Bone marrow derived growth factor for use in the treatment or prevention of fibrosis, hypertrophy or heart failure - Patent Application 20070122997

Abstract

The present invention relates to the protein myeloid-derived growth factor (MYDGF), or a nucleic acid encoding said protein, for use in the treatment or prevention of fibrosis and hypertrophy. The present invention also relates to the protein MYDGF, or a nucleic acid encoding said protein, for use in the treatment of heart failure. The present invention also relates to vectors comprising said nucleic acids, host cells expressing said nucleic acids, and methods for use in the treatment of fibrosis and hypertrophy and for use in the treatment of heart failure.

Description

[Technical Field]

[0001] The present invention relates to the protein myeloid-derived growth factor (MYDGF) or a nucleic acid encoding said protein for use in the treatment or prevention of fibrosis and hypertrophy. The present invention also relates to the protein MYDGF or a nucleic acid encoding said protein for use in the treatment or prevention of heart failure. The present invention also relates to vectors comprising said nucleic acids, host cells expressing said nucleic acids, and methods for use in the treatment of fibrosis and hypertrophy, and for use in the treatment or prevention of heart failure. [Background technology]

[0002] Myeloid-derived growth factor (MYDGF), also known as Factor 1, is a protein encoded by open reading frame 10 (C19Orf10) on human chromosome 19. The protein was described in 2007 as a novel secreted factor in the synovium in a proteomic analysis of so-called fibroblast-like synoviocytes (FLS cells). A correlation between the secretion of the protein and inflammatory diseases of the joints was speculated without any experimental or statistical evidence (Weiler et al., Arthritis Research and Therapy 2007, "The identification and characterization of a novel protein, c19orf10, in the synovium"). A corresponding patent application claims the protein as a therapeutic agent for the treatment of joints and for the diagnosis and monitoring of tissues undergoing proliferative changes (U.S. Patent Application Publication No. 2008 / 0004232A1, "Characterization of cl9orf10, a novel synovial protein"). Another scientific publication describes increased expression of the protein in hepatocellular carcinoma cells (Sunagozaka et al., International Journal of Cancer, 2010, "Identification of a secretory protein c19orf10 activated in hepatocellular carcinoma"). Recombinantly produced protein demonstrated a growth-promoting effect on cultured hepatocellular carcinoma cells. It should be noted that C19Orf10 was originally thought to be an interleukin and is therefore also referred to as IL-25, IL-27, and IL-27W. However, the terms "IL-25" and "IL-27" are used inconsistently in the art and are used to refer to a variety of different proteins. For example, U.S. Patent Application Publication No. 2004 / 0185049 refers to a protein as IL-27 and discloses its use in modulating immune responses.This protein is structurally distinct from factor 1 (compare the amino acid sequence of factor 1 according to SEQ ID NO: 1 with the amino acid sequence of "IL-27" according to UniProt: Q8NEV9). Similarly, European Patent Application Publication No. 2130547A1 refers to a protein as IL-25 and discloses its use in the treatment of inflammation. This protein is also referred to in the art as IL-17E, and is structurally distinct from factor 1 (compare the amino acid sequence of factor 1 according to SEQ ID NO: 1 with the amino acid sequence of "IL-25" according to UniProt: Q9H293).

[0003] WO 2014 / 111458 discloses Factor 1 for use in enhancing proliferation and inhibiting apoptosis of non-transformed tissues or cells, particularly for use in the treatment of acute myocardial infarction. Also disclosed are inhibitors of Factor 1 for medical use, particularly for use in the treatment or prevention of diseases in which angiogenesis contributes to the development or progression of the disease.

[0004] Korf-Klingebiel et al. [Nature Medicine, 2015, Vol. 21(2):140-149] report that C19Orf10 is secreted by bone marrow cells after myocardial infarction, and that the protein promotes cardiomyocyte survival and angiogenesis. The authors show that bone marrow-derived monocytes and macrophages endogenously produce this protein to protect and repair the heart after myocardial infarction and propose that it be named bone marrow-derived growth factor (MYDGF). Specifically, they report treatment with recombinant Mydgf to reduce scar size and contractile dysfunction after myocardial infarction.

[0005] Heart failure (HF) is a clinical syndrome with a poor prognosis that can develop in response to persistent hemodynamic stress, myocardial injury, or genetic mutations. Chronic inflammation contributes to the pathogenesis and progression of HF and has emerged as a therapeutic target (Adamo et al. Nat Rev Cardiol. 2020;17:269-285). The relationship between HF and inflammation is bidirectional and involves crosstalk between the heart, immune system, and peripheral organs. In the myocardium, cytokines and growth factors derived from inflammatory cells act on cardiac parenchymal and interstitial cells, promoting systolic dysfunction and adverse left ventricular (LV) remodeling (Bozkurt et al. Circulation. 1998;97:1382-1391; Ismahil et al. Circ Res. 2014;114:266-282; Sager et al. Circ Res. 2016;119:853-864; Hulsmans et al. J Exp Med. 2018;215:423-440; Bajpai et al. Nat Med. 2018;24:1234-1245).

[0006] In mice, acute cardiac pressure overload imposed by transverse aortic constriction (TAC) surgery induces an inflammatory response involving the innate and adaptive immune systems (Martini et al. Circulation. 2019;140:2089-2107). Signals emanating from stressed but viable cardiomyocytes trigger an inflammatory cascade (Suetomi et al. Circulation. 2018;138:2530-2544). Within hours, cardiac expression levels of proinflammatory cytokines and chemokines increase (Baumgarten et al. Circulation. 2002;105:2192-2197; Xia et al. Histochem Cell Biol. 2009;131:471-481), and within a week, most major immune cell subsets expand in size and / or show signs of activation in the pressure-overloaded heart (Xia 2009; Liao et al. Proc Natl Acad Sci U S A. 2018;115:E4661-E4669; Patel et al. JACC Basic Transl Sci. 2018;3:230-244).

[0007] Fibrosis refers to the formation of excess fibrous connective tissue in an organ or tissue during a reparative or reactive process, e.g., reactive, benign, or pathological conditions. The connective tissue deposited during fibrosis can interfere with or inhibit the normal structure and function of the underlying organ or tissue.

[0008] Hypertrophy refers to an increase in the volume of an organ or tissue due to enlargement of its constituent cells. Summary of the Invention

[0009] There remains a need for means and methods for treating hypertrophy and fibrosis. There also remains a need for means and methods for treating heart failure.

[0010] In a first aspect, the present invention provides myeloid-derived growth factor (MYDGF) or a fragment or variant thereof which exhibits the biological function of MYDGF for use in the treatment or prevention of fibrosis or hypertrophy.

[0011] According to one embodiment, the MYDGF protein or a fragment or variant thereof exhibiting the biological function of MYDGF is for use in the treatment or prevention of heart failure. According to a preferred embodiment, the heart failure is chronic heart failure. According to a further embodiment, the heart failure or chronic heart failure is heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), or heart failure with mid-range ejection fraction (HFmrEF).

[0012] According to a preferred embodiment, the MYDGF protein comprises SEQ ID NO: 1. Alternatively, the MYDGF protein comprises a fragment or variant of SEQ ID NO: 1, which exhibits a biological function of MYDGF, wherein said variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0013] According to a preferred embodiment, the fibrosis is cardiac, renal, pulmonary and / or hepatic fibrosis. According to one embodiment, the fibrosis is interstitial lung disease, preferably progressive fibrosing interstitial lung disease, more preferably idiopathic pulmonary fibrosis.

[0014] According to a preferred embodiment, the hypertrophy is cardiomyocyte hypertrophy.

[0015] According to a further aspect, the present invention provides a nucleic acid encoding the growth factor protein MYDGF or a fragment or variant thereof which exhibits the biological function of MYDGF for use in the treatment or prevention of fibrosis or hypertrophy.

[0016] According to a further aspect, the present invention provides a nucleic acid encoding the growth factor protein MYDGF, or a fragment or variant thereof which exhibits the biological function of MYDGF, for use in the treatment of heart failure.

[0017] According to one embodiment, the nucleic acid encodes an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1.

[0018] According to a further aspect, the present invention provides a vector comprising a nucleic acid of the invention for use in the treatment or prevention of fibrosis or hypertrophy.

[0019] According to yet another aspect, the present invention provides a vector comprising a nucleic acid of the present invention for use in the treatment of heart failure.

[0020] According to a further aspect, the present invention provides a host cell comprising a nucleic acid of the invention or a vector of the invention for use in the treatment or prevention of fibrosis or hypertrophy. Preferably, said host cell expresses said nucleic acid.

[0021] According to yet another aspect, the present invention provides a pharmaceutical composition for use in the treatment or prevention of fibrosis or hypertrophy, comprising a MYDGF protein, nucleic acid, vector or host cell of the present invention, and optionally a suitable pharmaceutical excipient.

[0022] According to yet another aspect, the present invention provides a pharmaceutical composition for use in improving cardiac function, comprising a MYDGF protein, nucleic acid, vector or host cell of the present invention, and optionally a suitable pharmaceutical excipient.

[0023] According to a preferred embodiment, the pharmaceutical composition for use is administered by oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intracoronary routes, preferably by one or more bolus injections and / or infusion.

[0024] According to a further aspect, the present invention provides a method for treating fibrosis, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of MYDGF or a fragment or variant thereof that exhibits a biological function of MYDGF.

[0025] According to one embodiment, MYDGF comprises SEQ ID NO: 1 or a fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0026] According to one embodiment, the fibrosis is cardiac, renal, pulmonary and / or hepatic fibrosis.

[0027] According to yet another embodiment, MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier.

[0028] According to a further aspect, the present invention provides a method for treating hypertrophy, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of MYDGF or a fragment or variant thereof which exhibits the biological function of MYDGF.

[0029] According to one embodiment, MYDGF comprises SEQ ID NO: 1 or a fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0030] According to a preferred embodiment, the hypertrophy is cardiomyocyte hypertrophy.

[0031] According to yet another embodiment, MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier.

[0032] According to a further aspect, the present invention provides a method for treating or preventing heart failure, comprising administering a therapeutically effective amount of the growth factor MYDGF or a fragment or variant thereof exhibiting the biological function of MYDGF to a patient in need thereof. According to a preferred embodiment, the heart failure is chronic heart failure. According to a further preferred embodiment, the heart failure or chronic heart failure is HFpEF or HFrEF, preferably HFpEF is stage C or stage D HFpEF, or HFrEF is stage C or stage D HFrEF.

