LONG NON-CODING RNA AS THERAPEUTIC TARGET IN CARDIAC DISORDERS AND CARDIAC REGENERATION

Abstract

The present invention relates to a long non-coding RNA as a therapeutic target in cardiac disorders.

Claims

1. A nucleic acid molecule comprising (i) the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5 or a functional fragment thereof, wherein a functional fragment of SEQ ID NO: 1, 2, 3, 4 or 5 increases viability and/or proliferation of cardiomyocytes, (ii) a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleic acid molecule of (i), wherein the percentage of identity is determined over the whole length of SEQ ID NO: 1, 2, 3, 4 or 5, or (iii) a nucleotide sequence which is complementary to the nucleotide sequence of the nucleic acid molecule of (i) or (ii) over the whole length of the two sequences, for use in a therapeutic or diagnostic method applied to the human or animal body.

2. The nucleic acid molecule of claim 1 comprising (i) the nucleotide sequence of SEQ ID NO: 1 or a functional fragment thereof, (ii) a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleic acid molecule of (i), or (iii) a nucleotide sequence which is complementary to the nucleotide sequence of the nucleic acid molecule of (i) or (ii)

3. The nucleic acid molecule of claim 1, which is an RNA molecule, optionally comprising at least one modified nucleotide building block.

4. The nucleic acid molecule of claim 1, which is a DNA molecule, optionally in operative linkage with an expression control sequence and optionally present on a vector, e.g. a plasmid or a viral vector.

5. The nucleic acid molecule of claim 1, which is conjugated to a heterologous moiety.

6. A genome editing composition which is adapted for activating endogenous expression of a nucleic acid molecule of SEQ ID NO: 1, 2, 3, 4 or 5 or a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5 wherein the percentage of identity is determined over the whole length of SEQ ID NO: 1, 2, 3, 4 or 5, in a eukaryotic cell or organism, particularly a mammalian cell or organism, more particularly in a human cell or organism.

7. The genome editing composition of claim 6, which comprises (i) a genome-editing enzyme such as a CRISPR/Cas enzyme, e.g. a CRISPR/Cas 9 or 13 enzyme, a transcription activator-like effector-based nuclease (TALEN), a zinc finger nuclease protein, a recombinase, a meganuclease, an Argonaute protein or (ii) a nucleic acid molecule coding for a genome-editing enzyme.

8. A pharmaceutical preparation comprising a nucleic acid molecule of claim 1 or a genome editing composition which is adapted for activating endogenous expression of a nucleic acid molecule of SEQ ID NO: 1, 2, 3, 4 or 5 or a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5 wherein the percentage of identity is determined over the whole length of SEQ ID NO: 1, 2, 3, 4 or 5, in a eukaryotic cell or organism, and a pharmaceutically acceptable carrier.

9. The pharmaceutical preparation of claim 8 wherein said pharmaceutically acceptable carrier is suitable for use in human medicine.

10. A method for treating a cardiac disorder comprising administering an active agent comprising (a) the nucleic acid molecule of claim 1, (b) a genome editing composition which is adapted for activating endogenous expression of a nucleic acid molecule of SEQ ID NO: 1, 2, 3, 4 or 5 or a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5 wherein the percentage of identity is determined over the whole length of SEQ ID NO: 1, 2, 3, 4 or 5, in a eukaryotic cell or organism, or (c) a pharmaceutical preparation comprising (a) or (b), to a patient in need of such treatment.

11. A method for the treatment of contractile dysfunction, cardiac decompensation or heart failure, and/or for use in cardioprotection or cardioregeneration, comprising administering (a) a nucleic acid molecule of claim 1, (b) a genome editing composition which is adapted for activating endogenous expression of a nucleic acid molecule of SEQ ID NO: 1, 2, 3, 4 or 5 or a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5 wherein the percentage of identity is determined over the whole length of SEQ ID NO: 1, 2, 3, 4 or 5, in a eukaryotic cell or organism, or (c) a pharmaceutical preparation comprising (a) or (b) to a patient in need of such treatment.

