Bone marrow-derived growth factors for the treatment of cardiogenic shock.

Abstract

The present invention relates to a protein, myeloid-derived growth factor (MYDGF), or a nucleic acid encoding said protein, for use in the treatment and / or prevention of cardiogenic shock. The present invention also relates to a vector containing said nucleic acid, a host cell expressing said nucleic acid, a pharmaceutical composition containing said protein, nucleic acid, vector, or host cell, all for use in the treatment and / or prevention of cardiogenic shock, and a method for treating and / or preventing cardiogenic shock. The present invention further relates to a method for producing an animal model of cardiogenic shock, and an animal obtained by said method.

Description

[Technical Field]

[0001] Array List This application contains a Sequence Listing which has been submitted electronically in xml format, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical field of the invention The present invention relates to a protein, bone marrow-derived growth factor (MYDGF), for use in the treatment and / or prevention of cardiogenic shock. The present invention also relates to a novel animal model of cardiogenic shock. [Background technology]

[0003] Background of the Invention Myeloid-derived growth factor (MYDGF), also known as factor 1, is a protein encoded by open reading frame 10 (C19Orf10) on human chromosome 19. This protein was reported in 2007 as a novel secretory factor in the synovium through proteomic analysis of so-called fibroblast-like synoviocytes (FLS cells). A correlation between the secretion of this protein and inflammatory joint diseases has been suggested, despite the lack of experimental or statistical evidence (Weiler et al., Arthritis Research and Therapy 2007, "The identification and characterization of a novel protein, c19orf10, in synovium"). A corresponding patent application claims this protein as a therapeutic agent for joints and for the diagnosis and monitoring of tissue changes undergoing growth alterations (US 2008 / 0004232 A1, "Characterization of c19orf10, a novel synoviolin protein"). Another scientific paper described increased expression of this 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 exhibited growth-promoting effects on cultured hepatocellular carcinoma cells. Because C19Orf10 was originally thought to be an interleukin, it has also been called IL-25, IL-27, and IL-27W. However, the terms "IL-25" and "IL-27" have not been used consistently in the art and have been used to designate various different proteins. For example, US 2004 / 0185049 refers to a protein called IL-27 and discloses its use to regulate 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, EP 2 130 547 A1 refers to a protein as IL-25 and discloses its use in treating inflammation. This protein, also known in the art as IL-17E, is structurally different 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).

[0004] WO 2014 / 111458 discloses Factor 1 for promoting proliferation and inhibiting apoptosis in non-transformed tissues or cells, particularly for the treatment of acute myocardial infarction. It also discloses inhibitors of Factor 1 for medical use, particularly for the treatment or prevention of diseases in which angiogenesis is involved in the development or progression of the disease.

[0005] Korf-Klingebiel et al. (Nature Medicine, 2015, Vol. 21(2):140-149) reported that C19Orf10 is secreted by bone marrow cells after myocardial infarction and that this protein promotes cardiomyocyte survival and angiogenesis. The authors showed that bone marrow-derived monocytes and macrophages endogenously produce this protein, which protects and repairs the heart after myocardial infarction and named it bone marrow-derived growth factor (MYDGF). Notably, treatment with recombinant mouse Mydgf has been reported to reduce scar size and contractile dysfunction after myocardial infarction.

[0006] WO 2021 / 148411 discloses MYDGF for use in the treatment or prevention of fibrosis, cardiac hypertrophy, and heart failure, a clinical syndrome with poor prognosis that develops in response to persistent hemodynamic overload, myocardial injury, or genetic mutations.

[0007] Cardiogenic shock (CS) is a life-threatening condition characterized by acute cardiac output reduction resulting from severe systolic and / or diastolic myocardial dysfunction, leading to arterial hypotension, pulmonary congestion, severe end-organ hypoperfusion, and impaired tissue oxygenation. CS is accompanied by insufficient blood flow to the extremities and vital organs, including the heart, liver, kidneys, and brain. Severe end-organ hypoperfusion and impaired tissue oxygenation lead to elevated blood lactate levels, which are used to diagnose and monitor patients with CS. Clinical criteria for defining CS are summarized, for example, in a review by Vahdatpour et al. (Journal of the American Heart Association, Vol. 8(8), 2019, e011991). Hypoperfusion and impaired cardiac oxygenation gradually worsen cardiac performance in CS, triggering a downward spiral of increasing hemodynamic instability that significantly increases mortality.

[0008] Although CS can develop as a complication of myocardial infarction, the pathophysiology of uncomplicated myocardial infarction (without cardiogenic shock) differs significantly from that of (generally massive) myocardial infarction with cardiogenic shock. Patients with uncomplicated myocardial infarction do not develop severe end-organ hypoperfusion and impaired tissue oxygenation, do not experience acute and progressive cardiac deterioration (a downward spiral), and do not experience the significantly higher acute mortality rate observed in patients with CS.

[0009] Similarly, patients with heart failure, cardiac fibrosis, and cardiac hypertrophy do not acutely present with severe end-organ hypoperfusion and impaired tissue oxygenation, nor do they experience the acute progressive deterioration of cardiac function (negative spiral), nor do they suffer from the significantly higher acute mortality rates observed in patients with CS.

[0010] Given the distinct pathophysiology of these conditions, medical therapies known to improve outcomes in patients with uncomplicated myocardial infarction and in patients with heart failure, cardiac fibrosis, and cardiac hypertrophy (e.g., angiotensin-converting enzyme inhibitors and beta-blockers) are not useful or even contraindicated in CS. Indeed, to date, no medical treatments have been found to improve survival in patients with CS.

[0011] Therefore, there is an urgent need for means and methods to treat and / or prevent cardiogenic shock, and appropriate animal models of cardiogenic shock are needed to further elucidate the underlying pathophysiology and define novel therapies for this condition.

[0012] Summary of the Invention

[0013] Summary of the Invention The present invention provides bone marrow-derived growth factor (MYDGF), or a fragment or variant thereof exhibiting the biological function of MYDGF, for use in the treatment and / or prevention of cardiogenic shock.

[0014] According to a preferred embodiment, the MYDGF protein comprises SEQ ID NO: 1. According to one embodiment, the MYDGF protein comprises a fragment or variant of SEQ ID NO: 1 that exhibits a biological function of MYDGF and comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0015] According to certain preferred embodiments, the MYDGF protein consists of SEQ ID NO:1 or SEQ ID NO:3.

[0016] According to a further aspect, the present invention provides a nucleic acid encoding MYDGF or a fragment or variant thereof exhibiting a biological function of MYDGF for use in the treatment and / or prevention of cardiogenic shock.

[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 present invention for use in the treatment and / or prevention of cardiogenic shock.

[0019] 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 and / or prevention of cardiogenic shock. Preferably, the host cell expresses the nucleic acid.

[0020] According to yet another aspect, the present invention provides a pharmaceutical composition comprising a MYDGF protein, nucleic acid, vector or host cell of the present invention, and optionally a suitable pharmaceutical excipient and / or carrier, for use in the treatment and / or prevention of cardiogenic shock.

[0021] According to a preferred embodiment, the pharmaceutical composition for use is administered orally, intravenously, subcutaneously, intramucosally, intraarterially, intramuscularly or intracoronarily, preferably by one or more bolus injections and / or by infusion injections.

[0022] According to a further aspect, the present invention provides a method of treating and / or preventing cardiogenic shock comprising administering to a patient in need thereof a therapeutically effective amount of a bone marrow-derived growth factor (MYDGF) protein.

[0023] According to one embodiment, the MYDGF protein comprises a fragment or variant of SEQ ID NO:1 that exhibits a biological function of MYDGF and comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO:1.

[0024] According to yet another aspect, MYDGF or a fragment or variant thereof is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier and / or excipient.

[0025] According to a further aspect, the present invention provides a method for treating and / or preventing cardiogenic shock, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a myeloid-derived growth factor (MYDGF) protein or a fragment or variant thereof. According to one embodiment, the MYDGF protein comprises a fragment or variant of SEQ ID NO: 1 that exhibits a biological function of MYDGF and comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO: 1.

[0026] According to a preferred embodiment, the pharmaceutical composition comprises suitable pharmaceutical excipients.

[0027] According to another embodiment, the pharmaceutical composition is administered by one or more bolus injections and / or by infusion.

[0028] According to a further aspect, the present invention provides a method for producing a non-human mammalian model of cardiogenic shock, the method comprising: (i) transiently ligating a coronary artery of the mammal; (ii) establishing reperfusion; and (iii) mechanically ventilating the mammal at an inspired oxygen concentration of about 0.18 or less.

