Lmna gene therapy constructs

Abstract

The present disclosure relates to nucleic acid molecules encoding Lamin A, as well as vectors and pharmaceutical compositions comprising such nucleic acid molecules for use in treatment of laminopathies.

Description

LMNA GENE THERAPY CONSTRUCTSCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 681,654, filed August 9, 2024, the disclosure of which is hereby incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The present invention relates to nucleic acid molecules encoding lamin A / C protein, vectors, adeno-associated virus (AAV), and pharmaceutical compositions comprising said nucleic acid molecules for use in treatment of laminopathies.BACKGROUND

[0003] Laminopathies are a group of rare genetic diseases caused by mutations in genes encoding for proteins associated with the nuclear envelope. Mutations in LMNA are responsible for diseases such as Emery-Dreifuss muscular dystrophy, dilative cardiomyopathy (DCM) and Progeria-Syndrome. Inheritance is typically autosomal-dominant, such that mutation of only one allele is sufficient for a disease phenotype.

[0004] Lamin A / C-associated forms of DCM are characterized by pronounced fibrosis, arrhythmia and reduced life expectancy (Paldino et al. Curr Cardiol. Rep. 2018, PubMed ID: 30105555). LMNA mutations can be found in approximately 6 to 10% of inherited DCM and are thought to be causative (Lu et al. Dis. Model Meeh. 2011, PubMed ID: 21810905; Mazzarotto et al. Circulation 2020, PubMed ID: 31983221).

[0005] The type V intermediate filaments, type A and B lamins, span the nuclear lamina (NL) below the inner nuclear membrane. The NL is not only important for the mechanical stability of the cell nucleus, but, through diverse interactions with associated proteins or even the DNA itself, the NL takes on various tasks, such as mechanotransduction, gene regulation and chromatin organization (Captur, G., et al., Heart, 2018. 104(6): 468-479). For example, extracellular-signal regulated kinases 1 and 2 (ERK1 / 2), relevant for proliferation signaling pathways, are bound in the NL and released in response to adequate stimuli, or heterochromatin, and thus inactive DNA sections are bound to the periphery and temporarily stored (“parked”) there (Worman, H.J. and G. Bonne, Exp Cell Res, 2007. 313(10): 2121-33; Foisner, Y.G.a.R. Lamins: Nuclear Intermediate Filament Proteins with Fundamental Functions in Nuclear Mechanics and Genome Regulation, 2023; van Steensel, B. and A.S. Belmont, Cell, 2017.169(5): 780-791; Crasto, S., I. My, and E. Di Pasquale, Front Physiol, 2020. 11 : 761). The type A lamins, lamins A and C, are encoded by the LMNA gene and arise through alternative splicing. Mutations in this LMNA gene lead to laminopathies. Due to the ubiquitous presence of lamins, laminopathies show manifestations in various tissues such as muscle, fat and nerve tissue and thus lead to complex syndromes and a wide range of symptoms (Captur, G., et al., Heart, 2018. 104(6): 468-479).

[0006] Laminopathies affect the heart with a penetrance of 85-100% and lead to DCM, which requires therapy at a young age. Together with the mechanical cardiac symptoms, such as impaired left ventricular pump function, there are also serious electrophysiological changes such as atrioventricular block (AV block) or ventricular tachycardia (Hasselberg, N.E., et al., Eur. Heart J., 2018. 39(10): 853-860). Since these electrophysiological changes are more common and often severe in laminopathies than in other forms of DCM, implantable cardioverter defibrillator (ICD) therapy is often indicated early in life for primary prophylaxis (Captur, G., et al., Heart, 2018. 104(6): 468-479; Crasto, S., I. My, and E. Di Pasquale, The Broad Spectrum of LMNA Cardiac Diseases: From Molecular Mechanisms to Clinical Phenotype. Front Physiol, 2020. 11 : 761). To date, there is no causal therapeutic approach for laminopathies and DCM patients are treated according to the standardized heart failure guidelines. If left untreated, these cardiac changes can lead to sudden cardiac death at a young age (Captur, G., et al., Heart, 2018. 104(6): 468-479).

[0007] Mutations in the LMNA gene are mostly missense mutations (0.43% vs. 0.01% truncating mutations; Mazzarotto F. et al., Circulation, 2020. 141 : 387-398) and show a large discrepancy between genotype and phenotype, even within the same family. More than 450 mutations have been described without clear concentration in specific regions of the gene (“hotspots”). The multitude of mutations pose a problem for gene editing attempts with classical CRISPR or CRISPR-based base editing.

[0008] Given that the majority of mutations are missense and act by a poison peptide mechanism, simple overexpression of wild-type lamin A is unlikely to be helpful in most cases. Cases with clear haploinsufficiency, i.e., truncating mutations, may be a rare exception. In addition, evidence suggests that simple overexpression of lamin A exerts deleterious consequences. For example, lamin A overexpression in transgenic mice led to sporadic occurrence of AV conduction defects (Frock, R.L., et al., 2012, PLoS One 7). It is not clear at this point whether the toxicity of overexpression of lamin A results from simply too much and / or from the unphy si ologi cal distribution of lamin A / C and / or disturbation of the normal stoichiometry of lamin A and C.SUMMARY

[0009] The following presents a simplified summary of various aspects of the present disclosure in order to provide a basic understanding of such aspects. This summary is not an extensive overview of the disclosure. It is intended to neither identify key or critical elements of the disclosure, nor delineate any scope of the particular embodiments of the disclosure or any scope of the claims. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

[0010] A first aspect of the present disclosure relates to a nucleic acid molecule for use in the treatment of laminopathy in a subject. In at least one embodiment, the nucleic acid molecule comprises a coding sequence that encodes: a small-interfering RNA (siRNA) targeting a portion of the endogenously expressed LMNA gene; and lamin A protein and / or lamin C protein that is resistant to the siRNA.

[0011] In at least one embodiment, the siRNA targets exon 2 of the endogenously expressed LMNA gene. In at least one embodiment, the siRNA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to either SEQ ID NO: 15 or SEQ ID NO: 18.

[0012] In at least one embodiment, the lamin A protein and / or lamin C protein corresponds to a wild-type lamin A protein and / or wild-type lamin C protein.

[0013] In at least one embodiment, the sequence encoding the lamin A protein and / or lamin C protein has a subset of introns removed therefrom. In at least one embodiment, the subset of introns includes introns 8 through 11, introns 8 through 10, introns 9 through 11, or shortened forms thereof.

[0014] In at least one embodiment, the coding sequence further comprises a promoter. In at least one embodiment, the promoter is a constitutive promoter or a tissue-specific promoter.

[0015] In at least one embodiment, the sequence encoding lamin A protein encodes an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 1.

[0016] In at least one embodiment, the laminopathy is caused by one or more mutations of the endogenously expressed LMNA gene.

[0017] In at least one embodiment, the one or more mutations of the LMNA gene causes haploinsufficiency for lamin A. In at least one embodiment, the one or more mutations of the LMNA gene causes a dominant-negative lamin A.

[0018] In at least one embodiment, the laminopathy is a laminopathy that affects striated muscle. In at least one embodiment, the laminopathy is selected from the group consisting of cardiomyopathy or muscular dystrophy.

[0019] In at least one embodiment, the laminopathy is a cardiomyopathy, such as dilated cardiomyopathy 1A.

[0020] A second aspect of the present disclosure relates to a vector comprising the nucleic acid molecule of any one of the aforementioned embodiments for use in the treatment of laminopathy of a subject.

[0021] In at least one embodiment, the coding sequence of the nucleic acid molecule comprises a promoter that is operably linked to the sequence encoding lamin A protein to effect the expression of lamin A in cells contacted with the vector when the promoter is active.

[0022] In at least one embodiment, the expression of lamin A protein from the vector increases the overall expression level of lamin A in cells affected by laminopathy contacted with the vector to at least 50% of the wild-type expression level. In at least one embodiment, the expression level of lamin A protein increases to between about 60% and about 300% of the wildtype expression level.

[0023] In at least one embodiment, the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes affected by laminopathy contacted with the vector by 5% to 200% compared to cardiomyocytes affected by laminopathy not contacted with the vector.

[0024] A third aspect of the present disclosure relates to an adeno-associated virus (AAV) for use in the treatment of laminopathy in a subject comprising the nucleic acid molecule of any one of the aforementioned embodiments. In at least one embodiment. In at least one embodiment, the AAV is serotype AAV6 or AAV9.

[0025] A fourth aspect of the present disclosure relates to a pharmaceutical composition for use in the treatment of laminopathy in a subject comprises the vector of any one of the aforementioned embodiments or the AAV of any one of the aforementioned embodiments, and a pharmaceutically acceptable carrier.

[0026] A fifth aspect of the present disclosure relates to a method of treating laminopathy in a subject comprises delivering a vector to tissue of the subject, the vector comprising a nucleic acid molecule that, when delivered to the tissue, causes the tissue to express: a small-interfering siRNA targeting a portion of the endogenously expressed LMNA gene; and wild-type lamin A protein and wild-type lamin C protein.

[0027] In at least one embodiment, the presence of the siRNA suppresses the expression of endogenous lamin A and lamin C proteins. In at least one embodiment, lamin A and lamin C proteins within the tissue are expressed in stochiometric or near stochiometric quantities.

[0028] In at least one embodiment, the siRNA suppresses the expression of each of the endogneous lamin A and lamin C proteins by greater than 10%, greater than 20%, greater than30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In at least one embodiment, the siRNA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to either SEQ ID NO: 15 or SEQ ID NO: 18.

[0029] Laminopathies are typically caused by mutations in the LMNA gene. About 500 different mutations have been described that are linked to laminopathy, over 150 of which are linked to cardiac laminopathies. Hence, in one embodiment, the laminopathy is caused by one or more mutations of the LMNA gene.

[0030] In one embodiment, the one or more mutations of the LMNA gene causes haploinsufficiency for Lamin A or the one or more mutations of the LMNA gene causes a dominant-negative Lamin A. For example, the mutation may lead to the expression of a truncated protein and therefore result in haploinsufficiency, or the mutation may be a miss-sense mutation that results in a dominant negative variant of the protein.

[0031] Laminopathies can be loosely categorized by their phenotype and affected systems. Generally, LMNA mutations affect one or more of striated muscle, adipose tissue, peripheral nerve or multiple systems with features of accelerated aging.

[0032] In one embodiment, the laminopathy is a laminopathy that affects striated muscle. Hence, in one embodiment, the laminopathy is selected from cardiomyopathy or muscular dystrophy. In one embodiment, the laminopathy may be Emery-Dreifuss muscular dystrophy, cardiomyopathy, limb-girdle muscular dystrophy or congenital muscular dystrophy. In a preferred embodiment, the laminopathy is cardiomyopathy, more preferably dilated cardiomyopathy.

[0033] The nucleic acid molecule of the invention may be comprised in a vector. Hence, in a further aspect, the present disclosure relates to a vector comprising the nucleic acid molecule any of the foregoing embodiments for use in the treatment of laminopathy in a subject.

[0034] In one embodiment, the vector is an expression vector further comprising a nucleic acid sequence encoding a promoter. In at least one embodiment, the promoter is operably linked to the nucleic acid sequence encoding lamin A protein and / or lamin C protein, resulting in expression of lamin A protein and / or lamin C protein in cells contacted with the vector when the promoter is active.

[0035] Mutations in LMNA commonly lead to haploinsufficiency and / or dominant-negative activity of the resulting mutant protein. Therefore, the levels of expression of the wild-type allele of lamin A in affected cells and tissues are usually greatly reduced compared to expression in wild-type cells or organisms. Hence, one objective of the invention is to raise the levels of wildtype Lamin A protein in the affected cells without inducing toxicity or protein aggregation.

[0036] Functionally, LMNA mutation phenotypes can affect the contractile force of affected cardiomyocytes, increase the risk of beating irregularities (arrhythmia) and increase heart rate in cardiomyocytes affected by laminopathy.

[0037] Thus, in one embodiment, the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes affected by laminopathy contacted with the vector by at least 5% compared to cardiomyocytes not contacted with the vector. In another embodiment, the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes contacted with the vector by 5% to 200% compared to cardiomyocytes affected by laminopathy not contacted with the vector.