[0033] According to one embodiment, MYDGF comprises SEQ ID NO: 1 or a fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0034] According to yet another embodiment, MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0035] [Figure 1] Bone marrow-derived growth factor inhibits transforming growth factor-β1 (TGFβ1)-stimulated SMAD phosphorylation in lung fibroblasts from patients with idiopathic pulmonary fibrosis. Phosphorylation of SMAD2 (Ser465 / 467) and SMAD3 (Ser423 / 425) (normalized to α-tubulin expression) in lung fibroblasts from patients with idiopathic pulmonary fibrosis cultured in the absence or presence of TGFβ1 and / or MYDGF. [Figure 2]Bone marrow-derived growth factor inhibits transforming growth factor-β1 (TGFβ1)-stimulated SMAD phosphorylation in left ventricular fibroblasts from patients with end-stage heart failure. Phosphorylation of SMAD2 (Ser465 / 467) and SMAD3 (Ser423 / 425) [normalized to expression of non-phosphorylated SMAD2 / 3] in left ventricular fibroblasts from patients with end-stage heart failure cultured in the absence or presence of TGFβ1 and / or Mydgf. [Figure 3] Bone marrow-derived growth factor inhibits transforming growth factor-β1 (TGFβ1)-stimulated SMAD phosphorylation in left ventricular fibroblasts from patients with end-stage heart failure. Phosphorylation of SMAD2 (Ser465 / 467) and SMAD3 (Ser423 / 425) (normalized to the expression of non-phosphorylated SMAD2 / 3) in left ventricular fibroblasts from patients with end-stage heart failure cultured in the absence or presence of TGFβ1 and / or MYDGF. [Figure 4] Mouse bone marrow-derived growth factor inhibits transforming growth factor-β1 (Tgfβ1)-stimulated phosphorylation of Smads in mouse embryonic fibroblasts. [Figure 5]MYDGF attenuates left ventricular (LV) remodeling during pressure overload. (A) Mydgf wild-type (WT) and knockout (KO) mice were subjected to transverse aortic constriction (TAC) or sham surgery (day 7). LV mass to tibia length ratios were measured on representative longitudinal histological sections (day 7; scale bar, 1 mm) and summary data from 6–15 mice per group. ***P<0.001 (one-way ANOVA with Dunnett's post-hoc test) vs. sham of the same genotype; #P<0.05, ##P<0.01 (t-test for two independent samples). (B) Cross-sectional area of ​​LV cardiomyocytes. Representative histological sections stained with wheat germ agglutinin (WGA; scale bar, 50 μm) and summary data from 3–7 mice per group. *<0.05, ***P<0.001 vs. sham of the same genotype (one-way ANOVA with Dunnett's post-hoc test); #P<0.05, ###P<0.001 (t-test for two independent samples). (C) Size of isolated ventricular cardiomyocytes. Typical phase-contrast microscopy images (day 7; scale bar, 100 μm) and summary data from 3–8 mice per group. ***P<0.001 vs. sham of the same genotype (statistical test); #P<0.05 (statistical test). (D) Typical LV pressure-volume loops 7 days after sham or TAC surgery. [Figure 6]Bone marrow-derived MYDGF attenuates left ventricular (LV) remodeling. (A-E) Bone marrow cells (BMCs) from Mydgf wild-type (WT) or knockout (KO) mice were transplanted into (→) KO or WT recipients. After bone marrow reconstitution, mice underwent transverse aortic constriction (TAC) surgery and were followed for 14 days. *P<0.05, **P<0.01 (t-test for two independent samples). (A) LV mass to tibia length ratio. 7–18 mice per group. (B) LV cardiomyocyte cross-sectional area. 4–5 mice per group. (C) LV isolectin B4 (IB4)+ endothelial cell density. 4–6 mice per group. (D) LV end-diastolic area (LVEDA) and LV end-systolic area (LVESA) determined by echocardiography. 5–10 mice per group. LVEDA: P<0.05, WT right arrow KO vs. KO right arrow KO. LVESA: P<0.01, WT right arrow KO vs. KO right arrow KO (t-test for two independent samples). (E) Factorial area change (FAC). Same animals as in (D). Circles represent individual mice. Horizontal bars represent the mean. *P<0.05, **P<0.01. (F) Experimental strategy used in (G-N) to overexpress MYDGF in inflammatory cells by lentiviral gene transfer (HSC stands for hematopoietic stem cells). (G) Representative immunoblots (out of four) showing MYDGF and alpha-tubulin expression in BMCs and splenocytes from WT recipient mice 6 weeks after transplantation, treated with or without 1 week of doxycycline following transplantation of lentivirally transduced BMCs. (H) Plasma levels of MYDGF in WT recipient mice 6 weeks after transplantation, treated with or without 1 week of doxycycline following transplantation of lentivirally transduced BMCs. (I-M) All animals were treated with doxycycline. (I) Representative immunoblots showing MYDGF and GAPDH expression in LVs in WT recipient mice 6 weeks after transplantation, treated with or without 1 week of doxycycline following transplantation of lentivirally transduced BMCs.(J-L, and N) *P<0.05, **P<0.01, ***P<0.001; ##P<0.01, ###P<0.001 vs. mock with the same lentivirus (two-way ANOVA with Tukey's post-hoc test). (J) LV mass to tibia length ratio. 6 mice per group. (K) LV cardiomyocyte cross-sectional area. 5 mice per group. (L) IB4+ endothelial cell density. 6-7 mice per group. (M) LVEDA and LVESA. 6-8 mice per group. LVEDA and LVESA: P<0.001, Lenti.Control TAC vs. Lenti.Control Mock. LVEDA: P<0.05, TAC Lenti.MYDGF vs. TAC Lenti.Control. LVESA: P<0.01, TAC Lenti.MYDGF vs. TAC Lenti.Control. (N) FAC. Same animal as (M). [Figure 7] Cardiomyocyte hypertrophy. Neonatal rat ventricular cardiomyocytes were stimulated with endothelin 1 (ET1, 100 nmol / L), angiotensin II (AngII, 100 nmol / L), insulin-like growth factor (IGF, 50 ng / mL), and / or MYDGF (100 ng / mL unless otherwise stated) for 24 h. (A) Representative immunofluorescence microscopy images. Scale bar, 50 mm. Summary data from 4–6 experiments. (B) Dose-response curves. Data from 4 experiments. Half-maximal inhibitory concentrations (IC50s) were calculated by 4-parameter logistic regression. Controls represent the size of unstimulated cells. (C) Protein content. Data from 3 experiments. (D) mRNA expression levels of Myh7 (beta-myosin heavy chain), Nppa (natriuretic peptide type A), and Gapdh determined by RT-qPCR. Data from 7–13 experiments. *P<0.05, ***P<0.001; #P<0.05, ##P<0.01, ###P<0.001 vs. unstimulated control (one-way ANOVA with Tukey's post-hoc test). [Figure 8]Phosphoproteomic analysis identifies PIM1 as a target of MYDGF signaling. (A-D) Phosphoproteomic analysis of neonatal rat ventricular cardiomyocytes (NRCMs) stimulated with endothelin 1 (ET1, 100 nmol / L) and / or MYDGF (100 ng / mL) for 8 hours. (A) Flowchart illustrating a bottom-up approach to infer kinase activity from phosphoproteomic data and prior knowledge of kinase-substrate interactions. (B) Principal component analysis of the phosphoproteomic dataset (four biological replicates per condition). (C) Histogram illustrating phosphoproteomic changes. Red bars represent all phosphosites significantly regulated by ET1 compared to unstimulated controls (n = 120, P < 0.05, log2 fold change > 1). Blue bars indicate the regulation of these sites in cells stimulated with ET1 + MYDGF compared with cells stimulated with ET1 alone. (D) Substrate-based inference of kinase activity in cells stimulated with ET1 + MYDGF compared with cells stimulated with ET1 alone. (E) PIM1 protein expression and (F) kinase activity in NRCMs stimulated with ET1 and / or MYDGF for 16 hours. Six experiments. *P<0.05, **P<0.01, ***P<0.001 (t-test for two independent samples). (G) Size of NRCMs after 24 hours of stimulation with ET1, MYDGF, and / or SMI4a (10 μmol / L). Three experiments. *P<0.05, #P<0.05 vs. control (one-way ANOVA with Tukey's post-hoc test). (H) Size of NRCMs transfected with scrambled (SCR) or PIM1 small interfering (si)RNA and stimulated with ET1 and / or MYDGF for 24 hours. Representative immunoblots show PIM1 and beta-actin expression after siRNA transfection. Four experiments. ***P<0.001, ##P<0.01 vs. control (one-way ANOVA with Tukey's post-hoc test). [Figure 9]MYDGF upregulates SERCA2a expression via PIM1. (A) Representative immunoblots and summary data showing the expression of PIM1, sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), and beta-actin in neonatal rat ventricular cardiomyocytes (NRCMs) stimulated with MYDGF (100 ng / mL). Four to five experiments were performed. *P<0.05 vs. baseline (one-way ANOVA with Dunnett's post-hoc test). (B) Representative immunoblots (out of three) showing the expression of SERCA2a and beta-actin in NRCMs stimulated with MYDGF and / or SM14a (10 μmol / L) for 16 hours. Where indicated, cells were first transfected with scrambled (SCR) or PIM1 small interfering (si) RNA. (C) Representative immunoblot and summary data showing the expression of SERCA2a and vinculin in the left ventricle (LV) of Mydgf wild-type (WT) and knockout (KO) mice subjected to sham or transverse aortic constriction (TAC) surgery. Nine mice per group. ***P<0.001 vs. sham of the same genotype (one-way ANOVA with Dunnett's post-hoc test); #P<0.05 (t-test for two independent samples). (D) Representative immunoblot and summary data showing the expression of SERCA2a, PIM1, and alpha-tubulin in cardiomyocytes isolated from WT and KO mice 7 days after sham or TAC surgery. Five to six mice per group. *P<0.05, ***P<0.001 vs. sham of the same genotype; #P<0.05, ##P<0.01 (two-way ANOVA with Tukey's post-hoc test). (E) Bone marrow cells from WT or KO mice (right arrow) were transplanted into lethally irradiated KO or WT recipients. After bone marrow reconstitution, mice underwent TAC surgery and were followed for 14 days. Representative immunoblot and summary data showing LV SERCA2a, PIM1, and alpha-tubulin expression. Eight mice per group. *P<0.05 (t-test for two independent samples). (F) Representative immunoblot and summary data showing LV SERCA2a, PIM1, and beta-actin expression 7 days after TAC.Mice were transplanted with Lenti.control or Lenti.MYDGF-transduced bone marrow cells and treated with doxycycline starting 1 week before surgery. ***P<0.001 (t-test for two independent samples). [Figure 10]MYDGF protein treatment. (A) Treatment regimen. After transverse aortic constriction (TAC) surgery, mice received a bolus injection of recombinant MYDGF (10 μg) into the left ventricular (LV) lumen, followed by subcutaneous infusion (10 μg / day) for 3 (B), 7 (C), or 42 days (DI). TAC-operated control mice were treated with diluent alone (bolus injection and infusion). (B) Plasma levels of MYDGF. Five to seven mice per group. ***P<0.001 (test). (C) Representative immunoblot and summary data showing expression of sarco / endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and alpha-tubulin in the LV. Five to seven mice per group. *P<0.05 (t-test for two independent samples). (D) LV end-diastolic area (LVEDA) and LV end-systolic area (LVESA) determined by serial echocardiography at 7 and 42 days after TAC (16–22 mice per group) or 7 days after sham surgery (9 mice). LVEDA: P<0.01, TAC (control and MYDGF) vs. sham at 28 days. LVESA: P<0.01, TAC (control and MYDGF) vs. sham at 7 and 28 days (one-way ANOVA with Dunnett's post-hoc test). LVEDA: P<0.01, MYDGF vs. control at 28 days; LVESA: P<0.001, MYDGF vs. control at 28 days (t-test for two independent samples). (E) Percent area change (FAC). Same animals as in (C). ***P<0.001 for all TAC groups (one-way ANOVA with Dunnett's post-hoc test); ##P<0.01, ###P<0.001 KO vs. WT (t-test for two independent samples). (F) LV mass to tibia length ratio at 28 days. 6–12 mice per group. (G) LV cardiomyocyte cross-sectional area at 28 days. 6 mice per group. (H) Isolectin B4 (IB4)+ endothelial cell density in the left ventricle at 28 days. 6 mice per group. (E–G) *P<0.05, **P<0.01, ***P<0.001 vs. sham (one-way ANOVA with Dunnett's post-hoc test); #P<0.05, ###P<0.001 (t-test for two independent samples).(I) Cumulative survival after TAC in 27 control mice and 17 MYDGF-treated mice. *P=0.05 (log-rank test). DETAILED DESCRIPTION OF THE INVENTION

[0036] Before describing the present invention in detail below, it is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0037] definition Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0038] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2004). ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). In addition, conventional methods of clinical cardiology, similarly described in the literature in the field, are also used (see, e.g., Braunwald's Heart Disease. A Textbook of Cardiovascular Medicine, 9 th Edition, P. Libby et al. eds., Saunders Elsevier Philadelphia, 2011).

[0039] Unless the context requires otherwise, throughout this specification and the claims that follow, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of stated integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0040] A nucleic acid molecule is understood as a polymeric macromolecule made from nucleotide monomers. A nucleotide monomer is composed of a nucleic acid base, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed by phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0041] The term "open reading frame" (ORF) refers to a sequence of nucleotides that can be translated into amino acids. Typically, such an ORF contains an initiation codon followed by a region that is usually a multiple of three nucleotides in length, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) in a given reading frame. Typically, ORFs are naturally occurring or artificially constructed, i.e., by genetic engineering means. ORFs encode proteins, which can be translated into amino acids that form a peptide chain.

[0042] The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modification. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes, and protein domains) can be further modified by chemical modification. This means that such chemically modified polypeptides contain chemical groups other than the 20 naturally occurring amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of a polypeptide can result in one or more advantageous properties compared to the parent polypeptide, such as enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to variants usable in the present invention include, but are not limited to: pegylation, glycosylation of a non-glycosylated parent polypeptide, covalent attachment to a small molecule therapeutic agent such as a glucagon-like peptide 1 agonist, including exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretins, and liraglutide, or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications applicable to variants usable in the present invention can occur co-translationally or post-translationally.

[0043] The term "amino acid" encompasses naturally occurring amino acids as well as amino acid derivatives. A hydrophobic, non-aromatic amino acid in the context of the present invention is preferably any amino acid having a Kyte-Doolittle hydrophobic index greater than 0.5, more preferably greater than 1.0, even more preferably greater than 1.5, and which is not aromatic. Preferably, a hydrophobic, non-aromatic amino acid in the context of the present invention is selected from the group consisting of the amino acids alanine (Kyte Doolittle hydrophobic index 1.8), methionine (Kyte Doolittle hydrophobic index 1.9), isoleucine (Kyte Doolittle hydrophobic index 4.5), leucine (Kyte Doolittle hydrophobic index 3.8) and valine (Kyte Doolittle hydrophobic index 4.2), or derivatives thereof having the Kyte Doolittle hydrophobic index defined above.

[0044] As used herein, the term "variant" refers to a polypeptide that differs from the polypeptide or fragment thereof from which it is derived by one or more changes in its amino acid sequence. The polypeptide from which a protein variant is derived is also known as the parent polypeptide. Similarly, the fragment from which a protein fragment variant is derived is known as the parent fragment. Typically, variants are constructed artificially, preferably by genetic engineering. Typically, the parent polypeptide is a wild-type protein or a wild-type protein domain. Furthermore, variants usable in the present invention may be derived from a homolog, ortholog, or paralog of the parent polypeptide, or an artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent polypeptide. The amino acid sequence change may be an amino acid exchange, insertion, deletion, N-terminal truncation, C-terminal truncation, or any combination of these changes, and may occur at one or more sites. In a preferred embodiment, a variant usable in the present invention exhibits a total of up to 23 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) amino acid sequence changes (i.e., exchanges, insertions, deletions, N-terminal truncations, and / or C-terminal truncations). The amino acid exchanges may be conservative, and / or semi-conservative, and / or non-conservative. In a preferred embodiment, a variant usable in the present invention differs from the protein or domain from which it is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid exchanges, preferably conservative amino acid changes.

[0045] Typical substitutions are between aliphatic amino acids, between amino acids with aliphatic hydroxyl side chains, between amino acids with acidic residues, between amide derivatives, between amino acids with basic residues or between amino acids with aromatic residues. Typical semi-conservative and conservative substitutions are as follows:

[0046] [Table 1]

[0047] A change from A, F, H, I, L, M, P, V, W, or Y to C is semi-conservative if the new cysteine ​​remains a free thiol. Furthermore, one skilled in the art will understand that one should not substitute a glycine at a sterically demanding position and that one should not introduce P into a protein moiety with alpha-helical or beta-sheet structure.

[0048] Alternatively, or in addition, a "variant" as used herein can be characterized by a degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 85% sequence identity to its parent polypeptide. Preferably, the polypeptide of interest and the reference polypeptide exhibit the indicated sequence identity over a contiguous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids, or over the entire length of the reference polypeptide. Preferably, the polynucleotide of interest and the reference polynucleotide exhibit the indicated sequence identity over a contiguous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides, or over the entire length of the reference polypeptide.

[0049] The term "at least 85% sequence identity" is used throughout this specification in reference to sequence comparisons of polypeptides and polynucleotides. This expression preferably refers to at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the respective reference polypeptide or with the respective reference polynucleotide.

[0050] A fragment of a protein includes an amino acid deletion, which may be an N-terminal truncation, a C-terminal truncation, or an internal deletion, or any combination thereof. Such variants including N-terminal truncations, C-terminal truncations, and / or internal deletions are referred to as "fragments" in the context of the present application. Fragments may be naturally occurring (e.g., splice variants) or may be artificially constructed, preferably by genetic engineering means. Preferably, a fragment (or deletion variant) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids at its N-terminus and / or at its C-terminus and / or internally, preferably at its N-terminus, its N-terminus and C-terminus, or its C-terminus, compared to the parent polypeptide.

[0051] When comparing two sequences and no reference sequence is specified for comparison to calculate the percentage of sequence identity, the sequence identity is calculated with reference to the longer of the two sequences being compared, unless specifically indicated otherwise.

[0052] Nucleotide and amino acid sequence similarity, i.e., percentage sequence identity, can be determined by sequence alignment. Such alignments can be performed using several algorithms known in the art, preferably using the mathematical algorithm of Karlin and Altschul [Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877], using hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or using the CLUSTAL algorithm [Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80] or the CLUSTALW2 algorithm [Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948.], which are available, for example, at http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html.Preferably, the CLUSTALW2 algorithm at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html is used, with the parameters used being the default parameters set at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html as follows: for the slow pairwise alignment option, Alignment type=Slow, protein weight matrix=Gonnet, gap open=10, gap extension=0,1, and protein weight matrix=Gonnet, gap open=10, gap extension=0,20, gap distances=5, No end gaps=no, output options: format=Aln w / numbers, Order=aligned.

[0053] The grade of sequence identity (sequence matching) may be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST protein searches are performed, for example, using the BLASTP program available at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. The preferred algorithm parameters used are the default parameters set on http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome as follows: Expect threshold=10, word size=3, max matches in a query range=0, matrix=BLOSUM62, gap costs=Existence:11 Extension:1, compositional adjustments=conditional compositional score matrix adjustment, and non-redundant protein sequences (nr) are used as a database to obtain amino acid sequences homologous to the polypeptides of factor 1 and factor 2.

[0054] To obtain gapped alignments for comparison purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, is used. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Sequence matching analysis may be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62) or Markov Random Fields. When percentages of sequence identity are referred to in this application, these percentages are calculated for the full length of the longer sequence unless otherwise specifically indicated.

[0055] The term "host" as used herein refers to a cell into which a nucleic acid of the invention (e.g., in the form of a plasmid or virus) has been placed. Such a host cell may be either a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a fungal cell, a plant cell, or an animal cell). The cell may be transformed or untransformed. The cell may be an isolated cell, for example, in a cell culture, or part of a tissue, which may itself be isolated or part of a more complex organic structure such as an organ or an individual.

[0056] The terms "bone marrow-derived growth factor," "MYDGF," "factor 1," "MYDGF polypeptide or protein," or "factor 1 polypeptide or protein" are used interchangeably and refer to the protein set forth in NCBI reference sequence NM_019107.3 (human homolog) and its mammalian homologs, particularly homologs from mouse or rat. The amino acid sequence of the human homolog is encoded in open reading frame 10 on human chromosome 19 (C19Orf10). Preferably, MYDGF and factor 1 protein refer to a protein comprising, consisting essentially of, or consisting of the core segment of human factor 1 having the amino acid sequence according to SEQ ID NO:1.

[0057] Whether a protein, variant, or fragment exhibits a biological function of MYDGF can be determined by any one of the tests described in the Examples below. According to the present invention, a peptide or protein exhibits a biological function of MYDGF if the results obtained with such peptide or protein achieve at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the effect of MYDGF reported for the indicated control, when compared with the results obtained with a MYDGF protein of the invention, as shown in at least one Example presented herein below.

[0058] As used herein, the terms "MYDGF" and "Mydgf" both refer to bone marrow-derived growth factor, where "MYDGF" is used in the present invention to refer to the human variant of bone marrow-derived growth factor and "Mydgf" is used to refer to the mouse variant of bone marrow-derived growth factor.

[0059] The term "improvement of cardiac function" as used herein refers to, for example, the improvement of systolic and / or diastolic cardiac function, which can be evaluated by, for example, echocardiography, cardiac magnetic resonance imaging, cardiac computed tomography or ventricular angiography.For example, the increase in left ventricular size and improvement in cardiac systolic function are indicators of the improvement of cardiac function, and can be measured, for example, as shown in Example 10 below.

[0060] The term "fibrosis" describes the formation of fibrous tissue as a reparative or reactive process, rather than as a normal constituent of an organ or tissue. It is used in, for example, the Farlex Partner Medical Dictionary, The American Heritage Medical Dictionary, or Pschyrembel Klinisches Woeterbuch, 261 st ed., 2007.

