Use of recombinant fibrinogen-like domain of angiopoietin-like 4 to treat adverse post-ischemic cardiac remodeling in patients who have undergone myocardial infarction

Abstract

Ischemic heart disease is a leading cause of death and reduced quality of life worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), many patients with MI develop chronic heart failure over time. We previously reported that human recombinant ANGPTL4 counteracts ischemia-induced vascular endothelial growth factor signaling and disruption of endothelial cell-cell adhesion, thereby inhibiting vascular permeability. We were able to demonstrate that ANGPTL4 administration before MI resulted in protection of the coronary capillary network, no-reflow syndrome, and reduced infarct size in mice. We also demonstrated that the therapeutic effects observed with ANGPTL4 under ischemic conditions were caused by the FLD fragment, not the CCD fragment (WO 2016 / 110498). To further examine the therapeutic potential of the FLD fragment of ANGPTL4 at the onset of reperfusion, we herein used a porcine model, a clinically relevant model of acute myocardial infarction that can be easily and safely translated into patient treatment. We demonstrated that local (antegrade) delivery of the FLD ANGPTL4 to infarcted porcine hearts can efficiently target the lesion site in a clinically relevant manner. A single administration of the FLD of ANGPTL4 improved cardiac function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments, coupled with complementary mouse studies, demonstrated myocardial protection. Thus, a single administration of the FLD of ANGPTL4 can reduce ischemia-reperfusion injury and protect against adverse postischemic cardiac remodeling and subsequent ischemic heart failure.

Description

[Technical Field]

[0001] Field of the invention: The present invention is in the field of medicine, particularly cardiology.

[0002] Background of the invention: Myocardial infarction (MI), the most common manifestation of cardiovascular disease, is associated with high mortality and morbidity (Mortality, GBD and C. Causes of Death, Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet, 2015. 385(9963): pp. 117-71). Nevertheless, considerable progress has been achieved in the early management of acute coronary thrombotic occlusion, including rapid mechanical restoration of coronary blood flow and antiplatelet therapy (Frangogiannis, NG, The inflammatory response in myocardial injury, repair, and remodeling. Nat Rev Cardiol, 2014. 11(5): pp. 255-65). Over the past few decades, a significant decrease in early mortality has been observed in patients with MI (Puymirat, E., et al., Association of changes in clinical characteristics and management with improvement in survival among patients with ST-elevation myocardial infarction. JAMA, 2012. 308(10): p. 998-1006).However, the long-term effects of ischemia-related cardiac damage remain a clinical and societal burden due to increased risk of arrhythmias, heart failure, and repeated hospitalizations (Lavoie, L., et al., Burden and Prevention of Adverse Cardiac Events in Patients with Concomitant Chronic Heart Failure and Coronary Artery Disease: A Literature Review. Cardiovasc Ther, 2016. 34(3): p. 152-60). Moreover, more efforts must be directed toward developing therapeutic approaches that target the pathophysiological pathways involved in postischemic cardiac remodeling. Human recombinant ANGPTL4 counteracts ischemia-induced vascular endothelial growth factor signaling and disruption of endothelial cell-cell adhesion, thereby inhibiting vascular permeability (Galaup, Ariane, et al. "Protection against myocardial infarction and no-reflow through preservation of vascular integrity by angiopoietin-like 4." Circulation 125.1 (2012): 140-149). When administered before MI, ANGPTL4 protects the coronary capillary network, prevents reflow, and reduces infarct size (WO 2011 / 089152 and WO 2016 / 110498). Subsequently, studies have shown that the reduction in infarct size does not correlate with improvement in ventricular remodeling (Piot C et al., 2008, N Engl J Med & Cung TT et al., 2015, N Engl J Med). Therefore, the effect of ANGPTL4 on postischemic cardiac remodeling has not been studied at all.

[0003] Summary of the Invention: The present invention is defined by the claims. In particular, the present invention relates to the use of recombinant fibrinogen-like domain of angiopoietin-like 4 to treat adverse post-ischemic cardiac remodeling in patients who have experienced myocardial infarction.