12. The method according to claim 10, wherein said patient is selected from the group consisting of: (i) patients having an increased risk for developing heart failure, (ii) patients suffering from (congestive) heart failure, e.g. patients having an increased risk of heart failure progression; (iii) post-myocardial infarction patients, (iv) patients with congenital heart diseases associated to cardiac hypertrophy, such as pulmonal vein stenosis, atrial or ventricular septum defects, and (v) patients suffering from hypertrophic cardiomyopathy.

13. The method according to claim 10, wherein said active agent is administered as a monotherapy or in combination with a further medicament selected from the group consisting of angiotensin-modulating agents, β-blockers, diuretics, aldosterone antagonists, vasodilators, ionotropic agents, and combinations thereof.

14. A cell, organ or a non-human organism transfected or transformed with a nucleic acid molecule of claim 1 or a genome editing composition which is adapted for activating endogenous expression of a nucleic acid molecule of SEQ ID NO: 1, 2, 3, 4 or 5 or a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5 wherein the percentage of identity is determined over the whole length of SEQ ID NO: 1, 2, 3, 4 or 5, in a eukaryotic cell or organism.

15. A cell, organ or a non-human organism having an increased endogenous expression of a nucleic acid molecule of SEQ ID NO: 1, 2, 3, 4 or 5 or of a nucleotide sequence which has an identity of at least about 70%, at least about 80%, at least about 90% or at least about 99% to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5 wherein the percentage of identity is determined over the whole length of SEQ ID NO: 1, 2, 3, 4 or 5, compared to a wild-type cell, organ or organism.

16. A method of detecting, diagnosing or monitoring a cardiac disorder comprising detecting a nucleic acid molecule of claim 1 (i) or (ii).

17. A reagent for detecting, diagnosing or monitoring a cardiac disorder comprising a reagent or reagent combination for detecting a nucleic acid molecule of claim 1 (i) or (ii).

18. The method according to claim 10, wherein said cardiac disorder is a cardiac hypertrophy-associated disorder.

19. The method according to claim 13, wherein the further medicament is entresto.

Description

EXAMPLES

Example 1

Identification of Foxo6os IncRNA as a Therapeutic Target

[0086] Using next generation sequencing technology for a genome-wide IncRNA screen in the developing mouse heart identified Foxo6os, a poly(A)+IncRNA, as a potential therapeutic target. The results are shown in FIG. 1:

[0087] (A) Pipeline to select candidates from RNA-seq data. (B) Genomic locus of Foxo6os in mouse (GRCm38/mm10) represented in UCSC Genome Browser. (C) Sequence conservation and coding potential of Foxo6os was analyzed by (C) PhyloCSF and (D) coding potential calculator 2(CPC2). Note that the positive peak only appears in Foxo6 but not in Foxo6os. For CPC2, Hotair, Xist and Gapdh were calculated as positive controls. (E) The cDNA sequence of Foxo6os was analyzed by NCBI ORF finder, and the predicted amino acid translated from the largest open reading frame (ORF) in Foxo6os was shown in (F) (blue box, ORF 12). (G) Subcellular distribution of Foxo6os revealed cytoplasmic localization in cardiomyocyte-like HL-1 cells. Neat1 and Hprt expression levels were measured as positive controls. (H) Cell-specific markers for each cell type from heart fractionation were validated by qPCR to determine the cell purity. Myh6 is the marker for cardiomyocyte (CM), Fsp1 is the marker for cardiac fibroblast (CF) and Pecam1 is the marker for endothelial cells (EC). (I) PolyA tail was detected in Foxo6os with Oligotex polyA tail kit and validated by qPCR. Data are mean ±SEM (N≥3 independent experiments). ***P<0.005 as calculated from student's t-test.

Example 2

Expression of Foxo6os IncRNA in mice under physiological and disease conditions.

