S100A1 protein for use in the treatment and prevention of progression of infarction

Abstract

The present invention relates to an S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid encoding the S100A1 protein or a biologically active fragment or variant thereof, a vector containing the nucleic acid, and a pharmaceutical composition comprising the protein or a biologically active fragment or variant thereof, the nucleic acid and / or the vector for use in the treatment and / or prevention of infarct progression in a subject in need of treatment and / or prevention of infarct progression.

Description

Technical Field

[0001] The present invention relates to nucleic acids encoding said proteins for use in the treatment and / or prevention of S100A1 protein and myocardial infarction progression in patients in need thereof, vectors comprising said nucleic acids, and respective pharmaceutical compositions.

Background Art

[0002] Muscle tissue is subdivided into skeletal muscle, cardiac muscle, and smooth muscle tissue, and can be considered the largest organ of vertebrates. Skeletal muscle and cardiac muscle belong to striated muscle tissue and share many functional aspects.

[0003] Myocardial ischemia caused by various causes, such as occlusion of coronary blood vessels, causes muscle necrosis, followed by aseptic inflammation and myocardial death. Many studies indicate that the long-term prognosis after such myocardial infarction is closely related to myocardial function, and that myocardial function is mainly determined by the size and location of the infarction.

[0004] Two complications are known to occur early after infarction and significantly increase the functional infarct size, which are infarct extension and infarct expansion. Myocardial extension is characterized by an increase in the mass of necrotic tissue, while myocardial expansion is accompanied by thinning and dilation of the infarct area. Infarct extension and infarct expansion are associated with increased mortality and morbidity in the early and late periods after myocardial infarction (Hochman and Bulkley, “Myocardial Infarct Expansion and Extension” in: Califf, R.M., Wagner, G.S. (eds) “Acute Coronary Care”. 1985 Springer, Boston, MA.). In infarct extension, the infarcted muscle tissue is ultimately replaced by fibrous scar due to secondary cardiomyocyte necrosis (M. B. Ratcliffe, "Non-Ischemic Infarct extension: a new type of infarct enlargement and a potential therapeutic target", Journal of American College of Cardiology, 40, 2002). As a result, the increasing scar continuously consumes the myocardial tissue in the border region and weakens the systolic performance of the heart. In hemodynamically loaded myocardium, both the innate and adaptive immune systems are continuously activated, and the border region of the infarct area is exposed to chronic hemodynamic overload and a persistent inflammatory environment by cytotoxic cytokines and chemokines, causing continuous secondary death of cardiomyocytes. Each process includes, but is not limited to, activation and persistence of the immune response based on B cells and T cells in the heart, such as infiltration and persistence of neutrophils, granulocytes, and macrophages after myocardial infarction (post-MI), and release of various interleukins, interferons, cytokines, and chemokines in the post-MI heart.Overall, this is in contrast to infarct expansion, which is distinguished as a result of thinning of scar tissue with the progression of ventricular dilation, and is associated with the progression of the infarct area (J. Wenk et al., "First Evidence of Depressed Contractility in the Border Zone of Human Myocardial Infarction", Ann Thorac Surg., 93, 2012; S. Bansal et al., "Activated T Lymphocytes are Essential Drivers of Pathological Remodeling in Ischemic Heart failure", Circ Heart Failure, 10, 2017; G. Garcia-Rivas et al., "The role of B cells in heart failure and implications for future immunomodulatory treatment strategies", 7, 2020; P. Westman et al., "Inflammation as a driver of adverse left ventricular remodeling after acute myocardial infarction", Journal of the American College of Cardiology, 67, 2017).

[0005] There are many treatment methods available for the treatment of myocardial infarction, but currently, there is no clinical therapy that suppresses the post-MI process and limits secondary damage to non-infarcted myocardium due to infarct progression (M. B. Ratcliffe, "Non-Ischemic Infarct extension: a new type of infarct enlargement and a potential therapeutic target", Journal of American College of Cardiology, 40, 2002). SUMMARY OF THE INVENTION

[0006] In view of the lack of appropriate means and methods for preventing or treating myocardial infarction progression, according to a first aspect, the present invention provides an S100A1 protein, a biologically active fragment or variant thereof, or a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof, for use in a patient in need of treatment and / or prevention of infarction progression, wherein the fragment or variant has at least 80% sequence identity with SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides a vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1, for use in a patient in need of treatment and / or prevention of infarction progression.

[0008] In a third aspect, the present invention provides a pharmaceutical composition for use in a patient in need of treatment and / or prevention of infarction progression, comprising (i) an S100A1 protein, a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1, or a nucleic acid encoding said S100A1 protein, fragment, or variant; (ii) a vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1; or (iii) a pharmaceutically acceptable salt of (i) or (ii). The pharmaceutical composition optionally further comprises a pharmaceutically acceptable excipient, carrier, and / or diluent.

[0009] According to one embodiment, an S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any of items 1 to 3, wherein the S100A1 protein has the sequence set forth in SEQ ID NO: 1.

[0010] According to a further embodiment, the vector is selected from the group consisting of plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores.

[0011] According to a preferred embodiment, the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors. More preferably, the adeno-associated virus (AAV) vector is selected from the group consisting of AAV5, AAV6, and AAV9, and preferably AAV5.

[0012] According to a further embodiment, the expression of the S100A1 protein is controlled by a heart tissue-specific promoter. Preferably, the heart tissue-specific promoter is selected from the group consisting of the cardiac actin enhancer / elongation factor 1 promoter, the cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin.

[0013] According to one embodiment, the protein, its biologically active fragment, nucleic acid, vector, or pharmaceutical composition is administered via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronal route.

[0014] According to a further embodiment, the protein, its biologically active fragment or variant, nucleic acid, vector, or pharmaceutical composition is administered via the intravenous route. Preferably, the protein, nucleic acid, vector, or pharmaceutical composition is administered retrogradely into the coronary system of the patient.

[0015] According to a preferred embodiment, the S100A1 protein, its biologically active fragment or variant, nucleic acid, vector, or pharmaceutical composition is administered to the patient between 1 day and 4 weeks after myocardial infarction has occurred after ischemia.

[0016] According to one embodiment, the intracellular level of S100A1 protein increases for at least 7 days.

[0017] According to a further embodiment, the vector is administered at a single dose of 1×10 13 vector genome copies (vgc).

[0018] In a fourth aspect, the present invention relates to a method for treating and / or preventing infarct progression in a patient in need thereof. The method comprises administering to the patient: (i) an S100A1 protein having at least 80% sequence identity with SEQ ID NO: 1, a biologically active fragment or variant thereof, or a nucleic acid encoding the S100A1 protein or a fragment or variant thereof; (ii) a vector comprising a nucleic acid encoding an S100A1 protein having at least 80% sequence identity with SEQ ID NO: 1 or a biologically active fragment thereof; or (iii) a pharmaceutically acceptable salt of (i) or (ii).

[0019] According to one embodiment, the S100A1 protein has the sequence set forth in SEQ ID NO: 1.

[0020] According to a preferred embodiment, the vector is selected from the group consisting of plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores. According to a preferred embodiment, the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors. According to a particularly preferred embodiment, the adeno-associated virus (AAV) vector is selected from the group consisting of AAV5, AAV6, and AAV9.

[0021] According to one embodiment, the expression of the S100A1 protein is controlled by a heart tissue-specific promoter. According to a preferred embodiment, the tissue-specific promoter is selected from the group consisting of a cardiac actin enhancer / elongation factor 1 promoter site, a cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin.

[0022] According to a further embodiment, the protein, its biologically active fragment, nucleic acid, vector, or pharmaceutical composition is administered via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronary route. According to a preferred embodiment, the protein, its biologically active fragment, nucleic acid, vector, or pharmaceutical composition is administered via the intravenous route. Preferably, the protein, nucleic acid, vector, or pharmaceutical composition is administered retrogradely into the coronary system of the patient.

[0023] According to a preferred embodiment, the protein, its biologically active fragment, vector, or pharmaceutical composition is administered to the patient between 1 day and 4 weeks after myocardial infarction in the patient becomes ischemic.

[0024] According to a further embodiment, the intracellular level of the S100A1 protein increases for at least 7 days.

[0025] According to one embodiment, the vector is administered at a single dose of 1x10 13 vector genome copies (vgc).

[0026] Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief Description of the Drawings

[0027]

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[0028] Sequence Array number 1 MGSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLDAQKDVDAVDKVMKELDENGDGEVDFQEYVVLVAALTVACNNFFWENS

[0029] Detailed Description of the Invention Before detailing the present invention below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein and may vary. Also, it should be understood that the terms used herein are for the purpose of describing particular embodiments only and are 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.

[0030] In the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety, whether above or below. No part of this specification shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being "incorporated by reference". In case of any conflict between the definitions or teachings of the incorporated references and the definitions or teachings described herein, the text of this specification shall prevail.

[0031] The elements of the present invention are described below. These elements are listed with specific embodiments, but it should be understood that they can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. This specification should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Further, all permutations and combinations of the elements described in this application should be considered to be disclosed by the description of this application, unless otherwise indicated by the context.