[0038] In one embodiment, expression of lamin A from the vector reduces the degree of beating irregularities by at least 5% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector. In at least one embodiment, the beating irregularities are detected by measuring cycle length variability using variability of beat-to-beat interval (RR scatter).

[0039] In another embodiment, expression of lamin A from the vector reduces beats per minute by at least 1% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector.

[0040] The vector of the invention may be packaged into a replication-deficient adeno- associated virus (AAV) or any other virus suitable for delivering gene therapy into cells.

[0041] Hence, a further aspect of the invention relates to an AAV for use in the treatment of laminopathy in a subject comprising or the nucleic acid molecule or the vector for use of the invention. In one embodiment, the AAV is serotype AAV6 or AAV9.

[0042] In one embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to at least 50% of WT expression. Preferably, the AAV for use is administered at a titer of about 10,000 to about 500,000 virus particles / cell.

[0043] Lastly, in one aspect the invention also provides a pharmaceutical composition for use in the treatment of laminopathy in a subject comprising the nucleic acid molecule for use, the vector for use or the AAV for use of the invention and a pharmaceutically acceptable carrier.DEFINITIONS

[0044] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0045] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of.” If hereinafter a group isdefined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0046] The terms “about” or “approximately” in the context of the present disclosure denote an interval of accuracy that the person of ordinary skill in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

[0047] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Embodiments of the present disclosure are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings.

[0049] FIG. l is a schematic overview of three designed constructs for the exogenous expression of lamin A and C, in accordance with at least one embodiment.

[0050] FIG. 2 is a scheme depicting the predicted regulatory regions in the LMNA introns (In) between exon 9 (E9) and exon 11 (El 1).

[0051] FIG. 3 A shows a Western blot analysis of 2D LMNA knockout hiPSC cardiomyocytes comparing non-transfected (NT) cells with cell transfected with mini-genes prepared in accordance with at least one embodiment.

[0052] FIG. 3B shows quantification of lamin C peptides by mass spectrometry of cell lysates from unrelated control ERC020 (Wild-type), LMNA knockout non-transfected (NT), or transfected with mini -genes 1, 2 or 3, in accordance with at least one embodiment.

[0053] FIG. 4 shows Western blot analysis of hiPSC-CM (ERC021) non-transfected (NT) or transfected with scrambled siRNA (scRNA), siRNA against GAPDH (sGAPDH), siRNA 3 and siRNA 4 against LMNA.

[0054] FIG. 5 illustrates target sequences of the siRNA 3 and siRNA 4 on the LMNA RNA and their respective tested siRNA sequences including their overhangs, in accordance with at least one embodiment.

[0055] FIG. 6 shows a plasmid construct designed for driving the expression of mini-gene 2res and siRNA 4, in accordance with at least one embodiment.

[0056] FIG. 7 shows a plasmid construct driving the expression of mini-gene 3res and siRNA 4, in accordance with at least one embodiment.

[0057] FIG. 8 shows Western blot images and quantification data demonstrating the expression levels of lamin A / C in ERC021 and LMNA KI hiPSC CM (R321Xhet) cells following transfection with plasmids containing siRNA4 or mini -gene 2.DETAILED DESCRIPTION

[0058] Embodiments of the present disclosure relate to nucleic acid molecules, vectors, and pharmaceutical compositions, along with methods of treatment utilizing the same, for the treatment of laminopathies. In certain embodiments, a nucleic acid molecule (e.g., in the form of a gene construct) may be used in an erase-replace gene therapy approach. For example, such embodiments may be used to erase complete endogenous lamin transcripts (endoLMNA) and at the same time replace it by an exogenously introduced, wild-type lamin A / C (res-wtLamin) that is resistant to degradation by mi / siRNA.

[0059] In principle, such a gene therapy approach represents a causal and mutation-spanning therapeutic approach, which should make it possible to treat laminopathies not only symptomatically but also causally by restoring the NL with its diverse functions. In contrast to previous approaches that result in overexpression of a lamin A cDNA, the embodiments described herein advantageously provide for expression of lamin A and C at normal, stochiometric ratios by using a mini-gene approach.

[0060] As used herein, the term “laminopathy” or “laminopathies” refers to a group of genetic disorders caused by mutations in genes encoding proteins of the nuclear Lamina. The nuclear Lamina is an intermediate filament network that provides scaffolding for the cell nucleus. Laminopathy belongs to a group of diseases termed “nuclear envelopathies” and disease-causing mutations may occur in nuclear envelope proteins including Emerin (encoded in humans by the gene EMD), MANI (encoded in humans by the gene LEMD3), Lamin A / C (encoded in humans by the gene LMNA), Lamin B 1 and 2 (encoded in humans by the genes LMNB1 and LMNB2) and Lamin B receptor (encoded in humans by the gene LBR). Thus, nuclear envelopathies involving Lamins are collectively called laminopathies.

[0061] The terms “nucleic acid” or “nucleic acid molecule” or “nucleic acid sequence” or “nucleotide sequence” are used interchangeably herein to refer to a biomolecule composed of nucleotides. The nucleic acid molecule can be comprised within an eukaryotic or prokaryotic organism, a eukaryotic or prokaryotic cell, a cell nucleus or a cell organelle, as part of a genome or as an individual molecule; or it can be comprised within a plasmid, a vector, an artificial chromosome; a nucleic acid can also exist outside of a cell, in vesicles, viruses or freely circulating, it can be isolated in a suitable composition, in a fixed or frozen tissue or cell culture, or dried. The nucleic acid can be synthesized or naturally occurring, i.e. isolated from nature.

[0062] The terms “encoded protein” or “encoded amino acid” refers a protein that includes a chain of amino acids, which results from a sequence that is encoded by a nucleic acid molecule comprising three-nucleotide codons.

[0063] Laminopathies can be loosely categorized by their phenotype and affected systems. Generally, LMNA mutations affect one or more of striated muscle, adipose tissue, peripheral nerve or multiple systems with features of accelerated aging. “Striated muscle” can be either cardiac muscle or skeletal muscle and the term refers to a muscle tissue that, as opposed to smooth muscle, features repeating functional units called sarcomeres. Hence, laminopathy phenotypes commonly affect both heart muscle and skeletal muscle.

[0064] In one embodiment, the laminopathy is a laminopathy that affects striated muscle. Hence, in one embodiment, the laminopathy is selected from Emery-Dreifuss muscular dystrophy, cardiomyopathy, limb-girdle muscular dystrophy IB and congenital muscular dystrophy. In a preferred embodiment, the laminopathy is cardiomyopathy, more preferably dilated cardiomyopathy 1A. Preferably, the laminopathy is not a peripheral neuropathy.

[0065] The term “muscular dystrophy” refers to a group of muscle disorders that are caused by genetic mutations, usually in genes encoding muscle cell-specific proteins. Muscular dystrophies cause progressive weakening and breakdown of muscle tissue which leads to a range of symptoms, including inability to walk, limited range of movement, respiratory difficulty, muscle spasms, deformations and cardiomyopathy. Muscular dystrophies associated with mutations in LMNA include autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, dilated cardiomyopathy (or “cardiomyopathy dilated 1 A”) and limb-girdle muscular dystrophy type IB.

[0066] The term “cardiomyopathy” refers to a disease or dysfunction of heart muscle.

[0067] Laminopathies caused by LMNA mutation with adipose tissue involvement, called partial lipodystrophy syndromes, include Dunnigan-type familial partial lipodystrophy, lipoatrophy with diabetes, hepatic steatosis, hypertrophic cardiomyopathy and leukomelanodermic papules, insulin resistance without lipoatrophy and mandibuloacral dysplasia.

[0068] LMNA mutations involving peripheral nerve tissue or “peripheral neuropathy” include Charcot-Marie-Tooth disorder type 2B1. Premature aging syndromes caused by LMNA mutations include Hutchinson-Gilford progeria syndrome, Restrictive dermopathy, variant progeroid disorders, mandibuloacral dysplasia (this disease has features of both lipodystrophy and progeria) and atypical Werner syndrome.

[0069] “Dilated cardiomyopathy” or “DCM” as used herein refers to a condition in which the heart becomes enlarged and fails to pump blood effectively, eventually resulting in heart failure.About 20-25% of cases of DCM are familial, i.e. they result from inherited mutations. Affected genes include LMNA encoding Lamin A / C, SCN5A encoding NaV1.5, TNNT2 encoding cardiac muscle Troponin, TTN encoding Titin, DES encoding Desmin, ACTC encoding cardiac muscle a-Actin, PLN encoding Phospholamban, and many others. DCM associated with mutations in LMNA is referred to as “dilated cardiomyopathy 1 A” or “CMD1 A”.

[0070] The “Lamin A” protein (UniProt accession number: P02545) referred to herein is one of two splice variants of the protein encoded by the LMNA gene, the other being Lamin C. Lamin A and C are type 5 intermediate filaments and localize to the nuclear Lamina, a stabilizing layer underneath the inner surface of the nuclear envelope. Nuclear Lamins have a variety of functions including structural support of the nuclear envelop, providing anchorage sites for chromatin, nuclear envelope assembly, DNA synthesis, transcription and apoptosis. Each Lamin subtype forms separate Lamin polymers and filaments in the nuclear Lamina. Additionally, Lamins can also be found in the nuclear interior in soluble, unpolymerized form.

[0071] LMNA encodes several splice isoforms, including Lamin A (664 amino acids) and Lamin C (576 amino acids). The two splice variants Lamin A and C share the first 566 amino acids but differ at the carboxyl-terminus, where Lamin A contains an additional two exons that are lacking in Lamin C. Nuclear Lamins are targeted to the inner nuclear membrane by nuclear localization motifs. Lamin A is first expressed as a precursor protein (PreLamin A), and then undergoes posttranslational modification. PreLamin A contains a CaaX motif which triggers famesylation and methylation, and also undergoes an additional endoproteolytic processing step at which the last 15 amino acids of the protein are clipped off, resulting in mature Lamin A. The “CaaX motif’ is a common terminal acid motif in cellular proteins and includes a cysteine residue followed by two aliphatic amino acid residues and another variable amino acid residue.

[0072] LMNA produces a variety of transcripts, encoding at least six known isoforms. The canonical sequence encodes a protein of 664 amino acids which is represented by the amino acid sequence according to SEQ ID NO: 1. The mature Lamin A protein after post-translational processing includes amino acids 1 to 646 of SEQ ID NO: 1 and is represented by the amino acid sequence according to SEQ ID NO: 2. A nucleic acid sequence encoding SEQ ID NO: 1 is represented by the nucleic acid sequence according to SEQ ID NO: 3.

[0073] In one embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence that is at least 70% identical to the amino acid according to SEQ ID NO: 1. In one embodiment, the Lamin A is wild-type Lamin A. In one embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid according to SEQ ID NO: 1. In one embodiment, the nucleic acid moleculeencodes an amino acid that comprises an amino acid sequence according to any one of SEQ ID NO: 1.

[0074] In another embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 2. In one embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid according to SEQ ID NO: 2. In one embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence according to any one of SEQ ID NO: 2.

[0075] In another embodiment, the nucleic acid of molecule comprises a nucleic acid molecule that is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 3. In another embodiment, the nucleic acid of molecule comprises a nucleic acid molecule that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence according to SEQ ID NO: 3. In another embodiment, the nucleic acid of molecule comprises a nucleic acid sequence according to SEQ ID NO: 3.

[0076] The nucleic acid of the invention may encode any wild-type isoform of Lamin A. Thus, in some embodiments, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence according to any one of SEQ ID NOS: 4-8. In one embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to any one of SEQ ID NO: 4-8. In one embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence according to any one of SEQ ID NO: 4-8.

[0077] Embodiments of the present disclosure were suprisingly and avantageously able to treat dilated cardiomyopathy and restore functional and structural properties of affected cardiomyocytes by introducing a nucleic acid molecule expressing lamin A and / or lamin C into affected cells.

[0078] In one embodiment, the nucleic acid molecule encodes an amino acid that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence according to any one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, or 8.