[0029] According to a preferred embodiment, the non-human mammal is a rodent, more preferably a mouse.

[0030] According to a preferred embodiment, the coronary artery is ligated for about 30 to about 90 minutes before reperfusion is established.

[0031] According to a preferred embodiment, the mammal is ventilated at an inspired oxygen concentration of about 0.16. [Brief explanation of the drawings]

[0032] [Figure 1]Figure 1: A: Schematic diagram of the cardiogenic shock model animal preparation. Eight- to ten-week-old male C57BL / 6 N mice were used to induce myocardial infarction (MI) by ligating the left proximal anterior descending artery for 60 minutes. Animals were subcutaneously administered 2 mg / kg butorphanol and 0.02 mg / kg atropine to suppress bronchial secretions. Anesthesia was induced with 3-4% isoflurane. After intubation, mice were mechanically ventilated, and anesthesia was maintained with 1-2% isoflurane. After reperfusion, a micromanometer-tipped conductance catheter was inserted through the right carotid artery to continuously record left ventricular (LV) pressure-volume (PV) loops. Mice were then artificially ventilated for 120 minutes at a fraction of inspired oxygen (FiO2) of 0.33 or 0.16. As shown in pilot experiments, an FiO of 0.33 was associated with normoxemia [nx; arterial oxygen partial pressure (PaO), 144 ± 16 mmHg; arterial oxygen saturation (SaO), 99 ± 1%], whereas an FiO of 0.16 resulted in mild hypoxemia (hx; PaO, 75 ± 16 mmHg; SaO, 89 ± 3%) (4–6 mice per group). Infarcted and sham-operated mice were then randomly ventilated with or without FiO and followed for 120 min, as described in Wang et al., Cardiovasc Res. 2021;117:2414–2415. B: Left ventricular pressure-volume loops taken after 120 min for sham-operated mice (sham) (dotted line) and mice with induced myocardial infarction (MI) (continuous line) ventilated under normoxic (nx) and hypoxic (hx) conditions. [Figure 2] Figure 2: Left ventricular end-systolic pressure (LVESP) (mmHg) (Figure 2A), cardiac output (mL / min) (Figure 2B), and arterial lactate concentration (mmol / L) (Figure 2C). Sham-operated mice (sham) (open circles) and myocardial infarction-induced mice (MI) (filled circles) were mechanically ventilated 120 min after surgery under normoxic (nx) or hypoxic (hx) conditions, respectively. MI mice mechanically ventilated with an FiO of 0.16 (MI-hx) gradually developed CS, defined by clinical features of CS, namely, hypotension (reduced LVESP), reduced cardiac output, and elevated lactate concentrations. MI mice mechanically ventilated with an FiO of 0.33 (MI-nx) remained hemodynamically stable. [Figure 3]Figure 3: Starting 60 minutes after the initiation of hypoxic mechanical ventilation, MI-hx mice were intravenously infused with dobutamine (5–7.5 ng / g / min via the left jugular vein, gradually increasing to an LVESP of 70 mmHg) (MI-hx, dobutamine). Saline-administered MI-hx mice served as controls (MI-hx, saline). Dobutamine improved the LVESP (Figure 3A) and cardiac output (Figure 3B) of MI-hx mice. Similar hemodynamic improvements are commonly observed in CS patients treated with dobutamine. [Figure 4] Figure 4: Phosphoproteome analysis of non-infarcted left ventricular myocardium using high-resolution mass spectrometry with unsupervised principal component analysis (6 animals per group). The four experimental groups were associated with distinct phosphoproteome signatures: Sham-nx = sham-operated mice with normoxia; Sham-hx = sham-operated mice with hypoxemia; MI-nx = infarcted mice with normoxia; MI-hx = infarcted mice with hypoxemia. [Figure 5]Figure 5: Volcano plot showing differences in regulated phosphorylation sites in non-infarcted left ventricular myocardium from myocardial infarction-induced CS (MI-hx) mice versus myocardial infarction-only (MI-nx) mice. The top 10 down- or up-regulated [p<0.05] phosphorylation sites are shown in black. MI-nx = infarcted mice with normoxemia; MI-hx = infarcted mice with hypoxemia. Abbreviations indicate proteins, and the amino acid position in parentheses indicates the phosphorylated residue of each protein. RIPR1 = Rho family interacting regulator of cell polarization 1. DDA1 = DET1- and DDB1-associated protein 1. OSB11 = oxysterol-binding protein-related protein 11. ODPA = pyruvate dehydrogenase E1 component subunit α. SCRIB = protein scribble homolog. SRF = serum response factor. KCNH2 = potassium voltage-gated channel subfamily H member 2. CSRP1 = cysteine-glycine-rich protein 1. MYH6 = Myosin-6. DP13 A = DCC-interacting protein 13 alpha. PDLI5 = PDZ and LIM domain protein 5. TITIN = Titin. HSPB1 = Heat shock protein beta 1. DESMIN = Desmin. LMNA = Prelamin A / C. XIRP1 = Xin actin-binding repeat-containing protein 1. TNNI3 = Troponin I. TIF1B = Transcription intermediary factor 1-beta. [Figure 6] Figure 6: Kaplan-Meier survival curve showing mortality over 120 min in mice in cardiogenic shock (MI-hx) treated with MYDGF or saline. [Figure 7] Figure 7: Bar graphs showing the results of PV loop recordings at the end of the 120-minute observation period. Heart rate (Figure 7A), left ventricular end-systolic volume (LVESV) (Figure 7B), left ventricular end-diastolic volume (LVEDV) (Figure 7C), cardiac output (Figure 7D), stroke volume (Figure 7E), and stroke work (Figure 7F) were measured when MYDGF or saline was administered to mice in cardiogenic shock (MI-hx). *p<0.05, **p<0.01, ***p<0.001. [Figure 8]Figure 8: Bar graphs showing the results of PV loop recording at the end of the 120-minute observation period. Left ventricular end-systolic pressure (LVESP) (Figure 8A), left ventricular end-diastolic pressure (LVEDP) (Figure 8B), left ventricular ejection fraction (LVEF) (Figure 8C), maximum rate of change in left ventricular pressure (dP / dtmax) (Figure 8D), minimum rate of change in left ventricular pressure (dP / dtmin) (Figure 8E), and left ventricular diastolic time constant (Tau) (Figure 8F) are from cardiogenic shock (MI-hx) mice administered MYDGF or saline. **p<0.01, ***p<0.001. [Figure 9] Figure 9: Bar graphs showing blood gas analysis results at the end of the 120-minute observation period. pH (Figure 9A), arterial oxygen tension (PaO2 (mmHg)) (Figure 9B), arterial oxygen saturation (SaO2 (%)) (Figure 9C), and lactate concentration (mmol / L) (Figure 9D) in cardiogenic shock (MI-hx) mice treated with MYDGF or saline. *P<0.05. [Figure 10] Figure 10: PV loop continuous recording results during a 120-minute observation period in the CS model. Cardiac output (mL / min) (Figure 10A) and left ventricular end-systolic pressure (LVESP) (mmHg) (Figure 10B) in MI-hx mice treated with saline (CS Saline) versus MI-hx mice treated with MYDGF (CS MYDGF). [Figure 11] Figure 11: Left ventricular risk area and infarct size determined at the end of the 120-minute observation period in the CS model. Saline-treated MI-hx mice (Saline) and MYDGF-treated MI-hx mice (MYDGF). Risk area / total LV area (unit: %) (Figure 11A), infarct size / risk area (unit: %) (Figure 11B). [Figure 12]Figure 12: Plasma concentrations of high-sensitivity cardiac troponin T (cTnT) and alanine aminotransaminase (ALT), a liver injury marker, at the end of a 120-minute observation period in the CS model. Left ventricular soluble nucleosome concentrations at the end of a 120-minute observation period in the CS model. cTnT (unit: ng / mL) (Figure 12A) and alanine aminotransaminase (Log2ALT, unit: U / L) (Figure 12B) were compared between saline-treated MI-hx mice (Saline) and MYDGF-treated MI-hx mice (MYDGF). Soluble nucleosome concentrations in the infarcted (I) versus non-infarcted (NI) regions of the left ventricle were measured by ELISA. The I / NI ratio is an indicator of cell death (Figure 12C). [Figure 13] Figure 13: Schematic representation of the treatment regimens used in the cardiogenic shock animal model in previous examples (Figures 6, 7, 8, 9, 10, 11 and 12). Abbreviations in the figure are as defined in Figure 1. [Figure 14] Figure 14: Kaplan-Meier survival curves showing mortality over 120 minutes of cardiogenic shock in wild-type mice (WT) and Mydgf gene-deficient (knockout) mice (MYDGF KO). [Figure 15] Figure 15: Cardiogenic shock exacerbates left ventricular tissue damage. Figure 15A shows the experimental setup. Figure 15B shows the experimental results, demonstrating that cardiogenic shock further exacerbates left ventricular tissue damage sustained in acute myocardial infarction. [Figure 16] Figure 16: Delayed treatment of cardiogenic shock. Figure 16A shows the experimental setup. Figure 16B shows the experimental results, demonstrating that cardiogenic shock can be treated by administering MYDGF after reperfusion has been established. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description of the Invention Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein. 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.