[0032] To carry out the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA technology are employed, which are described in the literature of the relevant field (for example, see Molecular Cloning: A Laboratory Manual, 2 nd nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). The elements of the present invention are described below. These elements are listed with specific embodiments, but it should be understood that they can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. This specification is to be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, all permutations and combinations of the elements described in this application should be considered to be disclosed by the description of this application, unless otherwise indicated by the context.

[0033] Definition Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0034] To carry out the present invention, unless otherwise stated, conventional methods of chemistry, biochemistry, cell biology, and recombinant DNA technology are employed, which are described in the literature of the relevant field (for example, see Molecular Cloning: A Laboratory Manual, 2 nd(See Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Furthermore, conventional clinical cardiology methods as described in the literature in this field are adopted (see, for example, Practical Methods in Cardiovascular Research, S. Dhein et al. eds., Springer Verlag Berlin Heidelberg, 2005).

[0035] Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted to mean including the stated integer or step or group of integers or steps but not to exclude any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the content clearly dictates otherwise. The term "about" when used in connection with a numerical value means a value within a range having a lower limit 5% less than the indicated value and an upper limit 5% greater than the indicated value.

[0036] As used herein, the term "and / or" means any one or both / all of the alternatives cited in the context of this term. For example, when referring to "treatment and / or prevention of myocardial infarction progression", this term is interpreted as follows: 1) treatment of myocardial infarction progression; 2) prevention of myocardial infarction progression; or 3) treatment and prevention of myocardial infarction progression.

[0037] In various aspects of the present invention, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins but is generally shorter in length.

[0038] In various aspects of the present invention, the term "polypeptide" refers to a single linear chain of amino acids joined by peptide bonds. A polypeptide can be one chain of a protein composed of multiple chains, or, if the protein is composed of one chain, the polypeptide can be the protein itself.

[0039] In the context of various aspects of the present invention, the term "protein" refers to a molecule containing a plurality of amino acid residues and / or one or more polypeptides that form secondary and tertiary structures, and further refers to a protein formed by a plurality of polypeptides that form a quaternary structure, i.e., a protein formed by a plurality of subunits. Non-peptide groups may be attached to proteins, and they may be referred to as prosthetic groups or cofactors.

[0040] The terms "polynucleotide" and "nucleic acid" are used synonymously and are understood to be single-stranded or double-stranded oligomers or polymers of deoxyribonucleotide bases or ribonucleotide bases or both. A description of a single strand of nucleic acid also defines (at least in part) the sequence of the complementary strand. Nucleic acids can be single-stranded or double-stranded, or can contain portions of both single-stranded and double-stranded sequences. In the context of various aspects of the present invention, the term nucleic acid includes cDNA, genomic DNA, recombinant DNA, cRNA, and mRNA. A nucleic acid can consist of an entire gene or a portion thereof, and a nucleic acid can also be a microRNA (miRNA) or a small interfering RNA (siRNA). The term "oligonucleotide", when used in the context of one of the different aspects of the present invention, refers to a nucleic acid having a length of up to about 50 nucleotides, for example 2 to about 50 nucleotides. A "nucleic acid" molecule is understood to be a macromolecule or oligomeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a pentose sugar (including but not limited to ribose and 2'-deoxyribose), and one to three phosphate groups. Usually, polynucleotides are 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), deoxyribonucleic acid (DNA), and mixtures thereof such as, for example, RNA-DNA hybrids. Nucleic acids can be obtained by any of biological, biochemical, or chemical synthetic methods, or methods known in the art. Nucleic acids can be chemically synthesized, for example, according to the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). An "aptamer" is a nucleic acid that binds to a polypeptide with high affinity. Aptamers can be isolated by selection methods from large pools of various single-stranded RNA molecules (see, for example, Jayasena (1999) Clin.Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol.Biol.Rep., 20, 97-107; US 5,582,981).Aptamers can also be synthesized and selected in mirror-image form, such as L-ribonucleotides (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). The form isolated in this way can enjoy the advantage of high stability because it is not degraded by naturally occurring ribonucleases. Nucleic acids are degraded by endonucleases, exonucleases, especially DNases and RNases present intracellularly. Therefore, it is advantageous to modify the nucleic acid to stabilize it against degradation so that a high concentration of the nucleic acid is maintained intracellularly over a long period (Beigelmanra (1995) Nucleic Acids Res. 23:3989-94). Usually, such stabilization can be achieved by introducing one or more internucleotide phosphate groups or by introducing one or more non-phosphate internucleotides. Suitable modified internucleotides are summarized in the above Uhlmann and Peyman (1990) (see also Beigelmanra (1995) Nucleic Acids Res. 23:3989-94). Modified phosphate internucleotide radicals and / or non-phosphate linkages in nucleic acids that can be employed in one of the uses according to the present invention include, for example, methylphosphonate, phosphorothioate, phosphoramidate, phosphorodithioate, and / or phosphate esters, while non-phosphate internucleotide analogs include, for example, siloxane linkages, carbonate linkages, carboxymethyl esters, acetamidate linkages, and / or thioether linkages. Also, this modification is also intended to improve the durability of the pharmaceutical composition that can be employed in one of the uses according to the present invention.

[0041] As used herein, the term "vector" refers to a protein, or polynucleotide, or a mixture thereof, into which or by which the proteins and / or nucleic acids contained therein can be introduced into cells. In the context of the present invention, the gene of interest encoded by the introduced polynucleotide is preferably expressed intracellularly upon introduction of the vector. Examples of suitable vectors include, but are not limited to, plasmids, cosmids, phages, viruses, or artificial chromosomes.

[0042] As used herein, the term "variant" refers to a polynucleotide or protein that is understood to be different from the polynucleotide or protein from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a protein or nucleic acid variant is derived is also known as the parent polypeptide or parent polynucleotide. The term "variant" includes "fragments" or "derivatives" of the parent molecule. Usually, a "fragment" is smaller in length or size compared to the parent molecule, and a "derivative" shows one or more sequence differences compared to the parent molecule. According to the present invention, the fragment of SEQ ID NO: 1 has a length of at least 9 amino acids. Preferred fragments of SEQ ID NO: 1 have a length of from 10 to 30 amino acids, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Particularly preferred fragments of SEQ ID NO: 1 have 20 amino acids.

[0043] Also included are modified molecules such as, but not limited to, translated and post-modified proteins (e.g., glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA. Also included, but not limited to, are mixtures of different molecules such as RNA-DNA hybrids. Usually, a variant is constructed artificially, preferably by genetic engineering techniques, while the parent polypeptide or polynucleotide is a wild-type protein or polynucleotide. However, naturally occurring variants are also understood to be encompassed by the term "variant" as used herein. Furthermore, variants useful in the present invention can also be derived from homologs, orthologs, paralogs, or artificially constructed variants of the parent molecule, provided that the variant exhibits at least one biological activity of the parent molecule, e.g., is functionally active.

[0044] As used herein, the term "variant" of a protein is understood to be a polypeptide that is different from the polypeptide from which it is derived by one or more changes in the amino acid sequence. The polypeptide from which a protein variant is derived is also referred to as the parent polypeptide. Usually, variants are constructed artificially, preferably by genetic engineering techniques. Usually, the parent polypeptide is a wild-type protein or a wild-type protein domain. In the context of the present invention, it is more preferred that the parent polypeptide is a consensus sequence of two or more wild-type polypeptides. Furthermore, variants that can be used in the present invention can also be derived from homologs, orthologs, paralogs of the parent polypeptide, or artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide. The changes in the amino acid sequence are amino acid exchanges, insertions, deletions, N-terminal truncations, or C-terminal truncations, or combinations thereof, and these changes may occur at one site or several sites. In a preferred embodiment, variants that can be used in the present invention exhibit a total number (up to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of changes in the amino acid sequence (e.g., exchanges, insertions, deletions, N-terminal truncations, and / or C-terminal truncations). Amino acid exchanges can be conservative and / or non-conservative.

[0045] Particularly preferred are semi-conservative and especially conservative amino acid substitutions in which the amino acid is replaced by a chemically related amino acid. Typical substitutions are between aliphatic amino acids, between amino acids with aliphatic hydroxyl side chains, between amino acids with acidic residues, between amide derivatives, between amino acids with basic residues, or between amino acids with aromatic residues. Typical semi-conservative and conservative substitutions are as follows: JPEG2025519230000001.jpg121153

[0046] A change from A, F, H, I, L, M, P, V, W, or Y to C is semi-conservative if the new cysteine remains in the free thiol form. Furthermore, one of ordinary skill in the art will understand that glycine at a sterically demanding position should not be substituted, and that P should not be introduced into portions of a protein having an α-helix structure or a β-sheet structure.

[0047] Alternatively or additionally, a "variant" as used herein can be characterized by a degree of sequence identity to the parent polypeptide or polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 80% sequence identity to its parent polypeptide. A polynucleotide variant in the context of the present invention exhibits at least 80% sequence identity to SEQ ID NO: 1. According to a preferred embodiment, the parent polypeptide is SEQ ID NO: 1.

[0048] The term "at least 80% sequence identity" is used throughout this specification with respect to the sequence comparison of polypeptides and polynucleotides. This expression preferably refers to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 SEQ ID NO: 1.

[0049] Variants may additionally or alternatively contain amino acid deletions, which may be N-terminal truncations, C-terminal truncations, or internal deletions, or any combination thereof. Such variants containing N-terminal truncations, C-terminal truncations, and / or internal deletions are referred to as "deletion variants" in the context of this application.

