancer. In some embodiments, the transcriptional regulatory elements described herein may additionally comprise 1-10, 2-9, 3-8 or 4-7 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at one or both of the 3’ or 5’ end. Thus, in some embodiments, the transcriptional regulatory element may comprise 1-10, 2-9, 3-8 or 4-7 nucleotides, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides upstream of the enhancer and promoter sequences. In some embodiments, the transcriptional regulatory element may comprise 1-10, 2-9, 3-8 or 4-7 nucleotides, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides downstream of the enhancer and promoter sequences, such as between the enhancer and promoter sequences and the nucleic acid encoding the expression product of interest.

[0031] Promoters

[0032] The term “promoter” as used herein is defined as a DNA sequence recognized by the transcriptional machinery of the cell, or protein regulators of gene transcription, required to initiate the specific transcription of a polynucleotide sequence. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. Promoters which cause a gene to be expressed only in certain tissues are commonly referred to as “tissue-specific promoters”. Preferred promoters for use in the transcriptional regulatory elements described herein are tissue-specific, preferably cardiac-specific. The term “promoter” as used herein encompasses variants derived from wildtype promoter sequences. The promoter may comprise a truncation at the 5’ end and / or the 3’ end of the wild-type promoter sequence. In some embodiments, a promoter may comprise the wild-type promoter and additional sequence flanking the wild-type promoter on either the 5’ or the 3’ end. In other words, a promoter may comprise a particular region derived from genomic sequence that includes the wild-type promoter sequence. The promoter may comprise a truncation at the 5’ end and additional flanking sequence at the 3’ end. The promoter may comprise additional flanking sequence at the 5’ end and a truncation at the 3’ end. Such promoter variants are often referred to based on the position of the first and last nucleotides with respect to the transcription start site.

[0033] A number of cardiac-specific promotors are known in the art, and include B-actin (-275 to +61), ACTA1, ACTC1, CKM, CMV IE (608), CryAB, cTnT(-555 to +38), cTnT-129 (269-201)neg, DES, FABP3, GNAQ (887), HSBP7, hTNNT2 (-502 to +42), Human EFla (230), MALAT1, MB, MHCK7 (771), Mybpc3, Myh6 containing EcoRI, Myh7, Myl2 containing EcoRI, Myl3, Original cTnT(442), RYR2, Synthetic hTNNT2 (455), Synthetic SPc5-12 (424), TNNC1, TNNI3, TNNT2 and Tpm1. In some aspects herein, the promoter is one of cTnT(-555 to +38), cTnT-129 (269-201)neg, hTNNT2(-502 to +42), Original cTnT (442), Synthetic hTNNT2 or TNNT2. In some aspects the promoter is one of cTnT-129 (269-201)neg, hTNNT2(-502 to +42), Original cTnT (442), Synthetic hTNNT2 or TNNT2. The promoter may be CMV IE (608) or cTnT (-555 to +38). The promoter may be hTNNT2 (-502 to -42) or Synthetic hTNNT2. In some preferred aspects, the promoter is the Synthetic hTNNT2 promoter.

[0034] In particularly preferred aspects, the promoter is a TNNT2 promoter, or a promoter derived from a TNNT2 promoter. TNNT2 is a gene that encodes the cardiac isoform of troponin T. It encodes the tropomyosinbinding subunit of the troponin complex, which is located on the thin filament of striated muscles and regulates muscle contraction in response to alterations in intracellular calcium ion concentration. The promotor of TNNT2 drives cardiac specific expression of the TNNT2 gene. Although the promoter may identical to the naturally occurring or wild-type TNNT2 promoter, various promoters have been developed based on the wild-type TNNT2 promoter including synthetic promoter sequences and promoter variants that drive cardiac expression from a smaller nucleic acid sequence. Troponin 2 promoters include the human TNNT2 promoter (hTNNT2), a TnT promoter, a synthetic hTNNT2 promoter, a truncated hTNNT2 (-502 to +42) promoter or a truncated cTnT (-555 to +38) promoter. In some aspects described herein, the promoter is a synthetic hTNNT2 promoter.

[0035] The human TNNT2 (hTNNT2) promoter, having SEQ ID NO: 7. The NCBI gene ID for the human TNNT2 gene is 7139. In some embodiments, the transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7.

[0036] In some aspects, the present disclosure relates to the cardiac troponin T promoter. The cTnT promoter is a 442 bp sequence encoded by SEQ ID NO: 5. The cardiac troponin T isoform (cTnT) is encoded by the TNNT2 gene. The NCBI gene ID for the chicken TNNT2 is 396433, updated on 17-Aug-2024. TNNT2 is expressed in cardiac muscle and, transiently, also in embryonic and neonatal skeletal muscles, including both slow- and fast-fiber-dominant muscles in avians and humans. As one of the 3 subunits of the troponin (troponin C, troponin T and troponin I) found in cardiac thin filaments, cardiac troponin T binds to tropomyosin to form the troponin-tropomyosin complex which plays an important role in regulating contractile function (Prasad et al., Gene Ther., 18(1): 43-52 (2010), herein incorporated by reference in its entirety). Mutational analysis of the cTnT promoter region showed that the -375 to +43 region (relative to the cTnT transcriptional start site) is sufficient to confer cardiomyocyte-specific gene expression (Ma et al., Am. J. Physiol. Cell Physiol. 286(3) :C556-64 (2004), herein incorporated by reference in its entirety). As used herein, “cTnT-129 (269-201)neg” refers to a promoter comprising the sequence from positions -269 to -201 with respect to the transcription start site of the chicken cTnT gene in the negative orientation, having the sequence of SEQ ID NO: 3. The cTnT-129 (269-201)neg promoter was first described in lannello RC, Mar JH, Ordahl CP J Biol Chem. 1991 ;266(5):3309-3316, which is herein incorporated by reference in its entirety. In some embodiments, the transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3.

[0037] As used herein, “Original cTnT(442)” refers to the full promoter sequence of the chicken cTnT gene, having the sequence of SEQ ID NO: 5. In some embodiments, the transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5.

[0038] As used herein, “cTnT (-555 to +38)” refers to a promoter comprising the 593 bp sequence from positions -555 to +38 with respect to the transcription start site of the chicken cTnT gene, having the sequence of SEQ ID NO: 9. In some embodiments, the transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. As used herein, “hTNNT2(-502 to +42)” refers to a promoter comprising the 544 bp sequence from positions -502 to +42 with respect to the transcription start site of the hTNNT2 gene, having the sequence of SEQ ID NO: 4. In some embodiments, the transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4.

[0039] The present disclosure also relates to a synthetic hTNNT2 promoter. As used herein, “synthetic hTNNT2” refers to a 455 bp promoter derived from the TNNT2 promoter, wherein non-conserved subregions that do not affect transcriptional activity have been deleted to reduce the overall size of the regulatory element. The synthetic hTNNT2 promoter is described in Kolwicz et al., Mol Ther. 2016;24(2):240-250, which is herein incorporated by reference in its entirety. In some embodiments, the transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6.

[0040] The present disclosure also relates to the cytomegalovirus intermediate early promoter (CMV IE, also referred to as CMV MIE). The CMV IE promoter controls the expression of the CMV major intermediate early genes, IE1 (UL123) and IE2 (UL122), and other auxiliary genes. In some embodiments, the disclosure relates to a CMV IE promoter. As used herein, “CMV IE (608)” refers to a promoter comprising the 608 bp sequence having SEQ ID NO: 8. The CMV IE (608) promoter comprises the CMV IE enhancer and the CMV IE promoter. In some embodiments, the transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8.

[0041] Enhancers

[0042] The term “enhancer” as used herein is defined as a DNA sequence recognized by the transcriptional machinery of the cell, or protein regulators of gene transcription, that function to increase transcription of a polynucleotide sequence. Enhancers are cis-acting regulatory modules (CRMs), that activate the expression of genes independent of their distance from and direction with respect to the promoter.

[0043] Enhancers comprise dense clusters of transcription factor binding sites (TFBS) and are bound by cell type-specific TFs, coregulators, chromatin modifiers, architectural proteins like Cohesin, Condensin and CTCF, other enzymes, and RNAPII. Owing to such large-scale protein assembly, enhancers are often nucleosome deficient, and thus are hypersensitive to nucleases reflecting DNA accessibility. Enhancers activate gene expression of genes that are located close by in 3D space within the nucleus and are widely believed to physically contact the target promoters to effect transcriptional activation (Panigrahi and O’Malley, Genome. Biol., 22: 108 (2021), herein incorporated by reference in its entirety. In some embodiments, enhancers increase gene expression to levels greater than that achieved when there is no enhancer. Enhancers may contribute to tissue-specific gene expression, such as cardiac specific gene expression. Enhancers include CS-CRM4 and SK-CRM3.

[0044] The CS-CRM4 enhancer was identified as a 192 bp cardiac-specific enhancer comprising binding sites for the transcription factors HNF3a, MEF2, SRF, NF1, RSRFC4, HNF3p, and HFH1, associated with the cardiac-specific gene Casq2 (Rincon 2015). The Casq2 gene encodes calsequestrin 2, the cardiac muscle calsequestrin family member. Calsequestrin is a protein that binds calcium acting as an internal calcium store in muscle. In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , and a binding site for one or more transcription factors selected from HNF3a, MEF2, SRF, NF1 , RSRFC4, HNF3p, and HFH1. Preferably, the enhancer comprises a binding site for at least two, at least three, at least four, at least five, at least six, or more preferably seven transcription factors selected from HNF3a, MEF2, SRF, NF1, RSRFC4, HNF3p, and HFH1.

[0045] The SK-CRM3 was identified as a 430 bp skeletal muscle-specific enhancer, comprising binding sites for the transcription factors E2A, CEBP, LRF, MyoD, SREBP, and Tall, associated with the TNNI1 gene (Sarcar 2019). The TNNI1 gene encodes troponin 11 , which is expressed in cardiac and skeletal muscle during early development, but is restricted to slow-twitch skeletal muscle fibres in adults. In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, and a binding site for one or more transcription factors selected from E2A, CEBP, LRF, MyoD, SREBP, and Tall. Preferably, the enhancer comprises a binding site for at least two, at least three, at least four, at least five, or more preferably six transcription factors selected from E2A, CEBP, LRF, MyoD, SREBP, and Tall.

[0046] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3.

[0047] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4.

[0048] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6.

[0049] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7.

[0050] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8.

[0051] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9.

[0052] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3.

[0053] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4.

[0054] In some embodiments, the transcriptional regulatory element comprises an enhancer having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, and a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12.

[0055] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13.

[0056] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14.

[0057] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15.

[0058] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16.

[0059] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17.

[0060] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18.

[0061] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19.

[0062] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 20.

[0063] In some embodiments, the transcriptional regulatory element comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 21. Cardiac-specific expression

[0064] The present disclosure relates to transcriptional regulatory elements that promote high and specific expression in cardiomyocytes. These transcriptional regulatory elements are therefore useful for driving cardiac-specific expression of a gene, such as a therapeutic gene, in gene therapies were expression of a gene in the heart is desirable.

[0065] As used herein, “gene expression” refers to the process by which information contained within a gene is made into a function via production of a protein encoded by that gene. Nucleic acid encoding the gene is initially transcribed to mRNA which is then expressed as protein. Gene and protein expression can be determined by means well known to the skilled person. Gene expression of a given protein can be evaluated using techniques that involve determining the level of mRNA encoding the protein by e.g. RT-qPCR, northern blot, etc. Protein expression can be evaluated e.g. by antibody-based methods including western blot, immunohisto / cytochemistry, flow cytometry, ELISA, or by reporter-based methods. In particular aspects, gene expression is determined as the number of copies of RNA encoding the GOL It may be expressed as the number of copies per ng RNA, or as the ratio of the copies of RNA to copies of DNA.

[0066] “Cardiac-specific expression” is used interchangeably herein with “expression in cardiac tissue”, and “expression in the heart” to refer to gene expression preferentially in cardiomyocytes, cardiac tissue or the heart, rather than other cell, tissue or organ types. As used herein, the term “cardiac-specific expression” refers to expression of the GOI in the heart, in the absence of (or negligible) expression of the GOI in one or more other tissues, or an increase in expression of the GOI in the heart but in the absence of increased expression of the GOI in other tissues.

[0067] In some embodiments, the transcriptional regulatory elements of the present disclosure are capable of increasing gene expression in cardiac tissue compared to a control transcriptional regulatory element, wherein the control transcriptional regulatory element comprises a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5, and does not comprise an enhancer. In some cases, the control transcriptional regulatory element consists of a promoter having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the promoter of the control transcriptional regulatory element is a cardiac-specific promoter. In some embodiments, the promoter of the control transcriptional regulatory element is the cTnT promoter.

[0068] The transcriptional regulatory elements of the present disclosure may result in 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold the expression of the gene in cardiac tissue compared to the expression of the gene with a control transcriptional regulatory element.

[0069] In some embodiments, cardiac tissue comprises the left ventricle, the right ventricle, and / or the atrium. In some embodiments, cardiac tissue comprises cells of the left ventricle, cells of the right ventricle, and / or cells of the atrium. Transcriptional regulatory elements described herein result in high and / or specific expression in at least one of cells of the left ventricle, cells of the right ventricle, and / or cells of the atrium. In some embodiments, the transcriptional regulatory element drives gene expression in cardiac tissue but does not drive gene expression in one or more tissues selected from: the liver, brain, cerebellum, diaphragm, eye, kidney, lung, muscle, ovary, spleen, testis, and uterus. In some embodiments, the transcriptional regulatory element drives gene expression in cardiac tissue, but does not drive gene expression in one or more tissues selected from: the cornea, lens, retina, choroid, sclera, optic nerve, testis, ovary, skeletal muscle, diaphragm, lymph node, cerebellum, cerebrum, brain stem, spinal cord (cervical), spinal cord (thoracic), dorsal root ganglion (cervical), dorsal root ganglion (thoracic), dorsal root ganglion (lumbar), kidney, lung, liver, and spleen.

[0070] In some embodiments, the transcriptional regulatory element drives gene expression in cardiac tissue, but does not drive gene expression in the liver, brain, cerebellum, diaphragm, eye, kidney, lung, muscle, ovary, spleen, testis, or uterus. In some embodiments, the transcriptional regulatory element drives gene expression in cardiac tissue, but does not drive gene expression (or results in negligible expression) in the cornea, lens, retina, choroid, sclera, optic nerve, testis, ovary, skeletal muscle, diaphragm, lymph node, cerebellum, cerebrum, brain stem, spinal cord (cervical), spinal cord (thoracic), dorsal root ganglion (cervical), dorsal root ganglion (thoracic), dorsal root ganglion (lumbar), kidney, lung, liver, or spleen. The transcriptional regulatory element may result in no or negligible expression of the gene in non-cardiac tissue. It may result in lower expression of the gene in non-cardiac tissue than the expression of the gene as compared to the expression of the gene with a control transcriptional regulatory element. It may result in 10% or lower, 20% or lower, 30% or lower, 40% or lower, 50% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower expression of the gene in non-cardiac tissue as compared to the expression of the gene in non-cardiac tissue with a control transcriptional regulatory element. For example, the transcriptional regulatory element of the disclosure may result in lower expression of the gene in liver tissue as compared to the expression of the gene with the control transcriptional regulatory element, such as 10% or lower, 20% or lower, 30% or lower, 40% or lower, 50% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower expression of the gene in liver tissue as compared to the control transcriptional regulatory element. The transcriptional regulatory element may result in 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less expression in non-cardiac tissue relative to the expression in cardiac tissue.

[0071] In some embodiments, the transcriptional regulatory element drives gene expression in cardiac tissue, but when the administration site is not the heart, the transcriptional regulatory element does not drive gene expression at the administration site. For example, the administration may be systemic administration, such as intravenous injection.

[0072] In some embodiments, the transcriptional regulatory elements of the present invention increase cardiacspecific gene expression independent of the viral copy number. For example, the transcriptional regulatory elements may increase gene expression in the heart without increasing gene expression in other tissues, even if other tissues receive a higher viral copy number than the heart. In some embodiments, the transcriptional regulatory elements of the present invention increase gene expression in the heart and do not increase gene expression in the liver, even when the liver receives a higher viral copy number compared to the heart. In some embodiments, the transcriptional regulatory elements are capable of increasing gene expression in the heart even when the viral copy number in the heart is 1.5 times, 2 times, 3 time, 5 times, or 10 times less than the viral copy number in the liver.

[0073] In some embodiments, the transcriptional regulatory sequences of the present invention increase cardiacspecific gene expression over a sustained period of time. The transcriptional regulatory elements may increase cardiac-specific gene expression for at least 2, 3, 4, 5, 6, 7, or 8 weeks after infection.

[0074] Preferably, the transcriptional regulatory elements of the present invention increase cardiac-specific gene expression for at least 2 months after infection.

[0075] Vectors

[0076] In some embodiments, the transcriptional regulatory element is comprised in a vector. The present disclosure provides a vector that comprises a transcriptional regulatory element according to the present disclosure. Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

[0077] A ‘vector’ as used herein refers to a nucleic acid used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may facilitate delivery of nucleic acid encoding a polypeptide according to the present disclosure into a cell. The vector may be an expression vector comprising elements required for expressing a polypeptide according to the present disclosure. The vector may comprise elements facilitating integration of nucleic acid encoding a polypeptide according to the present disclosure into the genomic DNA of the cell into which the vector is introduced.

[0078] The vector may be a vector for expression of the nucleic acid in the cell (i.e. the vector may be an expression vector). Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon (i.e. 3’ in the nucleotide sequence of the vector to the nucleotide sequence encoding the polypeptide) and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure. The term ‘operably linked’ may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and / or enhancer) are covalently linked in such a way that the expression of the selected nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the selected nucleic acid sequence. The resulting transcript(s) may then be translated into a polypeptide, e.g. a polypeptide according to the present disclosure.

[0079] In addition to the transcriptional regulatory elements disclosed herein, and a nucleic acid encoding a GOI, vectors may include one or more additional elements. For example, vectors may comprise nucleic acid sequences for regulating translation of protein from the nucleic acid encoding the gene of interest, secretion of the gene of interest or for downstream processing of the protein expressed from the gene of interest, such as a signal protein, tag or detectable tag such as a fluorescent sequence or barcode sequence.

[0080] In some embodiments, the vectors of the present disclosure comprise an intron (e.g. an MVM intron, pCI-Neo intron, an SV40 intron, or a beta-globulin intron) between the transcriptional regulatory element and the GOL In some embodiments, the intron is an MVM intron. An “intron” is an intervening sequence in a gene that does not encode a portion of the protein sequence. Thus, such sequences are transcribed into RNA but are then excised and are not translated.

[0081] The vectors may also include a Kozak sequence between the transcriptional regulatory element (or intron) and the GOI. A Kozak sequence is a nucleic acid motif that functions as

[0082] the protein translation initiation site. A number of suitable Kozak sequences will be readily appreciated by the skilled person and may be used in the vectors described herein.

[0083] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia virus vectors and herpesvirus vectors), transposonbased vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus etal., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 20164, 9, which are both hereby incorporated by reference in their entirety. In preferred embodiments, the vector is an adeno-associated virus vector or a lentiviral vector.

[0084] A vector may be selected based on tropism for a cell type / tissue / organ to which it is desired to deliver the nucleic acid. In some embodiments, a vector is selected based on tropism for a cardiomyocytes, cardiac tissue, or the heart.

[0085] In some embodiments it is desired to deliver nucleic acid encoding a polypeptide of the disclosure to muscle cells / tissue (e.g. cardiac muscle cells (card io myocytes) or tissue), and vectors having a tropism for such cells / tissue may be employed in such embodiments. In some embodiments, a vector may be cardiotropic. In some embodiments, a vector may be myotropic.

[0086] In some embodiments, the vector is an adeno-associated virus (AAV) vector. AAV vectors are among the most actively-investigated gene therapy vehicles, and are characterised by an excellent safety profile and high-efficiency transduction across a broad range of target cells / tissues. Adeno-associated virus vectors and their use to vector gene therapy is reviewed e.g. in Wang etal., Nat. Rev. Drug Discov. (2019) 18: 358-378 and Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272, both of which are hereby incorporated by reference in their entirety. In some embodiments, a vector may be an adeno-associated virus vector described in Wang etal., Nat. Rev. Drug Discov. (2019) 18: 358-378. In some embodiments, a vector may be an adeno-associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272.

[0087] In some embodiments, the AAV vector is a self-complementary adeno-associated virus (scAAV) vector. Self-complementary adeno-associated virus vectors are described e.g. in McCarty, Mol Ther. (2008) 16(10):1648-56, which is hereby incorporated by reference in its entirety. Conventional AAV have a single-stranded DNA genome, and depend on the DNA replication machinery of a transduced cell to synthesise the complementary strand, delaying transgene expression. By contrast, scAAV contain complementary sequences that spontaneously anneal upon infection, eliminating the requirement for DNA synthesis in the transduced host cell. Compared to classical, single-stranded AAV vectors, scAAV vectors have been shown to provide for accelerated onset of transgene expression, and an increased level of transgene expression.

[0088] In some embodiments, a vector may be an AAV vector of one of the following serotypes: AAV1 , AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV9, AAV9.45, AAV10 or AAVrh74.

[0089] In some embodiments, a vector may be a cardiotropic adeno-associated viral vector. In some embodiments, a vector may be an adeno-associated viral vector of one of the following serotypes: AAV1 , AAV8, AAV9, AAV9.45.

[0090] In some embodiments, a vector may be a myotropic adeno-associated viral vector. In some embodiments, a vector may be an adeno-associated viral vector of one of the following serotypes: AAV1 , AAV6, AAV7, AAV8, AAV9, AAV9.45.

[0091] In some embodiments, the vector is an AAV2 vector.

[0092] In some embodiments a vector comprises modification to increase binding to and / or transduction of a cell-type of interest ( / .e. as compared to the level of binding / transduction by the unmodified vector). In some embodiments modification is to a capsid protein.

[0093] In some embodiments a 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, which is hereby incorporated by reference in its entirety, e.g. a cell-targeting peptide shown in Table 1 , 2, 3 or 4 thereof.

[0094] In some embodiments a vector comprises a capsid protein comprising substitution of one or more tyrosine residues, e.g. one or more surface-exposed tyrosine residues. In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments a vector comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid as described in lida et al., Biomed Res Int. (2013) 2013: 974819, which is hereby incorporated by reference in its entirety.

[0095] In some embodiments, a vector may be an adeno-associated virus vector described in Buning and Srivastava, supra. In some embodiments, a vector may be an adeno-associated virus vector described in lida et al., supra.

[0096] A sequence for controlling expression of the nucleic acid may provide for expression of the nucleic acid in response to e.g. a given agent / signal. For example, expression may be under the control of an inducible promoter. The agent may provide for inducible expression of the nucleic acid in vivo by administration of the agent to a subject having been administered with a modified cell according to the present disclosure, or ex vivo / in vitro by administration of the agent to cells in culture ex vivo or in vitro.

[0097] In some embodiments a nucleic acid or vector according to the present disclosure may employ a conditional expression system for controlling expression of the nucleic acid encoding a polypeptide of the present disclosure by cells comprising the nucleic acid / vector. ‘Conditional expression’ may also be referred to herein as ‘inducible expression’, and refers to expression contingent on certain conditions, e.g. the presence of a particular agent. Conditional expression systems are well known in the art and are reviewed e.g. in Ryding et al. Journal of Endocrinology (2001) 171, 1-14, which is hereby incorporated by reference in its entirety.