[0034] 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).

[0035] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology and recombinant DNA technology, as described in the art (e.g., Molecular Cloning: A Laboratory Manual, 2004). nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Furthermore, references in this field (e.g., Braunwald's Heart Disease. A Textbook of Cardiovascular Medicine, 9 th Conventional clinical cardiology methods, as described in the cardiology journal, are employed (see also: The Journal of Cardiology, Vol. 1, No. 1, pp. 111-112, 2011).

[0036] Throughout this specification and the appended claims, unless the context clearly dictates otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or groups of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0037] The term "about" used in connection with a numerical value is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0038] As used herein, the term "and / or" is meant to refer to either or both / all of the alternatives recited in the context of the term.

[0039] Nucleic acid molecules, also called nucleic acids, are understood to be polymers made from nucleotide monomers. Nucleotide monomers consist of a nucleic acid base, a five-carbon sugar (such as ribose or 2'-deoxyribose), and one to three phosphate groups. Polynucleotides are typically 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.

[0040] The term "open reading frame" (ORF) refers to a sequence of nucleotides that can be translated into amino acids. Generally, such an ORF contains a start 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. ORFs usually occur naturally or are artificially constructed by genetic engineering means. ORFs encode proteins, and the amino acids that are translated form a peptide-linked chain.

[0041] The terms "protein" and "polypeptide" are used interchangeably herein to refer to a 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. That is, 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 provide advantageous properties compared to the parent polypeptide, such as one or more of improved stability, increased biological half-life, and 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 of a therapeutic small molecule such as exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretins, and glucagon-like peptide 1 agonists including 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.

[0042] The term "amino acid" includes naturally occurring amino acids and 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 hydrophobicity scale of greater than 0.5, more preferably greater than 1.0, and even more preferably greater than 1.5, and 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 hydrophobicity index 1.8), methionine (Kyte-Doolittle hydrophobicity index 1.9), isoleucine (Kyte-Doolittle hydrophobicity index 4.5), leucine (Kyte-Doolittle hydrophobicity index 3.8), and valine (Kyte-Doolittle hydrophobicity index 4.2), or derivatives thereof having the Kyte-Doolittle hydrophobicity index defined above.

[0043] 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 referred to 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 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 substitution, insertion, deletion, N-terminal truncation, C-terminal truncation, or a combination thereof, and these changes may occur at one or more positions. In a preferred embodiment, variants usable in the present invention exhibit up to 23 total mutations (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) in the amino acid sequence (i.e., substitutions, insertions, deletions, N-terminal truncations, C-terminal truncations, etc.). A particularly preferred variant of SEQ ID NO: 1 is shown in SEQ ID NO: 3, which exhibits one additional amino acid (glycine) at its N-terminus (+G variant). Amino acid substitutions may be conservative, semi-conservative, and / or non-conservative. In preferred embodiments, a variant that can be used 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 substitutions, preferably conservative amino acid changes.

[0044] Exemplary substitutions occur 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. Exemplary semi-conservative and conservative substitutions are as follows: TIFF2025530232000002.tif117158

[0045] Mutations of A, F, H, I, L, M, p, V, W, or Y to C are semi-conservative if the new cysteine ​​remains a free thiol. Furthermore, one skilled in the art will understand that glycines at sterically demanding positions should not be substituted and that P should not be introduced into portions of proteins with α-helical or β-sheet structure.

[0046] Alternatively or additionally, a "variant" as used herein may 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. The term "at least 85% sequence identity" is used throughout this specification in relation to sequence comparison of polypeptides and polynucleotides. This expression preferably means 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 to each reference polypeptide or each reference polynucleotide.

[0047] A fragment of a protein includes an amino acid deletion, which may be an N-terminal truncation, a C-terminal truncation, an internal deletion, or any combination thereof. Such proteins including N-terminal truncations, C-terminal truncations, and / or internal deletions are referred to herein as "fragments." Fragments may be naturally occurring (e.g., splice variants) or 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 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. Ebenhoch R. et al., 2019, suggested that receptor interaction is likely to occur at the front / protrusion on the top surface of MYDGF and that Tyr73 may be a key residue for receptor interaction.

[0048] When two sequences are compared and no reference sequence is specified for calculating percent sequence identity, the sequence identity is calculated based on the longer of the two sequences being compared, unless otherwise specified.

[0049] The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. Such alignments can be performed with several art-known algorithms, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, J. 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) (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 from http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html is used, where the parameters used are the default parameters set at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html: alignment type = Slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0,1 slow pairwise alignment option and protein weight matrix = Gonnet, gap open = 10, gap extension = 0,20, gap distance = 5, no end gaps = none, output options: format = Aln w / number, order = aligned.

[0050] The sequence identity grade (sequence match) can be calculated using BLAST, BLAT, BlastZ (or BlastX), etc. A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol:403-410. BLAST protein searches are performed using the BLASTP program available, for example, 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 at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome: expect threshold=10, word size=3, max matches within query=0, matrix=BLOSUM62, gap cost=presence: 11 extension: 1, composition adjustment=conditional composition score matrix adjustment is used in conjunction with the non-redundant protein sequence database (nr) to obtain amino acid sequences homologous to Factor 1 and Factor 2 polypeptides.

[0051] To obtain gapped alignments for comparison, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Sequence matching analysis can 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 over the entire length of the longer sequence, unless otherwise specified.

[0052] As used herein, the term "host cell" refers to a cell that harbors a nucleic acid of the invention (e.g., in the form of a plasmid or virus). Such a host cell may be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a fungal cell, a plant cell, an animal cell). The cell may be transformed or untransformed. The cell may be an isolated cell, for example, in cell culture, or part of a tissue that is itself isolated or is part of a more complex tissue structure such as an organ or an individual.

[0053] 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 mouse or rat homologs. The amino acid sequence of the human homolog is encoded in open reading frame 10 (C19Orf10) on human chromosome 19. Preferably, MYDGF and Factor 1 protein refer to proteins consisting of, consisting essentially of, or comprising the core segment of human Factor 1 having the amino acid sequence according to SEQ ID NO:1.

[0054] According to a further preferred embodiment, MYDGF and Factor 1 protein each refer to a protein comprising SEQ ID NO: 3. In a further preferred embodiment, MYDGF refers to a protein consisting essentially of SEQ ID NO: 3. In a further preferred embodiment, MYDGF refers to a protein consisting of SEQ ID NO: 3.

[0055] As used herein, MYDGF, as defined herein, and fragments and variants thereof, includes pharmaceutically acceptable salts thereof.

[0056] 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, compared to the results obtained with a MYDGF protein of the invention shown in at least one of the Examples set forth herein below, achieve at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the effect reported for MYDGF relative to the indicated control.

[0057] As used herein, the terms "MYDGF" and "Mydgf" both refer to bone marrow-derived growth factor.

[0058] The term "cardiogenic shock" refers to a state of end-organ hypoperfusion due to heart failure and the inability of the cardiovascular system to supply adequate blood flow to the extremities and vital organs. Patients in cardiogenic shock exhibit persistent hypotension (systolic blood pressure <80–90 mmHg, or mean arterial pressure 30 mmHg below baseline, or vasopressor or inotropic support to maintain SBP >90 mmHg, or mean arterial pressure <70 mmHg, or systolic blood pressure <100 mmHg despite adequate fluid resuscitation), with evidence of end-organ dysfunction (e.g., as evidenced by urine output <30 mL / h, or urine output <0.5 mL / kg, cold extremities, mottled skin, serum lactate >2 mmol / L, metabolic acidosis, or altered mental status), and the presence of adequate or elevated filling pressures (left ventricular [LV] end-diastolic pressure ≥15 mmHg, or right ventricular (RV) end-diastolic pressure ≥10–15 mmHg), and a markedly reduced cardiac index (m 2 less than 2.2 L / min per minute) (Vahdatpour et al, Journal of the American Heart Association, Vol 8(8), 2019, e011991).