[0004] Detailed description of the invention: Ischemic heart disease is a leading cause of death and reduced quality of life worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), many patients with MI develop chronic heart failure over time. We previously reported that human recombinant ANGPTL4 counteracts ischemia-induced vascular endothelial growth factor signaling and disruption of endothelial cell-cell adhesion, thereby inhibiting vascular permeability. We were able to demonstrate that ANGPTL4 administration before MI resulted in protection of the coronary capillary network, no-reflow syndrome, and reduced infarct size in mice. We also demonstrated that the therapeutic effects observed with ANGPTL4 under ischemic conditions were caused by the FLD fragment, not the CCD fragment (WO 2016 / 110498). To further examine the therapeutic potential of the FLD fragment of ANGPTL4 at the onset of reperfusion, we herein used a porcine model, a clinically relevant model of acute myocardial infarction that can be easily and safely translated into patient treatment. We demonstrated that local (antegrade) delivery of FLD ANGPTL4 to infarcted porcine hearts can efficiently target the lesion site in a clinically relevant manner. A single administration of FLD ANGPTL4 improved cardiac function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments, coupled with complementary mouse studies, demonstrated myocardial protection. Thus, a single administration of FLD ANGPTL4 can reduce ischemia-reperfusion injury and protect against adverse postischemic cardiac remodeling and subsequent ischemic heart failure. Furthermore, using intracoronary injection, we demonstrated reduced diffusion of the injected product to other organs.

[0005] Key definitions: As used herein, the term "polypeptide" has its common meaning in the art and refers to a polymer of amino acids of any length. The polymer may contain modified amino acids. These terms also encompass amino acid polymers that have been modified, either naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art, are also included in this definition.

[0006] As used herein, "polynucleotide," as used herein, refers to a polymer of nucleotides of any length, comprising ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA") as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified forms, for example, by alkylation and / or capping, as well as unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), whether spliced ​​or not, including tRNA, rRNA, hRNA, siRNA, and mRNA, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotidic backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, as well as other synthetic sequence-specific nucleic acid polymers (provided that the polymer contains nucleobases in a configuration that allows for base pairing and base stacking, e.g., as found in DNA and RNA). In some embodiments, a polynucleotide comprises an mRNA. In other aspects, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are substituted with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be substituted with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0007] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a predetermined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a predetermined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns, to the extent that nucleotide sequences that encode proteins may, in some versions, contain introns.

[0008] As used herein, the "percent identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % identity = number of identical positions / total number of positions × 100). Comparison of sequences and determination of the percent identity between two sequences can be achieved using a mathematical algorithm, such as the following: The percent identity between two amino acid sequences can be determined, for example, using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53). The percent identity between two nucleotide or amino acid sequences can also be determined, for example, using an algorithm such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with a BLOSUM62 matrix, a "Gap Open Penalty" of 10, a "Gap Extension Penalty" of 0.5, an "End Gap Penalty" of 10, and an "End Gap Extension Penalty" of 0.5. Generally, "percent identity" is a function of the number of matching positions divided by the number of compared positions multiplied by 100. For example, if 6 out of 10 sequence positions are identical between two compared sequences after alignment, the percent identity is 60%. Percent identity is typically determined over the entire length of the query sequence analyzed. Two sequences with the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications. According to the present invention, a first amino acid sequence having at least 90% identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence.

[0009] As used herein, the term "ANGPTL4" has its common meaning in the art and refers to angiopoietin-like protein 4, which is encoded by the ANGPTL4 gene. An exemplary human amino acid sequence for ANGPTL4 is SEQ ID NO:1. Human ANGPTL4 consists of 406 amino acids. Its protein structure is common to angiopoietins, with a signal peptide directing secretion, an amino-terminal coiled-coil domain (CCD), a linker, and a carboxy-terminal fibrinogen-like domain (FLD). ANGPTL4 undergoes processing to release a CCD-containing fragment and an FLD-containing fragment. The soluble CCD binds to lipoprotein lipase and converts the catalytically active dimeric form of the enzyme into an inactive monomer, while the soluble FLD regulates vascular biological properties. The CCD fragment consists of an amino acid sequence ranging from amino acid residue 22 to amino acid residue 170. The FLD fragment consists of the amino acid sequence ranging from the 186th amino acid residue to the 406th amino acid residue.

