COMPOUND DECREASING THE CONCENTRATION OF 2-HYDROXY-GLUTARATE

Abstract

The present invention relates to a compound decreasing the concentration of 2-hydroxy-glutarate (2HG) in a subject for use in treating, preventing, and/or preventing progression of cardiac remodeling, in particular cardiomyopathy and/or heart failure and to viral particles, compositions, uses and methods related thereto.

Claims

1-26. (canceled)

27. A method for treating, preventing, and/or preventing progression of cardiac remodeling, in particular cardiomyopathy and/or heart failure in a subject, comprising: (a) contacting the subject with a compound that decreases the concentration of 2-hydroxy-glutarate (2HG); and (b) thereby treating, preventing, and/or preventing progression of cardiac remodeling.

28. The method of claim 27, wherein the 2HG is L-2-hydroxy-glutarate (L2HG).

29. The method of claim 27, wherein the concentration is blood concentration and/or intracellular concentration in cells of the circulatory system.

30. The method of claim 27, wherein the concentration is in cells of the heart, preferably in cardiomyocytes.

31. The method of claim 27, wherein the compound is an enzyme catalyzing degradation of 2HG, preferably an 2HG-Dehydrogenase (2HGDH).

32. The method of claim 31, wherein the compound is an enzyme catalyzing degradation of L2HG, preferably an L2HG-Dehydrogenase (L2HGDH).

33. The method of claim 31, wherein the compound is an L2HGDH enzyme EC 1.1.99.2.

34. The method of claim 27, wherein the compound is a polynucleotide comprising an expressible sequence encoding a 2HGDH, preferably an L2HGDH.

35. The method of claim 27, wherein the compound is a polynucleotide comprising an expressible sequence encoding a 2HGDH, preferably an L2HGDH, comprised in a viral particle, preferably an adeno-associated virus (AAV) particle, more preferably in an AAV9 particle.

36. The method of claim 27, wherein the polynucleotide comprises (a) the nucleic acid sequence of SEQ ID NO:1 and/or 3 or a nucleic acid sequence at least 70% identical to at least one of SEQ ID NO:1 and/or 3; and/or (b) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and/or 4; or encoding a polypeptide comprising an amino acid sequence at least 70% identical to at least one of SEQ ID NO:2 and/or 4.

37. The method of claim 27, wherein the said polynucleotide comprises the nucleic acid sequence of SEQ ID NO:5 or a nucleic acid sequence at least 70% identical thereto; preferably comprises the nucleic acid sequence of SEQ ID NO:5, more preferably consists of the nucleic acid sequence of SEQ ID NO:5.

38. The method of claim 27, wherein the cardiac remodeling is caused by (i) arterial hypertension; (ii) congenital, age-related degenerative, or infection-related semilunar valve stenosis, in particular aortic valve stenosis; (iii) cardiomyopathy, in particular dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, left ventricular noncompaction, or restrictive cardiomyopathy; (iv) coronary heart disease; or (v) myocarditis.

39. The method of claim 27, wherein the subject is a mammal, preferably a human.

40. A viral particle comprising a polynucleotide comprising an expressible sequence encoding a 2HGDH, preferably an L2HGDH.

41. The viral particle of claim 40, wherein the viral particle is a virus-like particle.

42. The viral particle of claim 40, wherein the viral particle is an adeno-associated virus (AAV) particle, more preferably is an AAV9 particle.

43. The viral particle of claim 40, comprised in a pharmaceutical composition.

44. The viral particle according to claim 43, wherein the pharmaceutical composition is for use in treating, preventing, and/or preventing progression of cardiac remodeling, in particular cardiomyopathy and/or heart failure.

45. A kit comprising: (a) a polynucleotide comprising an expressible sequence encoding a 2HGDH, preferably an L2HGDH, and at least one viral packaging signal, and (b) a corresponding packaging helper polynucleotide and/or a corresponding packaging cell line.

Description

FIGURE LEGENDS

[0064] FIG. 1. (A) Gene expression analysis of L2HDGH in Dyrk1a over-expression transgenic mice model, Ang-II infusion mice model and TAC operated cardiac hypertrophy mice model. (B) Representative Western blot image of L2HGDH expression on protein level in heart homogenates of left ventricular 6 weeks subjected to sham/TAC and corresponding statistical quantification (C) (n=5-7 mice/group. *p<0.05; **p<0.01; ns=not significant).