[0059] As used herein, "treating," "treatment," or "therapy" of a 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 individuals who previously had the disorder, (f) reducing mortality after the onset of the disease or disorder, (g) curing, and (h) preventing the disease. The term "ameliorating" is also encompassed by the term "treating." Accordingly, the term "treatment and / or prevention" of a condition or disease referred to herein means that the condition or disease is treated, prevented, or both.

[0060] The term "stroke volume" refers to the volume of blood pumped out of the right or left ventricle with one contraction. It is the difference between the end-diastolic volume (EDV) and the end-systolic volume (ESV).

[0061] The term "stroke work" refers to the work done by the left or right ventricle to pump stroke volume into the aorta or pulmonary artery, respectively.

[0062] The term "cardiac output" refers to the volume of blood pumped out of the ventricles per unit time.

[0063] The term “dP / dt min " and "dP / dt max " and " represent the minimum and maximum rates of change in ventricular pressure, respectively. Peak dP / dt is used as an index of ventricular function.

[0064] The term "isovolumic relaxation constant" or "Tau" refers to the exponential decay of ventricular pressure during isovolumic relaxation. Also called the ventricular diastolic time constant.

[0065] The term "inspired oxygen concentration" or "FiO2" refers to the mole or volume fraction of oxygen in the inspired gas. Natural air contains 21% oxygen, which corresponds to an FiO2 of 0.21.

[0066] As used herein, the terms "subject," "individual," and "patient" are used interchangeably and refer to individuals such as humans, non-human primates (such as apes and monkeys, e.g., chimpanzees), birds, fish, livestock such as cows, sheep, pigs, goats, and horses, domestic mammals such as dogs and cats, and laboratory animals, including rodents such as mice, rats, and guinea pigs. The terms do not denote a particular age or sex. In a particular sense, the subject is a mammal. In a preferred sense, the subject is a human. A subject may be healthy or may be suffering from or suspected of suffering from one or more diseases. A subject suffering from or suspected of suffering from one or more diseases is also referred to as a patient.

[0067] These descriptions and definitions are valid throughout this application unless otherwise stated.

[0068] array The sequences used in the present invention are shown below. SEQ ID NO: 1 (amino acid sequence of human MYDGF lacking the 31 aa N-terminal signal peptide): VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL SEQ ID NO: 2 (amino acid sequence of MYDGF including the N-terminal signal peptide (shown in bold and underlined); UniProtKB - Q969H8): TIFF2025530232000003.tif26135 SEQ ID NO: 3 (amino acid sequence of a [+G] MYDGF variant in which a G residue precedes the N-terminal V residue at position +1 of mature human MYDGF): GVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL SEQ ID NO: 4 shows the nucleic acid sequence of human factor 1 encoding MYDGF of SEQ ID NO: 3 (NCBI gene number: 56005).

[0069] Aspects The elements of the present invention are described below. While these elements are listed with specific embodiments, it is 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 invention to only those embodiments explicitly described. The specification should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, all permutations and combinations of elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0070] This disclosure is the first to demonstrate the anti-cardiogenic shock effect of MYDGF. Specifically, this disclosure demonstrates that administration of MYDGF to a mouse model of cardiogenic shock alleviates symptoms associated with cardiogenic shock. Specifically, this disclosure demonstrates that administration of MYDGF to a mouse animal model of cardiogenic shock increases cardiac output, stroke work, stroke volume, ventricular end-systolic pressure, and ventricular ejection fraction compared to control mice administered saline. This disclosure further demonstrates that MYDGF successfully treats cardiogenic shock and extends overall survival (see Examples below).

[0071] Thus, in a first aspect, the present invention provides the protein myeloid-derived growth factor (MYDGF) or a fragment or variant thereof for use in the treatment and / or prevention of cardiogenic shock. According to a preferred embodiment, the fragment or variant of MYDGF exhibits a biological function of MYDGF.

[0072] According to a preferred embodiment, the cardiogenic shock to be treated or prevented is during the course of (acute) myocardial infarction (AMI, MI), preferably ST-elevation myocardial infarction (STEMI), more preferably cardiogenic shock during the course of MI or STEMI during the course of reperfusion after percutaneous coronary intervention (PCI).

[0073] 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% sequence identity to SEQ ID NO: 1.

[0074] In a preferred embodiment of this aspect of the invention, the protein comprises the amino acid sequence of SEQ ID NO: 1, a fragment or variant thereof having at least 85% sequence identity to SEQ ID NO: 1. According to a preferred embodiment, the MYDGF fragment or variant exhibits the biological function of MYDGF. 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 maintain the function of the polypeptide. Such information can be obtained, for example, from homologous gene sequences, which 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 across species, preferably mammals. In a particularly preferred embodiment of the invention, the MYDGF protein consists of, consists essentially of, or comprises the amino acid sequence of SEQ ID NO: 1 or 3, or a fragment or variant thereof. According to a preferred embodiment, the MYDGF fragment or variant 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.

[0075] N-terminal deletion mutants are also encompassed, which may, for example, delete one or more amino acids from amino acid positions 1 to 24 (based on SEQ ID NO: 1), ie, from the N-terminal conserved region.

[0076] C-terminal deletion mutants are also encompassed, which may, for example, delete one or more amino acids from amino acid positions 114 to 142 (based on SEQ ID NO: 1).

[0077] Alternatively, amino acids can be added to the MYDGF protein. Such additions include additions at the N-terminus, C-terminus, or within the amino acid sequence, or a combination thereof. Thus, the protein of the first aspect of the present invention may further comprise an additional amino acid sequence, for example, to stabilize or purify the resulting protein. Examples of such amino acids are 6xHis tags, myc tags, or FLAG tags, 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 additional amino acids, if any, such as the above-mentioned tags, one or more residual amino acids may remain at the N-terminus and / or C-terminus of the protein. MYDGF proteins and Mydgf proteins of the present invention include, for example, those described 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.

[0078] In some cases, it may be preferable to mutate protease cleavage sites within the MYDGF protein of the first aspect of the invention to stabilize the protein (see Segers et al., Circulation 2007, 2011). Those skilled in the art know how to determine potential proteolytic cleavage sites within a protein. For example, protein sequences can be submitted to websites that provide analyses, such as http: / / web.expasy.org / peptide_cutter / and http: / / pmap.burnham.org / proteases. Submitting the protein sequence according to SEQ ID NO: 1 to http: / / web.expasy.org / peptide_cutter / will determine the following rare (less than 10) cleavage sites:

[0079] [Table 1]

[0080] These sites can be modified to remove each identified protease recognition / cleavage sequence to increase the serum half-life of the protein.

[0081] The MYDGF protein may contain additional amino acid sequences, for example, to stabilize or purify the resulting protein. For example, it may be preferable to mutate a protease cleavage site within the MYDGF protein to stabilize the protein. Suitable proteolytic cleavage sites can be identified as described above.

[0082] The MYDGF protein or compositions containing this protein can be administered in vivo, ex vivo or in vitro, preferably in vivo.

[0083] Nucleic acid sequences can be optimized to enhance expression in host cells. Parameters to consider include C:G content, preferred codons, and avoidance of inhibitory secondary structures. These factors can be combined in various ways to obtain a nucleic acid sequence with enhanced expression in a particular host (see, e.g., Donnelly et al., International Publication No. WO 97 / 47358). Whether a particular sequence can enhance expression in a particular host requires empirical experimentation. Such experimentation involves measuring the expression of a potential nucleic acid sequence and, if necessary, modifying the sequence. Starting from a particular amino acid sequence and the known degeneracy of the genetic code, numerous different encoding nucleic acid sequences can be obtained. The degeneracy of the genetic code arises because almost every amino acid is encoded by a combination of different nucleotide triplets, or "codons." Translation of specific codons into specific amino acids is well known in the art (see, e.g., Lewin, GENES IV, p. 119, Oxford University Press, 1990). Accordingly, the present invention also provides nucleic acids encoding MYDGF or fragments or variants thereof for use in the treatment and / or prevention of cardiogenic shock. According to a preferred embodiment, the fragment or variant of MYDGF exhibits a biological function of MYDGF. According to a further preferred embodiment, the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1.