[0061] The term "hypertrophy" describes an abnormally strong increase in organ or tissue volume due to enlargement of its constituent cells. In sharp contrast to hyperproliferation, hypertrophy is characterized by an increase in tissue or organ volume that occurs solely through enlargement of existing cells rather than through rapid proliferation of cells by rapid division. The term "hypertrophy" as used herein may be used in any language other than that described in, for example, the Farlex Partner Medical Dictionary, The American Heritage Medical Dictionary, or Pschyrembel Klinisches Woeterbuch, 261 sted., 2007. Hypertrophy can be assessed or measured, for example, by comparing the surface area, cross-sectional area, or size of cells or tissues affected by hypertrophy with the surface area, cross-sectional area, or size of control cells or tissues not affected by hypertrophy, or by comparing the protein content of affected cells with a control. The hypertrophy treated according to the present invention is preferably pathological hypertrophy, for example, hypertrophy caused by ET1 and / or AngII, which are known to promote pathological types of cardiac hypertrophy.

[0062] The term "interstitial lung disease" or "ILD" is to be understood as described in Lederer et al., New England Journal of Medicine, 2018, Vol. 378(19):1811-1823, or van Cleemput, J. et al. Idiopathic Pulmonary Fibrosis for Cardiologists: Differential Diagnosis, Cardiovascular Comorbidities, and Patient Management; Adv Ther. 2019 Feb;36(2):298-317.

[0063] As used herein, the term "heart failure" in the context of "treatment and / or prevention of heart failure" should be understood as defined in the 2016 European Heart Association Guidelines for the Diagnosis and Treatment of Acute and Chronic Heart Failure (European Heart Journal, 2016; Vol. 37(27):2129-2200), and includes chronic heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), and heart failure with mildly reduced ejection fraction (HFmrEF). In the context of the present invention, the term is also intended to refer to HFpEF or HFrEF, particularly Stage C or D HFpEF and Stage C or D HFrEF as described in the 2017 ACC / AHH / HFSA Focused Update of the 2013 ACCF / AHA Guideline for the Management of Heart Failure (Journal of American College of Cardiology, 2017; Vol. 70(6):776-803). The description of the embodiments includes further definitions and explanations of terms used throughout this application, which descriptions and definitions are valid for the entire application unless otherwise stated.

[0064] array The sequences used in the present invention are listed below. SEQ ID NO: 1 (amino acid sequence of human factor 1, lacking the 31 amino acid N-terminal signal peptide): VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL SEQ ID NO:2 (amino acid sequence of the mouse homologue of factor 1, lacking the 24 amino acid N-terminal signal peptide): VSEPTTVPFDVRPGGVVHSFSQDVGPGNKFTCTFTYASQGGTNEQWQMSLGTSEDSQHFTCTIWRPQGKSYLYFTQFKAELRGAEIEYAMAYSKAAFERESDVPLKSEEFEVTKTAVSHRPGAFKAELSKLVIVAKAARSEL SEQ ID NO: 3 [amino acid sequence of human factor 1, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB-Q969H8]: [ka] SEQ ID NO: 4 [amino acid sequence of the mouse homologue of factor 1, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB-Q9CPT4]: [ka] SEQ ID NO: 5 shows the nucleic acid sequence of human factor 1 encoding MYDGF of SEQ ID NO: 3 (NCBI Gene ID: 56005). SEQ ID NO: 6 shows the nucleic acid sequence of mouse factor 1 encoding Mydgf of SEQ ID NO: 4 (NCBI Gene ID: 28106).

[0065] Embodiment Below, elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0066] The present inventors are the first to demonstrate the anti-fibrotic and anti-hypertrophic effects of MYDGF. The inventors particularly show that administration of MYDGF in a mouse model inhibits hypertrophy and fibrosis. These effects can be used, inter alia, to improve cardiac function. Thus, in a first aspect, the present invention provides the protein myeloid-derived growth factor (MYDGF), or a fragment or variant thereof exhibiting the biological function of MYDGF, for use in the treatment or prevention of fibrosis or hypertrophy.

[0067] In a second aspect, the present invention provides the protein bone marrow-derived growth factor (MYDGF) or a fragment or variant thereof exhibiting the biological function of MYDGF for use in the treatment or prevention of heart failure. According to a preferred embodiment, the heart failure is chronic heart failure or acute heart failure, wherein acute heart failure does not include myocardial infarction. According to a preferred embodiment, the acute heart failure is heart failure induced by acute pressure overload. Also provided is MYDGF or a fragment or variant thereof for this use, wherein the heart failure or chronic heart failure is heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), or heart failure with mildly reduced ejection fraction (HFmrEF). Without wishing to be bound by any theory, heart failure is treated or prevented by attenuating fibrosis and / or hypertrophy associated with heart failure and / or improving cardiac function.

[0068] In particularly preferred embodiments of the invention, the protein comprises the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. Preferably, the protein has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1.

[0069] In a preferred embodiment of this aspect of the invention, the protein comprises the amino acid sequence of SEQ ID NO: 1, or a fragment or variant thereof that exhibits the biological function of MYDGF and has at least 85% sequence identity to SEQ ID NO: 1. Those skilled in the art can determine, without undue burden, which positions in the parent polypeptide can be mutated and to what extent, and which positions must be maintained to preserve the polypeptide's functionality. Such information can be obtained, for example, from sequences of homologs that can be identified, aligned, and analyzed by bioinformatics methods well known in the art. Such analyses are exemplarily described in Example 7 and Figures 6 and 7 of WO 2014 / 111458. Mutations are preferably introduced into regions of the protein that are not completely conserved between species, preferably between mammals. In a particularly preferred embodiment of the invention, the MYDGF protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1, or a fragment or variant thereof that exhibits the biological function of MYDGF. Preferably, the protein has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1.

[0070] N-terminal deletion mutants are also encompassed, which may lack one or more amino acids, for example, from amino acid positions 1 to 24 (based on SEQ ID NO: 1), ie, the N-terminal conserved region.

[0071] C-terminal deletion mutants are also encompassed, which may lack one or more amino acids, for example, from amino acid positions 114 to 142 (based on SEQ ID NO: 1).

[0072] On the other hand, amino acids can be added to the MYDGF protein. Such additions include addition at the N-terminus, C-terminus, internal addition to the amino acid sequence, or a combination thereof. The protein of the first aspect of the present invention may further comprise an additional amino acid sequence, for example, for stabilizing or purifying the resulting protein. Examples of such amino acids are a 6xHis tag, a myc tag, or a FLAG tag, which are well known in the art and may be present at any position of the protein, preferably at the N-terminus or C-terminus. A particularly preferred additional sequence is a 6xHis tag. Preferably, the 6xHis tag is present at the C-terminus of the MYDGF protein. Depending on the expression system used and the presence, if any, of additional amino acids such as the above-mentioned tags, one or more remaining amino acids may remain at the N-terminus and / or C-terminus of the protein. It is emphasized that such artifacts may be present in the MYDGF and Mydgf proteins according to the present invention, as shown, for example, in Ebenhoch R. et al., Nat Commun. 2019 Nov 26;10(1):5379 and Polten F. et al., Anal Chem. 2019 Jan 15;91(2):1302-1308.

[0073] In order to stabilize the protein, it may be preferable to introduce mutations into the protease cleavage sites within the MYDGF protein of the first aspect of the present invention (see Segers et al. Circulation 2007, 2011). Those skilled in the art know how to determine potential protein cleavage sites within a protein. For example, the protein sequence can be submitted to a website that provides such analysis, such as http: / / web.expasy.org / peptide_cutter / or http: / / pmap.burnham.org / proteases. When the protein sequence according to SEQ ID NO: 1 is submitted to http: / / web.expasy.org / peptide_cutter / , the following cleavage sites with low cleavage frequency [less than 10] are determined:

[0074] [Table 2]

[0075] To increase the serum half-life of the protein, these sites may be altered to remove the recognition / cleavage sequences of each identified protease.

[0076] In the present invention, MYDGF has been shown to inhibit or prevent fibrosis, particularly cardiac fibrosis. Thus, the present invention provides MYDGF protein or fragments or variants thereof that exhibit the biological functions of MYDGF for use in the treatment or prevention of fibrosis.

[0077] Treating or preventing fibrosis in the context of the present invention means, for example, reducing the amount of fibrotic tissue or preventing or reducing the formation of fibrotic tissue, preferably by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to control tissue not treated with the active agent.

[0078] Without wishing to be bound by any theory, MYDGF inhibits transforming growth factor-beta, a universal profibrotic growth factor, thereby preventing and / or treating fibrosis.

[0079] In the present invention, MYDGF has been shown to inhibit or prevent hypertrophy, particularly hypertrophy of cardiomyocytes. Thus, the present invention provides MYDGF protein or a fragment or variant thereof exhibiting the biological function of MYDGF for use in treating or preventing hypertrophy. When cardiomyocyte hypertrophy is treated or prevented, the cardiomyocytes are preferably left or right ventricular cardiomyocytes or atrial cardiomyocytes.

[0080] Treatment and / or prevention of hypertrophy and fibrosis of cardiac tissue and / or cells, such as cardiomyocytes, can also be used to improve cardiac function. Thus, the present invention also provides MYDGF for use in improving cardiac function. Cardiac function within the meaning of the present invention relates to systolic and / or diastolic cardiac function, which can be assessed, for example, by echocardiography, cardiac magnetic resonance imaging, cardiac computed tomography, or ventricular angiography. A preferred method for assessing cardiac function is high-resolution 2D transthoracic echocardiography (e.g., as described in Lang et al., Eur Heart J Cardiovasc Imaging, 2015;16:233-270), using, for example, a 30 MHz linear transducer (Vevo 3100, VisualSonics) in mice. In such experiments, left ventricular (LV) end-diastolic area (LVEDA) and left ventricular end-systolic area (LVESA) are determined from the long-axis view. LVEDA (mm 2 ) is a two-dimensional approximation of the left ventricular end-diastolic volume; LVESA (mm 2) is a two-dimensional approximation of the left ventricular end-systolic volume. The fractional area change (FAC) is then calculated as a measure of systolic function, i.e., the pumping or contracting function of the heart [(LVEDA-LVESA) / LVEDA] x 100. Thus, improved cardiac function means an improvement in systolic and / or diastolic cardiac function, e.g., as measured by assessing FAC, of ​​at least 10% or more, e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, e.g., 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more, compared to the cardiac function of the same heart before treatment with MYDGF according to the present invention.

[0081] The MYDGF protein may further comprise an additional amino acid sequence, for example, for stabilizing or purifying the resulting protein. For example, to stabilize the protein, it is preferable to introduce a mutation into a protease cleavage site within the MYDGF protein. Suitable protease cleavage sites can be identified as described above.

[0082] The MYDGF protein or a composition containing said protein can be administered in vivo, ex vivo or in vitro, preferably in vivo.

[0083] The fibrotic or hypertrophic cells or tissues preferably belong to or are derived from a defined system of the individual's body, selected from the group consisting of the digestive system, endocrine system, excretory system, immune system, integumentary system, muscular system, nervous system, reproductive system, respiratory system, and skeletal system, or a combination thereof. Alternatively, the fibrotic or hypertrophic cells or tissues preferably belong to or are derived from a defined organ or organ of the individual's body, selected from the group consisting of the skin, bone, heart, cartilage, blood vessels, esophagus, stomach, intestines, glands, liver, kidney, lung, brain, and spleen. In a particularly preferred embodiment, the cells or tissues belong to or are derived from the heart.

[0084] Fibrotic or hypertrophied cells or tissues can be damaged or diseased cells or tissues.Preferably, the damage or disease is caused by genetic / hereditary disease or acquired disease, such as ischemia, reperfusion injury, inflammation, infection, trauma, mechanical stress, poisoning or surgery.In a particularly preferred embodiment, the damage is caused by infarction, particularly myocardial infarction.In another particularly preferred embodiment, the damage is caused by reperfusion injury.

[0085] In particularly preferred embodiments, the fibrotic cells or tissues are selected from the group consisting of cardiac, renal, pulmonary, and hepatic cells or tissues, most preferably cardiac cells or tissues. The results shown in Examples 15-18, based on cells from IPF patients, strongly suggest applicability to fibrosis in the context of ILD. The results shown in Examples 21 and 22, based on embryonic fibroblasts, also suggest applicability to fibrosis in other tissues, such as the kidney and liver.

[0086] In a preferred embodiment, the fibrosis is interstitial lung disease (ILD). According to a preferred embodiment, the ILD is progressive fibrotic interstitial lung disease, more preferably idiopathic pulmonary fibrosis (IPF). Thus, according to one embodiment, the MYDGF protein or its fragment or variant exhibiting the biological function of MYDGF is used for the prevention or treatment of interstitial lung disease as defined in Lederer et al., New England Journal of Medicine, 2018, Vol. 378(19):1811-1823, and van Cleemput, J. et al. Idiopathic Pulmonary Fibrosis for Cardiologists: Differential Diagnosis, Cardiovascular Comorbidities, and Patient Management, Adv Ther. 2019 Feb;36(2):298-317. According to further embodiments, the ILD is progressive fibrosing ILD (PF-ILD), in particular idiopathic nonspecific interstitial pneumonia (iNSIP), unclassifiable idiopathic interstitial pneumonia (unclassifiable IIP), idiopathic pneumonia with autoimmune features (IPAF), chronic hypersensitivity pneumonitis (CHP), environmental / occupational fibrosing lung disease, systemic sclerosis interstitial lung disease (SSc-ILD), or rheumatoid arthritis interstitial lung disease (RA-ILD).

[0087] In a particularly preferred embodiment, the cells or tissues undergoing hypertrophy are cardiac cells or tissues, more preferably cardiomyocytes.

[0088] In a further aspect, the present invention provides nucleic acids encoding the MYDGF protein or fragments or variants thereof that exhibit the biological function of MYDGF as described herein for use in the treatment or prevention of fibrosis or hypertrophy. The present invention also provides nucleic acids encoding the MYDGF protein or fragments or variants thereof that exhibit the biological function of MYDGF as described herein for use in the treatment or prevention of heart failure. Nucleic acids for use according to the invention preferably encode an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1.

[0089] Nucleic acid sequences can be optimized in an effort to enhance expression in a host cell. Parameters to consider include C:G content, preferred codons, and avoidance of inhibitory secondary structures. These factors can be combined in various ways in an attempt to obtain a nucleic acid sequence that enhances expression in a particular host (see, e.g., Donnelly et al., International Publication No. 97 / 47358). The ability of a particular sequence to enhance expression in a particular host involves some empirical experimentation. Such experimentation requires measuring the expression of promising nucleic acid sequences and altering the sequence as necessary. Starting with a particular amino acid sequence and the known degeneracy of the genetic code, many different encoding nucleic acid sequences can be obtained. The degeneracy of the genetic code arises because nearly all amino acids are encoded by multiple different combinations of nucleotide triplets, or "codons." It is well known in the art that specific codons translate into specific amino acids (see, e.g., Lewin, GENES IV, p. 119, Oxford University Press, 1990).

[0090] Nucleic acids for use according to the present invention may further comprise transcriptional or expression control elements arranged to control protein expression. Such nucleic acids with control elements are often referred to as expression systems. As used herein, the term "expression system" refers to a system designed to produce one or more gene products of interest. Typically, such systems are "artificial," i.e., engineered by genetic engineering techniques that can be used to produce the gene products of interest in vivo, in vitro, or ex vivo. The term "expression system" also encompasses expression of the gene products of interest, including transcription of a polynucleotide, mRNA splicing, translation into a polypeptide, co-translational and post-translational modification of the polypeptide or protein, and targeting of the protein to one or more intracellular compartments, secretion from the cell, and uptake of the protein into the same or another cell. This general description refers to expression systems for use in eukaryotic cells, tissues, or organisms. Expression systems for prokaryotic systems may differ, and construction of expression systems for prokaryotic cells is well known in the art.

