Method of treating LMNA dilated cardiomyopathy

Abstract

Disclosed is a method of treating LMNA Dilated Cardiomyopathy (DCM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a first recombinant Adeno-associated virus (rAAV) encoding Lamin C (rAAV-Lamin C) and / or a second rAAV encoding Lamin A (rAAV-Lamin A), wherein the rAAV- Lamin C comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO: 4, and the rAAV-Lamin A comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

Description

METHOD OF TREATING LMNA DILATED CARDIOMYOPATHYFIELD OF THE INVENTION

[0001] The present invention generally relates to a method of treating LMNA Dilated Cardiomyopathy LMNA DCM) In particular, the present invention relates to a method of treating LMNA DCM by overexpressing Lamin C and / or Lamin A in a subject in need thereof.BACKGROUND

[0002] Dilated cardiomyopathy (DCM) is a severe, non-ischemic heart disease which ultimately results in heart failure (HF). Decades of research on DCM have revealed diverse aetiologies. DCM is often caused by genetic pathogenesis, with an estimated prevalence up to 1: 250 [1], The DCM gene panel containing a list of over 100 genes provides a comprehensive genetic evaluation for patients with a personal or family history of hereditary DCM. Among them, the LMNA gene encoding Lamin A and Lamin C is the second most frequently mutated DCM gene [2, 3], Lamin A is initially expressed as prelamin A and undergoes a series of post-translational modifications to form mature Lamin A [4], Lamin C arises from alternative splicing. Lamin A and C are identical through their N- termini but diverge at the C-terminus with Lamin C being the shorter isoform, terminating in 6 unique amino acids. Furthermore, as Lamin C does not have the CAAX motif located at the C-terminal end of prelamin A, it lacks a post-translational modification unique to Lamin A.

[0003] LMNA DCM is a multifactorial and complex disease with no specific treatment thus far Many studies have demonstrated that perturbing candidates related to various dysregulated pathways ameliorate LMNA DCM. However, it is unknown whether these candidates could serve as potential therapeutic targets especially in long term efficacy. It is believed that defective function of LMNA leads to DCM wherein mechanistic traits and phenotypes are shared in global and especially cardiac specific knockout mouse models [5-7], Molecular mechanisms underlying LMNA DCM have been intensively studied. Currently, there are three main hypotheses for molecular mechanisms underlying LMNA related DCM, including dysregulated signaling pathways, lamina-chromatin interactions and lamina-cytoskeleton links [8-10]. Accordingly, many potential candidates have been shown to suppress LMNA DCM. Among them, suppression of mTor, Mapk, Pdgfsignaling pathways, inhibition of BET bromodomain, or disruption of laminacytoskeleton links have been proposed to suppress or ameliorate LMNA DCM by using various models including global / cardiac specific knockouts, LMNA mutants as well as patient specific iPS derived cardiomyocytes [6, 7, 11-13], However, it is unknown whether these candidates can effect comparable long term survival, nor have efforts been made to evaluate and compare their efficacy in suppression of LMNA DCM. Recently, the inventors produced a cardiac specific knockdown model for LMNA DCM (designated as Lmna DCM) which is comparable to cardiac specific Lmna knockouts

[0014] , Importantly, this new model does not require intensive crossing to generate compound genetically modified mice.

[0004] Autosomal recessive diseases have been successfully treated with recombinant adeno-associated virus (rAAV) mediated gene replacement, exemplified by approval of rAAV9-SMN gene therapy for spinal muscular atrophy (SMA), the leading genetic cause of infant mortality

[0015] , However, a gene therapy approach for heart failure (HF) was not successful at phase 2 clinical trial due to lack of efficacy, which highlights the importance of prior candidate evaluation [16, 17], Recently, AAV vector toxicity also emerged in some clinical trials especially at high dosage of virus administration needed for sufficient gene expression and virus distribution

[0018] . Besides toxicity of AAV vectors, the gene product itself should also be evaluated as, for example, ectopic expression of the KG P reporter induced DCM in a sensitive mouse strain

[0019] , Apart from recessive diseases, dominant cardiovascular diseases such as cardiomyopathy were also assessed by gene therapy. Proof of principle studies have been attempted by targeting the causal genes of cardiomyopathy including MHC, MYPBC3 wAMYL2 [20-22],

[0005] Therefore, there is a need to identify potential targets for effective treatment of LMNA DCM. There is also a need for an effective method of treating LMNA DCM by targeting potential targets, such as Lamin A and Lamin C. Here, to identify potential targets for the treatment of LMNA DCM, the efficacy of 14 candidates by modulating their gene expression using rAAV9 in Lmna DCM was evaluated. Lamin A and Lamin C produced by the Lmna gene were also included in the assessment since cardiac Lamin A partially rescued global Lmna knockouts

[0023] ,

[0006] In one aspect, the present disclosure refers to a method of treating LMNA Dilated Cardiomyopathy (DCM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a first recombinant Adeno-associated virus (rAAV) encoding Lamin C (rAAV-Lamin C) and / or a second rAAV encoding Lamin A (rAAV-Lamin A), wherein the rAAV-Lamin C comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, and the rAAV-Lamin A comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3.

[0007] In another aspect, the present disclosure refers to use of a first rAAV encoding Lamin C (rAAV-Lamin C) and / or a second rAAV encoding Lamin A (rAAV-Lamin A) in the manufacture of a medicament for treating LMNA Dilated Cardiomyopathy (DCM) in a subject in need thereof, wherein the rAAV-Lamin C comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, and the rAAV-Lamin A comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0008] Advantageously, the present disclosure discloses that supplementation of Lamin C and / or Lamin A significantly suppressed LMNA DCM and achieved prolonged survival of the subject treated. Particularly, Lamin C achieved long-term survival of afflicted subject by up to 2 years which highly correlated with reduced heart fibrosis, heart inflammation and DNA damage.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0010] Figure 1 depicts that effect of Lamin A and Lamin C on Lmna DCM in mice, (a and d) Experimental timeline showing timepoints of virus injection and echocardiogram. Sirius Red (SR) and H&E staining of paraffin heart sections and quantifications of Lmna DCM mice supplemented with control, Lamin A or Lamin C. Quantification of myocardial fibrosis of SR sections, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction. For complete heart images: magnification = 4 *, scale bar =1000 pm; for enlarged images: magnification = 20 scale bar = 100 pm. (b and e) Quantitative real-time PCR analyses of Nppa, Nppb, Collal and Colla2 in Lmna DCM mice supplemented with control, Lamin A or Lamin C, n = 5, Brown-Forsytheand Welch ANOVA test with Dunnett’s T3 correction, (c and f) Survival curve of Lmna DCM mice supplemented with Lamin A (Red) or Lamin C (Red) compared to Lmna DCM mice (Black) and Ctrl (Blue), n = 10, Log- rank (Mantel- Cox) test.

[0011] Figure 2 depicts RNAseq analysis of Lmna DCM with Lamin A or Lamin C upregulation, (a and b) Heat map representing color-coded expression level of genes that are significantly changed in Lmna DCM mice supplemented with (a) Lamin C or (b) Lamin A. Venn diagram of overlap between upregulated (red) and downregulated (green) genes in Lmna DCM with (a) Lamin C or (b) Lamin A upregulation compared to Lmna DCM mice. Mice were harvested four weeks after transduction, n = 3. (c) Venn diagram of overlap between Lmna DCM upregulated genes in Lamin A or Lamin C treatment, (d) Hallmark signature depicting top 10 dysregulated pathways of upregulated differentially expressed genes (DEGs) which cannot be reversed by Lamin A as designated by GSEA, arranged by NES.

[0012] Figure 3 depicts increased dose of Lamin A in wildtype mice, (a) Experimental timeline showing timepoints of virus injection and echocardiogram. SR and H&E staining of paraffin heart sections 4 weeks after transduction of EGFP control or Lamin A. Quantification of myocardial fibrosis of SR sections, virus dose, 2.0E+13 vg / kg, n = 5, two-tailed, unpaired T-test with Welch correction. For complete heart images: magnification = 4 scale bar =1000 pm; for enlarged images: magnification = 20 x, scale bar = 100 pm. (b, c) Quantitative real-time PCR analyses of Nppa, Nppb, Collal and Colla2 in mice transduced with EGFP control or Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction and Mann- Whitney test, (d) Western blot and quantitative analysis of phosphorylated Smad2 (p-Smad2) protein levels in mouse heart tissue of mice transduced with EGFP control or Lamin A, n = 5, two-tailed, unpaired T- test with Welch correction, (e-g) Paraffin heart sections (left) and quantifications (right) of (e) aSMA (red), (f) lba-1 (red), (g) CD3 (red), cTnl (green) and DAP1 (blue) positive cells in mice after transduction of EGFP control or Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction and Mann- Whitney test, scale bar = 50 pm.

[0013] Figure 4 depicts candidate selection for the treatment of Lmna DCM. (a) Volcano plot showing differentially expressed genes in heart samples of Lmna DCM mice. Downregulated genes are reflected in green and upregulated genes in red. (b) Hallmark signature depicting top 10 dysregulated pathways in Lmna DCM mice as designated by GSEA, arranged by Normalised Enrichment Score (NES). (c) Western blotand quantitative analysis of phospho-Smad2, phospho-Smad3, phospho-p38, phospho- p70S6K and phospho-mTOR protein levels in mouse heart tissues of control or Lmna DCM mice, n=5, two-tailed, unpaired T-test with Welch correction, (d) Schematic diagram of selected candidates in the cytoplasm and nucleus of cardiomyocyte as well as extracellular matrix (ECM).

[0014] Figure 5 depicts positive candidates extend the survival of Lmna DCM. (a) Comparison of EF (%) assessed by echocardiography in control and Lmna DCM mice supplemented with potential candidates compared to control (P value in green) or Lmna DCM (P value in black). Echocardiography was performed 3 weeks after transduction, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction, (b) Survival curve of Lmna DCM mice supplemented with selected candidates (Red) compared to Lmna DCM mice (Black), n = 10, Log- rank (Mantel- Cox) test.

[0015] Figure 6 depicts the evaluation of disease markers in Lmna DCM. (a and c) Heat map representing color-coded lo 2 fold change of the gene expression of Nppa, Nppb, Myh7, Col lai and Colla2, immunostaining (aSMA, Ibal, CD3, yH2AX, Ki67), Western blot (p-Smad2) and histology (fibrosis), (a) EF or (c) survival of Lmna DCM mice supplemented with selected candidates or control, n = 5. (b and d) Hierarchical clustering and correlation analysis of log2 fold change of selected markers with (b) EF or (d) survival of Lmna DCM mice supplemented with selected candidates or control by nonparametric Spearman correlation, n = 5.

[0016] Figure 7 depicts the upregulation of Lamin A and Lamin C in Lmna DCM mice. Evaluation of the upregulated level of Lamin A and Lamin C in vivo by western blot in mouse whole heart tissue lysates of control or Lmna DCM groups with control, Lamin A or Lamin C upregulation.

[0017] Figure 8 depicts the upregulation of Lamin A in mice, (a) Experimental timeline showing timepoints of virus injection and echocardiogram. Cardiac performance was assessed by echocardiogram at 5.5 weeks-old. SR and H&E staining of paraffin heart sections 3-4 weeks after transduction of EGFP control or Lamin A. Quantification of myocardial fibrosis of SR sections, virus dose, 1.0E+13 vg / kg, n = 5, Mann-Whitney test. For complete heart images: magnification = 4 x, scale bar =1000 pm; for enlarged images: magnification = 20 *, scale bar = 100 pm. (b, c) Quantitative real-time PCR analyses of Nppa, Nppb, Coll al and Colla2 in mice transduced with EGFP control or Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction and Mann-Whitney test.

[0018] Figure 9 depicts the upregulation of mature Lamin A by AAV leads to DCM and cardiac fibrosis, (a) SR and H&E staining of paraffin heart sections and quantifications of mice transduced with EGFP control or mature Lamin A. Quantification of myocardial fibrosis of SR sections, virus dose, 2.0E+13 vg / kg, n = 5, two-tailed, unpaired T-test with Welch correction For complete heart images: magnification = 4 x, scale bar =1000 pm; for enlarged images: magnification = 20 \ scale bar = 100 pm. (b, c) Quantitative real-time PCR analyses of Nppa, Nppb, Collal and Colla2 in mice transduced with EGFP control or mature Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction, (d) Western blot of p-Smad2 protein levels in mouse heart tissues of mice transduced with EGFP control or mature Lamin A, n = 5, two-tailed, unpaired T- test with Welch correction, (e-g) Paraffin heart sections (left) and quantifications (right) of (e) aSMA (red), (f) Iba-1 (red), (g) CD3 (red), cTnl (green) and DAPI (blue) positive cells in mice after transduction of EGFP control or mature Lamin A, n = 5, two-tailed, unpaired T-test with Welch correction, scale bar = 50 pm.