[0084] Nucleic acids for use according to the present invention may further include transcriptional or expression control elements positioned to control protein expression. Such nucleic acids and control elements together are often referred to as an expression system. As used herein, the term "expression system" refers to a system designed to produce one or more gene products of interest. Generally, such systems are "artificial," i.e., engineered by genetic engineering tools that can be used to produce the gene products of interest in vivo, in vitro, or ex vivo. The term "expression system" further encompasses expression of the gene products of interest, including transcription of a polynucleotide, splicing of mRNA, translation into a polypeptide, co-translational and post-translational modification of the polypeptide or protein, and targeting of the protein to one or more compartments within the cell, 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 vary, and construction of expression systems for prokaryotic cells is well known in the art.

[0085] The control elements present in a gene expression cassette generally 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 linked 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 that is recognized by RNA polymerase and mediates transcription of downstream regions. Preferred promoters are strong promoters that result in increased transcription levels. Examples of strong promoters include the human cytomegalovirus early promoter (CMV) and CMV with intron A (Chapman et al., Nucl. Acids Res. 19:3979-3986, 1991). Further examples of promoters include naturally occurring promoters such as the EF1α promoter, the mouse CMV promoter, the Rous sarcoma virus promoter, the SV40 early / late promoter, and the [β]-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).

[0086] 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 responsible for cleaving the transcribed RNA and adding a poly(A) tail to the RNA. The polyadenylation signal of higher eukaryotes contains an AAUAAA sequence approximately 11-30 nucleotides from the polyadenylation addition site. The AAUAAA sequence is involved in signal transduction of RNA cleavage (Lewin, Genes IV, Oxford University Press, NY, 1990). The poly(A) tail is important for mRNA processing, transport from the nucleus, translation, and stability.

[0087] Polyadenylation signals that can be used as part of a gene expression cassette include the minimal rabbit [β]-globin polyadenylation signal and the bovine growth hormone polyadenylation signal (BGH) (Xu et al., Gene 272:149-156, 2001, Post et al., U.S. Patent No. 5,122,458).

[0088] Examples of additional regulatory elements 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.

[0089] The expression of the MYDGF proteins or variants thereof of the present invention can be regulated. Such regulation can occur at many steps in gene expression. Possible regulatory steps include, but are not limited to, transcription initiation, promoter clearance, transcription elongation, splicing, nuclear transport, mRNA stability, translation initiation, translation efficiency, translation elongation, and protein folding. Other regulatory steps that affect the concentration of MYDGF polypeptides within cells affect the half-life of the protein. Such regulatory steps include, for example, controlled protein denaturation. Because the proteins of the present invention are secreted proteins, they can be directed into the secretory pathway of the host cell. The efficiency of secretion, along with regulatory steps related to expression and protein stability, regulates the concentration of the respective proteins outside the cell. "Extracellular" can mean, for example, but is not limited to, culture medium, tissue, intracellular matrix or space, or bodily fluids such as blood or lymph.

[0090] The control of the above-mentioned regulatory steps 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 control of the regulatory step is cell-type or tissue-type specific. Such cell-type or tissue-type specific regulation is preferably achieved by a regulatory step 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 the respective polypeptide is enhanced in the respective cell or tissue compared to other cell or tissue types, or that expression is restricted to the respective cell or tissue type. Cell-type or tissue-type specific promoter sequences are well known in the art and are available for a wide range of cell or tissue types.

[0091] Expression is not necessarily cell-type or tissue-type specific, but may vary depending on physiological conditions. Such conditions may be, for example, inflammation or trauma. Such physiological condition-specific expression can also be achieved by regulating all of the above regulatory steps. A preferred method for controlling physiological condition-specific expression is transcriptional regulation. For this purpose, wound- or inflammation-specific promoters can be used. Each promoter may be, for example, a naturally occurring sequence, derived from a gene specifically expressed during immune response and / or regeneration of damaged tissue. Another possibility is the use of artificial promoter sequences, constructed, for example, by combining two or more naturally occurring sequences.

[0092] The regulation can be cell-type or tissue-type specific or physiological condition specific, in particular cardiac-specific expression. Preferably, expression is cardiac-specific and / or wound-specific.

[0093] Another possibility for regulating the expression of the MYDGF protein or its variant of the present invention is 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. The conditional regulation of gene expression by the Tet operator and the Tet repressor is well known in the art, and many respective systems have been established for a wide range of prokaryotes and eukaryotes. Those skilled in the art know how to select an appropriate system and adapt it to the specific needs of each application.

[0094] In a particularly preferred embodiment, the use of the nucleic acids of the invention involves application to individuals or patients, preferably individuals or patients suffering from cardiogenic shock.

[0095] According to a further aspect, the present invention provides a vector comprising a nucleic acid or expression system as described herein for use in the treatment and / or prevention of cardiogenic shock.

[0096] As used herein, the term "vector" refers to a protein, polynucleotide, or mixture thereof, which is or can be introduced into a cell to contain proteins and / or nucleic acids. Preferably, the gene of interest encoded by the introduced polynucleotide is expressed in the vector or host cell upon introduction of the vector. Examples of suitable vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors such as λ phage, filamentous phage vectors, viral vectors, virus-like particles, bacterial spores, and the like.

[0097] In a preferred embodiment of the present invention, the vector is a viral vector, including, but 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.

[0098] In particularly preferred embodiments of the present invention, the vector is an adenovirus or adeno-associated virus (AAV) vector.

[0099] Nucleic acids encoding one or more MYDGF proteins or variants thereof of the present invention can be introduced into a host cell, tissue, or individual using a vector suitable for therapeutic administration. A suitable vector will be able to deliver the nucleic acid to target cells without causing unacceptable side effects.

[0100] In a particularly preferred embodiment, the use of a vector according to the invention comprises application to an individual in need thereof.

[0101] Vectors containing nucleic acids encoding MYDGF protein or fragments or variants thereof preferably exhibit the biological functions of MYDGF described above and are used in the treatment and / or prevention of cardiogenic shock.

[0102] According to a further aspect, the present invention provides a host cell comprising the vector described herein and expressing a nucleic acid encoding a MYDGF protein, or a fragment or variant thereof, for use in the treatment and / or prevention of cardiogenic shock. According to a preferred aspect, the fragment or variant of MYDGF exhibits a biological function of MYDGF.

[0103] According to a further aspect, the present invention provides a pharmaceutical composition comprising a MYDGF protein or a fragment or variant thereof, and optionally a suitable pharmaceutical excipient, for use in the treatment and / or prevention of cardiogenic shock. According to a preferred aspect, the fragment or variant of MYDGF exhibits a biological function of MYDGF.

[0104] As used herein, the term "suitable pharmaceutical excipient" refers to a pharmacologically inactive substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer, 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 liquids such as physiological saline solutions in water, and oils, including, but not limited to, those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions, aqueous dextrose, and aqueous glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Saline is a preferred carrier 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, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. Such suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

[0105] As used herein, the terms "pharmaceutical composition," "medicament," and "drug" are used interchangeably and refer to a substance and / or combination of substances used in the identification, prevention, and / or treatment of a tissue condition or disease.

[0106] "Pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, especially humans. The term "pharmaceutically acceptable salt" refers to a salt of a protein or peptide of the present invention. Suitable pharmaceutically acceptable salts include, for example, acid addition salts that may be formed by mixing a solution of the peptide of the present 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 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 appropriate organic ligands (e.g., ammonium, quaternary ammonium, amine cations, etc., formed with counter anions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates, and arylsulfonates).Examples of pharmaceutically acceptable salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, tartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, glucaptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylsanilate, hemisulfinate, heptanoate, hexanoate, hexylresorcinate, hydrabamate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, Acid salts include, but are not limited to, 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, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, acetate, succinate, tannate, tartrate, thioclate, tosylate, triethiodide, undecanoate, valerate, and the like (e.g., SM See Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, pp. 1-19 (1977).

[0107] The term "active ingredient" refers to the biologically active substance in a pharmaceutical composition or formulation, i.e., the substance that provides pharmaceutical value. Pharmaceutical compositions can contain one or more active ingredients, which can act in conjunction with one another or independently. Active ingredients can be formulated in neutral or salt form. Pharmaceutically acceptable salts include, but are not limited to, those formed with free amino groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0108] The terms "formulation" and "composition" are intended to include a formulation of an active compound with an encapsulating material as a carrier to provide a capsule in which the active ingredient is surrounded by the carrier, with or without other carriers.