[0050] The fragment may be a naturally occurring one (e.g., a splice variant), or may be artificially constructed, preferably by genetic engineering techniques. According to the present invention, the fragment contains at least 9 amino acids of SEQ ID NO: 1. Preferably, the fragment or deletion mutant has a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 amino acids, preferably at its N-terminus and / or internally, more preferably at its N-terminus, compared to the parental polypeptide of SEQ ID NO: 1. A particularly preferred fragment according to the present invention is the peptide consisting of amino acids Y75 to S94 of SEQ ID NO: 1. When the fragment sequence is compared with SEQ ID NO: 1, the percentage sequence identity is calculated based on the shorter of the two sequences being compared, i.e., the fragment. And the sequence identity is determined based on the overlap between the fragment and SEQ ID NO: 1. The percentage of sequence identity is preferably determined by sequence alignment. Such alignment can be performed with several technically known algorithms. For example, the grade of sequence identity (sequence match degree) can be calculated using BLAST (blastp) or EMBOSS Needle (EMBL - European Bioinformatics Institute). Each algorithm is incorporated into BLASTN and BLASTP of Altschul et al. (1990) J. Mol. Biol: 403 - 410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389 - 3402.

[0051] A fragment of SEQ ID NO:1 that further contains one or more amino acid substitutions within its amino acid sequence as compared to SEQ ID NO:1 is also called a "deletion mutant".

[0052] Additionally or alternatively, a deletion mutant may not be due to a structural deletion of each amino acid as described above, but may occur because these amino acids are inhibited or otherwise unable to perform their biological functions. Usually, such functional deletions result in changes in the chemical properties of the resulting protein (i.e., the exchange of hydrophobic amino acids for hydrophilic amino acids), changes in the post-translational modifications of the resulting protein (e.g., post-translational cleavage or glycosylation patterns), or changes in the secondary or tertiary structure of the protein (e.g., post-translational cleavage or glycosylation patterns), etc., and are caused by insertions into the amino acid sequence or exchanges of amino acid sequences that change the functional properties of the resulting protein, but are not limited thereto. Additionally or alternatively, functional deletions may occur due to transcriptional or post-transcriptional gene silencing (e.g., via siRNA), or the presence or absence of inhibitory molecules such as protein inhibitors or inhibitory antibodies.

[0053] In the context of the present invention, it is preferred that a protein or polypeptide being "functionally deleted" refers to the fact that the amino acids or nucleotides of the corresponding sequence are deleted or are present but do not perform their biological functions.

[0054] As used herein, "individual" means a mammal, reptile, or bird that can receive the benefits of the present invention. Preferably, the individual is selected from the group consisting of laboratory animals (e.g., mice, rats, or rabbits), domestic animals (e.g., guinea pigs, rabbits, horses, donkeys, cows, sheep, goats, pigs, chickens, ducks, camels, cats, dogs, turtles, snakes, or lizards), or primates including chimpanzees, bonobos, gorillas, and humans. It is particularly preferred that the "individual" is a human.

[0055] As used herein, the term "patient" refers to a mammal, reptile, or bird that may benefit from the prognosis, diagnosis, identification, or treatment of a disease or disorder. Preferably, the "patient" is selected from the group consisting of laboratory animals (e.g., mice, rats, or rabbits), domestic animals (e.g., including guinea pigs, rabbits, horses, donkeys, cows, sheep, goats, pigs, chickens, ducks, camels, cats, dogs, turtles, snakes, or lizards), or primates including chimpanzees, bonobos, gorillas, and humans. It is particularly preferred that the "patient" is a human.

[0056] The terms "disease" and "disorder" are used interchangeably herein and refer to an abnormal condition, particularly an abnormal medical condition such as a disease or injury where a tissue, organ, or individual is unable to perform its function efficiently. Usually, a disease is accompanied by specific symptoms or signs indicating its presence, but not necessarily. Thus, the presence of such symptoms or signs may indicate a tissue, organ, or individual suffering from a disease. Changes in these symptoms and signs may also suggest the progression of such a disease. The progression of a disease is typically characterized by an increase or decrease in symptoms and signs indicating "worsening" or "improvement" of the disease. "Worsening" of a disease is characterized by a decrease in the ability of a tissue, organ, or organism to perform its function efficiently, while "improvement" of a disease is generally characterized by an increase in the ability of a tissue, organ, or individual to perform its function efficiently. A tissue, organ, or individual at "risk of developing" a disease is in a healthy state but shows the potential for the disease to appear. Generally, the risk of developing a particular disease is associated with early or mild symptoms or signs of that disease. In such cases, the onset can still be prevented by treatment. Examples of diseases include, but are not limited to, traumatic diseases, inflammatory diseases, infectious diseases, and heart diseases. Heart disorders include, but are not limited to, post-ischemic systolic dysfunction, congestive heart failure, cardiogenic shock, septic shock, primary or secondary cardiomyopathy, cardiac valve dysfunction, ventricular disorders, and preferably myocardial infarction. Primary cardiomyopathy includes genetic cardiomyopathy and cardiomyopathy due to spontaneous mutation. Secondary cardiomyopathy includes ischemic cardiomyopathy due to arteriosclerosis, dilated cardiomyopathy due to myocardial infection or intoxication, hypertensive heart disease caused by pulmonary arterial hypertension and / or arterial hypertension, and diseases of the cardiac valves. The preferred medical indication to be treated by the present invention is myocardial infarction progression.

[0057] The "symptoms" of a disease are the implications of the disease that are prominently manifested by the tissues, organs, or organisms having such a disease, including but not limited to pain, weakness, tenderness, tension, stiffness, and spasms of the tissues, organs, or individuals. The "signs" or "signals" of a disease include but are not limited to the presence, absence, increase or elevation, decrease or decline, or other changes or variations of specific indicators such as biomarkers or molecular markers, or the manifestation, presence, and exacerbation of symptoms.

[0058] The disorder can be acquired or congenital. In this context, the term "acquired" means that the medical condition, i.e., the disorder, developed after birth. The acquired disorder referred to in the present invention is myocardial infarction. Congenital diseases include defects in the developing fetus that may be the result of genetic abnormalities, morphogenetic abnormalities, or chromosomal abnormalities. Although all genetic diseases or disorders are congenital, they may not manifest or be recognized until later in life. The congenital diseases referred to in the present invention are, for example, nemaline myopathy, myotubular myopathy, or centronuclear myopathy. Furthermore, in the context of the present invention, the heart disorder may be acute or chronic. For example, acute heart disease is acute heart failure, and acute skeletal muscle disease is rhabdomyolysis. Chronic myocardial disease is, for example, chronic heart failure. Heart diseases may be caused by muscle dysfunction associated with calcium circulation disorders and / or contractile performance disorders in muscle cells, preferably cardiomyocytes.

[0059] As used herein, "treating", "treatment", or "treat" a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the manifestation of symptoms characteristic of the disorder being treated; (c) suppressing the exacerbation of symptoms characteristic of the disorder being treated; (d) limiting or preventing the recurrence of the disorder in an individual who previously had the disorder; and (e) limiting or preventing the recurrence of symptoms in an individual who previously had symptoms of the disorder.

[0060] As used herein, "prevent", "preventing", "prevention", and "prophylaxis" of a disease or disorder mean preventing such disease or disorder from occurring in a patient.

[0061] As used herein, "administration" includes in vivo administration and direct administration to tissues ex vivo, such as to a vein graft.

[0062] "Effective amount" or "therapeutically effective amount" means an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a particular therapeutic agent will vary depending on factors such as the nature of the therapeutic agent, the route of administration, the size and type of animal to which the therapeutic agent is administered, and the purpose of the administration. The effective amount in the case of each individual can be determined empirically by one of ordinary skill in the art according to methods established in the art.

[0063] As used herein, the terms "pharmaceutical", "drug", and "medicine" mean substances and / or combinations of substances used for the identification, prevention, or treatment of a tissue condition or disease, and are used interchangeably.

[0064] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, particularly humans. The term "pharmaceutically acceptable salt" refers to salts of the proteins or peptides of the present invention. Suitable pharmaceutically acceptable salts include, for example, acid addition salts which can be formed by mixing a solution of a 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. Further, when the peptide bears an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium, amine cations formed with counter anions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates, arylsulfonates).Examples of pharmaceutically acceptable salts include, but are not limited to, 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, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylsulfanylate, hemisulfinate, heptanoate, hexanoate, hexylresorcinolate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, 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, subsulfate, succinate, tannate, tartrate, theoclate, tosylate, triethiodide, undecanoate, valerate, etc. (see, e.g., S.M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)).

[0065] The term "active ingredient" refers to a biologically active substance, i.e., a substance in a pharmaceutical formulation that provides a pharmaceutical value. A pharmaceutical composition may contain one or more active ingredients, and these active ingredients may act in relation to each other or independently. The active ingredient can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc., but are not limited thereto.

[0066] Whether a fragment or variant of the S100A1 protein of the present invention is biologically active can be determined, for example, by any one of the tests described in the following examples. According to a preferred embodiment, a fragment or variant of the S100A1 protein is compared with the results obtained using the S100A1 protein of the present invention shown in at least one of the examples shown hereinbelow, and the results obtained using such a fragment or variant are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the effects reported for the S100A1 protein against the indicated control, then it is biologically active.

