Proteins with cardioprotective activity

Abstract

To provide a protein, or a polynucleotide encoding the same, for use in treating or reducing the risk of cardiac disease.SOLUTION: Provided is a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and a fragment thereof, or a polynucleotide encoding the same.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the use of Chrdl1, Fam3c and Fam3b as medicaments, for example in the context of gene therapy or through administration as proteins such as recombinant or synthetic proteins, for the treatment or risk reduction of heart diseases. In particular, the present invention relates to the protection of the heart against the development of heart failure (HF) by maintaining cardiomyocyte viability. Conditions in which this medicament is effective include, but are not limited to, myocardial ischemia (myocardial infarction and reperfusion injury), cardiotoxic injury and genetically caused cardiomyopathies.

Background Art

[0002] Despite recent advances in cardiovascular surgery and treatment, cardiovascular disorders (CVDs) still account for approximately 30% of deaths worldwide, of which roughly 50% are due to ischemic heart disease, which according to the WHO is on the rise and will exceed 23 million by 2030 (www.who.int / cardiovascular_diseases / en / ). Coronary artery disease and myocardial infarction correspond to the main causes of heart failure (65 - 70% of cases). The prognosis of this condition remains poor, and the mortality rate is estimated to be 40% of patients at the fourth year after diagnosis (Owan, T.E. et al., Trends in prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med 355, 251 - 259 (2006)). An important component underlying the epidemic burden of HF is the inability of the myocardium to regenerate in adulthood. Cardiac trauma as a result of ischemia, hypertension, infection, inflammation or toxic injury typically leads to irreversible loss of cardiomyocytes (CMs), resulting in fibrosis and scarring (Laflamme, M.A. & Murry, C.E. Heart regeneration. Nature 473, 326 - 335 (2011); Xin, M., Olson, E.N. & Bassel-Duby, R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat Rev Mol Cell Biol 14, 529 - 541 (2013)).

[0003] Drug discovery in this area has been marginal in terms of efficacy since the mid-1990s, and all available drugs are small chemical molecules. Considering the current ESC guidelines for chronic HF with reduced ejection fraction (Ponikowski, P. et al., 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Eur J Heart Fail 18, 891-975 (2016)), all three drugs recommended for all patients (ACE inhibitors, β-blockers, and mineralocorticoid / aldosterone receptor antagonists) were all introduced into clinical practice in the 1970s or earlier (Gavras, H., Faxon, D.P., Berkoben, J., Brunner, H.R. & Ryan, T.J. Angiotensin converting enzyme inhibition in patients with congestive heart failure. Circulation 58, 770-776 (1978); Swedberg, K., Hjalmarson, A., Waagstein, F. & Wallentin, I. Prolongation of survival in congestive cardiomyopathy by beta-receptor blockade. Lancet 1, 1374-1376 (1979); Goldberger, E. Aldosterone and the Edema of Congestive Heart Failure. Am J Cardiol 15, 274 (1965)); among the drugs recommended only for selected patients, angiotensin II receptor blockers - ARBs date back to the mid-1990s (Gottlieb, S.S.Et al., Hemodynamic and neurohormonal effects of the angiotensin II antagonist losartan in patients with congestive heart failure. Circulation 88, 1602-1609 (1993)), and more recently LCZ6969 is based on a combination of the old ARB (valsartan) and the neprilysin inhibitor sacubitril, which also belong to the class of drugs developed in the late 1980s (Jhund, P.S. & McMurray, J.J. The neprilysin pathway in heart failure: a review and guide on the use of sacubitril / valsartan. Heart 102, 1342-1347 (2016)). The last drug recommended, I, which controls the heart rhythm. f Ivabradine, a class I channel inhibitor, was also developed in the mid-1990s (Thollon, C. et al., Electrophysiological effects of S 16257, a novel sino-atrial node modulator, on rabbit and guinea-pig cardiac preparations: comparison with UL-FS 49. Br J Pharmacol 112, 37-42 (1994)).

[0004] All studies on candidate biological factors (e.g., relaxin, natriuretic peptides, AVP antagonists) for these states shown by biochemical research have failed in phase III clinical trials (Teerlink, J.R. et al., Serelaxin in addition to standard therapy in acute heart failure: rationale and design of the RELAX-AHF-2 study. Eur J Heart Fail 19, 800-809 (2017); O'Connor, C.M. et al., Effect of nesiritide in patients with acute decompensated heart failure. N Engl J Med 365, 32-43 (2011); Matsuzaki, M., Hori, M., Izumi, T. & Fukunami, M. Efficacy and safety of tolvaptan in heart failure patients with volume overload despite the standard treatment with conventional diuretics: a phase III, randomized, double-blind, placebo-controlled study (QUEST study). Cardiovasc Drugs Ther 25 Suppl 1, S33-45 (2011); Wang, G. et al., Efficacy and Safety of 1-Hour Infusion of Recombinant Human Atrial Natriuretic Peptide in Patients With Acute Decompensated Heart Failure: A Phase III, Randomized, Double-Blind, Placebo-Controlled, Multicenter Trial. Medicine (Baltimore) 95, e2947 (2016)).

[0005] In particular, there are no drugs or treatments of any kind that protect the heart during acute ischemia and after myocardial infarction. When a patient has a myocardial infarction, cardiac cells gradually die due to the sudden lack of oxygen resulting from the occlusion of the coronary artery. If a patient undergoes a blood flow improvement procedure (percutaneous coronary angioplasty, angioplasty) within the first few hours after the infarction, a significant portion of the myocardium is preserved, but nevertheless, a large number of cardiac cells die irreversibly. Angioplasty itself promotes further damage due to the sudden oxygen flow that occurs after restoring blood perfusion. Since cardiac cells that contract in adulthood are unable to undergo significant regeneration, the lost portion of the myocardium is irreversibly repaired through scar formation, and this scar formation is a major decisive factor for heart failure in the long term.

[0006] Therefore, there remains a pressing need to provide drugs that can rescue cardiac cells immediately after the damage that causes cardiac cell loss and the resulting pathological tissue repair of the heart, particularly biological agents that mimic the intrinsic survival process. In particular, this need is relevant to the treatment of several conditions that cause HF, including myocardial infarction, reperfusion injury after angioplasty, cardiotoxic injury due to cancer chemotherapy, myocarditis, and cardiomyopathy of genetic and non-genetic causes.

[0007] Protecting cardiac cells from death is extremely effective. This is because it is thought to preserve the myocardium, enable the long-term maintenance of cardiac integrity and function, and avoid the occurrence of the deterioration of cardiac function that causes HF.

[0008] Despite the lack of a radical cure, significant progress has been made in understanding the cellular and molecular mechanisms that lead to tissue degeneration.

[0009] Therefore, there is also a need for new biological therapies that can particularly interfere with the different mechanisms of disease onset and progression and provide treatment opportunities.

[0010] Since the approval of recombinant insulin (Humulin®) in 1982, the number of biopharmaceutical drugs has increased exponentially over the past 30 years. Considering monoclonal antibodies, enzymes, receptor modulators, subunit vaccines, and peptides, well over 350 biopharmaceutical drugs are now clinically approved, and over 400 biopharmaceutical drugs are in clinical trials (Kinch, M.S. An overview of FDA-approved biologics medicines. Drug Discov Today 20, 393-398 (2015); Rader, R.A. (Re)defining biopharmaceutical. Nat Biotechnol 26, 743-751 (2008)).

[0011] In gene research, several landmark discoveries have been made through screening approaches. Since the 1980s, gene identification has been initially facilitated by the selection of genomic DNA libraries through hybridization, followed by the identification of cDNA by antibody screening in phage libraries. In the late 1980s and 1990s, functional screening of libraries in cultured cells led to the identification of several oncogenes and cellular receptors for animal viruses. The earliest approaches were based on the use of pooled libraries (typically cDNA libraries), where the desired factor was identified by phenotype-based selection. In the 2000s, with the advancement of robotics, library screening gradually shifted towards high-throughput screening (HTS) analysis based on the use of arrayed libraries. HTS has opened the way not only for the use of cDNA libraries but also for libraries of peptides, nucleic acids (Eulalio, A. et al., Functional screening identifies miRNAs inducing cardiac regeneration. Nature 492, 376 - 381 (2012)) and small molecules. Today, due to the progress of gene delivery, this field has advanced further, namely, it is possible to directly screen libraries in animals, thus progressing from in vitro biochemical or phenotypic selection to in vivo true functional selection.

Summary of the Invention

[0012] The inventors have utilized a unique procedure based on in vivo functional selection (FunSel) of factors that exhibit the desired function to identify factors with cardioprotective effects. The inventors' procedure is based on the use of a library of AAV vectors, which are sophisticated tools for highly efficient cardiac gene delivery.

[0013] Notably, factor identification by FunSel does not require the selected factor to play a role in a given tissue during normal physiological function, and thus expands the range of potential therapeutic proteins to all secreted factors encoded by the genome.

[0014] Surprisingly, the inventors have found that three factors, Chrdl1 (chordin-like protein 1), Fam3c, and Fam3b, exert quite different biological activities but commonly share a cardioprotective effect.

[0015] The three novel cardioprotective proteins, Chrdl1, Fam3c, and Fam3b, are highly homologous between mouse and human (93%, 94%, and 79%, respectively).