[0065] FIG. 2. Contractile function alterations in response to TAC/Sham. (A) Time-course monitoring of systolic function including ejection fraction and fractional shortening by echocardiography. Left ventricular end-diastolic diameter and left ventricular mass were determined as a measure of hypertrophic response in the different treatment groups. *p<0.05, **p<0.01, ***p<0.001: Sham versus AAV9-LUC+TAC. #<0.05, ##p<0.01, ###p<0.001: Sham versus AAV9-L2HGDH+TAC. § <0.05, §§ p<0.01, § § § p<0.001: AAV9-LUC+TAC versus AAV9-L2HGDH+TAC. (B) Representative short axis M-mode echocardiographs from 4 weeks post-TAC/Sham mice of the three groups.

[0066] FIG. 3. Histological analysis of hypertrophy and fibrosis, fibrosis markers expression. Statistical analysis was performed with one-way ANOVA multiple comparison including a Student-Newman-Keuls' post hoc analysis. (A) Gross morphology of heart sections from the mentioned treatment groups and corresponding representative images of HE stainings. (B) Statistical quantification of relative cardiomyocyte area in the depicted groups. (C) Masson's Trichrome staining showing extracellular matrix deposion in perivascular and interstitial areas. (D) Statistical quantification of the percentage of fibrosis (blue area) of at least 10 randomly selected areas in sections of myocardium in the indicated treatment groups. Scale bar represents 20 μm. (E) Gene expression analysis of fibrosis markers collagen 3a1 and TGF-β in cardiac tissue. mRNA levels were normalized to RPL32 as a housekeeping gene (n=14/14/12 mice in sham/AAV9-LUC/AAV9-L2HGDH groups, *** p<0.001; &p<0.05 vs. Sham control; #p<0.05 vs. AAV9-LUC+TAC).

[0067] FIG. 4. Measurement of Mitochondrial Superoxide Levels (MSLs) with MitoSOX in NRVCM. (A) Representative immunofluorescence images of cells belonging to the mentioned groups. Red represents MitoSOX fluorescence. DAPI (blue) was used to mark cell nuclei. Scale bars represent 20 μm. (B) Statistical quantification of MFIs (p<0.001 for the virus treatment and Interaction). 6-8 images/slide, n=5-6 slide/group. (&p<0.001 vs. Ad-LacZ+H2O group; #p<0.001 vs. Ad-LacZ+ET-1 group).

[0068] FIG. 5. Cell surface area measurement in NRVCM. (A) Representative immunofluorescence images of alpha-actinin staining (red) in cells receiving the four mentioned treatments. DAPI (blue) was used as a nuclear marker. Scale bar represents 50 μm. (B) Statistical quantification of cell surface area (p<0.001 for the virus infection treatment, hypertrophic stimulation treatment and interaction). (10 images/slide, n=3 slides/group. &p<0.001 vs. Ad-LacZ+H2O group; #p<0.001 vs. Ad-LacZ+ET-1 group).

[0069] FIG. 6. Pro-hypertrophic effects of 2-HG. (A) Representative immunofluorescence images alpha-actinin staining (red) and DAPI (blue). Scale bar represents 50 μm. (B) Statistical quantification of ANP and BNP mRNA levels, using RPL32 as a housekeeping gene. (10 images/slide, n=3 slides/group, **p<0.01. (C) Statistical quantification of relative level of 5-hmC following 2-HG application.

[0070] FIG. 7. L2HGDH is overexpressed in the mitochondria following AAV6 transduction. (A) Representative image of Western blot experiments using total NRVCMs protein lysates. GAPDH was used as an internal control, proving equal protein loading. (B) Statistical quantification of L2HGDH protein level in NRVCMs. Values were normalized to control non-transduced cells. (C) Illustrative confocal images of L2HGDH immunocytochemistry and Mitotracker, specifically labelling mitochondria. Scale bar represents 100 μm. (20 images/experimental group, n=4, ***p<0.001).

[0071] FIG. 8. 2HG acts as a pro-hypertrophic marker, while L2HGDH overexpression rescues the observed phenotype. (A) Representative images of α-actinin immunocytochemistry (red) performed using NRVCMs subjected to the mentioned treatments. Scale bar represents 25 μm. (B) Statistical quantification of cell area as a marker of pro-hypertrophic effect of L-2HG. (C, D) Gene expression analysis of ANP and BNP as hypertrophic markers, analysed by RT qPCR. RPL32 was used as a housekeeping gene. (40 images/experimental group, n=4, * p<0.05 **p<0.01).