[0091] The regulatory elements present in a gene expression cassette typically include: (a) a promoter transcriptionally linked to the nucleotide sequence encoding the polypeptide, (b) a 5' ribosome binding site operably linked to the nucleotide sequence, (c) a terminator attached to the 3' end of the nucleotide sequence, and (d) a 3' polyadenylation signal operably linked to the nucleotide sequence. Additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing may also be present. A promoter is a genetic element recognized by RNA polymerase that mediates transcription of a downstream region. A preferred promoter is a strong promoter that results in an increased level of transcription. Examples of strong promoters are the immediate early human cytomegalovirus promoter (CMV) and CMV containing intron A (Chapman et al., Nucl. Acids Res. 19:3979-3986, 1991). Further examples of promoters include naturally occurring promoters, such as the EF1 alpha promoter, the murine CMV promoter, the Rous sarcoma virus promoter, and the SV40 early / late promoter and the [beta]actin promoter; and artificial promoters, such as synthetic muscle-specific promoters and chimeric muscle-specific / CMV promoters (Li et al., Nat. Biotechnol. 17:241-245, 1999, Hagstrom et al., Blood 95:2536-2542, 2000).

[0092] The ribosome binding site is located at or near the start codon. Examples of preferred ribosome binding sites include CCACCAUGG, CCGCCAUGG, and ACCAUGG, where AUG is the start codon (Kozak, Cell 44:283-292, 1986). The polyadenylation signal is involved in cleavage of the transcribed RNA and addition of a poly(A) tail to the RNA. In higher eukaryotes, the polyadenylation signal contains an AAUAAA sequence 11 to 30 nucleotides from the polyadenylation addition site. The AAUAAA sequence is involved in signaling RNA cleavage (Lewin, Genes IV, Oxford University Press, NY, 1990). The poly(A) tail is important for mRNA processing, nuclear export, translation, and stability.

[0093] Polyadenylation signals that can be used as part of the gene expression cassette include the minimal rabbit [beta]globin polyadenylation signal and the bovine growth hormone polyadenylation signal (BGH) (Xu et al., Gene 272:149-156, 2001, Post et al., U.S. Pat. No. 5,122,458).

[0094] Examples of additional regulatory elements that may be present and are useful for enhancing or regulating gene expression or polypeptide processing include enhancers, leader sequences and operators.Enhancer regions increase transcription.Examples of enhancer regions include CMV enhancers and SV40 enhancers (Hitt et al., Methods in Molecular Genetics 7:13-30, 1995; Xu, et al., Gene 272:149-156, 2001).Enhancer regions can be associated with promoters.

[0095] The expression of the MYDGF protein or variant thereof of the present invention can be regulated. Such regulation can be achieved at many stages of gene expression. Possible regulatory steps include, but are not limited to, transcription initiation, promoter clearance, transcription elongation, splicing, nuclear export, mRNA stability, translation initiation, translation efficiency, translation elongation, and protein folding. Other regulatory steps that affect the intracellular concentration of MYDGF polypeptides affect the half-life of the protein. Such regulatory steps include, for example, regulating protein unfolding. Proteins of the present invention include secreted proteins, which can be directed into the secretory pathway of the host cell. Secretion efficiency, along with regulatory steps referring to expression and protein stability, regulates the extracellular concentration of the respective protein. Extracellular may refer to, for example, but not limited to, culture medium, tissue, intracellular matrix or space, or bodily fluids such as blood or lymph.

[0096] The above-mentioned regulatory control can be, for example, cell-type or tissue-type independent or cell-type or tissue-type specific. In a particularly preferred embodiment of the present invention, the regulatory control is cell-type or tissue-type specific. Such cell-type or tissue-type specific regulation is preferably achieved by a regulation that refers to the transcription of a nucleic acid. This transcriptional regulation can be achieved by using a cell-type or tissue-type specific promoter sequence. The result of this cell-type or tissue-type specific regulation can have various degrees of specificity. This means that the expression of a particular polypeptide is enhanced in a particular cell or tissue compared to other cell or tissue types, or that the expression is limited to a particular cell or tissue type. Cell- or tissue-type specific promoter sequences are well known in the art and are available for a wide range of cell or tissue types.

[0097] Expression is not necessarily cell-type or tissue-type specific, but may depend on the physiological state. Such states are, for example, inflammation or wounds. Such physiological state-specific expression can also be achieved by regulation at all of the regulatory stages mentioned above. A preferred mode of regulation for physiological state-specific expression is transcriptional regulation. For this purpose, wound- or inflammation-specific promoters can be used. The respective promoters can be, for example, naturally occurring sequences, which can be derived from genes that are specifically expressed during, for example, immune responses and / or wound tissue regeneration. Another possibility is the use of artificial promoter sequences, which can be constructed, for example, by combining two or more naturally occurring sequences.

[0098] The modulation can be cell-type or tissue-type specific and physiological state specific. In particular, the expression can be cardiac-specific. Preferably, the expression is cardiac-specific and / or wound-specific.

[0099] Another possibility for regulating the expression of the MYDGF protein or its variants according to the present invention is the conditional regulation of gene expression. To achieve conditional regulation, an operator sequence can be used. For example, the Tet operator sequence can be used to repress gene expression. Conditional regulation of gene expression using the Tet repressor together with the Tet operator is well known in the art, and many individual systems have been established for a wide range of prokaryotic and eukaryotic organisms. Those skilled in the art know how to choose a suitable system and adapt it to the specific needs of individual applications.

[0100] In a particularly preferred embodiment, the use of the nucleic acids according to the invention comprises application to individuals, preferably individuals suffering from fibrosis or hypertrophy.

[0101] According to a further aspect, the invention provides a vector comprising a nucleic acid or expression system as described herein for use in the treatment or prevention of fibrosis or hypertrophy, or for use in the treatment of heart failure.

[0102] As used herein, the term "vector" refers to a protein or polynucleotide, or a mixture thereof, that can be introduced into a cell, or that can introduce proteins and / or nucleic acids contained therein into a cell. In the context of the present invention, it is preferred that the gene of interest encoded by the introduced polynucleotide is expressed in the host cell upon introduction of the vector or vectors. Examples of suitable vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores.

[0103] In a preferred embodiment of the present invention, the vector is a viral vector.Suitable viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors and lentiviral vectors.

[0104] In particularly preferred embodiments of the invention, the vector is an adenovirus or adeno-associated virus (AAV) vector.

[0105] Nucleic acids encoding one or more MYDGF proteins or variants thereof according to the invention can be introduced into host cells, tissues or individuals using vectors suitable for therapeutic administration. Suitable vectors preferably are capable of delivering nucleic acids to target cells without causing unacceptable side effects.

[0106] In a particularly preferred embodiment, the use of the vector according to the invention comprises application to an individual in need thereof.

[0107] Vectors containing nucleic acids encoding the above-mentioned MYDGF proteins or fragments or variants thereof that exhibit the biological functions of MYDGF are preferably for use in the treatment or prevention of fibrosis or hypertrophy, or for use in the treatment of heart failure.

[0108] According to a further aspect, the present invention provides a host cell comprising a vector as described herein and expressing a nucleic acid encoding a MYDGF protein or a fragment or variant thereof that exhibits the biological function of MYDGF for use in the treatment or prevention of fibrosis or hypertrophy, or for use in the treatment of heart failure.

[0109] According to a further aspect, the present invention provides a pharmaceutical composition for use in the treatment or prevention of fibrosis or hypertrophy, or for use in the treatment of heart failure, comprising a MYDGF protein or a fragment or variant thereof which exhibits the biological function of MYDGF, and optionally a suitable pharmaceutical excipient.

[0110] The term "suitable pharmaceutical excipient," as used herein, refers to a pharmacologically inactive substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer solution, or vehicle, with which a therapeutically active ingredient is administered. A "pharmaceutical excipient," also referred to as a "pharmaceutical carrier," may be liquid or solid. Liquid carriers include, but are not limited to, sterile solutions, such as saline solutions in water and oils of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Saline solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. In a preferred embodiment of the present invention, the carrier is a suitable pharmaceutical excipient. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Such suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

[0111] "Pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans.

[0112] The term "composition" is intended to include a dosage form of an active compound that includes an encapsulating material as one carrier forming a capsule, in which the active component is surrounded by one carrier with or without other carriers, and thus the one carrier is associated with the active compound.

[0113] The term "active ingredient" refers to a biologically active substance, i.e., a substance that provides pharmaceutical value, in a pharmaceutical composition or dosage form. In the context of the present invention, the active ingredient is a MYDGF protein or a fragment or variant thereof that exhibits the biological function of MYDGF. A pharmaceutical composition may contain one or more active ingredients, which may act synergistically or independently of each other. The active ingredient can be formulated as a neutral or salt form. The salt form is preferably a pharmaceutically acceptable salt.

[0114] The term "pharmaceutically acceptable salt" refers to salts of the MYDGF polypeptides of the invention, including, but not limited to, fragments or variants thereof, as described herein. Suitable pharmaceutically acceptable salts include, for example, acid addition salts that can be formed by mixing a solution of a polypeptide of the invention with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Furthermore, when the peptide bears an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed using counteranions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates, and arylsulfonates).Illustrative examples of pharmaceutically acceptable salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, hydroxybenzo ... acid salt, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide Salt, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate These salts include, but are not limited to, acetate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, acetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S.M. Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, pp. 1-19 (1977)).

[0115] According to one embodiment, the active ingredient is administered to a cell, tissue, or individual in an effective amount. An "effective amount" refers to the amount of the active ingredient sufficient to achieve the intended purpose. The active ingredient may be a therapeutic agent. The effective amount of a given active ingredient will vary depending on parameters such as the nature of the ingredient, the route of administration, the size and species of the individual receiving the active ingredient, and the purpose of administration. The effective amount in each individual case may be determined empirically by one skilled in the art according to methods established in the art. As used in the context of the present invention, "administering" includes in vivo administration to an individual and direct administration to a cell or tissue in vitro or ex vivo.

[0116] In a preferred embodiment of the present invention, pharmaceutical compositions are customized for the treatment of disease or disorder.As used herein, "treat", "treating" or "treatment" of disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting or preventing the recurrence of the disorder in patients who have previously had the disorder; (e) limiting or preventing the recurrence of symptoms in patients who have previously shown symptoms of the disorder; (f) reducing the mortality rate after the onset of disease or disorder; (g) curing; and (h) preventing the onset of disease.The term "improving" is also included in the term "treating".As used herein, "preventing", "preventing" or "prevention" of disease or disorder means preventing such disease or disorder from occurring in patients.

[0117] In a particularly preferred embodiment of the present invention, treatment with a pharmaceutical composition according to the present invention comprises treatment of an individual in need of such treatment.

[0118] Pharmaceutical compositions contemplated by the present invention may be formulated in a variety of ways well known to those skilled in the art. For example, pharmaceutical compositions of the present invention may be in the form of a liquid, such as a solution, emulsion, or suspension. Preferably, pharmaceutical compositions of the present invention are formulated for parenteral administration, preferably intravenous, intraarterial, intramuscular, subcutaneous, transdermal, pulmonary, intraperitoneal, intracoronary, intracardiac, or mucosal administration, preferably intravenous, subcutaneous, or intraperitoneal. Formulations for oral or rectal administration are also possible. Preferably, pharmaceutical compositions of the present invention are in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salts or glucose to make the solution isotonic with blood. The aqueous solution should be suitably buffered, if necessary (preferably to a pH of 3 to 9, more preferably to a pH of 5 to 7). Pharmaceutical compositions are preferably in unit dosage form. In such form, the pharmaceutical composition is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the pharmaceutical composition, such as vials or ampoules.

[0119] The pharmaceutical compositions are preferably administered by intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary or intracardiac routes, including other routes of administration known in the art.

[0120] When the pharmaceutical composition is used as a treatment for an individual, the use of the pharmaceutical composition can replace or be administered in addition to standard treatment for the respective disease or condition. In the case of additional use, the pharmaceutical composition can be administered before, simultaneously with, or after the standard treatment.

[0121] More preferably, the pharmaceutical composition is administered one or more times, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 times. The duration of administration of the pharmaceutical is not limited. Preferably, administration does not exceed 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0122] A single dose of the pharmaceutical composition may constitute the total amount administered by itself, or each administration period may include one or more bolus injections and / or administration as an infusion.

[0123] According to a further aspect, the present invention provides a method for treating fibrosis, comprising administering to a patient in need thereof a therapeutically effective amount of MYDGF, or a fragment or variant thereof that exhibits the biological function of MYDGF, wherein the MYDGF preferably comprises SEQ ID NO: 1, or a fragment or variant thereof that exhibits the biological function of MYDGF of SEQ ID NO: 1. In this regard, the fragment or variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0124] In a preferred embodiment of the method of the present invention, the fibrosis is cardiac, renal, pulmonary and / or hepatic fibrosis. According to a preferred embodiment, the fibrosis is interstitial lung disease, more preferably progressive fibrosing interstitial lung disease, most preferably idiopathic pulmonary fibrosis.

[0125] According to a preferred embodiment of the method of the present invention, the MYDGF protein or a fragment or variant thereof that exhibits the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier.

[0126] According to a further aspect, the present invention provides a method for treating hypertrophy, said method comprising administering to a patient in need of treatment for hypertrophy a therapeutically effective amount of MYDGF, or a fragment or variant thereof that exhibits a biological function of MYDGF. In said method, the MYDGF preferably comprises SEQ ID NO: 1, or a fragment or variant thereof that exhibits a biological function of MYDGF of SEQ ID NO: 1. In this regard, the fragment or variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0127] According to a preferred embodiment of the method of the present invention, the hypertrophy is a hypertrophy of cardiomyocytes.

[0128] According to a preferred embodiment of the method of the present invention, the MYDGF protein or a fragment or variant thereof that exhibits the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier.

[0129] According to a preferred embodiment of the method of the present invention, the MYDGF protein or a fragment or variant thereof that exhibits the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier.

[0130] According to a further aspect, the present invention provides a method for treating or preventing heart failure, comprising administering to a patient in need thereof a therapeutically effective amount of the growth factor MYDGF or a fragment or variant thereof exhibiting the biological function of MYDGF, preferably wherein the heart failure is chronic heart failure. According to a preferred embodiment, in the method for treating heart failure, the heart failure or chronic heart failure is HFpEF or HFrEF, preferably HFpEF is stage C or stage D HFpEF, or HFrEF is stage C or stage D HFrEF. In this regard, the fragment or variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO:1.

[0131] According to a preferred embodiment of the method of the present invention, the MYDGF protein or a fragment or variant thereof that exhibits the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier. [Example]

[0132] The examples are designed to further illustrate and aid in a better understanding of the present invention, and should not be construed as limiting the scope of the invention in any way.

[0133] Materials and methods used in the examples Unless otherwise stated, the following materials and methods were used in the examples.

[0134] Endothelin 1 (ET1) and angiotensin II (AngII) were purchased from Sigma-Aldrich, mouse insulin-like growth factor 1 (IGF1) from R&D Systems, and SMI4a from Selleckchem (catalog number S8005). Antibodies were purchased from Abcam [sarcoplasmic reticulum Ca2 + ATPase 2a [SERCA2a], polyclonal, #ab91032; alpha-tubulin, clone EPR13478(B)], purchased from Cell Signaling Technology (beta-actin, clone 13E5; vinculin, clone E1E9V), Eurogentech (mouse MYDGF, polyclonal, custom-made) ( Korf-Klingebiel, 2015 ), and Thermo Fisher (PIM1, clone G.360.1).

[0135] Recombinant MYDGF. Recombinant mouse MYDGF with a C-terminal 8xHis tag was produced in HEK293-6E cells cultured in animal-origin-free, chemically defined, protein-free FreeStyle F17 expression medium (Gibco) (Karste et al. Methods Mol Biol. 2017;1586:313-324). Transfection efficiency was assessed by cotransfecting a control plasmid encoding GFP. Cell culture supernatant (6 L) was harvested by centrifugation and concentrated 15-fold by diafiltration against PBS containing 300 mmol / L NaCl (pH 7.4) (PBS / NaCl) in a Proflux M12 cross-flow ultrafiltration unit (Millipore) using a 5 kDa Pellicon 2 filter (Millipore). The concentrate was filtered using a 0.45 / 0.2 μm Sartobran 300 capsule filter (Sartorius) and sodium azide (0.05%) was added. MYDGF was captured using an AEKTA pure 25 M system (Cytiva) and a 5 mL HisTrap excel column (Cytiva). The column was washed with PBS / NaCl until the UV signal reached baseline. Fractions containing the recombinant protein, eluted with imidazole (30 mmol / L), were pooled and concentrated using a Vivaspin 20 (5,000 MWCO) ultrafiltration unit (Sartorius). The protein was further purified by size exclusion chromatography (SEC) using a HiLoad 26 / 600 Superdex 75 column (GE Healthcare). MYDGF eluted in a single peak and was concentrated to 2 mg / mL using a Vivaspin 20 (5,000 MWCO) ultrafiltration unit. Protein concentration was calculated using the molar extinction coefficient (1 mg / ml = Abs 280nm The total yield of the recombinant protein (5.95 mg / L) was characterized by trypsin fingerprinting using SDS-PAGE and mass spectrometry (Bruker). Protein samples were flash frozen in liquid nitrogen and stored at -80°C.