[0019] Figure 10 depicts the DNA damage levels in Lamin A DCM. (a) Representative images (left) and quantifications (right) of paraffin heart sections with immunostaining with for yH2AX (red), cTnl (green) and DAPI (blue), n = 5, two-tailed, unpaired T-test with Welch correction, scale bar = 5 pm. Arrow indicates yH2AX- positive CMs in Lamin A DCM

[0020] Figure 11 depicts the expression levels of selected candidates in Lrnna DCM. Quantitative real-time PCR analyses of Tgfbl, Smad2, Smad3, Atp2a2, Fgfl6, Mapkl4, Sunl, Bmp7, Ctgf Yyl, Yapl and Rptor expressions in control or Lmna DCM mice, n = 5, two-tailed, unpaired T-test with Welch correction and Mann-Whitney test.

[0021] Figure 12 depicts the knockdown efficacy of RNAi in vitro. 2-color system and quantification of shRNA knockdown efficacy of selected candidates. HEK293T cells co-transfected with selected candidates (green) and corresponding shRNAs or control shRNA (Red), n=4, two-tailed, unpaired T-test with Welch correction. Magnification = 4 x, scale bar=500 pm.

[0022] Figure 13 depicts the evaluation of Survival Rate for selected candidates in wildtype mice. Survival curve of wildtype mice supplemented with Ctrl shRNA (black), EGFP Ctrl (black) or selected candidates (red), n = 10.

[0023] Figure 14 depicts the upregulation level and RNAi efficacy in vivo for positive candidates. Evaluation of the upregulated level of positive candidates Yyl,Bmp7 and aYAPl and RNAi knockdown efficacy of positive candidates Smad3, Ctgf and Sun! in vivo by western blot in mouse heart tissues of control or Lmnci DCM groups. * indicates a non-specific band.

[0024] Figure 15 depicts the different dose of AAV9-shRNA. (a and b) Paraffin heart sections of (left) of (a) Sunl (red), (b) Yapl (red), cTnT (green) and DAPT (blue) and quantifications (right) of (a) Sunl intensity (red), (b) Yapl intensity (red) after transduction of 1E+13 vg / kg or 2E+13 vg / kg dose for (a) Sunl shRNA or (b) Yapl shRNA in control or Lmna DCM mice, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnet’s T3 correction, scale bar = 10 pm. Arrows indicate Sunl (a) or Yapl (b) positive CMs.

[0025] Figure 16 depicts the different dose of AAV9 overexpression, (a and b) Paraffin heart sections of (left) (a) Yapl (red), (b) Yyl (red), cTnT (green) and DAPT (blue) and quantifications (right) of (a) aYAPl intensity (red), (b) Yyl intensity (red) after transduction of 1E+13 vg / kg or2E+13 vg / kg dose for (a) aYAPl or(b) Yyl in control or Lmna DCM mice, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction, scale bar = 10 pm. Arrows indicate Yapl (a) or Yy l (b) positive CMs.

[0026] Figure 17 depicts the long-term cardiac performance of Lmna DCM supplemented with Lamin C or Sunl shRNA. (a and b) Evaluation of long-term cardiac performance by echocardiography in Lmna DCM mice supplemented with (a) Lamin C or (b) Sunl shRNA, n = 10.

[0027] Figure 18 depicts the Sunl distribution and nuclear protrusion in Lmna DCM. (a) Western blot of Sunl protein levels in mouse heart tissues of control or Lmna DCM mice, (b) Paraffin heart sections of (left) of Sunl (red), cTnl (green), Lamin A / C (teal) and DAPT (blue) and quantifications (right) of abnormal Sunl distribution and nuclear protrusion mlmna DCM, n=5, two-tailed, unpaired T-test with Welch correction. Arrows indicate Sunl positive CMs, white arrows indicate abnormal Sunl distribution in CMs. (c) Paraffin heart sections (left) of PCM1 (red), cTnl (green) and DAPI (blue) and quantifications (right) of nuclear protrusion in control or Lmna DCM mice supplemented with control or Sunl shRNA, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction, scale bar = 10 pm.

[0028] Figure 19 depicts Nesprinl distribution and nuclear shape in Lmna DCM. (a) Quantitative real-time PCR analyses of Synel (Nesprinl) in control or Lmna DCM mice, n = 5, two-tailed, unpaired T-test with Welch correction, (b) Western blot ofNesprinl protein levels in mouse heart tissues of control or Lmna DCM mice, (c) Paraffin heart sections of Sun! (red), Nesprinl (teal), cTnl (green) and DAPI (blue) positive cells, scale bar = 10 pm. (d) Paraffin heart sections of (left) of Nesprinl (red), cTnl (green) and DAPI (blue) and quantifications (right) of abnormal Nesprinl distribution and nuclear protrusion in LmnaDCM mice, n = 5, two-tailed, unpaired T-test with Welch correction, scale bar = 10 pm. Arrows indicate Nesprinl positive CMs, white arrows indicate abnormal Nesprinl distribution in CMs.

[0029] Figure 20 depicts that KASH domain suppresses Lmna DCM and cardiac fibrosis, (a and b) SR and H&E staining of paraffin heart sections and quantifications of Lmna DCM mice supplemented with control or KASH domain. Quantification of myocardial fibrosis of SR sections, n = 5, Brown-Forsythe and Welch ANOVA test with Dunnetf s T3 correction. For complete heart images: magnification = 4 *, scale bar =1000 pm; for enlarged images: magnification = 20 x, scale bar = 100 pm. (c) Quantitative realtime PCR analyses of Nppa, Nppb, Collal and Colla2, KASH domain in Lmna DCM mice supplemented with control or KASH domain, n = 5, Brown -Forsythe and Welch ANOVA test with Duimet’s T3 correction.

[0030] Figure 21 depicts LMNA Mutations. Schematic diagram of LMNA mutations depicted by green lines located along the Prelamin A and Lamin A / C. Diagram is adapted from Broers et al, 2006

[0063] .

[0031] Figure 22 depicts the evaluation of Survival Rate for Lamin C. Survival curve of Lamin C in wildtype mice, n = 10. Log- rank (Mantel- Cox) test.DETAILED DESCRIPTION

[0032] The present disclosure describes evaluating 14 potential candidates including Lmna gene products (Lamin A and Lamin C), key signaling pathways (Tgfp / Smad, mTor and Fgf / Mapk), calcium handling, proliferation regulators and modifiers of LINC complex function in a cardiac specific Lmna DCM model. Positive candidates for improved cardiac function were further assessed by survival analysis. Suppressive roles and mechanisms of these candidates in ameliorating Lmna DCM were dissected by comparing marker gene expression, Tgfp signaling pathway activation, fibrosis, inflammation, proliferation and DNA damage. Furthermore, transcriptome profiling compared the differences between Lamin A and Lamin C treatment.

[0033] Cardiac function was found to be restored by several positive candidates (Smad3, Yyl, Bmp7, Ctgf, aYAPl, Sunl, Lamin A, and Lamin C), which significantly correlated with suppression ofHF / fibrosis marker expression and cardiac fibrosis in Lmna DCM. Lamin C or Sunl shRNA administration achieved consistent, prolonged survival which highly correlated with reduced heart inflammation and DNA damage. Importantly, Lamin A treatment improved but could not reproduce long term survival, and Lamin A administration to healthy hearts itself induced DCM. Mechanistically, this lapse was identified as caused by a dose-dependent toxicity of Lamin A, which was independent from its maturation.

[0034] In vivo candidate evaluation revealed that supplementation of Lamin C or knockdown of Sunl significantly suppressed Lmna DCM and achieve prolonged survival. Conversely, Lamin A supplementation did not rescue long term survival and may impart detrimental cardiotoxicity risk. This study highlights a potential of advancing Lamin C and Lamin A (at a fine-tuned concentration) as therapeutic targets for the treatment of LMNA DCM.

[0035] In one aspect, the present disclosure refers to a method of treating LMNA Dilated Cardiomyopathy (DCM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a first recombinant Adeno-associated virus (rAAV) encoding Lamin C (rAAV-Lamin C) and / or a second rAAV encoding Lamin A (rAAV-Lamin A), wherein the rAAV-Lamin C comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, and the rAAV-Lamin A comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3.

[0036] In another aspect, the present disclosure refers to use of a first rAAV encoding Lamin C (rAAV-Lamin C) and / or a second rAAV encoding Lamin A (rAAV-Lamin A) in the manufacture of a medicament for treating LMNA Dilated Cardiomyopathy (DCM) in a subject in need thereof, wherein the rAAV-Lamin C comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, and the rAAV-Lamin A comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

[0037] As used herein the term "treatment", refers to any and all uses which remedy a disease state or symptoms of a disease such as LMNA DCM, prevent the establishment of disease such as LMNA DCM, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. Treatment may be effected prophylactically (prior to disease onset) or therapeutically (followingdisease diagnosis). For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i. e. , not worsening) state of condition, disorder or disease; delay or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state, remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a cellular response that is clinically significant, without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0038] The term “subject” refers to subjects of human or other mammal and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that “patient” does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (eg. sheep, cows, horses, donkeys, pigs), laboratory test animals (eg. rabbits, mice, rats, guinea pigs, hamsters), companion animals (eg. cats, dogs) and captive wild animals (eg. foxes, deer, dingoes). In one example, the subject is a human.

[0039] In some examples, the subject may be suffering from a cardiac disorder, such as LMNA DCM, heart failure, ischemia, myocardial infarction, congestive heart failure, arrhythmia, transplant rejection and the like. In another example, the subject has or is at risk for LMNA DCM or heart failure, e.g. a non-ischemic cardiomyopathy, mitral valve regurgitation, ischemic cardiomyopathy, or aortic stenosis or regurgitation.

[0040] Lamins are nuclear intermediate filament proteins, and the principal constituents of the nuclear lamina, the proteinaceous matrix underlying the inner nuclear membrane (INM). The lamina consists of the A-type lamins, consisting of 2 predominant forms, lamin A and lamin C, derived by alternate splicing of LMNA RNA. In this disclosure, it was found that treatment of LMNA DCM can be achieved by increasing expression of Lamin C and / or Lamin A in a subject using a gene delivery vector rAAV.

[0041] As used herein, the term "AAV" is a standard abbreviation for ad enoassociated virus. Adeno-associated virus (AAV) is a small non-pathogenic singlestranded DNA virus of the parvoviridae family. It grows only in cells in which certain functions are provided by a co-infecting helper virus. The approximately 4.7 kb genome of AAV consists of single stranded DNA of either plus or minus polarity. The ends of thegenome are short inverted terminal repeats (ITRs) which can fold into hairpin structures and serve as the origin of viral DNA replication. To date, at least thirteen serologically distinct AAVs have been identified and isolated from humans or primates. AAV has a few features have made it an attractive vector for gene transfer: AAV vectors possess a broad host range, transduce both dividing and non-dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes in the absence of a significant immune response to the transgene product in general. Moreover, both wild-type AAV and AAV vector particles are non-pathogenic (in contrast to adenoviral vectors). Viral particles are heat stable, resistant to solvents, detergents, changes in pH and temperature. The ITRs have been shown to be the only cis elements required for replication and packaging and may contain some promoter activities. No viral genes are encoded by AAV vectors. Vectors based on AAV thus emerged as those preferred for achieving truly stable transduction following in vivo administration. Recombinant adeno-associated virus (rAAV) based gene editing platform enables insertion, deletion or substitution of DNA sequences into the genomes of live mammalian cells. An emerging application of rAAV based genome editing is for gene therapy in patients, due to the accuracy and lack of off- target recombination events afforded by the approach.

[0042] As used herein, an "AAV vector" as used herein refers to a vector comprising one or more polynucleotides of interest (such as a Lamin A or Lamin C gene) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. AAV "rep" and "cap" genes are genes found in all AAV serotypes examined to date, encoding replication and encapsidation proteins, respectively. AAV rep and cap genes are also individually and collectively referred to as "AAV packaging genes".

[0043] An "AAV" or "AAV virion" or "AAV viral particle" or "AAV vector particle" or "AAV virus" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. When the viral particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a Lamin A or Lamin C gene to be delivered to a mammalian cell), it is typically referred to as a recombinant "AAV vector particle" or "AAV vector". Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.

[0044] The invention provides for methods of producing a recombinant adeno- associated virus (AAV) particle comprising the steps of: culturing a cell (for example, a mammalian cell, HEK293 cell) that has been transfected with any of the AAV vectors encoding Lamin C or Lamin A of this disclosure and recovering recombinant AAV particle from the supernatant of the transfected cell. The disclosure also discloses viral particles comprising any of the viral vectors of the invention encoding Lamin C or Lamin A, and cells (for example, a mammalian cell, HEK293 cell) comprising these viral vectors. As used herein, a "recombinant viral vector" such as a recombinant AAV, rAAV, refers to a viral vector comprising one or more heterologous gene products or sequences such as Lamin C or Lamin A gene Since many viral vectors exhibit size-constraints associated with packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e g., a helper virus or a packaging cell line carrying gene products necessary for replication and / or encapsidation).