[0016] These three factors protect against cardiomyocyte death induced by other types of injury, including ischemia and treatment with chemotherapeutic agents.

[0017] Accordingly, the present invention relates to the factors Chrdl1, Fam3c, and Fam3b for use as medicaments.

[0018] In one aspect, the present invention provides a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b, and fragments thereof, or a polynucleotide encoding the same, for use in the treatment or risk reduction of heart disease.

[0019] In another aspect, the present invention provides Chrdl1 or a fragment thereof, or a polynucleotide encoding the same, for use in the treatment or risk reduction of heart disease. In another aspect, the present invention provides Fam3c or a fragment thereof, or a polynucleotide encoding the same, for use in the treatment or risk reduction of heart disease. In another aspect, the present invention provides Fam3b or a fragment thereof, or a polynucleotide encoding the same, for use in the treatment or risk reduction of heart disease.

[0020] In one aspect, the present invention provides a method for treating or reducing the risk of heart disease, the method comprising administering to a subject in need thereof a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the same.

[0021] In some embodiments, its use reduces the risk of heart failure. In some embodiments, in a subject suffering from heart disease, the risk of heart failure is reduced. In some embodiments, in a subject at risk of heart disease, the risk of heart failure is reduced.

[0022] In one aspect, the present invention provides a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the same, for use in reducing the risk of heart failure.

[0023] In another aspect, the present invention provides Chrdl1 or a fragment thereof, or a polynucleotide encoding the same, for use in reducing the risk of heart failure. In another aspect, the present invention provides Fam3c or a fragment thereof, or a polynucleotide encoding the same, for use in reducing the risk of heart failure. In another aspect, the present invention provides Fam3b or a fragment thereof, or a polynucleotide encoding the same, for use in reducing the risk of heart failure.

[0024] In another aspect, the present invention provides a method for reducing the risk of heart failure, the method comprising administering to a subject in need thereof a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the same.

[0025] In some embodiments, in a subject suffering from heart disease, the risk of heart failure is reduced. In some embodiments, in a subject at risk of heart disease, the risk of heart failure is reduced.

[0026] In another aspect, the present invention provides a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b, and fragments thereof, or a polynucleotide encoding the same, for use in maintaining cardiomyocyte viability.

[0027] In another aspect, the present invention provides Chrdl1 or a fragment thereof, or a polynucleotide encoding the same, for use in maintaining cardiomyocyte viability. In another aspect, the present invention provides Fam3c or a fragment thereof, or a polynucleotide encoding the same, for use in maintaining cardiomyocyte viability. In another aspect, the present invention provides Fam3b or a fragment thereof, or a polynucleotide encoding the same, for use in maintaining cardiomyocyte viability.

[0028] In another aspect, the present invention provides a method of maintaining cardiomyocyte viability, the method comprising administering to a subject in need thereof a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b, and fragments thereof, or a polynucleotide encoding the same.

[0029] In some embodiments, cardiomyocyte viability is maintained in a subject suffering from a heart disease. In some embodiments, cardiomyocyte viability is maintained in a subject at risk of a heart disease.

[0030] One example of the amino acid sequence of Chrdl1 is SEQ ID NO: 1. One example of the nucleotide sequence of Chrdl1 is SEQ ID NO: 4.

[0031] One example of the amino acid sequence of Fam3c is SEQ ID NO: 2. One example of the nucleotide sequence of Fam3c is SEQ ID NO: 5.

[0032] One example of the amino acid sequence of Fam3b is SEQ ID NO: 3. One example of the nucleotide sequence of Fam3b is SEQ ID NO: 6.

[0033] In some embodiments, the protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. In some embodiments, the protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2. In some embodiments, the protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3.

[0034] In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 3.

[0035] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6.

[0036] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6.

[0037] Heart diseases can result, for example, from myocardial infarction, reperfusion injury after percutaneous coronary intervention (coronary angioplasty), hypertension, cardiotoxic injury (particularly due to cancer chemotherapy), myocarditis, and loss of cardiomyocytes as a result of genetic and non-genetic causes of cardiomyopathy.

[0038] In some embodiments, the heart disease is associated with myocardial ischemia. In some embodiments, the heart disease is associated with loss of cardiomyocytes.

[0039] In some embodiments, the heart disease is selected from myocardial infarction, reperfusion injury after myocardial infarction and as a result of percutaneous coronary intervention (coronary angioplasty), myocarditis, hypertension, cardiotoxic injury (particularly due to cancer chemotherapy), or cardiomyopathy.

[0040] In some embodiments, the heart disease is ventricular dysfunction.

[0041] In some embodiments, the heart is protected from myocardial infarction. In some embodiments, cardiac function is maintained after myocardial infarction or percutaneous coronary intervention (coronary angioplasty). In some embodiments, post-infarct fibrosis is reduced.

[0042] In some embodiments, the protein or polynucleotide protects the heart from myocardial infarction and / or other conditions that result in loss of cardiomyocytes, preferably maintains cardiac function, and reduces reparative fibrosis.

[0043] In some embodiments, heart failure is prevented.

[0044] In some embodiments, the protein is administered by direct protein delivery. In some embodiments, the polynucleotide is used in gene therapy.

[0045] In some embodiments, the protein is a recombinant protein.

[0046] In some embodiments, the protein is obtained from a bacterial, yeast, or mammalian cell culture. In some embodiments, the protein is obtained from Escherichia coli (E. coli), Pichia pastoris, or Chinese hamster ovary cells.

[0047] In some embodiments, the protein is glycosylated.

[0048] In some embodiments, the protein is a fusion protein. In some embodiments, the protein is an Fc fusion protein.

[0049] In some embodiments, the polynucleotide is in the form of a vector.

[0050] In some embodiments, the polynucleotide is in the form of a viral vector.

[0051] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector. In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector.

[0052] In some embodiments, the vector is an AAV2 vector.

[0053] In some embodiments, the vector is an AAV9 vector.

[0054] In some embodiments, the vector is an AAV8 vector.

[0055] In another aspect, the present invention provides a vector for use in the treatment or risk reduction of heart disease, comprising the polynucleotide disclosed herein. In another aspect, the present invention provides a vector for use in the risk reduction of heart failure, comprising the polynucleotide disclosed herein. In another aspect, the present invention provides a vector for use in the maintenance of cardiomyocyte viability, comprising the polynucleotide disclosed herein.

[0056] In some embodiments, the vector is a viral vector.

[0057] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector or an adenoviral vector. In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector.

[0058] In some embodiments, the vector is an AAV2 vector.

[0059] In some embodiments, the vector is an AAV9 vector.

[0060] In some embodiments, the vector is an AAV8 vector.

[0061] In some embodiments, the protein is administered parenterally. In some embodiments, the protein is administered intramyocardially. In some embodiments, the polynucleotide is administered parenterally. In some embodiments, the polynucleotide is administered intramyocardially.

[0062] In another aspect, the present invention provides a pharmaceutical composition comprising the protein disclosed herein and a pharmaceutically acceptable vehicle and / or excipient.

[0063] In another aspect, the present invention provides a pharmaceutical composition comprising the vector disclosed herein and a pharmaceutically acceptable vehicle and / or excipient.

[0064] In some embodiments, the composition is formulated for injection. In preferred embodiments, the composition is formulated for intramuscular injection or intravenous injection.

[0065] In another aspect, the present invention provides a pharmaceutical composition disclosed herein for use in the treatment or risk reduction of heart disease. In another aspect, the present invention provides a pharmaceutical composition disclosed herein for use in reducing the risk of heart failure. In another aspect, the present invention provides a pharmaceutical composition disclosed herein for use in maintaining cardiomyocyte viability.

[0066] In another aspect, the present invention provides a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof for use as a medicament, or a polynucleotide encoding the same.

Brief Description of the Drawings

[0067]

Figure 1A - B

Figure 1C - D

Figure 2A - B

Figure 2C - D

Figure 3

Figure 4

Figure 5A - C

Figure 5D - E

Figure 6

Figure 7A - C

Figure 7D

Figure 8

Mode for Carrying Out the Invention

[0068] As used herein, the terms "comprising", "comprises" and "comprised of" are synonymous with "including" or "includes"; or "containing" or "contains", and are inclusive or open-ended and do not exclude additional unrecited members, elements or steps. The terms "comprising", "comprises" and "comprised of" include the term "consisting of".

[0069] Protein Chrdl1 is a bone morphogenetic protein (BMP) extracellular inhibitor that is mainly expressed in mesenchymal-derived cell types, in retinal pericytes, and in nervous system cells (Sakuta, H. et al., Ventroptin: a BMP-4 antagonist expressed in a double-gradient pattern in the retina. Science 293, 111-115 (2001); Nakayama, N. et al., A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages. Dev Biol 232, 372-387 (2001); Chandra, A. et al., Neurogenesin-1 differentially inhibits the osteoblastic differentiation by bone morphogenetic proteins in C2C12 cells. Biochem Biophys Res Commun 344, 786-791 (2006); Coffinier, C., Tran, U., Larrain, J. & De Robertis, E.M. Neuralin-1 is a novel Chordin-related molecule expressed in the mouse neural plate. Mech Dev 100, 119-122 (2001)).