[0136] Mydgf-deficient mice. Mice with a genetic deletion of Mydgf (Mydgf tm1Kcw, Mouse Genome Informatics ID: 5688472) have been previously described. The animals (BALB / c × C57BL / 6J background) were backcrossed to the C57BL / 6N strain for 10 generations, and the background was maintained by heterozygous breeding. Littermates were used in all experiments. Wild-type and targeted alleles were detected by genomic PCR (Korf-Klingebiel, 2015).

[0137] Mouse Surgery and Functional Assessment. All surgical procedures were approved by the authorities in Hannover, Germany (Lower Saxony Consumer Protection and Food Safety Office). Mice were housed in individually ventilated cages in the central animal facility of the Hannover Medical School under a 12-h light / dark cycle. Food and water were provided ad libitum. Transverse aortic constriction (TAC) surgery (Rockman et al. Proc Natl Acad Sci U S A. 1991;88:8277-8281) was performed in 8- to 10-week-old C57BL / 6N mice. The animals were pretreated subcutaneously with 0.02 mg / kg atropine (B. Braun) and 200 mg / kg metamizole (Zentiva). Anesthesia was induced in an induction chamber using 3–4% isoflurane (Baxter). After oral intubation, mice were mechanically ventilated (Harvard Apparatus, Minivent Type 845), and anesthesia was maintained using 1.5–2% isoflurane. During surgery, mice were placed on a heating pad connected to a temperature controller (Foehr Medical Instruments) to maintain rectal temperature at 37°C. A left anterolateral thoracotomy was performed under an operating microscope. After separating the thymus and adipose tissue from the aortic arch, a 6-0 silk suture was placed between the brachiocephalic trunk and the left carotid artery and tied to a 26-gauge blunt needle. The knot was then tied and the needle was removed. After wound closure, mice were disconnected from the ventilator and allowed to recover in an incubator at 32°C. In sham-operated control mice, no ligature was tied around the aorta. Mice were subjected to a three-week swimming training protocol to induce physiological hypertrophy (Heineke, Methods Mol Biol. 2013;963:279-301).

[0138] Two-dimensional transthoracic echocardiography was performed in mice sedated with 1–2% isoflurane via a face mask using a 20–46 MHz linear transducer (MX400, Vevo 3100, VisualSonics). The pressure gradient across the aortic stenosis and the ratio of maximum blood velocity (Vmax) between the right and left common carotid arteries were determined by Doppler ultrasound immediately after TAC or sham surgery. LV end-diastolic area (LVEDA) and LV end-systolic area (LVESA) were recorded from the parasternal long-axis view. Percent area change (%) was calculated as [(LVEDA-LVESA) / LVEDA] × 100.

[0139] For LV pressure-volume measurements, mice were subcutaneously injected with 2 mg / kg butorphanol (Zoetis). Anesthesia was induced with 4% isoflurane. After oral intubation, mice were intraperitoneally injected with 0.8 mg / kg pancuronium (Actavis), and anesthesia was maintained with 2% isoflurane. A conductance catheter (SPR-839, Millar Instruments) tipped with a 1.4 F micromanometer was inserted into the left ventricle via the right carotid artery. Steady-state pressure-volume loops were sampled at a rate of 1 kHz and analyzed using LabChart 7 Pro software (ADInstruments).

[0140] Bone marrow transplantation. Bone marrow cells (BMCs) were flushed from the femurs and tibias of 7- to 9-week-old Mydgf WT or KO donor mice. Erythrocytes were removed by NH4Cl lysis. 7- to 9-week-old Mydgf WT or KO recipient mice were lethally irradiated (9.5 Gy) and then transplanted for 10 min. 6BMCs (106 BMCs) were transplanted via the tail vein. After transplantation, mice were treated with ciprofloxacin (Bayer) for 3 weeks (100 mg / L in drinking water). Transplantation surgery was performed 7–8 weeks after transplantation. In a control experiment, this protocol was applied to CD45.2 recipient mice, which were lethally irradiated and transplanted with BMCs obtained from congenic CD45.1 donor mice (B6.SJL-Ptprca Pepcb / BoyJ; Jackson Laboratory). After 8 weeks, more than 95% of blood leukocytes were CD45.1 high, as demonstrated by flow cytometry using CD45.1 (BioLegend, clone A20) (dilution, 1:16) and CD45.2 (BD Biosciences, clone 104) (dilution, 1:150) antibodies.

[0141] Lentiviral gene transfer. Lentivirus encoding full-length murine MYDGF under the control of the tetO / CMV promoter and an empty control virus were generated in HEK293T cells as previously described (Lachmann et al. Gene Ther. 2013;20:298-307). To enable conditional MYDGF overexpression in bone marrow-derived inflammatory cells, hematopoietic stem cells (HSCs) were isolated from 7- to 9-week-old R26-M2rtTA mice expressing the reverse tetracycline-controlled transactivator (rtTA-M2) protein under the control of the ubiquitously active Rosa26 promoter. For this purpose, lineage-negative (lin) HSCs were transfected using Miltenyi Biotec's Lineage Cell Depletion Kit. -Bone marrow cells were isolated by magnetic-activated cell sorting (MACS). As previously described (Lachmann 2013; Kustikova et al. Exp Hematol. 2014;42:505-515 e507), cells were expanded in serum-free StemSpan medium (Stem Cell Technologies) supplemented with cytokines (kit ligand, interleukin-3, interleukin-11, Flt3 ligand) and then transduced with HEK293T cell supernatant containing lentiviral vector particles in plates coated with RetroNectin (Takara Bio). WT recipient mice were lethally irradiated (9.5 Gy) and transduced with 1 × 10 Lenti. control or Lenti. MYDGF-transduced cells. 6 HSCs were transplanted via the tail vein. After transplantation, mice were treated with ciprofloxacin for 3 weeks. Sham or TAC surgery was performed 7 weeks after transplantation. To induce MYDGF expression in inflammatory cells derived from lentiviral-transduced HSCs, mice were treated with doxycycline (2 mg / ml in drinking water) for 1 week before surgery.

[0142] MYDGF protein treatment. An Alzet osmotic minipump (Model 2004, filled with 10 μg of MYDGF per 6 μL or diluent alone at an infusion rate of 0.25 μL / h for 28 days) was placed in a subcutaneous interscapular pocket immediately after TAC surgery. A single intraperitoneal bolus injection (10 μg of MYDGF or diluent alone) was then administered.

[0143] Adenovirus. Adenovirus encoding SERCA2A or the red fluorescent protein DsRed was generated using the AdEasy adenovirus vector system (Agilent Technologies). Five days before TAC surgery, mice were injected with adenovirus (1 × 10 via the tail vein). 10 Immediately after TAC, mice received a second injection of adenovirus.

[0144] MYDGF Targeted Liquid Chromatography / Multiple Reaction Monitoring Mass Spectrometry. MYDGF concentrations in EDTA plasma samples from mice and patients were determined by targeted liquid chromatography / multiple reaction monitoring mass spectrometry (LC / MRM-MS) (Polten et al. Anal Chem. 2019;91:1302-1308).

[0145] Fluorescence-activated cell sorting (FACS) and flow cytometry. Inflammatory cells were isolated from the left ventricle by enzymatic digestion and FACS (Hulsmans 2018; Korf-Klingebiel et al. Circ Res. 2019;125:787-801). Left ventricles were digested in PBS containing 1 mg / mL collagenase D (Roche), 2.4 mg / mL dispase (Gibco), and 100 U / mL deoxyribonuclease I (Sigma-Aldrich) for 30 min at 37°C. The cell suspension was filtered (40 μm cell strainer, Falcon), washed, and incubated for 5 min at 4°C in PBS containing 4% FCS, 2 mmol / L EDTA, and purified mouse CD16 / CD32 antibody (BD Biosciences, mouse BD Fc Block, clone 2.4G2) (1:55). The cells were then incubated for 20 min at 4°C with the following antibodies: CD45R / B220-PE (clone RA3-6B2) (1:500), CD90.2 / Thy-1.2-PE (clone 53-2.1) (1:2500), NK-1.1-PE (clone PK136) (1:500), CD49b / DX5-PE (clone DX5) (1:500), Ly6G-PE (clone 1A8) (1:500), and CD11b-Alexa Fluor 700 (clone M1 / 70) (1:50) from BD Biosciences; Ly6C-APC (clone 1G7.G10) (1:8) from Miltenyi Biotec; and CD45-Brilliant Violet from BioLegend. 570 (clone 30-F11) (1:33), F4 / 80-FITC (clone BM8) (1:33), CD3-PE / Cy7 (clone 17A2) (1:33), and CD19-PerCP / Cy5.5 (clone 6D5) (1:33). After washing, cells were sorted on a FACSAria IIu instrument (Becton Dickinson).Monocytes are CD45high CD11bhigh (CD45R / B220, CD90.2 / Thy-1.2, NK1.1, CD49b / DX5, Ly6G)low F4 / 80low Ly6Chigh; macrophages are CD45high CD11bhigh (CD45R / B220, CD90.2 / Thy-1.2, NK1.1, CD49b / DX5, Ly6G)low F4 / 80high or low Ly6Clow; neutrophils are CD45high CD11bhigh (CD45R / B220, CD90.2 / Thy-1.2, NK1.1, CD49b / DX5, Ly6G)high; and T cells are CD45high Inflammatory cells were identified as CD11 (CD45R / B220, CD90.2 / Thy-1.2, NK1.1, CD49b / DX5, Ly6G) high CD3 high CD19 low; and B cells were identified as CD45 high CD11 (CD45R / B220, CD90.2 / Thy-1.2, NK1.1, CD49b / DX5, Ly6G) high CD3 low CD19 high. For flow cytometry, inflammatory cells were incubated with the labeled antibodies listed above. Cells were then added to TruCOUNT tubes (BD Biosciences), counted on an LSR II flow cytometer (Becton Dickinson), and analyzed using FlowJo v10.6 software.

[0146] Isolation of endothelial cells and fibroblasts by MACS. LV myocardium was enzymatically digested with collagenase I (Worthington) and deoxyribonuclease I (Sigma-Aldrich). The cell suspension was filtered (30 μm cell strainer, Falcon), washed, incubated with CD45 MicroBeads, and applied to an LD column. The flow-through fraction of CD45 low cells was washed, incubated with CD146 MicroBeads, and applied to an LD column. Endothelial cells (CD45 low CD146 high) were eluted from the column and used for RNA isolation (RNeasy kit, Qiagen). The flow-through fraction of CD45 low CD146 low cells was washed, incubated with Feeder Removal MicroBeads, and applied to an LS column. Fibroblasts were eluted from the column and used for RNA isolation (all reagents and equipment were obtained from Miltenyi Biotec).

[0147] Tissue Collection and Analysis. Mice were sacrificed at different time points after TAC or sham surgery, and the left ventricle was removed. RNA was isolated using the RNeasy kit (Qiagen). Protein lysates were prepared in RIPA buffer. Midventricular slices were embedded in OCT compound (Tissue Tek), snap-frozen in liquid nitrogen, and stored at -80°C. 6-μm cryosections were prepared. Sections were stained with rhodamine-conjugated wheat germ agglutinin (WGA, Vector Laboratories) to highlight the boundaries of cardiomyocytes. The perimeters of 200–400 myocytes were traced and digitized to calculate the average cross-sectional area. Sections were stained with fluorescein-conjugated GSL I isolectin B4 (IB4, Vector Laboratories) to visualize capillaries. Images were acquired using a fluorescence microscope (Axio Observer.Z1). MYDGF was visualized in 6 μm frozen sections using a confocal fluorescence microscope (Leica DM IRB equipped with a TCS SP2 AOBS scan head) after staining with a Eurogentec polyclonal antibody (1:100) and an FITC-conjugated secondary antibody (Invitrogen, #A24532) (1:200). Sections were co-stained with a CD11b antibody (Invitrogen, clone M1 / 70) (1:200) and a Cy3-conjugated secondary antibody (Jackson ImmunoResearch, #712-165-153) (1:200). In pilot experiments, all secondary antibodies were found to give low background signals. Sections were stained with Sirius Red (Sigma-Aldrich), and interstitial collagen volume fraction was quantified using light microscopy.

[0148] Cardiomyocyte isolation and culture. Adult mouse ventricular cardiomyocytes (AMCMs) were isolated by enzymatic digestion using a protocol obtained from the Alliance for Cellular Signaling (http: / / www.signaling-gateway.org / data / cgi-bin / Protocols.cgi?cat=0). AMCMs were plated onto laminin-coated 6-well plates in 199 medium (Sigma-Aldrich) supplemented with 5% FCS and 10 mmol / L 2,3-butanedione monoxime (Sigma-Aldrich). Cell length and width were determined using a phase-contrast microscope and a digital image analyzer (AxioVision, Zeiss). Neonatal rat ventricular cardiomyocytes (NRCMs) were isolated from 1- to 3-day-old Sprague-Dawley rats by Percoll density gradient centrifugation (Shubeita et al. A paracrine mechanism for myocardial cell hypertrophy. J Biol Chem. 1990;265:20555-20562). NRCMs were plated overnight on gelatin-coated culture dishes in DMEM (Capricorn Scientific, 4 parts) and 199 medium (1 part) supplemented with 5% horse serum, 2.5% FCS, glutamine, and antibiotics. The cells were then switched to DMEM and 199 medium supplemented with glutamine and antibiotics only and stimulated with various drugs for 24 hours. NRCM surface area was determined by planimetry, and protein content was determined by Bradford assay (Bio-Rad). NRCMs were transfected with siRNA [50 pmol / 10 6 cells (50 pmol / 106 cells)].

[0149] Quantitative reverse transcription polymerase chain reaction (RT-qPCR). Total RNA was isolated from cells and tissues using the RNeasy RNA isolation kit (Qiagen) and then reverse transcribed into cDNA (SuperScript III reverse transcriptase, Thermo Fisher). mRNA concentrations were determined using TaqMan or SYBR green assays (annealing, 60°C for 1 minute; extension, 72°C for 1 minute) (all reagents were obtained from Thermo Fisher). Data were analyzed using a standard curve (TaqMan) or relative quantification (SYBR green).