[0079] The terms “sequence Identity”, “% sequence identity”, “% identity”, “% identical”, or “sequence alignment” are used interchangeably herein and refer to the comparison of a first nucleic acid sequence to a second nucleic acid sequence, or a comparison of a first amino acid sequence to a second amino acid sequence and is calculated as a percentage based on the comparison. The result of this calculation can be described as “percent identical” or “percentID.” A sequence identity may be determined by a program, which produces an alignment, and calculates identity counting both mismatches at a single position and gaps at a single position as non-identical positions in final sequence identity calculation. The sequence identity is determined over the entire length of the first and second nucleic acid sequence.

[0080] The terms “polypeptide”, “protein” or “peptide” are used interchangeably herein and refer to amino acid sequences of a variety of lengths. The term polypeptide may also refer to the primary, secondary, tertiary, quarternary or quinary structure or a protein. Polypeptides can be in uncharged forms or as salts, either unmodified or modified by glycosylation, side chain oxidation, phosphorylation, citrullination or transglutamination. In certain embodiments, the polypeptide is a full-length native protein. In other embodiments, the polypeptide is a smaller fragment of the full-length protein. In still other embodiments, the amino acid sequence is modified by additional substituents attached to the amino acid side chains, such as N- or C- terminal added protein tags, glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversion of the chains such as oxidation of sulfhydryl groups. Thus, the term “polypeptide” is intended to include the amino acid sequence of the full- length native protein, or a fragment thereof, subject to those modifications that do not significantly change its specific properties. In particular, the term “polypeptide” encompasses protein isoforms, i.e., variants that are encoded by the same gene, but that differ in their amino acid sequence or in other properties.

[0081] The “UniProt numbers” or “UniProf ’ or “UniProt Accession numbers” provided herein refer to the unique identifiers given to individual genes and proteins by the UniProt Consortium, which are available from their database at www.uniprot.org and commonly used as references in the field. UniProtKB (UniProt Knowledgebase) is a freely accessible database of protein sequence and functional information. The UniProt database includes manually annotated and reviewed entries (provided by the Swiss-Prot database) and automatically annotated and not manually reviewed entries (provided by TrEMBL database), many of which are derived from genome sequencing projects. TrEMBL includes translated coding sequences from the EMBL- Bank / GenBank / DDBJ nucleotide sequence database, and others.

[0082] In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding a promoter. In one embodiment, the promoter may be a promoter with constitutive activity in eukaryotic cells, or a promoter that is tissue specific.

[0083] The term “promoter” refers to a DNA sequence capable of starting and controlling the expression of a coding sequence or functional RNA. Typically, a coding sequence is located 3' to a promoter 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 evencomprise synthetic DNA segments. Typically, since the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. It is understood by a person skilled in the art that different promoters may direct the expression of a gene at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as constitutive promoters. On the other hand, promoters that cause a gene to be expressed in specific contexts only, e.g. based on the presence of specific factors, growth stages, temperatures, pH or the presence of specific metabolites etc. are understood as regulatable promoters.

[0084] The term “3' non-coding sequences,” “5’ non-coding sequences,” “3’ UTR,” or “5’ UTR” refers to DNA sequences located downstream of a coding sequence. The term “RNA transcript” refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. The term “mRNA” refers to messenger RNA, i.e., RNA that is without introns and that can be translated into proteins by the cell. The term “cDNA” refers to complementary DNA, which means a DNA that is generated from an RNA by reverse transcription.

[0085] The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. In the context of a promoter the term means that the coding sequence is under the transcriptional control of the promoter which regulates the expression of the coding sequence.

[0086] A “constitutive promoter” or “ubiquitous promoter” as used herein is a promoter active in vivo in all circumstances. The expression from a constitutive promoter is non-selective.

[0087] In contrast, a “selective” or “tissue-specific” promoter is a promoter that is specifically activated in one or more cell types or tissues and is not active in other cell types. For example, promoters of cardiac-specific genes are active in cardiac tissues, specifically in cardiomyocytes. Hence, in one embodiment, the promoter is a cardiac-specific promoter. In one embodiment, the cardiac-specific promoter is selected from the group consisting of myosin light chain 2 (MLC-2v) promoter, alpha myosin heavy chain (aMHC) promoter, beta myosin heavy chain (PMHC) promoter cardiac Troponin T (cTnT) promoter, myocyte-specific enhancer factor 2C (MEF2C) promoterand Nk2 homeobox 5 (Nkx2.5) promoter. Preferably, the promoter is selected from myosin light chain 2 (MLC-2v) promoter, alpha myosin heavy chain (aMHC) promoter, beta myosin heavy chain (PMHC) promoter cardiac Troponin T (cTnT) promoter, and myocyte-specific enhancer factor 2C (MEF2C) promoter. Most preferably, the promoter is a cardiac Troponin T promoter. In another embodiment, the promoter is a Lamin A / C promoter.

[0088] In another embodiment, the promoter may be a constitutive promoter, a tissuespecific promoter or an inducible promoter.

[0089] “Inducible promoters” are promoters that can be switched on and off. They can be chemically inducible, such as the tetracycline inducible Tet operator, lac operator, Gal4-UAS system or inducible Cre-lox system, temperature inducible, such as Hsp70 or Hsp90, or light inducible. Cardiac specific promoters can be combined with inducible enhancers or promoter elements for time and space specific inducible activity. Thus, in one embodiment, the promoter may be a cardiac specific inducible promoter.

[0090] The promoter may be an ubiquitous promoter or any suitable promoter for expression in the chosen system, for example CMV (cytomegalovirus), T7, lac operator, EFl alpha (elongation factor 1 alpha), chicken beta-Actin, CAG, Tet operator, EpIE2, SV40 (simian virus 40), MT (metallothioneine), UbC (ubiquitin c), RSV (rous sarcoma virus), AUG1, CMV / EFlalpha, FLD1 (formaldehyde dehydrogenase 1), TK (thymidine kinase), Gal4 UASZElb (galactose), trc, tac, AOX1 (alcohol oxidase 1), olyhedrin or copia promoter.

[0091] In one embodiment, the promoter is a constitutive promoter selected from the group consisting of CMV, CAG, Chicken beta-Actin, EFl alpha, CMV / EFlalpha, and SV40.

[0092] In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding an epitope tag, preferably selected from His-tag, GS-tag, FLAG-tag, c-Myc tag or HA-tag or a fluorescent tag, such as a fluorescent protein or FRET label, or a radiolabel or an antibody tag.

[0093] In another embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding a linker. The linker may be a fusion linker, a 2A peptide or an internal ribosome entry site (IRES). In one embodiment, the linker is selected from Gs linker, GGGGS linker, T2A, P2A, F2A, E2A and IRES.

[0094] Laminopathies are typically caused by mutations in the LMNA gene. About 500 different mutations have been described that are linked to laminopathy, distributed along the whole gene without any obvious hotspots, and over 150 of mutations are linked to cardiac laminopathies. Hence, in one embodiment, the laminopathy is caused by one or more mutations of the LMNA gene.

[0095] The term “mutation” as used herein refers to alterations in the nucleotide sequence of the genome of an organism. Specifically, the term “LMNA mutation” refers to alterations in the genomic DNA sequence of LMNA. Genes can for example be disrupted by large-scale genomic events, such as chromosomal deletions, rearrangements, inversions or crossovers. These large- scale events can lead to loss of heterozygosity if they only affect one allele, i.e. one copy of the gene. Small-scale mutations affect a gene in one or only a few nucleotides. Mutations that affect a gene in only one nucleotide are called point mutations. Small-scale or point mutations include insertions, deletions, and substitution mutations.

[0096] The following terms describe mutations on the nucleotide sequence level. “Insertions” refers to the addition of one or more nucleotides into the DNA, while “deletions” refers to the removal of one or more nucleotides from the DNA. Collectively, this type of mutation is also called “indel”. Indels can be caused by transposable elements, errors during replication or DNA damage. Indels typically cause frame-shifts in the reading frame of the gene and therefore lead to missense or nonsense mutations. Indels can also affect the splicing of mRNA if they occur in the splice sites, usually at the exon-intron boundaries, and involve the splice donator or splice acceptor nucleotides.

[0097] “Frame-shift” mutations occur if the indel involves a number of nucleotides that is not a multiple of three. Because of the three-nucleotide code, inserting or deleting a number of nucleotides that is not a multiple of three changes the amino acid sequence of the encoded protein downstream of the mutation. If such a severe change of sequence occurs early on in the protein sequence, this usually results in a nonfunctional protein. The nonfunctional protein may then be degraded by the cell.

[0098] A “missense” mutation causes the substitution of a different amino acid due to a change in the nucleotide codon sequence. A “nonsense” mutation refers to a mutation that results in a premature stop codon, which in turn often leads to a truncated version of the protein being expressed. Missense and nonsense mutations are also called “non-synonymous”, which means that they replace a codon with a codon that encodes a different amino acid, thereby changing the sequence.

[0099] A “synonymous” or “silent” mutation is a nucleotide mutation that does not change the encoded amino acid, due to the degenerate amino acid code. This typically occurs if only the third nucleotide is mutated, the so-called “wobble base”. In one embodiment, the nucleic acid sequence encoding wild-type lamin A and / or lamin C may comprise one or more synonymous mutations.

[0100] On the protein sequence level, mutations are usually described based on their functional effect. “Loss-of-function” mutations result in the gene product or protein having less or no function. A complete loss of function may be called a “null allele”. Loss of function mutations which result in the reduced expression or no expression of one of the two alleles of a gene cause haploinsufficiency if the reduced dosage of the protein is not enough for a normal or wild-type phenotype.

[0101] The term “haploinsufficiency” as used herein refers to a condition in which at least one of two alleles of a gene is mutated and wherein the combined gene product expression level of the wild-type and mutated alleles is not sufficient to produce a wild-type phenotype.

[0102] Gain-of-function” mutations change the gene product or protein such that either its effect is stronger (enhanced activation) or is replaced by a different or abnormal function. “Dominant-negative” mutations are mutations in which the altered gene product or protein acts antagonistically to the wild-type allele and adversely affects the wild-type gene product within the same cell. This occurs for example when a mutation in a protein removes a functional domain but retains DNA or protein-protein interaction domains. In this case, the protein can still bind its interaction partner but does not function, thereby effectively blocking the pathway from functioning normally.

[0103] Gain- and loss-of-function mutations can occur within the coding regions of a gene or within the regulatory regions, such as enhancer binding sites or promoter regions. Mutations in regulatory regions may lead to increased or decreased gene expression or changes in the spatiotemporal pattern of gene expression.

[0104] Mutations in LMNA typically include truncating mutations, which are generally associated with a more severe phenotype, and missense mutations (Crasto et al. Front Physiol 2018, PMID: 32719615). Mutant variants or the partial absence of Lamin A may lead to a disruption of the structure and function of the nuclear Lamina and disrupt both the import and export of molecules through the nuclear pores as well as transcription. Disruption of transcription may be due to the nuclear membrane’s function in assembly of densely packaged chromatin complexes in Lamin-associated domains (LADs), which result in a stable suppression of transcription in the affected chromosome segments. The nuclear membrane, and Lamin in particular, also play an important role in mechanotransduction, i.e. the transduction of mechanical stimuli and their effect on transcription (Crasto et al. Front Physiol 2018, PMID: 32719615). This could explain the fact that laminopathies predominantly affect tissues which experience mechanical stress, such as heart or skeletal muscle.

[0105] In one embodiment, the one or more mutations of the LMNA gene cause haploinsufficiency for Lamin A or the one or more mutations of the LMNA gene cause a dominant-negative Lamin A.

[0106] For example, the mutation may lead to the expression of a truncated protein and therefore result in haploinsufficiency, or the mutation may be a missense mutation that results in a dominant negative variant of the protein.

[0107] Methods of detecting mutations are known in the art and include, for example, sequencing of exons and intron-exon junctions of the LMNA gene or copy -number-variation studies.Vector for use in gene replacement therapy

[0108] The nucleic acid molecule of the invention can be packaged into one or more vectors, e.g., plasmids or viral vectors. In some embodiments, the vectors, e.g., plasmids or viral vectors, are delivered to the tissue of interest by, e.g., intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. Such delivery may be either via a single dose or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation / modification sought, the administration routes, the administration modes, the types of transformation / modification sought, etc.