[0098] The vector may be a vector comprising or consisting of the nucleic acid of SEQ ID NO: 10. The vector may be a vector comprising or consisting of the nucleic acid of SEQ ID NO: 96. The vector may be a vector comprising the nucleic acid sequence of SEQ ID NO: 11. The vector may be a vector comprising the nucleic acid sequence of SEQ ID NO: 15.

[0099] Formulations and Compositions

[0100] The present disclosure includes formulations and compositions that contain the transcriptional regulatory elements described herein, including formulations and compositions containing vectors such as AAV vectors containing the transcriptional regulatory elements. The compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g. wetting agents), masking agents or colouring agents (e.g. titanium oxide).

[0101] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0102] The pharmaceutical compositions / medicaments according to the present disclosure may be formulated for administration to a subject, e.g. administration via a route of administration as appropriate for the nature of the therapeutic agent and the disease to be treated / prevented. In some embodiments, a pharmaceutical composition / medicament may be formulated for parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral ortransdermal administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion.

[0103] Suitable formulations may comprise the relevant article in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via cannula) to a selected region of the human or animal body. The pharmaceutical compositions / medicaments may be provided in solid form, e.g. in lyophilised form. Medicaments and pharmaceutical compositions may be formulated for administration to a blood vessel, or to a tissue / organ of interest (e.g. a tissue / organ affected by the disease / condition (e.g. a tissue / organ in which symptoms of the disease / condition manifest).

[0104] The present disclosure also provides methods for producing pharmaceutical compositions / medicaments according to the present disclosure. Such methods may comprise mixing a polypeptide / nucleic acid / vector / cell described herein with a pharmaceutically-acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent. Such methods generally include the step of bringing into association the polypeptide / nucleic acid / vector / cell with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

[0105] The agents and compositions according to the present disclosure may be modified and / or formulated to facilitate delivery to, and / or uptake by, a cell / tissue of interest (e.g. cardiac cells / tissue).

[0106] Strategies for targeted delivery of such species are reviewed e.g. in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety.

[0107] In some embodiments, the agents and / or compositions may be encapsulated in a nanoparticle or a liposome. In some embodiments the agents and / or compositions may be (covalently or non-covalently) associated with a cell-penetrating peptide (e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier.

[0108] Nanoparticles may be organic, e.g. micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. Nanoparticles may be inorganic, e.g. such as nanotubes or metal particles, optionally with organic molecules added. In some embodiments, a nanoparticle is a nanoparticle 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, a nanoparticle is a PLGA, polypeptide, poly(p-amino ester), DOPE, p-cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosphoester nanoparticle.

[0109] In some embodiments, the agents and / or compositions comprise modification to incorporate one or more moieties facilitating delivery to, and / or uptake by, a cell type or tissue of interest (e.g. cardiac and / or skeletal muscle cells / tissue). In some embodiments, the agents and / or compositions are linked (e.g. chemically conjugated to) one or more moieties facilitating delivery to, and / or uptake by, a cell type or tissue of interest.

[0110] Moieties facilitating delivery to, and / or uptake by, cell types or tissues of interest are described e.g. in Benizri et al., Bioconjug Chem. (2019) 30(2): 366-383, which is hereby incorporated by reference in its entirety. Such moieties include e.g. N-acetylgalactosamine (GalNAc), a-tocopherol, cell-penetrating peptides, nucleic acid aptamers, antibodies and antigen-binding fragments / derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g. palmitic acid) and nucleolipid moieties. The agents and / or compositions of the present disclosure may be formulated in a sustained release delivery system, in order to release the polypeptide, nucleic acid, vector, cell or composition at a predetermined rate. Sustained release delivery systems may maintain a constant drug / therapeutic / prophylactic concentration for a specified period of time. In some embodiments, articles of the present disclosure are formulated in a liposome, gel, implant, device, or drug-polymer conjugate e.g. hydrogel.

[0111] Gene of interest (GOD

[0112] Transcriptional regulatory elements disclosed herein are useful for regulating the expression of a gene of interest. As used herein, a “gene of interest” (GOI) refers to any nucleic acid encoding an expression product of interest. The expression product may be a transgene or other expression product that is expressed under the control of the transcriptional regulatory elements according to the disclosure. The gene of interest will generally encode a protein of interest, such as a therapeutic protein or a marker protein such as a fluorescent protein, although expression of other expression products such nucleotide expression products such as microRNA (miRNA), small interfering RNA (siRNA), antisense RNA (ASO), messenger RNA (mRNA), self-amplifying RNA (saRNA) is also contemplated.

[0113] In particular embodiments, the gene of interest encodes a therapeutic protein. In such embodiments the transcriptional regulatory elements according to the disclosure can be used in gene therapy, wherein the expression of the GOI provides therapy for a disease or condition, i.e. the GOI is a therapeutic GOI that encodes a therapeutic peptide, therapeutic polypeptide or therapeutic polynucleotide. Expression of a therapeutic peptide or polypeptide may serve to restore or replace the function of the endogenous form of the peptide or polypeptide that is defective (i.e. gene replacement therapy). In other examples, expression of a therapeutic peptide or polypeptide, or polynucleotide, from the transgene serves to alter the levels and / or activity of one or more other peptides, polypeptides or polynucleotides in the host cell. Thus, according to particular embodiments, the expression of a transgene contained within a polynucleotide described herein in a host cell can be used to provide a therapeutic amount of a peptide, polypeptide or polynucleotide to ameliorate the symptoms of a disease or disorder. In some embodiments, the GOI may be a cardiac-specific gene or a gene suitable for gene therapy of the heart. In some embodiments, the GOI is gene encoding a LINC complex inhibiting protein or a focal adhesion protein.

[0114] In some embodiments, the nucleic acids and vectors comprising transcriptional regulatory elements according to the disclosure can be used to express a reporter gene, i.e., the GOI is a reporter gene. Reporter genes are genes that encode detectable markers, such as genes that encode enzymes that convert a substrate to a luminescent or coloured product (e.g. luciferase, (3-galactosidase etc) and genes that encode fluorescent markers (e.g. green fluorescent protein, red fluorescent protein etc).

[0115] Reference herein to a given GOI / protein generally refers to the canonical isoform of the human version of the protein, but also contemplates isoforms, fragments, variants (including mutants) and homologues thereof ( / .e. from other species, e.g. non-human mammalian species (e.g. a non-human primate, e.g. rhesus, cynomolgous; e.g. a rodent, e.g. rat or mouse)).

[0116] As used herein, a “fragment”, “variant” or “homologue” of a protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. the canonical isoform of the human protein). In some embodiments fragments / variants / isoforms / homologues may be characterised by ability to perform a function performed by the reference protein.

[0117] A “fragment” generally refers to a fraction of the reference protein. A “variant” generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein. An “isoform” generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein. A “homologue” generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. Homologues include orthologues.

[0118] A “fragment” may be of any length (by number of amino acids), although may optionally be at least 20% of the length of the reference protein (that is, 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.

[0119] Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property / activity of the reference protein, as determined by analysis by a suitable assay for the functional property / activity. Nucleic acid comprising the gene of interest may additionally comprise one or more non-polypeptide-encoding nucleotide sequence(s). Non-polypeptide-encoding nucleotide sequence(s) may be e.g. 5’ cap, 5’ UTR, 3’ UTR and / or PolyA tail sequences. A PolyA tail sequence may be the PolyA tail sequence of bovine growth hormone (bgh-PolyA).

[0120] LINC complex inhibiting polypeptide

[0121] In some embodiments, the GOI may encode a LINC complex inhibiting polypeptide. Herein, a “LINC complex inhibiting polypeptide” refers to a polypeptide that inhibits the LINC complex formation and / or function. A LINC complex inhibiting polypeptide may be used in the treatment of cardiomyopathies caused by mutation to LMNA. LINC complex inhibiting polypeptides are described in e.g. in WO 2019 / 143300 A1, WO 2021 / 010898 A1 and WO 2023 / 101607 A2, which are herein incorporated by reference in its entirety. In particular, when the cardiomyopathy is caused by mutation to LMNA, the GOI may be dominant-negative SUN1 (DNSUN1).

[0122] LINC complex inhibiting polypeptides according to the present disclosure may be based on a SUN domain-containing protein, e.g. selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO. In some embodiments, a LINC complex inhibiting polypeptide according to the present disclosure comprises an inhibitory region which is based on a SUN domain-containing protein, e.g. selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO.

[0123] LINC complex inhibiting polypeptides according to the present disclosure may comprise or consist essentially of an amino acid sequence having a high degree of sequence identity (e.g. at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of all or part of a SUN domain-containing protein, e.g. selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO. LINC complex inhibiting polypeptides according to the present disclosure may comprise an inhibitory region comprising or consisting of an amino acid sequence having a high degree of sequence identity (e.g. at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the amino acid sequence of all or part of a SUN domain-containing protein, e.g. selected from SUN1 , SUN2, SUN3, SUN5, SPAG4 and SUCO.

[0124] Such LINC complex inhibiting polypeptides preferably (i) retain the ability of the SUN domain-containing protein on which they are based to bind to a KASH domain-containing protein (e.g. one or more of Nesprin-1, Nesprin-2, Nesprin-3, Nesprin-4, KASH5 and LRMP), but (ii) lack, or display a reduced level of, one or more other properties of the SUN domain-containing protein on which they are based. In preferred embodiments, a LINC complex inhibiting polypeptide lacks or displays a reduced ability to bind to nuclear lamins and / or chromatin-binding proteins, and / or lacks or displays a reduced ability to associate with (e.g. localise to) the inner nuclear membrane, relative to the SUN domain-containing protein on which it is based.

[0125] In some embodiments, a LINC complex inhibiting polypeptide according to the present disclosure consists essentially of a human amino acid sequence. In some embodiments, the inhibitory region of a LINC complex inhibiting polypeptide according to the present disclosure consists essentially of a human amino acid sequence. As used herein, ‘a human amino acid sequence’ refers to an amino acid sequence that is encoded by nucleic acid of the genome of a human. That is, in some embodiments a LINC complex inhibiting polypeptide or the inhibitory region of a LINC complex inhibiting polypeptide consists essentially of an amino acid sequence having 100% amino acid sequence identity to an amino acid sequence encoded by the genome of a human subject. It will be appreciated that in some embodiments, the amino acid sequence encoded by the genome of a human subject is an amino acid sequence of a human SUN domain-containing protein (e.g. selected from SUN1, SUN2, SUN3, SUN5, SPAG4 and SUCO; e.g. SUN1 or SUN2). Such embodiments of LINC complex inhibiting polypeptides are contemplated in particular where administration to a human subject is intended, e.g. in the context of therapeutic / prophylactic intervention according to the present disclosure.

[0126] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a3 helix of the CC2 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a3 helix of the CC2 region of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a3 helix of the CC2 region of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0127] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the SUN domain of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the SUN domain of human SUNI. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the SUN domain of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25.

[0128] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a3 helix of the CC2 region and the SUN domain of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a3 helix of the CC2 region and the SUN domain of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an inhibitory region comprising an amino acid sequence corresponding to the a3 helix of the CC2 region and the SUN domain of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:27.

[0129] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a2 helix of the CC2 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a2 helix of the CC2 region of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a2 helix of the CC2 region of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0130] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a1 helix of the CC2 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a1 helix of the CC2 region of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a1 helix of the CC2 region of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31.

[0131] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC1 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC1 region of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC1 region of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33.

[0132] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC1 region and CC2 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC1 region and CC2 region of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC1 region and CC2 region of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35.

[0133] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC2 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC2 region of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the CC2 region of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37.

[0134] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a2 and a3 helices of the CC2 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a2 and a3 helices of the CC2 region of human SUN1. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to the a2 and a3 helices of the CC2 region of human SUN2. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39.

[0135] In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to part of the SMART coil-coiled 2 region, the CC1 region and CC2 region of a SUN domain-containing protein. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to positions 483 to 632 of human SUN1 (numbered according to SEQ ID NO: 40). In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence corresponding to positions 388 to 538 of human SUN2 (numbered according to SEQ ID NO: 41). In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42. In some embodiments, a LINC complex inhibiting polypeptide comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 43.

[0136] In some embodiments, a LINC complex inhibiting polypeptide, or an inhibitory region thereof, comprises or consists essentially of an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 44, 26, 45, 27, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84 or 85. In some embodiments, a LINC complex inhibiting polypeptide according to the present disclosure comprises an inhibitory region consisting essentially of an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 44, 65, 75, 64, 62, 63 or 66.

[0137] In some embodiments, a LINC complex inhibiting polypeptide according to the present disclosure comprises or consists essentially of an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86, 71 , 76, 70, 68, 69 or 72.

[0138] LINC complex inhibiting polypeptides according to the present disclosure may comprise one or more additional amino acids or sequences of amino acids. That is, the LINC complex inhibiting polypeptides may comprise one or more amino acids or sequences of amino acids in addition to the inhibitory region of the polypeptide (i.e. the region comprising an amino acid sequence corresponding to the a3 helix of the CC2 region and the SUN domain of a SUN domain-containing protein).

[0139] In some embodiments, LINC complex inhibiting polypeptides may comprise a signal peptide. A signal peptide may be provided N-terminal to the inhibitory region of a LINC complex inhibiting polypeptide according to the present disclosure. In some embodiments, a signal peptide is provided at the N-terminus of the amino acid sequence of the polypeptide. In some embodiments, the signal peptide is not preceded at the N-terminus of the polypeptide by any other amino acids. Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. The signal peptide may be present at the N-terminus of the peptide / polypeptide, and may be present in the newly synthesised peptide / polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature peptide / polypeptide. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176). In some embodiments, an N-terminal signal sequence is derived from a secretory protein or a type I transmembrane protein. In some embodiments, the secretory protein or type I transmembrane protein is selected from: human serum albumin, proinsulin, transferrin receptor, EGF receptor, pre-pro-opiomelanocortin, a carboxypeptidase, a complement protein, fibrinogen, a cytokine, a chemokine, fibrinogen, a pancreatic digestive enzyme (e.g. a protease, amylase or lipase) or an endoplasmic reticulum lumenal protein (e.g. a protein disulphide isomerase or GRP94). In some embodiments the N-terminal signal peptide is derived from human serum albumin.

[0140] In some embodiments, the signal peptide comprises a signal peptidase cleavage site. The signal peptidase cleavage site provides for removal of the signal peptide from the mature polypeptide. In some embodiments, a LINC complex inhibiting polypeptide according to the present disclosure comprises a signal peptidase cleavage site derived from a secretory protein or a type I transmembrane protein, e.g. a secretory protein or a type I transmembrane protein described hereinabove.

[0141] In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88.

[0142] In some embodiments, LINC complex inhibiting polypeptides may comprise a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The LINC complex inhibiting polypeptide may be covalently or non-covalently labelled with the detectable moiety.

[0143] In some embodiments, the detectable moiety is provided at the N-terminus of the polypeptide (either before or after processing of the polypeptide by signal peptidase to remove any signal peptide). In some embodiments, the detectable moiety is provided at the C-terminus of the polypeptide, e.g. downstream of (after) a sequence for preventing secretion of the polypeptide from a cell expressing the polypeptide (e.g. downstream of an ER retention motif).

[0144] In some embodiments, the detectable moiety is or comprises an epitope tag. In some embodiments, an epitope tag is selected from: a haemagglutinin A (HA), ALFA, histidine (His; e.g. 6XHis (SEQ ID NO: 89)), c-Myc, glutathione S-transferase (GST), green fluorescent protein (GFP), maltose-binding protein (MBP), FLAG, E, Biotin, Protein A, Protein G, streptavidin, T7, thioredoxin, V5, or vesicular stomatitis virus glycoprotein (VSV-G) tag. In some embodiments, the detectable moiety is or comprises a moiety having detectable activity, e.g. an enzymatic activity on a given substrate. Examples of such moieties include e.g. horseradish peroxidase (HRP) and luciferase moieties. In some embodiments, a LINC complex inhibiting polypeptide according to the present disclosure is provided with one of the following structures:

[0145] N-term-[signal peptide]-[inhibitory region comprising an amino acid sequence corresponding to the a3 helix of the CC2 region and the SUN domain of a SUN domain-containing protein]-[sequence preventing secretion of the polypeptide from a cell expressing the polypeptide]-C-term

[0146] N-term-[inhibitory region comprising an amino acid sequence corresponding to the a3 helix of the CC2 region and the SUN domain of a SUN domain-containing protein]-[sequence preventing secretion of the polypeptide from a cell expressing the polypeptide]-C-term

[0147] In some embodiments, LINC complex inhibiting polypeptides according to the present disclosure comprise one or more linker sequences between amino acid sequences. In some embodiments, a linker sequence has a length of 1-2, 1-3, 1-4, 1-5 or 1-10 amino acids. In some embodiments, a linker sequence may be provided at one or both ends of one or more of: an inhibitory region comprising an amino acid sequence corresponding to the a3 helix of the CC2 region and the SUN domain of a SUN domaincontaining protein; signal peptide; sequence preventing secretion of the polypeptide from a cell expressing the polypeptide; and / or detectable entity of the LINC complex inhibiting polypeptide.

[0148] Linker sequences are known to the skilled person, and are described e.g. in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and / or serine residues. In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues.

[0149] In preferred embodiments, the LINC complex inhibiting polypeptide is of a size (i.e. in terms of the number of amino acids making up the LINC complex inhibiting polypeptide) permitting delivery of the LINC complex inhibiting polypeptide as a gene therapy, i.e. in the form of nucleic acid encoding the polypeptide.

[0150] In some embodiments, the LINC complex inhibiting polypeptide has a size such that a polynucleotide encoding the polypeptide has a size (i.e. in terms of the number of nucleotides making up the polynucleotide) within the packaging limit of a vector for delivering the polynucleotide. In some embodiments, the LINC complex inhibiting polypeptide has a size such that a polynucleotide encoding the polypeptide has a size within the packaging limit of a vector described herein. In some embodiments, the LINC complex inhibiting polypeptide has a size such that a polynucleotide encoding the polypeptide has a size within the packaging limit of an adeno-associated virus (AAV) vector, e.g. an AAV vector described herein. In some embodiments, the LINC complex inhibiting polypeptide has a size such that a polynucleotide encoding the polypeptide has a size within the packaging limit of a scAAV vector. In some embodiments, the LINC complex inhibiting polypeptide consists of an amino acid sequence comprising fewer than 510 amino acids. In some embodiments, the LINC complex inhibiting polypeptide consists of an amino acid sequence comprising fewer than 457 amino acids.

[0151] In some embodiments, the LINC complex inhibiting polypeptide consists of an amino acid sequence comprising fewer than 600 amino acids, e.g. one of <550, <500, <450, <400, <350, <340, <330, <320, <310, <300, <290, <280, <270, <260, <250, <240, <230, <220 or <210 amino acids.

[0152] LINC complex inhibiting polypeptides according to the present disclosure may be prepared according to methods for the production of polypeptides known to the skilled person. Polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides / polypeptides can be synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety. Alternatively, antigen-binding molecules and polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety. Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety.

[0153] Focal adhesion proteins

[0154] Aspects and embodiments of the present disclosure relates concern increasing / upregulating the expression ( / .e. gene and / or protein expression) of focal adhesion proteins. As used herein, a ‘focal adhesion protein’ is a constituent protein of a focal adhesion.

[0155] Focal adhesion proteins include ponsin, LASP1, metavinculin, vinculin, VASP, vinexin, SORBS2, NEBL, paxillin, a-actinin, aE-catenin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, plastins (e.g. plastin 3), IQGAPs (e.g. IQGAP1), palladin, LPP, testin, zyxin, annexins (e.g. annexin A1, annexin A2, annexin A6, annexin A11 , annexin A5, annexin A7, annexin A8), TGM2, talins (e.g. talin 1 , talin 2), tensins (e.g. tensin 1, tensin 2, tensin 3), FHL3, FHL2, FAKs (e.g. FAK1), PIX, GITs (e.g. GIT1, GIT2), Hic-5, CSK, TRIP6, filamins (e.g. filamin A, filamin B, filamin C), parvins (e.g. parvin A, parvin B), ILK, kindlins (e.g. kindlin 2), Rsu-1 , LIMS1 , LIMS2, migfilin , constituent proteins of integrins (e.g. constituent proteins of a5p1 and avp3 integrins; e.g. CD49e, CD29, CD51 and CD61) and actins (e.g. p actin).