[0070] The name of Chrdl1 is derived from its sequence similarity to Cordin, another BMP inhibitor that was identified as a factor that dorsalizes Xenopus embryos. Chrdl1 has a different temporal and spatial expression pattern from Cordin, but both genes contain cysteine-rich units (CRs) called procollagen repeats, which are also present in various extracellular matrix proteins. CR1 and CR3 are responsible for Chrdl1-BMP binding. This protein binds to BMP4 with high affinity and has low affinity for binding to BMP5, BMP6, and BMP7.

[0071] Fam3b and Fam3c are two members of the family with sequence similarity 3 (FAM3) (Zhu, Y. et al., Cloning, expression, and initial characterization of a novel cytokine-like gene family. Genomics 80, 144-150 (2002)). Fam3b, also known as PANDER, is highly expressed in the pancreas, where it is involved in the regulation of glucose homeostasis and β-cell function (Robert-Cooperman, C.E., Wilson, C.G. & Burkhardt, B.R. PANDER KO mice on high-fat diet are glucose intolerant yet resistant to fasting hyperglycemia and hyperinsulinemia. FEBS Lett 585, 1345-1349 (2011); Robert-Cooperman, C.E. et al., Targeted disruption of pancreatic-derived factor (PANDER, FAM3B) impairs pancreatic beta-cell function. Diabetes 59, 2209-2218 (2010); Yang, J. et al., Mechanisms of glucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B) in pancreatic beta-cells. Diabetes 54, 3217-3228 (2005)).

[0072] Fam3c, also known as ILEI, is ubiquitously expressed. Fam3c induces inner ear cell proliferation (Pilipenko, V.V., Reece, A., Choo, D.I. & Greinwald, J.H., Jr. Genomic organization and expression analysis of the murine Fam3c gene. Gene 335, 159 - 168 (2004)), regulates bone differentiation (Bendre, A., Buki, K.G. & Maatta, J.A. Fam3c modulates osteogenic differentiation by down-regulating Runx2. Differentiation 93, 50 - 57 (2017)), and has a role in epithelial-mesenchymal transition (EMT) during cancer progression (Waerner, T. et al., ILEI: a cytokine essential for EMT, tumor formation, and late events in metastasis in epithelial cells. Cancer Cell 10, 227 - 239 (2006); Lahsnig, C. et al. ILEI requires oncogenic Ras for EMT of hepatocytes and liver carcinoma progression. Oncogene 28, 638 - 650(2009)). Restoration of Fam3c levels in the livers of obese diabetic mice improves insulin resistance and reduces fatty liver (Chen, Z. et al., Hepatic Activation of the FAM3C-HSF1-CaM Pathway Attenuates Hyperglycemia of Obese Diabetic Mice. Diabetes 66, 1185 - 1197 (2017); Chen, Z. et al., FAM3C activates HSF1 to suppress hepatic gluconeogenesis and attenuate hyperglycemia of type 1 diabetic mice. Oncotarget 8, 106038 - 106049 (2017)).

[0073] One example of the amino acid sequence of Chrdl1 is SEQ ID NO: 1 (human) - UniProt ID: Q9BU40-1. TIFF2025107171000001.tif32160 (SEQ ID NO: 1)

[0074] One example of the amino acid sequence of Fam3c is SEQ ID NO: 2 (human) - UniProt ID: Q92520-1. TIFF2025107171000002.tif21160 (SEQ ID NO: 2)

[0075] One example of the amino acid sequence of Fam3b is SEQ ID NO: 3 (human) - UniProt ID: P58499-1. TIFF2025107171000003.tif18160 (SEQ ID NO: 3)

[0076] One example of the nucleotide sequence encoding Chrdl1 (human) is SEQ ID NO: 4 (Chrdl1) - Seq ID: NM_001143981.1 (coding sequence). TIFF2025107171000004.tif31160TIFF2025107171000005.tif55160 (SEQ ID NO: 4)

[0077] One example of the nucleotide sequence encoding Fam3c is SEQ ID NO: 5 (Fam3c) - Seq ID: NM_014888.3 (coding sequence). TIFF2025107171000006.tif42161 (SEQ ID NO: 5)

[0078] One example of the nucleotide sequence serving as an example encoding Fam3b is SEQ ID NO: 6 (Fam3b) - Seq ID: NM_058186.3 (coding sequence). TIFF2025107171000007.tif44160 (SEQ ID NO: 6)

[0079] Any other polynucleotide encoding the above proteins is also included in the present invention.

[0080] The activities of the proteins and fragments thereof of the present disclosure can be readily determined by those skilled in the art. For example, suitable in vitro assays include: (a) protection from hydrogen peroxide-induced or doxorubicin-induced cell death, e.g., using a TUNEL assay or a caspase activation assay; (b) induction of autophagy, e.g., assaying for the formation of LC3-positive autophagosomes; and / or (c) for Chrdl1: reduction of BMP and TGFβ activities, e.g., activation of αSMA expression in cardiac fibroblasts in treatment with recombinant TGFβ, or reduction of SMAD1 / 5 / 8 phosphorylation in treatment with recombinant BMP4.

[0081] The inventors have surprisingly discovered hitherto unknown roles of Chrdl1, Fam3c, and Fam3b in promoting cardiomyocyte survival, which counteracts ischemia and other forms of heart damage, and thus exhibits its therapeutic activity in preventing heart failure.

[0082] According to the present invention, Chrdl1, Fam3c, and Fam3b effectively enhance cardiac function and reduce infarct size after intracardiac injection of viral vectors expressing these factors.

[0083] The inventors do not wish to be bound by theory or mechanism of action, but it is believed that the proteins Chrdl1, Fam3c, and Fam3b exert a therapeutic effect in the heart by preventing cardiomyocyte apoptosis and inducing cardiomyocyte autophagy. Collectively, this results in prevention of cardiac function, reduction of fibrosis and left ventricular pathological tissue repair, and induction of a beneficial expression pattern of genes associated with pathological heart tissue repair, e.g., an increase in Serca2a (sarcoplasmic reticulum / endoplasmic reticulum calcium ATPase 2a) and RYR2 (ryanodine receptor 2), and maintenance of the ratio of α-myosin heavy chain (αMHC) to β-myosin heavy chain (βMHC).

[0084] The mechanism by which Chrdl1, Fam3c, and Fam3b exert a cardioprotective effect on the ischemic heart is, for example, the maintenance of myocyte viability by preventing apoptotic cell death after intracardiac injection of a viral vector expressing the factors.

[0085] Chrdl1 and Fam3c may promote beneficial autophagy that counteracts cardiomyocyte cell death after myocardial infarction.

[0086] Chrdl1, Fam3c, and Fam3b may maintain myocyte viability by preventing apoptotic cell death after doxorubicin treatment.

[0087] The additional positive effect of Chrdl1 is to prevent cardiac fibroblast activation and cardiac fibrosis.

[0088] The heart is an organ that cannot undergo significant regeneration in adulthood. Therefore, cardiomyocyte integrity is maintained by autophagy, a mechanism that enables the regeneration of specific intracellular components including mitochondria. This mechanism is particularly relevant after myocardial infarction. This is because sudden ischemia or reperfusion after ischemia followed by percutaneous revascularization causes significant damage to mitochondria, which begin to generate chemical species that damage using oxygen (Yellon, DM., Hausenloy DJ. Myocardial reperfusion injury. N. Engl. J. Med. 357, 1121 - 1135 (2007); Gustafsson AB., Gottlieb RA. Circ. Res. 104(2), 150 - 158 (2009)). Therefore, autophagy and apoptosis are highly interconnected, and the former mechanism is activated after onset to remove damaged organelles as a protective response to avoid apoptotic cell death.

[0089] Thus, Chrdl1, Fam3c, and Fam3b can be used for heart protection and can thus reduce the risk of heart disease or heart failure. Heart protection may be by maintaining cardiomyocyte viability.

[0090] Protein delivery As an alternative to polynucleotide delivery, the proteins of the invention may be delivered by direct protein delivery.

[0091] The protein may be administered directly to a subject. In some embodiments, the protein is a fusion protein, preferably a fusion with a second protein that can increase the lifespan of the protein in the subject. For example, the protein may be an immunoglobulin Fc domain fusion protein.

[0092] Protein delivery may also be by vector delivery (Cai, Y. et al., (2014) Elife 3: e01911; Maetzig, T. et al., (2012) Curr. Gene Ther. 12: 389-409). Vector delivery involves engineering viral particles (e.g., lentiviral particles) to contain the protein to be delivered to cells. Thus, when the engineered viral particles enter cells as part of their natural life cycle, the protein contained in the particles is carried into the cells.

[0093] Protein delivery (Gaj, T. et al., (2012) Nat. Methods 9: 805-7) may be achieved, for example, by utilizing a vehicle (e.g., liposome).

[0094] Polynucleotide The polynucleotides of the present invention may comprise DNA or RNA, preferably DNA. The polynucleotides of the present invention may be single-stranded or double-stranded. It is understood by those skilled in the art that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it should be understood by those skilled in the art that nucleotide substitutions may be made using conventional techniques that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention so as to reflect the codon usage frequency of any particular host organism in which the polypeptides of the present invention will be expressed.

[0095] The nucleotide sequences of the present invention disclosed herein may or may not include a stop codon at their 3' ends, depending, for example, on their position in a bicistronic vector. Accordingly, this disclosure encompasses the sequence numbers disclosed herein with or without a stop codon.