[0072] FIG. 9. L2HGDH ameliorates 2HG-induced mitochondria dysfunction in NRVCMs. (A) Representative images showing live cell monitoring of mitochondrial membrane potential (red) at the mentioned time points and (B) statistical quantification of red fluorescence intensity. (C) Illustrative confocal images of NRVCMs stained with JC-1 as a marker for mitochondria function. Red fluorescence depicts polarized mitochondria, while 2HG induced depolarization causes a shift to detectable green fluorescence. (D) statistical quantification of mean green/red fluorescence intensities in the depicted treatment groups.

[0073] FIG. 10. 2HG induces fibroblast proliferation and collagen deposition in vitro. (A) Representative Western blot image showing collagen3a1 expression in total lysate isolated from primary cardiac fibrolasts. beta-actin was used as a loading control, proving equal protein loading. (B) Statistical quantification of collagen3a1 protein level. Non-treated fibroblasts were used as control. (C) Illustrative images showing Ki67 expression in fibroblasts by immunocytochemistry. DAPI was used as a nuclear marker. Scale bar represents 50 μm. (D) Quantification of the percentage of Ki67-positive nuclei in immunocytochemistry images. (20 images/experimental group, n=4, **p<0.01)

[0074] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1: MATERIALS AND METHODS

Data Integration to a Multi-Omics Data Set and Filter Approaches for Potential Drug Target Identification

[0075] To identify relations between genes and metabolites, ANOVA results of metabolomics and transcriptomics data sets were integrated to a multi-omics data set. Metabolites were mapped to human proteins based on protein associations (comprising mainly enzymes, transporters, and receptors) downloaded from the Human Metabolome Database (HMDB, www.hmdb.ca). Subsequently, human proteins were mapped to mouse genes using HomoloGene (www.ncbi.nlm.nih.gov/homologene) group identifiers. This relationship of functionally related metabolite-gene pairs was filtered for interesting common regulations by application of two filters. Filter 1_up: Significant upregulation of mRNA in all three time points relative to sham control and significant change of related metabolite in all three time points relative to sham control. The direction of metabolite change was either up- or downregulated, however, with consistent direction in all three time points. Filter 1_down: Significant downregulation of mRNA in all three time points relative to sham control and significant change of related metabolite in all three time points relative to sham control. The direction of metabolite change was either up- or downregulated, however, with consistent direction in all three time points.

[0076] Upregulation means a significant (p<0.05) fold change relative to sham control >1.1 and downregulation a significant (p<0.05) change relative to sham control <0.9.

Vector Cloning

[0077] The murine L2HGDH cDNA was amplified using the following primers with a NheI restriction site of each (tcagtcgctagcgccaccGTGGAGGGAGGGGA (SEQ ID NO:18)) and tcagtcgctagcCCTCTGCCACTCATAAC SEQ ID NO:19). 100 ng of murine cDNA was used to amplify the L2HGDH sequence following restriction with NheI, then inserted into a single stranded AAV genome plasmid (pSSV9) that contains the cardiac specific and CMV-enhanced MLC1500 promoter (Müller et al. 2007). Luciferase was used as control gene.

Vector Production and Quantification

[0078] Vector production was carried out as published earlier using the two plasmid system (Werfel et al. 2014). In brief, the AAV genome plasmid pSSV9-CMV-MLC1500-mL2HGDH was co-transfected with pDP9rs, that encodes the adenoviral helper genes for the serotype 9. After 72 hours, the vectors were harvested and purified with a discontinuous iodixanol gradient via ultracentrifugation. Quantification was done by qPCR.

RNA Isolation, cDNA Synthesis and Relative Quantification qRT-PCR

[0079] RNA was extracted and purified from frozen mouse heart using Trizol reagent (QIAzol lysis reagent, Qiagen) following manufacturer's instructions, followed by DnaseI digestion. Nucleic acid yields were analyzed photometrically (NanoDrop ND-1000, Spectrophotometer). 1.5 μg RNA was reverse transcribed into cDNA using Superscript III Kit (Invitrogen) and oligo(dT) Primers. cDNA synthesis was followed by RNA digestion using RNase H. qRT-PCR was executed using iTaq Universal SYBR Green Supermix (Thermo Fisher Scientific) and CFX96 Touch Real-Time PCR detection system (Bio Rad) as previously described (Doehner 2014). The following primers used for cDNA relative quantification are listed below.