[0010] [ka]

[0011] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc. In some preferred embodiments, the subject is a human.

[0012] As used herein, the term "myocardial infarction" has its common meaning in the art and relates to irreversible necrosis of the myocardium as a result of prolonged ischemia due to coronary thrombosis, i.e., the development of a clot in a major blood vessel serving the heart.

[0013] As used herein, the term "detrimental post-ischemic cardiac remodeling" has its common meaning in the art and refers to significant changes that occur after myocardial infarction, potentially detrimental to cardiac function. Cardiac remodeling involves molecular, cellular, and interstitial changes that clinically manifest as changes in cardiac size, shape, and function after myocardial infarction. For example, ventricular remodeling involves progressive enlargement of the ventricles with a decline in ventricular function. Myocardial function declines in the myocardium distant from the initial myocardial infarction. In particular, detrimental post-ischemic cardiac remodeling includes arrhythmias, cardiac dilation (assessed by left ventricular end-diastolic volume index per body surface area, or LVEDV), and cardiac dysfunction (left ventricular ejection fraction, or EF). Typically, detrimental post-ischemic cardiac remodeling is defined as a >20% increase in left ventricular end-diastolic volume (LVEDV) at 6 months compared to the initial assessment.

[0014] As used herein, the term "myocardial fibrosis" has its general meaning in the art and refers to a condition characterized by the excessive production and deposition of extracellular matrix (ECM) proteins within the myocardium, resulting in the disruption of normal tissue architecture, reduced tissue compliance, and mechanical and electrical dysfunction. Following acute myocardial infarction, the sudden loss of large numbers of cardiomyocytes induces an inflammatory response, ultimately resulting in the replacement of dead myocardium with a collagen-based scar.

[0015] As used herein, the terms "heart failure" or "HF" have their common meaning in the art and include congestive heart failure and / or chronic heart failure. Functional classification of heart failure is performed by the New York Heart Association's Cardiac Functional Classification (Criteria Committee, New York Heart Association. Diseases of the heart and blood vessels. Nomenclature and criteria for diagnosis, 6th ed. Boston: Little, Brown and co., 1964;114). This classification stages the severity of heart failure into four classes (I-IV). The classes (I-IV) are as follows: Class I: No limitation in any activity experienced; no symptoms with normal activity; Class II: slight mild limitation in activity; patient comfortable at rest or with light activity; Class III: significant limitation in any activity; patient comfortable only at rest; Class IV: discomfort with any physical activity and symptoms present at rest.

[0016] As used herein, the term "treatment" or "treating" refers to both preventative or prophylactic treatment, as well as curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients with a disease or diagnosed with a disease or medical condition, including the suppression of clinical recurrence. Treatment may be administered to patients with a medical disorder or a patient who may ultimately acquire a disorder in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the patient's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of disease treatment, e.g., a dosing pattern used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient early in the treatment regimen. An induction regimen may use (in part or in whole) a "loading regimen," which may involve administering a higher dose of drug than a physician uses during a maintenance regimen, administering a drug more frequently than a physician administers during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep a patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon the achievement of a specific predetermined criterion (e.g., disease manifestation, etc.)).

[0017] As used herein, the term "therapeutically effective amount" refers to a sufficient amount of the active ingredient to treat or reduce symptoms at a reasonable benefit / risk ratio applicable to any medical treatment. It is understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex, and diet of the subject; the administration time, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with the active ingredient; and similar factors well known in the medical arts. For example, it is well within the skill of one of ordinary skill in the art to start the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0018] Treatment method: A first object of the present invention relates to a method for treating adverse post-ischemic cardiac remodeling in a patient who has experienced a myocardial infarction, comprising administering to the patient a therapeutically effective amount of i) a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence ranging from amino acid residue 186 to amino acid residue 406 in SEQ ID NO:1, or ii) a polynucleotide encoding the same.