[0150] Sample preparation for proteomics and phosphoproteomics. After stimulation, NRCM (2 × 10 per replicate) were cultured. 6Cells (1000 cells) were washed twice with ice-cold PBS, solubilized in 400 μL of ice-cold RIPA buffer containing 24 mmol / L Tris-HCl (pH 7.6), 150 mmol / L NaCl, 1% NP-40, 1% sodium desoxycholate, 0.1% sodium dodecyl sulfate, cOmplete mini protease inhibitor cocktail (Roche), and PhosSTOP (Roche), and frozen overnight at -80°C. After thawing, samples were dispersed on ice using an IKA Ultra-Turrax and centrifuged at 14,000 g for 5 minutes at 4°C. The supernatant was transferred to a new reaction tube, and protein concentration was measured using the DC protein assay (Bio-Rad). For proteomics, 50 μg of protein was processed using SDS-PAGE and in-gel trypsin digestion as previously described (Polten et al. Anal Chem. 2019;91:1302-1308). Specifically, each gel lane was cut into six sections and digested separately to generate six peptide samples. For phosphoproteomics, a modified filter-aided sample preparation protocol was used (Nel et al. J Proteome Res. 2015;14:1637-1642). Briefly, 300 μg of protein was combined with 200 μL of urea buffer (8 mol / L urea, 0.1 mol / L Tris-HCl [pH 9.5]) and loaded onto a 10 kDa Amicon Ultra-0.5 mL centrifugal filter (Merck). The filter membrane was washed twice with 200 μL of urea buffer, and free cysteines were carbamidomethylated by adding 100 μL of 50 mmol / L iodoacetamide in urea buffer for 20 minutes in the dark. The filter membrane was then washed twice with 100 μL of urea buffer and twice with 100 μL of 40 mmol / L NH4HCO3. Trypsin digestion was performed in 120 μL of 40 mmol / L NH4HCO3 containing 7 μg of mass spectrometry-grade trypsin (Serva). After overnight digestion at 37°C, peptides were eluted twice with 40 μL of 40 mmol / L NH4HCO3.The flow-through fractions were combined and acidified by adding 12 μL of 10% trifluoroacetic acid (TFA) and then dried by vacuum centrifugation. Phosphopeptides were enriched using Thermo Scientific's Pierce Fe-NTA Phosphopeptide Enrichment Kit and Pierce TiO2 Phosphopeptide Enrichment Kit according to the manufacturer's protocol. Briefly, after equilibrating the Fe-NTA column, the dried digested peptides were dissolved in 200 μL of binding buffer, added to the column, and incubated at room temperature for 30 minutes. After centrifugation, the flow-through fractions were collected. After two washing steps with 100 μL of wash buffer A and two with 200 μL of wash buffer B, the flow-through and wash fractions were combined, dried by vacuum centrifugation, and loaded onto a TiO2 phosphopeptide enrichment spin tip. After washing, the phosphopeptides bound to the spin tip were eluted and dried by vacuum centrifugation. The phosphopeptides bound to the Fe-NTA column were eluted twice with 100 μL of elution buffer. The eluted fractions were combined and dried by vacuum centrifugation. The phosphopeptide eluates obtained from the Fe-NTA and TiO2-based enrichment steps were desalted using Pierce graphite spin columns (Thermo Fisher).

[0151] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) and data processing. Six peptide samples and two phosphopeptide samples per replicate were reconstituted in 30 μL of high-performance liquid chromatography loading buffer (2% acetonitrile, 0.1% TFA) each, as previously described (Junemann et al. Front Microbiol. 2018;9:3083), and analyzed separately using an UltiMate 3000 RSLCnano system (Thermo Scientific) interfaced to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Generated spectra were analyzed using MaxQuant software (version 1.6.1.0) applying the integrated Andromeda peptide search engine to the rat UniProt Knowledgebase (Tyanova et al. Nat Protoc. 2016;11:2301-2319; UniProt Consortium. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019;47:D506-D515). Propionamidation (C) (proteome analysis) or carbamidomethylation (C) (phosphoproteome analysis) were set as fixed modifications; phosphorylation (S / T / Y), oxidation (M), deamidation (N / Q), and N-terminal acetylation were set as variable modifications. Peptide lengths greater than six amino acids and up to two missed trypsin cleavages were allowed. The false discovery rate (FDR) threshold was set at 0.01 on both the peptide and protein levels, and a match-between-runs algorithm was applied to exclude potential contaminants and reverse database hits.Missing values ​​were imputed based on the normal distribution of all log2-transformed measured intensities, separately for each replicate, using Perseus software (version 1.6.1.3) applying a width of 0.3 and a downshift of 1.8 (Tyanova et al. Nat Methods. 2016;13:731-740). For phosphoproteome analysis, only phosphorylation sites with a localization probability greater than 0.75 were considered. Phosphorylation sites that were not detectable in all four replicates from at least one experimental condition were excluded. Phosphorylation site intensities were normalized to the abundance of the corresponding protein. Principal component analysis was based on all phosphorylation sites with an ANOVA p-value less than 0.05 calculated by Perseus. Clusters in the principal component space were defined based on linear regression of the replicate values. Unsupervised hierarchical clustering was performed using the ComplexHeatmap package in R, using median-adjusted phosphorylation site intensities and Euclidean distance metrics for row and column trees (Gu et al. Bioinformatics. 2016;32:2847-2849). For pairwise comparisons, differences in phosphorylation intensity were normalized to the differences in abundance of the corresponding proteins. Proteins that were missing quantification values ​​in the proteome for at least one condition or were not significantly regulated (2-sided unpaired t test P value > 0.05) were considered to have a protein abundance ratio of 1. Linear kinase motif enrichment analysis was performed using phosphorylation site annotations from the Perseus and PhosphoSitePlus databases (Hornbeck et al. Nucleic Acids Res. 2015;43:D512-520; Hogrebe et al. Nat Commun. 2018;9:1045). Kinase-substrate relationships were predicted based on sequence recognition motifs surrounding the measured phosphorylation sites.The kinase substrate motifs of significantly regulated phosphorylation sites were unambiguously enriched using Fisher's exact test. For significantly regulated kinases (Benjamini-Hochberg FDR < 0.02), all potential targets were extracted (kinase-substrate recognition motif positive, localization probability > 0.75, 2-tailed unpaired t test P value < 0.05) and the arithmetic mean regulation was determined (Hogrebe 2018).

[0152] Pim kinase activity assay. Pim kinase activity was measured using the Pim kinase enzyme system and ADP-Glo ​​kinase assay (Promega, #V4032).

[0153] Intracellular Ca 2+Measurement of concentration. AMCMs were plated onto laminin-coated coverslips in 199 medium supplemented with 5% FCS and 10 mmol / L 2,3-butanedione monoxime. After 3 hours, cells were switched to 199 medium, loaded with 1.5 μmol / L fura-2,AM (Invitrogen, #F1221) for 20 minutes at 37°C, washed twice for 15 minutes, and transferred to a custom-made perfusion chamber. Cells were electrically stimulated using a MyoPacer EP stimulator (IonOptix) under constant recirculation with an isotonic electrolyte solution containing 117 mmol / L NaCl, 5.7 mmol / L KCl, 1.2 mmol / L NaH2PO4, 1.2 mmol / L CaCl2, 1.25 mmol / L MgSO4, 10 mmol / L glucose, 5 mmol / L sodium pyruvate, 10 mmol / L creatine, and 20 mmol / L Hepes (pH 7.4) (Mutig et al. Mol Immunol. 2013;56:720-728). Quiescent, rod-shaped cardiomyocytes with well-defined striations were randomly selected to respond to stimulation (1 Hz, 15 V, 4 ms impulse duration). Ca concentrations in single cells were measured by measuring the fluorescence emitted at 510 nm after excitation with alternating wavelengths of 340 and 380 nm using a dual-excitation fluorescence photomultiplier system (IonOptix) as previously described (Mutig 2013; Dobson et al. Am J Physiol Heart Circ Physiol. 2008;295:H2364-2372). 2+ Transients were recorded. Average background fluorescence was recorded separately from groups of 10 cells not loaded with fura-2,AM and subtracted before calculating the 340 nm / 380 nm fluorescence ratio (R). Data from 20 calcium transients per cell were averaged. The magnitude of the fura-2,AM ratio, the maximum rate of increase in the fura-2,AM ratio, and the time constant (τ) of the decay of the fura-2,AM ratio were analyzed using IonWizard 6.5.

[0154] Analysis of sarcomere contraction and relaxation in single cells. AMCM were plated on laminin-coated coverslips in 199 medium supplemented with 5% FCS and 10 mmol / L 2,3-butanedione monoxime. After 3 hours, cells were switched to 199 medium, transferred to a custom-made perfusion chamber, and electrically stimulated as described above. A rectangular region of interest containing 15–20 sarcomeres per cell was defined, and sarcomere length changes were recorded using a variable-speed CCD video camera (MyoCam-S, IonOptix) connected to an inverted microscope (Olympus IX71). A fast Fourier transform (FFT) algorithm was used to record sarcomere length changes during electrically paced contractions. Data from 20–30 twitches per cell were averaged. Contraction amplitude, maximum shortening velocity, and maximum relaxation velocity were analyzed using IonWizard 6.5 software (IonOptix).

[0155] Human plasma sample. Severe high-gradient aortic stenosis (valve area 0.65 ± 0.05 cm). 2 EDTA-treated plasma samples were obtained from 11 patients (age range 76-86 years, 3 men and 8 women) scheduled to undergo elective transcatheter aortic valve implantation (TAVI) at the Hannover Medical School, with echocardiographic evidence of coronary artery disease (any luminal diameter stenosis >50%), active inflammation or malignancy, and a blood pressure of 30 ml / min / 1.73 m. 2 Patients with an estimated glomerular filtration rate below 1.0 or signs of cardiac decompensation were excluded. A second plasma sample was collected during a routine follow-up examination 3 months after TAVI. Additionally, EDTA plasma samples were obtained from 13 apparently healthy individuals (75-84 years old, 3 men and 10 women) recruited at the University of Heidelberg (Giannitsis et al. Clin Biochem. 2020;78:18-24). Plasma samples were stored at -80°C. All participants provided written informed consent, and the local ethics committee approved the study.

[0156] Statistical Analysis. Mouse litters were randomly assigned to different experimental groups. Based on visual inspection, the data were normally distributed, and the variances in different groups were similar. Due to the small sample size, we did not apply statistical tests for normality or identity of variance. Data are presented as mean ± sem unless otherwise stated. Two independent sample t-tests were used to compare two groups. For comparisons between more than two groups, one-way analysis of variance was used when there was one independent variable, and two-way analysis of variance was used when there were two independent variables. Dunnett's post hoc test was used for multiple comparisons using a single control group. Tukey's post hoc test was used to adjust for multiple comparisons. A two-sided P value of less than 0.05 was considered to indicate statistical significance. KCW had full access to all data in the study and takes responsibility for the integrity of the data and data analysis. [Example 1]

[0157] The MYDGF protein (human factor 1; C19orf10) was identified as detailed in WO 2014 / 111458. The nucleic acid sequence encoding human factor 1 is available under NCBI gene ID: 56005 (SEQ ID NO: 6). The amino acid sequence of human factor 1, including the N-terminal signal peptide, is detailed in SEQ ID NO: 3. In the examples, human MYDGF without the signal peptide was used and expressed as detailed in Ebenhoch R. et al., Crystal structure and receptor-interacting residues of MYDGF - a protein mediating ischemic tissue repair [Nat Commun. 2019 Nov 26;10(1):5379 and Polten et al. Plasma Concentrations of Myeloid-Derived Growth Factor in Healthy Individuals and Patients with Acute Myocardial Infarction as Assessed by Multiple Reaction Monitoring-Mass Spectrometry. Anal Chem. 2019 Jan 15;91(2):1302-1308].

[0158] Mouse homologues to human MYDGF used in the examples: The mouse Mydgf gene was expressed from the mouse DNA segment Chr17, Wayne State University 104 (D17Wsu104e). The nucleic acid sequence encoding mouse factor 1 is available under the NCBI reference sequence: NM_080837.2 (SEQ ID NO: 7). The amino acid sequence of mouse factor 1, including the N-terminal signal peptide, is detailed in SEQ ID NO: 4. Because the N-terminal signal peptide has no associated biological function, mouse Mydgf without the N-terminal peptide according to SEQ ID NO: 2 was used in the present invention. For this purpose, the cDNA sequence of mouse Mydgf (containing the endogenous N-terminal signal peptide and a C-terminal 6xHis tag) was cloned into the pFlpBtM-II plasmid vector and expressed in HEK293-6E cells (Meyer S, et al. Multi-host expression system for recombinant production of challenging proteins. PLoS One. 2013;8:e68674). Mouse Mydgf lacking the signal peptide was purified from conditioned cell supernatants using affinity and size-exclusion chromatography. For the purification of recombinant Mydgf, a 6xHis tag was added to the protein.

[0159] Sequence comparison between human MYDGF (SEQ ID NO: 1) and mouse Mydgf (SEQ ID NO: 2) reveals 92% sequence identity between the two amino acid sequences. [Example 2]

[0160] Mouse and human bone marrow-derived growth factors dose-dependently inhibit endothelin-1 (ET1)-stimulated hypertrophy (endpoint, cell area) of neonatal rat ventricular myocytes (NRVMs). NRVMs were treated with 100 nmol / L ET1 (purchased from Sigma-Aldrich and used herein and below) and / or recombinant mouse myeloid-derived growth factor or recombinant human myeloid-derived growth factor (MYDGF; produced in HEK293 cells and used herein and below; see Ebenhoch R. et al., Crystal structure and receptor-interacting residues of MYDGF - a protein mediating ischemic tissue repair. Nat Commun. 2019 Nov 26;10(1):5379 and Polten F. et al. Plasma Concentrations of Myeloid-Derived Growth Factor in Healthy Individuals and Patients with Acute Myocardial Infarction as Assessed by Multiple Reaction Monitoring-Mass Spectrometry. Anal Chem. 2019 Jan 26). 15;91(2):1302-1308 and used here and below) for 24 hours. ET1 is a peptide hormone and a prototypic inducer of cardiomyocyte hypertrophy in vitro and in vivo (reviewed in Heineke J & Molkentin JD. Regulation of cardiac hypertrophy by intracellular signaling pathways. Nat Rev Mol Cell Biol 2006;7:589-600). Cardiomyocyte hypertrophy was determined by planimetry (endpoint, cell area). The results are shown in Table 2 below.

[0161] [Table 3] [Example 3]

[0162] Mouse and human bone marrow-derived growth factors inhibit hypertrophy (endpoint, protein content) of neonatal rat ventricular myocytes (NRVMs) stimulated with endothelin-1 (ET1). NRVMs were stimulated for 24 hours with 100 nmol / L ET1 and / or 100 ng / mL recombinant mouse bone marrow-derived growth factor or recombinant human bone marrow-derived growth factor (i.e., Mydgf or MYDGF, respectively). The results are shown in Table 3 below.

[0163] [Table 4] [Example 4]

[0164] Mouse bone marrow-derived growth factor inhibits hypertrophy (endpoint, cell size) of adult rat ventricular myocytes (ARVMs) stimulated with endothelin 1 (ET1). ARVMs were stimulated with 100 nmol / L ET1 and / or 100 ng / mL recombinant mouse bone marrow-derived growth factor (i.e., Mydgf) for 24 hours. Cardiomyocyte hypertrophy was determined by morphometry (cell length and cell width) and planimetry (cell area). The results are shown in Table 4 below.

[0165] [Table 5] [Example 5]

[0166] Murine bone marrow-derived growth factor upregulates sarcoplasmic reticulum Ca in endothelin-1 (ET1)-stimulated neonatal rat ventricular myocytes (NRVMs). 2+Increased expression of the ATPase (Serca2a) protein. NRVMs were stimulated with 100 nmol / L ET1 and / or 100 ng / mL recombinant murine bone marrow-derived growth factor (i.e., Mydgf) for 24 hours. Serca2a and vinculin protein expression levels were then determined by immunoblotting. Serca2a is an essential regulator of calcium homeostasis in cardiomyocytes. In experimental models of heart failure and in human heart failure, Serca2a (SERCA2a in humans) expression in cardiomyocytes is decreased, thus promoting functional decline and heart failure. SERCA2a has therefore been proposed as a therapeutic target in heart failure (reviewed in Kawase Y & Hajjar RJ. The cardiac sarcoplasmic / endoplasmic reticulum calcium ATPase: a potent target for cardiovascular diseases. Nat Clin Pract Cardiovasc Med 2008;5:554-65). The results are shown in Table 5 below.

[0167] [Table 6] [Example 6]

[0168] Muscle / endoplasmic reticulum Ca 2+Downregulation of ATPase (Serca2a) abolishes the antihypertrophic effect of mouse bone marrow-derived growth factor in neonatal rat ventricular myocytes (NRVMs) stimulated with endothelin-1 (ET1). NRVMs were transfected with small interfering (si)RNA targeting Serca2a (purchased from Thermo Fisher Scientific, catalog number 4390771, ID: s132037) or control siRNA (Thermo Fisher Scientific, catalog number 4390843). NRVMs were then stimulated with 100 nmol / L ET1 and / or 100 ng / mL recombinant mouse bone marrow-derived growth factor (i.e., ) for 24 hours. Cardiomyocyte hypertrophy was determined by planimetry (endpoint, cell area). The results are shown in Table 6 below.

[0169] [Table 7] [Example 7]

[0170] Mouse bone marrow-derived growth factor promotes antifibrotic effects (endpoints: collagen 1A1 and connective tissue growth factor [Tgfβ1] mRNA expression) in fibroblasts isolated from neonatal rat ventricles (NRVFs) stimulated with transforming growth factor β1 (Tgfβ1). NRVFs were stimulated with recombinant mouse Tgfβ1 (2 ng / mL; purchased from R&D Systems and used here and below) and / or 100 ng / mL recombinant mouse bone marrow-derived growth factor (Mydgf) for 24 hours. TGFβ1 is a key inducer of organ fibrosis (reviewed in Border WA & Noble NA. Transforming growth factor beta in tissue fibrosis. N Engl J Med 1994;331:1286-92). The results are shown in Table 7 below.