[0088] Foxo6os expression was analyzed in mouse hearts. It was found that the expression increased under physiological conditions but decreased under disease conditions. The results are shown in FIG. 2:

[0089] (A) Volcano plot of differentially regulated IncRNAs from RNA-seq data of postnatal day 1 vs day 7 mouse hearts. Foxo6os is indicated in red. FC: fold change. (B) Validation of Foxo6os expression by qPCR in mouse organ panels (N=5). (C) Expression profile of Foxo6os was confirmed in different cell types isolated from mouse heart via qPCR, including cardiomyocyte (CM), cardiac fibroblast (CF) and endothelial cells (EC). (D) Validation of Foxo6os expression by qPCR from the mouse heart (N=5) of different ages as indicated. P: postnatal day. (E) Expression level of Foxo6os in aging mouse hearts (2 years old) and young mouse hearts (11 weeks old) (N=4 or more). The expression of Foxo6os was further measured in (F) cardiomyocyte isolated from 8 weeks TAC heart, and (G) transverse aortic constriction (TAC) mouse whole hearts (N=6) for different weeks as indicated. In addition to TAC disease model, the expression of Foxo6os was also measured in (H) doxorubicin (doxo, 5 mg/kg)-treated hearts (N=9) for 5 weeks and (I) ischemia-reperfusion injury (IRI, ischemia for 60 min and reperfusion for 24 h) heart (N=5 or more). Data are mean ±SEM (N 3 independent experiments). *P<0.05; **P<0.01; ***P<0.005 as calculated from student's t-test.

Example 3

Inhibition of Foxo6os Expression in Cardiomyocytes

[0090] It was found that inhibition of Foxo6os expression in cardiomyocytes by an siRNA leads to an apoptosis phenotype. The results are shown in FIG. 3:

[0091] (A) Two siRNAs against Foxo6os were designed and the knockdown efficiency was tested in HL-1 by qPCR. The si-Foxo6os-1 was later used for all the inhibition experiments. (B) HL-1 viability was measured by WST-1 assay after treated with si-Scramble or si-Foxo6os. (C) The expression of α-/β-MHC were measured by qPCR after HL-1 treated with si-Scramble or si-Foxo6os, and the ratio of α-/β-MHC was plotted. (D) GSEA analysis and heatmap from the HL-1 RNA-seq data of “si-Scramble” compared to “si-Foxo6os”. (E) Treated si-Foxo6os-1/si-Scramble to neonatal mouse cardiomyocytes (NMCMs) and the expression of Foxo6os was measured by qPCR (F) Apoptosis rate of NMCMs after si-Foxo6os/si-Scramble treatment was measured by TUNEL assay. Green: TUNEL-488; red: cardiac troponin T; blue: DAPI. Data are mean ±SEM (N≥3 independent experiments). *P<0.05; **P<0.01; n.s.: not significant as calculated from student's t-test.

Example 4

Knockout of Foxo6os Expression in HL-1 Cells

[0092] Foxo6os expression in HL-1 cells were knocked out by CRISPR/Cas9. It was found that knockout of a Foxo6os expression impairs cell proliferation. The results are shown in FIG. 4:

[0093] (A) Scheme of Foxo6os exons, CRISPR/Cas9 sgRNAs and PCR primers for genotyping. The two sgRNAs work synergistically to delete the whole Foxo6os gene locus. P1 and P2 are primers for genotyping. (B) Deletion of Foxo6os locus was validated by electrophoresis of PCR products. M: marker, WT: wildtype, KO: knockout. (C) Sequencing chromatograms showed the mutant junction site (indicated by red dash line) of CRISPRed HL-1 cells gDNA. (D) The p-gal staining (blue) was colocalized with dTomato positive cells, which meant the cells were transfected by CRISPR/Cas9 plasmid. (D) The HL-1 cells were transfected with dTomato-CRISPR/Cas9 plasmid for 48 h and sorted by dTomato fluorescence, the dTomato positive and negative cells were then cultured for 2 weeks.