[0067] The terms "formulation" and "composition" are intended to include a dosage form of an active compound with an encapsulating material as a carrier that provides a capsule in which the active ingredient, with or without other carriers, is surrounded by the carrier.

[0068] As used herein, the term "carrier" refers to, but is not limited to, a pharmacologically inert substance such as a diluent, excipient, or vehicle associated with the administration of a therapeutically active ingredient. Such pharmaceutical carriers may be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids such as physiological saline in water, and oils of petroleum origin, animal origin, plant origin, and synthetic origin such as peanut oil, soybean oil, mineral oil, and sesame oil. Physiological saline, aqueous glucose solution, and aqueous glycerol solution can also be employed as liquid carriers, particularly for injection solutions. Physiological 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.

[0069] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0070] The term "adjuvant" refers to an agent that increases, stimulates, activates, enhances, or modulates the immune response to the active ingredient of a composition, either at the cellular level or the humoral level. For example, an immunological adjuvant stimulates the immune system's response to an actual antigen but has no immunological effect itself. Examples of such adjuvants include inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponin or squalene), oily adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ, etc.), particulate adjuvants (e.g., immunostimulating complexes (ISCOMs), liposomes, or biodegradable microparticles), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A or muramyl peptides), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine), but are not limited thereto.

Mode for Carrying Out the Invention

[0071] The S100A1 protein is a member of the S100 protein family that is expressed in the myocardium, skeletal muscle, and brain. The present invention provides the S100A1 protein and its biologically active fragments and variants as novel therapeutic agents capable of preventing and treating the progression of infarcted regions of the heart. Without wishing to be bound by any theory, S100A1 and its biologically active fragments and variants exert their effects by suppressing sterile inflammation after MI and providing a molecular inotropic effect on the damaged heart. The inventors have surprisingly found that the S100A1 protein and its biologically active fragments and variants attenuate the activities of both the innate and adaptive immune systems in the heart after MI. The inventors have particularly shown that the therapeutic effects of the S100A1 protein and its biologically active fragments and variants are based on attenuating the activities of both innate and adaptive immune cells.

[0072] So far, the S100A1 protein has been suggested as a pure inotropic therapeutic agent in heart failure and cardiomyopathy after MI, because it has been shown that in heart failure, the myocardial level of S100A1 is decreased, and when S100A1 is delivered to cardiomyocytes, isometric muscle contraction increases, followed by an increase in the amount of calcium pumped into the sarcoplasmic reticulum (P. Most et al., "100A1: a novel inotropic regulator of cardiac performance. Transition from molecular physiology to pathophysiological relevance" Am J Physiol., 293, 2007; J. Ritterhoff and P. Most, "Targeting S100A1 in heart failure" Gene Therapy, 19, 2012). The use of S100A1 and its biologically active fragments and variants for the treatment and / or prevention of myocardial infarction progression has not been known so far and is similar to further medical uses defined by new medical indications.

[0073] According to a first aspect, the present invention provides an S100A1 protein, a biologically active fragment or variant thereof, or a nucleic acid encoding said S100A1 protein or a biologically active fragment or variant thereof for use in the treatment and / or prevention of infarct progression in a patient in need of treatment and / or prevention of infarct progression. The biologically active fragment or variant of the S100A1 protein has at least 80% sequence identity with SEQ ID NO: 1. Thus, each fragment or variant may have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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% identity with SEQ ID NO: 1. In the case of a short fragment or deletion variant of SEQ ID NO: 1, i.e., a peptide having a length between 9 and 15 amino acids of SEQ ID NO: 1, the fragment or deletion variant preferably has at least 88% sequence identity to SEQ ID NO: 1.

[0074] Examples of variants include, but are not limited to, S100A2, S100A3, S100A4, and other members of the S100 protein family. Each variant, including for example amino acid substitutions, preferably differs from SEQ ID NO: 1 at the C-terminus of the protein, more preferably at one or more of the most C-terminal 20 amino acids. These variants may be further truncated in that they also include deletions when compared to SEQ ID NO: 1. In such cases, they are also referred to as deletion variants of SEQ ID NO: 1.

[0075] The fragment of SEQ ID NO:1 according to the present invention preferably differs from SEQ ID NO:1 at the N-terminus or internally and contains one or more amino acid deletions. Particularly preferred fragments are peptides consisting of amino acids N65 to S94, V70 to S94, or Y75 to S94 of SEQ ID NO:1. The fragment may further contain one or more substitutions in the amino acid sequence as compared to SEQ ID NO:1. In such cases, the fragment is also referred to as a deletion mutant.

[0076] Preferably, the fragment or variant of SEQ ID NO:1 maintains or exhibits essentially the same biological function as the parent protein by SEQ ID NO:1. Maintaining or exhibiting essentially the same biological function as the parent protein by SEQ ID NO:1 means, in the present invention, interacting with the RyR calcium release channel and the sarcoplasmic reticulum calcium ATPase (SERCA), increasing SERCA activity, increasing the contractile function in cardiomyocytes and / or skeletal muscle cells, showing the inotropic effect of the full-length S100A1 protein (all described in Most P. et al., 2007, Am. J. Physiol. Regul. Integr. Comp. Physiol. 293:R568-577; Voelkers M. et al., 2007, Cell Calcium 41:135-143), and suppressing sterile inflammation after myocardial infarction.

[0077] However, according to a preferred embodiment, the S100A1 protein is a naturally occurring human S100A1 protein having the amino acid sequence of SEQ ID NO:1.

[0078] In a preferred embodiment, the protein further comprises one or more elements selected from the group consisting of a hydrophilic domain, a membrane permeability enhancing domain, one or more epitope tags, and a peptide targeting domain, preferably a hydrophilic domain, a membrane permeability enhancing domain, or a hydrophilic domain and a membrane permeability enhancing domain. These elements may be directly or indirectly linked to the N-terminus or C-terminus of the domain derived from the S100A1 protein. Preferably, the element is linked to the N-terminus.

[0079] In one embodiment, the protein further comprises an epitope tag and / or a peptide targeting domain. In another embodiment, the peptide further comprises one or more, e.g., one, two, three, or four elements selected from the group consisting of a hydrophilic domain, a membrane permeability enhancing domain, one or more epitope tags, and a peptide targeting domain.

[0080] An epitope is a part of a molecule to which an antibody binds. For the present invention, the epitope is preferably a peptide tag, such as a hemagglutinin-(HA-)tag, FLAG-tag, myc-tag, or poly-His-tag. Such epitope tags can be used, for example, to determine the location of the peptide of the present invention within a cell in order to determine whether the peptide traverses, i.e., crosses, the cell membrane and can be present inside an intact cell incubated with the peptide.

[0081] The peptide targeting domain according to the present invention can be any moiety suitable for targeting a peptide to a specific organ or specific cells in vivo. For example, the peptide targeting domain can be a peptide that specifically binds to a specific receptor specific to a particular cell or a particular organ. Preferably, the presence of a peptide targeting domain within the peptide according to the present invention enables the peptide to specifically target cells or organs of a patient administered systemically.

[0082] In an embodiment of the present invention, a nucleic acid encoding the S100A1 protein or a fragment or variant thereof is contained in a vector. Preferably, the vector is selected from the group consisting of plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores. The viral vector is particularly preferably selected from the group consisting of adenoviral vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors. In a preferred embodiment, the vector is integrated into the genome, preferably into the genome of cardiomyocytes. In a more preferred embodiment, the vector is an AAV selected from the group consisting of AAV5, AAV6, and AAV9. According to the most preferred embodiment, the nucleic acid encoding the S100A1 protein or a fragment or variant thereof is contained in an AAV5 vector.

[0083] In a preferred embodiment, the above vector induces the expression of the S100A1 protein or a fragment or variant thereof in cardiomyocytes. In a more preferred embodiment, the expression of the S100A1 protein is controlled by a heart tissue-specific promoter. Thus, according to a preferred embodiment, the vector further contains a heart tissue-specific promoter. Preferably, the heart tissue-specific promoter is selected from the group consisting of a cardiac actin enhancer / elongation factor 1 promoter, a cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin, but is not limited thereto.

[0084] In a preferred embodiment, the vector preferably has 1x10 12 ~1x10 14 vector genome copies, more preferably 3x10 10 vgc, or about 3x10 11 vgc / kg body weight (vgc / kg BW) ~ about 3.3x10 12 vgc / kg BW, preferably about 3.3x1011 It is a viral vector administered at a dose of vgc / kg BW.

[0085] In a preferred embodiment of the present invention, the intracellular level of S100A1 protein increases in at least 30% of the cells of an individual's heart tissue. The size and amount of heart cells expressing S100A1 protein depend on the state of the underlying disease. However, within the treated heart region, it is preferred that the intracellular level of S100A1 increases as described herein.

[0086] In an embodiment of the present invention, the S100A1 protein or a fragment or variant thereof, or a nucleic acid encoding the S100A1 protein or a fragment or variant thereof, as described herein, is administered via an oral, intravenous, intramucosal, intraarterial, intrathecal, or intracoronal route. According to a particularly preferred embodiment, the protein, nucleic acid, vector, or pharmaceutical composition is administered retrogradely to the coronary system of the patient.