[0109] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and may be used herein interchangeably with the term “recombinant nucleic acid molecule”. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. The term “expression vector” means a vector capable of directing expression of a particular nucleotide sequence in an appropriate host cell. An expression vector comprises a regulatory nucleic acid element operably linked to a nucleic acid of interest, which is optionally operably linked to a termination signal and / or other regulatory element.

[0110] Examples of other expression vectors and host cells are the pET vectors (NOVAGEN), pGEX vectors (Amersham Pharmacia), and pMAL vectors (New England labs. Inc.) for protein expression in E. coli host cells such as BL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3) (NOVAGEN); the strong CMV promoter-based pcDNA3.1 (INVITROGEN) and pCIneo vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviral vector vectors pADENO X, pAd5F35, pLP- ADENO-X-CMV (CLONTECH), pAd / CMV / V5-DEST, pAd-DEST vector (INVITROGEN) for adenovirus- mediated gene transfer and expression in mammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for use with the RETRO-X™ system from Clontech for retroviral-mediated gene transfer and expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (INVITROGEN) for lentivirus-mediated gene transfer and expression in mammalian cells; adenovirus-associated virus expression vectors such as pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (STRATAGENE) for adeno- associated virus- mediated gene transfer and expression in mammalian cells.[OHl] The term “coding sequence” refers to a DNA sequence which codes for a specific amino acid sequence. The term “regulatory sequence” refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0112] The term “expression” or “gene expression” as used herein refers to the process of synthesis of a gene product, preferably a functional RNA or protein. Gene expression generally comprises DNA transcription, optionally RNA processing and in the case of protein-expressing genes, RNA translation.

[0113] The nucleic acid molecules of the embodiments described herein may be comprised in a vector. Hence, in another aspect, the invention relates to a vector comprising the nucleic acid molecule of the invention for use in the treatment of laminopathy in a subject.

[0114] In one embodiment, the vector comprises a nucleic acid molecule encoding an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 1. In one embodiment, the vector comprises a nucleic acid molecule encoding an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 1.

[0115] In another embodiment, the vector comprises a nucleic acid molecule encoding an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 2. In one embodiment, the vector comprises a nucleic acid molecule encoding an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 2.

[0116] In another embodiment, the vector comprises a nucleic acid molecule comprising a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 3. In another embodiment, the vector comprises a nucleic acid molecule comprising a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence according to SEQ ID NO: 3. In another embodiment, the vector comprises a nucleic acid molecule comprising a nucleic acid sequence according to SEQ ID NO: 3.

[0117] The vector may comprise a nucleic acid molecule encoding any wild-type isoform of lamin A. Thus, in some embodiments, the vector comprises a nucleic acid molecule encoding anamino acid sequence that is at least 70% identical to the amino acid sequence according to any one of SEQ ID NOS: 4-8. In one embodiment, the vector comprises a nucleic acid molecule encoding an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to any one of SEQ ID NOS: 4-8. In one embodiment, the vector comprises a nucleic acid molecule encoding an amino acid sequence according to any one of SEQ ID NOS: 4-8.

[0118] In one embodiment, the vector comprises a nucleic acid molecule encoding an amino acid sequence according to any one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, or 8.

[0119] In one embodiment, the vector is an expression vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding lamin A and further comprising a nucleic acid sequence encoding a promoter. In a preferred embodiment, the nucleic acid sequence encoding the promoter is operably linked to the nucleic acid sequence encoding lamin A. Typically, contacting the cells with the vector results in expression of lamin A in said cells. Preferably, expression of lamin A in cells contacted with the vector occurs when the promoter is active.

[0120] A promoter is considered “active” when gene expression, i.e. transcription, from the promoter occurs. This may occur when the promoter is bound by RNA polymerase and / or activators and / or enhancers. Promoter activity can be measured by methods known in the art, such as determining the gene product, mRNA or protein, or expressing a reporter gene product.

[0121] As used herein, the term “level” refers to the quantity of a specific molecule in a biological sample. The term “expression” or “gene expression” as used herein refers to the process of synthesis of a gene product, preferably a functional RNA or protein. Gene expression generally comprises DNA transcription, optionally RNA processing and in the case of proteinexpressing genes, RNA translation.

[0122] “Expression”, “expression level”, “gene expression” or “gene expression level” can be measured by determining mRNA (transcription) or protein (translation) levels. Methods to measure mRNA or protein levels are known in the art and may involve relative or absolute quantification. For example, absolute quantification of mRNA can be achieved by RNA sequencing, while relative quantification of mRNA can be achieved by quantitative PCR; absolute quantification of protein can be achieved by mass spectrometry or enzyme-linked immunosorbent assay (ELISA), while relative quantification and / or presence or absence of protein can be achieved by immunoblotting (Western Blot) or immunohistochemistry. Gene expression can also be measured using reporter assays such as luciferase assay. The level may be determined numerically, or it may be determined by measuring a secondary signal, such asfluorescence intensity. The level may also be expressed binary by presence or absence of the measured molecule, i.e. the level may be positive or negative.

[0123] Since mutations in LMNA may lead to haploinsufficiency or dominant-negative activity of the resulting mutant protein, the expression levels of functional lamin A expression in affected cells are usually reduced compared to wild-type expression. This may be due to expression of a non-functional allele of lamin A, or due to expression of a dominant-negative allele of lamin A. Since lamins polymerize into filaments, dominant-negative lamins can also affect the functionality of wild-type lamins expressed in the same cell, thereby further reducing the concentration or level or available functional lamins. This may occur for example through aggregation of lamin. Hence, one objective of the invention is to raise the levels of wild-type lamin A protein in the affected cells.

[0124] Thus, in one aspect of the invention, the expression of lamin A from the vector increases the overall expression level of lamin A in cells affected by laminopathy contacted with the vector to at least 50% of the wild-type expression level.

[0125] In one embodiment, the expression of lamin A from the vector increases the overall expression level of lamin A in cells affected by laminopathy contacted with the vector to at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, or at least 300%, of the wild-type expression level.

[0126] In one embodiment, the expression of lamin A from the vector increases the overall expression level of lamin A in cells affected by laminopathy contacted with the vector to no more than 300% of wild-type expression level. Hence, in one embodiment, the expression level of lamin A increases to between about 50% and about 300% of the wild-type expression level.

[0127] In another embodiment, the expression level of lamin A increases to between about 50% and about 150% of the wild-type expression level. In another embodiment, the expression level of lamin A increases to between about 50% and about 200% of the wild-type expression level. In another embodiment, the expression level of lamin A increases to between about 50% and about 250% of the wild-type expression level. In another embodiment, the expression level of lamin A increases to between about 100% and about 150% of the wild-type expression level. In another embodiment, the expression level of lamin A increases to between about 100% and about 200% of the wild-type expression level. In another embodiment, the expression level of lamin A increases to between about 100% and about 250% of the wild-type expression level. Inanother embodiment, the expression level of lamin A increases to between about 100% and about 300% of the wild-type expression level.

[0128] In one embodiment, the expression level is determined on the mRNA level. In another embodiment, the expression level is determined on the protein level.

[0129] “Wild-type”, “control” or “reference” gene expression, mRNA or protein levels are determined by a control sample, cell or organisms, or by averaging the expression levels from multiple control samples, cells or organisms. In the context of the present invention, the term “wild-type” or “control” refers to a cell or organism that is healthy or a sample from a subject that is healthy or to a cell or organism with a specific disease other than laminopathy or a sample from a subject that has been diagnosed with a specific disease other than laminopathy.

[0130] In one embodiment, the term “wild-type” when describing lamin A refers to lamin A or an isoform of lamin A, expressed by a wild-type cell or organism or detected in a sample of a healthy subject, without any changes in amino acid sequence. In another embodiment, wild-type lamin A is encoded by a nucleotide sequence of the LMNA gene present in the genome of a wild-type cell or organism or a healthy subject, without any mutations or comprising one or more synonymous / silent mutations.

[0131] Functionally, LMNA mutation phenotypes can affect the contractile force of cardiomyocytes in subject, increases the risk of beating irregularities (arrhythmia) and increases heart rate in a subject with laminopathy.

[0132] The “contractile force” of cardiomyocytes refers to the force that is produced by myosin in muscle cells in order to slide the myofilaments of the cardiac muscle past each other and generate contractions. Reduced contractile force is a common phenotype of cardiomyopathies. Cardiomyocytes need to generate synchronized contractions in order to produce a heartbeat. Contractions are triggered by electrical stimuli in the form of a cardiac action potential, which results in the release of calcium from the sarcoplasmic reticulum, which in turn causes myofibrils to slide past each other in a process called excitation-contraction coupling. Myofibrils are composed of actin filaments and myosin motor proteins, which are organized into sarcomeres and are the contractile units of muscle cells. After hydrolysis of ATP by myosin, force is produced upon sequential conformational changes of the motor triggered by rebinding to F-actin.

[0133] Thus, in one embodiment, the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes affected by laminopathy contacted with the vector by at least 5% compared to cardiomyocytes not contacted with the vector. In another embodiment, the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes affected by laminopathy contacted with the vector by at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% compared to cardiomyocytes not contacted with the vector.

[0134] In another embodiment, the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes contacted with the vector by 5% to 200% compared to cardiomyocytes affected by laminopathy not contacted with the vector. In another embodiment, the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes contacted with the vector by 10% to 200%, by 20% to 200%, by 30% to 200%, by 40% to 200%, by 50% to 200%, by 60% to 200%, by 70% to 200%, by 80% to 200%, by 90% to 200%, by 100 to 200%, 110% to 200%, by 120% to 200%, by 130% to 200%, by 140% to 200%, by 150% to 200%, 10% to 150%, by 20% to 150%, by 30% to 150%, by 40% to 150%, by 50% to 150%, by 60% to 150%, by 70% to 150%, by 80% to 150%, by 90% to 150%, by 100 to 150%, by 10% to 100%, by 20% to 100%, by 30% to 100%, by 40% to 100% or by 50% to 100% compared to cardiomyocytes affected by laminopathy not contacted with the vector.

[0135] The term “arrhythmia” as used herein refers to an abnormal heart rhythm. Arrhythmias can be classified by changes in beating rate and beating regularity, and the region of their origin (supraventricular and ventricular; e.g. sinoatrial node, atria, AV node and ventricle). The normal beating activity of the heart origins in the sinoatrial node (called sinus rhythm). The heart can beat in regular sinus rhythm that is too fast (sinus tachycardia) or too slow (sinus bradycardia). Tachycardia is defined as a resting heart rate greater than 100 beats per minute (bpm) while bradycardia is defined as a resting heart rate of less than 60 bpm. The beating activity of the heart can also come from another site than the sinoatrial node, which is generally pathological. Examples are the atria (atrial arrhythmia), the AV node (junctional arrhythmia) and the ventricle (ventricular arrhythmia). Generally, supraventricular arrhythmias are less dangerous than ventricular arrhythmias because the AV node normally filters fast electrical activity from the atria and therefore does not allow the ventricle to beat too fast. Some examples of arrhythmia include sick sinus syndrome, conduction block (leading to bradycardia) and premature heartbeats (can be both supraventricular and ventricular), atrial fibrillation, atrial flutter, supraventricular tachycardia, ventricular tachycardia, ventricular fibrillation (leading to tachycardic disturbances). Ventricular fibrillation is the most severe arrhythmia and, without intervention, leads to death in minutes.

[0136] In one embodiment, the presence of arrhythmia in cardiomyocytes affected by laminopathy is detected by determining the RR scatter.