[0156] Ponsin (also known as CAP and SORBS1) is the protein identified by UnitProtKB: Q9BX66; the canonical isoform of human ponsin has the amino acid sequence of Q9BX66-1 (v3, 2011-01-11; SEQ ID NO: 90). LASP1 (also known as LIM and SH3 domain protein 1) is the protein identified by UnitProtKB: Q14847; the canonical isoform of human LASP1 has the amino acid sequence of Q14847-1 (v2, 1998-01-01; SEQ ID NO: 91). Human metavinculin has the amino acid sequence of P18206-1 (v4, 2007-01-23; SEQ ID NO: 92). Human vinculin (also known as VCL) has the amino acid sequence of P18206-2 (SEQ ID NO: 93). VASP (also known as vasodilator-stimulated phosphoprotein) is the protein identified by UnitProtKB: P50552; the canonical isoform of human VASP has the amino acid sequence of P50552-1 (v3, 2007-01- 23). Vinexin (also known as S0RBS3) is the protein identified by UnitProtKB: 060504; the canonical isoform of human vinexin has the amino acid sequence of 060504-1 (v2, 2011-01-11). SORBS2 is the protein identified by UnitProtKB: 094875; the canonical isoform of human SORBS2 has the amino acid sequence of 094875-1 (v3, 2008-07-22). NEBL (also known as nebulette, LIM-NEBL) is the protein identified by UnitProtKB: 076041 ; the canonical isoform of human NEBL has the amino acid sequence of 076041-1 (v1, 1998-11-01). Paxillin (also known as PXN) is the protein identified by UnitProtKB: P49023; the canonical isoform of human paxillin has the amino acid sequence of P49023-1 (v3, 2011-01-11). A-actinin (also known as ACTN1) is the protein identified by UnitProtKB: H0YJW3; the canonical isoform of human a-actinin has the amino acid sequence of H0YJW3 (v2, 2012-02-22). AE-catenin (also known as CTNNA1) is the protein identified by UnitProtKB: P35221 ; the canonical isoform of human aE-catenin has the amino acid sequence of P35221-1 (v1, 1994-02-01). Calponin (also known as CNN1) is the protein identified by UnitProtKB: Q53FP8; the canonical isoform of human aE-catenin has the amino acid sequence of Q53FP8 (v1, 2005-05-24). PDLIM1 (also known as PDZ And LIM Domain 1) is the protein identified by UnitProtKB: 000151; the canonical isoform of human PDLIM1 has the amino acid sequence of 000151 (v4, 2007-01-23). PDLIM5 (also known as PDZ And LIM Domain 5) is the protein identified by UnitProtKB: Q96HC4; the canonical isoform of human PDLIM5 has the amino acid sequence of Q96HC4-1 (v5, 2011-01-11). PDLIM7 (also known as PDZ And LIM Domain 7; LMP-1) is the protein identified by UnitProtKB: Q9NR12; the canonical isoform of human PDLIM7 has the amino acid sequence of Q9NR12-1 (v1, 2000-10-01). Caldesmon (also known as CALD1) is the protein identified by UnitProtKB: Q05682; the canonical isoform of human caldesmon has the amino acid sequence of Q05682-1 (v3, 2011-06-28). Plastin 3 (also known as PLS3) is the protein identified by UnitProtKB: P13797; the canonical isoform of human plastin 3 has the amino acid sequence of P13797-1 (v4, 2009-04-14). IQGAP1 is the protein identified by UnitProtKB: Q05DN7; the canonical isoform of human IQGAP1 has the amino acid sequence of Q05DN7-1 (v1, 2006-11-14). Palladin (also known as PLLD) is the protein identified by UnitProtKB: Q8WX93; the canonical isoform of human palladin has the amino acid sequence of Q8WX93-1 (v3, 2010-11-30). LPP (also known as lipoma-preferred partner) is the protein identified by UnitProtKB: Q93052; the canonical isoform of human LPP has the amino acid sequence of Q93052-1 (v1 , 1997-02-01). Testin (also known as TES) is the protein identified by UnitProtKB: Q9UGI8; the canonical isoform of human palladin has the amino acid sequence of Q9UGI8-1 (v1 , 2000-05-01). Zyxin (also known as ZYX) is the protein identified by UnitProtKB: Q15942; the canonical isoform of human zyxin has the amino acid sequence of Q15942-1 (v1 , 1997-11-01). Annexin A1 (also known as ANXA1) is the protein identified by UnitProtKB: P04083; the canonical isoform of human annexin A1 has the amino acid sequence of P04083-1 (v2, 2007-01-23). Annexin A2 (also known as ANXA2) is the protein identified by UnitProtKB: P07355; the canonical isoform of human annexin A2 has the amino acid sequence of P07355-1 (v2, 2007-01-23). Annexin A6 (also known as ANXA6) is the protein identified by UnitProtKB: P08133; the canonical isoform of human annexin A6 has the amino acid sequence of P08133-1 (v3, 2007-01-23). Annexin A11 (also known as ANXA11) is the protein identified by UnitProtKB: P50995; the canonical isoform of human annexin A11 has the amino acid sequence of P50995-1 (v1 , 1996-10-01). Annexin A5 (also known as ANXA5) is the protein identified by UnitProtKB: P08758; the canonical isoform of human annexin A5 has the amino acid sequence of P08758-1 (v2, 2007-01-23). Annexin A7 (also known as ANXA7) is the protein identified by UnitProtKB: P20073; the canonical isoform of human annexin A7 has the amino acid sequence of P20073-1 (v3, 2008-11-25). Annexin A8 (also known as ANXA8) is the protein identified by UnitProtKB: P13928; the canonical isoform of human annexin A8 has the amino acid sequence of P13928-1 (v3, 2008-11-25). TGM2 (also known as protein-glutamine gammaglutamyltransferase 2) is the protein identified by UnitProtKB: P21980; the canonical isoform of human TGM2 has the amino acid sequence of P21980-1 (v2, 2002-01-23). Talin 1 (also known as TLN1) is the protein identified by UnitProtKB: Q9Y490; the canonical isoform of human talin 1 has the amino acid sequence of Q9Y490-1 (v3, 2005-11-08). Talin 2 (also known as TLN2) is the protein identified by UnitProtKB: Q9Y4G6; the canonical isoform of human talin 2 has the amino acid sequence of Q9Y4G6-1 (v4, 2009-05-05). Tensin 1 (also known as TNS1) is the protein identified by UnitProtKB: Q9HBL0; the canonical isoform of human tensin 1 has the amino acid sequence of Q9HBL0-1 (v3, 2022-12-14). Tensin 2 (also known as TNS2) is the protein identified by UnitProtKB: Q63HR2; the canonical isoform of human tensin 2 has the amino acid sequence of Q63HR2-1 (v2, 2007-06-26). Tensin 3 (also known as TNS3) is the protein identified by UnitProtKB: Q68CZ2; the canonical isoform of human tensin 3 has the amino acid sequence of Q68CZ2-1 (v2, 2007-07-24). FHL3 (also known as four and a half LIM domains protein 3) is the protein identified by UnitProtKB: Q13643; the canonical isoform of human FHL3 has the amino acid sequence of Q13643-1 (v4, 2008-10-14). FHL3 (also known as four and a half LIM domains protein 2) is the protein identified by UnitProtKB: Q14192; the canonical isoform of human FHL2 has the amino acid sequence of Q14192-1 (v3, 2006-10-17). FAK1 (also known as focal adhesion kinase 1, and PTK2) is the protein identified by UnitProtKB: Q05397; the canonical isoform of human FAK1 has the amino acid sequence of Q05397-1 (v2, 1998-07-15). PIX (also known as ARHGEF7) is the protein identified by UnitProtKB: Q14155; the canonical isoform of human PIX has the amino acid sequence of Q14155-1 (v2, 2004-07-19). GIT1 (also known as ARF GTPase-activating protein GIT1) is the protein identified by UnitProtKB: Q9Y2X7; the canonical isoform of human GIT1 has the amino acid sequence of Q9Y2X7-1 (v2, 2004-03-15). GIT2 (also known as ARF GTPase-activating protein GIT2) is the protein identified by UnitProtKB: Q14161 ; the canonical isoform of human GIT2 has the amino acid sequence of Q14161-1 (v2, 2001-10-18). Hic-5 (also known as TGFB1I1) is the protein identified by UnitProtKB: 043294; the canonical isoform of human Hic-5 has the amino acid sequence of 043294-1 (v2, 2007-06-26). CSK (also known as tyrosine-protein kinase CSK) is the protein identified by UnitProtKB: P41240; the canonical isoform of human CSK has the amino acid sequence of P41240-1 (v1 , 1995-02-01). TRIP6 (also known as thyroid receptor-interacting protein 6) is the protein identified by UnitProtKB: Q15654; the canonical isoform of human TRIP6 has the amino acid sequence of Q15654-1 (v3, 2002-05-15). Filamin A (also known as FLNA) is the protein identified by UnitProtKB: P21333; the canonical isoform of human filamin A has the amino acid sequence of P21333-1 (v4, 2007-01-23). Filamin B (also known as FLNB) is the protein identified by UnitProtKB: 075369; the canonical isoform of human filamin B has the amino acid sequence of 075369-1 (v2, 2010-05-18). Filamin C (also known as FLNC) is the protein identified by UnitProtKB: Q14315; the canonical isoform of human filamin C has the amino acid sequence of Q14315-1 (v3, 2009-07-28). Parvin A (also known as PARVA) is the protein identified by UnitProtKB: Q9NVD7; the canonical isoform of human parvin A has the amino acid sequence of Q9NVD7-1 (v1 , 2000-10-01). Parvin B (also known as PARVB) is the protein identified by UnitProtKB: Q9HBI1 ; the canonical isoform of human parvin B has the amino acid sequence of Q9HBI1-1 (v1, 2001-03-01). Kindlin 2 (also known as FERMT2) is the protein identified by UnitProtKB: Q96AC1; the canonical isoform of human kindlin 2 has the amino acid sequence of Q96AC1-1 (v1, 2001-12-01). Rsu-1 (also known as Ras suppressor protein 1) is the protein identified by UnitProtKB: Q15404; the canonical isoform of human Rsu-1 has the amino acid sequence of Q15404-1 (v2, 2007-01-23). LIMS1 (also known as LIM and senescent cell antigen-like domains 1) is the protein identified by UnitProtKB: P48059; the canonical isoform of human LIMS1 has the amino acid sequence of P48059-1 (v4, 2007-01-23). LIMS2 (also known as LIM and senescent cell antigen-like domains 2) is the protein identified by UnitProtKB: Q7Z4I7; the canonical isoform of human LIMS2 has the amino acid sequence of Q7Z4I7-1 (v1, 2003-10-01). Migfilin (also known as FBLIM1) is the protein identified by UnitProtKB: Q8WUP2; the canonical isoform of human migfilin has the amino acid sequence of Q8WUP2-1 (v2, 2007-02-06). CD49e (also known as ITGA5) is the protein identified by UnitProtKB: P08648; the canonical isoform of human CD49e has the amino acid sequence of P08648 -1 (v2, 2002-10-10). CD29 (also known as ITB1) is the protein identified by UnitProtKB: P05556; the canonical isoform of human CD29 has the amino acid sequence of P05556-1 (v2, 2008-12-16). CD51 (also known as ITAV) is the protein identified by UnitProtKB: P06756; the canonical isoform of human CD51 has the amino acid sequence of P06756-1 (v2, 2007-04-03). CD61 (also known as ITB3) is the protein identified by UnitProtKB: P05106; the canonical isoform of human CD61 has the amino acid sequence of P05106-1 (v2, 2007-02-06). p actin (also known as ACTB) is the protein identified by UnitProtKB: P60709; the canonical isoform of human p actin has the amino acid sequence of P60709-1 (v1, 1988-04-01).

[0157] Reference herein to a given protein generally refers to the canonical isoform of the human version of the protein, but also contemplates isoforms, fragments, variants (including mutants) and homologues thereof ( / .e. from other species, e.g. non-human mammalian species (e.g. a non-human primate, e.g. rhesus, cynomolgous; e.g. a rodent, e.g. rat or mouse)). For example, reference herein to ponsin / SORBSI includes both human ponsin isoform 1 (SORBS1iso1; Q9BX66-1, the amino acid sequence of which is shown in SEQ ID NO: 90) and human ponsin isoform 5 (SORBS1iso5; Q9BX66-5, the amino acid sequence of which is shown in SEQ ID NO: 94).

[0158] Horton etal., Nat Cell Biol. (2015) 17(12):1577-1587 categorises proteins of focal adhesions into four theoretical modules based on known signalling axes and links from integrin to actin. Focal adhesion proteins in module 1 include LASP1, VASP, a-actinin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, plastins (e.g. plastin 3), IQGAPs (e.g. IQGAP1), palladin, LPP, testin, zyxin, annexins (e.g. annexin A1, annexin A2, annexin A6, annexin A11 , annexin A5, annexin A7, annexin A8) and TGM2. Focal adhesion proteins in module 2 include metavinculin, vinculin, ponsin, vinexin and talins (e.g. talin 1 , talin 2). Focal adhesion proteins in module 3 include tensins (e.g. tensin 1 , tensin 2, tensin 3), FHL3, FHL2, FAKs (e.g. FAK1), PIX, GITs (e.g. GIT1 , GIT2), Hic-5, CSK and TRIP6. Focal adhesion proteins in module 4 include filamins (e.g. filamin A, filamin B, filamin C), parvin (e.g. parvin A, parvin B), ILK, kindlins (e.g. kindlin 2), Rsu-1, LIMS1, LIMS2, migfilin.

[0159] Horton etal., Nat Cell Biol. (2015) 17(12):1577-1587 also categorises proteins of focal adhesions based on their reported functional properties. Adaptor proteins include ponsin, metavinculin, vinculin, vinexin, SORBS2, paxillin, palladin, LPP, testin, zyxin, talins (e.g. talin 1, talin 2), tensins (e.g. tensin 1, tensin 2, tensin 3), FHL3, FHL2, TRIP6, Hic-5, parvins (e.g. parvin A, parvin B), and kindlins. Actin and actin regulator proteins include LASP1, VASP, a-actinin, filamins (e.g. filamin A, filamin B, filamin C), and actins (e.g. p actin). Adhesion receptor proteins include CD49e, CD29, CD51 and CD61. Kinase proteins include FAKs (e.g. FAK1), CSK and ILK. GTPase / GTPase regulator proteins include GIT (e.g. GIT1, GIT2).

[0160] Horton etal., Nat Cell Biol. (2015) 17(12):1577-1587 also identifies actin-binding proteins of focal adhesions. Actin-binding proteins include LASP1, metavinculin, vinculin, VASP, a-actinin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, plastins (e.g. plastin 3), IQGAPs (e.g. QGAP1), palladin, talins (e.g. talin 1, talin 2), tensins (e.g. tensin 1, tensin 2, tensin 3), FHL3, parvins (e.g. parvin A, parvin B) and filamins (e.g. filamin A, filamin B, filamin C).

[0161] In some embodiments, a focal adhesion protein according to the present disclosure is selected from: ponsin, LASP1, metavinculin, vinculin, VASP, vinexin, SORBS2, NEBL, paxillin, a-actinin, aE-catenin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, a plastin (e.g. plastin 3), an IQGAP (e.g. IQGAP1), palladin, LPP, testin, zyxin, an annexin (e.g. annexin A1 , annexin A2, annexin A6, annexin A11 , annexin A5, annexin A7, annexin A8), TGM2, a talin (e.g. talin 1 , talin 2), a tensin (e.g. tensin 1 , tensin 2, tensin 3), FHL3, FHL2, a FAK (e.g. FAK1), PIX, a GIT (e.g. GIT1, GIT2), Hic-5, CSK, TRIP6, a filamin (e.g. filamin A, filamin B, filamin C), a parvin (e.g. parvin A, parvin B), ILK, a kindlin (e.g. kindlin 2), Rsu-1, LIMS1, LIMS2, migfilin, a constituent protein of an integrins (e.g. a constituent protein of a5p1 integrin or avp3 integrin; e.g. CD49e, CD29, CD51 , CD61) and an actin (e.g. p actin). In some embodiments, a focal adhesion protein according to the present disclosure is selected from: ponsin, LASP1 , metavinculin, vinculin, VASP, vinexin, SORBS2, NEBL, paxillin, a-actinin, aE-catenin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, a plastin (e.g. plastin 3), an IQGAP (e.g. IQGAP1), palladin, LPP, testin, zyxin, an annexin (e.g. annexin A1 , annexin A2, annexin A6, annexin A11 , annexin A5, annexin A7, annexin A8), TGM2, a talin (e.g. talin 1 , talin 2), a tensin (e.g. tensin 1 , tensin 2, tensin 3), FHL3, FHL2, a FAK (e.g. FAK1), PIX, a GIT (e.g. GIT1, GIT2), Hic-5, CSK, TRIP6, a filamin (e.g. filamin A, filamin B, filamin C), a parvin (e.g. parvin A, parvin B), ILK, Rsu-1, LIMS1, LIMS2, a constituent protein of an integrins (e.g. a constituent protein ofa5p1 integrin oravp3 integrin; e.g. CD49e, CD29, CD51, CD61) and an actin (e.g. p actin).

[0162] In some embodiments, a focal adhesion protein is selected from: LASP1, VASP, a-actinin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, a plastin (e.g. plastin 3), an IQGAP (e.g. IQGAP1), palladin, LPP, testin, zyxin, an annexin (e.g. annexin A1 , annexin A2, annexin A6, annexin A11 , annexin A5, annexin A7, annexin A8), TGM2, metavinculin, vinculin, ponsin, vinexin, SORBS2, NEBL and a talin (e.g. talin 1, talin 2). In some embodiments, a focal adhesion protein is selected from: LASP1, VASP, a-actinin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, a plastin (e.g. plastin 3), an IQGAP (e.g. IQGAP1), palladin, LPP, testin, zyxin, an annexin (e.g. annexin A1 , annexin A2, annexin A6, annexin A11 , annexin A5, annexin A7, annexin A8) and TGM2. In some embodiments, a focal adhesion protein is selected from: metavinculin, vinculin, ponsin, vinexin, SORBS2, NEBL and a talin (e.g. talin 1, talin 2).

[0163] In some embodiments, a focal adhesion protein is an adaptor protein, actin or an actin regulator protein. In some embodiments, a focal adhesion protein is selected from: ponsin, metavinculin, vinculin, vinexin, SORBS2, NEBL, paxillin, palladin, LPP, testin, zyxin, a talin (e.g. talin 1 , talin 2), a tensin (e.g. tensin 1 , tensin 2, tensin 3), FHL3, FHL2, TRIP6, Hic-5, a parvin (e.g. parvin A, parvin B), a kindlin (e.g. kindlin 2), LASP1, VASP, a-actinin, a filamin (e.g. filamin A, filamin B, filamin C) and an actin (e.g. p actin). In some embodiments, a focal adhesion protein is selected from: ponsin, metavinculin, vinculin, vinexin, S0RBS2, NEBL, paxillin, palladin, LPP, testin, zyxin, a talin (e.g. talin 1 , talin 2), a tensin (e.g. tensin 1 , tensin 2, tensin 3), FHL3, FHL2, TRIP6, Hic-5, a parvin (e.g. parvin A, parvin B), LASP1, VASP, a-actinin, a filamin (e.g. filamin A, filamin B, filamin C) and an actin (e.g. p actin).

[0164] In some embodiments, a focal adhesion protein is an adaptor protein. In some embodiments, a focal adhesion protein is selected from: ponsin (e.g. SORBS'! iso1 or SORBS'! iso5), metavinculin, vinculin, vinexin, SORBS2, NEBL, paxillin, palladin, LPP, testin, zyxin, a talin (e.g. talin 1 , talin 2), a tensin (e.g. tensin 1, tensin 2, tensin 3), FHL3, FHL2, TRIP6, Hic-5, a parvin (e.g. parvin A, parvin B) and a kindlin (e.g. kindlin 2). In some embodiments, a focal adhesion protein is selected from: ponsin (e.g.

[0165] SORBS'! iso1 or SORBS1iso5), metavinculin, vinculin, vinexin, SORBS2, NEBL, paxillin, palladin, LPP, testin, zyxin, a talin (e.g. talin 1 , talin 2), a tensin (e.g. tensin 1 , tensin 2, tensin 3), FHL3, FHL2, TRIP6, Hic-5, and a parvin (e.g. parvin A, parvin B).

[0166] In some embodiments, a focal adhesion protein is actin or an actin regulator protein. In some embodiments, a focal adhesion protein is selected from: LASP1 , VASP, a-actinin, a filamin (e.g. filamin A, filamin B, filamin C) and an actin (e.g. p actin).

[0167] In some embodiments, a focal adhesion protein is an actin-binding protein. In some embodiments, a focal adhesion protein is selected from: LASP1, metavinculin, vinculin, VASP, a-actinin, calponin, PDLIM1, PDLIM5, PDLIM7, caldesmon, a plastin (e.g. plastin 3), an IQGAP (e.g. IQGAP1), palladin, a talin (e.g. talin 1 , talin 2), a tensin (e.g. tensin 1 , tensin 2, tensin 3), FHL3, a parvin (e.g. parvin A, parvin B) and a filamin (e.g. filamin A, filamin B, filamin C).

[0168] In some embodiments, a focal adhesion protein is selected from: ponsin (e.g. SORBS'! iso1 or SORBS'! iso5), LASP1, metavinculin and vinculin. In some embodiments, a focal adhesion protein is selected from: ponsin (e.g. SORBS'! iso1 or SORBS'! iso5), LASP1 and metavinculin. In some embodiments, a focal adhesion protein is ponsin (e.g. SORBS'! iso1 or SORBS'! iso5). In some embodiments, a focal adhesion protein is LASP1. In some embodiments, a focal adhesion protein is metavinculin or vinculin. In some embodiments, a focal adhesion protein is metavinculin. In some embodiments, a focal adhesion protein is ponsin (e.g. SORBS'! iso1 or SORBS'! iso5) or metavinculin. In some embodiments, a focal adhesion protein according to the present disclosure is not kindlin 2. In some embodiments, a focal adhesion protein according to the present disclosure is not migfilin. In some embodiments, a focal adhesion protein according to the present disclosure is not a kindlin. In some embodiments, a focal adhesion protein according to the present disclosure is not kindlin 2 and is not migfilin. In some embodiments, a focal adhesion protein according to the present disclosure is not a kindlin and is not migfilin.

[0169] In some embodiments, a focal adhesion protein according to the present disclosure is not aE-catenin. In some embodiments, a focal adhesion protein is not p actin. In some embodiments, a focal adhesion protein is not vinculin. In some embodiments, a focal adhesion protein according to the present disclosure is not a catenin. In some embodiments, a focal adhesion protein according to the present disclosure is not an actin. In some embodiments, a focal adhesion protein is not aE-catenin, is not p actin and is not vinculin. In some embodiments, a focal adhesion protein is not a catenin, is not an actin and is not vinculin.

[0170] In some embodiments, the focal adhesion protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:90, 94 or 92. In some embodiments, the focal adhesion protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1 or 94. In some embodiments, the focal adhesion protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 90. In some embodiments, the focal adhesion protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 94. In some embodiments, the focal adhesion protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 91. In some embodiments, the focal adhesion protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 92. In some embodiments, the focal adhesion protein comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 93.

[0171] Particular Exemplary Embodiments

[0172] Particular embodiments of vectors and polynucleotides according to the present invention are described herein.

[0173] In some embodiments, the vector or polynucleotide comprises (in 5’ to 3’ order):

[0174] (i) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 1 ,

[0175] (ii) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 6,

[0176] (iii) a nucleotide sequence encoding an amino acid sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to a sequence selected from SEQ ID NOs: 22 to 86, or 90 to 94.

[0177] In some embodiments, the vector or polynucleotide comprises (in 5’ to 3’ order):

[0178] (i) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 1 , (ii) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 6,

[0179] (iii) a nucleotide sequence encoding an amino acid sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 80.

[0180] In some embodiments, the vector or polynucleotide comprises (in 5’ to 3’ order):

[0181] (i) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 1 ,

[0182] (ii) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 6,

[0183] (iii) a nucleotide sequence encoding an amino acid sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 90.

[0184] In some embodiments, the vector or polynucleotide comprises (in 5’ to 3’ order):

[0185] (i) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 1 ,

[0186] (ii) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 6,

[0187] (iii) a nucleotide sequence encoding an amino acid sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 91.

[0188] In some embodiments, the vector or polynucleotide comprises (in 5’ to 3’ order):

[0189] (i) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 1 ,

[0190] (ii) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 6,

[0191] (iii) a nucleotide sequence encoding an amino acid sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 92.

[0192] In some embodiments, the vector or polynucleotide comprises (in 5’ to 3’ order): (i) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 1,

[0193] (ii) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 6,

[0194] (iii) a nucleotide sequence encoding an amino acid sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 93.

[0195] In some embodiments, the vector or polynucleotide comprises (in 5’ to 3’ order):

[0196] (i) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 1,

[0197] (ii) a nucleotide sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 6,

[0198] (iii) a nucleotide sequence encoding an amino acid sequence comprising or consisting of a sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 94.

[0199] In some embodiments, the vector or polynucleotide comprises an intron after the nucleotide sequence of (ii), optionally followed by a Kozak sequence. In some embodiments the intron selected from: an MVM intron, pCI-Neo intron, an SV40 intron, and a beta-globulin intron. In some embodiments, the vector or polynucleotide comprises an intervening sequence in between the nucleotide sequence of (i) and the nucleotide sequence of (ii), optionally wherein the intervening sequences encodes a restriction enzyme recognition site. In some embodiments, the vector or polynucleotide comprises a polyA sequence after the nucleotide sequence of (iii).