TABLE-US-00001 Gene Sequence SEQ ID NO: GAPDH forward:  6 5′-ATGTTCCAGTATGACTCCACTCACG-3′ reverse:  7 5′-GAAGACACCAGTAGACTCCACGACA-3′ ANP forward:  8 5′-ACCTGCTAGACCACCTGGAGGAG-3′ reverse:  9 5′-CCTTGGCTGTTATCTTCGGTACCGG-3′ β-MHC forward: 10 5′-TGCAAAGGCTCCAGGTCTGAGGGC-3′ reverse: 11 5′-GCCAACACCAACCTGTCCAAGTTC-3′ RCAN1 forward: 5′-GTTGGCTGGAAACAAGTAG-3′ 12 reverse: 5′-GGTCTCTTCATTCTCTCC-3′ 13 Col3 forward: 5′-TGGCCCAGCTGGTGACAAGG-3′ 14 reverse: 5′-CAGCAGGGCCCTTTCCTCCC-3′ 15 L2HGDH forward: 5′-AGGGAAAGGAGATTCGGTGT-3′ 16 reverse: 5′-GGGCGTAAAGTGAACTCCAA-3′ 17

[0080] Two technical replicates were considered for each reaction. GAPDH served as a housekeeping gene, and values were normalized to the sham group.

Protein Isolation and Western Blot Analysis

[0081] Protein was extracted from cardiac tissue using RIPA buffer containing a mixture of protease inhibitors, followed by Western blot analysis as previously described with slight alterations (Heckmann et al. 2016). A rabbit anti-L2HGDH antibody was used (LS-C165661-400, LS Bio, Washington, USA) and incubated at a dilution of 1:1000. Images were analysed using ImageLab (Bio-Rad, California, USA).

Histological Analysis

[0082] Heart tissue was fixed in 4% PFA overnight at 4° C. and embedded in paraffin. For cell surface area measurement, the sections were stained Hematoxylin and Eosin staining as published before (Fischer et al. 2008). For visualization of extracellular matrix deposition, sections were subjected to Masson's Trichrome staining as previously described (Bickelhaupt et al. 2017). Images were taken in random areas of the left ventricle using a bright field microscope (Leica DM500, Leica Microsystems, Mannheim, Germany). Cell surface area was analysed using Image J (NIH, Bethesda, Md., USA) and the average value of each group was calculated. Interstitial fibrosis and perivascular fibrosis were quantified using Image Dx (Reveal Biosciences, San Diego, Calif.).

Study Protocol and Animal Handling

[0083] 8 weeks old male wild-type mice (C57BL/6NCrl—Charles River) were randomly assigned to treatment (AAV9-L2HGDH, n=16) or vector control group (AAV9-LUC, n=19) with a dose of 10.sup.12 vector genomes/mouse (injected into the tail vein) for both groups. Another 15 mice served as sham control without any injection. Two weeks after injection, mice in the AAV9-L2HGDH and AAV9-LUC groups were subjected to TAC surgery (deAlmeida et al. 2010). A 27-gauge needle was used for inducing the stenosis. Successful ligation was confirmed by echocardiography measurements of the innominate artery/left common carotid artery flow velocity ratio during 48-72 hours after TAC surgery. Prior to TAC procedure as well as every two weeks after TAC, left ventricular function was monitored using echocardiography (Vevo2100 System), under anaesthesia. 6 weeks after TAC, mice were sacrificed and tissue samples were harvested. All clinical parameters were measured and analysed by blinded investigators. Mice were housed in pathogen-free conditions with controlled temperature and humidity and day/night rhythm of 12:12 hours. A complete diet of Rod 16-A (LASvendi, Soest, Germany) and water were served ad libitum. All animal procedures were administered according to the Directive 2010/63/EU of the European Parliament and the German animal protection code. Permission was approved by the Regierungspräsidium Karlsruhe, Germany (G122/12).

NRVCM Isolation and Treatments

[0084] NRVCMs were isolated and prepared as published previously (Rangrez et al. 2013). Briefly, left ventricles from 1-2 days old Wistar rats (Charles River) were harvested and cut in pH 7.4 buffer containing 120 mmol/liter NaCl, 20 mmol/liter Hepes, 8 mmol/liter NaH.sub.2PO.sub.4, 6 mmol/liter glucose, 5 mmol/liter KCl, and 0.8 mmol/liter MgSO.sub.4. The separation of single cardiomyocyte from cut tissue mass, was achieved by enzymatic digestion with 0.6 mg/ml pancreatin (Sigma) at 37° C. and 0.5 mg/ml collagenase type 11 (Worthington). Cell suspension was filtered by a cell strainer. Newborn calf serum was added to stop enzymatic digestion. In order to separate cardiomyocytes from fibroblasts, a Percoll gradient (GE Healthcare) centrifugation step was performed. Afterwards, cardiomyocytes were cultured in DMEM medium containing 10% fetal calf serum (FCS), 2 mM penicillin/streptomycin, and L-glutamine (PAA Laboratories).