Example 5

Overexpression of Foxo6os in HL-1 Cells Via CRISPR/dCas Activation

[0094] Using the CRISPRa system, Foxo6os was overexpressed in HL-1 cells via CRISPR/dCas activation. The results are shown in FIG. 5:

[0095] Three different sgRNAs were used to activate Foxo6os expression and validated by (A) qPCR and (B) RNA FISH. Red: Foxo6os probe, Blue: nucleus (DAPI). Both of the (C) cell viability and (D) ratio of α/β-MHC are increased after overexpression of Foxo6os. (E) GSEA analysis and heatmap from the RNA-seq of “Foxo6os sgRNA 4-1 group” compared to “no sgRNA ctrl group”. (F) Important genes that selected from GSEA analysis were further validated by qPCR. Data are mean ±SEM (N≥3 independent experiments). *P<0.05; **<0.01; ***P<0.005 as calculated from student's t-test.

[0096] Overexpression of Foxo6os could also be carried out both endogenously and exogenously. The results are shown in FIG. 6:

[0097] (A) Scheme of how CRISPRa system works. Foxo6os was also successfully overexpressed by (B) transfection of in vitro transcripted (IVT) Foxo6os RNA into HL-1 cells and (C) adeno-associated virus serotype 6 (AAV6)-mediated overexpression in NMCMs.

Example 6

Identification of Human Transcript AC093151.3 as a Potential Locus-Conserved Transcript

[0098] Human transcript AC093151.3 was identified as a locus-conserved transcript for mouse Foxo6os. The results are shown in FIG. 7:

[0099] (A) Scheme of mouse chromosome 4 and human chromosome 1. The location of Foxo6os, its potential human homolog AC093151.3 and their neighboring genes were plotted. The graph was not plotted to scale. (B) The expression of four AC093151.3 transcripts was validated via qPCR in different human cell lines, hiPSCs, hiPSC-CMs, HCFs and HUVECs. The expression of AC093151.3 transcript 201 was further measured by qPCR in (C) hiPSC-CMs treated with and without ISO+PE treatment (100 pM) for 24 h or 72 h and (D) hiPSC-CMs under normoxia and 0.1% O.sub.2 hypoxia condition for 24 h to 72 h. Data are mean ±SEM (N 3 independent experiments). *P<0.05; **P<0.01; ***P<0.005 as calculated from student's t-test. hiPSC-CM: human induced pluripotent stem cell-derived cardiomyocyte, HCFs: human cardiac fibroblasts, HUVECs: human umbilical vein endothelial cells. ISO: isoproterenol, PE: phenylephrine.

[0100] Further, the human AC093151.3 transcript was confirmed via gel electrophoresis and sequencing. The results are shown in FIG. 8:

[0101] (A) Two different primers were designed to detect AC093151.3 transcript 201 in hiPSC-CMs and the size of PCR product was indicated. (B) The

[0102] AC093151.3 PCR product was extracted, sequenced and BLASTed in UCSC genome browser (shown as “Your Seq”).

Example 7

Transcription Factors of Foxo6os

[0103] Lists of potential transcription factors that bind to promoter of mouse Foxo6os and human AC093151.3 are shown in FIG. 9:

[0104] The sequence of mouse Foxo6os promoter and human AC093151.3 promoter (1 kp upstream of the transcription start site) were extracted from Ensembl and analyzed by JASPAR, among all of the 1011 TFs, 163 TFs were predicted for (A) mouse Foxo6os and 344 TFs were predicted for (B) human AC093151.3. Note that only the top 25 TFs were shown in the list and Mzf1 was indicated in red box. (C) Chromatin immunoprecipitation (ChIP) was performed against Mzf1 and control IgG followed by qPCR and then the qPCR product was validated by gel electrophoresis.