[0200] Diseases / conditions to be treated / prevented

[0201] Aspects of the present invention relate to the treatment of conditions which benefit from expression of a gene in cardiac tissue and / or in the heart, and in particular in cardiomyocytes. The condition may be a condition characterised by cardiomyopathy, dilated cardiomyopathy or cardiac muscular dystrophy.

[0202] Treatment of cardiomyopathies is described in more detail in PCT / SG2024 / 078705, the entire contents of which are incorporated by reference herein.

[0203] Subjects

[0204] A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in humans or animals (veterinary use). The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.

[0205] A subject may have (e.g. may have been diagnosed with) a disease or condition described herein (e.g. a cardiomyopathy), may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation of one or more markers of such a disease / condition, e.g. as described hereinabove. A subject may be suspected of having or suffering from a disease / condition described herein based on the presence of other symptoms indicative of the disease / condition in the subject or in a cell / tissue / organ of the subject. A subject may be considered at risk of developing the disease / condition because of genetic predisposition or other risk factors for the disease.

[0206] In some embodiments, a subject comprises a mutation according to any embodiment described herein. In some embodiments, the subject comprises a mutation giving rise to a disease / condition described herein. In some embodiments, methods according to the present disclosure may comprise determining whether a subject has a disease / condition described herein. In some embodiments the methods comprise diagnosing a disease / condition described herein. Determining whether a subject has a disease / condition described herein may comprise analysing a subject for one or more symptoms / correlates of the disease / condition. Genetic factors may be assayed by methods known to those of ordinary skill in the art, including PCR based and sequencing assays. By determining the presence of genetic factors, e.g. in a sample obtained from a subject, a diagnosis may be confirmed, and / or a subject may be classified as being at risk of developing a disease / condition described herein, and / or a subject may be identified as being suitable for treatment with an agent / composition described herein.

[0207] Assays may be performed in vitro on a sample obtained from a subject, or following processing of a sample obtained from a subject. The sample obtained from a subject may be of any kind. A biological sample may be taken from any tissue or bodily fluid, e.g. a blood sample, blood-derived sample, serum sample, lymph sample, semen sample, saliva sample, synovial fluid sample. A blood-derived sample may be a selected fraction of a patient’s blood, e.g. a selected cell-containing fraction or a plasma or serum fraction. A sample may comprise a tissue sample or biopsy; or cells isolated from a subject.

[0208] Sequence identity

[0209] As used herein, ‘sequence identity” refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame etal. 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, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0210] Sequences

[0211] SEQ ID NO: DESCRIPTION SEQUENCE

[0212] CS-CRM4 AGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATACCTTACATAG 1 CTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTCAGGCTAA AAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATTCGCTGATAC AGTCTGTCCGA SK-CRM3 ATGGAGACAATCCATGAATTCCTGAGATGCTTGGCTGGTATTAGATTTTATGGGCAGCTGC TTATTCTTAGGGCTCTGCTTCTCCAAAGACACTGAGGAAGTCCAAAGGaAACACCAGCTG GCGAAGAGCCACCTCCAGGCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCC

[0213] 2 AGAGGGCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGTGGGCATTT GGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCAACTGTCCCCATTGTGGGGCCCT ACCTTCCAAGGCCGGTTTACACCTCCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCT CACTGACAAATGCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGCTC TGACACCCCTCAG

[0214] cTnT-129 (269- GTTTTGCTATTTTAAACCCGTCGGACGGAGATACGTGAGTGCCCGAGGGGCTGACACAAG 201)neg CCAGCCAGTCCGCTGCTGCCAAAATAGCAGCTCACAAGTGTTGCATTCCTCTCTGGGCGC 3 CGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGTGGGCGCCGGGGGGACCTTAAAG CCTCTGCCCCCCAAGGAGCCCTTCCCAGACAGCCGCCGGCACCCACCGCTCCGTGGGA C CTCAGTCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTACCCCCAGCATCAGTTCAAGT GGAGCAGCACATAACTCTTGCCCTCTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAG TGTCTTGGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCTGGCCCTCCCAGGCCTG GGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGA

[0215] 4 hTNNT2(-502 to CTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCACAT +42) GGGCTTATATGGCGTGGGGTACATGTTCCTGTAGCCTTGTCCCTGGCACCTGCCAAAATA GCAGCCAACACCCCCCACCCCCACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCAC ATTCCTCCCTCCGCAGGGCTGGCTCACCAGGCCCCAGCCCACATGCCTGCTTAAAGCCC TCTCCATCCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCAGGATCTGT CGGCAG CGGGCCCCCCCTCGAGGTCGGGATAAAAGCAGTCTGGGCTTTCACATGACAGCATCTGG GGCTGCGGCAGAGGGTCGGGTCCGAAGCGCTGCCTTATCAGCGTCCCCAGCCCTGGGA GGTGACAGCTGGCTGGCTTGTGTCAGCCCCTCGGGCACTCACGTATCTCCGTCCGACGG

[0216] 5 Original cTnT (442) GTTTAAAATAGCAAAACTCTGAGGCCACACAATAGCTTGGGCTTATATGGGCTCCTGTGG GGGAAGGGGGAGCACGGAGGGGGCCGGGGCCGCTGCTGCCAAAATAGCAGCTCACAA GTGTTGCATTCCTCTCTGGGCGCCGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGT GGGCGCCGGGGGGACCTTAAAGCCTCTGCCCCCCAAGGAGCCCTTCCCAGACAGCCGC CGGCACCCACCGCTCCGTGGGACGATCCCCGA CTGCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCA AGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATA GCTTATCTGCTAGCCTGCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCCTCTGCT

[0217] 6 Synthetic hTNNT2 TTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCATATCTG CTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCACATGGGCTTATATGGGGCAC CTGCCAAAATAGCAGCCAACACCCCCCCCTGTCGCACATTCCTCCCTGGCTCACCAGGC CCCAGCCCACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGTCCCCGCTGA GACTGAGCAGACGCCTCCAGGATCTGTCGGCAGCT TTGACTGGAAGTTCTACCTTGTATCTGGCCTCCTGTAGCAGTTTCAGTCCATTCCCTGTGA GGAGGGTGTGCCACATGGCTTTGGGGGTCATGGAGAAGACCCACCTTGCAGATGTCCTC ACTGGGGCTGGCAGAGCCGGCAACCTGCCTAAGGCTGCTCAGTCCATTAGGAGCCAGTA GCCTGGAAGATGTCTTTACCCCCAGCATCAGTTCAAGTGGAGCAGCACATAACTCTTGCC CTCTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAGTGTCTTGGAGGTTGCCTTCTGC

[0218] 7 TNNT2 CCCCCAACCCTGCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATC AGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCATATCTGCTCTG GTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCACATGGGCTTATATGGCGTGGGGTAC ATGTTCCTGTAGCCTTGTCCCTGGCACCTGCCAAAATAGCAGCCAACACCCCCCACCCCC ACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCACATTCCTCCCTCCGCAGGGCTGG CTCACCAGGCCCCAGCCCACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAG TCCCCGCTGAGACTGAGCAGACGCCTCCAGGATCTGTCGGCAGCTGCTGTTCTG CMV IE (608) GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGC 8 CCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC

[0219]

[0220] AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATT AGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCG GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTG GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTC AGATC

[0221] cTnT (-555 to +38) AGATCTGCTCTTGCTTGTTTGGCTTTGGGTAGGACTTGATTTTCTTTAGGACTGGGTTGTT TTCTAATAAAAGTGGCTCCTTCTGAAACGTTCCTGCTCGGCTGGGACCTGAAGGACTCGC ATCCCATCATCCTGCAAGAGGAAAAAAGCCGAAATTTAAGACAAATCTCTCAGGGTGGGG GATAAAAGAAGTCTGGGCTTTCACATGACAGCATCTGGGGCTGCAGCAGAGGGTCGGGT CCGAAGCGCTGCCTTATCAGCGTCCCCAGCCCTGGGAGGTGACAGCTGGCTGGCTTGTG TCAGCCCCTCGGGCACTCACGTATCTCCGTCCGACGGGTTTAAAATAGCAAAACTCTGAG GCCACACAATAGCTTGGGCTTATATGGGCTCCTGTGGGGGAAGGGGGAGCACGGAGGG GGCCGGGGCCGCTGCTGCCAAAATAGCAGCTCACAAGTGTTGCATTCCTCTCTGGGCGC CGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGTGGGCGCCGGGGGGACCTTAAAG CCTCTGCCCCCCAAGGAGCCCTTCCCAGACAGCCGCCGGCACCCACCGCTCCGTGGGA C NVC-001 CTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT TTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCAT CACTAGGGGTTCCTGCGGCCGCCCTAGGAGTAGAAAAACAGCCAAGCTAGGGAGGCTGG GAGGCCAAGCCCCAGATACCTTACATAGCTCTGCTCAGCCTCTGTCTCATTAGGAACTCC ATTTTTAGGATGCAGTTGTTTCAGGCTAAAAATAAATCATGCAATGAATAAAAAAGTTAGAT ACGACACTGTAGAGGGATTCGCTGATACAGTCTGTCCGATGTACACTGCTCCCAGCTGGC CCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGT TTATGTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGCTAGCCT GCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAG AGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGC TTATCTGAGCAGCTGGAGGACCACATGGGCTTATATGGGGCACCTGCCAAAATAGCAGCC AACACCCCCCCCTGTCGCACATTCCTCCCTGGCTCACCAGGCCCCAGCCCACATGCCTG CTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTC CAGGATCTGTCGGCAGCTGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAG AAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTT ACTGACATCCACTTTGCCTTTCTCTCCACAGGCCACCATGAAGTGGGTAACCTTTATTTCC CTTCTCTTCCTCTTCTCCTCTGCCTATTCCGGTGAGCAACTCCTGCCCACAGTGGAGCAC CTCCAGCTGGAGCTGGATCAGCTAAAGTCAGAGCTGTCCAGCTGGCGACATGTGAAGAC TGGCTGTGAGACAGTGGATGCTGTACAAGAAAGAGTGGATGTGCAAGTCAGAGAAATGGT GAAACTCCTGTTTAGTGAAGATCAGCAAGGGGGTTCTCTGGAACAGCTGCTGCAGAGGTT CTCATCACAGTTTGTGAGCAAAGGGGACTTGCAGACAATGCTGAGGGACCTGCAGCTGC AGATCCTGAGGAATGTCACCCACCATGTTTCTGTGACCAAGCAGCTCCCAACCTCAGAAG CTGTGGTGTCTGCTGTGTCTGAGGCTGGGGCATCTGGAATAACAGAGGCACAAGCAAGG GCCATTGTGAACTCTGCCTTGAAGCTGTATTCCCAAGATAAGACTGGGATGGTGGACTTT GCTCTGGAATCTGGTGGTGGCAGCATCTTGAGTACTAGGTGTTCTGAAACTTATGAAACC AAAACTGCACTGATGAGTCTGTTTGGGATCCCACTGTGGTACTTCTCACAGTCCCCAAGG GTGGTCATCCAGCCTGACATTTACCCTGGTAACTGCTGGGCATTTAAAGGCTCCCAGGGG TACCTGGTGGTGAGGCTCTCCATGATGATCCACCCAGCTGCCTTCACTCTGGAGCACATC CCTAAGACACTGTCACCAACAGGCAACATCAGCTCTGCCCCCAAGGACTTTGCTGTCTAT GGATTAGAAAATGAGTATCAGGAAGAAGGGCAGCTTCTGGGACAGTTCACATATGATCAG GATGGGGAGTCACTCCAGATGTTCCAGGCCCTGAAAAGACCTGATGACACAGCTTTCCAA ATAGTGGAACTTAGGATTTTTTCTAACTGGGGCCATCCTGAGTATACCTGTCTGTATAGGT TCAGAGTTCATGGGGAACCTGTCAAGAAAGATGAGTTGTAGATCAACCTCTGGATTACAAA ATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGC TGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGT ATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCG TGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGT CAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCC GCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGT GTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCAACTGGATCCT GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCTCTCAATCCAGCGGACCTCCCTTCCC GAGGCCTTCTGCCGGTTCTGCGGCCTCTCCCGCGTCTTCGCTTTCGGCCTCCGACGAGT CGGATCTCCCTTTGGGCCGCCTCCCCGCCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTT GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA TGCGGTGGGCTCTATGGGTCGACGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCA CTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACG CCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCC AGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATACCTTACATAG CS-CRM4 / TNNT2 CTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTCAGGCTAA

[0222] AAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATTCGCTGATAC

[0223]

[0224] AGTCTGTCCGATGTACATTGACTGGAAGTTCTACCTTGTATCTGGCCTCCTGTAGCAGTTT CAGTCCATTCCCTGTGAGGAGGGTGTGCCACATGGCTTTGGGGGTCATGGAGAAGACCC ACCTTGCAGATGTCCTCACTGGGGCTGGCAGAGCCGGCAACCTGCCTAAGGCTGCTCAG TCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTACCCCCAGCATCAGTTCAAGTGGAGC AGCACATAACTCTTGCCCTCTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAGTGTCTT GGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGC TGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCC CTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCACATGGGCTTA TATGGCGTGGGGTACATGTTCCTGTAGCCTTGTCCCTGGCACCTGCCAAAATAGCAGCCA ACACCCCCCACCCCCACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCACATTCCTC CCTCCGCAGGGCTGGCTCACCAGGCCCCAGCCCACATGCCTGCTTAAAGCCCTCTCCAT CCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCAGGATCTGTCGGCAG CTGCTGTTCTG CS-CRM4 / cTnT- AGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATACCTTACATAG 129 (269-201 )neg CTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTCAGGCTAA AAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATTCGCTGATAC AGTCTGTCCGATGTACAGTTTTGCTATTTTAAACCCGTCGGACGGAGATACGTGAGTGCC CGAGGGGCTGACACAAGCCAGCCAGTCCGCTGCTGCCAAAATAGCAGCTCACAAGTGTT GCATTCCTCTCTGGGCGCCGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGTGGGC GCCGGGGGGACCTTAAAGCCTCTGCCCCCCAAGGAGCCCTTCCCAGACAGCCGCCGGC ACCCACCGCTCCGTGGGAC CS- AGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATACCTTACATAG CRM4 / hTNNT2(- CTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTCAGGCTAA 502 to +42) AAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATTCGCTGATAC AGTCTGTCCGATGTACACTCAGTCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTACCC CCAGCATCAGTTCAAGTGGAGCAGCACATAACTCTTGCCCTCTGCCTTCCAAGATTCTGG TGCTGAGACTTATGGAGTGTCTTGGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCT GGCCCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGC TGGTTTATGTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAGC AGCTGGAGGACCACATGGGCTTATATGGCGTGGGGTACATGTTCCTGTAGCCTTGTCCCT GGCACCTGCCAAAATAGCAGCCAACACCCCCCACCCCCACCGCCATCCCCCTGCCCCAC CCGTCCCCTGTCGCACATTCCTCCCTCCGCAGGGCTGGCTCACCAGGCCCCAGCCCACA TGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGCAGA CGCCTCCAGGATCTGTCGGCAG CS-CRM4 / Original AGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATACCTTACATAG cTnT (442) CTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTCAGGCTAA AAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATTCGCTGATAC AGTCTGTCCGATGTACACGGGCCCCCCCTCGAGGTCGGGATAAAAGCAGTCTGGGCTTT CACATGACAGCATCTGGGGCTGCGGCAGAGGGTCGGGTCCGAAGCGCTGCCTTATCAGC GTCCCCAGCCCTGGGAGGTGACAGCTGGCTGGCTTGTGTCAGCCCCTCGGGCACTCAC GTATCTCCGTCCGACGGGTTTAAAATAGCAAAACTCTGAGGCCACACAATAGCTTGGGCT TATATGGGCTCCTGTGGGGGAAGGGGGAGCACGGAGGGGGCCGGGGCCGCTGCTGCC AAAATAGCAGCTCACAAGTGTTGCATTCCTCTCTGGGCGCCGGGCACATTCCTGCTGGCT CTGCCCGCCCCGGGGTGGGCGCCGGGGGGACCTTAAAGCCTCTGCCCCCCAAGGAGCC CTTCCCAGACAGCCGCCGGCACCCACCGCTCCGTGGGACGATCCCCGA CS- AGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATACCTTACATAG CRM4 / Synthetic CTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTCAGGCTAA hTNNT2 AAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATTCGCTGATAC AGTCTGTCCGATGTACACTGCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCCTCT GCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCATAT CTGCTCTGGTTTTAAATAGCTTATCTGCTAGCCTGCTCCCAGCTGGCCCTCCCAGGCCTG GGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGA CTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCACAT GGGCTTATATGGGGCACCTGCCAAAATAGCAGCCAACACCCCCCCCTGTCGCACATTCCT CCCTGGCTCACCAGGCCCCAGCCCACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTC ACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCAGGATCTGTCGGCAGCT SK-CRM3 / CMV IE ATGGAGACAATCCATGAATTCCTGAGATGCTTGGCTGGTATTAGATTTTATGGGCAGCTGC (608) TTATTCTTAGGGCTCTGCTTCTCCAAAGACACTGAGGAAGTCCAAAGGaAACACCAGCTG GCGAAGAGCCACCTCCAGGCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCC AGAGGGCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGTGGGCATTT GGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCAACTGTCCCCATTGTGGGGCCCT ACCTTCCAAGGCCGGTTTACACCTCCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCT CACTGACAAATGCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGCTC TGACACCCCTCAGTGTACAGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAAT GACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACT TGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACA CCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACG TCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCC

[0225]

[0226] CGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCGTTTAGTGAACCGTCAGATC

[0227] SK-CRM3 / cTnT- ATGGAGACAATCCATGAATTCCTGAGATGCTTGGCTGGTATTAGATTTTATGGGCAGCTGC 129 (269-201 )neg TTATTCTTAGGGCTCTGCTTCTCCAAAGACACTGAGGAAGTCCAAAGGaAACACCAGCTG GCGAAGAGCCACCTCCAGGCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCC AGAGGGCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGTGGGCATTT GGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCAACTGTCCCCATTGTGGGGCCCT ACCTTCCAAGGCCGGTTTACACCTCCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCT CACTGACAAATGCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGCTC TGACACCCCTCAGTGTACAGTTTTGCTATTTTAAACCCGTCGGACGGAGATACGTGAGTG CCCGAGGGGCTGACACAAGCCAGCCAGTCCGCTGCTGCCAAAATAGCAGCTCACAAGTG TTGCATTCCTCTCTGGGCGCCGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGTGGG CGCCGGGGGGACCTTAAAGCCTCTGCCCCCCAAGGAGCCCTTCCCAGACAGCCGCCGG CACCCACCGCTCCGTGGGAC SK-CRM3 / cTnT (- ATGGAGACAATCCATGAATTCCTGAGATGCTTGGCTGGTATTAGATTTTATGGGCAGCTGC 555 to +38) TTATTCTTAGGGCTCTGCTTCTCCAAAGACACTGAGGAAGTCCAAAGGaAACACCAGCTG GCGAAGAGCCACCTCCAGGCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCC AGAGGGCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGTGGGCATTT GGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCAACTGTCCCCATTGTGGGGCCCT ACCTTCCAAGGCCGGTTTACACCTCCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCT CACTGACAAATGCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGCTC TGACACCCCTCAGTGTACAAGATCTGCTCTTGCTTGTTTGGCTTTGGGTAGGACTTGATTT TCTTTAGGACTGGGTTGTTTTCTAATAAAAGTGGCTCCTTCTGAAACGTTCCTGCTCGGCT GGGACCTGAAGGACTCGCATCCCATCATCCTGCAAGAGGAAAAAAGCCGAAATTTAAGAC AAATCTCTCAGGGTGGGGGATAAAAGAAGTCTGGGCTTTCACATGACAGCATCTGGGGCT GCAGCAGAGGGTCGGGTCCGAAGCGCTGCCTTATCAGCGTCCCCAGCCCTGGGAGGTG ACAGCTGGCTGGCTTGTGTCAGCCCCTCGGGCACTCACGTATCTCCGTCCGACGGGTTT AAAATAGCAAAACTCTGAGGCCACACAATAGCTTGGGCTTATATGGGCTCCTGTGGGGGA AGGGGGAGCACGGAGGGGGCCGGGGCCGCTGCTGCCAAAATAGCAGCTCACAAGTGTT GCATTCCTCTCTGGGCGCCGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGTGGGC GCCGGGGGGACCTTAAAGCCTCTGCCCCCCAAGGAGCCCTTCCCAGACAGCCGCCGGC ACCCACCGCTCCGTGGGAC SK- ATGGAGACAATCCATGAATTCCTGAGATGCTTGGCTGGTATTAGATTTTATGGGCAGCTGC CRM3 / hTNNT2(- TTATTCTTAGGGCTCTGCTTCTCCAAAGACACTGAGGAAGTCCAAAGGaAACACCAGCTG 502 to +42) GCGAAGAGCCACCTCCAGGCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCC AGAGGGCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGTGGGCATTT GGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCAACTGTCCCCATTGTGGGGCCCT ACCTTCCAAGGCCGGTTTACACCTCCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCT CACTGACAAATGCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGCTC TGACACCCCTCAGTGTACACTCAGTCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTAC CCCCAGCATCAGTTCAAGTGGAGCAGCACATAACTCTTGCCCTCTGCCTTCCAAGATTCT GGTGCTGAGACTTATGGAGTGTCTTGGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAG CTGGCCCTCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACA GCTGGTTTATGTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGA GCAGCTGGAGGACCACATGGGCTTATATGGCGTGGGGTACATGTTCCTGTAGCCTTGTC CCTGGCACCTGCCAAAATAGCAGCCAACACCCCCCACCCCCACCGCCATCCCCCTGCCC CACCCGTCCCCTGTCGCACATTCCTCCCTCCGCAGGGCTGGCTCACCAGGCCCCAGCCC ACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGC AGACGCCTCCAGGATCTGTCGGCAG SK-CRM3 / TNNT2 ATGGAGACAATCCATGAATTCCTGAGATGCTTGGCTGGTATTAGATTTTATGGGCAGCTGC TTATTCTTAGGGCTCTGCTTCTCCAAAGACACTGAGGAAGTCCAAAGGaAACACCAGCTG GCGAAGAGCCACCTCCAGGCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCC AGAGGGCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGTGGGCATTT GGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCAACTGTCCCCATTGTGGGGCCCT ACCTTCCAAGGCCGGTTTACACCTCCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCT CACTGACAAATGCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGCTC TGACACCCCTCAGTGTACATTGACTGGAAGTTCTACCTTGTATCTGGCCTCCTGTAGCAG TTTCAGTCCATTCCCTGTGAGGAGGGTGTGCCACATGGCTTTGGGGGTCATGGAGAAGA CCCACCTTGCAGATGTCCTCACTGGGGCTGGCAGAGCCGGCAACCTGCCTAAGGCTGCT CAGTCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTACCCCCAGCATCAGTTCAAGTGG AGCAGCACATAACTCTTGCCCTCTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAGTG TCTTGGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCTGGCCCTCCCAGGCCTGGG TTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGACTG TTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCACATGGG CTTATATGGCGTGGGGTACATGTTCCTGTAGCCTTGTCCCTGGCACCTGCCAAAATAGCA GCCAACACCCCCCACCCCCACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCACATT CCTCCCTCCGCAGGGCTGGCTCACCAGGCCCCAGCCCACATGCCTGCTTAAAGCCCTCT CCATCCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCAGGATCTGTCG GCAGCTGCTGTTCTG SK- ATGGAGACAATCCATGAATTCCTGAGATGCTTGGCTGGTATTAGATTTTATGGGCAGCTGC CRM3 / Synthetic TTATTCTTAGGGCTCTGCTTCTCCAAAGACACTGAGGAAGTCCAAAGGaAACACCAGCTG hTNNT2 GCGAAGAGCCACCTCCAGGCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCC

[0228]

[0229] AGAGGGCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGTGGGCATTT GGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCAACTGTCCCCATTGTGGGGCCCT ACCTTCCAAGGCCGGTTTACACCTCCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCT CACTGACAAATGCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGCTC TGACACCCCTCAGTGTACACTGCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCCT CTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCAT ATCTGCTCTGGTTTTAAATAGCTTATCTGCTAGCCTGCTCCCAGCTGGCCCTCCCAGGCC TGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTATGTTGCAT GACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGACCAC ATGGGCTTATATGGGGCACCTGCCAAAATAGCAGCCAACACCCCCCCCTGTCGCACATTC CTCCCTGGCTCACCAGGCCCCAGCCCACATGCCTGCTTAAAGCCCTCTCCATCCTCTGCC TCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCAGGATCTGTCGGCAGCT

[0230] Human SUN1 CC2 ITEAQARAIVNSALKL

[0231] a3

[0232] Human SUN2 CC2

[0233] a3 VTEEQVHHIVKQALQR

[0234] Human SUN1 SUN GGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLWRLSM domain MIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQAL KRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPV

[0235] Human SUN2 SUN GASVISTRCSETYETKTALLSLFGIPLWYHSQSPRVILQPDVHPGNCWAFQGPQGFAWRLSA domain RIRPTAVTLEHVPKALSPNSTISSAPKDFAIFGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQA PTMATYQVVELRILTNWGHPEYTCIYRFRVHGEPA SUN1(616-812) GITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFS QSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVY GLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFR VHGEPVK SUN2(522-717) GVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYHS QSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAIF GFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRILTNWGHPEYTCIYRFRVH GE PAH

[0236] Human SUN1 CC2 TSEAVVSAVSEA

[0237] a2

[0238] Human SUN2 CC2 AAASLSLTLQKE

[0239] a2

[0240] Human SUN1 CC2 KGDLQTMLRDLQLQILRNVTHHVSV

[0241] a1

[0242] Human SUN2 CC2

[0243] a1 REEMQAQLRELESKILTHVAEMQGK

[0244] Human SUN1 CC1 GEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGG SLEQLLQR

[0245] Human SUN2 CC1 QESFQESSVKELRRLED QLAGLQQELAALALKQSSVAEEVGLLPQQIQAVRDDVESQFPAWISQFLA

[0246] Human SUN1 CC1, GEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGG CC2 a1 , CC2 a2, SLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGIT CC2 a3 EAQARAIVNSALKL

[0247] Human SUN2 CC1, QESFQESSVKELRRLEDQLAGLQQELAALALKQSSVAEEVGLLPQQIQAVRDDVESQFPAWIS CC2 a1 , CC2 a2, QFLARGGGGRVGLLQREEMQAQLRELESKILTHVAEMQGKSAREAAASLSLTLQKEGVIGVT CC2 a3 EEQVHHIVKQALQR

[0248] Human SUN1 CC2 KGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKL a1, CC2 a2, CC2

[0249] a3

[0250] Human SUN2 CC2 REEMQAQLRELESKILTHVAEMQGKSAREAAASLSLTLQKEGVIGVTEEQVHHIVKQALQR a1, CC2 a2, CC2

[0251] a3

[0252] Human SUN1 CC2 TSEAVVSAVSEAGASGITEAQARAIVNSALKL

[0253] a2, CC2 a3

[0254] Human SUN2 CC2 AAASLSLTLQKEGVIGVTEEQVHHIVKQALQR

[0255] a2, CC2 a3

[0256] Human SUN1 MDFSRLHMYSPPQCVPENTGYTYALSSSYSSDALDFETEHKLDPVFDSPRMSRRSLRLATTA CTLGDGEAVGADSGTSSAVSLKNRAARTTKQRRSTNKSAFSINHVSRQVTSSGVSHGGTVSL QDAVTRRPPVLDESWIREQTTVDHFWGLDDDGDLKGGNKAAIQGNGDVGAAAATAHNGFSC SNCSMLSERKDVLTAHPAAPGPVSRVYSRDRNQKCDDCKGKRHLDAHPGRAGTLWHIWAC AGYFLLQILRRIGAVGQAVSRTAWSALWLAVVAPGKAASGVFWWLGIGWYQFVTLISWLNVF LLTRCLRNICKFLVLLIPLFLLLAGLSLRGQGNFFSFLPVLNWASMHRTQRVDDPQDVFKPTTS RLKQPLQGDSEAFPWHWMSGVEQQVASLSGQCHHHGENLRELTTLLQKLQARVDQMEGGA AGPSASVRDAVGQPPRETDFMAFHQEHEVRMSHLEDILGKLREKSEAIQKELEQTKQKTISAV GEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGG SLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGIT EAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQS PRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGL ENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVH GEPVK

[0257]

[0258] Human SUN2 MSRRSQRLTRYSQGDDDGSSSSGGSSVAGSQSTLFKDSPLRTLKRKSSNMKRLSPAPQLGP SSDAHTSYYSESLVHESWFPPRSSLEELHGDANWGEDLRVRRRRGTGGSESSRASGLVGR KATEDFLGSSSGYSSEDDYVGYSDVDQQSSSSRLRSAVSRAGSLLWMVATSPGRLFRLLYW WAGTTWYRLTTAASLLDVFVLTRRFSSLKTFLWFLLPLLLLTCLTYGAWYFYPYGLQTFHPALV SWWAAKDSRRPDEGWEARDSSPHFQAEQRVMSRVHSLERRLEALAAEFSSNWQKEAMRL ERLELRQGAPGQGGGGGLSHEDTLALLEGLVSRREAALKEDFRRETAARIQEELSALRAEHQ QDSEDLFKKIVRASQESEARIQQLKSEWQSMTQESFQESSVKELRRLEDQLAGLQQELAALA LKQSSVAEEVGLLPQQIQAVRDDVESQFPAWISQFLARGGGGRVGLLQREEMQAQLRELESK ILTHVAEMQGKSAREAAASLSLTLQKEGVIGVTEEQVHHIVKQALQRYSEDRIGLADYALESGG ASVISTRCSETYETKTALLSLFGIPLWYHSQSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARI RPTAVTLEHVPKALSPNSTISSAPKDFAIFGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPT MATYQWELRILTNWGHPEYTCIYRFRVHGEPAH

[0259] Human SUN1

[0260] partial SMART coil- LEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMV coiled 2, CC1 , CC2 KLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVS a1, CC2 a2, CC2 AVSEAGASGITEAQARAIVNSALKL

[0261] a3

[0262] Human SUN2

[0263] partial SMART coil- IQQLKSEWQSMTQESFQESSVKELRRLEDQLAGLQQELAALALKQSSVAEEVGLLPQQIQAV coiled 2, CC1 , CC2 RDDVESQFPAWISQFLARGGGGRVGLLQREEMQAQLRELESKILTHVAEMQGKSAREAAASL a1, CC2 a2, CC2 SLTLQKEGVIGVTEEQVHHIVKQALQR

[0264] a3

[0265] SUN1 (360-812); DDPQDVFKPTTSRLKQPLQGDSEAFPWHWMSGVEQQVASLSGQCHHHGENLRELTTLLQKL DNSUN1 QARVDQMEGGAAGPSASVRDAVGQPPRETDFMAFHQEHEVRMSHLEDILGKLREKSEAIQK ELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREM VKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAW SAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSL FGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNIS SAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHP EYTCLYRFRVHGEPVK SUN2(258-717); DEGWEARDSSPHFQAEQRVMSRVHSLERRLEALAAEFSSNWQKEAMRLERLELRQGAPGQ DNSUN2 GGGGGLSHEDTLALLEGLVSRREAALKEDFRRETAARIQEELSALRAEHQQDSEDLFKKIVRA SQESEARIQQLKSEWQSMTQESFQESSVKELRRLEDQLAGLQQELAALALKQSSVAEEVGLL PQQIQAVRDDVESQFPAWISQFLARGGGGRVGLLQREEMQAQLRELESKILTHVAEMQGKSA REAAASLSLTLQKEGVIGVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYE TKTALLSLFGIPLWYHSQSPRVILQPDVHPGNCWAFQGPQGFAWRLSARIRPTAVTLEHVPK ALSPNSTISSAPKDFAIFGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRIL TNWGHPEYTCIYRFRVHGEPAH DNSUN1 w / SP and MKWVTFISLLFLFSSAYSRGVFRRDGPALDDPQDVFKPTTSRLKQPLQGDSEAFPWHWMSG KDEL VEQQVASLSGQCHHHGENLRELTTLLQKLQARVDQMEGGAAGPSASVRDAVGQPPRETDF MAFHQEHEVRMSHLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSE LSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQT MLRDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTG MVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQ GYLVVRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDG ESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL MKWVTFISLLFLFSSAYSRGVFRRDGPALDEGWEARDSSPHFQAEQRVMSRVHSLERRLEAL AAEFSSNWQKEAMRLERLELRQGAPGQGGGGGLSHEDTLALLEGLVSRREAALKEDFRRET AARIQEELSALRAEHQQDSEDLFKKIVRASQESEARIQQLKSEWQSMTQESFQESSVKELRRL DNSUN2 w / SP and EDQLAGLQQELAALALKQSSVAEEVGLLPQQIQAVRDDVESQFPAWISQFLARGGGGRVGLL KDEL QREEMQAQLRELESKILTHVAEMQGKSAREAAASLSLTLQKEGVIGVTEEQVHHIVKQALQRY SEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYHSQSPRVILQPDVHPGNCWA FQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAIFGFDEDLQQEGTLLGKFTY DQDGEPIQTFHFQAPTMATYQWELRILTNWGHPEYTCIYRFRVHGEPAHKDEL SHEDTLALLEGLVSRREAALKEDFRRETAARIQEELSALRAEHQQDSEDLFKKIVRASQESEA RIQQLKSEWQSMTQESFQESSVKELRRLEDQLAGLQQELAALALKQSSVAEEVGLLPQQIQA SUN2(324-717) VRDDVESQFPAWISQFLARGGGGRVGLLQREEMQAQLRELESKILTHVAEMQGKSAREAAA (Zhou et al., 2012) SLSLTLQKEGVIGVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALL SLFGIPLWYHSQSPRVILQPDVHPGNCWAFQGPQGFAWRLSARIRPTAVTLEHVPKALSPNS TISSAPKDFAIFGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQWELRILTNWGHP EYTCIYRFRVHGEPAH LVSRREAALKEDFRRETAARIQEELSALRAEHQQDSEDLFKKIVRASQESEARIQQLKSEWQS MTQESFQESSVKELRRLEDQLAGLQQELAALALKQSSVAEEVGLLPQQIQAVRDDVESQFPA SUN2(335-717) WISQFLARGGGGRVGLLQREEMQAQLRELESKILTHVAEMQGKSAREAAASLSLTLQKEGVI (Sosa et al., 2012) GVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYHS QSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAIF GFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRILTNWGHPEYTCIYRFRVH GE PAH MTQESFQESSVKELRRLEDQLAGLQQELAALALKQSSVAEEVGLLPQQIQAVRDDVESQFPA SUN2(398-717) WISQFLARGGGGRVGLLQREEMQAQLRELESKILTHVAEMQGKSAREAAASLSLTLQKEGVI (Zhou et al., 2012) GVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYHS

[0266] QSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAIF

[0267]

[0268] GFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRILTNWGHPEYTCIYRFRVH GE PAH SSVAEEVGLLPQQIQAVRDDVESQFPAWISQFLARGGGGRVGLLQREEMQAQLRELESKILT SUN2(433-717) HVAEMQGKSAREAAASLSLTLQKEGVIGVTEEQVHHIVKQALQRYSEDRIGLADYALESGGAS (Zhou et al., 2012) VISTRCSETYETKTALLSLFGIPLWYHSQSPRVILQPDVHPGNCWAFQGPQGFAWRLSARIR PTAVTLEHVPKALSPNSTISSAPKDFAIFGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTM ATYQVVELRILTNWGHPEYTCIYRFRVHGEPAH RGGGGRVGLLQREEMQAQLRELESKILTHVAEMQGKSAREAAASLSLTLQKEGVIGVTEEQV SUN2(467-717) HHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYHSQSPRVILQ (Wang et al., 2012) PDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAIFGFDEDLQ QEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRILTNWGHPEYTCIYRFRVHGEPAH AAASLSLTLQKEGVIGVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETK SUN2(507-717) TALLSLFGIPLWYHSQSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKAL (Sosa et al., 2012) SPNSTISSAPKDFAIFGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRILTN WGHPEYTCIYRFRVHGEPAH TLQKEGVIGVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLF SUN2(514-717) GIPLWYHSQSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTIS (Sosa et al., 2012) SAPKDFAIFGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQWELRILTNWGHPEY TCIYRFRVHGEPAH VIGVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYH SUN2(520-717) SQSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAI (Zhou et al., 2012) FGFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQWELRILTNWGHPEYTCIYRFRV HGEPAH IGVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYHS SUN2(521 -717) QSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAIF (Sosa et al., 2012) GFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRILTNWGHPEYTCIYRFRVH GE PAH DDSKGMHRPGPLPPSPPPKVDHKASQWPQESDMGQKVASLSAQCHNHDERLAELTVLLQKL QIRVDQVDDGREGLSLWVKNWGQHLQEMGTIEPPDAKTDFMTFHHDHEVRLSNLEDVLRKL

[0269] Sun1 (458-913); TEKSEAIQKELEETKLKAGSRDEEQPLLDRVQHLELELNLLKSQLSDWQHLKTSCEQAGARIQ DnSunl (Crisp et ETVQLMFSEDQQGGSLEWLLEKLSSRFVSKDELQVLLHDLELKLLQNITHHITVTGQAPTSEAI al., 2006) VSAVNQAGISGITEAQAHIIVNNALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALLSL FGVPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLWRLSMKIYPTTFTMEHIPKTLSPTGNIS SAPKDFAVYGLETEYQEEGQPLGRFTYDQEGDSLQMFHTLERPDQAFQIVELRVLSNWGHP EYTCLYRFRVHGEPIQ SRFVSKDELQVLLHDLELKLLQNITHHITVTGQAPTSEAIVSAVNQAGISGITEAQAHIIVNNALK

[0270] Sun1 (668-913) (Xu LYSQDKTGMVDFALESGGGSILSTRCSETYETKTALLSLFGVPLWYFSQSPRWIQPDIYPGN et al., 2018) CWAFKGSQGYLVVRLSMKIYPTTFTMEHIPKTLSPTGNISSAPKDFAVYGLETEYQEEGQPLG RFTYDQEGDSLQMFHTLERPDQAFQIVELRVLSNWGHPEYTCLYRFRVHGEPIQ LLLLLGAGVSLWGQGNFFSLLPVLNWTAMQPTQRVDDSKGMHRPGPLPPSPPPKVDHKASQ WPQESDMGQKVASLSAQCHNHDERLAELTVLLQKLQIRVDQVDDGREGLSLWVKNWGQHL QEMGTIEPPDAKTDFMTFHHDHEVRLSNLEDVLRKLTEKSEAIQKELEETKLKAGSRDEEQPL

[0271] Sun2(410-731) LDRVQHLELELNLLKSQLSDWQHLKTSCEQAGARIQETVQLMFSEDQQGGSLEWLLEKLSSR (Nie et al., 2016) FVSKDELQVLLHDLELKLLQNITHHITVTGQAPTSEAIVSAVNQAGISGITEAQAHIIVNNALKLYS QDKTGMVDFALESGGGSILSTRCSETYETKTALLSLFGVPLWYFSQSPRVVIQPDIYPGNCWA FKGSQGYLWRLSMKIYPTTFTMEHIPKTLSPTGNISSAPKDFAVYGLETEYQEEGQPLGRFTY DQEGDSLQMFHTLERPDQAFQIVELRVLSNWGHPEYTCLYRFRVHGEPIQ PSPPPKVDHKASQWPQESDMGQKVASLSAQCHNHDERLAELTVLLQKLQIRVDQVDDGREG LSLWVKNVVGQHLQEMGTIEPPDAKTDFMTFHHDHEVRLSNLEDVLRKLTEKSEAIQKELEET KLKAGSRDEEQPLLDRVQHLELELNLLKSQLSDWQHLKTSCEQAGARIQETVQLMFSEDQQG

[0272] Sun2(458-731) GSLEWLLEKLSSRFVSKDELQVLLHDLELKLLQNITHHITVTGQAPTSEAIVSAVNQAGISGITE (Jahed et al., 2016) AQAHIIVNNALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALLSLFGVPLWYFSQSPR WIQPDIYPGNCWAFKGSQGYLVVRLSMKIYPTTFTMEHIPKTLSPTGNISSAPKDFAVYGLET EYQEEGQPLGRFTYDQEGDSLQMFHTLERPDQAFQIVELRVLSNWGHPEYTCLYRFRVHGE PIQ ASLSAQCHNHDERLAELTVLLQKLQIRVDQVDDGREGLSLWVKNVVGQHLQEMGTIEPPDAK TDFMTFHHDHEVRLSNLEDVLRKLTEKSEAIQKELEETKLKAGSRDEEQPLLDRVQHLELELNL

[0273] Sun2(482-731) LKSQLSDWQHLKTSCEQAGARIQETVQLMFSEDQQGGSLEWLLEKLSSRFVSKDELQVLLHD (Nie et al., 2016) LELKLLQNITHHITVTGQAPTSEAIVSAVNQAGISGITEAQAHIIVNNALKLYSQDKTGMVDFALE SGGGSILSTRCSETYETKTALLSLFGVPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLWRL SMKIYPTTFTMEHIPKTLSPTGNISSAPKDFAVYGLETEYQEEGQPLGRFTYDQEGDSLQMFH TLERPDQAFQIVELRVLSNWGHPEYTCLYRFRVHGEPIQ HHHGENLRELTTLLQKLQARVDQMEGGAAGPSASVRDAVGQPPRETDFMAFHQEHEVRMS HLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCE TVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNV SUN1 (404-812) THHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSI LSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHP AAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPD DTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVK HQEHEVRMSHLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSS SUN1 (455-812) WRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTML

[0274] RDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMV

[0275]

[0276] DFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGY LVVRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESL QMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVK GEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGG SLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGIT SUN1 (495-812) EAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQS PRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGL ENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVH GEPVK QRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAWSAVSEAGASGITEAQARA SUN1 (562-812) IVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQP DIYPGNCWAFKGSQGYLVVRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQE EGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVK TSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETK SUN1(601-812) TALMSLFGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLS PTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFS NWGHPEYTCLYRFRVHGEPVK DKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRVVIQPDIYPGNCWAF SUN1 (636-812) KGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTY DQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVK HHHGENLRELTTLLQKLQARVDQMEGGAAGPSASVRDAVGQPPRETDFMAFHQEHEVRMS HLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCE SUN1 (404-812) TVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNV w / KDEL THHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSI LSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHP AAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPD DTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL HQEHEVRMSHLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSS WRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTML SUN1 (455-812) RDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMV w / KDEL DFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGY LVVRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESL QMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL GEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGG SLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVSAVSEAGASGIT SUN1 (495-812) EAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQS w / KDEL PRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGL ENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVH GEPVKKDEL QRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAWSAVSEAGASGITEAQARA SUN1 (562-812) IVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQP w / KDEL DIYPGNCWAFKGSQGYLVVRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQE EGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKK DEL TSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETK SUN1(601-812) TALMSLFGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLS w / KDEL PTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFS NWGHPEYTCLYRFRVHGEPVKKDEL GITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFS SUN1(616-812) QSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVY w / KDEL GLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFR VHGEPVKKDEL GVTEEQVHHIVKQALQRYSEDRIGLADYALESGGASVISTRCSETYETKTALLSLFGIPLWYHS SUN2(522-717) QSPRVILQPDVHPGNCWAFQGPQGFAVVRLSARIRPTAVTLEHVPKALSPNSTISSAPKDFAIF w / KDEL GFDEDLQQEGTLLGKFTYDQDGEPIQTFHFQAPTMATYQVVELRILTNWGHPEYTCIYRFRVH GEPAHKDEL SUN1 (483-812) LEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMV KLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVS AVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLF GIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISS APKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPE YTCLYRFRVHGEPVK SUN1 (483-812) LEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMV w / KDEL KLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVVS AVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLF GIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISS APKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPE YTCLYRFRVHGEPVKKDEL SUN1 (360-812) MKVWTFISLLFLFSSAYSDDPQDVFKPTTSRLKQPLQGDSEAFPWHWMSGVEQQVASLSGQ w / SP, KDEL CHHHGENLRELTTLLQKLQARVDQMEGGAAGPSASVRDAVGQPPRETDFMAFHQEHEVRM SHLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGC ETVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRN

[0277]

[0278] VTHHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGS ILSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHP AAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPD DTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL SUN1 (404-812) MKWVTFISLLFLFSSAYSHHHGENLRELTTLLQKLQARVDQMEGGAAGPSASVRDAVGQPPR w / SP, KDEL ETDFMAFHQEHEVRMSHLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQ LKSELSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKG DLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAWSAVSEAGASGITEAQARAIVNSALKLYSQD KTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFK GSQGYLVVRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYD QDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL SUN1 (455-812) MKWVTFISLLFLFSSAYSHQEHEVRMSHLEDILGKLREKSEAIQKELEQTKQKTISAVGEQLLP w / SP, KDEL TVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQVREMVKLLFSEDQQGGSLEQLL QRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAWSAVSEAGASGITEAQARA IVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQP DIYPGNCWAFKGSQGYLVVRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQE EGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKK DEL SUN1 (495-812) MKWVTFISLLFLFSSAYSGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQERVDVQ w / SP, KDEL VREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPT SEAWSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKT ALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSP TGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSN WGHPEYTCLYRFRVHGEPVKKDEL SUN1 (483-812) MKWVTFISLLFLFSSAYSLEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCET w / SP, KDEL VDAVQERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVT HHVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSIL STRCSETYETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPA AFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDD TAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL SUN1 (562-812) MKWVTFISLLFLFSSAYSQRFSSQFVSKGDLQTMLRDLQLQILRNVTHHVSVTKQLPTSEAVV w / SP, KDEL SAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETYETKTALMSL FGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNIS SAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHP EYTCLYRFRVHGEPVKKDEL SUN1 (601-812) MKWVTFISLLFLFSSAYSTSEAWSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALE w / SP, KDEL SGGGSILSTRCSETYETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLVVRL SMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQ ALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL SUN1 (616-812) MKWVTFISLLFLFSSAYSGITEAQARAIVNSALKLYSQDKTGMVDFALESGGGSILSTRCSETY w / SP, KDEL ETKTALMSLFGIPLWYFSQSPRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPK TLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELR IFSNWGHPEYTCLYRFRVHGEPVKKDEL SUN1 (636-812) MKWVTFISLLFLFSSAYSDKTGMVDFALESGGGSILSTRCSETYETKTALMSLFGIPLWYFSQS w / SP, KDEL PRWIQPDIYPGNCWAFKGSQGYLWRLSMMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGL ENEYQEEGQLLGQFTYDQDGESLQMFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVH GEPVKKDEL DDPQDVFKPTTSRLKQPLQGDSEAFPWHWMSGVEQQVASLSGQCHHHGENLRELTTL LQKLQARVDQMEGGAAGPSASVRDAVGQPPRETDFMAFHQEHEVRMSHLEDILGKLR EKSEAIQKELEQTKQKTISAVGEQLLPTVEHLQLELDQLKSELSSWRHVKTGCETVDAVQ SUN1(360-812) ERVDVQVREMVKLLFSEDQQGGSLEQLLQRFSSQFVSKGDLQTMLRDLQLQILRNVTH w / KDEL HVSVTKQLPTSEAVVSAVSEAGASGITEAQARAIVNSALKLYSQDKTGMVDFALESGGG SILSTRCSETYETKTALMSLFGIPLWYFSQSPRVVIQPDIYPGNCWAFKGSQGYLVVRLS MMIHPAAFTLEHIPKTLSPTGNISSAPKDFAVYGLENEYQEEGQLLGQFTYDQDGESLQ MFQALKRPDDTAFQIVELRIFSNWGHPEYTCLYRFRVHGEPVKKDEL