[0109] The term "carrier," as used herein, refers to a pharmacologically inert substance, such as a diluent, excipient, or vehicle, with which a therapeutically active ingredient is administered, but is not limited to such a substance. Such pharmaceutical carriers can be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids such as saline solutions in water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions, aqueous dextrose solutions, and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Saline solutions are preferred when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0110] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The term "composition" is preferably intended to include a formulation of the active compound with a carrier, e.g., an encapsulating material, to provide a capsule in which the active ingredient is surrounded by the carrier, with or without other carriers.

[0111] According to one embodiment, the active ingredient is administered to a cell, tissue, or individual in a therapeutically effective amount. A "therapeutically effective amount" is an amount of the active ingredient sufficient to achieve its 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 type of the individual receiving the active ingredient, and the purpose of administration. The effective amount in a particular case can be determined empirically by one skilled in the art according to methods established in the art. As used herein, "administration" includes in vivo administration to an individual as well as direct administration to a cell or tissue in vitro or ex vivo.

[0112] In a preferred embodiment of the present invention, the pharmaceutical composition is customized for the treatment of a disorder, in particular for the treatment of cardiogenic shock. In a further preferred embodiment of the present invention, the pharmaceutical composition is customized for the prevention of a disorder, in particular for the prevention of cardiogenic shock. According to a particularly preferred embodiment, the pharmaceutical composition is customized for the prevention and treatment of cardiogenic shock. In a particularly preferred embodiment of the present invention, treatment with the pharmaceutical composition according to the present invention includes treatment of an individual in need of such treatment and / or prevention of cardiogenic shock in an individual in need of such treatment.

[0113] Pharmaceutical compositions contemplated by the present invention can be formulated in a variety of ways known to those skilled in the art. For example, pharmaceutical compositions of the present invention may be in liquid form, 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, intramyocardial, or mucosal administration, preferably intravenous, subcutaneous, or intraperitoneal. Formulations for oral or anal 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, for example, sufficient salts or glucose to make the solution isotonic with blood. The aqueous solution should be suitably buffered (preferably pH 3-9, more preferably pH 5-7), if necessary. Pharmaceutical compositions are preferably in unit dosage form. In such form, the pharmaceutical composition is subdivided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form can be a sachet, which contains discrete quantities of the pharmaceutical composition, such as vials or ampoules.

[0114] The pharmaceutical compositions are preferably administered via intravenous, intraarterial, intrathecal, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary or intracardiac routes, although other routes of administration known in the art are also contemplated.

[0115] When a pharmaceutical composition is used as a treatment for an individual or in the prevention of a disease or disorder, the use of the pharmaceutical composition can replace standard treatment or prevention for the respective disease or condition, or can be administered in addition to standard treatment. In the case of additional use of the pharmaceutical composition, the pharmaceutical composition can be administered before, simultaneously with, or after standard treatment and / or prevention.

[0116] It is more preferred to administer the pharmaceutical composition once or more than once, 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 administration period of the pharmaceutical composition is not limited. Preferably, administration does not exceed 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0117] Single doses of the pharmaceutical composition may independently form the entire amount of the dosage, or each period of administration may comprise one or more bolus injections and / or administration as an infusion.

[0118] According to a further aspect, the present invention provides a method for treating and / or preventing cardiogenic shock, comprising administering a therapeutically effective amount of MYDGF, or a fragment or variant thereof, to a patient in need thereof. Suitable MYDGF proteins, fragments, or variants thereof include those described in the first aspect. According to a preferred embodiment, the fragment or variant of MYDGF exhibits a biological function of MYDGF. In said method, 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 preferably comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

[0119] Administration can be carried out, for example, as described for the first aspect. According to a preferred embodiment of this method of the invention, the MYDGF protein or fragment or variant thereof is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier and / or excipient.

[0120] According to a further aspect, the present invention provides a method for producing a non-human mammalian model of cardiogenic shock, comprising (i) transiently ligating a coronary artery of the mammal, (ii) establishing reperfusion, and (iii) mechanically ventilating the mammal at a fraction of inspired oxygen (FiO) of about 0.18 or less. The temporary ligation can be performed by methods known to those skilled in the art as suitable for blocking or significantly reducing blood flow. According to a preferred embodiment, the ligation is performed by placing and tightening a surgical thread or wire around the coronary artery, thereby blocking or substantially blocking blood flow through the artery. Reperfusion is preferably established by reestablishing blood flow, e.g., by opening the surgical thread or wire and allowing blood to flow through the artery. Mechanical ventilation of the mammal is performed by standard means known in the art, such as mechanical ventilation commonly used in mammalian surgery.

[0121] This method does not specifically treat animals but rather induces a state similar to cardiogenic shock. Cardiogenic shock is characterized by, for example, decreased ventricular end-systolic pressure (VESP), decreased cardiac output, and increased arterial lactate concentration compared to healthy controls or compared to controls before the procedure. This new nonhuman mammalian model of cardiogenic shock has the advantage of using small animals, such as rodents, which are well established in laboratory practice. It does not require the use of larger animals, such as pigs, which can increase feed, shelter, and husbandry costs and complicate investigations. This new model also makes it possible to study, for example, genetically modified mice to investigate the molecular mechanisms of cardiogenic shock. Genetically modified mice are easily generated and are already available in the research community or commercially available from sources such as the Jackson Laboratory. However, generating larger genetically modified animals, such as pigs, is extremely challenging, and only a limited number of genetically modified large animals are available.

[0122] Thus, in a preferred embodiment, the non-human mammal is a rodent, and in a particularly preferred embodiment, the non-human animal is a mouse.

[0123] In a preferred embodiment, the coronary artery is the proximal left anterior descending coronary artery. Alternatively, any other major coronary artery whose transient ligation reduces ventricular end-systolic pressure (VESP) and increases arterial lactate concentration can be used.

[0124] In a further preferred embodiment, the coronary artery is ligated for about 30 to about 90 minutes before reperfusion is established. In a further preferred embodiment, the coronary artery is ligated for a time period selected from a range having a lower limit of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 minutes and an upper limit of about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes, or any combination thereof. More preferably, the coronary artery is ligated for about 45 to 70 minutes, and most preferably for about 60 minutes.

[0125] In a further preferred embodiment, the mammal is ventilated at a fraction of inspired oxygen (FiO2) between about 0.18 and about 0.12. In a further preferred embodiment, the mammal is ventilated at a fraction of inspired oxygen selected from a range having a lower limit of about 0.12, 0.13, 0.14, 0.15, 0.16, or 0.17 and an upper limit of about 0.13, 0.14, 0.15, 0.16, 0.17, or 0.18, and any combination thereof. More preferably, the mammal is ventilated at a fraction of inspired oxygen between about 0.17 and about 0.14, and most preferably about 0.16.

[0126] The present invention also provides a model animal obtained by carrying out the method for producing a non-human mammalian model of cardiogenic shock. [Example]

[0127] Example The examples are presented to further illustrate and understand the present invention and are not to be construed as limiting the scope of the invention in any way.

[0128] Materials and methods used in the examples: Unless otherwise stated, the following materials and methods were used in the examples.

[0129] Recombinant MYDGF The [+G]MYDGF mutant (HEK) used in the examples has a G residue before the N-terminal V residue at position +1 of mature human MYDGF. This mutant has the sequence shown in SEQ ID NO: 3 and was produced as described in Polten, F. et al. (2019), Anal Chem, 91, 1302-1308, page 1303, first column and Figure S1, and Ebenhoch, R. et al. (2019), Nat Commun, 10, 5379, page 8, left column.

[0130] Mouse surgery and functional assessment All surgical procedures were approved by the authorities in Hannover, Germany (Niedersaechsisches Landesamt fuer Verbraucherschutz und Lebensmittelsicherheit). All animal experiments conformed to the guidelines of the EU Laboratory Animal Protection Directive 2010 / 63 on the protection of animals used for scientific purposes. 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 available ad libitum. During surgery, mice were placed on a heating pad connected to a thermostat (Foehr Medical Instruments) to maintain a rectal temperature of 37°C.

[0131] statistical analysis Litters of mice were randomly assigned to each experimental group. Visual inspection revealed that the data were normally distributed, with similar variances across groups. Data are presented as mean ± standard error. Two-group comparisons were performed using two independent samples t-tests. For comparisons of 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 with a single control group. Tukey's post hoc test was used to adjust for multiple comparisons. A two-tailed P value of less than 0.05 was considered statistically significant. KCW had full access to all data in this study and takes responsibility for the integrity of the data and data analysis.