[0045] The rAAV as disclosed herein comprises a serotype selected from the group consisting of AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and a chimeric AAV vector. In one example, the rAAV is rAAV type 9.

[0046] In one example, rAAV encodes a mouse Lamin C gene. rAAV-Lamin C (mouse) has a sequence of SEQ ID NO:2 (AAV cTNT mouse Lamin C, 5978 bp): ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatc taccagggtaatggggatcctctagaactatagctagaattcgcccttacgggccccccctcgaggtcgggataaaagcagtc tgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagccctggg aggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgag gccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaa atagcagctcacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccgg ggggaccttaaagcctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccg aagctctagagctttattgcggtagtttatcacagttaaattgctaacgcagtcagtgcttctgacacaacagtggtcgtgaggca ctgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagttcaattacagctctta aggctagagtacttaatacgactcactataggctagcATGGAGACCCCgtcacagcggcgcgccacccgcagtgg ggcgcaggccagctctaccccactgtcgcccactcggatcacccggctgcaggagaaggaggacctgcaggagctcaatg accgcctggccgtgtacatcgatcgcgtgcgttccctggagaccgagaacgcggggctgcgccttcgcatcactgagtctga agaggtggtcagccgagaggtgtccggcatcaaggcggcctacgaggccgagctgggggatgcccgcaagacccttgatt ctgtggccaaggagcgcgcccgcctccagctagagctgagcaaagtgcgtgaggagttcaaggagctgaaggctcgcaac accaagaaggagggggacttgttggctgcgcaggcccggctcaaggacctcgaggctcttctcaactccaaggaagctgcc ctgagcactgctctcagtgagaagcgcacattggagggcgagctccatgacctgcgggggcaggtagccaagcttgaggc ggccctgggagaggctaagaagcagcttcaggatgagatgctgaggcgagtggatgctgagaacaggctacagacgctga aggaggagcttgacttccagaagaacatttacagcgaggaactgcgtgagaccaagcgccggcatgagacgcggcttgtgg agatcgataacgggaagcagcgagagttgagagccggctggcagatgccctgcaggagctgcgggctcagcatgaggac caggtggaacagtataagaaggagctagaaaagacatactccgccaagctggataatgccaggcagtctgctgagaggaac agcaacctcgtgggggctgcccatgaggaactgcagcagtctcgaatccgcattgacagcctctcggcccagctcagccag ctccaaaagcagttggcagccaaggaggcaaagctgcgtgacctggaggactcgctggcccgtgagcgcgataccagccg gcgcctgctggctgagaaagagcgagagatggcggagatgcgggcgaggatgcagcagcagctggacgagtaccagga gctgctggacatcaagctggccctggacatggagatccatgcctatcgaaagctgctggagggcgaggaggagaggctgc gcctgtcccccagccctacctcgcagcgcagccgtggccgcgcctcctcccactcatcccagtctcagggtggaggcagcg tcaccaaaaagcgcaagctggagtcttccgagagccggagcagcttctcgcagcatgctcgcactagcgggcgtgtggcgg tagaggaagtcgatgaagagggaaagttcgtgcggctgcgcaacaagtccaacgaggaccagtccatgggcaactggcag atcaggcgtcagaatggtgacgatcctttgatgacctatcgcttcccaccgaagttcaccctaaaggctgggcaggtggtgac gatctgggcttcaggagctggggccacccatagcccccctactgacttggtgtggaaggcgcagaacacctggggctgtgg gagcagccttcgcaccgctctcatcaactccactggagaagaagtggccatgcgcaagctggtgcgctcactgaccatggtt gaggacaatgaggatgacgacgaggatggagaagagctcctccatcaccaccgtgtgagtgGCAGCCGCCGCT GAggtacCTGGAGGCTTGCTGAAGGCTGTATGCTCGAGACGTAACGTCTCGCA GGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGTCGACccgggcggc ctcgaggacggggtgaactacgcctgaggatccgatctttttccctctgccaaaaattatggggacatcatgaagccccttgag catctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaagcaattcgttga tctgaatttcgaccacccataatacccattaccctggtagataagtagcatggcgggttaatcattaactacaaggaacccctagt gatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggcttt gcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactg ggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgca ccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcataagcgcggcgggtg tggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaa acttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaat agtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggccta ttggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggg gaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgctt caataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttg ctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac agcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatta tcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcaca gaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactta cttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgg gaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaa actattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccactt ctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagca ctggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagac agatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaactt catttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagc gtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatac caaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcc tgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag cggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgt gagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcg attttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccg cagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgc gcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtg agttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttc acacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg

[0047] In another example, rAAV encodes a human Lamin C gene. rAAV-Lamin C (human) has a sequence of SEQ ID NO:4 (AAV cTNT human Lamin C, 5972 bp):ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatc taccagggtaatggggatcctctagaactatagctagaattcgcccttacgggccccccctcgaggtcgggataaaagcagtc tgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagccctggg aggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgag gccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaa atagcagctcacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccgg ggggaccttaaagcctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccg aagctctagagctttattgcggtagtttatcacagttaaattgctaacgcagtcagtgcttctgacacaacagtggtcgtgaggca ctgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttg cgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagtcaattacagctctta aggctagagtacttaatacgactcactataggctagcatggagaccccgtcccagcggcgcgccacccgcagcggggcgc aggccagctccactccgctgtcgcccacccgcatcacccggctgcaggagaaggaggacctgcaggagctcaatgatcgct tggcggtctacatcgaccgtgtgcgctcgctggaaacggagaacgcagggctgcgccttcgcatcaccgagtctgaagagg tggtcagccgcgaggtgtccggcatcaaggccgcctacgaggccgagctcggggatgcccgcaagacccttgactcagta gccaaggagcgcgcccgcctgcagctggagctgagcaaagtgcgtgaggagtttaaggagctgaaagcgcgcaataccaa gaaggagggtgacctgatagctgctcaggctcggctgaaggacctggaggctctgctgaactccaaggaggccgcactga gcactgctctcagtgagaagcgcacgctggagggcgagctgcatgatctgcggggccaggtggccaagcttgaggcagcc ctaggtgaggccaagaagcaacttcaggatgagatgctgcggcgggtggatgctgagaacaggctgcagaccatgaagga ggaactggacttccagaagaacatctacagtgaggagctgcgtgagaccaagcgccgtcatgagacccgactggtggagat tgacaatgggaagcagcgtgagtttgagagccggctggcggatgcgctgcaggaactgcgggcccagcatgaggaccag gtggagcagtataagaaggagctggagaagacttattctgccaagctggacaatgccaggcagtctgctgagaggaacagc aacctggtgggggctgcccacgaggagctgcagcagtcgcgcatccgcatcgacagcctctctgcccagctcagccagctc cagaagcagctggcagccaaggaggcgaagcttcgagacctggaggactcactggcccgtgagcgggacaccagccgg cggctgctggcggaaaaggagcgggagatggccgagatgcgggcaaggatgcagcagcagctggacgagtaccaggag cttctggacatcaagctggccctggacatggagatccacgcctaccgcaagctcttggagggcgaggaggagaggctacgc ctgtcccccagccctacctcgcagcgcagccgtggccgtgcttcctctcactcatcccagacacagggtgggggcagcgtca ccaaaaagcgcaaactggagtccactgagagccgcagcagcttctcacagcacgcacgcactagcgggcgcgtggccgtg gaggaggtggatgaggagggcaagtttgtccggctgcgcaacaagtccaatgaggaccagtccatgggcaattggcagatc aagcgccagaatggagatgatcccttgctgacttaccggttcccaccaaagttcaccctgaaggctgggcaggtggtgacgat ctgggctgcaggagctggggccacccacagcccccctaccgacctggtgtggaaggcacagaacacctggggctgcggg aacagcctgcgtacggctctcatcaactccactggggaagaagtggccatgcgcaagctggtgcgctcagtgactgtggttg aggacgacgaggatgaggatggagatgacctgctccatcaccaccacgtgagtggtagccgccgctgaggtacCTGGAGGCTTGCTGAAGGCTGTATGCTCGAGACGTAACGTCTCGCAGGACACAAGG CCTGTTACTAGCACTCACATGGAACAAATGTCGACccgggcggcctcgaggacggggtga actacgcctgaggatccgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaat aaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaagcaattcgttgatctgaatttcgaccaccc ataatacccattaccctggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactc cctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctca gtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtt acccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccca acagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgca gcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttcccc gtcaagctctaaatcgggggctccctttagggttccgatttagtgcttacggcacctcgaccccaaaaaacttgattagggtgat ggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttcc aaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgag ctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaa cccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa ggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgc tggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttg agagtttcgccccgaagaacgttttccaatgatgagcacttttaaagtctgctatgtggcgcggtatatcccgtattgacgccg ggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacgg atggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcg gaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaat gaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaa ctacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttc cggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggta agccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggt gcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaagga tctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggttt gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtg tagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggg gggtcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttc ctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaat gcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggc accccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatg accatgattacgccagatttaattaaggccttaattagg

[0048] In another example, rAAV encodes a mouse Lamin A gene. rAAV-Lamin A (mouse) has a sequence of SEQ ID NO:1 (AAV cTNT mouse Lamin A, 6338 bp): ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctactatc taccagggtaatggggatcctctagaactatagctagaattcgcccttacgggccccccctcgaggtcgggataaaagcagtc tgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagccctggg aGCGTGTGACCGGCGGCTCTAGAggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatc tccgtccgacgggTACGGGAGGTACTTGGAGCGGCtttaaaatagcaaaactctgaggccacacaatag cttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaaatagcagctcac aagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaaa gcctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccgaagctctagagct ttattgcggtagtttatcacagttaaattgctaacgcagtcagtgcttctgacacaacagtggtcgtgaggcactgggcaggtaa gtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgatagg cacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagttcaattacagctcttaaggctagagtact taatacgactcactataggctagcatggagaccccgtcacagcggcgcgccacccgcagtggggcgcaggccagctctacc ccactgtcgcccactcggatcacccggctgcaggagaaggaggacctgcaggagctcaatgaccgcctggccgtgtacatc gatcgcgtgcgttccctggagaccgagaacgcggggctgcgccttcgcatcactgagtctgaagaggtggtcagccgagag gtgtccggcatcaaggcggcctacgaggccgagctgggggatgcccgcaagacccttgattctgtggccaaggagcgcgc ccgcctccagctagagctgagcaaagtgcgtgaggagttcaaggagctgaaggctcgcaacaccaagaaggagggggact tgttggctgcgcaggcccggctcaaggacctcgaggctcttctcaactccaaggaagctgccctgagcactgctctcagtgag aagcgcacattggagggcgagctccatgacctgcgggggcaggtagccaagcttgaggcggccctgggagaggctaaga agcagcttcaggatgagatgctgaggcgagtggatgctgagaacaggctacagacgctgaaggaggagcttgacttccaga agaacatttacagcgaggaactgcgtgagaccaagcgccggcatgagacgcggcttgtggagatcgataacgggaagcag cgagagtttgagagccggctggcagatgccctgcaggagctgcgggctcagcatgaggaccaggtggaacagtataagaa ggagctagaaaagacatactccgccaagctggataatgccaggcagtctgctgagaggaacagcaacctcgtgggggctgc ccatgaggaactgcagcagtctcgaatccgcattgacagcctctcggcccagctcagccagctccaaaagcagttggcagccaaggaggcaaagctgcgtgacctggaggactcgctggcccgtgagcgcgataccagccggcgcctgctggctgagaaag agcgagagatggcggagatgcgggcgaggatgcagcagcagctggacgagtaccaggagctgctggacatcaagctggc cctggacatggagatccatgcctatcgaaagctgctggagggcgaggaggagaggctgcgcctgtcccccagccctacctc gcagcgcagccgtggccgcgcctcctcccactcatcccagtctcagggtggaggcagcgtcaccaaaaagcgcaagctgg agtcttccgagagccggagcagcttctcgcagcatgctcgcactagcgggcgtgtggcggtagaggaagtcgatgaagagg gaaagttcgtgcggctgcgcaacaagtccaacgaggaccagtccatgggcaactggcagatcaggcgtcagaatggtgac gatcctttgatgacctatcgcttcccaccgaagttcaccctaaaggctgggcaggtggtgacgatctgggcttcaggagctggg gccacccatagcccccctactgacttggtgtggaaggcgcagaacacctggggctgtgggagcagccttcgcaccgctctca tcaactccactggagaagaagtggccatgcgcaagctggtgcgctcactgaccatggttgaggacaatgaggatgacgacg aggatggagaagagctcctccatcaccaccgtggttcccactgcagcggctcgggggaccccgctgagtacaacctgcgct cacgcaccgtgctgtgcgggacgtgtgggcagcctgctgacaaggctgccggtggagcgggagcccaggtgggcggatc catctcctctggctcttctgcctccagtgtcacagtcactcgaagcttccgcagtgtggggggcagtgggggtggcagcttcgg ggacaacctagtcacccgctcctacctcctgggcaactccagtccccggagccagagctcccagaactgcagcatcatgtaa ggtacCTGGAGGCTTGCTGAAGGCTGTATGCTGAAATCGCTGATTTGTGTAGTC GTTTTGGCCACTGACTGACGACTACACATCAGCGATTTCAGGACACAAGGC CTGTTACTAGCACTCACATGGAACAAATGTCGACccgggcggcctcgaggacggggtgaa ctacgcctgaggatccgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaata aaggaaatttattttcattgcaatagtgtgttggaatttttgtgtctctcactcggaagcaattcgttgatctgaatttcgaccaccca taatacccattaccctggtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactcc ctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagt gagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttac ccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaac agttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagc gtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatg gttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttcca aactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagc tgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaac ccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaag gaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgct ggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttg agagtttcgccccgaagaacgttttccaatgatgagcacttttaaagtctgctatgtggcgcggtattatcccgtattgacgccg ggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcg gaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaat gaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaa ctacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttc cggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggta agccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggt gcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaagga tctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggttt gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtg tagccgtagttaggccaccactcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggg gggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagct tccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttc ctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaat gcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggc accccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatg accatgattacgccagatttaattaaggccttaattagg