[0096] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or lifespan of the polynucleotides of the present invention.

[0097] Polynucleotides, such as DNA polynucleotides, can be made recombinantly, synthetically, or by any means available to those skilled in the art. The polynucleotides may be cloned by standard techniques.

[0098] Longer polynucleotides are generally made using recombinant means, for example, using polymerase chain reaction (PCR) cloning techniques. This involves creating a pair of primers (e.g., about 15 - 30 nucleotides) adjacent to the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that cause amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture using an agarose gel), and recovering the amplified DNA. The primers can be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

[0099] Vector A vector is a tool that enables or facilitates the introduction of substances from one environment to another.

[0100] In one aspect, the present invention provides a vector comprising the polynucleotide of the present invention. In a preferred embodiment, the vector is a viral vector. In some embodiments, the vector is an adeno - associated virus (AAV) vector, a retroviral vector, a lentiviral vector or an adenoviral vector, preferably an AAV vector.

[0101] Adeno - associated virus (AAV) vector In one aspect, the present invention provides an AAV vector comprising the polynucleotide of the present invention.

[0102] Preferably, the AAV vector is in the form of AAV vector particles.

[0103] In some embodiments, the AAV vector particles contain the AAV2 genome. In some embodiments, the AAV vector particles contain the AAV9 genome. In some embodiments, the AAV vector particles contain the AAV8 genome.

[0104] In some embodiments, the AAV vector particles comprise an AAV9 capsid protein. In some embodiments, the AAV vector particles comprise an AAV8 capsid protein.

[0105] In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV9 capsid protein (AAV2 / 9). In other embodiments, the AAV vector particles comprise an AAV2 genome and an AAV8 capsid protein (AAV2 / 8).

[0106] Methods for preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

[0107] The AAV vector may comprise an AAV genome or a fragment or derivative thereof.

[0108] It is known that AAV can package a genome of up to 5.2 kb in size (Dong, J.Y. et al. (1996) Human Gene Therapy 7: 2101-2112).

[0109] The AAV genome is a polynucleotide sequence, and the sequence may encode functions required for the production of AAV particles. These functions include functions that operate in the replication and packaging cycle of AAV in host cells, such as capsid formation of the AAV genome into AAV particles. Naturally occurring AAV is replication-defective and depends on the supply of a trans helper function for the completion of the replication and packaging cycle. Therefore, the AAV genome of the AAV vector of the present invention is typically replication-defective.

[0110] The AAV genome may be in either a single-stranded form, either positive-sense or negative-sense, or a double-stranded form. The use of the double-stranded form enables bypassing the DNA replication step in target cells, and thus can promote the expression of the transgene.

[0111] The AAV genome may be obtained from any naturally occurring serotype, isolate or clade of AAV. Thus, the AAV genome may be the entire genome of a naturally occurring AAV. As is known to those skilled in the art, naturally occurring AAVs can be classified according to various biological systems.

[0112] Generally, AAVs are referred to in terms of their serotypes. Serotypes correspond to variant subtypes of AAV, and the variant subtypes have unique reactivity that can be used to distinguish them from other variant subtypes by the expression profile of their capsid surface antigens. Typically, a virus having a particular AAV serotype does not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype.

[0113] AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, and also include recombinant serotypes such as Rec2 and Rec3 recently identified from the brains of primates. Any of these AAV serotypes can be used in the present invention.

[0114] In some embodiments, the AAV vector particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rec2 or Rec3 AAV vector particles.

[0115] In some embodiments, the AAV may be of the AAV1, AAV2, AAV5, AAV7, AAV8 or AAV9 serotype.

[0116] In some embodiments, the AAV may be of the AAV9 or AAV8 serotype.

[0117] In some embodiments, the AAV is of the AAV9 serotype. In other embodiments, the AAV is of the AAV8 serotype.

[0118] The capsid protein may be a mutant capsid protein as disclosed in WO 2008 / 124724 (which is incorporated herein by reference).

[0119] In some embodiments, the AAV vector comprises an AAV8 capsid having a Y733F mutation.

[0120] An overview of AAV serotypes can be found in Choi et al. (2005) Curr. Gene Ther. 5:299-310 and Wu et al. (2006) Molecular Therapy 14:316-27. The sequence of the AAV genome for use in the present invention, or elements of the AAV genome including the ITR sequence, rep or cap gene, may be derived from the following accession numbers for the AAV full genome sequence: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

[0121] AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally occurring AAV, and typically refers to a phylogenetic group of AAV that can be traced back to a common ancestor and includes all of its descendants. Additionally, AAV may be referred to in terms of a specific isolate, i.e., a genetic isolate of a specific AAV found in nature. The term genetic isolate describes a population of AAV in which there has been limited genetic mixing with other naturally occurring AAV, thereby defining a population that is distinct to the extent that it can be recognized at the genetic level.

[0122] One of ordinary skill in the art can select a suitable serotype, clade, clone, or isolate of AAV for use in the present invention based on their common general knowledge. For example, the AAV5 capsid has been shown to efficiently transduce primate cone photoreceptors, as demonstrated by the success of correcting hereditary color vision defects (Mancuso et al. (2009) Nature 461:784-7).

[0123] The AAV serotype determines the tissue specificity of AAV infection (or affinity). Thus, suitable AAV serotypes for use in AAV administered to a patient according to the present invention are those that have a natural affinity for target cells within the heart or those that have a high infection efficiency for target cells within the heart.

[0124] Typically, the AAV genome of a naturally occurring serotype, isolate or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequence functions in cis to provide an origin of replication and enables integration and excision of the vector from the cell's genome. In preferred embodiments, one or more ITR sequences flank the nucleotide sequence encoding the protein of the invention. The AAV genome typically also contains packaging genes, such as the rep and / or cap genes that encode the packaging functions of the AAV particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins, such as VP1, VP2 and VP3 or variants thereof. These proteins constitute the capsid of the AAV particle. Capsid variants are discussed below.

[0125] A promoter is operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76:5567-5571). For example, the p5 and p19 promoters are typically used to express the rep gene, while the p40 promoter is typically used to express the cap gene.

[0126] As discussed above, the AAV genome used in the AAV vectors of the present invention may, therefore, be the entire naturally occurring AAV genome. For example, AAV vectors or vector particles may be prepared in vitro by using vectors containing the entire AAV genome. However, although such vectors are in principle administrable to patients, this is rarely actually carried out. Preferably, the AAV genome is derivatized for administration to patients. Such derivatization is standard in the art, and the present invention includes the use of any known derivatives of the AAV genome, and derivatives that can be produced by applying techniques known in the art. The derivatization of the AAV genome and AAV capsid is outlined in Coura and Nardi (2007) Virology Journal 4:99, as well as Choi et al., and Wu et al. (supra).

[0127] Derivatives of the AAV genome include any terminal truncation or modification of the AAV genome that enables the expression of the transgene from the AAV vectors of the present invention in vivo. Typically, it is possible to greatly truncate the AAV genome such that it contains a minimal viral sequence while retaining the above function. This is preferred for safety reasons to reduce the risk of recombination between the vector and the wild-type virus and to avoid inducing a cellular immune response due to the presence of viral gene proteins in the target cells.

[0128] Typically, the derivative includes at least one inverted terminal repeat (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be chimeric ITRs or variant ITRs. Suitable variant ITRs are those having a deletion of the trs (terminal resolution site). This deletion enables the continuous replication of the genome for producing a single-stranded genome containing both the coding sequence and the complementary sequence, i.e., a self-complementary AAV genome. This enables bypassing DNA replication in the target cells and, for that reason, accelerated transgene expression.

[0129] One or more ITRs are preferably adjacent at both ends to the nucleotide sequence encoding the protein of the present invention. Inclusion of one or more ITRs preferably promotes concatemer formation of the vector of the present invention in the nucleus of the host cell, for example, after conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerase. Formation of such episomal concatemers protects the vector construct during the lifespan of the host cell, thereby enabling sustained expression of the transgene in vivo.

[0130] In a preferred embodiment, the ITR element is the only sequence retained from the native AAV genome in the derivative. Thus, the derivative preferably does not contain the native genomic rep and / or cap genes and any other sequences of the native genome. This is preferred for the reasons described above and also to reduce the possibility of integration of the vector into the host cell genome. Furthermore, by reducing the size of the AAV genome, flexibility can be increased in incorporating other sequence elements (e.g., regulatory elements) into the vector in addition to the transgene.

[0131] Thus, the following portions: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes, may be removed in the derivatives of the present invention. However, in some embodiments, the derivative may further contain one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a specific site on human chromosome 19 at a high frequency and shows a negligible frequency of random integration such that retention of the vector's integration ability is acceptable in a therapeutic setting.

[0132] When the derivative contains a capsid protein, i.e., VP1, VP2, and / or VP3, the derivative may be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the present invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e., pseudotyped vectors).