[0105] The transcription factor Mzf1 was shown to be essential for transcription of Foxo6os. The results are shown in FIG. 10:

[0106] (A) Predicted transcription factor Mzf1 binding site by JASPAR. (B) The expression of Foxo6os, Foxo6 and Mzf1 were measured by qPCR after treatment of siRNA against Mzf1 in HL-1 cells. (C) HL-1 viability was measured by WST-1 assay after treated with si-Scramble or si-Mzf1. (D) Chromatin immunoprecipitation (ChIP) was performed against Mzf1 and control IgG followed by qPCR with primers specific to the Foxo6os promoter region in HL-1 cells. (E) RNA pulldown (N=6) and qPCR was performed in HL-1 to detect the protein candidates that potentially interact with Foxo6os. (F) The proteins/peptides that were detected by mass spectrometry was shown as dot plot. The red curve indicated the p value (<0.05) and q value (<0.05). Only the candidates that meet the selection criteria were further analyzed and the two green dots are Cirbp and Rbm3. (G) The 37 protein candidates were further analyzed by STRING and shown as protein-protein interaction network. Unless indicated individually, the data are mean ±SEM (N 3 independent experiments). *P<0.05; **P<0.01; ***P<0.005 as calculated from student's t-test.

Example 8

Foxo6os Knockout Mouse Model

[0107] A Foxo6os knockout mouse model was generated via Cre-LoxP recombination. The experimental protocol and the results are shown in FIG. 11:

[0108] (A) Scheme of Foxo6os gene locus and loxP site. Both exons of Foxo6os will be deleted after recombinase Cre induction. The graph was not plotted to scale. (B) Gel electrophoresis showed the different genotypes after PCR with primer set F1+R1 for Foxo6os floxed/Myh6-Cre mice. Upper panel (from left to right): homozygous floxed (product size: 655 bp), heterozygous floxed (product size: 527/655 bp) and wild type (product size: 527 bp). Lower panel: genotype of Myh6-Cre (product size: 595 bp). (C) Gel electrophoresis showed the genotype for Foxo6os floxed/CMV-Cre mice. After CMV-Cre induction, the whole Foxo6os gene locus was deleted. The representative gel images shown here are heterozygous mice with wildtype allele (527 bp, primer F1+R1) and knockout allele (148 bp, primer F1+R2).

Example 9

Expression of Human Foxo6os Analog in Human Patients

[0109] Strikingly, the expression of AC093151.3 was also significantly reduced in heart samples obtained from patients with heart failure (HF) compared to healthy human heart tissue. The results are shown in FIG. 12. The expression of AC093151.3 transcript 201 was measured by qPCR in HF patients (N=11) vs. healthy control (N=10). FC: fold change. *P<0.05 as calculated from student's t-test.

Example 10

Modulation of Human Foxo6os Analog Expression in Human Cardiomyocytes

[0110] Similar to Foxo6os modulation in HL-1 cells, we modulated AC093151.3 expression by introducing siRNA and adeno-associated virus serotype 6 (AAV6) in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs).

[0111] FIG. 13A shows the expression level of AC093151.3 (left) and cell viability (right) in hiPSC-CMs treated with siRNA against AC093151.3 and 0.1% O.sub.2 hypoxic condition for 48 h. FIG. 13B shows the expression level of AC093151.3 (left) and cell viability (right) in hiPSC-CMs treated with AAV6 overexpressing AC093151.2 (1×10.sup.4 multiplicity of infection) and 0.1% 02 hypoxic condition for 48 h. FC: fold change. *P<0.05; **P <0.01; ***P<0.005 as calculated from two-way ANOVA.

[0112] The expression of AC093151.3 was significantly down-regulated after siRNA treatment (FIG. 13A, left) and up-regulated after AAV6 treatment (FIG. 13B, left). Notably, The AC093151.2 expression was even higher after overexpression under hypoxic condition, suggesting the potential beneficial effects of AC093151.3 in diseased state.