[0171] [Table 8] [Example 8]

[0172] Mouse bone marrow-derived growth factor promotes antifibrotic effects (endpoint, α-smooth muscle actin [SMA] promoter activity) in fibroblasts isolated from neonatal rat ventricles (NRVFs) stimulated with transforming growth factor β1 (Tgfβ1). NRVFs were transfected with a reporter plasmid encoding firefly luciferase under the control of the human αSMA promoter fragment (-259 / +51 base pairs) and then stimulated with recombinant mouse Tgfβ1 (2 ng / mL) and / or 100 ng / mL recombinant mouse bone marrow-derived growth factor (Mydgf) for 24 hours. The results are shown in Table 8 below.

[0173] [Table 9] [Example 9]

[0174] Human bone marrow-derived growth factor partially attenuates gene expression changes induced by transforming growth factor β1 (Tgfβ1) in fibroblasts (NRVFs) isolated from neonatal rat ventricles. NRVFs were incubated with recombinant mouse Tgfβ1 (2 ng / mL) and / or 100 ng / mL recombinant human bone marrow-derived growth factor (MYDGF) for 4 hours. PolyA RNA was isolated, converted to cDNA, and library preparation was performed, followed by gene expression profiling using next-generation sequencing. Reads were aligned to the rat reference genome, the number of reads mapping to each gene was counted, and differential genes across different treatment conditions (shown in Table 9) were computed using the limma package in Bioconductor, applying a corrected p-value cutoff of 0.1 and a fold-change cutoff of 1.5. Twenty-six genes downregulated by Tgfβ1 were upregulated by MYDGF, and 30 genes induced by Tgfβ1 were downregulated by MYDGF.

[0175] [Table 10-1] [Table 10-2] [Example 10]

[0176] Murine bone marrow-derived growth factor protein therapy promotes antihypertrophic and antifibrotic effects in mice subjected to transverse aortic constriction (TAC). C57BL6 / N wild-type mice underwent TAC surgery (originally described in Rockman HA et al. Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo mouse model of cardiac hypertrophy. Proc Natl Acad Sci USA 1991;88:8277-81). TAC exposes the left ventricle to prolonged pressure overload, leading to left ventricular (LV) hypertrophy and interstitial fibrosis (reviewed in Houser SR et al. Animal models of heart failure: A scientific statement from the American Heart Association. Circ Res 2012;111:131-50). Control mice underwent sham surgery (thoracotomy without aortic constriction). Immediately after TAC, mice received an intraperitoneal bolus injection of 10 μg of recombinant murine bone marrow-derived growth factor (Mydgf). Then, mice were treated with subcutaneous infusion of Mydgf (10 μg / day via osmotic minipump) for 7 days. Additional TAC mice were injected and infused with vehicle only (0.9% NaCl). After 42 days, LV hypertrophy was determined gravimetrically (endpoint, LV mass / body weight using a commercially available reference balance); LV interstitial fibrosis was quantified by Sirius Red staining. The results are shown in Table 10 below.

[0177] [Table 11] [Example 11]

[0178] Murine bone marrow-derived growth factor protein therapy improves cardiac function in mice subjected to transverse aortic constriction (TAC). C57BL6 / N wild-type mice underwent TAC surgery. Control mice underwent sham surgery (thoracotomy without aortic constriction). Immediately after TAC, mice received an intraperitoneal bolus injection of 10 μg of recombinant murine bone marrow-derived growth factor (Mydgf). Mice were then treated with subcutaneous infusion of Mydgf (10 μg / day via osmotic minipump) for 7 days. Additional TAC mice were injected and infused with vehicle only (0.9% NaCl). After 42 days, mice underwent high-resolution 2D transthoracic echocardiography using a 30 MHz linear transducer (Vevo 3100, VisualSonics). Left ventricular (LV) end-diastolic area (LVEDA) and left ventricular end-systolic area (LVESA) were determined from the long-axis view. The fractional area change (FAC) was calculated as a measure of systolic function [(LVEDA-LVESA) / LVEDA] x 100. The results are shown in Table 11 below.

[0179] [Table 12] [Example 12]

[0180] Protein therapy with murine bone marrow-derived growth factor reduces sarcoplasmic reticulum Ca in left ventricular cardiomyocytes isolated from mice subjected to transverse aortic constriction (TAC). 2+Increased expression of ATPase (Serca2a) protein. C57BL6 / N wild-type mice underwent TAC surgery. Control mice underwent sham surgery (thoracotomy without aortic constriction). Immediately after TAC, mice received an intraperitoneal bolus injection of 10 μg of recombinant mouse bone marrow-derived growth factor (Mydgf). Mice were then treated with subcutaneous infusion of Mydgf (10 μg / day via osmotic minipump) for 7 days. Additional TAC mice were injected and infused with vehicle alone (0.9% NaCl). Seven days later, Serca2a and β-actin protein expression levels were measured by immunoblotting in isolated left ventricular cardiomyocytes. The results are shown in Table 12 below.

[0181] [Table 13] [Example 13]

[0182] Mouse bone marrow-derived growth factor protein therapy reduces left ventricular cardiomyocyte hypertrophy in mice subjected to transverse aortic constriction (TAC). C57BL6 / N wild-type mice underwent TAC surgery. Control mice underwent sham surgery (thoracotomy without aortic constriction). Immediately after TAC, mice received an intraperitoneal bolus injection of 10 μg of recombinant mouse bone marrow-derived growth factor (Mydgf). Mice were then treated with subcutaneous infusion of Mydgf (10 μg / day via osmotic minipump) for 7 days. Additional TAC mice were injected and infused with vehicle only (0.9% NaCl). After 42 days, the cross-sectional area of ​​left ventricular cardiomyocytes was determined in tissue sections stained with wheat germ agglutinin. The results are shown in Table 13 below.

[0183] [Table 14] [Example 14]

[0184] Mouse bone marrow-derived growth factor gene therapy improves cardiac function and promotes antihypertrophic and antifibrotic effects in mice subjected to transverse aortic constriction (TAC). C57BL6 / N wild-type mice were lethally irradiated and transplanted with bone marrow stem cells transduced with lentivirus encoding mouse bone marrow-derived growth factor (Mydgf) or green fluorescent protein (GFP control). This lentiviral system has been previously described (Magnusson M et al. HOXA10 is a critical regulator for hematopoietic stem cells and erythroid / megakaryocyte development. Blood 2007;109:3687-96). Six weeks after transplantation, mice underwent TAC surgery. Control mice underwent sham surgery (thoracotomy without aortic constriction). Forty-two days later, mice underwent high-resolution two-dimensional transthoracic echocardiography using a 30 MHz linear transducer (Vevo 3100, VisualSonics). Left ventricular (LV) end-diastolic area (LVEDA) and left ventricular end-systolic area (LVESA) were determined from the long-axis view. The fractional area change (FAC) was calculated as a measure of systolic function [(LVEDA-LVESA) / LVEDA] x 100. Left ventricular end-diastolic posterior wall thickness, a measure of left ventricular hypertrophy, was determined from the short-axis view. LV interstitial fibrosis was quantified by Sirius red staining. The results are shown in Table 14 below.

[0185] [Table 15] [Example 15]

[0186] Human bone marrow-derived growth factor inhibits transforming growth factor-β1 (TGFβ1)-stimulated SMAD phosphorylation in lung fibroblasts from patients with idiopathic pulmonary fibrosis. Lung fibroblasts from two patients with idiopathic pulmonary fibrosis were stimulated with 2 ng / mL recombinant human TGFβ1 (purchased from R&D Systems, used herein and below) for 15, 30, or 60 minutes in the absence or presence of 100 ng / mL recombinant human bone marrow-derived growth factor (MYDGF; produced in HEK293 cells and used herein and below). SMAD phosphorylation (activation) was determined by immunoblotting. The SMAD signaling pathway is an essential mediator of the profibrotic effects of TGFβ1 (reviewed in Walton KL et al. Targeting TGFβ-mediated SMAD signaling for the prevention of fibrosis. Front Pharmacol 2017;8:461). The results are shown in Table 15 below.

[0187] [Table 16] [Example 16]

[0188] Human bone marrow-derived growth factor inhibits the migration of lung fibroblasts from patients with idiopathic pulmonary fibrosis. Lung fibroblasts from two patients with idiopathic pulmonary fibrosis were grown to confluency. The monolayers were scratched with a 200 μL pipette tip, washed, and cultured for 16 hours in the absence or presence of 2 ng / mL human transforming growth factor β1 (TGFβ1) and / or 100 ng / mL recombinant human bone marrow-derived growth factor (MYDGF). Digital phase-contrast images were captured before (0 hours) and after (16 hours) stimulation. The percent recovery was calculated as [(cell-free area at 0 hours) - cell-free area at 16 hours) / cell-free area at 0 hours] × 100. The results are shown in Table 16 below.

[0189] [Table 17] [Example 17]

[0190] Murine bone marrow-derived growth factor inhibits the migration of lung fibroblasts from patients with idiopathic pulmonary fibrosis. Lung fibroblasts from two patients with idiopathic pulmonary fibrosis were grown to confluency. The monolayer was scratched with a 200 μL pipette tip, washed, and cultured for 16 hours in the absence or presence of 2 ng / mL human transforming growth factor β1 (TGFβ1) and / or 100 ng / mL recombinant murine bone marrow-derived growth factor (Mydgf). Digital phase-contrast images were captured before (0 hours) and after (16 hours) stimulation. The percent recovery was calculated as [(cell-free area at 0 hours - cell-free area at 16 hours) / cell-free area at 0 hours] × 100. The results are shown in Table 17 below.

[0191] [Table 18] [Example 18]

[0192] Human bone marrow-derived growth factor inhibits transforming growth factor-β1 (TGFβ1)-stimulated SMAD phosphorylation in lung fibroblasts from patients with idiopathic pulmonary fibrosis. Lung fibroblasts from patients with idiopathic pulmonary fibrosis were stimulated with 2 ng / mL recombinant human TGFβ1 for 5, 15, 30, or 60 minutes in the absence or presence of 100 ng / mL recombinant human bone marrow-derived growth factor (MYDGF). SMAD phosphorylation (activation) was determined by immunoblotting. SB431542 (10 μmol / L; purchased from Sigma-Aldrich and used here and below), a small molecule inhibitor of the ALK5 / TGFβ type I receptor, was used as a positive control. The results are shown in Figure 1. [Example 19]

[0193] Mouse bone marrow-derived growth factor inhibits transforming growth factor-β1 (TGFβ1)-stimulated SMAD phosphorylation in left ventricular fibroblasts from patients with end-stage heart failure. Left ventricular lung fibroblasts from patients with end-stage heart failure were stimulated with 2 ng / mL recombinant human TGFβ1 for 30 minutes in the absence or presence of 100 ng / mL recombinant mouse bone marrow-derived growth factor (Mydgf). SMAD phosphorylation (activation) was determined by immunoblotting. SB431542 (10 μmol / L), a small molecule inhibitor of the ALK5 / TGFβ type I receptor, was used as a positive control. The results are shown in Figure 2. [Example 20]

[0194] Human bone marrow-derived growth factor inhibits transforming growth factor-β1 (TGFβ1)-stimulated SMAD phosphorylation in left ventricular fibroblasts from patients with end-stage heart failure. Left ventricular pulmonary fibroblasts from patients with end-stage heart failure were stimulated with 2 ng / mL recombinant human TGFβ1 for 30 minutes in the absence or presence of 100 ng / mL recombinant human bone marrow-derived growth factor (MYDGF). SMAD phosphorylation (activation) was determined by immunoblotting. SB431542 (10 μmol / L), a small molecule inhibitor of the ALK5 / TGFβ type I receptor, was used as a positive control. The results are shown in Figure 3. [Example 21]

[0195] Mouse bone marrow-derived growth factor inhibits mouse embryonic fibroblast (MEF) migration. MEFs were grown to confluency. The monolayer was scratched with a 200 μL pipette tip, washed, and cultured for 16 hours in the absence or presence of 2 ng / mL mouse transforming growth factor β1 (Tgfβ1) and / or 100 ng / mL recombinant mouse bone marrow-derived growth factor (Mydgf). Digital phase-contrast images were captured before (0 hours) and after (16 hours) stimulation. The percent recovery was calculated as [(cell-free area at 0 hours - cell-free area at 16 hours) / cell-free area at 0 hours] × 100. The results are shown in Table 21 below.

[0196] [Table 19] [Example 22]

[0197] Mouse bone marrow-derived growth factor inhibits transforming growth factor β1 (Tgfβ1)-stimulated Smad phosphorylation in mouse embryonic fibroblasts (MEFs). MEFs were stimulated with 2 ng / mL recombinant mouse Tgfβ1 for 30 minutes in the absence or presence of 100 ng / mL recombinant mouse bone marrow-derived growth factor (Mydgf). Smad phosphorylation (activation) was determined by immunoblotting. The results are shown in Figure 4. [Example 23]

[0198] Inflammatory cell-derived MYDGF attenuates cardiac remodeling during pressure overload. To explore the function of MYDGF during pressure overload, Mydgf KO mice and their WT littermates were subjected to transcatheter arterial coronary artery (TAC). Mydgf KO mice thrive and develop normally and do not exhibit any overt cardiovascular phenotypes at baseline (Korf-Klingebiel 2015). The pressure gradient across the site of aortic constriction and the ratio of peak blood flow velocities between the left and right common carotid arteries were similar in WT and KO mice. Despite comparable aortic constriction severity, KO mice developed more pronounced LV hypertrophy than WT mice (Figure 5A), and a greater increase in cardiomyocyte size was evident by histology and single-cell examination at 7 and 42 days after TAC (Figures 5B and 5C). Compared to WT mice, KO mice showed a stronger decline in Myh6 (alpha myosin heavy chain) mRNA expression at day 7, a greater increase in Myh7 (beta myosin heavy chain) mRNA at days 7 and 42, a greater increase in Nppa (natriuretic peptide type A) mRNA at day 42, and a similar increase in Nppb (natriuretic peptide type B) mRNA at both time points.

[0199] LV pressure-volume measurements showed that KO mice developed more pronounced systolic and diastolic dysfunction after TAC than WT controls (FIG. 5D; Table 22).

[0200] [Table 20]

[0201] Additionally, we generated bone marrow chimeric mice to specifically address the importance of inflammatory cell-derived MYDGF in modulating LV hypertrophy and cardiac function during pressure overload. Transplantation of WT bone marrow cells (BMCs) into KO mice inhibited LV hypertrophy (Figure 6A) and cardiomyocyte hypertrophy (Figure 6B) after TAC challenge, enhanced myocardial capillaryization (Figure 6C), and attenuated LV dilation (Figure 6D) and systolic dysfunction (Figure 6E). Conversely, transplantation of KO BMCs into WT mice enhanced hypertrophy, reduced capillary density, and exacerbated LV remodeling and systolic dysfunction (Figure 6A–6E). Using lentiviral gene transfer, we enabled inducible, inflammatory cell-specific overexpression of MYDGF in WT mice (Figure 6F).