[0133] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide one or more desired functionalities to the AAV vector. Thus, these derivatives may exhibit improved gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered affinity range, and / or improved targeting of specific cell types as compared to AAV vectors containing a naturally occurring AAV genome, e.g., the AAV2 AAV genome. The improvement in gene delivery efficiency may be achieved by improving receptor or coreceptor binding at the cell surface, improving internalization, improving transport into the cell and nucleus, improving uncoating of the viral particle, and improving conversion of the single-stranded genome into the double-stranded form. Also, the improvement in efficiency may be related to an altered affinity range or targeting of a specific cell population such that the vector dose is not diluted by administration to tissues that do not require it.

[0134] Chimeric capsid proteins include those produced by recombination between the capsid-encoding sequences of two or more naturally occurring AAV serotypes. This may be carried out, for example, by co-transfecting a non-infectious capsid sequence of one serotype with a capsid sequence of a different serotype and selecting for a capsid sequence with desired properties using a markerless rescue method that utilizes directed selection. The capsid sequences of the different serotypes can be altered by homologous recombination intracellularly to generate novel chimeric capsid proteins.

[0135] Chimeric capsid proteins also include those produced by manipulating the capsid protein sequence to transfer a specific capsid protein domain, surface loop, or specific amino acid residue between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes.

[0136] Shuffled or chimeric capsid proteins may likewise be produced by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be produced by randomly fragmenting the sequences of related AAV genes, for example, sequences encoding capsid proteins of multiple different serotypes, and then subsequently reconstructing the fragments in a self-priming polymerase reaction where crossovers can also occur in regions having sequence homology. A library of hybrid AAV genes produced by shuffling the capsid genes of several serotypes in this way can be screened to identify viral clones having the desired functionality. Similarly, a diverse library of mutants may be produced by randomly mutating the AAV capsid gene using error-prone PCR, and this may then be selected for the desired properties.

[0137] The sequence of the capsid gene may likewise be genetically modified to introduce specific deletions, substitutions, or insertions with respect to the native wild-type sequence. In particular, the capsid gene may be modified by insertion of the sequence of an unrelated protein or peptide into the open reading frame of the capsid coding sequence or at the N- and / or C-terminus of the capsid coding sequence.

[0138] The non-related protein or peptide may advantageously act as a ligand for a specific cell type, thereby providing improved binding to target cells or improving the targeting specificity of the vector for a specific cell population. Further, the non-related protein may be one that promotes the purification of virus particles as part of the production process, i.e., an epitope or an affinity tag. The site of insertion is typically selected so as not to interfere with other functions of the virus particle, such as the internal translocation and transport of the virus particle. Those skilled in the art can identify suitable sites for insertion based on their common general knowledge. Specific sites are disclosed by Choi et al. (supra).

[0139] The present invention further encompasses the provision of AAV genome sequences in an order and arrangement different from that of the native AAV genome. The present invention also encompasses the substitution of one or more AAV sequences or genes with a chimeric gene consisting of a sequence from another virus or sequences derived from two or more viruses. Such chimeric genes may consist of sequences derived from two or more related viral proteins of different virus species.

[0140] The AAV vector of the present invention may take the form of a nucleotide sequence comprising an AAV genome or a derivative thereof and a sequence encoding the protein of the present invention.

[0141] The AAV particles of the present invention include a transcapsidated form in which an AAV genome or derivative having ITRs of one serotype is packaged within a capsid of a different serotype. The AAV particles of the present invention also include a mosaic form in which a mixture of unmodified capsid proteins derived from two or more different serotypes constitutes the virus capsid. The AAV particles also include a chemically modified form carrying a ligand adsorbed on the capsid surface. For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0142] The AAV vector may contain multiple copies (e.g., 2, 3, etc.) of the nucleotide sequences referred to herein.

[0143] In some embodiments, the polynucleotide further comprises one or more AAV ITRs. In preferred embodiments, the polynucleotide further comprises two AAV ITRs. In some embodiments, the polynucleotide comprises one AAV ITR at its 5' end and further comprises one AAV ITR at its 3' end. In some embodiments, the AAV ITR is an ITR of AAV2, AAV9 or AAV8.

[0144] Promoters and regulatory sequences The polynucleotide or vector of the present invention may also contain elements that enable the expression of the nucleotide sequence encoding the protein of the present invention in vitro or in vivo. These may sometimes be referred to as expression control sequences. Thus, the polynucleotide or vector typically contains an expression control sequence (e.g., including a promoter sequence) operably linked to the nucleotide sequence encoding the protein of the present invention in a functional form.

[0145] Any suitable promoter may be used, and the selection can be readily made by those skilled in the art. The promoter sequence may be constitutively active (i.e., operable in any host cell background), or may be active only in a specific host cell environment, resulting in targeted expression of the transgene in a specific cell type (e.g., a tissue-specific promoter). The promoter may exhibit inducible expression in response to the presence of another factor, e.g., a factor present in the host cell. In any case, when the vector is administered for treatment, the promoter is preferably functional in the target cell background.

[0146] In preferred embodiments, the promoter is a liver-specific promoter. In preferred embodiments, the promoter is the liver-specific hAAT promoter.

[0147] The liver-specific hAAT promoter can confer selective specificity for hepatocytes. In the present invention, it is shown that when the vector is administered through the portal vein, each circulating factor secreted from the liver can protect the heart after injury.

[0148] Suitable promoters include the chicken β-actin (CBA) promoter, optionally in combination with a cytomegalovirus (CMV) enhancer element. An example of a promoter for use in the present invention is the CAG promoter.

[0149] In some embodiments, the promoter is the CMV promoter.

[0150] The polynucleotide or vector of the present invention may similarly contain one or more additional regulatory sequences that can act before or after transcription. The regulatory sequence may be part of the locus of the native transgene or a heterologous regulatory sequence. The polynucleotide or vector of the present invention may contain a part of the 5'-UTR or 3'-UTR derived from the native transgene transcript.

[0151] The regulatory sequence is any sequence that acts to facilitate the expression of the transgene, i.e., to increase the expression of the transcript, to improve the nuclear export of mRNA, or to enhance its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

[0152] Suitable enhancers include the WPRE regulatory element. Suitable polyA signals include the bovine growth hormone polyA signal.

[0153] Additional regulatory sequences can be easily selected by those skilled in the art.

[0154] Method of administration Various routes of administration and techniques can be utilized, among which, parenteral techniques such as intravenous, intracardiac, intra-arterial injection, catheter insertion, etc. can be used. The average amount of the active agent varies and should be carried out especially based on the recommendation and prescription of a qualified physician.

[0155] The protein, polynucleotide or vector of the present invention may be administered systemically (e.g., by peripheral intravenous injection) or may be administered locally or regionally.

[0156] Preferably, the protein is administered by a parenteral route, especially an intravenous, intra-arterial, or intramuscular route.

[0157] Administration of the polynucleotide encoding the protein disclosed herein can be achieved by gene therapy (see, for example, WO 2013 / 093870).

[0158] According to the present invention, Chrdl1, Fam3c and Fam3b are active even when reaching the infarcted heart through the systemic circulation.

[0159] Pharmaceutical compositions and infusion solutions The medicaments of the present invention, such as proteins, polynucleotides or vectors, may be formulated into pharmaceutical compositions. These compositions may contain, in addition to the said medicaments, pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers or other substances well known in the art. Such substances should be non-toxic and should not interfere with the effect of the active ingredient. The exact nature of the said carrier or other substances can be determined by those skilled in the art according to the route of administration.

[0160] Depending on the selected route of administration, the composition can be in solid or liquid form suitable for oral, parenteral, intravenous, or intra-arterial administration. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions usually contain a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. It may contain physiological saline, magnesium chloride, dextrose or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol. In some cases, a surfactant, such as 0.001% poloxamer (PF68), may be used.

[0161] In the case of injection at the affected site, the active ingredient may be in the form of an aqueous solution that does not contain pyrogens and has appropriate pH, isotonicity, and stability. A person skilled in the art can successfully prepare an appropriate solution using an isotonic vehicle, such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0162] In the case of delayed release, the medicament may be included in a pharmaceutical composition formulated for sustained release, such as microcapsules formed from a biocompatible polymer or a liposome carrier system by methods known in the art.

[0163] Such compositions are well known in the art (see, for example, Remington’s Pharmaceutical Sciences; last edition, Mack Pub).

[0164] Method of treatment In the context of the present invention, references to prevention more generally evoke prophylactic treatment, but it should be understood that all references to treatment in this specification include curative, palliative, and prophylactic treatment. Treatment may also include preventing the progression of the severity of the disease in some cases.

[0165] Treatment of mammals, especially humans, is preferred. However, treatment of both humans and animals is within the scope of the present invention.

[0166] The dosing regimen, dosage, and posology are determined according to the physician's experience, the disease to be treated, and the condition of the patient.

[0167] The proteins and / or polynucleotides of the present invention can be administered alone or in combination with each other.

[0168] As used herein, the term "combination", or the terms "in combination", "used in combination", or "combined preparation" can mean administering two or more agents simultaneously, sequentially, or separately in combination.

[0169] As used herein, the term "simultaneous" means that the agents are administered concurrently, i.e., at the same time.

[0170] As used herein, the term "sequential" means that the agents are administered one after another.

[0171] As used herein, the term "separately" means that the agents are administered independently of each other, but within a time interval in which the agents can be combined, preferably showing a synergistic effect. Thus, in "separate" administration, one agent can be administered after the other, for example, within 1 minute, within 5 minutes, within 10 minutes.