[0049] In another example, rAAV encodes a human Lamin A gene. rAAV-Lamin A (human) has a sequence of SEQ ID NO:3 (AAV cTNT human Lamin A, 6248 bp): ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctactatc taccagggtaatggggatcctctagaactatagctagaattcgcccttacgggccccccctcgaggtcgggataaaagcagtc tgggctttcacatgacagcatctggggctgcggcagagggtcgggtccgaagcgctgccttatcagcgtccccagccctggg aggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtttaaaatagcaaaactctgag gccacacaatagcttgggcttatatgggctcctgtgggggaagggggagcacggagggggccggggccgctgctgccaaa atagcagctcacaagtgttgcattcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccgg ggggaccttaaagcctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggacgatccccg aagctctagagctttattgcggtagtttatcacagttaaattgctaacgcagtcagtgctctgacacaacagtggtcgtgaggca ctgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagttcaattacagctctta aggctagagtacttaatacgactcactataggctagcatggagaccccgtcccagcggcgcgccacccgcagcggggcgc aggccagctccactccgctgtcgcccacccgcatcacccggctgcaggagaaggaggacctgcaggagctcaatgatcgct tggcggtctacatcgaccgtgtgcgctcgctggaaacggagaacgcagggctgcgccttcgcatcaccgagtctgaagagg tggtcagccgcgaggtgtccggcatcaaggccgcctacgaggccgagctcggggatgcccgcaagacccttgactcagta gccaaggagcgcgcccgcctgcagctggagctgagcaaagtgcgtgaggagtttaaggagctgaaagcgcgcaataccaa gaaggagggtgacctgatagctgctcaggctcggctgaaggacctggaggctctgctgaactccaaggaggccgcactga gcactgctctcagtgagaagcgcacgctggagggcgagctgcatgatctgcggggccaggtggccaagcttgaggcagcc ctaggtgaggccaagaagcaacttcaggatgagatgctgcggcgggtggatgctgagaacaggctgcagaccatgaagga ggaactggacttccagaagaacatctacagtgaggagctgcgtgagaccaagcgccgtcatgagacccgactggtggagat tgacaatgggaagcagcgtgagtttgagagccggctggcggatgcgctgcaggaactgcgggcccagcatgaggaccag gtggagcagtataagaaggagctggagaagacttattctgccaagctggacaatgccaggcagtctgctgagaggaacagc aacctggtgggggctgcccacgaggagctgcagcagtcgcgcatccgcatcgacagcctctctgcccagctcagccagctc cagaagcagctggcagccaaggaggcgaagcttcgagacctggaggactcactggcccgtgagcgggacaccagccgg cggctgctggcggaaaaggagcgggagatggccgagatgcgggcaaggatgcagcagcagctggacgagtaccaggag cttctggacatcaagctggccctggacatggagatccacgcctaccgcaagctcttggagggcgaggaggagaggctacgc ctgtcccccagccctacctcgcagcgcagccgtggccgtgcttcctctcactcatcccagacacagggtgggggcagcgtca ccaaaaagcgcaaactggagtccactgagagccgcagcagctctcacagcacgcacgcactagcgggcgcgtggccgtg gaggaggtggatgaggagggcaagtttgtccggctgcgcaacaagtccaatgaggaccagtccatgggcaattggcagatc aagcgccagaatggagatgatcccttgctgacttaccggttcccaccaaagttcaccctgaaggctgggcaggtggtgacgat ctgggctgcaggagctggggccacccacagcccccctaccgacctggtgtggaaggcacagaacacctggggctgcggg aacagcctgcgtacggctctcatcaactccactggggaagaagtggccatgcgcaagctggtgcgctcagtgactgtggttg aggacgacgaggatgaggatggagatgacctgctccatcaccaccacggctcccactgcagcagctcgggggaccccgct gagtacaacctgcgctcgcgcaccgtgctgtgcgggacctgcgggcagcctgccgacaaggcatctgccagcggctcagg agcccaggtgggcggacccatctcctctggctcttctgcctccagtgtcacggtcactcgcagctaccgcagtgtggggggc agtgggggtggcagcttcggggacaatctggtcacccgctcctacctcctgggcaactccagcccccgaacccagagcccc cagaactgcagcatcatgtaaggtacCTGGAGGCTTGCTGAAGGCTGTATGCTCGAGACGT AACGTCTCGCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATG TCGACccgggcggcctcgaggacggggtgaactacgcctgaggatccgatctttttccctctgccaaaaattatggggac atcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctca ctcggaagcaattcgttgatctgaatttcgaccacccataatacccattaccctggtagataagtagcatggcgggttaatcatta actacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggt cgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgt aatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcgg cgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctt tcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttta cggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttga cgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataa gggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgctta caatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatga gacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgg gtacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaa gttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgact tggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatga gtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggat catgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtag caatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggagg cggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtg ggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcat atatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa cgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgct gcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactg gcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcc tacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgat agttaccggataaggcgcagcggtcgggctgaacggggggtcgtgcacacagcccagcttggagcgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaag cggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcg ccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggccttttt acggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttga gtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaat acgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagt gagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgg aattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg

[0050] The amino acid sequences of the Lamin C and Lamin A are as follows:

[0051] SEQ ID NO: 5 (mouse Lamin A):METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLRLRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARNTKKEGDLLAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTLKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRL SP SPTSQRSRGRAS SHS SQSQGGGS VTKKRKLES SESRS SFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIRRQNGDDPLMTYRFPPKFTLKAGQWTTWASGAGATHSPPTDLVWKAQNTWGCGSSLRTALTNSTGEEVAMRKLVRSLTMVEDNEDDDEDGEELLHHHRGSHCSGSGDPAEYNLRSRTVLCGTCGQPADKAAGGAGAQVGGSISSGSSASSVTVTRSFRSVGGSGGGSFGDNL VTRS YLLGNS SPRSQ SSQNC SIM

[0052] SEQ ID NO:6 (mouse Lamin C):METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLRLRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARNTKKEGDLLAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTLKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIHAYRKLLEGEEERLRL SP SPTSQRSRGRAS SHS SQSQGGGS VTKKRKLES SESRS SFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIRRQNGDDPLMTYRFPPKFTLKAGQVVT1WASGAGATHSPPTDLVWKAQNTWGCGSSLRTALINSTGEEVAMRKLVRSLTMVEDNEDDDEDGEELLHHHRVSGSRR

[0053] SEQ ID NO:7 (human Lamin A):

[0054] METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLRLRITESEEWSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARNTKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAK EAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLAL DMEIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTKKRKLEST ESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDP LLTYRFPPKFTLKAGQWTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRTA LINSTGEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLR SRTVLCGTCGQP ADKAS ASGSGAQVGGPISSGSS AS SVTVTRS YRS VGGSGGGS FGDNLVTRSYLLGNSSPRTQSPQNCSIM

[0055] SEQ ID NO: 8 (human Lamin C):METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETE NAGLRLRITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKV REEFKELKARNTKKEGDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELH DLRGQVAKLEAALGEAKKQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEEL RETKRRHETRLVEIDNGKQREFESRLADALQELRAQHEDQVEQYKKELEKTYS AKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQLQKQLAAKEAKLR DLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLALDMEIH AYRKLLEGEEERLRL SP SPTSQRSRGRAS SHS SQTQGGGS VTKKRKLESTESRS S FSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQIKRQNGDDPLLTY RFPPKFTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRTALINST GEEVAMRKLVRSVTVVEDDEDEDGDDLLHHHHVSGSRR

[0056] By overexpressing Lamin C and / or Lamin A in the subject, restored cardiac function and prolonged survival can be achieved. Overexpressing Lamin C and / or Lamin A can be done via administering a therapeutically effective amount of a first rAAV encoding Lamin C (rAAV-Lamin C, having nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4) and / or a second rAAV encoding Lamin A (rAAV-Lamin A, having nucleotide sequence of SEQ ID NO: 1 or SEQ LD NO:3). As used herein the term "therapeutically effective amount” includes within its meaning a non-toxic but sufficient amount of an agent such as rAAV-Lamin C and / or rAAV-Lamin A to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effectiveamount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

[0057] The rAAV-Lamin C and / or rAAV-Lamin A helps transfect Lamin C and / or Lamin A gene into a cell inside the subject's body. The term “transfection” is used to refer to the uptake of foreign DNA by a cell . A cell has been “transfected” when exogenous DNA (such as Lamin C and / or Lamin A DNA) has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art to introduce one or more exogenous DNA moieties, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells. The term captures chemical, electrical, and viral-mediated transfection procedures. In this invention, specifically transfection is done via a viral vector such as rAAV.

[0058] To transfect Lamin C and / or Lamin A genes into cells of the subject as disclosed herein, the rAAV-Lamin C and / or rAAV-Lamin A may be administered with a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to the subject. Useful carriers are well known in the art, and include, e.g., water, buffered w water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

[0059] In one example, the rAAV after administering into the subject only expresses the encoded gene such as Lamin C and / or Lamin A in the heart. The rAAV vector can include conventional control elements or sequences which are operably linked to the transgene (such as Lamin C or Lamin A) in a manner which permits its transcription, translation and / or expression in a cell infected with the rAAV. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e ., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / ortissue-specific, are known in the art and may be utilized. As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product (such as Lamin C or Lamin A gene) operably linked to the promoter / regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the b-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e g., acute phase, a particular differentiation state of the cell, or in replicating cells only. A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product (such as the Lamin C or Lamin A gene), causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0060] In one example, the rAAV comprises the native promoter for the transgene (such as Lamin C or Lamin A) will be used. The native promoter may be preferred when it is desired that expression of the transgene (such as Lamin C or Lamin A) should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue- specific manner, or in response to specific transcriptional stimuli. In another example, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0061] In another example, the rAAV comprises a tissue- specific promoter, for example, a cardiomyocyte (CM) specific promoter to allow expression of Lamin C and / or Lamin A only in the CM. In another example, the cardiomyocyte-specific promoter is derived from the troponin T gene of the chicken. The TNNT2 gene encodes cardiactroponin T, a protein exclusively expressed in heart muscle. Sequence of the CM specific promotor is: SEQ ID NO:73: cgggccccccctcgaggtcgggataaaagcagtctgggctttcacatgacagcatctggggctgcggcagagggtcgggtc cgaagcgctgccttatcagcgtccccagccctgggaggtgacagctggctggcttgtgtcagcccctcgggcactcacgtatc tccgtccgacgggtttaaaatagcaaaactctgaggccacacaatagcttgggcttatatgggctcctgtgggggaaggggga gcacggagggggccggggccgctgctgccaaaatagcagctcacaagtgttgcattcctctctgggcgccgggcacattcct gctggctctgcccgccccggggtgggcgccggggggaccttaaagcctctgccccccaaggagcccttcccagacagccg ccggcacccaccgctccgtgggacgatccccga

[0062] Other suitable viral vectors which can be used to deliver Lamin C and / or Lamin A to the subject can be selected from the group comprising adenovirus, helper-dependent adenovirus, retrovirus (such as Moloney murine leukemia viruses and HIV-based viruses), or hem agglutinating virus of Japan-liposome (HVJ) complex.