[0202] In mice transplanted with Lenti.MYDGF-transduced BMCs, the addition of doxycycline to drinking water increased MYDGF protein expression in BMCs (5.8 ± 2.2-fold compared with Lenti.MYDGF without doxycycline) and splenocytes (9.1 ± 2.7-fold compared with Lenti.MYDGF without doxycycline) (Figure 6G). After TAC surgery, doxycycline-treated Lenti.MYDGF mice had higher plasma concentrations of MYDGF (Figure 6H) and greater LV MYDGF expression levels (Figure 6I) than doxycycline-treated Lenti. control mice. Doxycycline-treated Lenti.MYDGF mice developed milder LV hypertrophy after TAC than doxycycline-treated Lenti.control mice (Figure 6J), with smaller cardiomyocytes (Figure 6K), slightly higher capillary density (P = 0.11, Figure 6L), less LV dilation (Figure 6M), and preserved systolic function (Figure 6N). Therefore, we conclude that inflammatory cell-derived MYDGF is necessary and sufficient to limit maladaptive hypertrophy and remodeling during prolonged pressure overload. [Example 24]

[0203] Phosphoproteomic analysis identifies PIM1 as a target of MYDGF signaling in cardiomyocytes. An in vitro hypertrophy model was established to assess whether MYDGF directly targets cardiomyocytes. 24-hour ET1 stimulation increased the size (Figure 7B), protein content (Figure 7C), and mRNA gene expression of Myh7 and Nppa (Figure 7D) in neonatal rat ventricular cardiomyocytes (NRCMs). Recombinant MYDGF alone had no effect on these endpoints, whereas cotreatment with MYDGF completely prevented the hypertrophic response to ET1; the antihypertrophic effect of MYDGF was concentration-dependent and saturable at a half-maximal inhibitory concentration of 7.6 ng / mL (Figure 7B). MYDGF similarly inhibited AngII-induced hypertrophy but did not attenuate hypertrophy in response to IGF1 (Figure 7A). In contrast to the pathological type of cardiac hypertrophy promoted by ET1 and AngII, insulin-like growth factor 1 (IGF1) promotes a physiological type of cardiac hypertrophy in neonatal rat cardiomyocytes that is not attenuated by MYDGF.

[0204] To identify signaling intermediates activated by MYDGF, we performed phosphoproteome analysis followed by computational substrate-based kinase activity inference (Figure 8A) (Hogrebe et al. Nat Commun. 2018;9:1045; Strasser et al. Integr Biol. 2019;11:301-314). High-content LC-MS / MS data were collected 8 hours after NRCMs were stimulated with ET1 in the absence or presence of MYDGF. 423 of 2,308 quantified phosphorylation sites distributed across 1,110 distinct proteins were differentially phosphorylated across the four experimental conditions when scaled for the corresponding protein abundance differences.

[0205] Unsupervised hierarchical clustering and principal component analysis (Figure 8B) showed that biological replicates from each condition clustered together, thereby confirming the reproducibility of the workflow. The four conditions were well separated in principal component space, indicating that they were associated with distinct phosphoproteome signatures. Using Euclidean distance as a metric of similarity, we observed that NRCMs co-treated with ET1 and MYDGF displayed an intermediate phenotype that fell between ET1 and unstimulated controls (Figure 8B). Indeed, MYDGF reversed many of the phosphoproteome changes induced by ET1: phosphorylation sites that were more highly phosphorylated in ET1-treated cells (compared to unstimulated controls) tended to be less highly phosphorylated in cells co-stimulated with ET1 and MYDGF (compared to ET1 alone), and vice versa (Figure 8C). We applied our prior knowledge of kinase-substrate interactions (Hornbeck et al. Nucleic Acids Res. 2015;43:D512-520) to the phosphoproteomic dataset to infer changes in kinase activity induced by MYDGF in ET1-treated cells. Based on the differentially regulated phosphorylation sites and their respective enrichment of kinase substrate motifs, MYDGF was predicted to alter the activity of multiple protein kinases, including potent activation of the serine / threonine kinase PIM1 (Figure 8D).

[0206] PIM1 and its isoforms, PIM2 and PIM3, have similar substrate preferences but distinct tissue expression profiles (Selten et al. Cell. 1986;46:603-611; Qian et al. J Biol Chem. 2005;280:6130-6137; Nawijn et al. Nat Rev Cancer. 2011;11:23-34), with PIM1 being the predominant isoform in the heart (Muraski et al. Nat Med. 2007;13:1467-1475). PIM kinases are constitutively active and regulated at the level of protein expression (Nawijn 2011). Previous studies have shown that overexpression of PIM1 protected NRCMs from ET1-induced hypertrophy (Muraski 2007), thereby identifying PIM1 as a potential candidate mediating the effects of MYDGF in cardiomyocytes. Substantiating our phosphoproteomic data, MYDGF increased PIM1 expression (Figure 8E) and kinase activity (Figure 8F) in unstimulated and ET1-stimulated NRCMs. The antihypertrophic effects of MYDGF were suppressed by the small molecule PIM kinase inhibitor SMI4a (Beharry et al. Mol Cancer Ther. 2009;8:1473-1483; Xia et al. J Med Chem. 2009;52:74-86) (Figure 8G) and siRNA-mediated downregulation of PIM1 (Figure 8H), demonstrating that MYDGF indeed signals through PIM1. [Example 25]

[0207] MYDGF enhances SERCA2a expression in cardiomyocytes via PIM1. Ca2+, which delays contraction and relaxation of cardiomyocytes, is a key factor influencing cardiomyocyte contraction and relaxation. 2+ Abnormalities in cycling are common in the hypertrophied and failing heart (Houser et al. J Mol Cell Cardiol. 2000;32:1595-1607). Sarco / endoplasmic reticulum Ca 2+ Decreased expression and / or activity of ATPase 2a (SERCA2a) inhibits Ca transport into the sarcoplasmic reticulum. 2+This leads to a reduction in Ca sequestration in heart failure. 2+ This may contribute to the dysregulation of SERCA2a expression (Luo et al. Circ Res. 2013;113:690-708). Because PIM1 has previously been reported to stimulate SERCA2a expression (Muraski et al. Nat Med. 2007;13:1467-1475), we explored whether SERCA2a abundance is regulated by MYDGF. Indeed, increased PIM1 expression in MYDGF-stimulated NRCMs was accompanied by increased Serca2a protein expression (Figure 9A). Inhibition of PIM1 or siRNA-mediated downregulation of PIM1 prevented the increase in Serca2a, indicating that MYDGF regulates SERCA2a expression via PIM1 (Figure 9B).

[0208] In vivo, SERCA2a protein levels in LV myocardium were comparable between sham-operated WT and Mydgf KO mice (Figure 9C). After TAC surgery, SERCA2a expression in WT mice was still maintained at day 7 (-17%, P = 0.14) but decreased at day 42. At both time points, SERCA2a expression in KO mice was significantly lower than that in WT mice (Figure 9C). Protein expression levels of PIM1 and SERCA2a in isolated LV cardiomyocytes from sham-operated WT and KO mice were similar (Figure 9D). PIM1 expression in cardiomyocytes after TAC increased in WT mice but not in KO mice. The inability of KO mice to upregulate PIM1 after TAC correlated with a significant decrease in SERCA2a abundance in cardiomyocytes (Figure 9D).

[0209] To specifically clarify whether inflammatory cell-derived MYDGF regulates the expression of PIM1 and SERCA2a in pressure-overloaded hearts, we subjected Mydgf bone marrow chimeric transgenic mice and bone marrow conditional transgenic mice to TAC surgery. The abundance of PIM1 and SERCA2a in the LV after TAC was enhanced by transplanting WT BMCs into Mydgf KO mice, whereas it was reduced by transplanting KO BMCs into WT mice (Figure 9E). Furthermore, the expression levels of PIM1 and SERCA2a in the LV of doxycycline-treated Lenti.MYDGF mice were higher than those of doxycycline-treated Lenti.control mice (Figure 9F). We conclude that inflammatory cell-derived MYDGF is necessary and sufficient to enhance the expression of PIM1 and SERCA2a in the pressure-overloaded left ventricle. [Example 26]

[0210] We explored the therapeutic potential of MYDGF during pressure overload. After TAC surgery, mice were treated with recombinant MYDGF for 28 days using subcutaneously implanted osmotic minipumps to ensure continuous protein delivery (10 μg / day) (Figure 10A). Three days after TAC, MYDGF plasma concentrations in MYDGF-treated mice were higher than in control mice treated with diluent alone (Figure 10B). Seven days after TAC, MYDGF-treated mice showed more abundant SERCA2a protein expression in isolated ventricular cardiomyocytes (Figure 10C), and the LV dilation (Figure 10D) and systolic dysfunction (Figure 10E) that these animals developed over the 28-day period were less pronounced. The anti-remodeling effect was associated with an attenuated hypertrophic response (Figure 10F) and associated smaller cardiomyocytes (Figure 10G), increased capillary density in the LV myocardium (Figure 10H), and a significant survival benefit (Figure 10I).

[0211] In particular, Examples 23, 24, 25, and 26 demonstrate the protection of mice from pressure overload-induced heart failure by MYDGF. Acute pressure overload induced by TAC surgery led to the emergence of monocytes and macrophages as the major MYDGF-producing cell types, and induced a rapid increase in MYDGF abundance in the left ventricle. Circulating CCR2 high Monocyte recruitment and differentiation and proliferation of cardiac resident macrophages led to a significant expansion of the macrophage pool during pressure overload.

[0212] After TAC challenge, Mydgf KO mice developed severe LV hypertrophy with larger cardiomyocytes than wild-type mice. The greater hypertrophy in the KO mice was due to increased Ca 2+ It was characterized by impaired cycling and sarcomere function, more prominent fetal gene activation, reduced capillary density, increased interstitial fibrosis, and LV dilation and systolic and diastolic dysfunction, all hallmarks of a maladaptive response to pressure overload.

[0213] Exercise training did not induce an expansion of myeloid cells in the heart. Thus, MYDGF expression was relatively low in the left ventricles from trained and untrained mice, and trained Mydgf KO mice developed physiological hypertrophy similar to their wild-type littermates.

[0214] Acting on cardiomyocytes, MYDGF reduces cell hypertrophy and enhances SERCA2a expression, thereby reducing Ca 2+ Improved cycling and sarcomere function. Phosphoproteomics identified the constitutively active serine / threonine kinase PIM1 as a potential downstream target of MYDGF. Indeed, inhibiting or downregulating PIM1 abolished the antihypertrophic and SERCA2a-inducing effects of MYDGF in cultured cardiomyocytes.

[0215] Recombinant MYDGF treatment was shown to mediate beneficial effects on LV geometry, systolic function, and survival during sustained afterload stress. The present disclosure relates, for example, to the following: [1] Myeloid-derived growth factor (MYDGF) or a fragment or variant thereof exhibiting the biological function of MYDGF for use in the treatment or prevention of fibrosis or hypertrophy. [2] MYDGF or a fragment or variant thereof which exhibits the biological function of MYDGF for use in the treatment or prevention of heart failure, preferably chronic heart failure. [3] MYDGF or a fragment or variant thereof for use according to [2] above, wherein the heart failure or chronic heart failure is heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), or heart failure with mildly reduced ejection fraction (HFmrEF). [4] (i) SEQ ID NO: 1; or (ii) a fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF MYDGF for use according to [1], [2] or [3] above, comprising an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1. [5] The growth factor MYDGF for use according to any one of [1] or [4] above, wherein the fibrosis is cardiac and / or pulmonary fibrosis, preferably the fibrosis is interstitial lung disease, preferably progressive fibrosing interstitial lung disease, more preferably idiopathic pulmonary fibrosis. [6] The growth factor MYDGF for use according to [1] or [4] above, wherein the hypertrophy is hypertrophy of cardiomyocytes. [7] A nucleic acid encoding the growth factor MYDGF or a fragment or variant thereof which exhibits the biological function of MYDGF for use in the treatment or prevention of fibrosis or hypertrophy. [8] A nucleic acid encoding the growth factor MYDGF or a fragment or variant thereof which exhibits the biological function of MYDGF for use in the treatment or prevention of heart failure. [9] A nucleic acid for use according to [7] or [8] above, which encodes an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1.

[10] A vector comprising the nucleic acid according to [7] or [8] above for use in treating or preventing fibrosis or hypertrophy.

[11] A vector comprising the nucleic acid according to [8] or [9] above, for use in treating or preventing heart failure.

[12] A host cell comprising and expressing the nucleic acid according to [7] or the vector according to

[10] for use in treating or preventing fibrosis or hypertrophy.

[13] A host cell comprising and expressing the nucleic acid according to [8] or the vector according to

[11] for use in the treatment or prevention of heart failure.

[14] A pharmaceutical composition for use in the treatment or prevention of fibrosis or hypertrophy, comprising the MYDGF described in any one of [1], [4] to [6] above, the nucleic acid described in [6] or [8] above, the vector described in [9] above, or the host cell described in

[11] above, and optionally containing a suitable pharmaceutical excipient.

[15] A pharmaceutical composition for use in the treatment or prevention of heart failure, comprising the MYDGF described in any one of [2] to [6] above, the nucleic acid described in [8] or [9] above, the vector described in

[11] above, or the host cell described in

[13] above, and optionally containing a suitable pharmaceutical excipient.

[16] The pharmaceutical composition for use according to

[14] or

[15] above, which is administered by oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intracoronary route.

[17] The pharmaceutical composition for use according to

[16] above, wherein the administration is by one or more bolus injections and / or infusion.

[18] A method for treating fibrosis, comprising administering to a patient in need thereof a therapeutically effective amount of MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF.

[19] The MYDGF (i) SEQ ID NO: 1; or (ii) a fragment or variant thereof that exhibits the biological function of MYDGF of SEQ ID NO: 1 The method of claim 18, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[20] The method according to

[18] , wherein the fibrosis is cardiac and / or pulmonary fibrosis, and preferably the fibrosis is interstitial lung disease, preferably progressive fibrosing interstitial lung disease, more preferably idiopathic pulmonary fibrosis.

[21] The method of

[18] , wherein the MYDGF or a fragment or variant thereof exhibiting the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier.

[22] A method for treating hypertrophy, comprising administering to a patient in need thereof a therapeutically effective amount of MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF.

[23] The MYDGF (i) SEQ ID NO: 1; or (ii) a fragment or variant thereof that exhibits the biological function of MYDGF of SEQ ID NO: 1 The method of claim 22, wherein the fragment or variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[24] The method according to

[22] above, wherein the hypertrophy is hypertrophy of cardiomyocytes.

[25] The method of

[22] , wherein the MYDGF or a fragment or variant thereof exhibiting the biological function of MYDGF is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier.

[26] A method for treating or preventing heart failure, comprising administering to a patient in need thereof a therapeutically effective amount of the growth factor MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF, preferably wherein the heart failure is chronic heart failure.

[27] The method for treating heart failure according to

[26] , wherein the heart failure or chronic heart failure is HFpEF or HFrEF, and preferably the HFpEF is stage C or stage D HFpEF, or the HFrEF is stage C or stage D HFrEF.

[28] The MYDGF (i) SEQ ID NO: 1; or (ii) a fragment or variant thereof that exhibits the biological function of MYDGF of SEQ ID NO: 1 The method according to

[26] or

[27] , wherein the fragment or variant comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

Claims

1. 1. A pharmaceutical composition comprising bone marrow-derived growth factor (MYDGF) or a fragment or variant thereof for use in the treatment or prevention of fibrosis, comprising: A pharmaceutical composition, wherein the fragment or variant of MYDGF is capable of increasing the expression of SERCA2a and comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:

1.

2. (i) SEQ ID NO: 1; or (ii) a fragment or variant of SEQ ID NO: 1 that is capable of increasing the expression of SERCA2a 2. The pharmaceutical composition for use according to claim 1, comprising:

3. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the fibrosis is cardiac and / or pulmonary fibrosis.

4. A pharmaceutical composition for use as described in claim 3, wherein the fibrosis is interstitial lung disease.

5. A pharmaceutical composition for use as described in claim 4, wherein the interstitial lung disease is progressive fibrosing interstitial lung disease.

6. A pharmaceutical composition for use as described in claim 5, wherein the progressive fibrosing interstitial lung disease is idiopathic pulmonary fibrosis.

7. A pharmaceutical composition comprising a nucleic acid encoding a fragment or variant thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 1, capable of increasing expression of the growth factor MYDGF or SERCA2a, for use in the treatment or prevention of fibrosis.

8. 8. The pharmaceutical composition for use according to claim 7, wherein the nucleic acid encodes an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

1.

9. A pharmaceutical composition comprising a vector containing the nucleic acid of claim 7 or 8 for use in the treatment or prevention of fibrosis.

10. A pharmaceutical composition comprising a nucleic acid according to claim 7 or 8 or a vector according to claim 9 and a host cell expressing said nucleic acid for use in the treatment or prevention of fibrosis.

11. A pharmaceutical composition for use according to any one of claims 1 to 10, further comprising suitable pharmaceutical excipients.

12. The pharmaceutical composition for use according to any one of claims 1 to 11, administered by oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intracoronary route.

13. 13. The pharmaceutical composition for use according to claim 12, wherein said administration is by one or more bolus injections and / or by infusion.

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