[0085] NRVCMs were infected with 50 MOI (multiplicity of infection) adenovirus per cell in serum-free DMEM the next day after seeding. After 24 hours, the mediums were changed. ET-1 was applied to serum-free medium to a final concentration of 200 μmol/l for 24 hours.

[0086] L-α-Hydroxyglutaric acid disodium salt (2-HG, Sigma-Aldrich) was applied to serum-free medium to a concentration of 2 mM. Cells were harvested 2 days after treatment.

[0087] In additional experiments, NRVCMs were transduced with AAV6 vectors, either overexpressing L2HGDH or luciferase (Luc) as control at the M01=10.sup.5 vp/cell. Three days after transduction L-2HG (Sigma-Aldrich) was applied to cell culture medium to a final concentration of 2 mM. Cells were harvested 2 days after treatment.

Measurement of Mitochondrial ROS Production

[0088] MitoSOX was applied to culture medium to a final concentration of 0.5 μM and cells were incubated for 15 minutes at 37° C. Afterwards, cells were washed with PBS and fixed with PFA. The fluorescence (red) was detected (Ex: 400 nm, Em at 590 nm) using confocal microscopy (Zeiss LSM 800). Mean fluorescence intensity (MFI) was calculated from the total fluorescence intensity detected from viable cells and divided by the number of cells using the software generated by Image J software (NIH, Bethesda, Md., USA).

NRVCM Staining with Mitotracker

[0089] Mitotracker Orange (Thermo Fischer Scientific) was added to cell culture medium to a concentration of 25 nmol/L. Afterwards, cells were incubated at 37° C. for 30 min, washed with PBS and fixed with 4% PFA for 5 min.

Determination of Mitochondrial Membrane Potential

[0090] NRVCMs were maintained in μ-Slide 8-wells, (Ibidi), suitable for live cell imaging. Cells were incubated with tetramethylrhodamine (TMRE) at 37° C. to a concentration of 50 nmol/L for 30 min. Afterwards, cells were washed with warm PBS and media was replaced with FluoroBrite DMEM (Thermo Fischer Scientific). Cells were imaged within 30 min (Ex: 549; Em: 574 nm).

[0091] Similarly, JC-1 was applied to cell culture medium to a concentration of 1 μmol/L and cells were incubated at 37° C. for 30 min. After washing with warm PBS, cells were imaged in FluoroBrite DMEM (J-monomers Ex:485; Em:535/J-aggregates Ex: 530; Em:590) using a confocal microscope (LSM800, Zeiss). Mean fluorescence intensity was analysed using ImageJ.

In Vitro Cell Surface Area Measurement

[0092] Cardiomyocytes were fixed with 4% PFA and blocked in 2.5% BSA in PBS including 0.1% Triton. Next NRVCMs were incubated with monoclonal mouse anti-α-actinin (1:200 in 2.5% BSA in PBS; Sigma) antibody for 1 hour at room temperature. After washing with PBS, secondary antibody conjugated to Alexa Fluor-546 (Thermo Fisher Scientific) together with nuclear stain DAPI (Sigma-Aldrich) at a dilution of 1:200 or 1:1000 respectively in 2.5% BSA in PBS were applied and incubated for 1 h. Afterwards, coverslips were mounted using FluorPreserve reagent (Merck Millipore). Florescence graphs were collected with Keyence fluorescence microscope BZ-9000 at ×10 magnification (objective: CFI Plan Apochromat λ×10; Nikon) and BZ-II viewer software (Keyence, version 2.1). 10 pictures were taken from each coverslip. BZ-II Analyzer (version 2.1) were used to process and analyze the graphs. HybridCellCount module and fluorescence intensity singe-extraction mode were applied for cell size measurement.

Assessment of Hydroxymethylation Status of Cytosine

[0093] Epigenase 5mC-Hydroxylase TET Activity/Inhibition Assay Kit was employed to analyse Tet2 activity in nuclear extracts isolated from NRVCMs after 2-HG treatment. Cell fractionation was performed according to standard REAP protocol (Suzuki et al. 2010). The measurement of hydroxymethylcytosine (hmC) amount was performed according to manufacturer's instructions. 50 μg of nuclear protein was used per sample.