[0113] In line, our WST-1 results showed that the hiPSC-CM viability decreased to 80% after knockdown of AC093151.3 under normoxic condition and decreased to 65% under hypoxic condition (FIG. 13A, right). In contrast, the hiPSC-CM viability significantly increased from 61% to 73% after AAV6-mediated overexpression of AC093151.2 under hypoxic condition (FIG. 13B, right).

[0114] All the data above indicates that transcript AC093151.3 is indeed a functionally conserved transcript and could serve as a therapeutic target to rescue ischemic heart disease.

LIST OF REFERENCES

[0115] 1. Barry, S. P.; Townsend, P. A. (2010). What causes a broken heart-Molecular insights into heart failure. Int Rev Cell Mol Biol 284, 113-179. [0116] 2. Datta, S. R.; Brunet, A.; Greenberg, M. E. (1999). Cellular survival: a play in three Akts. Genes Dev. 13, 2905-2927. [0117] 3. DeBosch, B. J.; Muslin, A. J. (2008). Insulin signaling pathways and cardiac growth. J Mol Cell Cardiol. 44, 855-864. [0118] 4. Doppler, S.A.; Werner, A.; Barz, M.; Lahm, H.; Deutsch, M.-A., Dreβen, M.; Schiemann, M.; Voss, B.; Gregoire, S.; Kuppusamy, R.; Wu, S. M.; Lange, R.; Kranke, M. (2014). Myerloid Zinc Finger 1 (Mzf1) Differentially Modulates Murine Cardiogenesis by Interacting with an Nk×2.5 Cardiac Enhancer. PLoS ONE 9 12: e113775. [0119] 5. Frescas, D.; Valenti, L.; Accili, D. (2005). Nuclear trapping of the forkhead transcription factor FoxO1 via Sirt1-dependent deacetylation promotes expression of glucogenetic genes. J Biol Chem. 280, 20589-20595. [0120] 6. Glas, D. J. (2010). P13 kinase regulation of skeletal muscle hypertrophy and atrophy. Curr Top Microbiol Immunol. 346, 267-278. [0121] 7. Hill, J. A.; Olson, E. N (2008). Cardiac Plasticity. N Engl J Med. 358, 1370-1380. [0122] 8. McMullen, J. R.; Shioi, T.; Huang, W. Y.; Zhang, L.; Tarnayski, O.; Bisping, E.; Schinke, M.; Kong, S.; Sherwood, M. C.; Brown, J. et al. (2004). The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase (p110alpha) pathway. J Biol Chem. 279, 4782-4793. [0123] 9. Ni, Y. G.; Berenji, K.; Wang, N.; Oh, M.; Sachan, N.; Dey, A.; Cheng, J.; Lu, G.; Morris, D. J.; Castrillon, D. H. et al. (2006). Foxo transcription factors blunt cardiac hypertrophy by inhibiting calcineurin signaling. Circulation. 114, 1159-1168. [0124] 10. Ronnebaum, S. M.; Patterson, C. (2010). The foxO family in cardiac function and dysfunction. Annu Rev Physiol. 72, 81-94. [0125] 11. Skurk, C.; Izumiya, Y.; Maatz, H.; Razeghi, P.; Shiojima, I.; Sandri, M.; Sato, K.; Zeng, L.; Schiekofer, S.; Pimentel, D. et al. (2005). The FOXO3a transcription factor regulates cardiac myocyte size downstream of AKT signaling. J Biol Chem. 280, 20814-23. [0126] 12. Zhang, N.; Zhou, Y.; Yuan, Q.; Gao, Y.; Wang, Y.; Wang, X.; Cui, X.; Xu, P.; Ji, C.; Guo, X.; You, L.; Gu, N.; Zeng, Y. (2018). Dynamic transcriptome profile in db/db skeletal muscle reveal critical roles for long noncoding RNA. Int. J. Biochem. Cell Biol. 104, 14-24.