[0172] Variants, derivatives, analogs, homologs, and fragments In addition to the specific proteins and nucleotides described herein, the present invention also encompasses the use of their variants, derivatives, analogs, homologs, and fragments.

[0173] In the context of the present invention, a variant of any given sequence is a sequence in which the residues (amino acid residues or nucleic acid residues) of that particular sequence are modified such that the polypeptide or polynucleotide in question substantially retains its function. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one residue present in a naturally occurring protein.

[0174] As used herein, the term "derivative" with respect to a protein or polypeptide of the present invention includes any substitution, mutation, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to its sequence, provided that the resulting protein or polypeptide substantially retains at least one of its intrinsic functions.

[0175] As used herein, the term "analogue" with respect to a polypeptide or polynucleotide includes any mimetic, i.e., a chemical compound that retains at least one of the intrinsic functions of the polypeptide or polynucleotide being mimicked.

[0176] Typically, amino acid substitutions may be, for example, 1, 2 or 3 to 10 or 20 substitutions, provided that the modified sequence substantially retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0177] The proteins used in the present invention may similarly have deletions, insertions, or substitutions of amino acid residues that result in silent changes and yield functionally equivalent proteins. Planned amino acid substitutions can be carried out based on similarity in terms of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, as long as their endogenous function is retained. For example, amino acids with a negative charge include aspartic acid and glutamic acid, amino acids with a positive charge include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.

[0178] Conservative substitutions can be carried out, for example, according to the following table. Amino acids within the same block in the second column, and preferably amino acids within the same row in the third column, can be substituted for each other: TIFF2025107171000008.tif38160

[0179] As used herein, the term "homolog" means a substance having a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be regarded as equivalent to "identity".

[0180] A homologous sequence can include an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical, preferably at least 95% or 97% or 99% identical to the target sequence. Typically, the homolog includes the same active site and the like as the target amino acid sequence. Homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), but in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0181] The homologous sequence may include a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the target sequence, preferably at least 95% or 97% or 99% identical. Homology can also be considered in terms of similarity, but in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0182] Preferably, when referring to a sequence having percent identity to any one of the sequence numbers detailed herein, it refers to a sequence having the stated percent identity over the entire length of the recited sequence number.

[0183] Homology comparisons can be performed visually or, more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or percent identity between two or more sequences.

[0184] Percent homology may be calculated for contiguous sequences, i.e., one sequence is aligned with another and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are only performed for a relatively small number of residues.

[0185] This is a very simple and consistent method, but for example, in sequence pairs that are otherwise identical, one insertion or deletion in the nucleotide sequence may cause the subsequent codon to be excluded from the alignment, thus not considering the possibility that a global alignment may result in a significant decrease in percent homology. As a result, most sequence comparison methods are designed to generate an optimal alignment that takes into account insertions and deletions that can be considered without unduly penalizing the overall homology score. This is achieved by attempting to insert "gaps" in the sequence alignment to maximize local homology.

[0186] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment so that, for the same number of identical amino acids, sequence alignments with as few gaps as possible (reflecting a higher relatedness between the two compared sequences) achieve a higher score than sequence alignments with many gaps. Typically, an "affine gap cost" is used that imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty, of course, produces an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for the gap and -4 for each extension.

[0187] Calculation of the maximum percent identity, therefore, requires first the generation of an optimal alignment taking into account a gap penalty. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res. 12:387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package of comparison tools (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS comparison tool set. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool called BLAST 2 Sequences can also be used for comparison of protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0188] The final percent identity can be measured in terms of identity, but the alignment process itself is typically not based on all-or-none pair-wise comparisons. Instead, a scaled similarity score matrix is usually used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix (the default matrix for the programs in the BLAST program suite). The GCG Wisconsin programs typically use the public default values, or this if a custom symbol comparison table is supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or the default matrix such as BLOSUM62 for other software.

[0189] If the software generates an optimal alignment, it is possible to calculate the percent identity, preferably the percent sequence identity. The software typically does this as part of the sequence comparison and yields a numerical result.

[0190] A "fragment" of Chrdl1, Fam3c or Fam3b is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide that is functionally or, for example, important in an assay. Thus, a "fragment" refers to an amino acid sequence or nucleic acid sequence that is part of a full-length polypeptide or full-length polynucleotide.

[0191] Such variants may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. When an insertion is made, synthetic DNA encoding the insert may be made along with 5' and 3' flanking regions corresponding to the naturally occurring sequences on both sides of the insertion site. The flanking regions contain convenient restriction sites corresponding to sites in the naturally occurring sequences, so that the sequences can be cut with an appropriate enzyme(s) and the synthetic DNA ligated to the cut site. The encoded protein is produced by then expressing the DNA according to the present invention. These methods are merely illustrative of many standard techniques known in the art for manipulating DNA sequences, and other known techniques are equally applicable.

[0192] One skilled in the art will understand that all features of the invention disclosed herein can be combined without departing from the scope of the disclosed invention.

[0193] Various preferred features and embodiments of the invention are described herein by way of non-limiting example.

[0194] In the practice of the present invention, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology are used, which are within the capabilities of those skilled in the art. Such techniques are described in the literature. For example, see Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley and Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley and Sons; Polak, J.M. and McGee, J.O’D (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. These general texts are hereby incorporated by reference into this specification, each as if fully set forth herein.

[0195] [Examples] [Example 1] FunSel, an in vivo selection procedure for identifying novel heart therapeutics for myocardial infarction Recently, the inventors developed FunSel(1,2), a novel procedure for the in vivo functional identification of novel therapeutic agents against mutant phenotypes, which the inventors applied herein for the purpose of identifying factors that ensure heart protection after myocardial infarction (MI). This is based on the use of adeno-associated virus (AAV) vectors, which are sophisticated tools for highly efficient heart gene transfer(3).

[0196] Briefly, the inventors generated an arrayed library of genes corresponding to the secretome, defined as a subset of proteins secreted into the extracellular environment of cells. Using a computer-based approach, 2033 unique proteins that lack any transmembrane domain or intracellular localization signal and thus are potentially secreted from cells were identified in the genome. Due to the size of the AAV genome, which was restricted to 4.5 kb for cloning, and the availability of cDNA clones, the number of cDNAs suitable for cloning was restricted to 1198. The coding regions of these genes were individually cloned into the pAAV pGi backbone plasmid under the control of the constitutive CMV IE promoter and confirmed by sequencing. Each clone was uniquely identified by a unique 10-nt barcode that could be PCR amplified and sequenced (Figure 1A).

[0197] FunSel is based on the following strategy. A pool of AAV plasmids from the library, each encoding one specific factor and identified by a unique barcode, was used for the batch production of AAV serotype 9 (AAV9) vectors (for a total of 24 pools, each composed of 50 factors of similar size). 8-week-old CD1 mice (n = 9 animals for each AAV9 pool) were subjected to myocardial infarction induced by permanent ligation of the left descending coronary artery, which represents a selective stimulus in this context. Immediately after MI, each AAV9 pool of vectors was administered at a multiplicity such that each vector would in principle enter different cells (10^ 10(The viral genome) was injected in vivo into the peri-infarctual region of the left ventricle (LV) infarct. Three weeks later, the vector inserts were recovered from the surviving LV tissue, and the frequency of each vector was determined by barcode next-generation sequencing (NGS) and compared to the frequency found in control animals (n = 6 animals per pool) injected with the same AAV9 pool but not undergoing MI. Most myocytes die after infarction, but if the cells express a protective factor, they survive selectively. Thus, barcode enrichment indicates positive gene selection (beneficial effect), and reduced representation indicates negative selection (neutral or harmful effect) (experimental design in Figure 1B).

[0198] Figure 1C reports the cumulative results obtained from the in vivo screening of 1,198 factors. In the graph, the frequency of each factor recovered from the heart after MI is reported as the ratio to the frequency of the same factor without selective treatment. Based on Z-score calculations, the inventors selected the top 200 performers and performed four additional screenings for these factors (4 pools of 50 vectors). The 11 resulting factors had a Z-score of 1.96 or more and were competitively enriched (P < 0.05) (Figure 1D). To support the robustness of the FunSel approach, among the hits of this second-round screening, the inventors found a few known cardioprotective factors and unknown factors somehow related to cardiomyocyte biology. These factors include Mdk, a pleiotropic molecule that plays a protective role against cardiac injury, and Rin1, a well-described antifibrotic agent that can reduce ROS production, apoptosis, and inflammation in the infarcted heart. Notably, however, among the top performers were three new proteins for which no current information was available and no studies had been conducted regarding their association with cardioprotection. These are chordlin-like 1 (Chrdl1) and two members of the family with sequence similarity 3, namely, Fam3b and Fam3c. These factors were selected for further individual investigations to evaluate their efficacy and mechanism of action.

[0199] [Example 2] Effectiveness of Chrdl1, Fam3c and Fam3b in maintaining cardiac integrity and function after myocardial infarction in mice Based on the FunSel results, the inventors determined to verify and characterize the effects of selected top-secreted factors (Chrdl1, Fam3c and Fam3b) expressed as individual AAV2 / 9 vectors against myocardial infarction in the mouse heart for the specific purpose of evaluating the ability of each factor to counteract or reduce ischemic injury and promote cardiac function.