Statistical Analysis

[0094] All metabolite profiling data and mRNA data were log 10-transformed before further analysis to achieve an approximate normal distribution. Missing values were not imputed for univariate analysis. Univariate analysis was performed by ANOVA (analysis of variance) using R with package nlme and the following linear model with metabolite (or gene)˜treatment+timepoint+treatment:timepoint+body weight as fixed effects. ANOVA models were read out concerning t-statistics results comprising estimates, t-values, and p-values. Significance level was set to an alpha-error of 5%. The multiple test problem was addressed by calculating the false discovery rate (FDR) using the Benjamini & Hochberg method (Benjamini and Hochberg 1995).

[0095] For the in vivo gene transfer study, the statistical analysis was performed in Sigma Plot 5 software using a one-way ANOVA and Student-Newman-Keuls' post hoc analysis (comparison between more than two groups) or unpaired t-test with Welch's correction (between two groups). For in vitro study, two-way ANOVA was performed including a Turkey's post hoc analysis when significant interaction was noted. P-values less than 0.05 were considered significant.

EXAMPLE 2: RESULTS

Reduced L2HDGH Expression in Various Cardiac Hypertrophy and Heart Failure Mice Models

[0096] We first analysed the expression pattern of L2HDGH in various models of cardiac hypertrophy. Gene expression and protein analysis allowed us to confirm a stable L2HGDH downregulation in the analysed models as compared to controls. Notably, Dyrk1a TG mice model exhibited a 52% decrease in L2HGDH mRNA level, whereas Ang-II infusion mice had 35% down-regulation in L2HGDH mRNA level (FIG. 1A). In addition, we could find a significant decrease in L2GHDH mRNA and protein levels were found in TAC operated cardiac hypertrophy mice versus sham control (FIGS. 1A, 1B and 1C)

L2HGDH Over-Expression Alleviate Contractile Dysfunction after TAC

[0097] To analyse the effect of L2HGDH on heart function in a pressure-overload model of heart hypertrophy and failure in mice, the vector was injected 2 weeks prior to TAC. Follow-up echocardiography revealed significant improvement in systolic function (EF and FS) and myocardial hypertrophy (LV Mass) in the AAV9-L2HGDH group compared to AAV9-LUC group (FIG. 2). In contrast to mice receiving gene therapy, AAV9-LUC treated animals presented with marked myocardial dilatation proven by increased LVEDD at 4 and 6 weeks post-TAC, as depicted in FIG. 2A. FIG. 2B shows exemplary echocardiographic tracings.

L2HGDH Over-Expression Attenuates Pathological Remodeling after TAC

[0098] Quantification of cardiomyocyte surface area in hematoxylin/eosin (HE) staining of cardiac sections of mice subjected to TAC revealed a significant reduction in AAV9-L2HGDH treated mice compared to AAV-LUC controls (p<0.001) suggesting an attenuating effect on cardiac hypertrophy (FIGS. 3A and 3B), as observed in echocardiography.

[0099] In addition to myocyte hypertrophy, pressure overload induces intense remodeling, characterized by extracellular matrix deposition. Therefore, to assess whether L2HGDH overexpression has an effect in this context, we performed Masson's trichrome staining. As shown in FIGS. 3C and 3D, intense fibrosis was observed in hearts isolated from AAV9-LUC control mice, while AAV9-mediated L2HGDH over-expression prior to TAC significantly decreased the area of collagen deposition, both in perivascular (p<0.05) and interstitial areas (p<0.05). Additionally, fibrosis markers were analyzed on mRNA level in heart tissue. As expected, TAC resulted in a significant up-regulation of mRNA levels of transforming growth factor beta (TGF-beta) and collagen type III alpha 1 chain (COL3a1). On the other hand, when mice were injected with AAV9-L2HGDH prior to TAC, a significant decrease of TGF-beta and COL3a1 on mRNA level was detected (FIG. 3E) as compared to control AAV9-LUC injected mice.

L2HGDH Overexpression Decreases the Mitochondrial Reactive Oxygen Species Production

[0100] To uncover the molecular and functional mechanisms controlling L2HGDH-mediated protection, we performed in vitro experiments using primary neonatal rat ventricular cardiomyocytes (NRVCMs). As expected, ET-1 stimulation caused a significant increase in MitoSOX fluorescence level, corresponding to increased ROS production as compared to control. However, L2HGDH overexpression could normalize the generation of ROS, as pictured in FIG. 4.

L2HGDH Overexpression Decreases ET-1 Induced Cardiomyocyte Hypertrophy In Vitro

[0101] To confirm the effect of L2HGDH of cardiomyocyte hypertrophy in vitro, we analysed NRVCMs size following transduction and ET-1 stimulation. Indeed, cardiomyocytes treated with L2HGDH-overexpressing vector presented with marked reduction in hypertrophic growth, as compared to controls (FIG. 5).