[0279] Signal Peptide MKWVTFISLLFLFSSAYSRGVFRRD

[0280] Minimal human MKWVTFISLLFLFSSAYS

[0281] serum albumin

[0282] signal peptide

[0283] 6xHis tag HHHHHH

[0284] Human ponsin MSSECDGGSKAVMNGLAPGSNGQDKATADPLRARSISAVKIIPVKTVKNASGLVLPTDMDLTK isoform 1 ICTGKGAVTLRASSSYRETPSSSPASPQETRQHESKPGLEPEPSSADEWRLSSSADANGNAQ (SORBS1iso1; PSSLAAKGYRSVHPNLPSDKSQDATSSSAAQPEVIVVPLYLVNTDRGQEGTARPPTPLGPLG Q9BX66-1 v3, CVPTIPATASAASPLTFPTLDDFIPPHLQRWPHHSQPARASGSFAPISQTPPSFSPPPPLVPPA 2011-01-11) PEDLRRVSEPDLTGAVSSTDSSPLLNEVSSSLIGTDSQAFPSVSKPSSAYPSTTIVNPTIVLLQH NREQQKRLSSLSDPVSERRVGEQDSAPTQEKPTSPGKAIEKRAKDDSRRWKSTQDLSDVS MDEVGIPLRNTERSKDWYKTMFKQIHKLNRDTPEENPYFPTYKFPELPEIQQTSEEDNPYTPT YQFPASTPSPKSEDDDSDLYSPRYSFSEDTKSPLSVPRSKSEMSYIDGEKWKRSATLPLPAR SSSLKSSSERNDWEPPDKKVDTRKYRAEPKSIYEYQPGKSSVLTNEKMSRDISPEEIDLKNEP WYKFFSELEFGKPPPKKIWDYTPGDCSILPREDRKTNLDKDLSLCQTELEADLEKMETLNKAP SANVPQSSAISPTPEISSETPGYIYSSNFHAVKRESDGAPGDLTSLENERQIYKSVLEGGDIPL

[0285]

[0286] QGLSGLKRPSSSASTKDSESPRHFIPADYLESTEEFIRRRHDDKEKLLADQRRLKREQEEADIA ARRHTGVIPTHHQFITNERFGDLLNIDDTAKRKSGSEMRPARAKFDFKAQTLKELPLQKGDIVY IYKQIDQNWYEGEHHGRVGIFPRTYIELLPPAEKAQPKKLTPVQVLEYGEAIAKFNFNGDTQVE MSFRKGERITLLRQVDENWYEGRIPGTSRQGIFPITYVDVIKRPLVKNPVDYMDLPFSSSPSRS ATASPQFSSHSKLITPAPSSLPHSRRALSPEMHAVTSEWISLTVGVPGRRSLALTPPLPPLPEA SIYNTDHLALSPRASPSLSLSLPHLSWSDRPTPRSVASPLALPSPHKTYSLAPTSQASLHMNG DGGVHTPSSGIHQDSFLQLPLGSSDSVISQLSDAFSSQSKRQPWREESGQYERKAERGAGE RGPGGPKISKKSCLKPSDVVRCLSTEQRLSDLNTPEESRPGKPLGSAFPGSEAEQTERHRGG EQAGRKAARRGGSQQPQAQQRRVTPDRSQTSQDLFSYQALYSYIPQNDDELELRDGDIVDV MEKCDDGWFVGTSRRTKQFGTFPGNYVKPLYL

[0287] Human LASP1 MNPNCARCGKIVYPTEKVNCLDKFWHKACFHCETCKMTLNMKNYKGYEKKPYCNAHYPKQS (Q14847-1 v2, FTMVADTPENLRLKQQSELQSQVRYKEEFEKNKGKGFSVVADTPELQRIKKTQDQISNIKYHE 1998-01-01) EFEKSRMGPSGGEGMEPERRDSQDGSSYRRPLEQQQPHHIPTSAPVYQQPQQQPVAQSYG GYKEPAAPVSIQRSAPGGGGKRYRAVYDYSAADEDEVSFQDGDTIVNVQQIDDGWMYGTVE RTGDTGMLPANYVEAI

[0288] Human MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKETVQT metavinculin TEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTSDLLLTFDE (P18206-1 v4, AEVRKIIRVCKGILEYLTVAEWETMEDLVTYTKNLGPGMTKMAKMIDERQQELTHQEHRVML 2007-01-23) VNSMNTVKELLPVLISAMKIFVTTKNSKNQGIEEALKNRNFTVEKMSAEINEIIRVLQLTSWDED AWASKDTEAMKRALASIDSKLNQAKGWLRDPSASPGDAGEQAIRQILDEAGKVGELCAGKER REILGTCKMLGQMTDQVADLRARGQGSSPVAMQKAQQVSQGLDVLTAKVENAARKLEAMTN SKQSIAKKIDAAQNWLADPNGGPEGEEQIRGALAEARKIAELCDDPKERDDILRSLGEISALTS KLADLRRQGKGDSPEARALAKQVATALQNLQTKTNRAVANSRPAKAAVHLEGKIEQAQRWID NPTVDDRGVGQAAIRGLVAEGHRLANVMMGPYRQDLLAKCDRVDQLTAQLADLAARGEGES PQARALASQLQDSLKDLKARMQEAMTQEVSDVFSDTTTPIKLLAVAATAPPDAPNREEVFDER AANFENHSGKLGATAEKAAAVGTANKSTVEGIQASVKTARELTPQVVSAARILLRNPGNQAAY EHFETMKNQWIDNVEKMTGLVDEAIDTKSLLDASEEAIKKDLDKCKVAMANIQPQMLVAGATSI ARRANRILLVAKREVENSEDPKFREAVKAASDELSKTISPMVMDAKAVAGNISDPGLQKSFLD SGYRILGAVAKVREAFQPQEPDFPPPPPDLEQLRLTDELAPPKPPLPEGEVPPPRPPPPEEKD EEFPEQKAGEVINQPMMMAARQLHDEARKWSSKPGIPAAEVGIGVVAEADAADAAGFPVPPD MEDDYEPELLLMPSNQPVNQPILAAAQSLHREATKWSSKGNDIIAAAKRMALLMAEMSRLVR GGSGTKRALIQCAKDIAKASDEVTRLAKEVAKQCTDKRIRTNLLQVCERIPTISTQLKILSTVKAT MLGRTNISDEESEQATEMLVHNAQNLMQSVKETVREAEAASIKIRTDAGFTLRWVRKTPWYQ

[0289] Human vinculin MPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVAAVQAAVSNLVRVGKETVQT (P18206-2) TEDQILKRDMPPAFIKVENACTKLVQAAQMLQSDPYSVPARDYLIDGSRGILSGTSDLLLTFDE AEVRKIIRVCKGILEYLTVAEWETMEDLVTYTKNLGPGMTKMAKMIDERQQELTHQEHRVML VNSMNTVKELLPVLISAMKIFVTTKNSKNQGIEEALKNRNFTVEKMSAEINEIIRVLQLTSWDED AWASKDTEAMKRALASIDSKLNQAKGWLRDPSASPGDAGEQAIRQILDEAGKVGELCAGKER REILGTCKMLGQMTDQVADLRARGQGSSPVAMQKAQQVSQGLDVLTAKVENAARKLEAMTN SKQSIAKKIDAAQNWLADPNGGPEGEEQIRGALAEARKIAELCDDPKERDDILRSLGEISALTS KLADLRRQGKGDSPEARALAKQVATALQNLQTKTNRAVANSRPAKAAVHLEGKIEQAQRWID NPTVDDRGVGQAAIRGLVAEGHRLANVMMGPYRQDLLAKCDRVDQLTAQLADLAARGEGES PQARALASQLQDSLKDLKARMQEAMTQEVSDVFSDTTTPIKLLAVAATAPPDAPNREEVFDER AANFENHSGKLGATAEKAAAVGTANKSTVEGIQASVKTARELTPQVVSAARILLRNPGNQAAY EHFETMKNQWIDNVEKMTGLVDEAIDTKSLLDASEEAIKKDLDKCKVAMANIQPQMLVAGATSI ARRANRILLVAKREVENSEDPKFREAVKAASDELSKTISPMVMDAKAVAGNISDPGLQKSFLD SGYRILGAVAKVREAFQPQEPDFPPPPPDLEQLRLTDELAPPKPPLPEGEVPPPRPPPPEEKD EEFPEQKAGEVINQPMMMAARQLHDEARKWSSKGNDIIAAAKRMALLMAEMSRLVRGGSGT KRALIQCAKDIAKASDEVTRLAKEVAKQCTDKRIRTNLLQVCERIPTISTQLKILSTVKATMLGRT NISDEESEQATEMLVHNAQNLMQSVKETVREAEAASIKIRTDAGFTLRWVRKTPWYQ

[0290] Human ponsin MSSECDGGSKAVMNGLAPGSNGQDKATADPLRARSISAVKIIPVKTVKNASGLVLPTDMDLTK isoform 5 ICTGKGAVTLRASSSYRETPSSSPASPQETRQHESKPDEWRLSSSADANGNAQPSSLAAKGY (SORBS1 iso5; RSVHPNLPSDKSQDATSSSAAQPEVIVVPLYLVNTDRGQEGTARPPTPLGPLGCVPTIPATAS Q9BX66-5) AASPLTFPTLDDFIPPHLQRWPHHSQPARASGSFAPISQTPPSFSPPPPLVPPAPEDLRRVSE PDLTGAVSSTDSSPLLNEVSSSLIGTDSQAFPSVSKPSSAYPSTTIVNPTIVLLQHNREQQKRL SSLSDPVSERRVGEQDSAPTQEKPTSPGKAIEKRAKDDSRRVVKSTQDLSDVSMDEVGIPLR NTERSKDWYKTMFKQIHKLNRDTPEENPYFPTYKFPELPEIQQTSEDDDSDLYSPRYSFSEDT KSPLSVPRSKSEMSYIDGEKVVKRSATLPLPARSSSLKSSSERNDWEPPDKKVDTRKYRAEP KSIYEYQPGKSSVLTNEKMSRDISPEEIDLKNEPWYKFFSELEFGKPPPKKIWDYTPGDCSILP REDRKTNLDKDLSLCQTELEADLEKMETLNKAPSANVPQSSAISPTPEISSETPGYIYSSNFHA VKRESDGAPGDLTSLENERQIYKSVLEGGDIPLQGLSGLKRPSSSASTKDSESPRHFIPADYLE STEEFIRRRHDDKEKLLADQRRLKREQEEADIAARRHTGVIPTHHQFITNERFGDLLNIDDTAK RKSGSEMRPARAKFDFKAQTLKELPLQKGDIVYIYKQIDQNWYEGEHHGRVGIFPRTYIELLPP AEKAQPKKLTPVQVLEYGEAIAKFNFNGDTQVEMSFRKGERITLLRQVDENWYEGRIPGTSR QGIFPITYVDVIKRPLVKNPVDYMDLPFSSSPSRSATASPQQPQAQQRRVTPDRSQTSQDLFS YQALYSYIPQNDDELELRDGDIVDVMEKCDDGWFVGTSRRTKQFGTFPGNYVKPLYL MGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSAFRCNTQQ

[0291] Human CX43 PGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLYLAHVFYVMRKEEKLNKKEEELKVAQT DGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFEVAFLLIQWYIYGFSL

[0292] (P17302 v2, 2007- SAVYTCKRDPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNIIELFYVFFKGVKDRVKGK 01-23) SDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNY

[0293] NKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIV

[0294]

[0295] DQRPSSRASSRASSRPRPDDLEI AGTAGAAAAACAGCCAAGCTAGGGAGGCTGGGAGGCCAAGCCCCAGATACCTTACA TAGCTCTGCTCAGCCTCTGTCTCATTAGGAACTCCATTTTTAGGATGCAGTTGTTTCA GGCTAAAAATAAATCATGCAATGAATAAAAAAGTTAGATACGACACTGTAGAGGGATT CGCTGATACAGTCTGTCCGAtgtacactgctcccagctggccctcccaggcctgggtgctggcctctgcttatc aggattctcaagagggacagctggttatgttgcatgactgttccctgcatatctgctctggttaaatagctatctgctagcctg ctcccagctggccctcccaggcctgggtgctggcctctgcttatcaggatctcaagagggacagctggttatgtgcatgac tgttccctgcatatctgctctggttttaaatagctatctgagcagctggaggaccacatgggctatatggggcacctgccaaaa tagcagccaacacccccccctgtcgcacattcctccctggctcaccaggccccagcccacatgcctgctaaagccctctcc atcctctgcctcacccagtccccgctgagactgagcagacgcctccaggatctgtcggcagctaggtacgatagcgcctcca gctaaaagagctaaaagaggtaagggtttaagggacggtggttggtggggtataacgttaattacctgttttacaggcctg aaatcacttggttttaggtggGCCACCATGCCAGTGTTTCATACGCGCACGATCGAGAGCATC CTGGAGCCGGTGGCACAGCAGATCTCCCACCTGGTGATAATGCACGAGGAGGGCG AGGTGGACGGCAAAGCCATTCCTGACCTCACCGCGCCCGTGGCCGCCGTGCAGGC GGCCGTCAGCAACCTCGTCCGGGTTGGAAAAGAGACTGTTCAAACCACTGAGGATC AGATTTTGAAGAGAGATATGCCACCAGCATTTATTAAGGTTGAGAATGCTTGCACCAA GCTTGTCCAGGCAGCTCAGATGCTTCAGTCAGACCCTTACTCAGTGCCTGCTCGAG ATTATCTAATTGATGGGTCAAGGGGCATCCTCTCTGGAACATCAGACCTGCTCCTTA CCTTCGATGAGGCTGAGGTCCGTAAAATTATTAGAGTTTGCAAAGGAATTTTGGAATA TCTTACAGTGGCAGAGGTGGTGGAGACTATGGAAGATTTGGTCACTTACACAAAGAA TCTTGGGCCAGGAATGACTAAGATGGCCAAGATGATTGACGAGAGACAGCAGGAGC TCACTCACCAGGAGCACCGAGTGATGTTGGTGAACTCGATGAACACCGTGAAAGAG TTGCTGCCAGTTCTCATTTCAGCTATGAAGATTTTTGTAACAACTAAAAACTCAAAAAA CCAAGGCATAGAGGAAGCTTTAAAAAATCGCAA I l l i ACTGTAGAAAAAATGAGTGCT GAAATTAATGAGATAATTCGTGTGTTACAACTCACCTCTTGGGATGAAGATGCCTGG GCCAGCAAGGACACTGAAGCCATGAAGAGAGCATTGGCCTCCATAGACTCCAAACT GAACCAGGCCAAAGGTTGGCTCCGTGACCCTAGTGCCTCCCCAGGGGATGCTGGT GAGCAGGCCATCAGACAGATCTTAGATGAAGCTGGAAAAGTTGGTGAACTCTGTGC AGGCAAAGAACGCAGGGAGATTCTGGGAACTTGCAAAATGCTAGGGCAGATGACTG ATCAAGTGGCTGACCTCCGTGCCAGAGGACAAGGATCCTCACCGGTGGCCATGCAG AAAGCTCAGCAGGTATCTCAGGGTCTGGATGTGCTCACAGCAAAAGTGGAAAATGC AGCTCGCAAGCTGGAAGCCATGACCAACTCAAAGCAGAGCATTGCAAAGAAGATCG PL785 (CS-CRM4 ATGCTGCTCAGAACTGGCTTGCAGATCCAAATGGTGGACCGGAAGGAGAAGAGCAG ATTCGAGGTGCTTTGGCTGAAGCTCGGAAAATAGCAGAATTATGTGATGATCCTAAA

[0296] Syn-hTNNT2 GAAAGAGATGACATTCTACGTTCCCTTGGGGAAATATCTGCTCTGACTTCTAAATTAG Mvcn-A) CAGATCTACGAAGACAGGGGAAAGGAGATTCTCCAGAGGCTCGAGCCTTGGCCAAA CAGGTGGCCACGGCCCTGCAGAACCTGCAGACCAAAACCAACCGGGCTGTGGCCA ACAGCAGACCGGCCAAAGCAGCTGTACACCTTGAGGGCAAGATTGAGCAAGCACAG CGGTGGATTGATAATCCCACAGTGGATGACCGTGGAGTCGGTCAGGCTGCCATCCG GGGGCTTGTGGCCGAAGGGCATCGTCTGGCTAATGTTATGATGGGGCCTTATCGGC AAGATCTTCTCGCCAAGTGTGACCGAGTGGACCAGCTGACAGCCCAGCTGGCTGAC CTGGCTGCCAGAGGGGAAGGGGAGAGTCCTCAGGCACGAGCACTTGCATCTCAGC TCCAAGACTCCTTAAAGGATCTAAAAGCTCGGATGCAGGAGGCCATGACTCAGGAA GTGTCAGATGTTTTCAGCGATACCACAACTCCCATCAAGCTGTTGGCAGTGGCAGCC ACGGCGCCTCCTGATGCGCCTAACAGGGAAGAGGTATTTGATGAGAGGGCAGCTAA CTTTGAAAACCATTCAGGAAAGCTTGGTGCTACGGCCGAGAAGGCGGCTGCGGTTG GTACTGCTAATAAATCAACAGTGGAAGGCATTCAGGCCTCAGTGAAGACGGCCCGA GAACTCACACCCCAGGTGGTCTCGGCTGCTCGTATCTTACTTAGGAACCCTGGAAAT CAAGCTGCTTATGAACATTTTGAGACCATGAAGAACCAGTGGATCGATAATGTTGAA AAAATGACAGGGCTGGTGGACGAAGCCATTGATACCAAATCTCTGTTGGATGCTTCA GAAGAAGCAATTAAAAAAGACCTGGACAAGTGCAAGGTAGCTATGGCCAACATTCAG CCTCAGATGCTGGTTGCTGGGGCAACCAGTATTGCTCGTCGGGCCAACCGGATCCT GCTGGTGGCTAAGAGGGAGGTGGAGAATTCCGAGGATCCCAAGTTCCGTGAGGCT GTGAAAGCTGCCTCTGATGAATTGAGCAAAACCATCTCCCCGATGGTGATGGATGCA AAAGCTGTGGCTGGAAACATTTCCGACCCTGGACTGCAAAAGAGCTTCCTGGACTCA GGATATCGGATCCTGGGAGCTGTGGCCAAGGTCAGAGAAGCCTTCCAACCTCAGGA GCCTGACTTCCCGCCGCCTCCACCAGACCTTGAACAACTCCGACTAACAGATGAGC TTGCTCCTCCCAAACCACCTCTGCCTGAAGGTGAGGTCCCTCCACCTAGGCCTCCA CCACCAGAGGAAAAGGATGAAGAGTTCCCTGAGCAGAAGGCCGGGGAGGTGATTAA CCAGCCAATGATGATGGCTGCCAGACAGCTCCATGATGAAGCTCGCAAATGGTCCA GCAAGCCGGGCATCCCAGCCGCTGAGGTGGGTATAGGTGTTGTAGCTGAGGCAGA TGCGGCCGATGCTGCTGGCTTCCCTGTCCCCCCTGACATGGAAGACGATTACGAAC CTGAGCTGCTGTTAATGCCATCCAATCAGCCGGTCAACCAGCCCATTCTGGCCGCG GCTCAGTCCTTGCATCGGGAAGCTACCAAGTGGTCTAGTAAGGGCAATGACATCATT GCAGCAGCCAAGCGCATGGCTCTGCTGATGGCTGAGATGTCTCGGCTGGTAAGAG GGGGCAGTGGTACCAAGCGGGCACTCATTCAGTGTGCCAAGGACATCGCCAAGGC CTCAGATGAGGTGACTCGGTTGGCCAAGGAGGTTGCCAAGCAGTGCACAGATAAAC GGATTAGAACCAACCTCTTACAGGTATGTGAGCGAATCCCAACCATAAGCACCCAGC TCAAAATCCTGTCCACAGTGAAGGCCACCATGCTGGGCCGGACCAACATCAGTGAT

[0297]