[0063] The recombinant AAV encoding Lamin C or Lamin A described herein can be used to express Lamin C or Lamin A to treat LMNA DCM. In another example, the recombinant AAV encoding Lamin C or Lamin A described herein may be used to treat other types of cardiac diseases, including heart failure, heart muscle disease, cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, viral infection, wounds, hypertensive heart disease, valvular disease, congenital heart disease, myocardial infarction, congestive heart failure, arrhythmias, diseases resulting in remodeling of the heart, etc. Diseases of the heart can be due to any reason, such as for example, damage to cardiac tissue such as a loss of contractility (e.g., as might be demonstrated by a decreased ejection fraction). The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease, while some cause remain undefined and such patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC).

[0064] In one example, to treat LMNA DCM, the rAA V -Lamin C and / or rAA V -Lamin A are to be administered to the subject by intravenous injection. Lamin C and Lamin A will be expressed in the serum, and other tissue / organs such as the heart, and cardiomyocytes (CM). An intravenous infusion of the rAAV-Lamin C and / or rAAV- Lamin A in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, can be used.

[0065] In another example, to treat LMNA DCM the rAAV-Lamin C and / or rAAV- Lamin A are administered via local delivery. The terms "local administration" or"local delivery," refer to delivery that does not rely upon transport of the rAAV-Lamin C and / or rAAV-Lamin A to its intended target tissue via the vascular or lymphatic system from a site of administration that is remote from the intended target tissue. The rAAV- Lamin C and / or rAAV-Lamin A is delivered directly to its intended target tissue or in the vicinity thereof, e g. by injection or implantation. It will be appreciated that a small amount of the delivered agent may enter the vascular system and may ultimately reach the target tissue via the vascular system. Thus, in another example, the rAAV-Lamin C and / or rAAV-Lamin A is to be administered locally, to the heart of the subject. The rAAV-Lamin C and / or rAAV-Lamin A may be administered directly to a target site such as heart by, for example, surgical delivery to the heart, inj ection to the heart, or by catheter to a site accessible by a blood vessel. Other methods of delivery may be diffusion from a device impregnated with the rAAV-Lamin C and / or rAAV-Lamin A

[0066] The rAAV-Lamin C and / or rAAV-Lamin A may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously). The rAAV- Lamin C and / or rAAV-Lamin A are preferably formulated in a sterilized pyrogen-free form.

[0067] The precise amount of rAAV-Lamin C and / or rAAV-Lamin A which is therapeutically effective, and the route by which rAAV-Lamin C and / or rAAV-Lamin A is best administered, can be readily determined by one of ordinary skill in the art by comparing the administered level of rAAV-Lamin C and / or rAAV-Lamin A to the concentration required to have a therapeutic effect.

[0068] Whether intravenous injection or heart administration, the rAAV-Lamin C and / or rAAV-Lamin A modulate (specifically, increase) the expression or amounts of Lamin C and / or Lamin A in the target cell such as a cardiomyocyte The amount of the heterologous protein Lamin C or Lamin A expressed in the subject (e.g., serum, or cardiomyocytes (CM) of the subject) can vary. In some examples, the amount of the Lamin C or Lamin A protein expressed in the CM of the subject can be increased by 10%- 100%, 10-30%, 20-40%, 30-60%, 40-70%, 50-80%, 60-90%, 70-100%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times, or higher, as compared to the amount without administration of the rAAV-Lamin C and / or rAAV-Lamin A. A skilled artisan will understand that the level of increase of expression level of Lamin Cand / or Lamin A for the method to be effective can vary depending on non-limiting factors such as the particular protein of interest and the subj ect receiving the treatment, and an effective amount of the protein to achieve a desired therapeutic effect can be readily determined by a skilled artisan using conventional methods known in the art without undue experimentation.

[0069] In one example, the viral vector used in the invention methods, rAAV-Lamin C, is administered at a concentration of 1.0E13 to 1.1E14 vector genomes per kilogram of body weight (vg / kg) virus particles. The administered rAAV-Lamin C has a concentration selected from the group consisting of about 1.0E13 vg / kg, about 2.0E13 vg / kg, about 3.OE13 vg / kg, about 4.0E13 vg / kg, about 5.0E13 vg / kg, about 6.0E13 vg / kg, about 7.0E13 vg / kg, about 8.0E13 vg / kg, about 9.0E13 vg / kg, about 1.0E14 vg / kg, and about 1.1E14 vg / kg, and any ranges in between.

[0070] In another example, the viral vector used in the invention methods, rAAV- Lamin A, is administered at a concentration of I .0EI3 to 2.0E13 vg / kg virus particles. The administered rAAV-Lamin A has a concentration selected from the group consisting of about 1.0E13 vg / kg, about I.1E13 vg / kg, about I.2E13 vg / kg, about 1.3E13 vg / kg, about 1.4E13 vg / kg, about 1.5E13 vg / kg, about 1.6E13 vg / kg, about 1.7E13 vg / kg, about 1.8E13 vg / kg, about 1.9E13 vg / kg, and about 2.0E13 vg / kg, and any ranges in between. rAAV-Lamin A should be used at a lower concentration compared to rAAV-Lamin C due to its dose-dependent toxicity.

[0071] In one example, the viral vector used in the invention methods, rAAV-Lamin C and / or rAAV-Lamin A, is administered in a single dose. In another example, the rAAV- Lamin C and / or rAAV-Lamin A, is administered at multiple doses selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more doses. The skilled person will be able to determine the optimal doses depending on the subject condition, disease progression, and response to the treatment.

[0072] In one example, the viral vector used in the invention methods, rAAV-Lamin C, is administered for a duration of up to 2 years, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. rAAV-Lamin C extends the survival of the administered subject by up to 2 years, for example about 1 months, about 2 months, about3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months. None of the current literatures discloses such a long term survi val effect for LMNA DCM treatment.

[0073] In another example, the viral vector used in the invention methods, rAAV- Lamin A, is administered for a duration of up to 2 months, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. rAAV-Lamin A extends the survival of the administered subject by up to 2 months, for example about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks.

[0074] When administered with multiple doses over a period as disclosed herein, the rAAV-Lamin C and / or rAAV-Lamin A are delivered using different serotypes of rAAV as disclosed herein, selected from the group consisting of AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and a chimeric AAV vector.

[0075] In one example, only rAAV-Lamin C is administered to treat LMNA DCM. In another example, only rAAV-Lamin A is administered to treat LMNA DCM. In another example, a combination of rAAV-Lamin C and rAAV-Lamin A are administered in order to treat LMNA DCM. In one example, the molar ratio of Lamin A expressed from rAAV Lamin A to Lamin C expressed from rAAV-Lamin C is at most 1 :1. In another example, the molar ratio of Lamin A expressed from rAAV-Lamin A to Lamin C expressed from rAAV-Lamin C is selected from the group consisting of about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1:10, about 1:20, about 1:30, about 1 :50, about 1 :80, about 1 :100, about 1:200, about 1 :500, and about 1 : 1000.

[0076] Disclosed also is a composition of recombinant adeno-associated virus (rAAV) vectors, comprising a first rAAV vector encoding Lamin A and a second rAAV vector encoding Lamin C, wherein the molar ratio of Lamin A expressed from rAAVLamin A to Lamin C expressed from rAAV-Lamin C is at most 1:1

[0077] Disclosed also is a composition of virus particles, comprising a first virus particle comprising the rAAV-Lamin A as disclosed herein, and a second virus particlecomprising the rAAV- Lamin C as disclosed herein, wherein the molar ratio of Lamin A expressed from rAAV-Lamin A to Lamin C expressed from rAAV-Lamin C is at most 1 : 1.

[0078] Disclosed also is a pharmaceutical composition comprising the composition of rAAV vectors or rAAVs as disclosed herein or the composition of virus particles as disclosed herein, and a pharmaceutically acceptable carrier.

[0079] A treatment can be evaluated by assessing the effect of the treatment on a parameter related to heart function and animal survival, for example the expression of HF / fibrosis markers as well as fibrosis which are utilised commonly in most HF related research, upregulation of inflammatory cytokines, DNA damage, and term of subject survival. Therapeutic activity includes treatment of a disease or condition, including both prophylactic and ameliorative treatment. Treatment of a disease or condition can include improvement of a disease or condition by any amount, including prevention, amelioration, and elimination of the disease or condition. For example, both in vivo and in vitro methods can be used, including for example, clinical evaluation, EC50, and IC50 assays, and dose response curves.

[0080] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a primer” includes a plurality of primers, including mixtures and combinations thereof.

[0081] As used herein, the term “comprising” means “including.” Variations of the word "comprising", such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a composition “comprising” X may consist exclusively of X or may include one or more additional unrecited components.

[0082] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0083] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0084] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0085] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0086] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0087] Other embodiments are within the following claims and non-limiting examples.EXAMPLES

[0088] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the disclosure.

[0089] Example 1- Methods

[0090] Animal protocols

[0091] Animal procedures were performed in accordance with the Singapore National Advisory Committee for Laboratory Animal Research guidelines. All mice were housed in animal care facilities and studied using protocols approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore. The methods used in the study conformed to the Guidelines on the Care and Use of Animals for Scientific Purposes (NACLAR, Singapore, 2004) as well as the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011). Male C57BL / 6JINV (Jax) mice were used in this study. A power calculation was performed to estimate sample size required to demonstrate significant improvement (error alpha 0.05, power 0.8). Details of virus injection is performed as previously described

[0014] , For heart harvesting, each mouse was euthanized by cervical dislocation under deep anaesthesia with 5% isoflurane and the heart was exposed by opening of the chest. 15% KC1 was then injected into the inferior vena cava to achieve asystole at diastole, and the heart was rapidly isolated. Half of the apex was isolated and immersed it in RNALater (Qiagen, 76104) for RNA extraction. The other half of the apex was snap frozen in liquid nitrogen for protein extraction. For histology experiments, the remaining section of the heart was fixed in 4% paraformaldehyde for 24 hours and subsequently embedded in paraffin. A dose of 2.0EI3 vg / kg was injection for Lnrna DCM and 1 0E13 vg / kg was injected for candidates.

[0092] Echocardiogram (Echo)

[0093] To measure the cardiac dimensions and function of mice after virus transduction, echocardiography was conducted by using a 40 MHz-550S probe on the VisualSonics Vevo 2100 machine. During the echo and analysis, the researchers were blinded to the animal group allocation and identities. To obtain accurate measurements, LV tracings were averaged from at least three consecutive heartbeats of M-mode, and LVDD (LV diastolic dimensions), LVWT (LV posterior wall thickness), EF (ejection fraction), and FS (fractional shortening) were obtained from short axis images.

[0094] Cell culture and transfection

[0095] Transfection of shRNA constructs and other plasmids were done in HEK293T cells (ATCC CRL-1573™, RRID: CVCL 0063). Two transfection methods, PEI (Polysciences. Inc, 24765-2) and Lipofectamine 3000 (Invitrogen L3000015), were used to transfect the cells based on the instructions provided by the manufacturers.

[0096] Recombinant adeno-associated virus (rAAV) production, purification and titration, transduction of mice using rAA V

[0097] To produce rAAVs, HEK293T cells were transiently triple transfected with rAAV viral vector with gene, helper plasmid pAdAF6, and plasmid pAAV2 / 9 (Penn Vector Core). The viruses were harvested three days post-transfection, and purified using Optiprep density gradient medium (Sigma, D-1556) before being stored at -80°C. To determine virus titration, a forward primer (gataaaagcagtctgggctttcaca) and a reverse primer (gagcccatataagcccaagctattg) were used to target the rAAV cTnT promoter region, and quantified by qPCR to determine physical titers. rAAV vectors were generated in HEK293T cells using a triple plasmid transfection system that included rAAV viral vector with gene to be delivered, helper plasmids pAdAF6 and plasmid pAAV2 / 9 (Penn Vector Core). The AAV constructs were propagated in Stbl3 competent cells (Thermo Fisher Scientific). After three days, viruses were harvested from cell pellets and purified using Optiprep density gradient medium (Sigma). Centrifugal filters (Milipore) were used to concentrate the virus, which was then aliquoted and stored at -80 °C. To determine the physical titer, qPCR was performed using primers targeting the rAAV genome-containing particle and the cTnT promoter region.

[0098] To transduce mice with the rAAVs, institutional guidelines for cryoanesthesia and secure pups was followed An insulin syringe with 50 pl of AAV at a dose of 1.0 or 2.0 x 1013vg / kg or other doses specified in the results was prepared. The needle was positioned at the xiphoid process and the skin and diaphragm was punctured at a 40-45° angle, avoiding vital organs. AAV was slowly injected into the thoracic cavity.