[0087] According to one embodiment, the protein, its biologically active fragment or variant, vector, or pharmaceutical composition is administered to the patient between 1 day and 4 weeks after myocardial infarction has occurred after ischemia. Preferably, the protein, its biologically active fragment or variant, vector, or pharmaceutical composition is administered to the patient between 1 week and 2 weeks after myocardial infarction has occurred after ischemia.

[0088] In a further embodiment of the present invention, the intracellular level of S100A1 protein increases for at least 7 days, more preferably for at least 10 days, even more preferably for at least 14 days. Alternatively, the intracellular level of S100A1 protein increases for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or for a longer period. This is preferably achieved as a result of a single administration or repeated administrations. A single administration is preferred.

[0089] In an embodiment of the present invention, an individual or patient has had, has, or is at risk of developing a heart disease. Preferably, the heart disease is myocardial infarction.

[0090] According to a further aspect, the present invention provides a pharmaceutical composition comprising (i) an S100A1 protein, a biologically active fragment thereof or a nucleic acid encoding the S100A1 protein, (ii) a vector comprising a nucleic acid encoding an S100A1 protein having at least 80% sequence identity with SEQ ID NO: 1 or a biologically active fragment or variant thereof, or (iii) any pharmaceutically acceptable salt of (i) or (ii). The pharmaceutical composition is for use in the treatment and / or prevention of infarct progression in a patient in need thereof.

[0091] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the S100A1 protein or a fragment or variant thereof described herein, the nucleic acid or vector of the present invention, which are also referred to herein as "active ingredients". In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the active ingredient, preferably in a purified form, together with a suitable amount of a carrier and / or excipient so as to provide a form suitable for proper administration to a patient. The dosage form of the composition must be suitable for the mode of administration. In the case of intravenous administration, the carrier is preferably an aqueous carrier. According to one embodiment, such an aqueous carrier can confer improved properties, such as improved solubility, efficacy, and / or improved immunotherapy, when combined with the antigen-binding polypeptide of the present invention.

[0092] According to one embodiment, the pharmaceutical composition can contain additional therapeutic agents or pharmacologically active substances, such as adjuvants and / or additional active ingredients, but is not limited thereto, in a pharmaceutically or physiologically acceptable dosage form selected to be appropriately administered according to the selected mode of administration.

[0093] According to one embodiment, the pharmaceutical composition can take forms such as solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release dosage forms, etc. To prepare the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier can be either solid or liquid, preferably liquid. Liquid compositions include solutions, suspensions, and emulsions, for example, water, physiological saline, glucose aqueous solution, glycerol solution, or water / propylene glycol solution. In the case of parenteral injection (e.g., intravenous injection, intra-arterial injection, intramedullary injection, subcutaneous injection, intraperitoneal injection, intradermal injection, and intrathecal injection), the liquid preparation can be formulated, for example, as a solution in an aqueous polyethylene glycol solution. Physiological saline is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0094] According to one embodiment, the pharmaceutical composition is in unit dosage form. In such a form, the composition can be subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form can be a packaged composition, and the package contains a discontinuous amount of the composition, such as powder in a packaged tablet, capsule, vial, or ampoule. Also, the unit dosage form can be a capsule, an injection vial, a tablet, a cachet, a lozenge itself, or any number of these packaged together. The pharmaceutical composition can optionally contain a small amount of wetting agent or emulsifier, or a pH buffer.

[0095] The form, route of administration, dosage, and regimen of the pharmaceutical composition will, of course, depend on the condition to be treated, the severity of the disease, the age, weight, and gender of the patient, the desired treatment period, etc. The pharmaceutical composition can be in any appropriate form according to the desired method of administration to the patient. The preferred mode of administration is retrograde administration to the patient's coronary system.

[0096] According to one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle in a dosage form injectable into a patient. These are in particular isotonic, sterile, physiological saline (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a mixture of such salts), or a dried, in particular lyophilized, composition which can, optionally by addition of sterile water or physiological saline, form an injectable solution.

[0097] To prepare the pharmaceutical composition, an effective amount of the active ingredient can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0098] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; dosage forms containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the preparation of sterile injectable solutions or dispersions. In each case, the form must be sterile and must be fluid to the extent that it can be easily injected with a syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Solutions of the active compound as the free base or as a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal storage and use conditions, these formulations contain preservatives to prevent the growth of microorganisms.

[0099] According to one embodiment, the S100A1 protein or a fragment or variant thereof, the nucleic acid or vector of the present invention described herein can be formulated into a pharmaceutical composition in neutral or salt form using a pharmaceutically acceptable salt.

[0100] Aseptic injectable solutions are prepared by incorporating the required amount of the active compound into a suitable solvent, along with several of the other ingredients enumerated above if required, and then filtering the solution sterilizing it. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients of the above enumerated ingredients. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is by vacuum drying and freeze drying techniques which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered solution.

[0101] According to a preferred embodiment, the pharmaceutical composition is administered by oral, intravenous, intramucosal, intraarterial, intrathecal, or intracoronary routes. Intravenous administration is preferred. In the case of intravenous administration, it is particularly preferred that the protein, nucleic acid, vector, or pharmaceutical composition be administered retrogradely into the coronary system of the patient.

[0102] In a preferred embodiment, administration of the protein, biologically active fragment thereof, nucleic acid, vector, or pharmaceutical composition of the present invention results in an increase of about 50-fold in the concentration of S100A1 protein in the myocardium of the individual as compared to the concentration of S100A1 in the myocardium of a healthy individual. Thus, preferably, the concentration of S100A1 protein in the myocardium is increased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold as compared to the concentration of S100A1 in the myocardium of a healthy individual.

[0103] According to a further aspect, the present invention relates to a method for treating and / or preventing infarct progression in a patient in need thereof, the method comprising administering to the patient (i) an S100A1 protein, a biologically active fragment or variant thereof, or a nucleic acid encoding said S100A1 protein, fragment or variant, (ii) a vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof, or (iii) a pharmaceutically acceptable salt of (i) or (ii). A biologically active fragment or variant of the S100A1 protein has at least 80% sequence identity with SEQ ID NO: 1. Thus, each fragment or variant may have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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% identity with SEQ ID NO: 1. Non-limiting examples of such fragments or variants include, but are not limited to, S100A2, S100A3, S100A4 and other members of the S100 protein family. Each fragment and variant preferably differs from SEQ ID NO: 1 at the C-terminus of the protein, more preferably at one or more of the most C-terminal 20 amino acids. Preferably, a fragment or variant of SEQ ID NO: 1 further maintains or exhibits essentially the same biological function as the parent protein according to SEQ ID NO: 1.

[0104] However, according to a preferred embodiment, the S100A1 protein is a naturally occurring human S100A1 protein having the amino acid sequence of SEQ ID NO: 1.

[0105] In a preferred embodiment, the protein further comprises one or more elements selected from the group consisting of a hydrophilic domain, a membrane permeability enhancing domain, one or more epitope tags, and a peptide targeting domain, preferably a hydrophilic domain, a membrane permeability enhancing domain, or a hydrophilic domain and a membrane permeability enhancing domain. These elements may be directly or indirectly linked to the N-terminus or C-terminus of the domain derived from the S100A1 protein. Preferably, the element is linked to the N-terminus.

[0106] In one embodiment, the protein further comprises an epitope tag and / or a peptide targeting domain. In another embodiment, the peptide further comprises one or more, for example, one, two, three, or four elements selected from the group consisting of a hydrophilic domain, a membrane permeability enhancing domain, one or more epitope tags, and a peptide targeting domain.

[0107] An epitope is a part of a molecule to which an antibody binds. For the present invention, the epitope is preferably a peptide tag, such as a hemagglutinin-(HA-)tag, FLAG-tag, myc-tag, or poly-His-tag. Such epitope tags can be used, for example, to determine the location of the peptide of the present invention within a cell to determine whether the peptide crosses, i.e., traverses, the cell membrane and can be present inside an intact cell incubated with said peptide.

[0108] The peptide targeting domain according to the present invention can be any moiety suitable for targeting a peptide to a specific organ or specific cells in vivo. For example, the peptide targeting domain can be a peptide that specifically binds to a specific receptor specific to a specific cell or specific organ. Preferably, the presence of the peptide targeting domain within the peptide according to the present invention enables the peptide to specifically target cells or organs of a patient administered systemically.

[0109] In an embodiment of the present invention, a nucleic acid encoding an S100A1 protein or a fragment or variant thereof is contained in a vector. Preferably, the vector is selected from the group consisting of plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores. The viral vector is particularly preferably selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retrovirus vectors, and lentivirus vectors. In a preferred embodiment, the vector is integrated into the genome, preferably the genome of cardiomyocytes. In a more preferred embodiment, the vector is an AAV selected from the group consisting of AAV5, AAV6, and AAV9. According to the most preferred embodiment, the nucleic acid encoding the S100A1 protein or a fragment or variant thereof is contained in an AAV5 vector.

[0110] In a preferred embodiment, the above vector induces the expression of the S100A1 protein or a fragment or variant thereof in cardiomyocytes. In a more preferred embodiment, the expression of the S100A1 protein is controlled by a heart tissue-specific promoter. Thus, according to a preferred embodiment, the vector further contains a heart tissue-specific promoter. Preferably, the heart tissue-specific promoter is selected from the group consisting of the cardiac actin enhancer / elongation factor 1 promoter, the cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin, but is not limited thereto.