[0200] Eight-week-old CD1 mice were subjected to MI and simultaneously injected with AAV2 / 9 vectors expressing Chrdl1, Fam3c, Fam3b or a control empty vector (1×10 11 vg / animal; n = 8 per group) into the peri-infarct area of the LV. From the inventors' previous experience, this procedure has been shown to result in efficient myocardial transduction and a 1-month continuous expression of the transgene (1,5). The cardiac function of the animals was monitored by echocardiography on days 15, 30 and 60 after MI.

[0201] As reported in Figure 2, AAV2 / 9-mediated overexpression of Chrdl1, Fam3c, or Fam3b resulted in the successful maintenance of the left ventricular ejection fraction (LVEF) in infarcted mice compared to control-treated animals (Figure 2A). LVEF values began to improve significantly on day 15 after MI and were maintained over time (60 days after MI: AAV9-Chrdl1 39.96 ± 2.67%, AAV2 / 9-Fam3c 39.02 ± 2.53%, and AAV2 / 9-Fam3b 32.50 ± 2.70% compared to 19.86 ± 0.98% in animals receiving the control vector, P < 0.001 for all treatments). At both 30 and 60 days after MI, diastolic left ventricular volume (Vd) was significantly larger in control animals than in treated mice, as expected at the onset of heart failure (Figure 2B) (60 days after MI: AAV2 / 9-Chrdl1 101.22 ± 10.31 μL, AAV2 / 9-Fam3c 107.31 ± 8.85 μL, and AAV2 / 9-Fam3b 115.16 ± 11.03 μL compared to 168.64 ± 7.47 μL in animals receiving the control vector, P < 0.001 for all treatments).

[0202] Upon myocardial infarction, cardiac fibroblasts proliferate, differentiate into myofibroblasts, and produce extracellular matrix to create a scar that replaces the gap created by cardiomyocyte loss. This often results in myocardial stiffness, pathological tissue repair, dilation, and dysfunction of the left ventricle. Morphometric analysis of trichrome-stained heart sections on day 60 showed that AAV2 / 9-Chrdl1-, AAV2 / 9-Fam3c-, and AAV2 / 9-Fam3b-treated mice demonstrated significant maintenance of LV contractile tissue and reduction in the fibrotic area (infarct size: AAV9-Chrdl1 6.88 ± 1.60%, AAV2 / 9-Fam3c 10.19 ± 2.05%, AAV2 / 9-Fam3b 13.35 ± 1.79% of the LV in control animals; Figure 2C).

[0203] Finally, by wheat germ agglutinin (WGA) staining, it was revealed that the cardiomyocyte hypertrophy response was not induced by Chrdl1, Fam3c, or Fam3b overexpression in treated animals as the fiber cross-sectional area did not increase compared to the control (Figure 2D).

[0204] Collectively, these results indicate that AAV2 / 9-mediated cardiac overexpression of Chrdl1, Fam3c, or Fam3b after acute ischemia promotes cardiomyocyte viability, reduces infarct size, and maintains cardiac function after myocardial infarction.

[0205] [Example 3] AAV2 / 9-mediated expression of Chrdl1, Fam3c, or Fam3b counteracts pathological left ventricular tissue remodeling associated with the development of heart failure in mice Two months after MI, the inventors also examined the effect of each secreted factor on the expression of a set of genes previously associated with LV pathological tissue remodeling (overexpression of β-myosin heavy chain (βMHC) and decreased levels of α-myosin heavy chain (αMHC), sarcoplasmic reticulum / endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) and ryanodine receptor 2 (RYR2)).

[0206] Total RNA was extracted from LV tissue and analyzed by qRT-PCR using TaqMan probes specific for the genes examined. Consistent with the echocardiographic and morphometric findings, AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, and AAV2 / 9-Fam3b counteracted the characteristic pattern of gene expression typically associated with pathological LV tissue remodeling observed in AAV2 / 9 control mice, reducing the level of β-MHC and increasing the levels of α-MHC, SERCA2a, and RYR2 (Figure 3, panels A–D).

[0207] [Example 4] All three factors maintain tissue viability that protects cardiomyocytes from cell death, and both Chrdl1 and Fam3c promote beneficial autophagy in the heart after MI. To date, the available information on the Chrdl1, Fam3c, and Fam3b factors has been insufficient, and the reason why these factors exert a cardioprotective effect after myocardial infarction cannot be explained at all. Therefore, the inventors initiated an investigation into the possible biological mechanisms that may mediate their activity. Since the FunSel approach is based on the selection of factors based on cardiomyocyte survival, the first set of experiments was conducted by comparing the hearts of infarcted adult CD1 mice treated with three AAV vectors (1×10 11 vg / animal; n = 5 per group) with controls to test the level of cell death from apoptosis (which is usually extremely high 2 days after coronary artery occlusion (6)). Fam3b, Fam3c, and, in particular, Chrdl1 were extremely effective in preventing apoptotic cell death in the infarcted heart when evaluated by nuclear TUNEL (TdT-mediated dUTP nick end labeling) staining on snap-frozen heart sections 2 days after MI (% of positive TUNEL nuclei: AAV2 / 9-Chrdl1 4.01 ± 1.21%, AAV2 / 9-Fam3c 10.33 ± 1.43%, AAV2 / 9-Fam3b 19.83 ± 3.01%, vs. 30.67 ± 4.38% in control animals) (quantification in Figure 4A).

[0208] The heart is an organ that cannot undergo significant regeneration during adulthood. Therefore, the integrity of cardiomyocytes is maintained by autophagy, a mechanism that enables the regeneration of specific intracellular components, particularly mitochondria. This mechanism is particularly relevant after myocardial infarction. This is because sudden ischemia or reperfusion after ischemia followed by percutaneous revascularization causes significant damage to mitochondria, and mitochondria begin to generate chemical species that damage using oxygen (7). Not surprisingly, therefore, autophagy and apoptosis are highly interconnected, and the former mechanism is activated after injury to remove damaged organelles as a protective response to avoid apoptotic cell death (8).

[0209] To evaluate the induction of autophagy after acute cardiac ischemia, another group of adult infarcted CD1 mice was injected with AAV2 / 9 vectors expressing Chrdl1, Fam3c, Fam3b or control vectors (1×10 11 vg / animal; n = 5 per treatment). Two days after MI, the inventors found an increase in the conversion of soluble LC3-I protein to lipidated LC3-II, particularly in the hearts of Chrdl1- and Fam3c-treated mice, which was associated with autophagosome formation (representative blots and quantification for hearts of AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, AAV2 / 9-Fam3b and AAV2 / 9-control in Figures 4B and 4C, respectively).

[0210] To directly visualize autophagic flux in infarcted hearts, the inventors previously generated an AAV2 / 9 vector expressing a monomeric red fluorescent protein (mRFP)-enhanced green fluorescent protein (EGFP) tandem fluorescent-tagged LC3 protein derived from the ptfLC3 plasmid, where green fluorescence is sensitive to the pH difference between neutral and acidic autophagosomes but red fluorescence is not (9). This vector was administered together with AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, AAV2 / 9-Fam3b or AAV2 / 9-control (1×10 11 vg / animal; n = 5 per treatment) immediately after MI. Two days later, the number of yellow, LC3-positive vesicles, and particularly the number of vesicles showing only red fluorescence, were significantly increased in the peri-infarct region of the LV of hearts injected with AAV9-Chrdl1 and Fam3c, indicating that these two factors stimulate autophagic flux in vivo (quantification of yellow and red puncta for each treatment is reported in Figure 4D).

[0211] Collectively, these results show that AAV2 / 9-mediated cardiac overexpression of Chrdl1, Fam3c, Fam3b maintains cardiomyocyte viability by preventing apoptotic cell death and, in particular, Fam3c and Chrdl1 promote beneficial autophagy in the heart.

[0212] [Example 5] When expressed specifically in tissues mediated by AAV8, circulating Chrdl1, Fam3c, and Fam3b produced and secreted by the liver counteract pathological left ventricular tissue repair after myocardial infarction. In contrast to being expressed endogenously using viral vectors, to evaluate whether circulating Chrdl1, Fam3b, and Fam3c are active after reaching the heart from the circulation, the inventors developed a strategy in which each of the three factors is expressed by the liver and secreted into the circulation before myocardial infarction (Figure 5A). More specifically, the inventors performed parenchymal injection (5×10 11 vg / animal; n = 6 per group) using AAV vector serotype 8 (AAV2 / 8) in adult CD1 mice. This vector selectively introduced genes into hepatocytes. In these vectors, the factors were expressed under the control of the human α-1 antitrypsin (hAAT) promoter, which ensures specific expression only in liver parenchymal cells (10). Seven days after administration, when the liver was actively producing each factor and releasing its administrable amount into the circulation (Figure 5B), myocardial infarction was induced by ligating the left descending coronary artery.