2-HG Acts as a Pro-Hypertrophic Stimulus In Vitro and Increases the Amount of 5-Hydroxymethylcytosine (5-hmC)

[0102] To determine whether the aforementioned results are a direct effect of increased 2-HG production due to L2HGDH downregulation in hypertrophic conditions, we applied 2-HG directly to NRVCMs and analysed the effect on cell size. As shown in FIG. 6A, 2-HG addition to cell culture medium leg to a dramatic increase in cell size as compared to non-treated cells. Moreover, we could observe a marked increase in fetal gene program, represented by ANP and BNP (FIG. 6B). Finally, it is well known that pathological cardiac hypertrophy is characterized by epigenetic alterations such as a shift towards a neonatal 5-hydroxymethylcytosine (5-hmC) distribution pattern. 2-HG treatment resulted in increased epigenetic 5-hmC deposition (FIG. 6C), further supporting a causal role of 2-HG in pathological hypertrophy (Greco et al. 2016).

AAV6-Mediated L2HGDH Overexpression in Cardiomyocytes is Translocated into the Mitochondria

[0103] First, we aimed to determine the degree of L2HGDH overexpression in NRVCMs following AAV6 transduction. As shown in FIG. 7A, B, Western blot experiments prove an 8-fold increased L2HGDH protein level in lysates of primary cells transduced with AAV6-L2HGDH. Taking into account that L2HGDH is primarily located within the inner membrane of the mitochondria (Rzem et al. Proc Natl Acad Sci USA; 101:16849-54), we next performed colocalization studies of L2HGDH and Mitotracker. As proven in FIG. 7C, confocal images could clearly demonstrate that the overexpressed L2HGDH is located in the mitochondria after NRVCM transduction.

L2HGDH Overexpression Ameliorates 2HG Induced Cardiomyocyte Hypertrophy In Vitro

[0104] We next wanted to establish the role of 2HG in cardiac hypertrophy in vitro. 2HG application caused a marked induction of hypertrophy in NRVCMs, as proven by increased cell size and mRNA levels of well-established pro-hypertrophic markers (FIG. 8). On the other hand, transduction with L2HGDH-overexpressing AAV6 prior to metabolite addition to culture medium led to a marked decreased hypertrophic growth and reduced mRNA levels of ANP and BNP.

L2HGDH Overexpression Blunts Mitochondrial Dysfunction Caused by 2HG

[0105] Given that mitochondrial dysfunction is one of the main drivers of pathological hypertrophy (Zhou et al. J Clin Invest 128:3716-26), we further evaluated mitochondrial activity in NRVCMs following L-2HG treatment. As shown is FIG. 9 A, B, 2HG caused a time-dependent decrease in TMRE fluorescence, proving a likewise reduction in mitochondrial membrane potential. In stark contrast, L2HGDH-overexpressing NRVCMs presented with increased mitochondrial activity as compared to Luc transduced cells. To further corroborate these findings, we next performed JC-1 stainings and measured the ratio of green (depolarized mitochondria) to red (polarized mitochondria) mean fluorescence intensity. Our results demonstrate that 2HG treatment resulted in mitochondrial depolarization, while L2HGDH overexpression preserved its function (FIG. 9 C,D).

L-2HG Induces Fibroblast Proliferation and Collagen Synthesis

[0106] We further addressed whether L-2HG affects other resident cells in the myocardium. Fibroblasts respond to stress stimuli by increasing their rate of proliferation and synthesis of extracellular matrix, further leading to development of pathological fibrosis (Moore-Morris et al. J Mol Med 93:823-30). Our data underlines that L-2HG application to primary rat neonatal fibroblasts induces a significant increased collagen3a1 expression (FIG. 10A, B). Moreover, L-2HG treatment intensified fibroblasts proliferation, as proven by a dramatic increased Ki67-positive nuclei following treatment (FIG. 10C,D).

LITERATURE

[0107] 1. Ponikowski, P., et al., 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur J Heart Fail, 2016. 18(8): p. 891-975. [0108] 2. Goldberg L R. In the clinic. Heart failure. Ann Intern Med. 2010 Jun. 1; 152(11): ITC 61-15 [0109] 3. Müller, O J., et al., Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in cardiac hypertrophy and failure. Cardiovasc Res. 2018 Nov. 9. doi: 10.1093/cvr/cvy274. [Epub ahead of print] [0110] 4. Kranendijk, M., et al., Progress in understanding 2-hydroxyglutaric acidurias.