[0099] Vector construction

[0100] For shRNA candidates, primers designed to target 21 base-pair gene-specific regions (Invitrogen) were inserted at the miR-155 backbone of the AAN-cTnT-EGFP vector. The sequences of shRNAs are as follows:

[0101] For vectors used in gain of function studies, EGFP in AAV -cTnT-EGFP vector was replaced by Serca, Fgfl 6, Bmp7, Yyl, Lamin A or C (RNAi resistant forms with three silent mutations at the target region). AAV-cTnT-SFlag-AFAPf S127A (aYAPl) was from Addgene (Plasmid #86558, RRID: Addgene_86558). The cloning primers are as follows:

[0102] RNAseq Library Preparation and Next Generation Sequencing

[0103] To establish RNAseq libraries, total RNA was extracted from apex of male mice (n = 3 per group). RNA library was prepared using NEBNext® Ultra™ II Directional RNA library prep kit for Illumina (NEB #E7760) according to NEB’s protocol. Libraries were sequenced using the Illumina NovaSeq 6000 sequencing system and paired-end 150 bp reads were generated for analysis. Raw sequencing reads were filtered to remove reads with adapter contamination, those with uncertain and low-quality nucleotides to obtain clean reads. RNAseq data were aligned to GRCm38 / mml0 reference genome by using HISAT2

[0024] , Following that, a reference-based approached was used to assemble the mapped reads of each sample using StringTie

[0025] , To count the reads numbers mapped to each gene, FeatureCounts

[0026] was used. The FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method was used to calculate the expression of each gene. Differentially expressed genes were selected on thebasis of adjusted p value (adjP). RNA-seq data was deposit (GSE240745). Potential genetic candidates (adjP < 0.001) were identified from Lmna DCM control group compared to Control, Lamin C or Lamin A treated group. TBtools was used togenerate the volcano plot

[0027] , The genes that were expressed differently were submitted to Morpheus, where they were subject to Hierarchical clustering and displayed in a heatmap that was color-coded. The Hallmark gene sets were evaluated using Gene Set Enrichment Analysis (GSEA, Broad Institute).

[0104] Quantitative real-time PCR (qPCR)

[0105] The levels of transcription were measured using qPCR. Following RNA extraction using Trizol, cDNA was synthesized using Pure-NA™ First Strand cDNA Synthesis Kit (Research Instrument, KR01-100). All qPCR was carried out by KAPA SYBR Fast qPCR Master Mix kit (KAPA Biosystems, KR0389) and relative expression levels were quantified using -ACT. Transcription data were normalized to Ctcf expression. All qPCR primers are listed as follows:SEQID NO:33,34SEQID NO:35,36SEQID NO:37,38SEQID NO: 39,40SEQID NO:41,42SEQID NO:43,44SEQID NO:45,46SEQID NO:47,48SEQID NO:49,50SEQID NO:51,52SEQID NO:53,54SEQID NO:55,56SEQID NO:57,58SEQID NO:59,60SEQID NO:61,62SEQID NO:63,64SEQID NO:65,66SEQID NO:67,68SEQID NO: 69, 70SEQID NO:71,72

[0106] Histological and immunostaining analysis

[0107] Heart sample collection and staining were as described previously

[0014] , Quantification of fibrosis was calculated as the red-stained areas relative to total ventricular area, using NIS-Elements software (Nikon). For antigen retrieval of aSMA,Ki67, Ibal, Yapl, Yyl, cTnl and pH2AX, samples were boiled in citrate buffer (pH 6.0). As for CD3, Sunl and Nesprinl, samples were boiled in EDTA (pH 9.0) for antigen retrieval. The following primary antibodies were used: aSMA (Sigma, A5228, RRID:AB_262054), Ki67 (Abeam, ab!5580, RRID:AB_443209), CD3 (Dako A045201, RRID:AB_2335677), Phospho-Histone H2A X (Seri 39) (Cell Signaling Technology, #9718, RRID:AB_2118009), Ibal (Wako Laboratory Chemicals (019-19741, RRID:AB_839504), Sunl (gift from Dr. Brian Burke), YAP1 (Cell Signaling Technology, #14074, RRID:AB_2650491), YY1 (Abeam, ab38422, RRID:AB_778962), Lamin A / C (Cell Signaling Technology, 2032, RRID: AB 10694918), Nesprinl (Abeam, abl92234, RRID_AB_2917992), PCM1 (Sigma, HPA023374), RRID: AB_1855073 and cTnl (Abeam, ab8295, RRID :AB_306445). DAPI was used for nuclear staining (ThermoFisher, DI 306, RRID: AB 2629482). To check for signal specificity, speciesspecific secondary antibody only controls were used. For each heart sample, investigators unaware of group identities counted the total number of positive signals from three crosssections, and this data was then standardized to the total number of nuclei or total ventricular area.

[0108] Western Blots

[0109] Heart tissue collection and western blot were performed as previously described

[0014] , The following primary antibodies were used: phospho-Smad2 (Ser465 / Ser467) (Cell Signaling Technology, 18338, RRID:AB_2798798), phospho- Smad3 (Abeam, ab52903, RRID:AB 882596), Smad 2 / 3 (Cell Signaling Technology, 5678, RRID :AB_10693547), phospho-p38 (Cell Signaling Technology, 9211, RRID:AB_331641), p38 (Cell Signaling Technology, 9212, RRID:AB_330713), phospho-p70S6K (Cell Signaling Technology, 9234, RRID:AB_2269803), p70S6K, (Cell Signaling Technology, 9209, RRID:AB_2269804), phospho-mTOR (Cell Signaling Technology, 5536, RR1D:AB_1O691552), mTOR (Cell Signaling Technology, 2972, RRID:AB_330978), Lamin A / C (Cell Signaling Technology, 2032, RRID:AB_10694918), Ctgf (Santa Cruz Biotechnology, Inc, sc-365970, RRID:AB_10917259), Yyl (Thermofsher, PA5-29171, RRID AB 778962), Bmp7 (Proteintech group, Inc, 12221-1-AP), YAP1 (Cell Signaling Technology, 14074, RRFD:AB_2650491), Sunl (Abeam, ab 103021, RRID: AB 2890137), Nesprinl (Abeam, ab!92234, RRID AB 2917992) and Lamin Bl (Abeam, ab!6048, RRID:AB_443298). The secondary antibody used were Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, HRP (Thermofisher, A16035, RRID: AB 2534709) and m-IgGK BP-HRP (Santa Cruz Biotechnology, sc-516102, RRID: AB 2687626). Protein levels on the blots were detected using the enhanced chemiluminescence system (GE Healthcare, RPN2106) according to the manufacturer’s instructions. Protein band intensity was quantified using Image J (NTH, 1.52e RRID:SCR_003070) and protein levels were normalized to Lamin Bl for mouse hearts.

[0110] Statistical Analyses

[0111] Prism 10.0.2 (GraphPad Software, La Jolla, California, RRID:SCR_002798) was used to conduct statistical analysis. Normality of sample distribution was assessed by the Shapiro-Wilk normality test. For data with two groups, a two-tailed, unpaired T- test with Welch correction was performed for data that followed a normal Gaussian distribution whereas Mann-Whitney test was performed for data that depart from normality. Brown-Forsythe and Welch ANOVA test with Dunnetfs T3 multiple comparisons were performed for multiple groups. Survival curve was generated according to the Kaplan-Meier method and P value was calculated using Log- rank (Mantel- Cox) test. Quantitative data were shown as mean ± SD. ns, non-significant.

[0112] Example 2- Results

[0113] Lmna gene products suppress Lmna DCM[001 14] To assess whether mna gene products suppress Lmna DCM, Lamin A was administered via rAAV9 upon the development of Lmna DCM mice. HF markers Nppa and Nppb, fibrosis markers Collal and Colla2, fibrosis as well as cardiac function were significantly improved by Lamin A compared to EGFP control (Figure la, b, Table 1). To further assess whether improvement of cardiac function can be translated into long term protection in Lmna DCM, survival analysis for Lamin A treatment was performed. Unexpectedly, the protective effect of Lamin A on Lmna DCM lapsed starting at 2 months (Figure 1c). Supplementation with Lamin C, the other product produced by the Lmna gene, preserved heart function, supressed HF / fibrosis marker genes, and imparted longterm survival of Lmna DCM mice (Figure Id-f, Table 1). The difference between Lamin A and Lamin C treatment was not due to excessive overexpression, as Lamin A and Lamin C dose and detection in cardiac tissue were comparable (Figure 7).

[0115] Further, to evaluate the long-term protective effects of Lamin C in LMNA DCM, survival analysis was conducted. As can be seen in Figure 22, mice administered with rAAV9 encoded Lamin C were evaluated for their survival rate. It was found thatsupplementation of Lamin C suppressed Lmna DCM and achieved long term survival of at least one year. Lamin C treatment significantly extended median survival rate to 21.3 months.

[0116] Table 1. Effect of potential candidates on Lmna DCM in mice Echocardiography of Lmna DCM mice with Lamin A (top panel) and Lamin C (bottom panel) upregulation at a dose of 1E+13 vg / kg assessed at 5.5 weeks. P value represents comparisons to Lmna DCM, Brown-Forsythe and Welch ANOVA test with Dunnetf s T3 correction. LVDD, left ventricular diastolic dimension; LVWT, LV wall thickness; EF, ejection fraction; FS, fractional shortening.

[0117] Difference between Lamin A and Lamin C for the treatment of Lmna DCM

[0118] To dissect the difference between Lamin A and Lamin C treatment, the transcriptional profiles of various groups including control, Lmna DCM, Lamin A treated, and Lamin C treated were compared by RNAseq Hierarchical clustering compared differentially expressed genes (DEGs, P < 0.001) among the different groups. The Lamin C treated group was closely clustered with the control group, in which up to 80% of DEGs in Lmna DCM were reversed by Lamin C (Figure 2a). In contrast, less than 40% of DEGs in Lmna DCM were reversed by Lamin A (Figure 2b). The list of upregulated DEGs that cannot be reversed by Lamin A was further analysed using GSEA (Figure 2c, d). Significantly upregulated gene sets identified “Epithelial Mesenchymal transition” as a top enriched set, which is associated with fibrosis. Consistent with this, cardiac fibrosis in Lmna DCM was not reduced to a similar level by Lamin A as compared to Lamin C (Figure la, d). Upregulated gene sets were also enriched in “Inflammatory response”,“Allograft rejection” and “TNFa Signaling ViaNFkB”, suggesting cardiac inflammation remains activated in Lamin A treated hearts.

[0119] Both Lamin A and its mature form induce DCM in wildtype mice

[0120] The impaired protection of Lamin A in Lmna DCM and detection of retained fibrosis and inflammatory gene expression by RNAseq analysis, hinted at a detrimental impact of Lamin A following its administration. The specific detrimental effect of Lamin A supplementation was tested by application of an equivalent Lamin A dose to wildtype mice. Although EF was not significantly altered, HF and fibrosis markers, as well as fibrosis were modestly upregulated by Lamin A (Table 2, Figure 8). Since AAV mediated gene delivery is among the most common tools in current gene therapy, it is crucial to dissect the mechanism of this AAV-Lamin A induced toxicity. An increased dose of Lamin A resulted in significantly impaired cardiac function, enlarged heart chamber of the left ventricle (LV) and reduced LV wall thickness coupled with interstitial fibrosis (Figure 3a, Table 3). HF markers Nppa and Nppb as well as fibrosis markers Collal and Colla2 were significantly upregulated by Lamin A compared to EGFP control (Figure 3b, c). Consistent with cardiac fibrosis, Lamin A transduced hearts showed a significant upregulation of p-Smad2 and the myofibroblast marker aSMA (Figure 3d, e). Additionally, Lamin A induced cardiac inflammation is indicated by increased presence of infiltrating Tba-1+ macrophage and CD3+ T cells (Figure 3f, g) Taken together, these results demonstrate that supplementation of Lamin A by AAV is detrimental and induces DCM in a dose dependent manner (designated as Lamin A DCM). It was noted that accumulation of prelamin A was observed in Lmna transduced mice, suggesting that prelamin A is not properly processed upon upregulation of Lamin A by AAV (Figure 3d). It was hypothesised that this accumulation of prelamin A in CMs could drive detrimental heart function.

[0121] Table 2. Effect of potential candidates in wildtype mice. Echocardiography of potential candidates Lamin A at a dose of 1E+13 vg / kg assessed at 5.5 weeks. P value represents comparisons to EGFP control, two-tailed, unpaired T-test with Welch correction and Mann-Whitney test.

[0122] Table 3. Effect of Lamin A on wildtype mice. Effect of Lamin A at a dose of 2E+13 vg / kg assessed by echocardiography at 5.5 weeks. P value represents comparisons to EGFP control, two-tailed, unpaired T-test with Welch correction.