[0132] Example 1: Establishment of an animal model of cardiogenic shock. Myocardial infarction (MI) was induced in 8-10 week-old male C57BL / 6 N mice by transient ligation of the left proximal anterior descending coronary artery for 60 minutes. To suppress bronchial secretions, animals were subcutaneously administered 2 mg / kg butorphanol and 0.02 mg / kg atropine. Anesthesia was induced with 3-4% isoflurane. After intubation, mice were mechanically ventilated, and anesthesia was maintained with 1-2% isoflurane. After left thoracotomy, the left anterior descending coronary artery (LAD) was ligated with a 7-0 prolene slipknot (Ethicon, catalog number EH7405) to induce ischemia, which was then removed 1 hour later to induce reperfusion. Sham-operated mice did not undergo ligation around the LAD. After coronary reperfusion, a micromanometer-tipped conductance catheter was inserted through the right carotid artery to record left ventricular pressure-volume loops. At the end of the protocol, arterial blood was collected from the left ventricle.

[0133] In a pilot study, coronary artery ligation alone only slightly reduced LV end-systolic pressure (LVESP) and did not increase arterial lactate, a biomarker of peripheral hypoperfusion and tissue hypoxia in cardiogenic shock (CS). Based on the premise that the combination of myocardial infarction and hypoxic mechanical ventilation may induce CS, we compared different fractions of inspired oxygen (FiO2) for mechanical ventilation of mice after reperfusion (medical O2 mixed with medical N2). During isoflurane anesthesia, infarcted and sham-operated mice were randomly ventilated in normoxemia (nx; arterial oxygen tension [PaO2], 144 ± 16 mmHg; arterial oxygen saturation [SaO2], 99 ± 1%) with an FiO2 of 0.33, or in mild hypoxemia (hx; PaO2, 75 ± 16 mmHg; SaO2, 89 ± 3%; 4–6 mice per group) with an FiO2 of 0.16 (Fig. 1A).

[0134] An example of a pressure-volume loop recorded after 120 min is shown (Figure 1B). Over the course of 120 min, LVESP (Figure 2A) and cardiac output (Figure 2B) gradually declined in MI-hx mice (MI with CS) but remained stable in MI-nx mice (MI without CS). After 120 min, MI-hx mice developed arterial hyperlactatemia (Figure 2C) and exhibited more severe systolic and diastolic dysfunction than MI-nx mice (LV ejection fraction, 18 ± 2 vs. 33 ± 2%, P < 0.001; LV end-diastolic pressure, 14 ± 1 vs. 9 ± 1 mmHg, p = 0.005). Heart rate was unaffected (475 ± 16 vs. 486 ± 8 min). -1 Mortality was higher in MI-hx mice (11 of 23 mice died) than in MI-nx mice (1 of 12 died, p = 0.031).

[0135] Intravenous dobutamine, an established symptomatic treatment for patients with CS (Vahdatpour et al. Journal of American Heart Association, Vol 8(8), 2019, e011991), increased LVESP (Figure 3A) and cardiac output (Figure 3B) and reduced arterial lactate (5.6±0.6 vs 11.1±0.7 mmol / L, p<0.001, 4-6 animals per group).

[0136] As a use case for this model, high-resolution mass spectrometry was used to define the phosphoproteome signature in non-infarcted LV myocardium after 120 minutes. Among 1,264 proteins, 9,004 phosphorylation sites were detected. Principal component analysis revealed that the four groups (Sham-nx = sham-operated mice with normoxia; Sham-hx = sham-operated mice with hypoxemia; MI-nx = infarcted mice with normoxia; MI-hx = infarcted mice with hypoxemia) were associated with distinct phosphoproteome signatures (Figure 4). Reflecting the observation that hypoxemia by itself does not alter cardiac performance (Figures 2A and 2B), the phosphoproteome signatures of sham-Hx and sham-nx mice overlapped (Figure 4). Conversely, the phosphoproteome signatures of MI-hx and MI-nx mice were significantly different (Figure 4), with 72 distinct phosphorylation sites being regulated (Figure 5).

[0137] A more detailed description is provided in Wang Y, Polten F, Jaeckle F, Korf-Klingebiel M, Kempf T, Bauersachs J, Freitag-Wolf S, Lichtinghagen R, Pich A, Wollert KC. A mouse model of cardiogenic shock. Cardiovasc Res. 2021;117:2414-2415, incorporated herein by reference in its entirety. The animal model of the present invention is the first of its kind and reproduces key features of CS in patients, including severe systolic and diastolic dysfunction, low cardiac output and hypotension, elevated arterial lactate levels, hemodynamic response to dobutamine, and high mortality. This model provides a platform for exploring the molecular pathophysiology of CS and developing much-needed therapies.

[0138] Example 2: The MYDGF protein (human factor 1; C19orf10) was identified as detailed in WO2014 / 111458. The nucleic acid sequence encoding human factor 1 is available at NCBI Gene ID: 56005 (SEQ ID NO: 4). The amino acid sequence of human factor 1, including the N-terminal signal peptide, is detailed in SEQ ID NO: 2. In the examples, the human [+G] MYDGF mutant without the signal peptide according to SEQ ID NO: 3 was used and expressed as described in detail 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).

[0139] Example 3: Acute myocardial infarction was induced in C57BL6 / N mice. They were pretreated with butorphanol (2 mg / kg, subcutaneously (sc)) and atropine (0.02 mg / kg, sc) to reduce bronchial secretions, followed by transient ligation of the left anterior descending coronary artery for 60 minutes. Human MYDGF (10 μg in 100 μL) or saline (control) was injected as a bolus into the left ventricular cavity during reperfusion. After reperfusion, a micromanometer-tipped conductance catheter was inserted through the right carotid artery, and left ventricular pressure-volume (PV) loops were continuously recorded. Cardiogenic shock (CS) was then induced by hypoxic mechanical ventilation (FiO2 0.16), and mice were continuously infused with MYDGF (5 μg / h, infusion rate: 2 μL / min) or saline (control) into the left jugular vein for 120 minutes. After 120 minutes, arterial blood (anticoagulated with heparin) was collected from the left ventricle and immediately analyzed for blood gases and lactate. Another blood sample was collected, treated with EDTA, and centrifuged at 3,500 g and 4°C for 10 minutes to obtain plasma. Figure 13 shows a schematic representation of this process (abbreviations in Figure 13 are as defined in Figure 1; sc = subcutaneous; iv = intravenous).

[0140] The results are shown in Figures 6 to 12. As shown in Figure 6, MYDGF significantly improved the survival rate of mice with cardiogenic shock.

[0141] The experiments further demonstrate that administration of MYDGF improves left ventricular systolic and diastolic function (Figures 7A-7F, and PV loop recordings in Figures 8A-8F).

[0142] MYDGF further reverses acidosis and reduces lactate levels, two hallmarks of cardiogenic shock (blood gas analysis in Figure 9).

[0143] MYDGF has also been shown to prevent the decline in cardiac output and LVESP, two additional features of cardiogenic shock (continuous PV loop recordings in Figures 10A and 10B).

[0144] This study also demonstrated that MYDGF treatment reduced infarct size (Figures 11A and 11B). To this end, left ventricles were removed at the end of the observation period, and the area at risk and infarct size were measured by Evans blue staining and 2,3,5-triphenyltetrazolium chloride (TTC) staining (method described in Korf-Klingebiel et al., Nat Med. 2015;21:140-149).

[0145] Figures 12A and 12B show the effects of MYDGF treatment on plasma troponin and alanine transaminase (ALT) concentrations measured at the end of the 120-minute observation period. Plasma troponin is an indicator of cardiac damage, while ALT reflects end-organ (liver) damage. MYDGF treatment of CS mice reduces both plasma troponin and alanine transaminase levels. The effect of MYDGF on cell death in the infarcted region was determined at the end of the 120-minute observation period by measuring soluble nucleosomes released as a result of cell death. Measurements were performed using a cell death detection ELISA commercially available from Roche. Figure 12C displays the ratio of soluble nucleosomes in the infarcted region (I) to the non-infarcted region (NI) as an indicator of cell death. It demonstrates that MYDGF significantly reduced the amount of soluble nucleosomes and, consequently, reduced cell death in the infarcted region.

[0146] Example 4: MYDGF knockout mice were obtained as described by Korf-Klingebiel et al. (Nature Medicine, 2015, Vol. 21(2):140-149). Cardiogenic shock (CS) was induced in wild-type (WT) and MYDGF knockout (KO) mice as described in Example 1. The mortality rates of WT and MYDGF knockout mice were compared during a 120-minute observation period. As shown in Figure 14, the mortality rate was significantly higher in KO mice, indicating that endogenous MYDGF has a protective effect in cardiogenic shock.