[0137] “Heart rate variability” or abnormal heart rhythm is measured by measuring the variation in the beat-to-beat interval of the heart beat with the help of an electrocardiogram (ECG). Heart rate variability is also called “cycle length variability” or “R-R variability”. Herein, “R” refers to the point of the peak of the QRS complex of the electrocardiogram (ECG) wave, and the “RR interval” is the interval between successive Rs. The RR interval is also sometimes called NN interval to indicate that the heart beat is “normal”. The “QRS complex” is a combination of three graphical deflections seen on a typical ECG, wherein the “R” deflection is an upward peak or “wave” located between the Q and S downward deflections. Methods used to detect heart beats are known in the art and include ECG, blood pressure, ballistocardiograms and the pulse wave signal derived from a photoplethysmograph (PPG).

[0138] In one embodiment, expression of lamin A from the vector reduces the degree of beating irregularities by at least 5% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector, wherein the beating irregularities are detected by measuring cycle length variability based on RR intervals.

[0139] In another embodiment, expression of lamin A from the vector reduces the degree of beating irregularities by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector, wherein the beating irregularities are detected by measuring cycle length variability based on RR intervals.

[0140] Methods of measuring cycle length variability using RR intervals are known in the art. The most commonly used method is a time-domain method called “SDNN” or standard deviation of NN / RR intervals. This method calculates the standard deviation of the average RR intervals calculated over a 24 hour period, or over a shorter period, in order to represent total variability in the heart rate cycle. Another common time-domain method is the root mean square of successive differences or “RMSSD”. This method calculates the square root of the mean of the squares of successive differences in RR intervals. “NN50” or “NN20” methods analyze the number of pairs of successive NNs / RRs that differ by more than 50 ms or 20 ms, respectively.

[0141] Geometric methods can also be used to detect cycle length variability based on RR intervals. An RR scatter plot may be used, also termed “Poincare plot”. The Poincare plot plots RR interval 1 against RR interval 2, RR interval 2 against RR interval 3, and so on. In short, the X-axis of the scatter plot shows RRn (ms) and the Y-axis of the scatter plot shows RRn+1 (ms). Hence, the plot shows how well each RR interval predicts the next, i.e. the variability betweenone RR interval and the next. A greater spread or scatter of values indicates an increased heart rate variability.

[0142] In one embodiment, measuring cycle length variability based on RR intervals means measuring cycle length variability using RR scatter.

[0143] Hence, in one embodiment, expression of lamin A from the vector reduces the degree of beating irregularities by at least 5 % in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector, wherein the beating irregularities are detected by measuring cycle length variability using RR scatter.

[0144] In another embodiment, expression of lamin A from the vector reduces the degree of beating irregularities by at least 10 %, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector, wherein the beating irregularities are detected by measuring cycle length variability using RR scatter.

[0145] In another embodiment, expression of lamin A from the vector reduces beats per minute by at least 1% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector.

[0146] In another embodiment, expression of lamin A from the vector reduces beats per minute by at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector.

[0147] In one embodiment, expression of lamin A from the vector for use of the invention reduces the degree of beating irregularities by at least 5 % and / or beats per minute by at least 1% in cardiomyocytes affected by laminopathy contacted with the vector compared to cardiomyocytes affected by laminopathy not contacted with the vector, wherein the beating irregularities are detected by measuring cycle length variability using RR scatter.Delivery of gene replacement therapy into cells

[0148] The nucleic acid molecule or the vector of the invention can be delivered by various delivery systems such as adeno-associated viruses (AAV), lentiviruses, adenoviruses, and other viral vectors, or methods, such as transfection, transduction, infection, gene transfer, gene editing, nucleofection or electroporation.

[0149] In certain embodiments, the delivery is via adeno-associated viruses (AAV). Adeno- associated viruses are replication-defective, nonenveloped viruses with a linear single-stranded DNA genome. Gene therapy vectors using AAV can infect cells without integration into the host cell genome. At least eleven serotypes of AAV have been identified so far, most of which can infect cells from multiple tissue types. Tissue specificity is determined by the capsid serotype and gene therapy vectors are selected according to their tropism. While the most commonly used serotype is AAV2, the optimal serotype for muscle cell infections are AAV1, AAV6, AAV7, AAV8, and AAV9.

[0150] In some embodiments, the delivery is via a recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, a modified AAV vector may be used for delivery. Modified AAV vectors can be based on one or more of several capsid types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV8.2. AAV9, AAV rhlO, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV6) and pseudotyped AAV (e.g., AAV2 / 8, AAV2 / 5 and AAV2 / 6). Exemplary AAV vectors and techniques that may be used to produce rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. Appl. Microbiol.Biotechnol. 2018, PMID: 29204900; Zhong et al. J. Genet. Syndr. Gene Ther. 2012, PMID: 23264889).

[0151] Hence, a further aspect of the invention relates to an AAV for use in the treatment of laminopathy in a subject comprising or the nucleic acid molecule for use of the invention or the vector for use of the invention. In one embodiment, the AAV is an AAV with a serotype selected from the group consisting of serotype AAV1, AAV2, AAV6, AAV7, AAV8, and AAV9. Preferably, the AAV is serotype AAV6 or AAV9.

[0152] Preferably, the AAV comprises an expression cassette comprising a promoter and a nucleic acid molecule of the invention, encoding lamin A.

[0153] In one embodiment, the AAV comprises a nucleic acid molecule encoding an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 1. In one embodiment, the AAV comprises a nucleic acid molecule encoding an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 1.

[0154] In another embodiment, the AAV comprises a nucleic acid molecule encoding an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 2. In one embodiment, the AAV comprises a nucleic acid molecule encoding an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO: 2.

[0155] In another embodiment, the AAV comprises a nucleic acid molecule comprising a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 3. In another embodiment, the AAV comprises a nucleic acid molecule comprising a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence according to SEQ ID NO: 3. In another embodiment, the AAV comprises a nucleic acid molecule comprising a nucleic acid sequence according to SEQ ID NO: 3.

[0156] The AAV may comprise a nucleic acid molecule encoding any wild-type isoform of lamin A. Thus, in some embodiments, the AAV comprises a nucleic acid molecule encoding an amino acid sequence that is at least 70% identical to the amino acid sequence according to any one of SEQ ID NOS: 4-8. In one embodiment, the AAV comprises a nucleic acid molecule encoding an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to any one of SEQ ID NOS: 4-8. In one embodiment, the AAV comprises a nucleic acid molecule encoding an amino acid sequence according to any one of SEQ ID NOS: 4-8.

[0157] In one embodiment, the AAV comprises a nucleic acid molecule encoding an amino acid sequence according to any one of SEQ ID NOs: 1, 2, 4, 5, 6, 7, or 8, preferably wherein the AAV is serotype AAV6 or AAV9.

[0158] In one embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to at least 50% of wild-type expression level. In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, or at least 300%, of the wild-type expression level.

[0159] In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to between about 50% and about 300% of the wild-type expression level.

[0160] In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to between about 50% and about 150% of the wild-type expression level. In another embodiment, the AAV for use is administered at a titer suitable to induce expressionof lamin A to between about 50% and about 200% of the wild-type expression level. In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to between about 50% and about 250% of the wild-type expression level. In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to between about 100% and about 150% of the wild-type expression level. In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to between about 100% and about 200% of the wild-type expression level. In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to between about 100% and about 250% of the wild-type expression level. In another embodiment, the AAV for use is administered at a titer suitable to induce expression of lamin A to between about 100% and about 300% of the wild-type expression level.

[0161] In one embodiment, the expression level is determined on the mRNA level. In another embodiment, the expression level is determined on the protein level.

[0162] The term “virus particles” or “virus particle units” as used herein refers to a complete virus particle, also known as virion, comprising a nucleic acid surrounded by a protective coat of protein called a capsid.

[0163] In one embodiment, the AAV for use is administered at a titer resulting in about 10,000 to about 500,000 virus parti cles / cell, preferably wherein the cell is a cardiomyocyte. In another embodiment, the AAV for use is administered at a titer resulting in about 100,000 to about 500,000 virus particles / cell, preferably wherein the cell is a cardiomyocyte.

[0164] The AAV can be administered in a single dose containing at least 1 x 1010virus particles of adeno-associated viruses per kg of body weight of the subject. In one embodiment, the AAV can be administered in a single dose containing at least 1 x 1012virus particles of adeno-associated viruses per kg of body weight of the subject.

[0165] In one embodiment, the AAV is administered to a subject at a dose between lx 1012to 2 x 1014virus particles per kg body weight. In one embodiment, the AAV is administered to a subject at a dose of at least about 1 x 1012, at least about 2 x 1012, at least about 5 x 1012, at least about 8 x 1012, at least about 1 x 1013, at least about 2 x 1013, at least about 5 x 1013, at least about 8 x 1013, at least about 1 x 1014, or at least about 2 x 1014virus particles per kg body weight.

[0166] In one embodiment, the AAV for use in the treatment of laminopathy in a subject comprising the nucleic acid molecule for use of the invention or the vector for use of the invention, is administered at a titer suitable to induce expression of lamin A to at least 50% of WT expression, and / or wherein the AAV for use is administered at a titer about 10,000 to about500,000 virus particles / cell, preferably wherein the AAV is serotype AAV6 or AAV9, optionally wherein the cell is a cardiomyocyte.

[0167] In one embodiment, the invention relates to a method of treatment of laminopathy in a human or non-human subject in need thereof comprising administering to said subject a nucleic acid encoding a lamin A polypeptide, a vector comprising a nucleic acid encoding a lamin A polypeptide or an AAV comprising a nucleic acid encoding a lamin A polypeptide. In one embodiment, the laminopathy is dilated cardiomyopathy type 1 A.

[0168] In one embodiment, the invention relates to the use of a nucleic acid encoding a lamin A polypeptide, a vector comprising a nucleic acid encoding a lamin A polypeptide or an AAV comprising a nucleic acid encoding a lamin A polypeptide in the manufacture of a medicament.

[0169] In one embodiment, the invention relates to the use of a nucleic acid encoding a lamin A polypeptide, a vector comprising a nucleic acid encoding a lamin A polypeptide or an AAV comprising a nucleic acid encoding a lamin A polypeptide in the manufacture of a medicament for the treatment of laminopathy. In one embodiment, the laminopathy is dilated cardiomyopathy type 1A.

[0170] In a further aspect, the invention also relates to a pharmaceutical composition comprising the nucleic acid molecule encoding lamin A for use, the vector comprising a nucleic acid molecule encoding lamin A for use or an AAV comprising a nucleic acid encoding lamin A for use, and optionally a pharmaceutically acceptable carrier.

[0171] In one embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation caninclude standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). In one embodiment, other ingredients can be added to pharmaceutical formulations, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, di saccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

[0172] The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine.

[0173] In one embodiment, the nucleic acid molecule for use of the invention, the vector for use of the invention, the AAV for use of the invention or the pharmaceutical composition of the invention are administered locally. Preferably, the nucleic acid molecule for use of the invention, the vector for use of the invention, the AAV for use of the invention or the pharmaceutical composition of the invention are administered to muscle tissue. More preferably, the nucleic acid molecule for use of the invention, the vector for use of the invention, the AAV for use of the invention or the pharmaceutical composition of the invention are administered to heart muscle tissue. In one embodiment, the nucleic acid molecule for use of the invention, the vector for use of the invention, the AAV for use of the invention or the pharmaceutical composition of the invention may be administered to heart muscle tissue by cardiac transfusion. Cardiac transfusion may be achieved by enabling direct access to the cardiac muscle.

[0174] In another embodiment, the nucleic acid molecule for use of the invention, the vector for use of the invention, the AAV for use of the invention or the pharmaceutical composition of the invention are administered systemically. Systemic routes of administration may include inhalation, injection or infusion. Systemic administration refers to a route of administration that affects the entire body, usually by administration into the circulatory system. Administration may be via injection, preferably via intravenous, intracardiac or intramuscular injection.