[0111] Journal of inherited metabolic disease, 2012. 35(4): p. 571-587. [0112] 5. Moroni, I., et al., L-2-hydroxyglutaric aciduria and brain malignant tumors A predisposing condition? Neurology, 2004. 62(10): p. 1882-1884. [0113] 6. Shim, E.-H., et al., L-2-Hydroxyglutarate: an epigenetic modifier and putative oncometabolite in renal cancer. Cancer discovery, 2014. 4(11): p. 1290-1298. [0114] 7. Latini, A., et al., Mitochondrial energy metabolism is markedly impaired by D-2-hydroxyglutaric acid in rat tissues. Molecular genetics and metabolism, 2005. 86(1): p. 188-199. [0115] 8. Fu, X., et al., 2-Hydroxyglutarate inhibits ATP synthase and mTOR signaling. Cell metabolism, 2015. 22(3): p. 508-515. [0116] 9. Intlekofer, A. M., et al., Hypoxia induces production of L-2-hydroxyglutarate. Cell metabolism, 2015. 22(2): p. 304-311. [0117] 10. Oldham W M, Clish C B, Yang Y, Loscalzo J. Hypoxia-Mediated Increases in L-2-hydroxyglutarate Coordinate the Metabolic Response to Reductive Stress. Cell Metab. 2015, 22(2):291-303.

[0118] 11. Bensaad, K. and A. L. Harris, Hypoxia and metabolism in cancer, in Tumor Microenvironment and Cellular Stress. 2014, Springer. p. 1-39. [0119] 12. Müller, O. J., et al., Targeting the heart with gene therapy-optimized gene delivery methods. Cardiovasc Res, 2007. 73(3): p. 453-62.

[0120] 13. Werfel, S., et al., Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase. Cardiovasc Res, 2014. 104(1): p. 15-23. [0121] 14. Heckmann M B, Bauer R, Jungmann A, Winter L, Rapti K, Strucksberg K-H, Clemen C S, Li Z, Schröder R, Katus H A, Müller OJ. AAV9-mediated gene transfer of desmin ameliorates cardiomyopathy in desmin-deficient mice. Gene Ther 2016; 23:673-679. [0122] 15. Fischer, A. H., et al., Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc, 2008. 2008: p. pdb prot4986. [0123] 16. Bickelhaupt, S., et al., Effects of CTGF Blockade on Attenuation and Reversal of Radiation-Induced Pulmonary Fibrosis. J Natl Cancer Inst, 2017. 109(8). [0124] 17. deAlmeida, A. C., et al., Transverse aortic constriction in mice. J Vis Exp, 2010(38). [0125] 18. Rangrez, A. Y., et al., Dysbindin is a potent inducer of RhoA-SRF-mediated cardiomyocyte hypertrophy. J Cell Biol, 2013. 203(4): p. 643-56. [0126] 19. Suzuki, K., et al., REAP: A two minute cell fractionation method. BMC Res Notes, 2010. 3: p. 294. [0127] 20. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Source J R Stat Soc Ser B 1995; 57:289-300. [0128] 21. Greco C M, Kunderfranco P, Rubino M, Larcher V, Carullo P, Anselmo A, Kurz K, Carell T, Angius A, Latronico M V, Papait R, Condorelli G. DNA hydroxymethylation controls cardiomyocyte gene expression in development and hypertrophy. Nat Commun. 2016; 7:12418. [0129] 22. Rzem R, Veiga-da-Cunha M, Noel G, et al.: A gene encoding a putative FAD-dependent L-2-hydroxyglutarate dehydrogenase is mutated in L-2-hydroxyglutaric aciduria. P Natl Acad Sci USA 2004; 101:16849-54. [0130] 23. Zhou B, Tian R: Mitochondrial dysfunction in pathophysiology of heart failure. J Clin Invest 2018; 128:3716-26. [0131] 24. Moore-Morris T, Guimaraes-Camboa N, Yutzey K E, Puceat M, Evans S M: Cardiac fibroblasts: from development to heart failure. J Mol Med 2015; 93:823-30. [0132] 25. Ban et al.: A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-InducibleFactor-1 by Regulating Mitochondrial Respiration, PLoS One, doi:10.1371/journal.pone.0162568 (2016) [0133] 26. Shelar et al.: Biochemical and Epigenetic Insights into L-2-Hydroxyglutarate, a Potential Therapeutic Target in Renal Cancer, Clin Cancer Res 24(24):6433; doi:10.1158/1078-0432. CCR-18-1727 (2018)