[0298] GAGGAGTCTGAGCAGGCCACAGAGATGCTGGTTCACAATGCCCAGAACCTCATGCA GTCTGTGAAGGAGACTGTGCGGGAAGCTGAAGCTGCTTCAATCAAAATTCGAACAG ATGCTGGATTTACACTGCGCTGGGTTAGAAAGACTCCCTGGTACCAGTAGaataaaaga tcCttattttcattGgatctgtgtgttggttttttgtgtg cgggccccccctcgaggtcgggataaaagcagtctgggcttcacatgacagcatctggggctgcggcagagggtcgggt ccgaagcgctgcctatcagcgtccccagccctgggaggtgacagctggctggctgtgtcagcccctcgggcactcacgta tctccgtccgacgggttaaaatagcaaaactctgaggccacacaatagcttgggcttatatgggctcctgtgggggaaggg ggagcacggagggggccggggccgctgctgccaaaatagcagctcacaagtgtgcatcctctctgggcgccgggcac attcctgctggctctgcccgccccggggtgggcgccggggggacctaaagcctctgccccccaaggagcccttcccagac agccgccggcacccaccgctccgtgggacgatccccgaGCCACCATGCCAGTGTTTCATACGCGCAC GATCGAGAGCATCCTGGAGCCGGTGGCACAGCAGATCTCCCACCTGGTGATAATGC ACGAGGAGGGCGAGGTGGACGGCAAAGCCATTCCTGACCTCACCGCGCCCGTGGC CGCCGTGCAGGCGGCCGTCAGCAACCTCGTCCGGGTTGGAAAAGAGACTGTTCAAA CCACTGAGGATCAGA I l l i GAAGAGAGATATGCCACCAGCATTTATTAAGGTTGAGA ATGCTTGCACCAAGCTTGTCCAGGCAGCTCAGATGCTTCAGTCAGACCCTTACTCAG TGCCTGCTCGAGATTATCTAATTGATGGGTCAAGGGGCATCCTCTCTGGAACATCAG ACCTGCTCCTTACCTTCGATGAGGCTGAGGTCCGTAAAATTATTAGAGTTTGCAAAG GAATTTTGGAATATCTTACAGTGGCAGAGGTGGTGGAGACTATGGAAGATTTGGTCA CTTACACAAAGAATCTTGGGCCAGGAATGACTAAGATGGCCAAGATGATTGACGAGA GACAGCAGGAGCTCACTCACCAGGAGCACCGAGTGATGTTGGTGAACTCGATGAAC ACCGTGAAAGAGTTGCTGCCAGTTCTCATTTCAGCTATGAAGATTTTTGTAACAACTA AAAACTCAAAAAACCAAGGCATAGAGGAAGCTTTAAAAAATCGCAA I l l i ACTGTAGA AAAAATGAGTGCTGAAATTAATGAGATAATTCGTGTGTTACAACTCACCTCTTGGGAT GAAGATGCCTGGGCCAGCAAGGACACTGAAGCCATGAAGAGAGCATTGGCCTCCAT AGACTCCAAACTGAACCAGGCCAAAGGTTGGCTCCGTGACCCTAGTGCCTCCCCAG GGGATGCTGGTGAGCAGGCCATCAGACAGATCTTAGATGAAGCTGGAAAAGTTGGT GAACTCTGTGCAGGCAAAGAACGCAGGGAGATTCTGGGAACTTGCAAAATGCTAGG GCAGATGACTGATCAAGTGGCTGACCTCCGTGCCAGAGGACAAGGATCCTCACCGG TGGCCATGCAGAAAGCTCAGCAGGTATCTCAGGGTCTGGATGTGCTCACAGCAAAA GTGGAAAATGCAGCTCGCAAGCTGGAAGCCATGACCAACTCAAAGCAGAGCATTGC AAAGAAGATCGATGCTGCTCAGAACTGGCTTGCAGATCCAAATGGTGGACCGGAAG GAGAAGAGCAGATTCGAGGTGCTTTGGCTGAAGCTCGGAAAATAGCAGAATTATGT GATGATCCTAAAGAAAGAGATGACATTCTACGTTCCCTTGGGGAAATATCTGCTCTG ACTTCTAAATTAGCAGATCTACGAAGACAGGGGAAAGGAGATTCTCCAGAGGCTCGA PL872 (cTNT- GCCTTGGCCAAACAGGTGGCCACGGCCCTGCAGAACCTGCAGACCAAAACCAACC GGGCTGTGGCCAACAGCAGACCGGCCAAAGCAGCTGTACACCTTGAGGGCAAGATT

[0299] Mvcn-F) GAGCAAGCACAGCGGTGGATTGATAATCCCACAGTGGATGACCGTGGAGTCGGTCA GGCTGCCATCCGGGGGCTTGTGGCCGAAGGGCATCGTCTGGCTAATGTTATGATGG GGCCTTATCGGCAAGATCTTCTCGCCAAGTGTGACCGAGTGGACCAGCTGACAGCC CAGCTGGCTGACCTGGCTGCCAGAGGGGAAGGGGAGAGTCCTCAGGCACGAGCAC TTGCATCTCAGCTCCAAGACTCCTTAAAGGATCTAAAAGCTCGGATGCAGGAGGCCA TGACTCAGGAAGTGTCAGATGTTTTCAGCGATACCACAACTCCCATCAAGCTGTTGG CAGTGGCAGCCACGGCGCCTCCTGATGCGCCTAACAGGGAAGAGGTATTTGATGAG AGGGCAGCTAACTTTGAAAACCATTCAGGAAAGCTTGGTGCTACGGCCGAGAAGGC GGCTGCGGTTGGTACTGCTAATAAATCAACAGTGGAAGGCATTCAGGCCTCAGTGA AGACGGCCCGAGAACTCACACCCCAGGTGGTCTCGGCTGCTCGTATCTTACTTAGG AACCCTGGAAATCAAGCTGCTTATGAACATTTTGAGACCATGAAGAACCAGTGGATC GATAATGTTGAAAAAATGACAGGGCTGGTGGACGAAGCCATTGATACCAAATCTCTG TTGGATGCTTCAGAAGAAGCAATTAAAAAAGACCTGGACAAGTGCAAGGTAGCTATG GCCAACATTCAGCCTCAGATGCTGGTTGCTGGGGCAACCAGTATTGCTCGTCGGGC CAACCGGATCCTGCTGGTGGCTAAGAGGGAGGTGGAGAATTCCGAGGATCCCAAGT TCCGTGAGGCTGTGAAAGCTGCCTCTGATGAATTGAGCAAAACCATCTCCCCGATG GTGATGGATGCAAAAGCTGTGGCTGGAAACATTTCCGACCCTGGACTGCAAAAGAG CTTCCTGGACTCAGGATATCGGATCCTGGGAGCTGTGGCCAAGGTCAGAGAAGCCT TCCAACCTCAGGAGCCTGACTTCCCGCCGCCTCCACCAGACCTTGAACAACTCCGA CTAACAGATGAGCTTGCTCCTCCCAAACCACCTCTGCCTGAAGGTGAGGTCCCTCCA CCTAGGCCTCCACCACCAGAGGAAAAGGATGAAGAGTTCCCTGAGCAGAAGGCCG GGGAGGTGATTAACCAGCCAATGATGATGGCTGCCAGACAGCTCCATGATGAAGCT CGCAAATGGTCCAGCAAGCCGGGCATCCCAGCCGCTGAGGTGGGTATAGGTGTTGT AGCTGAGGCAGATGCGGCCGATGCTGCTGGCTTCCCTGTCCCCCCTGACATGGAAG ACGATTACGAACCTGAGCTGCTGTTAATGCCATCCAATCAGCCGGTCAACCAGCCCA TTCTGGCCGCGGCTCAGTCCTTGCATCGGGAAGCTACCAAGTGGTCTAGTAAGGGC AATGACATCATTGCAGCAGCCAAGCGCATGGCTCTGCTGATGGCTGAGATGTCTCG GCTGGTAAGAGGGGGCAGTGGTACCAAGCGGGCACTCATTCAGTGTGCCAAGGAC ATCGCCAAGGCCTCAGATGAGGTGACTCGGTTGGCCAAGGAGGTTGCCAAGCAGTG CACAGATAAACGGATTAGAACCAACCTCTTACAGGTATGTGAGCGAATCCCAACCAT AAGCACCCAGCTCAAAATCCTGTCCACAGTGAAGGCCACCATGCTGGGCCGGACCA

[0300]

[0301] ACATCAGTGATGAGGAGTCTGAGCAGGCCACAGAGATGCTGGTTCACAATGCCCAG AACCTCATGCAGTCTGTGAAGGAGACTGTGCGGGAAGCTGAAGCTGCTTCAATCAA AATTCGAACAGATGCTGGATTTACACTGCGCTGGGTTAGAAAGACTCCCTGGTACCA GTAGaataaaagatcCttatttcattGgatctgtgtgttggtttttgtgtg

[0302]

[0303] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0304] The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0305] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0306] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ 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.

[0307] It must be noted that, as used in the 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 the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0308] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0309] Methods described herein may be performed in vitro or in vivo. In some embodiments, methods described herein are performed in vitro. The term ‘in vitro’ is intended to encompass experiments with cells in culture whereas the term ‘in vivo’ is intended to encompass experiments with intact multi-cellular organisms.

[0310] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0311] Figure 1 shows (A) a schematic of the reporter construct used in the high-throughput promoter screen, comprising an NGS barcode, and a summary of the screening approach; (B) results from the high- throughput promoter screen, when each promoter of interest was combined with either no enhancer or the CS-CRM4, or the SK-CRM3 enhancers; and (C) the enhancer / promoter combinations that resulted in increased cardiac-specific gene expression. Figure 2 shows a (A) schematic of the constructs used for further testing of the enhancer / promoter combinations that increase cardiac-specific gene expression, and (B) a summary of the methods used to quantify gene expression and viral integration.

[0312] Figure 3 shows (A) transgene expression in terms of transgene mRNA copies per ng RNA in the heart 2 weeks and 2 months after infection, for the enhancer and promoter combinations tested in Example 2. Details of the constructs are provided in Figure 2A; and (B) viral genome copy number and the transgene expression in terms of transgene mRNA copies / ng RNA and RNA / DNA ratio, in the heart and liver, 2 weeks and 2 months post-infection for the full set of constructs tested in Example 2; and (C) the viral genome copy number and the transgene expression in terms of transgene mRNA copies / ng RNA and RNA / DNA ratio for the constructs in the heart, liver, brain, cerebellum, diaphragm, eye, kidney, lung, muscle, ovary, spleen, and uterus, 2 months post-infection.

[0313] Figure 4 shows (A) a schematic of the therapeutic construct used for mouse studies validating the cardiac-specific gene expression driven by the candidate enhancer / promoter combinations; (B) the viral copy number of the AAV vector and (C) transgene mRNA expression in the heart, liver, brain, cerebellum, diaphragm, eye, kidney, lung, muscle, spleen, testis, ovary, and uterus. One-way ANOVA with Tukey’s multiple comparisons test has been performed to confirm that expression in the heart is significantly higher than in all other organs tested (p < 0.0001).

[0314] Figure 5 shows the results of studies in cynomolgus monkeys, validating the cardiac-specific gene expression driven by the candidate enhancer / promoter combinations, including the vector copy number in various tissues 8 weeks (A) and 26 weeks (C) after infection, and transgene mRNA expression at 8 weeks (B) and 26 weeks (D) after infection. Heart samples from 9 different regions (5LV2, 3LV2 and 1LV2 from the left ventricle; 5RV2, 3RV2 and 1RV2 from the right ventricle; 5S5-2, 3S3-2 and 1S1-2 from the septum) were analyzed for each animal at each timepoint.

[0315] Figure 6 shows (A) echocardiography and (B) electrocardiography results for LMNA-DCM mice treated with NVC-001 or control.

[0316] Figure 7 shows a Kaplan-Meier survival curve for LMNA-DCM mice treated with NVC-001. A log-rank (Mantel-Cox) test was performed to confirm that mice treated with NVC-001 show significantly improved survival compared to control mice (p < 0.0001).

[0317] Figure 8 shows (A) the AAV construct used to measure in vivo expression of luciferase, driven by the CS-CRM4 / Syn-hTNNT2 enhancer / promoter combinations; and (B) representative bioluminescence imaging 13 weeks post-AAV treatment

[0318] Figure 9 shows (A) Western blot analysis of SORBS1 and SORBS1-iso5 expression in wildtype and DspDCM mice. Top: Representative immunoblots showing SORBS1 (250 kD), SORBS1-iso5 (150 kD), and Gapdh (37 kD, loading control) protein levels in various mouse samples. (B) Quantification of S0RBS1 and SORBS1-iso5 protein levels expressed as fold change over WT-SORBS1 (C) Western blot analysis of MVCN expression in wildtype mice. Representative immunoblots showing Vcl (150 kD), MVCN (100 kD), and Beta-actin (37 kD, loading control) protein levels in WT + PBS and WT + MVCN groups. (D) Quantification of MVCN protein levels expressed as fold change over WT-PBS. Statistical test: Log-rank (Mantel-Cox) test. Pairwise comparison to DspDCM + PBS.

[0319] Figure 10 shows superior transgene expression in adult-like human iPSC-derived cardiomyocytes using selected enhancer-promoter combination. (A) Experimental timeline: human iPSCs were differentiated (24 d), matured (21 d), and transduced with AAV6 vectoring different constructs; cells were analyzed 7 d after AAV transduction. (B) Immunoblot of metavinculin (Mvcn) expression in human adult-like cardiomyocytes driven by a construct comprising the CS-CRM4 / syn-hTNNT2 (CS-CRM4 syn-hTNNT2 Mvcn) compared with the reference cTNT (chicken cardiac troponin T)-Mvcn constructs (MvcnA-E). GAPDH, loading control. (C) Densitometric quantification of Mvcn normalized to GAPDH, shown as fold expression relative to cTNT-Mvcn of column 1. (D) Schematics of the constructs used in panels A-C.

[0320] Figure 11 shows superior transgene expression in mouse heart using selected enhancer-promoter combination. (A) Experimental timeline: mice were injected with AAV vectors comprising the promoterenhancer combination (‘MvcnA’, PL785) or the promoter only (‘MvcnF’, PL872) and hearts were harvested 2 weeks after AAV transduction. (B) Relative vector copy number normalized to Tert. (C) Relative transgene expression normalized to Ctcf. (D) Transgene expression relative to vector copy number. (E) Immunoblot of metavinculin (Mvcn) expression in mouse hearts. a-Tubulin, loading control.

[0321] (F) Schematics of the constructs used in panels A-E.

[0322] Examples

[0323] Example 1 : High-throughput promoter screen to identify candidate enhancer-promoter combinations for cardiac-specific expression

[0324] 3 cardiac-specific enhancer sequences (CMV-enh, CS-CRM4 and SK-CRM3) and 30 cardiac-specific promoter sequences (B-actin (-275 to +61), ACTA1 , ACTC1 , CKM, CMV IE (608), CryAB, cTnT(-555 to +38), cTnT-129 (269-201)neg, DES, FABP3, GNAQ (887), HSBP7, hTNNT2 (-502 to +42), Human EFla (230), MALAT1, MB, MHCK7 (771), Mybpc3, Myh6 containing EcoRI, Myh7, Myl2 containing EcoRI, Myl3, Original cTnT(442), RYR2, Synthetic hTNNT2 (455), Synthetic SPc5-12 (424), TNNC1, TNNI3, TNNT2 and Tpm1) were identified and paired in various combinations to obtain 120 different enhancerpromoter combinations (including a condition where no enhancer was used).

[0325] Each combination was tagged with a unique barcode and subsequently cloned into an AAV vector backbone expressing a gene of interest. AAV generated from all combinations were pooled and administered into wild-type mice via intravenous injection to enable systemic dissemination of the virus. Organs were harvested from mice at 2 different timepoints (2 weeks or 2 months after AAV injection), and DNA and RNA from each organ was analyzed by next-generation sequencing (NGS) to quantify RNA expression of the gene of interest, normalized to DNA vector copy number. The results for 90 of the combinations are shown in Figure 1B. The use of unique barcodes within each vector construct enabled quantitative comparison of the relative strength of expression driven by each enhancer-promoter combination within each organ.

[0326] 1.11dentification of enhancer-promoter combinations driving high gene expression in the heart Gene expression in the heart was first analyzed to identify the following enhancer-promoter combinations that drove high gene expression in the heart (Figure 1B):

[0327] CS-CRM4 / cTnT (-555 to +38)

[0328] CS-CRM4 / cTnT-129 (269-201 )neg

[0329] CS-CRM4 / hTNNT2 (-502 to +42)

[0330] CS-CRM4 / original cTnT(442)

[0331] CS-CRM4 / synthetic hTNNT2

[0332] CS-CRM4 / TNNT2

[0333] SK-CRM3 / cTnT-129 (269-201 )neg

[0334] SK-CRM3 / hTNNT2 (-502 to +42)

[0335] SK-CRM3 / synthetic hTNNT2

[0336] SK-CRM3 / TNNT2

[0337] No enhancer / hTNNT2 (-502 to +42)

[0338] No enhancer / synthetic hTNNT2

[0339] No enhancer / TNNT2.

[0340] Notably, the combination of an enhancer sequence with a promoter sequence did not consistently result in an increase in gene expression in the heart compared with the promoter sequence alone. For example, while CS-CRM4 / synthetic hTNNT2 drove higher gene expression in the heart compared with synthetic hTNNT2 alone, CS-CRM4 / MALAT1 drove lower gene expression compared with MALAT1 alone. This underscores the need for experimental validation to identify potential synergistic effects between various enhancer and promoter combinations.

[0341] 1.2 Identification of enhancer-promoter combinations driving cardiac-specific gene expression Next, off-target gene expression was analyzed, focusing particularly on expression in the liver, which is known to receive high levels of AAV vector. The following enhancer-promoter combinations showed the greatest differential gene expression between the heart and the liver (Figure 1C):

[0342] CS-CRM4 I cTnT-129 (269-201 )neg

[0343] CS-CRM4 I hTNNT2 (-502 to +42)

[0344] CS-CRM41 original cTnT(442)

[0345] CS-CRM41 synthetic hTNNT2

[0346] SK-CRM3 I cTnT-129 (269-201 )neg

[0347] SK-CRM3 I hTNNT2 (-502 to +42)

[0348] SK-CRM31 synthetic hTNNT2

[0349] SK-CRM3 I TNNT2

[0350] No enhancer I hTNNT2 (-502 to +42)

[0351] No enhancer I synthetic hTNNT2

[0352] No enhancer / TNNT2 1.3 Materials and methods

[0353] Design and generation of AAV vectors

[0354] 30 cardiac-specific promoter sequences were paired individually with 1 of 3 cardiac-specific enhancer sequences, or used without any enhancer sequence, to generate 120 different enhancer-promoter combinations. Each combination was cloned individually into an AAV vector backbone expressing a gene of interest, together with a unique barcode downstream of the promoter sequence. Cloned AAV vectors were pooled and used to generate AAV viral stocks for in vivo experiments.

[0355] In vivo studies in mice

[0356] Mice were administered with pooled AAV viral stocks via intravenous injection (retro-orbital) to enable systemic dissemination of the virus. At two different timepoints (2 weeks and 2 months) after AAV injection, mice were sacrificed and the following organs were harvested: heart (left ventricle, right ventricle, atria), aorta, liver, skeletal muscle, diaphragm, lung, brain (without cerebellum), cerebellum, testis, eye, kidney, spleen.

[0357] DNA and RNA extraction and NGS library preparation

[0358] DNA and RNA were extracted from each organ. Barcode sequences were amplified by PCR using unique index sequence combinations for each organ. Samples were then pooled and sent to a commercial vendor for NGS.

[0359] NGS analysis

[0360] NGS raw sequencing data was analyzed to identify and quantify barcode sequences for DNA and RNA samples from each organ. Each barcode count was normalized to the total barcode counts, then further normalized to the respective barcode counts in the virus input to account for differences in the starting virus pool. Finally, normalized RNA barcode counts were divided by normalized DNA barcode counts to account for differences in transduction efficiency in different organs.

[0361] Example 2: Confirmation of high and cardiac-specific expression driven by candidate enhancer-promoter combinations

[0362] Based on the data from Example 1 , the following enhancer-promoter combinations were selected for a confirmatory in vivo study to validate the results of the high-throughput screen:

[0363] • No enhancer I synthetic hTNNT2 (Syn-hTNNT2 only)

[0364] • CS-CRM4 I synthetic hTNNT2 (CS-CRM4 / Syn-hTNNT2)

[0365] • SK-CRM3 I synthetic hTNNT2 (SK-CRM4 / Syn-hTNNT2)

[0366] • CS-CRM4 I hTNNT2 (-502 to +42) (CS-CRM4 / hTNNT2)

[0367] A control construct that uses the cTnT promoter alone, which is a commonly used cardiac-specific promoter in many studies, was included for comparison. The constructs tested are summarized in Figure 2A. 2.1 1n vivo validation of high and cardiac-specific gene expression driven by candidate enhancerpromoter combinations

[0368] Wild-type mice were intravenously injected with individual vectors expressing a gene of interest driven by one of the selected candidate enhancer-promoter combinations. 5 mice were injected per construct per time point, with 2e10viral genomes per gram of body weight (vg / g). Organs were harvested from mice at 2 different timepoints (2 weeks or 2 months after AAV injection, from age-matched mice), and DNA and RNA from each organ was extracted and analyzed by qPCR or RT-qPCR, respectively, using primers specific for the gene of interest, to quantify vector DNA copy number and transgene mRNA expression (Figure 2B).

[0369] The large majority of selected enhancer-promoter combinations drove high gene expression in the heart at both 2 weeks and 2 months after AAV injection, significantly outperforming the cTnT promoter (Figures 3A and 3B). Importantly, very low off-target gene expression in the liver and other organs analyzed was observed with all candidates, confirming the cardiac specificity of gene expression driven by these enhancer-promoter combinations (Figure 3C). These data confirmed the results obtained from the high-throughput screen described in Example 1.

[0370] 2.2 Materials and methods

[0371] Design and generation of AAV vectors

[0372] Each enhancer-promoter combination was cloned individually into an AAV vector backbone expressing a gene of interest. Cloned AAV vectors were used individually to generate AAV viral stocks for in vivo experiments.

[0373] In vivo studies in mice

[0374] AAV viral stocks were administered into mice via intravenous injection (retro-orbital) to enable systemic dissemination of the virus. At two different timepoints (2 weeks and 2 months) after AAV injection, mice were sacrificed and the following organs were harvested: heart (left ventricle, right ventricle, atria), liver, skeletal muscle, diaphragm, lung, brain (without cerebellum), cerebellum, testis, eye, kidney, spleen.

[0375] Analysis of vector copy number and transgene mRNA expression

[0376] DNA and RNA were extracted from each organ. Vector DNA copy number and transgene mRNA expression were quantified by qPCR or RT-qPCR, respectively, using primers specific for the gene of interest. Vector DNA copy number was normalized to input gDNA and expressed as copies per diploid genome, assuming that each diploid genome contains 6 pg of gDNA. Transgene mRNA expression was normalized to input RNA and expressed as copies per ng RNA.

[0377] Example 3: Demonstration of high and cardiac-specific expression of DNSUN1 driven by final enhancerpromoter combination in mouse and non-human primates

[0378] Based on the data from Examples 1 and 2, the combination of the CS-CRM4 enhancer and synthetic hTNNT2 (Syn-hTNNT2) was selected for inclusion into NVC-001 , which is designed to drive cardiacspecific expression of the DNSUN1 transgene. The final version of the NVC-001 vector is shown in Figure 4A, comprising the CS-CRM4 enhancer, the Syn-hTNNT2 promoter, a p-globin / IgG chimeric intron from pCI-Neo vector (pCI-Neo intron), an optimized Kozak sequence, the DNSUN1 transgene sequence, the woodchuck hepatitis virus posttranscriptional regulatory element, mutated to abolish expression of woodchuck hepatitis virus-X protein (WPREmut6 element), and a bovine growth hormone polyadenylation signal (BgH-PolyA). Expression of DNSUN1 from NVC-001 was assessed in two different species, mouse and cynomolgus monkey, to validate the activity of the CS-CRM4 / Syn-hTNNT2 combination.

[0379] 3.1 CS-CRM4 enhancer and Syn-hTNNT2 promoter combination drives cardiac-specific transqene expression in mice

[0380] Wild-type mice were intravenously injected with NVC-001 , which expresses the DNSUN1 transgene driven by CS-CRM4 and Syn-hTNNT2. Organs were harvested from mice at 6 weeks after AAV injection, and DNA and RNA from each organ was extracted and analyzed by qPCR or RT-qPCR, respectively, using primers specific for the NVC-001 construct, to quantify vector DNA copy number and transgene mRNA expression.

[0381] Vector DNA was detected across most organs analyzed, with the highest concentration in the liver as expected (Figure 4B). However, transgene mRNA expression was most highly expressed in the heart, with significantly lower expression in the liver and other off-target organs analyzed (Figure 4C). This confirms the high and cardiac-specific expression driven by the CS-CRM4 / Syn-hTNNT2 enhancerpromoter combination.