[0175] In one embodiment, treatment with the nucleic acid for use of the invention, the vector for use of the invention, the AAV for use of the invention or the pharmaceuticalcomposition of the invention may lead to an up-regulation of proteins involved in cardiac contraction. In another embodiment, treatment with the nucleic acid for use of the invention, the vector for use of the invention, the AAV for use of the invention or the pharmaceutical composition of the invention may lead to an up-regulation of one or more proteins selected from Dystrophin, muscular LMNA-interacting protein, Cofilin 2, LIM domain -binding protein 3, lamin B2, lamin Bl, dual specificity phosphatase 3, four and a half LIM domains protein 1, PDZ and LIM domain protein 1, LIM only protein 7, Actin-binding LIM protein 1, Emerin, SUN domain-containing protein 1, PDZ and LIM domain protein 3, Na+ / K+-ATPase subunit 1, Na+ / K+-ATPase subunit al, Troponin I, Tubulin P-3 chain, Myosin-binding protein C, Caveolin 1, Titin, Myosin 7, Troponin C, Plakophilin 2, A-actin 1, Junction Plakoglobin, Desmoplakin, Desm oglein 2, Catenin a3, Ryanodine receptor 2, and Desmin. Preferably, treatment with the nucleic acid for use of the invention or the vector for use of the invention leads to an upregulation of one or more proteins selected from lamin Bl, lamin B2, Emerin and SUN domaincontaining protein 1.ILLUSTRATIVE EXAMPLES

[0176] The following examples are set forth to assist in understanding the disclosure and should not be construed as specifically limiting the invention described and claimed herein. Such variations of the invention, including the substitution of all equivalents now known or later developed, which would be within the purview of those skilled in the art, and changes in formulation or minor changes in experimental design, are to be considered to fall within the scope of the invention incorporated herein.

[0177] As would be appreciated by those of ordinary skill in the art, one or more protocols, procedures, assays, or analyses may be performed according to, or adapted from, International Application No. PCTZEP2022 / 085962, filed December 14, 2022, the disclosure of which is hereby incorporated by reference herein in its entirety.Design and tests of different LMNA mini-gene constructs

[0178] LMNA mini-genes were constructed in view of the packaging limits of AAV vectors. Thus, in the various constructs, some introns or parts of them were deleted. FIG. l is a schematic overview of three designed constructs for the exogenous expression of lamin A and C. Each box indicates an exon of the LMNA gene, with the central lines representing included introns. Specifically, FIG. 1 shows the construction of exemplary LMNA mini-genes, illustrating the deletion of different introns and / or reduction of intron sizes.

[0179] Mini -gene 1 contained intron 8, intron 9, intron 10, but not intron 11. Mini -gene 2 contained intron 9, intron 10, and intron 11. Mini -gene 3 contained intron 8, parts of intron 9, parts of intron 10, and intron 11.

[0180] The parts of introns 9 and 10 are shown in FIG. 2, which is a schematic overview of mini -gene 3 with the remaining regulatory regions of intron 9 and 10. Regulatory regions are depicted as “don” (donor site) and “Acept” (acceptor site). In intron 10, small square boxes labeled 1 to 6 denote predicted splice donor sites with low scores. Small boxes labeled “top” positioned in or near introns 9 and 10 represent predicted splice donor sites with the highest scores. Conversely, the box labeled “top Acept” and box labeled “acTopl” within introns 9 and 10 highlight the predicted splice acceptor sites with the highest scores. The designation of “top scores” corresponds to sequence alignments exhibiting high similarity to canonical splice sequences. Rectangular boxes within introns 9 and 10 (“INR”) delineate critical intronic regions implicated in efficient splicing mechanisms.

[0181] SEQ ID NOS: 9-11 provide the full sequences of mini-genes 1-3, respectively, with the target sequences indicated: SEQ ID NO: 14 corresponds to the target sequence of siRNA 3, and SEQ ID NO: 17 corresponds to the target sequence of siRNA 4. The respective sequences of mini-genes 2 and 3 resistant to siRNA 4 (final sequences) are shown in SEQ ID NOS: 12 (“mini -gene 2res”) and 13 (“mini-gene 3res”), respectively.

[0182] The mini-genes were cloned in a plasmid vector pcDNA, which drives the expression of the mini-gene by a TNNT2 promoter and also contains a miR cassette driving the respective siRNA (SEQ ID NO: 9).

[0183] LMNA knockout human-induced pluripotent stem cell cardiomyocytes (hiPSC CMs) were transfected with the plasmids by liposomal transfection. FIG. 3 A shows Western blot analysis of 2D LMNA knockout hiPSC cardiomyocytes either non-transfected (NT) or transfected with 1 pg per well plasmids mini -gene (MG) 1, 2 or 3, and unrelated control ERC020. Expression was driven by a CMV promoter. Blots were stained for MYBPC3 (-150 kDa), lamin A and C (-74 kDa and 62 kDa) or GAPDH (-37 kDa). Expression of lamin A and C was determined by comparison to the two housekeeping proteins MYBPC3 and GAPDH. Note the absence of lamin bands in LMNA-KO cells non-transfected (NT). Mini-gene 1 gave only rise to lamin C (lower band of the doublet), while mini-gene 2 and 3 gave rise to roughly equivalent amounts of lamin A and C. FIG. 3B shows quantification of lamin C compared to the wild-type control ERC021. Notably, among the constructs tested, mini-gene 2 restored lamin A and C expression to levels and banding patterns that most closely mirrored those observed in wild-type cardiomyocytes, suggesting that this construct provides the most physiological relevant rescue of LMNA function.Testing of different siRNAs and construction of vector

[0184] Different siRNAs (21-mers) against different exons within the LMNA gene were designed using publicly available web tools. A total of 6 different siRNAs were tested by liposomal transfection into wild-type hiPSC-CMs, and were compared with a scrambled siRNA (scRNA) and a commercially available siRNA against GAPDH. FIG. 4 shows the effect of transfection of wild-type hiPSC-CM (ERC021) with scrambled siRNA (scRNA), siRNA against GAPDH (sGAPDH), siRNA 3, and siRNA 4. The blots were stained with Ponceau red to analyze even loading and with antibodies against MYBPC3 and GAPDH as loading controls and lamin A / C. The histograms show quantification of Western blot bands normalized to GAPDH. Stars indicate overloaded bands not considered in the quantification. Cells were subjected to Western blot analysis 96 hours after transfection. The group treated with siRNA 4 (which targeted exon 2 of LMNA) was the most effective, leading to a decrease in lamin A and lamin C protein by about 60-70%. The target sequences of the endogneous LMNA mRNA and the respective sequences of siRNA 3 (SEQ ID NO: 15 and SEQ ID NO: 16 complement) and siRNA 4 (SEQ ID NO: 18 and SEQ ID NO: 19 complement) are shown in FIG. 5, where overhangs are indicated in bold underline.

[0185] FIG. 6 shows a plasmid construct designed for driving the expression of mini-gene 2res and siRNA 4. The construct includes the TNNT2 promoter (Troponin T) to drive the expression of the LMNA construct resistant to siRNA 4 by wobble base mutation of target region (grey box “resistant” in exon 2) and the siRNA 4 sequence embedded in miR30 backbone. FIG. 7 shows a plasmid construct driving the expression of mini-gene 3res and siRNA 4. It is believed that such constructs could beneficially result in stochiometric, or near- stochiometric, expression of exogenous lamin A and lamin C protein while reducing or eliminating endogenous expression of lamin A and lamin C in cardiomyocytes when delivered thereto.

[0186] Previously tested siRNAs were introduced into a plasmid vector and an miRNA expression cassette. ERC021 (wild-type control) and LMNA KI hiPSC CM (R321Xhet) cells were prepared in 2D cultures and were transfected with the plasmid containing siRNA4 or minigene 2. Western blots for loading control MYBPC3 and lamin A / C are shown in FIG. 8, top. As expected, lower total lamin A / C was observed in the heterozygous R321X line compared to ERC021, and quantification demonstrated a similar result (FIG. 8, bottom). Notably, transfection with mini-gene 2 resulted in increased lamin A / C and a normal A / C ratio, thus supporting the data in FIG. 3B.

[0187] In the foregoing description, numerous specific details are set forth, such as specific materials, dimensions, processes parameters, etc., to provide a thorough understanding of the present invention. The particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The words “example” or “exemplary” are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the words “example” or “exemplary” is simply intended to present concepts in a concrete fashion. As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or”. That is, unless specified otherwise, or clear from context, “X includes A or B” is intended to mean any of the natural inclusive permutations. That is, if X includes A; X includes B; or X includes both A and B, then “X includes A or B” is satisfied under any of the foregoing instances. Reference throughout this specification to “an embodiment”, “certain embodiments”, or “one embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “an embodiment”, “certain embodiments”, or “one embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[0188] The present invention has been described with reference to specific exemplary embodiments thereof. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.SEQUENCES