[0019] In particular, the method of the present invention is suitable for protecting against or reducing damage to the myocardium after myocardial infarction, after, during, or before ischemic reperfusion. More particularly, the method of the present invention is particularly suitable for reducing post-ischemic left ventricular remodeling. Even more particularly, the method of the present invention is suitable for increasing the left ventricular ejection fraction (LVEF), inhibiting left ventricular dilation, reducing left ventricular end-systolic volume, reducing left ventricular end-diastolic volume, restoring left ventricular dysfunction, and / or improving myocardial contractility.

[0020] In some embodiments, the methods of the present invention are particularly suited to preventing myocardial fibrosis.

[0021] The methods of the present invention are also suitable for preventing heart failure in patients who have experienced a myocardial infarction.

[0022] In some embodiments, a polypeptide or polynucleotide of the invention is administered to a patient having one or more signs or symptoms of acute myocardial infarction injury. In some embodiments, the patient has one or more signs or symptoms of myocardial infarction, such as a mid-thoracic pressure sensation, fullness, or squeezing; chest pain radiating to the jaw or teeth and / or back; difficulty breathing or shortness of breath; epigastric discomfort with or without nausea and vomiting; and excessive sweating or diaphoresis.

[0023] In some embodiments, the polypeptide or polynucleotide of the invention is administered simultaneously with or sequentially (i.e., before or after) a revascularization procedure performed on a patient. In some embodiments, the polypeptide or polynucleotide of the invention is administered to a patient before, during, and after the revascularization procedure. In some embodiments, the polypeptide or polynucleotide of the invention is administered to a patient as a bolus dose immediately prior to the revascularization procedure. In some embodiments, the polypeptide or polynucleotide of the invention is administered continuously to a patient during and after the revascularization procedure. In some embodiments, the polypeptide or polynucleotide of the invention is administered to a patient for a period selected from the group consisting of at least 3 hours after the revascularization procedure; at least 5 hours after the revascularization procedure; at least 8 hours after the revascularization procedure; at least 12 hours after the revascularization procedure; or at least 24 hours after the revascularization procedure. In some embodiments, the revascularization procedure is selected from the group consisting of percutaneous coronary intervention; balloon angioplasty; insertion of a bypass graft; insertion of a stent; directional coronary atherectomy; treatment with one or more thrombolytic agents; and removal of an obstruction.

[0024] In some embodiments, it is contemplated that the polypeptides of the present invention may be modified to improve therapeutic efficacy. Such modifications of therapeutic compounds can be used to reduce toxicity, increase circulation time, or alter biodistribution. For example, combination with various drug carrier vehicles that modify biodistribution can significantly reduce the toxicity of potentially important therapeutic compounds. A strategy for improving drug potential is the use of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve cellular uptake patterns, alter permeability through physiological barriers, and modify clearance rates from the body. Water-soluble polymers have been synthesized containing drug moieties as terminal groups, as part of the backbone, or as pendant groups on the polymer chain to achieve either targeted or sustained-release effects. Polyethylene glycol (PEG) is widely used as a drug carrier given its high biocompatibility and ease of modification. Conjugation of various drugs, proteins, and liposomes has been shown to improve residence time and reduce toxicity. PEG can be linked to active agents through hydroxyl groups at the ends of the chains and by other chemical methods; however, PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids have been explored as novel biomaterials that retain the biocompatibility of PEG but have the added advantage of multiple attachment points per molecule (providing greater drug loading) and can also be synthetically designed to suit a variety of applications.

[0025] In some embodiments, the polypeptides of the present invention are fused to the Fc domain of an immunoglobulin. Suitable immunoglobulins include IgG, IgM, IgA, IgD, and IgE. IgG and IgA are preferred, and IgG, e.g., IgG1, is most preferred. The Fc domain may be a complete Fc domain or a function-conservative variant thereof. The polypeptides of the present invention may be linked to the Fc domain by a linker. The linker may consist of approximately 1 to 100, preferably 1 to 10, amino acid residues.