[0147] Example 5: For example, as shown in Figure 10, cardiac function continues to deteriorate during cardiogenic shock. This deterioration was thought to be caused by the continued worsening of left ventricular tissue damage during cardiogenic shock. To test this hypothesis, acute myocardial infarction was induced by transient ligation of the left anterior descending coronary artery for 60 minutes. After reperfusion, CS was induced by hypoxic mechanical ventilation (FiO2 0.16; MI / shock present). Infarcted control mice received normal mechanical ventilation (FiO2 0.33; MI / shock absent). Figure 15A shows an outline of the experimental setup.

[0148] Left ventricles were removed 60 min after reperfusion and at the end of the experiment (five mice without MI / shock and five mice with MI / shock at each time point), and cell death (soluble nucleosomes) was measured in the infarcted (I) and non-infarcted (NI) regions using a Roche cell death detection ELISA. The I / NI ratio was reported as an index of cell death. The results are shown in Figure 15B. The soluble nucleosome concentration in the infarcted region increased throughout the observation period in both groups (apoptotic cell death requires time to occur). The increase in soluble nucleosome concentration was significantly more pronounced in mice with MI / shock than in mice without MI / shock, indicating that MI further exacerbates the left ventricular tissue damage caused by acute MI.

[0149] Example 6: As shown in Example 5, cardiogenic shock further exacerbates left ventricular tissue damage caused by acute myocardial infarction. Therefore, we examined whether delayed MYDGF therapy could alleviate the symptoms of cardiogenic shock and improve cardiac function. To this end, mice were transiently ligated for 60 minutes to induce acute myocardial infarction. After reperfusion, a micromanometer-tipped conductance catheter was inserted into the right carotid artery, and left ventricular pressure-volume (PV) loops were continuously recorded. Cardiogenic shock was then induced by hypoxic mechanical ventilation (FiO20.16). Starting 60 minutes after reperfusion, mice were continuously infused into the left jugular vein with either (i) human MYDGF (5 μg / h, infusion rate: 2 μL / min) or (ii) saline (control). Blood samples (anticoagulated with heparin) were collected from the left ventricle until the end of the experiment, and blood gas and lactate analyses were performed immediately. Three mice with MI / shock received delayed MYDGF therapy and three mice with MI / shock received delayed saline. Figure 16A shows a schematic of the experimental set-up.

[0150] The results are shown in Figure 16B. One of the saline-treated control mice died. In surviving animals, cardiac function (e.g., left ventricular ejection fraction, LVEF) and peripheral tissue perfusion (e.g., lactate concentration) were better in MYDGF-treated mice than in saline-treated control mice. These data indicate that delayed MYDGF therapy also has beneficial effects in cardiogenic shock.

[0151] item The items of the present invention are as follows: Section 1. 1. A bone marrow derived growth factor (MYDGF), or a fragment or variant thereof exhibiting the biological function of MYDGF, for use in the treatment and / or prevention of cardiogenic shock. Section 2. MYDGF, (i) SEQ ID NO: 1; or (ii) a fragment or variant of SEQ ID NO: 1 that exhibits a 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; Item 1. MYDGF for use according to Item 1, comprising: Section 3. MYDGF for use according to item 1, wherein the MYDGF protein consists of SEQ ID NO: 1 or SEQ ID NO: 3. Section 4. A nucleic acid encoding MYDGF or a fragment or variant thereof exhibiting the biological function of MYDGF for use in the treatment of cardiogenic shock. Section 5. Item 5. The nucleic acid for use according to Item 4, wherein the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1. Section 6. A vector comprising the nucleic acid according to item 5 for use in the treatment and / or prevention of cardiogenic shock. Section 7. A host cell comprising the nucleic acid of paragraph 5 or the vector of paragraph 6 for use in the treatment and / or prevention of cardiogenic shock. Section 8. A pharmaceutical composition comprising the MYDGF protein of any one of paragraphs 1 to 3 or a fragment or variant thereof exhibiting the biological function of MYDGF, the nucleic acid of paragraph 4 or 5, the vector of paragraph 6, or the host cell of paragraph 7, for use in the treatment and / or prevention of cardiogenic shock. Section 9. Item 9. The pharmaceutical composition for use according to item 8, wherein the pharmaceutical composition is administered via oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intracoronary route. Section 10. Item 10. The pharmaceutical composition for use according to item 9, wherein the administration is via one or more bolus injections and / or infusion. Section 11. A method for treating and / or preventing cardiogenic shock, comprising administering a therapeutically effective amount of MYDGF or a fragment or variant thereof that exhibits the biological function of MYDGF to a patient in need thereof. Section 12. A method for treating and / or preventing cardiogenic shock, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a bone marrow-derived growth factor (MYDGF) protein or a fragment or variant thereof. Section 13. MYDGF, (i) SEQ ID NO: 1; or (ii) a fragment or variant of SEQ ID NO: 1 that exhibits a 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; Item 13. The method according to item 11 or 12, comprising: Section 14. 14. The method of any one of paragraphs 11 to 13, wherein MYDGF or a fragment or variant thereof is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier and / or excipient. Section 15. 1. A method of producing a non-human mammalian model of cardiogenic shock, comprising: (i) transient ligation of a mammalian coronary artery; (ii) establishing reperfusion; and (iii) artificially ventilating a mammal with an inspired oxygen concentration of about 0.18 or less. A method comprising: Section 16. 16. The method of paragraph 15, wherein the non-human mammal is a rodent, preferably the non-human mammal is a mouse. Section 17. 17. The method of paragraph 15 or 16, wherein the coronary artery is ligated for about 30 to about 90 minutes before reperfusion is established. Section 18. 18. The method of any one of paragraphs 15 to 17, wherein the mammal is ventilated at an inspired oxygen concentration of about 0.16.

Claims

1. Bone marrow derived growth factor (MYDGF), or a fragment or variant thereof exhibiting the biological function of MYDGF, for use in the treatment and / or prevention of cardiogenic shock.

2. MYDGF, (i) SEQ ID NO: 1; or (ii) 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; MYDGF for use according to claim 1, comprising:

3. MYDGF for use according to claim 1, wherein the MYDGF protein consists of SEQ ID NO: 1 or SEQ ID NO:

3.

4. A nucleic acid encoding MYDGF or a fragment or variant thereof exhibiting the biological function of MYDGF for use in the treatment and / or prevention of cardiogenic shock.

5. 5. The nucleic acid for use according to claim 4, wherein the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

1.

6. A vector comprising the nucleic acid of claim 5 for use in the treatment and / or prevention of cardiogenic shock.

7. A host cell comprising, and preferably expressing, a nucleic acid according to claim 5 or a vector according to claim 6 for use in the treatment and / or prevention of cardiogenic shock.

8. A pharmaceutical composition comprising a MYDGF protein described in any one of claims 1 to 3 or a fragment or variant thereof exhibiting the biological function of MYDGF, a nucleic acid described in any one of claims 4 or 5, a vector described in claim 6, or a host cell described in claim 7, for use in the treatment and / or prevention of cardiogenic shock.

9. 9. The pharmaceutical composition for use according to claim 8, wherein the pharmaceutical composition is administered via oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intracoronary routes.

10. 10. The pharmaceutical composition for use according to claim 9, wherein administration is via one or more bolus injections and / or infusion.

11. A method for treating and / or preventing cardiogenic shock, 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.

12. A method for treating and / or preventing cardiogenic shock, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a bone marrow-derived growth factor (MYDGF) protein or a fragment or variant thereof.

13. MYDGF, (i) SEQ ID NO: 1; or (ii) 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.

13. The method of claim 11 or 12, comprising:

14. 14. The method of any one of claims 11 to 13, wherein MYDGF or a fragment or variant thereof is administered by one or more bolus injections and / or infusion, preferably in a pharmaceutically acceptable carrier and / or excipient.

15. 1. A method of producing a non-human mammalian model of cardiogenic shock, comprising: (i) transiently ligating a coronary artery in a mammal; (ii) establishing reperfusion, and (iii) ventilating the mammal at an inspired oxygen concentration of about 0.18 or less. A method comprising:

16. 16. The method of claim 15, wherein the non-human mammal is a rodent, preferably the non-human mammal is a mouse.

17. 17. The method of claim 15 or 16, wherein the coronary artery is ligated for about 30 to about 90 minutes before reperfusion is established.

18. 18. The method of any one of claims 15 to 17, wherein the mammal is ventilated at an inspired oxygen concentration of about 0.16.

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