[0189] Sequences included in this description in this section “Sequences” rule over the sequences of the sequence listing according to WIPO ST.26 standard in case there are conflicts.SEQ ID NO: 1 - PreLamin A wild-type amino acid sequence (isoform A) METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLR LRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARN TKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGE AKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQRE FESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQ SRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQ QQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRTALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRS YLLGNS SPRTQ SPQNC SIMSEQ ID NO: 2 - Mature lamin A wild-type amino acid sequenceMETPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLR LRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARN TKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGE AKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQ SRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQ QQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGG SVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKR QNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRT ALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRS YSEQ ID NO: 3 - LMNA wild-type nucleic acid sequenceATGGAGACCCCGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCCACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGCGGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCATCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAGGCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCTGCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCAAGAAGGAGGGTGACCTGATAGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTGAACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGAGCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGAAGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATGAAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGCGCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGCCGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTATAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTGAGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGCATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGCGAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGCTGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGACGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACCGCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAGCGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACCAAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCACTAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCTGCGCAACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGATGATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTGACGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAAGGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTGGGGAAGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAGGATGAGGATGGAGATGACCTGCTCCATCACCACCACGGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAGAGCCCCCAGAACTGCAGCATCATGTAASEQ ID NO: 4 - Lamin A wild-type isoform C amino acid sequenceMETPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLR LRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARN TKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGE AKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQRE FESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQ SRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGG SVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKR QNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRT ALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHVSGSRRSEQ ID NO: 5 - Lamin A wild-type isoform A DeltalO amino acid sequenceMETPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLR LRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARN TKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGE AKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQRE FESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQ SRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQ QQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKR QNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRT ALINSTGEGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSAS SVTVTRSYRSVGGSGGGSFGDNLVTRSYLLGNSSPRTQSPQNCSIMSEQ ID NO: 6 - Lamin A wild-type isoform 4 amino acid sequenceMGNSEGCNTKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRTALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRSYLLGNSSPRTQSPQNCSIIQEMGMRWEVEEGRRKVSLSCLPSEQ ID NO: 7 - Lamin A wild-type isoform 5 amino acid sequenceMDLEAWDPHLEPDAEAMVDGNTKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKR TLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEE LRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLD NARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARER DTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRL SPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATH SPPTDLVWKAQNTWGCGNSLRTALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLH HHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQVGGPISSGSSASSVTV TRSYRSVGGSGGGSFGDNLVTRSYLLGNSSPRTQSPQNCSIMSEQ ID NO: 8 - Lamin A wild-type isoform 6 amino acid sequenceMETPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLR LRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARN TKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGE AKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQRE FESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQ SRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQ QQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKR QNGDDPLLTYRFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRT ALINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASASGSGAQSPQNCSIMSEQ ID NO: 9 - LMNA mini-gene 1 (siRNA 3 target sequence in bold-underline; siRNA 4 target sequence in bold-italic)ATGGAGACCCCGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCCACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGCGGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCATCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAGGCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCTGCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCAAGAAGGAGGGTGACCTGATAGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTGAACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGAGCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGAAGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATGAAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGCGCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGCCGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTATAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTGAGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGCATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGCGAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGCTGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGACGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACCGCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAGCGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACCAAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCACTAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCTGCGCAACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGATGATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTGACGGTGAGTGGCAGGGCGCTTGGGACTCTGGGGAGGCCTTGGGTGGCGATGGGAGCGCTGGGGTAAGTGTCCTTTTCTCCTCTCCAGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAAGGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTGGGGAAGTAAGTAGGCCTGGGCCTGGCTGCTTGCTGGACGAGGCTCCCCCTGATGGCCAACATCGGAGCCAGCTGCCCCCAACCCAAGTTTGCCAATTCAGGGCCCCTTTCTAGAGCTCTCTGTTGCAGGCTCCAGACTTCTCCACCCAGTAGGCAAACCAAAAGATGCTTCCTCAACAGCACAAGGGGTGGAAGTTAGACAGTGAGGATTGTTAAAGGCAGAGCCATACTCCTACCCGGAGAGCTTGACAGTGTCCCTCTGGGGTGGAAATGAGTTCCTTAGCTCCATCACCACAGAGGACAGAGTAAGCAGCAGGCCGGACAAAGGGCAGGCCACAAGAAAAGTTGCAGGTGGTCACTGGGGTAGACATGCTGTACAACCCTTCCCTGGCCCTGACCCTTGGACCTGGTTCCATGTCCCCACCAGGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAGGATGAGGATGGAGATGACCTGCTCCATCACCACCACGTGAGTGGTAGCCGCCGCTGAGGCCGAGCCTGCACTGGGGCCACCCAGCCAGGCCTGGGGGCAGCCTCTCCCCAGCCTCCCCGTGCCAAAAATCTTTTCATTAAAGAATGTTTTGGAACTTTACTCGCTGGCCTGGCCTTTCTTCTCTCTCCTCCCTATACCTTGAACAGGGAACCCAGGTGTCTGGGTGCCCTACTCTGGTAAGGAAGGGAGTGGGAACTTTCTGATGCCATGGAATATTCCTGTGGGAGCAGTGGACAAGGGTCTGGATTTGTCTTCTGGGAAAGGGAGGGGAGGACAGACGTGGGGCATGCCCGCCCTGCCTCTCTCCCCCATTCTTGTTGCATGCATATCCTCTCATTTCCCTCATTTTTCCTGCAAGAATGTTCTCTCTCATTCCTGACCGCCCCTCCACTCCAATTAATAGTGCATGCCTGCTGCCCTACAAGCTTGCTCCCGTTCTCTCTTCTTTTCCTCTTAAGCTCAGAGTAGCTAGAACAGAGTCAGAGTCACTGCTCTGGTTCTCTGTCCCCAAGTCTTCCTGAGCCTTCTCCCCTTTTATGTCTTCCCTCTCCTCCTCCGGGCCCCTAGCCTCCCAAACCCCCATTGCCCGCTGGCTCCTTGGGCACAGAACCACACCTTCCTGCCTGGCGGCTGGGAGCCTGCAGGAGCCTGGAGCCTGGTTGGGCCTGAGTGGTCAGTCCCAGACTCGCCGTCCCGCCTGAGCCTTGTCTCCCTTCCCAGGGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAGAGCCCCCAGAACTGCAGCATCATGTAASEQ ID NO: 10 - LMNA mini-gene 2 (siRNA 3 target sequence in bold-underline; siRNA 4 target sequence in bold-italic)ATGGAGACCCCGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCCACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGCGGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCATCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAGGCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCTGCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCAAGAAGGAGGGTGACCTGATAGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTGAACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGAGCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGAAGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATGAAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGCGCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGCCGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTATAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTGAGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGCATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGCGAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGCTGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGACGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACCGCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAGCGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACCAAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCACTAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCTGCGCAACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGATGATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTGACGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAAGGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTGGGGAAGTAAGTAGGCCTGGGCCTGGCTGCTTGCTGGACGAGGCTCCCCCTGATGGCCAACATCGGAGCCAGCTGCCCCCAACCCAAGTTTGCCAATTCAGGGCCCCTTTCTAGAGCTCTCTGTTGCAGGCTCCAGACTTCTCCACCCAGTAGGCAAACCAAAAGATGCTTCCTCAACAGCACAAGGGGTGGAAGTTAGACAGTGAGGATTGTTAAAGGCAGAGCCATACTCCTACCCGGAGAGCTTGACAGTGTCCCTCTGGGGTGGAAATGAGTTCCTTAGCTCCATCACCACAGAGGACAGAGTAAGCAGCAGGCCGGACAAAGGGCAGGCCACAAGAAAAGTTGCAGGTGGTCACTGGGGTAGACATGCTGTACAACCCTTCCCTGGCCCTGACCCTTGGACCTGGTTCCATGTCCCCACCAGGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAGGATGAGGATGGAGATGACCTGCTCCATCACCACCACGTGAGTGGTAGCCGCCGCTGAGGCCGAGCCTGCACTGGGGCCACCCAGCCAGGCCTGGGGGCAGCCTCTCCCCAGCCTCCCCGTGCCAAAAATCTTTTCATTAAAGAATGTTTTGGAACTTTACTCGCTGGCCTGGCCTTTCTTCTCTCTCCTCCCTATACCTTGAACAGGGAACCCAGGTGTCTGGGTGCCCTACTCTGGTAAGGAAGGGAGTGGGAACTTTCTGATGCCATGGAATATTCCTGTGGGAGCAGTGGACAAGGGTCTGGATTTGTCTTCTGGGAAAGGGAGGGGAGGACAGACGTGGGGCATGCCCGCCCTGCCTCTCTCCCCCATTCTTGTTGCATGCATATCCTCTCATTTCCCTCATTTTTCCTGCAAGAATGTTCTCTCTCATTCCTGACCGCCCCTCCACTCCAATTAATAGTGCATGCCTGCTGCCCTACAAGCTTGCTCCCGTTCTCTCTTCTTTTCCTCTTAAGCTCAGAGTAGCTAGAACAGAGTCAGAGTCACTGCTCTGGTTCTCTGTCCCCAAGTCTTCCTGAGCCTTCTCCCCTTTTATGTCTTCCCTCTCCTCCTCCGGGCCCCTAGCCTCCCAAACCCCCATTGCCCGCTGGCTCCTTGGGCACAGAACCACACCTTCCTGCCTGGCGGCTGGGAGCCTGCAGGAGCCTGGAGCCTGGTTGGGCCTGAGTGGTCAGTCCCAGACTCGCCGTCCCGCCTGAGCCTTGTCTCCCTTCCCAGGGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAGGTGAGTTGTCTCTGCTTTGTCTCCAAATCCTGCAGGCGGGTCCCTGGTCATCGAGGGGTAGGACGAGGTGGCCTTGCAGGGGGGAGAGCCTGCCTTCTCTTCCGCAGCCCGGGGGAGTGGGAGCCTCCTCCCCACAGCCTGAGTCCTAGACAGCCCACCTCTGCATCCTGCCCCTCTTGTCTGAGCCCCAGACTGGAGGGCAGGGGCAGGGCTGGAGTGTGAGGGATGGGGGAGATGCTACCTCCCTTCTAGGGGCCAGGGGAGGGAGGGTCTGGGTCCAGGCCCTGCTGCTCACACCTCTCTCCTCTGTTTTCTCTCTTAGAGCCCCCAGAACTGCAGCATCATGTAASEQ ID NO: 11 - LMNA Mini-gene 3 (siRNA 3 target sequence in bold-underline; siRNA 4 target sequence in bold-italic)ATGGAGACCCCGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCCACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGCGGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCATCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAGGCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCTGCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCAAGAAGGAGGGTGACCTGATAGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTGAACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGAGCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGAAGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATGAAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGCGCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGCCGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTATAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTGAGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGCATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGCGAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGCTGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGACGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACCGCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAGCGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACCAAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCACTAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCTGCGCAACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGATGATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTGACGGTGAGTGGCAGGGCGCTTGGGACTCTGGGGAGGCCTTGGGTGGCGATGGGAGCGCTGGGGTAAGTGTCCTTTTCTCCTCTCCAGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAAGGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTGGGGAAGTAAGTAGGCCTGGGCCTGGCTGCTTGCTGGACGAGGCTCCCCCTGATGGCCAACATCGGAGCCAGCTGCCCCCAACCCAAGTTTGCCAATTCAGGGCCCCTTAGAGCTCTCTGTTGCAGGCTCCAGACTTCTCCAGCTCCATCACCACAGAGGACAGAGTAAGCAGCAGGCCGGACAAAGGGCAGGCCACAAGAAAAGTTGCAGGTGGTCACTGGGGTAGACATGCTGTACAACCCTTCCCTGGCCCTGACCCTTGGACCTGGTTCCATGTCCCCACCAGGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAGGATGAGGATGGAGATGACCTGCTCCATCACCACCACGTGAGTGGTAGCCGCCGCTGAGGCCGAGCCTGCACTGGGGCCACCCAGCCAGGCCTGGGGGCAGCCTCTCCCCAGCCTCCCCGTGCCAAAAATCTTTTCATTAAAGAATGTTTTGGAACTTTACTCGCTGGCCTGGCCTTTCTTCTCTCTCCTCCCTATACCTTGAACAGGGAACCCAGGTGTCTGGGTGCCCTACTCTGGTAAGGAAGGGAGTGGGAACTTTCTGATGCCATGGAATATTCCTGTGGGAGCAGTGGACAAGGGTCTGGATTTGTCTTCTGGGAAAGGGAGGGGAGGACAGACGTGGGGCATGCCCGCCCTGCTACAAGCTTGCTCCCGTTCTCTCTTCTTTTCCTCTTAAGCTCAGAGTAGCTAGAACAGAGTCAGAGTCACTGCTCTGGTTCTCTGTCCCCAAGTCTTCCTGAGCCTTCTCCCCTTTTATGTCTTCCCTCTCCTCCTCCGGGCCCCTAGCCTCCCAAACCCCCATTGCCCGCTGGCTCCTTGGGCACAGAACCACACCTTCCTGCCTGGCGGCTGGGAGCCTGCAGGAGCCTGGAGCCTGGTTGGGCCTGAGTGGTCAGTCCCAGACTCGCCGTCCCGCCTGAGCCTTGTCTCCCTTCCCAGGGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAGGTGAGTTGTCTCTGCTTTGTCTCCAAATCCTGCAGGCGGGTCCCTGGTCATCGAGGGGTAGGACGAGGTGGCCTTGCAGGGGGGAGAGCCTGCCTTCTCTTCCGCAGCCCGGGGGAGTGGGAGCCTCCTCCCCACAGCCTGAGTCCTAGACAGCCCACCTCTGCATCCTGCCCCTCTTGTCTGAGCCCCAGACTGGAGGGCAGGGGCAGGGCTGGAGTGTGAGGGATGGGGGAGATGCTACCTCCCTTCTAGGGGCCAGGGGAGGGAGGGTCTGGGTCCAGGCCCTGCTGCTCACACCTCTCTCCTCTGTTTTCTCTCTTAGAGCCCCCAGAACTGCAGCATCATGTAASEQ ID NO: 12 - Final full sequence of the LMNA Mini-gene 2resGAGACCCCGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCCACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGCGGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCATCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAGGCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCTGCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCAAAAAAGAAGGCGATCTGATCGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTGAACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGAGCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGAAGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATGAAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGCGCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGCCGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTATAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTGAGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGCATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGCGAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGCTGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGACGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACCGCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAGCGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACCAAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCACTAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCTGCGCAACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGATGATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTGACGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAAGGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTGGGGAAGTAAGTAGGCCTGGGCCTGGCTGCTTGCTGGACGAGGCTCCCCCTGATGGCCAACATCGGAGCCAGCTGCCCCCAACCCAAGTTTGCCAATTCAGGGCCCCTTTCTAGAGCTCTCTGTTGCAGGCTCCAGACTTCTCCACCCAGTAGGCAAACCAAAAGATGCTTCCTCAACAGCACAAGGGGTGGAAGTTAGACAGTGAGGATTGTTAAAGGCAGAGCCATACTCCTACCCGGAGAGCTTGACAGTGTCCCTCTGGGGTGGAAATGAGTTCCTTAGCTCCATCACCACAGAGGACAGAGTAAGCAGCAGGCCGGACAAAGGGCAGGCCACAAGAAAAGTTGCAGGTGGTCACTGGGGTAGACATGCTGTACAACCCTTCCCTGGCCCTGACCCTTGGACCTGGTTCCATGTCCCCACCAGGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAGGATGAGGATGGAGATGACCTGCTCCATCACCACCACGTGAGTGGTAGCCGCCGCTGAGGCCGAGCCTGCACTGGGGCCACCCAGCCAGGCCTGGGGGCAGCCTCTCCCCAGCCTCCCCGTGCCAAAAATCTTTTCATTAAAGAATGTTTTGGAACTTTACTCGCTGGCCTGGCCTTTCTTCTCTCTCCTCCCTATACCTTGAACAGGGAACCCAGGTGTCTGGGTGCCCTACTCTGGTAAGGAAGGGAGTGGGAACTTTCTGATGCCATGGAATATTCCTGTGGGAGCAGTGGACAAGGGTCTGGATTTGTCTTCTGGGAAAGGGAGGGGAGGACAGACGTGGGGCATGCCCGCCCTGCCTCTCTCCCCCATTCTTGTTGCATGCATATCCTCTCATTTCCCTCATTTTTCCTGCAAGAATGTTCTCTCTCATTCCTGACCGCCCCTCCACTCCAATTAATAGTGCATGCCTGCTGCCCTACAAGCTTGCTCCCGTTCTCTCTTCTTTTCCTCTTAAGCTCAGAGTAGCTAGAACAGAGTCAGAGTCACTGCTCTGGTTCTCTGTCCCCAAGTCTTCCTGAGCCTTCTCCCCTTTTATGTCTTCCCTCTCCTCCTCCGGGCCCCTAGCCTCCCAAACCCCCATTGCCCGCTGGCTCCTTGGGCACAGAACCACACCTTCCTGCCTGGCGGCTGGGAGCCTGCAGGAGCCTGGAGCCTGGTTGGGCCTGAGTGGTCAGTCCCAGACTCGCCGTCCCGCCTGAGCCTTGTCTCCCTTCCCAGGGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAGGTGAGTTGTCTCTGCTTTGTCTCCAAATCCTGCAGGCGGGTCCCTGGTCATCGAGGGGTAGGACGAGGTGGCCTTGCAGGGGGGAGAGCCTGCCTTCTCTTCCGCAGCCCGGGGGAGTGGGAGCCTCCTCCCCACAGCCTGAGTCCTAGACAGCCCACCTCTGCATCCTGCCCCTCTTGTCTGAGCCCCAGACTGGAGGGCAGGGGCAGGGCTGGAGTGTGAGGGATGGGGGAGATGCTACCTCCCTTCTAGGGGCCAGGGGAGGGAGGGTCTGGGTCCAGGCCCTGCTGCTCACACCTCTCTCCTCTGTTTTCTCTCTTAGAGCCCCCAGAACTGCAGCATCATGTAASEQ ID NO: 13 - Final full sequence of the LMNA mini-gene 3resGAGACCCCGTCCCAGCGGCGCGCCACCCGCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCCACCCGCATCACCCGGCTGCAGGAGAAGGAGGACCTGCAGGAGCTCAATGATCGCTTGGCGGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCAGGGCTGCGCCTTCGCATCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCTACGAGGCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCTGCAGCTGGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCAAAAAAGAAGGCGATCTGATCGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTGAACTCCAAGGAGGCCGCACTGAGCACTGCTCTCAGTGAGAAGCGCACGCTGGAGGGCGAGCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGCAGCCCTAGGTGAGGCCAAGAAGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTGCAGACCATGAAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGCGCCGTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGCCGGCTGGCGGATGCGCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTATAAGAAGGAGCTGGAGAAGACTTATTCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTGAGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGGAGCTGCAGCAGTCGCGCATCCGCATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGCAGCCAAGGAGGCGAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTGCTGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGACGAGTACCAGGAGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACCGCAAGCTCTTGGAGGGCGAGGAGGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAGCGCAGCCGTGGCCGTGCTTCCTCTCACTCATCCCAGACACAGGGTGGGGGCAGCGTCACCAAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCTCACAGCACGCACGCACTAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCTGCGCAACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGATGATCCCTTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTGACGGTGAGTGGCAGGGCGCTTGGGACTCTGGGGAGGCCTTGGGTGGCGATGGGAGCGCTGGGGTAAGTGTCCTTTTCTCCTCTCCAGATCTGGGCTGCAGGAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAAGGCACAGAACACCTGGGGCTGCGGGAACAGCCTGCGTACGGCTCTCATCAACTCCACTGGGGAAGTAAGTAGGCCTGGGCCTGGCTGCTTGCTGGACGAGGCTCCCCCTGATGGCCAACATCGGAGCCAGCTGCCCCCAACCCAAGTTTGCCAATTCAGGGCCCCTTAGAGCTCTCTGTTGCAGGCTCCAGACTTCTCCAGCTCCATCACCACAGAGGACAGAGTAAGCAGCAGGCCGGACAAAGGGCAGGCCACAAGAAAAGTTGCAGGTGGTCACTGGGGTAGACATGCTGTACAACCCTTCCCTGGCCCTGACCCTTGGACCTGGTTCCATGTCCCCACCAGGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTGACTGTGGTTGAGGACGACGAGGATGAGGATGGAGATGACCTGCTCCATCACCACCACGTGAGTGGTAGCCGCCGCTGAGGCCGAGCCTGCACTGGGGCCACCCAGCCAGGCCTGGGGGCAGCCTCTCCCCAGCCTCCCCGTGCCAAAAATCTTTTCATTAAAGAATGTTTTGGAACTTTACTCGCTGGCCTGGCCTTTCTTCTCTCTCCTCCCTATACCTTGAACAGGGAACCCAGGTGTCTGGGTGCCCTACTCTGGTAAGGAAGGGAGTGGGAACTTTCTGATGCCATGGAATATTCCTGTGGGAGCAGTGGACAAGGGTCTGGATTTGTCTTCTGGGAAAGGGAGGGGAGGACAGACGTGGGGCATGCCCGCCCTGCTACAAGCTTGCTCCCGTTCTCTCTTCTTTTCCTCTTAAGCTCAGAGTAGCTAGAACAGAGTCAGAGTCACTGCTCTGGTTCTCTGTCCCCAAGTCTTCCTGAGCCTTCTCCCCTTTTATGTCTTCCCTCTCCTCCTCCGGGCCCCTAGCCTCCCAAACCCCCATTGCCCGCTGGCTCCTTGGGCACAGAACCACACCTTCCTGCCTGGCGGCTGGGAGCCTGCAGGAGCCTGGAGCCTGGTTGGGCCTGAGTGGTCAGTCCCAGACTCGCCGTCCCGCCTGAGCCTTGTCTCCCTTCCCAGGGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAGGTGAGTTGTCTCTGCTTTGTCTCCAAATCCTGCAGGCGGGTCCCTGGTCATCGAGGGGTAGGACGAGGTGGCCTTGCAGGGGGGAGAGCCTGCCTTCTCTTCCGCAGCCCGGGGGAGTGGGAGCCTCCTCCCCACAGCCTGAGTCCTAGACAGCCCACCTCTGCATCCTGCCCCTCTTGTCTGAGCCCCAGACTGGAGGGCAGGGGCAGGGCTGGAGTGTGAGGGATGGGGGAGATGCTACCTCCCTTCTAGGGGCCAGGGGAGGGAGGGTCTGGGTCCAGGCCCTGCTGCTCACACCTCTCTCCTCTGTTTTCTCTCTTAGAGCCCCCAGAACTGCAGCATCATGTAASEQ ID NO: 14 - siRNA 3 target sequenceAAGCGCCAGAATGGAGATGATSEQ ID NO: 15 - siRNA 3 sequence with overhangAUCAUCUCCAUUCUGGCGCUUSEQ ID NO: 16 - siRNA 3 complementary sequence with overhangGCGCCAGAAUGGAGAUGAUCCSEQ ID NO: 17 - siRNA 4 target sequenceAAGAAGGAGGGTGACCTGATAGCSEQ ID NO: 18 - siRNA 4 sequence with overhangUAUCAGGUCACCCUCCUUCUUSEQ ID NO: 19 - siRNA 4 complementary sequence with overhang GAAGGAGGGUGACCUGAUAGC