[0026] According to the present invention, polypeptides of the present invention can be produced by conventional automated peptide synthesis or recombinant expression. General principles for designing and producing proteins are well known to those of skill in the art. Polypeptides of the present invention can be synthesized in solution or on a solid support by conventional techniques. A variety of automated synthesizers are commercially available. Polypeptides of the present invention can also be synthesized by solid-phase techniques using an exemplary peptide synthesizer, such as the Model 433A manufactured by Applied Biosystems Inc. The purity of any given protein produced by automated peptide synthesis or by recombinant methods can be determined using reverse-phase HPLC synthesis. The chemical authenticity of each peptide can be established by any method known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology can be used in which a nucleotide sequence encoding the protein of choice is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression, as described herein below. A variety of expression vector / host systems can be utilized to contain and express the peptide or protein coding sequence. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; microorganisms such as yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus; see Ghosh et al., 2002); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Mammalian cells useful for producing recombinant proteins include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g., COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Mammalian host systems for the expression of recombinant proteins are also well known to those skilled in the art.Host cell lines can be selected for their particular ability to process expressed proteins or produce certain post-translational modifications useful for providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves the "prepro" form of a protein, can also be important for correct insertion, folding, and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, and WI38, have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be selected to ensure the correct modification and processing of introduced foreign proteins.

[0027] In some embodiments, a polynucleotide of the invention is messenger RNA (mRNA).

[0028] In some embodiments, the polynucleotide is inserted into a vector, such as a viral vector. As used herein, the term "viral vector" refers to a virion or virus particle that functions as a nucleic acid delivery vehicle and contains a vector genome packaged within the virion or virus particle. Typically, the vector is a viral vector that is an adeno-associated virus (AAV), a retroviral vector, a bovine papilloma virus, an adenovirus vector, a vaccinia virus, or a polyoma virus. In some embodiments, the viral vector is a retroviral vector. As used herein, the term "retroviral vector" refers to a vector that contains structural and functional genetic elements primarily derived from a retrovirus. In some embodiments, retroviral vectors of the invention are derived from a retrovirus selected from the group consisting of an alpharetrovirus (e.g., avian leukosis virus), a betaretrovirus (e.g., mouse mammary tumor virus), a gammaretrovirus (e.g., murine leukemia virus), a deltaretrovirus (e.g., bovine leukemia virus), an epsilonretrovirus (e.g., walleye dermal sarcoma virus), a lentivirus (e.g., HIV-1, HIV-2), and a spumavirus (e.g., human spumavirus). In some embodiments, retroviral vectors of the invention are viral particles of replication-defective retroviruses capable of delivering exogenously imported genetic RNA in place of retroviral mRNA.

[0029] Thus, the present invention encompasses the use of virus-like particles. As used herein, the terms "virus-like particle" or "VLP" refer to structures that resemble virus particles but lack the viral genome, are replication-incompetent, and lack pathogenicity. Particles typically contain at least one structural protein from a virus. Preferably, only one structural protein is present. Most preferably, other nonstructural components of the virus are absent. Thus, virus-like particles can spontaneously self-assemble in vitro under appropriate conditions by viral structural proteins, eliminating genetic material and potential replication. Virus-like particles, with diameters of approximately 20-150 nm, also possess nanometer-sized material characteristics, such as a large surface area, surface-accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), an immobile spatial structure, and good biocompatibility. Thus, assembled virus-like particles have great potential as delivery systems for specifically transporting various cargoes. In some embodiments, one or more of the zinc finger motifs of the Gag protein are replaced with one or more RNA-binding domains. In some embodiments, the RNA-binding domain is a coat protein of MS2 bacteriophage, PP7 phage, or Q3 phage, a prophage HK022 Nun protein, a U1A protein, or an hPum protein. More preferably, the RNA-binding domain is a coat protein of MS2 bacteriophage or PP7 phage. Even more preferably, the RNA-binding domain is a coat protein of MS2 bacteriophage. These embodiments are particularly suitable for packaging mRNA encoding an ANGPTL4 FLD polypeptide into a VLP. Thus, in some embodiments, the mRNA encoding the ANGPTL4 FLD polypeptide enclosed in the viral particles of the present invention comprises at least one encapsidation sequence. By "encapsidation sequence" is meant an RNA motif (sequence and three-dimensional structure) that is specifically recognized by the RNA-binding domain. Preferably, the encapsidation sequence is a stem-loop motif.Even more preferably, the encapsidation sequence of the retroviral particle is, for example, the stem-loop motif of the RNA of the MS2 bacteriophage or the PP7 phage. The stem-loop motif, more particularly the stem-loop motif of the RNA of the MS2 bacteriophage or the PP7 phage, may be used alone or repeated several times, preferably 2 to 25 times, more preferably 2 to 18 times, for example 6 to 18 times. In some embodiments, the invention encompasses the use of LentiFlash® technology, which is based on non-integrating lentiviral particles constructed using a bacteriophage coat protein and its cognate 19 nt stem loop to replace the native lentiviral Psi packaging sequence to achieve active mRNA packaging into lentiviral particles (Prel A, Caval V, Gayon R, Ravassard P, Duthoit C, Payen E, Maouche-Chretien L, Creneguy A, Nguyen TH, Martin N, Piver E, Sevrain R, Lamouroux L, Leboulch P, Deschaseaux F, Bouille P, Sensebe L, Pages JC. Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles. Mol Ther Methods Clin Dev. 2015 Oct 21;2:15039. doi: 10.1038 / mtm.2015.39. PMID: 26528487; PMCID: PMC4613645).