[0111] In a preferred embodiment, the vector is preferably 1x10 11 ~1x10 14 vector genome copies (vgc), preferably 2x10 11 ~1x10 13 (vgc), or about 3.3x10 10 vgc / kg body weight (vgc / kg BW) to about 3.3x10 12vgc / kg BW, preferably about 3.3x10 11 It is a viral vector administered at a dose of vgc / kg BW.

[0112] In a preferred embodiment of the present invention, the intracellular level of the S100A1 protein is increased in at least 30% of the cells of an individual's heart tissue. The size and amount of heart cells expressing the S100A1 protein depend on the underlying disease state. However, within the treated heart region, it is preferred that the intracellular level of S100A1 is increased as described herein.

[0113] In an embodiment of the present invention, the S100A1 protein, or a fragment or variant thereof, or a nucleic acid encoding the S100A1 protein, or a fragment or variant thereof as described herein, is administered via an oral, intravenous, intramucosal, intraarterial, intrathecal, or intracoronal route. According to a particularly preferred embodiment, the protein, nucleic acid, vector, or pharmaceutical composition is administered retrogradely into the coronary system of the patient.

[0114] According to one embodiment, the protein, a biologically active fragment or variant thereof, vector, or pharmaceutical composition is administered to the patient between 1 day and 4 weeks after myocardial infarction has occurred in the patient after ischemia. Preferably, the protein, a biologically active fragment or variant thereof, vector, or pharmaceutical composition is administered to the patient between 1 week and 2 weeks after myocardial infarction has occurred in the patient after ischemia.

[0115] In a further embodiment of the present invention, the intracellular level of the S100A1 protein is increased for at least 7 days, more preferably for at least 10 days, even more preferably for at least 14 days. Alternatively, the intracellular level of the S100A1 protein is increased for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or for a longer period. This is preferably achieved as a result of a single administration or repeated administrations. A single administration is preferred.

[0116] In embodiments of the present invention, an individual or patient has had, has, or is at risk of developing a heart disease. Preferably, the heart disease is myocardial infarction.

[0117] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the S100A1 protein or fragment or variant thereof described herein, a nucleic acid, or a vector of the invention, which are also referred to herein as "active ingredients". In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the active ingredient, preferably in a purified form, together with a suitable amount of a carrier and / or excipient so as to provide a form suitable for appropriate administration to a patient. The dosage form of the composition must be suitable for the mode of administration. In the case of intravenous administration, the carrier is preferably an aqueous carrier. According to one embodiment, such an aqueous carrier can confer improved properties, such as improved solubility, effectiveness, and / or improved immunotherapy, when combined with the antigen-binding polypeptide of the invention.

[0118] According to one embodiment, the pharmaceutical composition can contain additional therapeutic agents or pharmacologically active substances, such as adjuvants and / or additional active ingredients, in a pharmaceutically or physiologically acceptable dosage form selected to be appropriately administered according to the selected mode of administration, but is not limited thereto.

[0119] According to one embodiment, the pharmaceutical composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release dosage form, etc. For preparing the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier can be solid or liquid, preferably liquid. Liquid compositions include solutions, suspensions, and emulsions, for example, water, physiological saline, glucose aqueous solution, glycerol solution, or water / propylene glycol solution. In the case of parenteral injection (e.g., intravenous injection, intra-arterial injection, intramedullary injection, subcutaneous injection, intraperitoneal injection, intradermal injection, and intrathecal injection), the liquid formulation can be formulated, for example, as a solution in an aqueous polyethylene glycol solution. Physiological saline is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0120] According to one embodiment, the pharmaceutical composition is in unit dosage form. In such a form, the composition can be subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form can be a packaged composition, the package containing a discrete amount of the composition, such as a powder in a packaged tablet, capsule, vial or ampoule. Also, the unit dosage form can be a capsule, an injection vial, a tablet, a cachet, a lozenge per se, or any number of these packaged together. The pharmaceutical composition can optionally contain a small amount of a wetting agent or emulsifying agent, or a pH buffering agent.

[0121] The form, route of administration, dosage, and regimen of the pharmaceutical composition will of course depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, the desired treatment period, etc. The pharmaceutical composition can be in any suitable form depending on the desired method of administration to the patient. A preferred mode of administration is retrograde administration to the patient's coronary system.

[0122] According to one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable vehicle as an injectable form for the patient. These are in particular isotonic, sterile, physiological saline (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a mixture of such salts), or a dried, in particular lyophilized, composition which can in some cases be made into an injectable solution by adding sterile water or physiological saline.

[0123] To prepare the pharmaceutical composition, an effective amount of the active ingredient can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0124] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions; dosage forms containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the preparation of sterile injectable solutions or dispersions. In each case, the form must be sterile and must be fluid to the extent that it can be easily injected with a syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Solutions of the active compounds as the free base or as pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal storage and use conditions, these formulations contain preservatives to prevent the growth of microorganisms.

[0125] According to one embodiment, the S100A1 protein or a fragment or variant thereof, nucleic acid, or vector of the present invention described herein can be formulated into a pharmaceutical composition in neutral or salt form using a pharmaceutically acceptable salt.

[0126] Sterile injectable solutions are prepared by incorporating the required amount of the active compound into a suitable solvent, with or without several of the other ingredients enumerated above, and then filtering the solution sterilize. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is by vacuum drying and lyophilization techniques to obtain a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered solution.

[0127] According to a preferred embodiment, the pharmaceutical composition is administered by oral, intravenous, intramucosal, intraarterial, intrathecal, or intracoronal routes. Intravenous administration is preferred. In the case of intravenous administration, it is particularly preferred that the protein, nucleic acid, vector, or pharmaceutical composition be administered retrogradely into the coronary system of the patient.

[0128] In a preferred embodiment, administration of the protein, biologically active fragment, nucleic acid, vector, or pharmaceutical composition of the invention results in an increase of about 50-fold in the concentration of S100A1 protein in the myocardium of an individual compared to the concentration of S100A1 in the myocardium of a healthy individual. Thus, preferably, the concentration of S100A1 protein in the myocardium is increased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold compared to the concentration of S100A1 in the myocardium of a healthy individual.

[0129] The present invention further relates to the following items: Item 1: For use in a patient in need of treatment and / or prevention of infarct progression, an S100A1 protein, a biologically active fragment or variant thereof, or a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof, wherein the fragment or variant has at least 80% sequence identity with SEQ ID NO: 1. Item 2: A vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1 for use in a patient in need of treatment and / or prevention of infarct progression. Item 3: A pharmaceutical composition comprising (i) an S100A1 protein, a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1, or a nucleic acid encoding said S100A1 protein, fragment, or variant; (ii) a vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1; or (iii) a pharmaceutically acceptable salt of (i) or (ii). A pharmaceutical composition for use in a patient in need of treatment and / or prevention of infarct progression, optionally further comprising a pharmaceutically acceptable excipient, carrier, and / or diluent. Item 4: An S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any one of items 1 to 3, wherein the S100A1 protein has the sequence set forth in SEQ ID NO: 1. Item 5: A vector or a pharmaceutical composition for use according to item 2, 3, or 4, wherein the vector is selected from the group consisting of a plasmid vector, a cosmid vector, a phage vector such as lambda phage, a filamentous phage vector, a viral vector, a virus-like particle, and a bacterial spore. Item 6: A vector or a pharmaceutical composition for use according to item 5, wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus (AAV) vector, an alphavirus vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, a vesicular stomatitis virus vector, a retrovirus vector, and a lentivirus vector. Item 7: A vector or a pharmaceutical composition for use according to item 6, wherein the adeno-associated virus (AAV) vector is selected from the group consisting of AAV5, AAV6, and AAV9, preferably AAV5. Item 8: A vector or a pharmaceutical composition for use according to any one of items 2 to 7, wherein the expression of the S100A1 protein is controlled by a heart tissue-specific promoter. Item 9: A vector or a pharmaceutical composition for use according to item 8, wherein the heart tissue-specific promoter is selected from the group consisting of a cardiac actin enhancer / elongation factor 1 promoter, a cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin. Item 10: An S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any one of items 1 to 9, which is administered via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronal route. Item 11: The S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to item 10, which is administered via the intravenous route, preferably by retrograde administration into the coronary system of a patient. Item 12: The S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any one of items 1 to 11, which is administered between 1 day and 4 weeks after myocardial infarction has occurred in a patient. Item 13: The S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any one of items 1 to 12, wherein the intracellular level of the S100A1 protein increases for at least 7 days. Item 14: The vector or pharmaceutical composition for use according to any one of items 2 to 13, which is administered at a single dose of 1 x 10 13 vector genome copies (vgc). Item 15: A method comprising administering to a patient the following for use in a patient in need of treatment and / or prevention of infarct progression: (i) The S100A1 protein, a biologically active fragment or variant thereof, or a nucleic acid encoding the S100A1 protein or a fragment or variant thereof, (ii) A vector containing a nucleic acid encoding the S100A1 protein or a biologically active fragment thereof, or (iii) A pharmaceutically acceptable salt of (i) or (ii), wherein the fragment or variant has at least 80% sequence identity with SEQ ID NO: 1. Item 16: The method according to item 15, wherein the S100A1 protein has the sequence defined in SEQ ID NO: 1. Item 17: The method according to item 15, wherein the vector is selected from the group consisting of plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores. Item 18: The method according to item 17, wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus (AAV) vector, an alphavirus vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, a vesicular stomatitis virus vector, a retrovirus vector, and a lentivirus vector. Item 19: The method according to item 18, wherein the adeno-associated virus (AAV) vector is selected from the group consisting of AAV5, AAV6, and AAV9. Item 20: The method according to item 15, wherein the expression of the S100A1 protein is controlled by a heart tissue-specific promoter. Item 21: The method according to item 20, wherein the tissue-specific promoter is selected from the group consisting of a cardiac actin enhancer / elongation factor 1 promoter, a cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin. Item 22: The method according to item 15, wherein the protein, its biologically active fragment, nucleic acid, vector, or pharmaceutical composition is administered via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronal route. Item 23: The method according to item 22, wherein the protein, its biologically active fragment, nucleic acid, vector, or pharmaceutical composition is administered via the intravenous route, preferably by retrograde administration into the coronary system of the patient of the protein, nucleic acid, vector, or pharmaceutical composition. Item 24: The method according to item 15, wherein the protein, its biologically active fragment, vector, or pharmaceutical composition is administered to the patient between 1 day and 4 weeks after myocardial infarction has occurred after ischemia. Item 25: The method according to item 15, wherein the intracellular level of the S100A1 protein increases for at least 7 days. Item 26: The vector is administered at a single dose of 1x10 13 vector genome copies (vgc). The method according to item 15.