Claims

1. CLAIMS1. A nucleic acid molecule for use in the treatment of laminopathy in a subject, the nucleic acid molecule comprising a coding sequence that encodes: a small-interfering RNA (siRNA) targeting a portion of the endogenously expressed LMNA gene; and lamin A protein and / or lamin C protein that is resistant to the siRNA sequence.

2. The nucleic acid molecule of claim 1, wherein the siRNA targets exon 2 of the endogenously expressed LMNA gene.

3. The nucleic acid molecule of claim 1, wherein the siRNA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to either SEQ ID NO: 15 or SEQ ID NO: 18.

4. The nucleic acid molecule of any one of claims 1-3, wherein the lamin A protein and / or lamin C protein corresponds to a wild-type lamin A protein and / or wild-type lamin C protein.

5. The nucleic acid molecule of any one of claims 1-3, wherein the sequence encoding the lamin A protein and / or lamin C protein has a subset of introns removed therefrom.

6. The nucleic acid molecule of claim 5, wherein the subset of introns comprises introns 8 through 11, introns 8 through 10, introns 9 through 11, or shortened forms thereof.

7. The nucleic acid molecule of any one of claims 1-6, wherein the coding sequence further comprises a promoter, and wherein the promoter is a constitutive promoter or a tissue-specific promoter.

8. The nucleic acid molecule of any one of claims 1-7, wherein the sequence encoding lamin A protein encodes an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID NO: 1.

9. The nucleic acid molecule of any one of claims 1-8, wherein the laminopathy is caused by one or more mutations of the endogenously expressed LMNA gene.

10. The nucleic acid molecule of claim 9, wherein the one or more mutations of the LMNA gene causes haploinsufficiency for lamin A or wherein the one or more mutations of the LMNA gene causes a dominant-negative lamin A.

11. The nucleic acid molecule of of any one of claims 1-10, wherein the laminopathy is a laminopathy that affects striated muscle, preferably wherein the laminopathy is selected from the group consisting of cardiomyopathy or muscular dystrophy.

12. The nucleic acid molecule of claim 11, wherein the laminopathy is a cardiomyopathy, preferably dilated cardiomyopathy 1 A.

13. A vector comprising the nucleic acid molecule of any one of claims 1-12, for use in the treatment of laminopathy in a subject.

14. The vector according to claim 13, wherein the coding sequence of the nucleic acid molecule comprises a promoter that is operably linked to the sequence encoding lamin A protein to effect the expression of lamin A in cells contacted with the vector when the promoter is active.

15. The vector of claim 14, wherein the expression of lamin A protein from the vector increases the overall expression level of lamin A in cells affected by laminopathy contacted with the vector to at least 50% of the wild-type expression level, preferably wherein the expression level of lamin A protein increases to between about 60% and about 300% of the wild-type expression level.

16. The vector for use according to claim 14, wherein the expression of lamin A from the vector increases the absolute force produced by cardiomyocytes affected by laminopathy contacted with the vector by 5% to 200% compared to cardiomyocytes affected by laminopathy not contacted with the vector.

17. An adeno-associated virus (AAV) for use in the treatment of laminopathy in a subject comprising the nucleic acid molecule of any one of claims 1-12, preferably wherein the AAV is serotype AAV6 or AAV9.

18. A pharmaceutical composition for use in the treatment of laminopathy in a subject comprising the vector of claim 13 or the AAV of claim 17, and a pharmaceutically acceptable carrier.

19. A method of treating laminopathy in a subject comprising delivering a vector to tissue of the subject, the vector comprising a nucleic acid molecule that, when delivered to the tissue, causes the tissue to express: a small-interfering RNA (siRNA) targeting a portion of the endogenously expressed LMNA gene; and wild-type lamin A protein and wild-type lamin C protein.

20. The method of claim 19, wherein the presence of the siRNA supresses the expression of endogenous lamin A and lamin C proteins, and wherein lamin A and lamin C proteins within the tissue are expressed in stochiometric or near stochiometric quantities.

21. The method of claim 20, wherein the siRNA suppresses the expression of each of the endogneous lamin A and lamin C proteins by greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

22. The method of claim 19, wherein the siRNA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to either SEQ ID NO: 15 or SEQ ID NO: 18.