[0030] In some embodiments, vectors of the invention contain "control sequences," which collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region containing DNA regulatory sequences, where the regulatory sequence is derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0031] In some embodiments, the polypeptides or polynucleotides of the present invention can be conjugated to at least one other molecule. Typically, the molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs. In some embodiments, the polypeptides or polynucleotides of the present invention are formulated using one or more lipid-based structures, including, but not limited to, liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16). Liposomes are artificially prepared vesicles that may be primarily composed of a lipid bilayer and can be used as a delivery vehicle for administering pharmaceutical formulations. Liposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by thin aqueous compartments; small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve liposome attachment to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes can also contain low or high pH to improve delivery of pharmaceutical formulations. As a non-limiting example, liposomes, such as synthetic membrane vesicles, are prepared by the methods, apparatus, and devices described in U.S. Patent Application Publication Nos. 20130177638, 20130177637, 20130177636, 20130177635, 20130177634, 20130177633, 20130183375, 20130183373, and 20130183372.In some embodiments, the liposomes are formed from liposomes capable of delivering small molecule drugs, such as 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (described in U.S. Patent Application Publication No. 20100324120), and DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.), without limitation. The polypeptides or polynucleotides of the invention can be encapsulated by liposomes and / or can be contained in an aqueous core, which can then be encapsulated by liposomes (see WO 2012031046, WO 2012031043, WO 2012030901, and WO 2012006378 and U.S. Patent Application Publication Nos. 20130189351, 20130195969, and 20130202684).In some embodiments, polynucleotides of the invention are formulated using stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for in vitro and in vivo oligonucleotide delivery (Wheeler et al. Gene Therapy. 1999 6:271-281; ​​Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; see U.S. Patent Application Publication No. 20130122104).

[0032] In some embodiments, the polypeptide or polynucleotide of the invention is administered by intracoronary injection.

[0033] Typically, less than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg / kg of polypeptide is administered as a bolus by intracoronary injection after recanalization. More particularly, after recanalization, approximately 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, 51, 53, 54, 55, 56, 57, 58, 59, or 60 μg / kg of polypeptide is administered as a bolus via intracoronary injection.

[0034] Typically, the active ingredient (i.e., polypeptide or polynucleotide) of the present invention is combined with a pharmaceutically acceptable excipient and, optionally, a sustained-release matrix, such as a biodegradable polymer, to form a pharmaceutical composition. The terms "pharmaceutical" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant of any type.

[0035] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0036] [Figure 1] Comparison of infarct size, expressed as a percentage of the area at risk, between control pigs and pigs injected as a bolus with FLD at a concentration of 50 μg / kg, subjected to 60 min of ischemia and 1 month of reperfusion. Panel a shows infarcts in n=6 pigs, and panel b shows quantification. [Figure 2]Comparison of left ventricular function assessed by electrocardiogram at 3 days and 1 month after reperfusion in pigs injected with FLD compared with control animals. [Figure 3] Comparison of left ventricular interstitial fibrosis assessed by Sirius Red (a) and Masson's Trichrome (b) 1 month after reperfusion in pigs injected with FLD compared with control animals.