[0382] 3.2 CS-CRM4 enhancer and Syn-hTNNT2 promoter combination drives cardiac-specific transqene expression in cynomolgus monkeys

[0383] Wild-type cynomolgus monkeys were intravenously injected with two variants of NVC-001 , both expressing DNSUN1 driven by the CS-CRM4 enhancer and Syn-hTNNT2 promoter. Organs were harvested from animals at 8 weeks and 26 weeks after AAV injection, and DNA and RNA from each organ was extracted and analyzed by qPCR or RT-qPCR, respectively, using primers specific for the NVC-001 construct, to quantify vector DNA copy number and transgene mRNA expression.

[0384] As with the mouse, vector DNA was detected across most organs analyzed, with the highest concentration in the liver as expected (Figure 5A and 5C). However, transgene mRNA expression was most highly expressed in the heart, with significantly lower expression in the liver and other off-target organs analyzed (Figure 5B and 5D). Transgene expression remained high and specific to the heart for up to 26 weeks after AAV injection (Figure 5D). This confirms that the CS-CRM4 / Syn-hTNNT2 combination drives high and cardiac-specific expression across different species.

[0385] 3.3 Materials and Methods

[0386] In vivo studies in mice

[0387] Mice were administered with NVC-001 via intravenous injection (retro-orbital) to enable systemic dissemination of the virus. 6 weeks after AAV injection, mice were sacrificed and the following organs were harvested: heart, liver, skeletal muscle, diaphragm, lung, brain (without cerebellum), cerebellum, testis, ovary, uterus, eye, kidney, spleen. In vivo studies in cynomolgus monkeys

[0388] Cynomolgus monkeys with administered with NVC-001 via intravenous infusion (forelimb subcutaneous vein) to enable systemic dissemination of the virus. At two different timepoints (8 weeks and 26 weeks) after AAV injection, animals were sacrificed and the following organs were harvested: heart, ocular tissues, administration site, gonads (testis, ovary), skeletal muscle, diaphragm, lymph node, brain (cerebrum, cerebellum, brainstem), spinal cord, dorsal root ganglia, kidney, lung, liver, spleen.

[0389] Analysis of vector copy number and transgene mRNA expression

[0390] DNA and RNA were extracted from each organ. Vector DNA copy number and transgene mRNA expression were quantified by qPCR or RT-qPCR, respectively, using primers specific for the gene of interest. Vector DNA copy number was normalized to input gDNA and expressed as copies per diploid genome for mice (assuming that each diploid genome contains 6 pg of gDNA), and copies per ug gDNA for cynomolgus monkeys. Transgene mRNA expression was normalized to input RNA and expressed as copies per ng RNA for mice, and copies per ug RNA for cynomolgus monkeys.

[0391] Example 4: High and cardiac-specific expression of DNSUN1 driven by enhancer-promoter combination treats LMNA cardiomyopathy

[0392] To demonstrate that the CS-CRM4 / Syn-hTNNT2 enhancer-promoter combination drives functionally relevant gene expression levels in the heart, NVC-001 was used to treat a mouse model of dilated cardiomyopathy (DCM) that has previously been shown to be rescued by expression of DNSUN1 in the heart. This mouse model involves cardiac-specific deletion of the LMNA gene induced by administration of tamoxifen, which results in mice rapidly developing key features of LMNA-related DCM in humans, including atrial and ventricular conduction-system disease, severely reduced ejection fraction and endstage heart failure, with death ensuing in less than 6 weeks (Chai et al., 2021).

[0393] 4.1 DNSUN1 expression driven by CS-CRM4 / Syn-hTNNT2 enhancer-promoter combination preserves cardiac function and extends survival in LMNA-DCM mice

[0394] LMNA-DCM mice were treated with a clinically relevant dose of NVC-001 (5e13 vg / kg) two days after disease induction. Heart function in untreated control LMNA-DCM mice decreased overtime, with significant reduction in ejection fraction and fractional shortening, as measured by echocardiography (Figure 6A), and development of cardiac conduction delays (increase in PR and QRS intervals) and a decrease in heart rate (RR interval), as measured by electrocardiography (ECG) (Figure 6B). In contrast, LMNA-DCM mice treated with NVC-001 showed stabilization of ejection function over the duration of the study after an initial decline, while cardiac conduction-system disease progression was halted, with mice showing normal PR and QRS intervals throughout the study. This preservation of heart function was accompanied by a significantly prolonged median survival of greater than 4-fold compared with untreated LMNA-DCM mice (Figure 7). Collectively, these data demonstrate that the level of DNSUN1 expression driven by the CS-CRM4 / Syn-hTNNT2 combination is sufficient to treat disease in LMNA-DCM mice.

[0395] 4.2 Materials and methods

[0396] Induction of dilated cardiomyopathy in LMNA-DCM mice LMNA-DCM mice were generated by crossing Lmnaflox / floxmice with the tamoxifen-inducible MerCreMer (MCM) allele, which drives expression of Cre recombinase specifically in cardiomyocytes. LMNA-DCM mice were injected with tamoxifen via intraperitoneal injection at a dose of 40 mg / kg to induce deletion of Lmna in the heart, which leads to the development of dilated cardiomyopathy. Control wild-type mice harbor the Lmnaflox / floxallele without the MCM allele, and therefore remain functionally wild-type (without deletion of Lmna in the heart or development of dilated cardiomyopathy) upon tamoxifen administration.

[0397] 1.5 days (for male mice) and 2.5 days (for female mice) after disease induction, LMNA-DCM mice were administered with NVC-001 via intravenous injection (retro-orbital). Mice were monitored by echocardiography and electrocardiography throughout the study on Days 0, 14, 21, 28, 38, 42, and 56 after disease induction, and every 4 weeks thereafter until the end of the study. The study was terminated on D134 (male mice) and D135 (female mice) after disease induction.

[0398] Echocardiography in mice

[0399] Echocardiography was performed with the Prospect T1 ultrasound systems from S-Sharp Inc. One day before ultrasound examination, animals were shaved to remove fur from the thoracic region. On the day of examination, animals were placed under anesthesia using 1.5% isoflurane mixed with oxygen. B-mode and M-mode readings were obtained while the heart rates of the animals ranged from 450 bpm to 350 bpm. The cardiac package of the Prospect T1 software version 3.1.34.2884 (S-Sharp Inc.) was utilized to calculate fractional shortening (FS) and ejection fraction (EF) from the parasternal long axis. For the diastolic and systolic state, measurements of the left ventricular interior diameter, interventricular septum, and left ventricular posterior wall were obtained from the parasternal short axis.

[0400] Electrocardiography in mice

[0401] Non-invasive electrocardiography (ECG) was performed using the ecgTUNNEL system (emka Technologies) with minimal filtering. Animals were placed under anesthesia using 1.5% isoflurane mixed with oxygen for 2 minutes prior to the ECG recording, after which animals were transferred onto the ECG platform with continuous sedation throughout the procedure. A 6-lead ECG was recorded using lox Software (emka Technologies). ECG recordings were taken for a period of 0.5-1 minute per animal. Recorded ECG data were analyzed using ecgAUTO v3.5.5.28 software (emka Technologies). A library of normal waves was built using the Day 0 waveforms as a baseline. ECG segments were quantified for heart rate (HR), ventricular rate (RR intervals), atrioventricular (AV) conduction time (PR interval) and ventricular depolarization time (QRS duration). Two hundred heartbeats per tracing were analyzed. Only clearly discernable ECG parameters were included in the analysis for each animal.

[0402] Example 5: Enhancer-promoter combination drives cardiac-specific expression of other transqenes Examples 1 , 2, 3 and 4 demonstrate the functionality of the CS-CRM4 / Syn-hTNNT2 enhancer-promoter combination in driving high and cardiac-specific expression of the DNSUN1 transgene in various contexts in both mice and cynomolgus monkeys. The following example demonstrates that this functionality extends to genes other than DNSUN1. 5.1 CS-CRM4 / Syn-hTNNT2 drives cardiac-specific expression of firefly luciferase in mice

[0403] A version of NVC-001 that expresses firefly luciferase instead of DNSUN1 was cloned and used to determine the kinetics of transgene expression from the AAV vector. This vector, NVC-Luciferase, contains the same regulatory elements as NVC-001 , with luciferase expression driven by CS-CRM4 / Syn-hTNNT2 (Figure 8A). Wild-type mice were administered with NVC-Luciferase via intravenous injection (retro-orbital) to enable systemic dissemination of the virus. Luciferase expression in mice was monitored by in vivo bioluminescence imaging for 13 weeks after AAV injection. Despite the known high tropism of the AAV to the liver, the highest expression of luciferase was observed in the left thoracic region corresponding to the position of the heart, with significantly lower expression observed in the upper abdominal region corresponding to the position of the liver (Figure 8B). This result demonstrates that luciferase expression driven by the CS-CRM4 / Syn-hTNNT2 combination is cardiac-specific.

[0404] 5.2 Materials and methods

[0405] Injection of NVC-Luciferase into mice and in vivo bioluminescence imaging

[0406] Wild-type mice were administered with NVC-Luciferase via intravenous injection (retro-orbital). In vivo bioluminescence imaging was performed on animals at 0.5 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks and 13 weeks post-injection, using the I VIS Spectrum In vivo Imaging System (Revvity). Prior to in vivo imaging, fur was removed from the chest and abdomen of study animals.

[0407] Animals were then injected with 200 pl of luciferin substrate (15 mg / ml in PBS) via intraperitoneal injection, 10 minutes before in vivo imaging. Imaging was performed and analyzed using Living Image software version 4.7.4 (PerkinElmer) with default program settings.

[0408] Example 6: Robust expression of SORBS1, SORBS1 iso5 and MVCN in cardiac tissue

[0409] 6.1 Detecting protein expression of SORBS1, SORBS1 iso5 and MVCN in cardiac tissue

[0410] The inventors assessed the cardiac expression of three other therapeutics in an independent transgene expression experiment, where the therapeutic candidates were injected into WT mice (DspFF-McmWT). All candidates demonstrated robust myocardial expression (Figures 9A, 9B, 9C, and 9D). Notably, SORBS1 iso5, which is driven by CS-CRM4 / Syn-hTNNT2, exhibited, on average, double the expression of SORBS1 , which is driven by the Syn-TNNT2 promoter alone, at the same dose (Figure 9B). MVCN expression was found to be approximately 12 times higher than endogenous levels (Figure 9D).

[0411] 6.2 Materials and Methods

[0412] Cloning of A AV9 expression constructs and production of AAV9

[0413] The human sequences of Metavinculin (UniProt P18206), SORBS1 (UniProt Q9BX66-1), and SORBS1-iso5 (Uniprot Q9BX66-5; SEQ ID NO:94) were obtained by gene synthesis, restriction digested, and ligated into an AAV transfer plasmid. MVCN and SORBS1-iso5 were operably linked to a transcriptional regulatory element comprising CS-CRM4 enhancer and Syn-hTNNT2 promoter, as used in DNSUN1 construct of Example 5. SORBS1 was operably linked to the Syn-hTNNT2 promoter alone, as the length of the transgene precludes the inclusion of the CS-CRM4 enhancer sequence without going over the size limit of a typical AAV vector. Western Blot

[0414] The samples were first minced into extremely small pieces using a scalpel blade while kept on ice.

[0415] Subsequently, they were lysed using the following lysis buffer, with the reagents in bold added fresh to the buffer:

[0416] Tris 50mM

[0417] NaCI 500mM

[0418] SDS 0.4%

[0419] EDTA 5mM

[0420] Trition-X 2%

[0421] DTT 1mM

[0422] Protease Inhibitor cocktail 1x

[0423] ddH2O to make up volume

[0424]

[0425] To ensure complete lysis, the samples were homogenized. Samples were treated with benzonase and sonicated to remove any remaining traces of DNA. Finally, the samples were centrifuged to remove membranes, and the resulting supernatant was transferred to a new tube. Protein concentration was determined using the BCA method, and 45 pg of protein was loaded onto a gradient SDS pre-cast gel (4-15% Mini-PROTEAN™ TGX Stain-Free™ Protein Gels, Bio-Rad).

[0426] The separated proteins were transferred from the gel onto a nitrocellulose membrane using a wet transfer system at a constant voltage of 115V for a duration of 100 minutes. Membranes were subsequently blocked for 1 h using blocking buffer contains TBS-T (Tris-buffered saline with Tween-20) and 5% milk at room temperature, to block non-specific binding. Primary antibodies were diluted in blocking buffer, applied to the membranes and incubated overnight at 4°C with gentle agitation. The following primary antibodies were used at the indicated dilutions:

[0427] 1. anti-Dsp (Proteintech, 1 :2000)

[0428] 2. anti-Vinculin (Sigma, 1 :4000)

[0429] 3. anti-Lasp1 (Proteintech 1:1000)

[0430] 4. anti-Sorbs1 (Atlas antibodies 1 :1000)

[0431] 5. anti-HSPB7 (Proteintech 1 :1000)

[0432] 6. anti-Fermt2 (Proteintech 1:1000)

[0433] 7. anti-Fblim1 (Atlas antibodies 1 :1000)

[0434] 8. anti-ALFAtag (Nanotag Biotechnologies GmbH, 1:1000)

[0435] 9. anti-Beta-Actin (Abeam, 1:10000)

[0436] Membranes were washed multiple times with a washing buffer (TBS-T) at room temperature to eliminate unbound primary antibody. Subsequently, HRP-conjugated secondary antibodies were diluted in 5% milk in TBS-T at a 1 :10,000 dilution and incubated for 1 h at room temperature with gentle agitation. The chemiluminescence signal was then detected by exposing the membrane to SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher) for 1 minute and capturing the signal using the ChemiDoc Imaging System from Bio-Rad. Example 7: Enhancer-promoter combination drives superior transgene expression in adult-like human induced pluripotent stem cell (IPS -derived cardiomyocytes

[0437] Examples 1-6 demonstrate the functionality of the CS-CRM4 / syn-hTNNT2 enhancer-promoter combination in driving robust and cardiac-specific expression of various transgenes in both mice and cynomolgus monkeys. The present example demonstrates that this functionality extends to human cells.

[0438] 7.1 Comparison of MVCN protein expression in adult-like human iPSC-derived cardiomyocytes driven by different regulatory elements

[0439] The inventors assessed expression of metavinculin (Mvcn) in adult-like human iPSC-derived cardiomyocytes under the control of two different regulatory elements: the chicken cardiac troponin T promoter (cTNT), serving as a reference cardiac-specific promoter, and the CS-CRM4 / syn-hTNNT2 enhancer-promoter combination. A schematic representation of the experimental timeline is provided in Figure 10A, with schematic representations of the constructs used shown in Figure 10D. Notably, Mvcn expression driven by CS-CRM4 / syn-hTNNT2 was approximately 27-fold higher compared with the reference constructs (Figures 10B and 10C).

[0440] 7.2 Materials and Methods

[0441] Differentiation and maturation of human iPSC-derived cardiomyocytes

[0442] A human iPSC line derived from the PGP1 donor (Personal Genome Project) was used. The iPSC line was directed to differentiate into beating cardiomyocytes (CMs) using a method adapted from a published protocol (Lian et al. 2013) and metabolically matured following a published protocol (Feyen et al. 2020). Cells were transduced with AAV6 at MOI 1.5E5 GC / cell and lysed 7 days after transduction.

[0443] Production ofAAVG

[0444] AAV6 was produced by the triple transfection method and purified using methods adapted from published protocols (Arden and M. Metzger 2016; Strobel et al. 2015).

[0445] Western Blot

[0446] The method for Western Blot is described in Example 6.2 herein.

[0447] Example 8. Superior transgene expression in mouse heart using selected enhancer-promoter combination at both transcript and protein levels

[0448] Example 2 demonstrates that the CS-CRM4 / syn-hTNNT2 enhancer-promoter combination drives superior transgene expression compared with the canonical cardiac-specific promoter cTNT in mouse hearts, with markedly increased mRNA levels. Example 6 further shows robust myocardial protein expression driven by CS-CRM4 / syn-hTNNT2. The present example demonstrates that, at similar vector copy numbers, the superior mRNA expression achieved with CS-CRM4 / syn-hTNNT2 compared to cTNT directly translates into correspondingly higher protein levels. 8.1 1n vivo validation of superior transqene mRNA and protein expression driven by candidate enhancerpromoter combination

[0449] A schematic representation of the experimental timeline is provided in Figure 11A, with schematic representations of the constructs used shown in Figure 11 F. Wild-type mice were intravenously injected with AAV viral stocks expressing different metavinculin variants driven by either cTNT or the CS-CRM4 / syn-hTNNT2 enhancer-promoter combination. Three mice were injected per construct at 5E10viral genomes per gram of body weight (vg / g). Hearts were harvested 2 weeks after AAV9 injection, and DNA, RNA, and proteins were extracted and analyzed by qPCR, RT-qPCR, and Western blot, respectively. Primers specific for the gene of interest were used to quantify vector DNA copy number and transgene mRNA expression, while protein expression was assessed using metavinculin-specific antibodies by Western blot (Figure 11E).

[0450] The selected enhancer-promoter combination drove high gene expression in the heart at 2 weeks after AAV injection, significantly outperforming the cTNT promoter at both transcript and protein levels (Figures 11B-E).

[0451] 8.2 Materials and Methods

[0452] The methods for in vivo studies in mice and analysis of vector copy number and transgene mRNA expression are described in Example 2.2 herein. The method for Western blot is described in Example 6.2 herein.

[0453] References

[0454] Rincon MY, Sarcar S, Danso-Abeam D, et al. Genome-wide computational analysis reveals cardiomyocyte-specific transcriptional Cis-regulatory motifs that enable efficient cardiac gene therapy. Mol Ther. 2015;23(1):43-52.

[0455] Sarcar S, Tulalamba W, Rincon MY, et al. Next-generation muscle-directed gene therapy by in silico vector design. Nat Commun. 2019; 10(1 ):492.

[0456] Jefferies JL and Towbin JA, Dilated Cardiomyopathy. Lancet 375: 752-762 (2010).

[0457] R. E. Hershberger, Hedges DJ, Morales A, Dilated cardiomyopathy: the complexity of a diverse genetic architecture. Nat Rev Cardiol 10: 531-547 (2013).

[0458] Herman DS, Lam L, Taylor MRG, et al., Truncations of titin causing dilated cardiomyopathy. N Engl J Med 366: 619-628 (2012).

[0459] Tayal U, Prasad S, and Cook SA, Genetics and genomics of dilated cardiomyopathy and systolic heart failure. Genome Med 9: 20 (2017). Arden, Erik, and Joseph M. Metzger. 2016. “Inexpensive, Serotype-Independent Protocol for Native and Bioengineered Recombinant Adeno-Associated Virus Purification.” Journal of Biological Methods 3 (2): 1.

[0460] Feyen, Dries A.M., Wesley L. McKeithan, Arne A.N. Bruyneel, et al. 2020. “Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.” Cell Reports 32 (3): 107925.

[0461] Lian, Xiaojun, Jianhua Zhang, Samira M Azarin, et al. 2013. “Directed Cardiomyocyte Differentiation from Human Pluripotent Stem Cells by Modulating Wnt / p-Catenin Signaling under Fully Defined Conditions.” Nature Protocols 8 (1): 162-75.

[0462] Strobel, Benjamin, Felix D. Miller, Wolfgang Rist, and Thorsten Lamia. 2015. “Comparative Analysis of Cesium Chloride- and lodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.” Human Gene Therapy Methods 26 (4): 147-57.

Claims

1. Claims:

1. A transcriptional regulatory element comprising an enhancer and a promoter, wherein:3.i. the enhancer is selected from CS-CRM4 and SK-CRM3; and4.ii. the promoter is selected from Synthetic hTNNT2, hTNNT2(-502 to +42), cTnT-129 (269- 201)neg, Original cTnT (442), TNNT2, CMV IE (608) and cTnT (-555 to +38).

2. The transcriptional regulatory element according to claim 1 , wherein:6.i. the enhancer is CS-CRM4 and the promoter is selected from Synthetic hTNNT2, Original cTnT (442), cTnT-129 (269-201)neg, hTNNT2(-502 to +42) and TNNT2; or7.ii. the enhancer is SK-CRM3 and the promotor is selected from cTnT-129 (269-201)neg, hTNNT2(-502 to +42), synthetic hTNNT2, and TNNT2.

3. The transcriptional regulatory element according to claim 2, wherein the enhancer is CS-CRM4 and the promotor is Synthetic hTNNT2.

4. The transcriptional regulatory element according to any one of the preceding claims wherein:10.i. CS-CRM4 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 1 ;11.and / or12.ii. SK-CRM3 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 2;13.and / or14.Hi. Synthetic hTNNT2 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 6; and / or15.iv. hTNNT2(-502 to +42) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 4; and / or16.v. cTnT-129 (269-201)neg is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 3; and / or17.vi. Original cTnT (442) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 5; and / or18.vii. TNNT2 is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO: 7; and / or19.viii. CMV IE (608) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO:20.8; and / or21.ix. cTnT (-555 to +38) is a nucleic acid comprising at least 70% sequence identity to SEQ ID NO:9.

5. The transcriptional regulatory element according to any one of the preceding claims comprising a restriction enzyme recognition site arranged between the enhancer and the promoter.

6. The transcriptional regulatory element according to claim 5 which comprises or consists of a sequence selected from any one of SEQ ID NOs: 11 to 21.

7. A polynucleotide comprising the transcriptional regulatory element according to any one of claims 1 to 6 and a nucleic acid encoding an expression product.

8. A vector comprising the transcriptional regulatory element according to any one of claim 1 to 6 and a gene of interest (GOI).

9. The vector according to claim 8, wherein the gene of interest encodes a therapeutic protein.

10. The vector according to claim 8 or claim 9, wherein the gene of interest encodes a LINC complex inhibiting polypeptide or a focal adhesion protein.

11. The vector according to claim 10, wherein the LINC complex inhibiting polypeptide comprises or consists essentially of any one of SEQ ID NOs: 22 to 86; or the focal adhesion protein comprises or consists essentially of any one of SEQ ID NOs: 90 to 94.

12. The vector according to any one of claims 9 to 11 further comprising at least one, several, or all elements selected from an intron, a Kozak sequence, a signal peptide, and a polyA sequence.

13. The vector according to any one of claims 8 to 12, wherein the vector is an adeno-associated virus (AAV) vector.

14. The vector according to claim 13, wherein the vector comprises or consists of SEQ ID NO: 10 or SEQ ID NO: 96.

15. Use of the transcriptional regulatory element according to any one of claims 1 to 6 to induce cardiac-specific gene expression of a GOI.

16. An isolated cell comprising the transcriptional regulatory element, polynucleotide or the vector according to any one of claims 1 to 13.

17. The vector according to any one of claims 8 to 14 for use in a method of medical treatment or prophylaxis.

18. The vector according to any one of claims 8 to 14 for use in a method of treating or preventing a cardiomyopathy.

19. Use of the vector according to any one of claims 1 to 13 in the manufacture of a medicament for use in a method treating or preventing a cardiomyopathy.

20. A method of treating or preventing a cardiomyopathy, the method comprising administering to a subject a therapeutically or prophy lactically effective amount of the vector of any one of claims 8 to 19.

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