Examples

[0130] Examples are for further explaining the present invention and providing a better understanding. These are not to be construed as limiting the scope of the present invention in any sense.

[0131] Example 1 Myocardial distribution of rAAV5, rAAV6, and rAAV9 after retrograde intravenous administration to the cardiac target in pigs As a clinically applicable route of administration (ROA), it was investigated whether retrograde intravenous administration (CRID) by a catheter targeting the heart with rAAV5 would result in an appropriate in vivo cardiac transduction pattern in the porcine heart. For this purpose, an rAAV5 vector with a luciferase (luc) reporter gene was employed, the expression of which was controlled by a previously published cardiac-specific MLC-2v-driven promoter (P. Raake et al., "AAV6-BARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model", Eur Heart J., 34, 2013). The biodistribution of rAAV5-luc was systematically evaluated by luminescence measurement of luc reporter gene activity in porcine hearts and additional organ samples 4 weeks after CRID in healthy pigs. These results were benchmarked against CRID of rAAV6-luc and rAAV9-luc, taking into account that, for example, the ability to introduce into the porcine diseased heart with the hs100a1 gene has been previously demonstrated (P. Most et al., "Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model", Science Translational Medicine, 3, 2011; C. Weber, "Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model", 21, 2014). Either rAAV5-luc, rAAV6-luc, or rAAV9-luc was administered to anesthetized and ventilated pigs at 1.0×10 13VGC was administered. As part of a reproducible standard operating procedure (SOP) for CRID, the appropriate position of the retrograde injection catheter in the anterior interventricular vein (AIV) proximal to the coronary sinus outflow was recorded using digital fluoroscopic images. Subsequently, to exclude immediate leakage into the systemic circulation and avoid selective migration of rAAV into smaller venous branches of the AIV during rAAV injection, contrast agent was administered retrogradely into the occluded venous catheter. At the same time, the appropriate position of the coronary balloon catheter was confirmed to avoid immediate retrograde flushing of the myocardial capillary system during retrograde rAAV introduction. For each animal, digital fluoroscopic images of each part of the CRID SOP were recorded and saved.

[0132] Figures 1A and B are representative fluoroscopic images recording the aforementioned steps before CRID. Figures 1C and D show the main findings regarding the in vivo distribution of rAAV5, rAAV6, and rAAV9 in the heart and non-heart by systematic luminometer evaluation of tissue luc activity. For the average myocardial luc activity level, rAAV5 showed approximately 30-fold higher reporter gene activity than rAAV9, which is widely used in preclinical heart GTMP development (rAAV5 167.179 ± 16.083 RLU / mg protein vs. rAAV9 5.084 ± 0.835 RLU / mg protein; data are shown as mean ± SEM, P < 0.01 rAAV5 vs. rAAV9). Figures 1E and F show that the CRID SOP significantly reduced the luc activity of each rAAV serotype tested in the liver, lung, kidney, and skeletal muscle. These results indicate that rAAV5, in particular, is a preferred vector for heart-targeted hs100a1 gene therapy via CRID as a clinically applicable ROA in a heart disease model closely resembling human pathophysiology.

[0133] Example 2 Effect of rAAV5-hs100a1 on cardiac performance and injury in a cardiac injury pig model evaluated by CMR The MI size was reassessed 2 weeks after the experimental MI by LGE and further by global native T1-weighing, and only pigs with an MI size exceeding 14% of the LV were alternately assigned to the rAAV5-luc control and the rAAV5-hs100a1 intervention. Each animal received a single dose of 1.0×10 13 vgc of either vector as a benchmark for previous rAAV9-hs100a1 and rAAV6-hs100a1 studies. The rAAV5 CRID SOP was performed using blinded experimenters to the quality measures and intervention types established herein, as described in these studies (P. Most et al., "Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model", Science Translational Medicine, 3, 2011; C. Weber, "Therapeutic safety of high myocardial expression levels of molecular inotrope S100A1 in a preclinical heart failure model", 21, 2014). The design of the intervention study is shown in Figure 2A. Three months after the rAAV5-based gene therapy, the CMR SOP was reapplied blindly to determine changes in cardiac performance and MI size within and between both groups. LV-EF and MI mass were the co-primary endpoints. The data obtained were analyzed simultaneously, and animal enrollment was stopped when one of the endpoints reached significance compared to the control.

[0134] Figure 2A shows the overall outline of the experiment, and Figure 2B shows that treatment with rAAV5-hs100a1 at 2 weeks after MI prevented the progression of total LV MI mass that occurred in the control group until the end of the 12-week follow-up period, as evaluated by LGE signal and T1 relaxation time. In the simultaneous analysis of changes in LV-EF based on CMR, it was revealed that the LV-EF was significantly increased in the rAAV5-hs100a1 treatment group compared to the rAAV5-luc treatment group (Figure 2D). To support the implementation of the standardized MI size cut-off and CMR protocol, the significance of both co-primary endpoints had already been achieved with animal numbers of 5 or less per group. From these results, it was concluded that rAAV5 is an appropriate rAAV serotype for sufficient delivery of the hs100a1 gene to diseased hearts of human size and subsequent long-term improvement of cardiac performance. The protective effect of hs100a1 against MI progression, which has not yet been reported, sheds new light on the therapeutic potential of hs100a1. This is because the anti-inflammatory effect of S100A1 improves the overall cardiac contractile performance after MI, as demonstrated by the positive change in LF-EF, thereby halting MI progression.

[0135] Example 3 Expression analysis of S100A1, exploratory safety and toxicity assessment after cardiac targeting delivery of rAAV5-hs100a1 First, the cardiac expression of the therapeutic hs100a1 transgene was determined by semi-quantitative PCR to distinguish human and porcine S100A1 gene expression and confirm effective myocardial gene delivery by cardiac-targeted CRID. Subsequently, rAAV5 off-target organ transduction observed in Figures 1E and F (in vivo distribution of rAAV5) prompted the analysis of hs100a1 expression outside the heart.

[0136] Figure 3A shows the experimental setup. Three months after rAAV5-hs100a1 gene therapy, a whole-body assessment of vector distribution (black-bordered box) was performed on biological samples from each animal. Figure 3B confirmed that the transgene expression levels were similar in the control and rAAV5-s100a1-treated hearts after CRID SOP. For example, detection of hs100a1 mRNA in the liver, lung, kidney, or CNS suggests that rAAV5 that was not absorbed by the heart during CRID SOP may ultimately reach the systemic circulation and result in off-target delivery (Figure 3C). Figure 3D confirmed that hs100a1 mRNA was not detected in other organs outside the heart, supporting the idea that the heart-biased promoter included hs100a1 expression in the heart despite off-target delivery of rAAV5. The animals' behavior, body temperature, as well as food intake and excreta were regularly examined, and no abnormalities were found during the 12-week observation period after treatment. From these results, it was concluded that rAAV5 and catheter-based CRID are suitable for delivering sufficient hs100a1 gene copies to the diseased porcine heart at clinically appropriate doses to exert the demonstrated therapeutic effects on LV-EF% and MI progression.