[0037] Example: Evaluation of the cardioprotective effect of FLD of ANGPTL4 in a porcine model of IR injury: a translational approach. The inventors previously demonstrated that the therapeutic effects observed with ANGPTL4 under ischemic conditions were mediated by the FLD fragment, not the CCD fragment (WO 2016 / 110498). The next step was to study FLD in a large animal model of myocardial infarction. Because most patients undergo primary percutaneous coronary intervention today, the inventors decided to change the administration route and inject FLD directly into the coronary circulation during an intracoronary permeabilization procedure as a translational approach. This offers several advantages: i) concentrating FLD at its target, i.e., the coronary circulation; ii) reducing the amount of FLD circulating peripherally, i.e., reducing the likelihood of non-cardiac effects and thereby reducing the occurrence of potential side effects and toxicities; iii) simplifying the protein administration scheme, i.e., a single intracoronary bolus at the time of reperfusion, with no further injections; and iv) reducing the total amount of FLD required per subject. Therefore, FLD was administered intracoronarily as a 50 μg / kg bolus after recanalization (data not shown). As shown in Figure 1a, infarct size measured using TTC / Evans blue after 1 month was significantly reduced by injection of 50 μg / kg FLD at the time of reperfusion. We next quantified infarct size, expressed as a percentage of the area at risk, and demonstrated a statistically significant reduction in infarct size between control and FLD-injected pigs (Figure 1b). This procedure also allowed us to measure left ventricular function, assessed by electrocardiography, 3 days and 1 month after reperfusion, demonstrating that ejection fraction was preserved in FLD-injected pigs, whereas the corresponding baseline was significantly affected in control animals (Figure 2). Interstitial fibrosis was also reduced in the infarct area in FLD-treated hearts (Figure 3).

[0038] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into the present disclosure.

Claims

1. A method for treating adverse post-ischemic myocardial remodeling in a patient who has experienced a myocardial infarction, comprising administering to the patient a therapeutically effective amount of i) a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence ranging from amino acid residue 186 to amino acid residue 406 in SEQ ID NO: 1, or ii) a polynucleotide encoding the same.

2. 2. A method according to claim 1 for reducing post-ischemic left ventricular remodeling, in particular for increasing the left ventricular ejection fraction (LVEF) and / or for inhibiting left ventricular dilation and / or for reducing left ventricular end-systolic volume and / or for reducing left ventricular end-diastolic volume and / or for restoring left ventricular dysfunction and / or for improving myocardial contractility.

3. 10. The method of claim 1 for preventing myocardial fibrosis in patients who have experienced a myocardial infarction.

4. 10. The method of claim 1 for preventing heart failure in patients who have experienced a myocardial infarction.

5. The method according to any one of claims 1 to 4, wherein the polypeptide or polynucleotide is administered simultaneously or sequentially (ie before or after) a revascularization procedure performed on the patient.

6. 6. The method of claim 5, wherein the polypeptide or polynucleotide is administered as a bolus dose to the patient immediately prior to the revascularization procedure.

7. 6. The method of claim 5, wherein the polypeptide or polynucleotide is administered continuously to the patient during and after the revascularization procedure.

8. The method of any one of claims 1 to 7, wherein the polynucleotide is messenger RNA (mRNA).

9. The method of claim 2, wherein the polynucleotide is inserted into a vector.

10. 10. The method of any one of claims 1 to 9, wherein the polypeptide or polynucleotide is conjugated to at least one other molecule selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs.

11. 11. The method of any one of claims 1 to 10, wherein the polypeptide or polynucleotide is formulated using one or more lipid-based structures selected from the group consisting of liposomes, lipoplexes, or lipid nanoparticles.

12. 12. The method of any one of claims 1 to 11, wherein less than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg / kg of polypeptide is administered as a bolus by intracoronary injection after recanalization.

13. The method of claim 12, wherein after recanalization, approximately 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, 51, 53, 54, 55, 56, 57, 58, 59, or 60 μg / kg of the polypeptide is administered as a bolus via intracoronary injection.

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