[0123] Lamin A, initially expressed as prelamin A, undergoes farnesylation and cleavage by Zmpste24 to generate mature Lamin A [28, 29], The complete processing of famesylated prelamin A to mature Lamin A is crucial as the accumulation of farnesyl- prelamin A is toxic. Importantly, mice expressing prelamin A only in the absence of Lamin C developed DCM, suggesting that prelamin A accumulation and fameysylation contribute to cardiomyopathy development

[0030] , To test this possibility, an AAV vector expressing a mature form of Lamin A was constructed, bypassing prelamin A synthesis and farnesylation steps. Transduction of mature Lamin A also led to impaired cardiac contraction, enlarged LV chamber size, reduced LV wall thickness and interstitial fibrosis (Figure 9a, Table 4). Furthermore, a significant increase in HF / fibrosis markers in the mature Lamin A DCM mice wase observed compared to controls (Figure 9b-e). Consistently, these mice showed significant upregulation of fibrotic mediators as well as cardiac inflammation markers (Figure 9f-g). Therefore, upregulation of either pre- or mature Lamin A induced DCM pathogenesis, suggesting that the detrimental effect is caused by the Lamin A product alone and not by impaired prelamin A processing.

[0124] Table 4. Effect of cardiac specific upregulation of mature Lamin A. Effect of mature Lamin A at a dose of 2E+13 vg / kg assessed by echocardiography at 5.5 weeks. P value represents comparisons to EGFP control, two-tailed, unpaired T-test with Welch correction.

[0125] Lamin A / C preserves the integrity of the nucleus and DNA. DNA damage is commonly observed in EMNA DCM and Lmtici animal models [31-34], To further dissect the molecular mechanisms of Lamin A upregulation, DNA damage marker yH2AX in the hearts of Lamin A DCM was examined. Interestingly, yH2AX was significantlyincreased in the CMs of Lamin A DCM, indicating that Lamin A upregulation induces DNA breaks in mouse hearts and contributes to the detrimental effects of Lamin A on heart function (Figure 10).

[0126] Selection of potential candidates for the treatment of Lmna DCM

[0127] LMNA gene replacement is able to treat LMNA DCM caused by haploinsufficiency through compensation for the loss of functional LMNA. However, LMNA DCM caused by dominant negative mutations are not amenable to gene replacement. To evaluate other potential candidates, the generated RNAseq data for Lmna DCM were analysed. Significant dysregulated genes were detected by Gene Set Enrichment Analysis (GSEA, Broad Institute) (Figure 4a, b). Dysregulated gene sets identified hallmark signature associated signaling pathways including KRas, Tgfb and mTord , many of which have been shown to be deregulated in various LMNA related models. To validate these pathways in Lmna DCM, the key factors in these pathways in heart tissues were examined by Western blot. Consistent with previous studies, it was observed that Tgfp and mTor signaling pathways were dysregulated in Lmna DCM [11, 35] (Figure 4c). However, the p-p38 / Mapk pathway did not change significantly. Based on the pathway analysis / validation as well as previous studies, candidates including Tgfbl, Smad2, Smad3 for Tgf signaling pathway, Raptor for mTor signaling pathway, L'gfl6 and Mapkl4 for Mapk signaling pathway, Serca2 for calcium handling and Sunl for the linker of nucleoskeleton and cytoskeleton (LINC) were selected to be evaluated. The recently studied genes such as Bmp7, Ctgf, Yyl, Yapl were also included in the comparison [14, 36] (Figure 4d). Their gene expression in heart tissue of Lmna DCM were further examined. The expression of Tgfbl, Fgfl6, Bmp7, Ctgf, Serca2, Smad2, Raptor and Yapl were dysregulated in Lmna DCM Other signalling downstream molecules or transcriptional regulators such as Smad3, Mapkl4, Yyl and Sunl, were not transcriptionally regulated in Lmna DCM (Figure 11).

[0128] Positive candidates extend survival of Lmna DCM

[0129] To evaluate the functions of these selected candidates in Lmna DCM, their expression by rAAV9 was modulated. The knockdown efficacy in vitro was assessed by a 2-color system developed previously

[0037] (Figure 12). At least 75 percent knockdown was achieved for loss-of-function candidates. All candidates were assessed in Lmna DCM. The effects of each candidate on cardiac function were compared using echocardiography (Figure 5a, Table 5). These candidates alone did not affect cardiacfunction and survival of wildtype mice in the observed time window (Table 6, Figure 13). Among them, Smad3 shRNA, Yyl, combination of Bmp7 and Ctgf shRNA (Bmp7- Ctgf shRNA), trYAPl, Sunl shRNA, Lamin A, and Lamin C, can significantly improve the ejection fraction (EF) in Lmna DCM. Their upregulation or knockdown efficacy in vivo was validated by Western Blot (Figure 14). Selected candidates including Smad2, Tgfbl, Mapkl4, Smad3, Yyl, Yapl and Sunl were included to assess the effect of dosage on gene expression. Echocardiography results showed that increased dosage of the selected candidates did not change their effect on cardiac function (Table 7). In addition, the knockdown and overexpression efficacy of Sunl, Yapl and Yyl at different dosages was examined by immunostaining (Figures 15 and 16) To further assess whether improvement of cardiac function can be translated into long term protection in Lmna DCM, survival analysis for all positive candidates was performed (Figure 5b). Two negative candidate shRNAs Tgfbl and Mapkl4 were included for comparison. Consistently, knockdown of these two negative candidates did not prolong survival for Lmna DCM. Although cardiac EF in Lmna DCM was significantly improved to ~ 35% upon Smad3 shRNA treatment, the median survival was not significantly extended, suggesting improved cardiac function does not always extend to prolonged animal survival. Yyl, uYAPl or Bmp7-Clgf shRNA extended the median survival of Lmna DCM by a modest 15 - 30%. Strikingly, Sunl shRNA extended survival of Lmna DCM by at least 10-fold. Importantly, cardiac function in Lmna DCM was preserved by Sunl shRNA, similar to Lamin C (Figure 17). As a key component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, Sunl plays a pivotal role in linking the nuclear lamina to the cytoskeleton by bridging nuclear lamins with cytoskeletoninteracting KASH domain proteins [9].

[0130] Table 5. Effect of potential candidates on Lmna DCM in mice Echocardiography of Lmna DCM mice supplemented with control or potential candidates Sunl shRNA, Bmp7-Ctgf shRNA, Lamin C, aYAPl, Lamin A, Smad3 shRNA, Yyl, Ctgf shRNA, Bmp7, Fgfl6, Smad2 shRNA, Mapkl4 shRNA, Yapl shRNA, Tgfbl shRNA, Raptor shRNA or Serca2a at a dose of 1E+13 vg / kg assessed at 5.5 weeks. P value represents comparisons to Lmna DCM, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction and Kruskal- Wallis test. LVDD, left ventricular diastolic dimension; LVWT, LV wall thickness; EF, ejection fraction; FS, fractional shortening. *One mouse died before echocardiography.

[0131] Table 6. Effect of potential candidates in wildtype mice. Echocardiography of potential candidates Lamin C, Sunl, Bmp7-Ctgf shRNA, aYAPl, SmcidS shRNA, Yyl, Ctgf shRNA, Bmp7, Fgfl6, Smadl shRNA, Mapkl4 shRNA, Yapl shRNA, Tgfbl shRNA, Raptor shRNA or Serca2a or control at a dose of 1E+13 vg / kg assessed at 5.5 weeks. P value represents comparisons to control, Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 correction.

[0132] Table 7. Effect of selected candidates at higher dose on Lmna DCM mice. Echocardiography of Lmna DCM mice supplemented with control or candidates Smad2 shRNA, Yapl shRNA, Tgfbl shRNA, Mapkl4 shRNA, Smad3 shRNA, Sunl shRNA, Yyl, &YAP1 or control at a dose of 2E+13 vg / kg assessed at 5.5 weeks. P value represents comparisons to control, Brown -Forsythe and Welch ANOVA test with Dunnett’s T3 correction. *One mouse died before echocardiography.

[0133] To dissect the roles of Sunl in Lmna DCM, Sunl expression and protein levels were first assessed. No significant change of Sunl was detected in Lmna DCM compared to that in control mice (Figures 11, 15a and 18a). Similar to Lamin A / C, Sunl signal was detected around the nuclei in control cardiac cells (Figure 18b). Interestingly, the number of cardiomyocyte (CM) nuclei with abnormal Sunl distribution was significantly increased in Lmna DCM compared to those in the controls (Figure 18b), suggesting that Lmna knockdown perturbs Sunl distribution. Analysis of the nuclear shape of CM with abnormally distributed Sunl revealed a significant increase of nuclear protrusion when compared to CM with normal Sunl distribution (Figure 18c). Importantly, the number of nuclei with abnormal nuclear protrusion in Lmna DCM mice was significantly reduced by Sunl shRNA (Figure 18c), indicating that knockdown of Sunl preserved nuclear integrity of the CMs in Lmna DCM.

[0134] Nesprins interact with Sun! via their conserved K ASH domain

[0038] , To further elucidate the role of LINC complex in Lmna DCM, Nesprins were investigated and no significant changes in Synel (coding for Nesprin-1) expression and protein levels were found (Figure 19a and b). Sunl and Nesprin-1 were co-localized in the nucleus (Figure19c). Interestingly, a significant increase of abnormal Nesprin-1 distribution in CM nuclei in Lmna DCM compared to controls was observed (Figure 19d), indicating that Lamin A / C knockdown also induces abnormal distribution of Nesprin-1. Furthermore, the proportion of nuclear protrusion with abnormally distributed Nesprin-1 was significantly increased compared to those with normal Nesprin-1 distribution in Lmna DCM CMs (Figure 19d)

[0135] The inventors' earlier work suggested that mutating the KASH domain of Nesprin-1 could ameliorate Lmna DCM

[0039] , To perturb the interaction between Sunl and Nesprins in Lmna DCM mice, the KASH domain of Nesprin-1 was introduced to compete for binding with Sunl Interestingly, upregulation of the KASH domain suppressed Lmna DCM. The EF of Lmna DCM mice was significantly improved by KASH domain expression (Table 8). Furthermore, cardiac fibrosis was significantly reduced in Lmna DCM mice with KASH domain upregulation (Figure 20a, b). Consistently, a significant decrease in HF markers Nppa and Nppb as well as fibrosis markers Collal and Colla2 was observed with KASH treatment (Figure 20c). Taken together, these results indicate that the LINC complex is a key target for treatment of Lmna DCM.

[0136] Table 8. Effect of KASH Domain on Lmna DCM Effect of KASH domain at a dose of 1.0E+13 vg / kg on Lmna DCM mice at 5.5 weeks. P value represented comparisons Lmna DCM + Ctrl, Brown-Forsythe and Welch ANOVA test with Durmetf s T3 correction. LVDD, left ventricular diastolic dimension; LVWT, LV wall thickness; EF, ejection fraction; FS, fraction shortening.

[0137] Additional markers are required to evaluate candidates for the treatment of Lmna DCM.

[0138] For a comprehensive understanding of whether the candidates and markers were related to the treatment of Lmna DCM, EF was aligned with commonly used markers including Nppa and Nppb for HF markers, and Colal and Cola2 for fibrosis related markers (Figure 6a). Additionally, picrosirius red staining for fibrosis level,aSMA staining for myofibroblast activation, p-Smad2 level for the activation of Tgfp signaling pathway, Ibal and CD3 staining for the infiltration of macrophages and T cells, yH2AX staining for DNA damage, and Ki67 staining for cardiac cell proliferation were incorporated. Most markers except Ki67 were negatively correlated with cardiac function (Spearman’s correlation, -0.640 ~ -0.921, P < 0.05 ~ 0.001). HF and fibrosis markers as well as fibrosis level ranked closely with EF (Figure 6b). However, these commonly used markers in most HF studies were not sufficient to predict the long-term efficacy for the treatment. The lapses of protective effect of the candidates were associated with an increase of markers including yH2AX, Ibal and CD3. The correlation of the markers with animal survival for the treatment of Lmna DCM was further analyzed (Figure 6c) Similar to EF, most markers except Ki67 and p-Smad2 were negatively correlated to animal survival (Spearman’s correlation, -0.782 ~ -0.927, P < 0.05 ~ 0.001). In addition to HF and fibrosis markers, top ranking markers included immune (CD3) and DNA damage markers (yH2AX) which had a highly negative correlation with animal survival, suggesting that these additional markers are important to evaluate candidates for the treatment of Lmna DCM (Figure 6d).