[0137] Example 4 Weighted gene co-expression network analysis indicates positive cardioprotection and attenuation of the inflammatory gene program after rAAV5-hs100a1-based gene therapy To obtain further molecular clues for excellent research results and to explain the complex biological processes underlying the prevention of LV-MI progression and the improvement of LV-EF by rAAV5-hs100a1 treatment, an unbiased network-based analytical approach was adopted. For this purpose, 12 weeks after gene transfer, bulk RNA sequencing was performed on LV myocardial tissue samples obtained from each enrolled animal. Next, the recently published WGCNA R software package (P. Langfelder & S. Horvath, "WGCNA: an R package for weighted correlation network analysis", BMC Bioinformatics, 9, 2008) was applied to perform weighted gene co-expression network analysis (WGCNA) to capture clusters of highly co-expressed gene ensembles called modules. Subsequently, the module-trait relationship (MTR) was calculated by pairwise correlation between each module eigengene (ME) and the directed changes in LV-MI and LV-EF after MI. Only the modules with significant and strong correlations (>0.8 and < -0.8) with LV-MI progression and / or LV-EF were further processed in in silico pathway databases such as Reactome, GEO, and KEGG to infer molecular pathways potentially mechanistically related to the therapeutic effect mediated by rAAV5-hs100a1.

[0138] Figure 4A shows the experimental setup. Three months after rAAV5-hs100a1 gene therapy, bulk RNAseq of myocardial tissue and subsequent WGCNA (black boxed frame) were performed. Figure 4B showing the MTR matrix indicates that the turquoise and cyan MEs are significantly and strongly correlated with LV-EF and LV-MI progression, respectively. More specifically, in our model, the ME turquoise showed a negative correlation with the change in LV-EF. Subsequently, in silico pathway analysis was performed using the Reactome, KEGG, and GO term pathway databases, and the turquoise-colored ME's "neutrophil degranulation", "T cell receptor signaling", "signaling by B cell receptor", "downstream signaling events of B cell receptor", "Fc epsilon receptor signaling", and "Fc epsilon receptor signaling-mediated NF-kB activation", which are inflammatory and immune signaling pathways, were significantly overexpressed (Figure 4C). Thus, the in silico test infers that the activities of gene networks related to neutrophils and T and B cells are low in the rAAV5-hs100a1-treated porcine heart where LV-EF was significantly enhanced after MI.

[0139] Furthermore, the MTR matrix showed a strong negative correlation between ME cyan and the surrogate T1 change of LV-MI CMR. Subsequently, when functional annotation was performed using the Reactome, KEGG, and GO term pathway databases, it was revealed that the pathways of cyan ME, "calcium signaling", "mitochondrial translation initiation, elongation, termination", as well as "activation of genes in response to endoplasmic reticulum stress by ATF4", "regulation of gene expression by PERK", "unfolded protein response", and "signaling by retinoic acid", "ligand binding and activation of fibroblast growth factor receptor 2" pathways were significantly overexpressed (Figures 4D and 4E). Therefore, the transcriptome data analysis of this system infers the beneficial cooperation of myocardial calcium circulation, maintenance of mitochondrial energy balance, stress response, and cardioprotective pathways in the myocardium treated with rAAV5-hs100a1, and the LV-MI progression after MI was completely suppressed.

[0140] Example 5 Treatment of LV infarct mice with rAAV5-hs100a1 attenuates the activities of the innate and adaptive immune systems in the myocardium. The previously published mouse LV-MI model (Most et al., "Cardiac S100A1 protein levels determine contractile performance and propensity toward heart failure after myocardial infarction", 114, 2006) was used to confirm important findings inferred from the signaling pathway analysis in many of our animal post-MI models. The experimental setup is shown in Figure 5A. Intramyocardial injection was performed immediately after experimental MI. Four weeks after gene therapy, LV-EF% and inflammatory gene expression were determined by echocardiography and RT-PCR (black-bordered boxes). Specifically, prior to ligation of the LAD, a total of 2×10 11 vgc of rAAV5-hs100a1 and rAAV5-luc were injected into the LV wall of the mouse heart (Figure 5B). Four weeks after treatment, the effects of rAAV5-hs100a1 and the control on LV performance after MI and the size of MI were determined by echocardiography and immunohistology (J. Takagawa et al., "Myocardial infarct size measurement in mouse chronic infarction model: comparison of area- and length-based approaches", H Appl Physiol, 102, 2007), respectively. Since the effect of S100A1 gene therapy on immune system activity in remodeled and dysfunctional hearts had not been reported yet, RT-PCR analysis focused on the myocardial abundance of transcripts of marker genes related to innate and adaptive immune system activities.

[0141] Figures 5C and 5D show that rAAV5-hs100a1 treatment improved LV-EF% and reduced MI size in a mouse model compared to the control group after 4 weeks. Similar to many animal models after MI that received rAAV5-hs100a1, mice after MI that received acute intramyocardial injection of rAAV5-hs100a1 showed low levels of various marker genes for the presence of myocardial macrophages, neutrophils, and T cells, as well as inflammatory cytokines, including, for example, cd68, cxcr2, and cd4, and il-1b, tnf, and inf-g (Figures 5E and F).

[0142] The examples particularly show that S100A1 is an effective, safe, and specific therapeutic means for combating myocardial infarction progression.

Claims

1. For use in a patient in need of treatment and / or prevention of infarct progression, an S100A1 protein, a biologically active fragment or variant thereof, or a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof, wherein the fragment or variant has at least 80% sequence identity with SEQ ID NO:

1.

2. A vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1 for use in a patient in need of treatment and / or prevention of infarct progression.

3. A pharmaceutical composition comprising: (i) an S100A1 protein, a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1, or a nucleic acid encoding said S100A1 protein, fragment, or variant; (ii) a vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment or variant thereof having at least 80% sequence identity with SEQ ID NO: 1; or (iii) a pharmaceutically acceptable salt of (i) or (ii) for use in a patient in need of treatment and / or prevention of infarct progression, optionally further comprising a pharmaceutically acceptable excipient, carrier, and / or diluent.

4. An S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition according to any one of claims 1 to 3 for use, wherein the S100A1 protein has the sequence set forth in SEQ ID NO:

1.

5. The vector or pharmaceutical composition according to claim 2, 3, or 4 for use, wherein the vector is selected from the group consisting of plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores.

6. The vector or pharmaceutical composition according to claim 5 for use, wherein the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors.

7. A vector or pharmaceutical composition for use according to claim 6, wherein the adeno-associated virus (AAV) vector is selected from the group consisting of AAV5, AAV6, and AAV9, preferably AAV5.

8. A vector or pharmaceutical composition for use according to any one of claims 2 to 7, wherein the expression of the S100A1 protein is controlled by a heart tissue-specific promoter.

9. A vector or pharmaceutical composition for use according to claim 8, wherein the heart tissue-specific promoter is selected from the group consisting of a cardiac actin enhancer / elongation factor 1 promoter, a cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin.

10. An S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any one of claims 1 to 9, which is administered via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronal route.

11. An S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to claim 10, which is administered via an intravenous route, preferably retrogradely administered into the coronary system of a patient.

12. An S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any one of claims 1 to 11, which is administered between 1 day and 4 weeks after myocardial infarction has occurred in a patient.

13. An S100A1 protein, a biologically active fragment or variant thereof, a nucleic acid, a vector, or a pharmaceutical composition for use according to any one of claims 1 to 12, wherein the intracellular level of the S100A1 protein is increased for at least 7 days.

14. The vector is 1x10 １３ A vector or pharmaceutical composition for use according to any one of claims 2 to 13, wherein the vector is administered in a single dose of vector genome copies.

15. A method comprising administering to a patient in need of treatment and / or prevention of infarct progression the following for treatment and / or prevention of infarct progression in the patient: (i) an S100A1 protein, a biologically active fragment or variant thereof, or a nucleic acid encoding an S100A1 protein or a fragment or variant thereof, (ii) a vector comprising a nucleic acid encoding an S100A1 protein or a biologically active fragment thereof, or (iii) a pharmaceutically acceptable salt of (i) or (ii), wherein the fragment or variant has at least 80% sequence identity with SEQ ID NO:

1.

16. The method according to claim 15, wherein the S100A1 protein has the sequence defined in SEQ ID NO:

1.

17. The method according to claim 15, wherein the vector is selected from the group consisting of plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores.

18. The method according to claim 17, wherein the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retroviral vectors, and lentiviral vectors.

19. The method according to claim 18, wherein the adeno-associated virus (AAV) vector is selected from the group consisting of AAV5, AAV6, and AAV9.

20. The method according to claim 15, wherein the expression of the S100A1 protein is controlled by a heart tissue-specific promoter.

21. The method according to claim 20, wherein the tissue-specific promoter is selected from the group consisting of cardiac actin enhancer / elongation factor 1 promoter, cytomegalovirus enhancer / myosin light chain ventricular 2 promoter, and troponin.

22. The method according to claim 15, wherein the protein, its biologically active fragment, nucleic acid, vector, or pharmaceutical composition is administered via an oral, intravenous, intramucosal, intraarterial, intramuscular, or intracoronal route.

23. The method according to claim 22, wherein the protein, its biologically active fragment, nucleic acid, vector, or pharmaceutical composition is administered via the intravenous route, preferably the protein, nucleic acid, vector, or pharmaceutical composition is retrogradely administered into the coronary system of the patient.

24. The method according to claim 15, wherein the protein, its biologically active fragment, vector, or pharmaceutical composition is administered to the patient between 1 day and 4 weeks after myocardial infarction has become ischemic.

25. The method according to claim 15, wherein the intracellular level of the S100A1 protein increases for at least 7 days.

26. The method according to claim 15, wherein the vector is administered at a single dose of 1 x 10 １３ vector genome copies (vgc).

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