[0139] Example 3- Discussion

[0140] Candidates related to key pathways in Lmna DCM including Tgip / Smad, Fgf / Mapk and mTor have been systematically evaluated. Among them, the Tgip / Smad signaling pathway is commonly activated in Lmna DCM as well as many other HF models

[0040] , Smad3 plays an essential role in heart remodelling in MI or TAC-induced heart failure models, although it plays different roles in CFs and CMs [41-43], Consistent with MI or TAC models, CM specific reduction of Smad3 ameliorated the impaired cardiac function in Lmna DCM mice. However, cardiac fibrosis, markers for HF / fibrosis and activation of myofibroblasts were only modestly reduced. Critically, this protective effect quickly lapsed, resulting in no significant improvement of survival of Lmna DCM mice. p38a MAP kinase and mTor signaling pathways are activated in / . / ; / / / i7H222|1 H’22l‘ or Lmna -I- mouse models. Inhibition of these pathways by p38a inhibitor ARRY-371797 or mTOR inhibitor rapamycin prevents cardiac dilation, improves cardiac function and / or modestly extends survival of diseased mice [11, 44, 45], However, knockdown of Mapkl4 (coding for p38a), Raptor (a key component for mTORCl signaling) or overexpression of Fg[16 (a downstream gene of Gata4) specifically in CMs did not significantly suppress Lmna DCM, suggesting their protective roles might be contributedby non-CMs or are specific to certain disease models

[0046] , Moreover, modulation of both Bmp7 and Ctgf, the secreted factors regulated by Yyl, improved cardiac function and extended survival by ~ 30%. However, Lmna DCM mice died quickly in a short time window after treatment, suggesting modulation of signaling pathways has a broad but modest therapeutic benefit for Lnma DCM Similarly, modulation of calcium handling and proliferation related genes has a limited beneficial role in Lnrna DCM.

[0141] Lamin A, Lamin C and the LINC complex component Sunl were evaluated as therapeutic candidates for Lnrna DCM. These three proteins are co-localized and interact in the nuclear envelope. Lamin A and Lamin C are the products of the Lmna gene via alternative splicing. Lamin A / C forms a matrix to preserve the strength and integrity of CM nuclei. Although Lamin A has a unique C-terminal modification and maturation process compared to Lamin C, Lamin A and Lamin C appear to be functionally equal and interchangeable because Lamin A or Lamin C only knockout mice are normal in general [47, 48], Prelamin A only transgenic mice instead succumbed to DCM

[0030] , Consistent with Lamin C only transgenic mice, supplementation of Lamin C suppressed Lmna DCM and achieved long term survival of at least one year. Although Lamin A supplementation also prolonged survival, this protection lapsed, with median survival limited to 110 days. Dose-dependent Lamin A toxicity was identified as the novel pathological mediator of this effect. This is inconsistent with previous results that transgenic mice expressing Lamin A alone in the absence of Lamin C had no disease phenotypes

[0048] , One possible explanation for this discrepancy is that rAAV9 mediated upregulation of transgenes is acute and rapid in comparison to classic transgenic techniques. In addition, it is speculated that there is a need to maintain a specific ratio between Lamin A and Lamin C levels in the CMs, given that endogenous Lamin C is the dominant form in CM compared to Lamin A

[0014] , It is possible that Lamin A upregulation, resulting in an increased Lamin A / C ratio, led to detrimental effects. Hence, the necessity to ensure a careful balance of cellular Lamin A levels has important implications for LMNA DCM gene therapy approaches. It is noted that this is consistent with a reported higher frequency of misshapen nuclei in mouse embryonic fibroblasts of Lamin A only mice

[0048] , Lamin A upregulation induced DNA damage, however, the exact mechanisms of how Lamin A upregulation resulted in DCM is yet unknown and warrants further study. Sunl interacts with Lamin A / C, linking nucleoskeleton to cytoskeleton via KASH domain proteins such as Nesprin-1 . Perturbing this interaction a. Sunl shRNA or KASH overexpression significantly suppressed LmnaDCM and achieved long term survival, which is consistent with a protective role of dominant negative Sunl in a DCM model induced by cardiac specific Lmna knockout [7], Sunl- / - or mutating the KASH domain of Nesprin-1 also ameliorates Lnma null and progeroid Lnma^9mutant mice, suggesting that Sun 1 / Nesprin-l -containing LINC complexes can serve as a therapeutic target for other Lnma related diseases [39, 49, 50] A comparable beneficial effect between Sunl knockdown and Lamin C gene replacement for Lnma DCM advances a potential of translational research for LMNA DCM treatment. In addition, this finding implies that perturbing the LINC complex can achieve a similar efficacy as gene replacement for Lmna DCM, which can be potentially extended to the treatment for LMNA DCM caused by different pathological variants. To date, more than 400 LMNA mutations have been described (http : / Avw w ,umd. be / LMN A / ) and among them, 165 unique mutations distributed along the entire LMNA gene have been linked to cardiomyopathy

[0051] , Missense mutations in LMNA have been proposed to act mainly through a dominant negative pathway while truncating variants likely result in haploinsufficiency [52, 53], Only a handful of mutations are unique to Lamin A, the majority of which are located on the C terminus of prelamin A, a hotspot for progeria where DCM mutations are rare [51, 52, 54] (Figure 21). Hence, Lamin C gene replacement is suitable for the treatment of LMNA DCM with haploinsufficiency mechanism through compensation for the loss of functional Lamin A / C in which the majority of mutations affect both Lamin A and C. On the other hand, the rescue role of Sunl knockdown or KASH overexpression will extend to LMNA DCM caused by dominant negative mutations.

[0142] Various pathological markers were compared to heart function and animal survival. Heart function was negatively correlated with the expression of HF / fibrosis markers as well as fibrosis which are utilised commonly in most HF related research. Importantly, tests for B-type natriuretic peptide (BNP, coded by NPPB) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are extensively utilized as diagnostic and prognosis biomarkers for HF in clinical settings due to their highly sensitivity and specificity. However, these common markers alone cannot predict the long-term efficacy of treatments for Lmna DCM. Activation of resident or infiltration of blood-derived immune cells are involved in pathological inflammatory pathways and tissue reparative processes in HF [55-57], Upregulation of inflammatory cytokines contributes to the pathogenesis of LMNA -cardiomyopathy patients and affects the severity of cardiacphenotype

[0058] , Reduced immune cell numbers correlated with long term animal survival following treatment by Sunl shRNA or Lamin C supplementation in Lmna DCM. Whether other types of immune cells, resident or infiltrated are also related to heart function and animal survival requires further investigation. DNA damage was observed in CMs of Lmna DCM as well as LMNA mutant models Suppression of DNA damage was also highly correlated with animal survival, suggesting that maintenance of DNA / nuclei integrity is important for long term efficacy of Lmna DCM therapy.

[0143] In this study, AAV9 as well as a CM specific promoter were used to modulate gene expression specifically in CMs. Besides cardiac tropism, AAV9 has a broad tropism to other organs including neuron, liver and lung

[0059] , AAV9’s liver tropism also facilitates CRISPR-based gene therapy for liver targets related to cardiovascular disease including atherosclerotic cardiovascular disease (PCSK9)

[0060] However, AAV9 has limited access to cardiac fibroblasts, smooth muscle cells, and endothelial cells which impedes application to non-CMs in hearts. Recently AAV9 capsid modification enhanced potency for skeletal muscle which could extend applications to muscular disease such as Duchenne muscular dystrophy

[0061] , Since the present Lmna DCM model is also cardiac specific, the evaluation of selected candidates specifically in CMs highlights a potential of advancing positive candidates to the treatment of cardiac specific LMNA DCM

[0062] , Whether these candidates also benefit other LMNA related disease requires further validation.

[0144] It is found that Lamin C and the LINC complex are promising gene therapy targets for LMNA DCM.

[0145] Here, 14 potential therapeutic candidates in Lmna DCM were evaluated. Heart function, animal survival and pathological cardiac markers were analyzed and compared, revealing Lamin C and Sunl as viable therapeutic targets, and an unforeseen risk of Lamin A toxicity. This study further provides a simple platform that can be adapted to evaluate and compare other potential candidates for LMNA DCM.

[0146] This study demonstrates that gene replacement or LINC complex perturbation has a profound and beneficial impact on LMNA DCM, providing a solid foundation for the therapy development. These findings suggest that AAV-mediated gene therapy could be a potential option for the treatment of LMNA DCM.

[0147] List of abbreviationsLMNA Lamin A / C geneHF Heart failure DCM Dilated cardiomyopathy rAAV Recombinant Adeno-associated virus SMA Spinal muscular atrophy IACUC Institutional Animal care and Use Committee NACLAR Guidelines on the Care and Use of Animals for ScientificPurposesI.VDD Left ventricular diastolic dimensions LVWT Left ventricular wall thickness EF Ejection fraction FS Fractional shortening GSEA Gene Set Enrichment Analysis qPCR Quantitative real-time PCR iPSc Induced pluripotent stem cells LINC Linkers of nucleoskeleton and cytoskeleton CM Cardiomyocytes MI Myocardial infarction TAC Transverse aortic constriction TgfP Transforming growth factor beta Fgf Fibroblast growth factors Mapk Mitogen-activated protein kinases Yyl Yin Yang 1 Bmp7 Bone morphogenetic protein 7 Ctgf Connective tissue growth factor aYAPl Activated yes-associated protein 1 Sunl Sadi and UNC84 Domain containing 1 Pdgf Platelet-derived growth factor mTor Mammalian target of rapamycin BET Bromodomain and extraterminal MI 1C Major histocompatibility complex MYBPC3 Myosin binding protein C3 MYL2 Myosin light chain 2 KASH Klarsicht, ANC-1, Syne homology Nppa Natriuretic Peptide A Nppb Natriuretic Peptide B Myh7 Myosin heavy chain 7 Atp2a2 Sarcoplasmic / endoplasmic reticulum calcium ATPase 2(Serca2)Ctcf CCCTC-binding factor aSMA Actin alpha 2, smooth muscle CD3 Cluster of differentiation 3 Ibal Allograft inflammatory factor 1 (Aifl) PCM1 Pericentriolar material 1 yH2AX Gamma-H2A.X variant histone cTnl Cardiac Troponin I PCSk9 Proprotein convertase subtilisin / kexin type 9

[0148] Industrial Applicability

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Claims

Claims1. A method of treating LMNA Dilated Cardiomyopathy (DCM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a first recombinant Adeno-associated virus (rAAV) encoding Lamin C (rAAV- Lamin C) and / or a second rAAV encoding Lamin A (rAAV -Lamin A), wherein the rAAV-Lamin C comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, and the rAAV-Lamin A comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID N0:3.2 Use of a first rAAV encoding Lamin C (rAAV-Lamin C) and / or a second rAAV encoding Lamin A (rAAV-Lamin A) in the manufacture of a medicament for treating LMNA Dilated Cardiomyopathy (DCM) in a subject in need thereof, wherein the rAAV-Lamin C comprises a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, and the rAAV-Lamin A comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID N0:3.

3. The method of claim 1 or the use of claim 2, wherein the rAAV comprises a serotype selected from the group consisting of AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 1 1 , avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and a chimeric AAV vector.

4. The method of claim 1 or the use of claim 2, wherein rAAV-Lamin C is to be administered for a period up to 2 years using different serotypes of claim 3, and achieves long term survival of the subject of up to 2 years.5 The method of claim 1 or the use of claim 2, wherein rAAV-Lamin A is administered for a period up to 2 months using different serotypes of claim 3, and achieves survival of the subject up to 2 months.

6. The method or the use of claim 3, wherein the rAAV is rAAV type 9.

7. The method or the use of claim 3, wherein the rAAV comprises a cardiomyocyte (CM) specific promoter.

8. The method claim 1 or the use of claim 2, wherein the subject is human.

9. The method of any one of claims 1 and 3-8, or the use of any one of claims 2-8, wherein about 1.0E13 to 2.0E13 vector genomes per kilogram of body weight (vg / kg) rAAV- Lamin A are to be administered.

10. The method of any one of claims 1 and 3-8, or the use of any one of claims 2-8, wherein about 1.0E13 to 1.1E14 vg / kg rAAV-Lamin C are to be administered.

11. The method of any one of claims 1 and 3-10, or the use of any one of claims 2-10, wherein the rAAV-Lamin C and / or rAAV-Lamin A is administered in a single-dose.12 The method of any one of claims and 3-10, or the use of any one of claims 2-10, wherein the rAAV-Lamin C and / or rAAV-Lamin A is administered in multiple doses using different serotypes of claim 3.

13. The method of any one of claims 1 and 3-12, or the use of any one of claims 2-12, wherein the rAAV-Lamin C and / or rAAV-Lamin A are to be administered to the subject by intravenous injection14. The method of any one of claims 1 and 3-12, or the use of any one of claims 2- 12, wherein the rAAV-Lamin C and / or rAAV-Lamin A are to be administered to the heart of the subject.

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