[0213] As reported in Figure 5C, AAV2 / 8-mediated liver production of Chrdl1, Fam3c, or Fam3b successfully maintained the left ventricular ejection fraction (LVEF) of infarcted mice compared to control-treated mice. The LVEF values began to improve significantly on the 15th day after MI and were maintained over time (60 days after MI: 28.77±1.66% for AAV2 / 8-Chrdl1, 28.09±1.61% for AAV2 / 8-Fam3c, and 31.22±1.40% for AAV8-Fam3b compared to 20.05±1.47% in animals receiving the control vector). Two months after MI, as expected, the diastolic LV volume was significantly larger in control animals compared to treated mice (Figure 5D) (60 days after MI: 150.2±10.2 μL for AAV2 / 8-Chrdl1, 137.9±16.8 μL for AAV2 / 8-Fam3c, and 134.7±9.2 μL for AAV2 / 8-Fam3b compared to 193.2±11.6 μL in animals receiving the control vector).

[0214] Finally, also in this experiment, morphometric analysis of trichrome-stained day 60 heart sections showed that AAV8-Chrdl1, AAV2 / 8-Fam3c, and AAV2 / 8-Fam3b-treated mice exhibited a significant reduction in the fibrotic area (infarct size: AAV2 / 8-Chrdl1 13.6 ± 3.1%, AAV2 / 8-Fam3c 15.0 ± 3.4%, AAV2 / 8-Fam3b 13.2 ± 2.7%, vs. 28.7 ± 3.3% of the LV in control animals; Figure 5E).

[0215] Collectively, these results demonstrate that each of these circulating factors is therapeutically expressed from the liver and protects cardiomyocytes from ischemic injury after MI and improves cardiac function. This represents a pre-efficacy study prior to injection of Chrdl1, Fam3c, and Fam3b as recombinant proteins.

[0216] [Example 6] Recombinant Chrdl1, Fam3c, and Fam3b protect cardiomyocytes against doxorubicin-induced cell death To evaluate the potential effect of Chrdl1, Fam3c, and Fam3b in maintaining cell viability during toxic injury, the corresponding recombinant proteins were tested in primary neonatal rat ventricular cardiomyocytes treated with the chemotherapeutic drug doxorubicin (Figure 6).

[0217] Treatment with 100 ng / mL of Chrdl1, Fam3c, or Fam3b recombinant protein significantly abrogated caspase 3 / 7 activation (as a measure of apoptotic cell death) 20 hours after doxorubicin treatment (1 and 1.5 μM).

[0218] [Example 7] Chrdl1 prevents fibroblast activation and cardiac fibrosis after myocardial infarction When MI occurs, cardiac fibroblasts proliferate, differentiate into myofibroblasts, stimulate collagen deposition, and create scars that replace the gaps created by cardiomyocyte loss. Interestingly, infarcted hearts overexpressing Chrdl1 not only had very small scars but also did not undergo pathological tissue repair and dilation two months after MI. This suggested a specific effect of Chrdl1 on scar formation in addition to its effect on cardiomyocyte survival.

[0219] Transforming growth factor-β1 (Tgfβ1) is highly expressed in scars after MI and is an important inducer of collagen deposition and fibroblast differentiation into myofibroblasts (11). To evaluate whether Chrdl1 can regulate the fibroblast differentiation conversion induced by Tgfβ1, primary adult mouse cardiac fibroblasts were treated for 3 days with different doses of Tgfβ1 (1 - 10 - 50 ng / mL) in the presence (100 ng / mL) or absence of recombinant Chrdl1 (Figure 7A). Tgfβ1 induced a significant dose-dependent increase in collagen α-1(I) (Col1α1) and α-Sma expression, and Chrdl1 blunted this effect (Figure 7B).

[0220] To further investigate the effect of Chrdl1 in the in vivo fibrotic response, the hearts of Collα1(I)-EGFP mice (a transgenic mouse model in which EGFP is expressed only in fibroblasts (12)) were transduced with AAV2 / 9-control or AAV2 / 9-Chrdl1, and MI was induced. The hearts of Col1α1(I)-EGFP mice overexpressing Chrdl1 showed significantly attenuated cardiac fibrosis and reduced collagen 1α1 and α-SMA expression (Figure 7C). These data were also confirmed by q-PCR quantifying the transcript levels of Col1α1, α-SMA, Tgfβ1, and MMP9 in CD1 mice three days after infarction (Figure 7D).

[0221] [Example 8] Expression of Chrdl1, Fam3c, and Fam3b protects mice from doxorubicin-induced cardiotoxicity and death Despite its effectiveness as an anticancer drug, anthracyclines (including doxorubicin) can induce both acute and chronic cardiotoxicity (Swain, S.M. et al., (2003) Cancer 97: 2869-2879). In particular, the cumulative amount of these drugs can cause left ventricular systolic dysfunction and heart failure. The reported incidence of ventricular dysfunction as a result of treatment with these drugs is approximately 10% of patients (Cardinale, D. (2015) Circulation 131: 1981-1988), and the majority of cases occur within the first year of treatment. Currently, there is no standard treatment to prevent anthracycline-induced cardiotoxicity (Zamorano, J.L. et al., (2016) Eur Heart J 37: 2768-2801).

[0222] Six-week-old female C57 / BL6 mice were intramyocardially injected with 30 μL of an AAV9 vector preparation expressing Chrdl1, Fam3c, or Fam3b using a 30G needle syringe. One week later, doxorubicin was intraperitoneally administered at a concentration of 4 mg / kg (cumulative amount: 24 mg / kg) on days 0, 2, 5, 8, 10, and 12 according to a protocol established for chronic treatment (Li M. et al., (2018) Circulation 138: 696-711). Mice were followed by echocardiography at 0, 6, and 8 weeks of age.

[0223] Each of Chrdl1, Fam3c, and Fam3b showed strong protective activity against drug-induced death (Figure 8A). A significant cardioprotective effect was subsequently observed with treatment using any of the three factors, as shown by significant protection against both the deterioration of left ventricular (LV) ejection fraction (Figure 8B) and the adverse effect on LV internal diameter (Figure 8C).

[0224] References TIFF2025107171000009.tif120164TIFF2025107171000010.tif27165

[0225] All publications mentioned in the above specification are hereby incorporated by reference into this specification. Various modifications and variations of the disclosed agents, compositions, uses, and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention will be apparent to those skilled in the art, but are intended to be within the scope of the following claims.

Claims

1. A pharmaceutical composition for treating or reducing the risk of heart disease, comprising Fam3b or a polynucleotide encoding same.

2. 2. The pharmaceutical composition of claim 1, wherein the cardiac disease is associated with cardiac ischemia or loss of cardiac muscle cells.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the cardiac disease is selected from myocardial infarction, consequences of myocardial infarction, reperfusion injury after percutaneous coronary intervention, myocarditis, hypertension, cardiotoxic damage, or cardiomyopathy.

4. A pharmaceutical composition for maintaining cardiomyocyte viability, comprising Fam3b or a polynucleotide encoding same.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the Fam3b comprises the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence consisting of amino acids at positions 30 to 235 of SEQ ID NO:

3.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the polynucleotide encodes Fam3b and comprises the nucleotide sequence of SEQ ID NO: 6; or the polynucleotide encodes Fam3b and comprises the nucleotide sequence consisting of nucleotides 88 to 708 of SEQ ID NO:

6.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the heart is protected from myocardial infarction, cardiac function is maintained after myocardial infarction or percutaneous coronary intervention, or fibrosis after infarction is reduced.

8. 8. The pharmaceutical composition of claim 1, wherein heart failure is prevented.

9. 9. The pharmaceutical composition of claim 1, wherein the Fam3b is glycosylated.

10. 10. The pharmaceutical composition of claim 1, wherein the Fam3b is a fusion protein.

11. 11. The pharmaceutical composition of claim 1, wherein the Fam3b is an Fc fusion protein.

12. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the polynucleotide is in the form of a vector, optionally a viral vector.

13. 13. The pharmaceutical composition of claim 12, wherein the polynucleotide is in the form of an adeno-associated virus (AAV) vector.

14. A pharmaceutical composition for treating or reducing the risk of heart disease, comprising a vector containing a polynucleotide encoding Fam3b.

15. 15. The pharmaceutical composition of claim 14, wherein the vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.

16. 12. A pharmaceutical composition according to any one of claims 1 to 11, comprising Fam3b and a pharmaceutically acceptable vehicle and / or excipient.

17. 17. The pharmaceutical composition of claim 16, formulated for injection.

18. 16. A pharmaceutical composition according to claim 14 or 15, comprising the vector and a pharmaceutically acceptable vehicle and / or excipient.

19. Use of Fam3b, or a polynucleotide encoding same, in the manufacture of a medicament for treating or reducing the risk of heart disease.

20. 20. The use according to claim 19, wherein the cardiac disease is associated with cardiac ischemia or loss of cardiac muscle cells.

21. 21. The use of claim 19 or 20, wherein the cardiac disease is selected from myocardial infarction, consequences of myocardial infarction, reperfusion injury after percutaneous coronary intervention, myocarditis, hypertension, cardiotoxic damage, or cardiomyopathy.

22. Use of Fam3b, or a polynucleotide encoding same, in the manufacture of a medicament for maintaining cardiomyocyte viability.

23. The use according to any one of claims 19 to 22, wherein the Fam3b comprises the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence consisting of amino acids 30 to 235 of SEQ ID NO:

3.

24. The use according to any one of claims 19 to 23, wherein the polynucleotide encodes Fam3b and comprises the nucleotide sequence of SEQ ID NO: 6; or the polynucleotide encodes Fam3b and comprises the nucleotide sequence consisting of nucleotides 88 to 708 of SEQ ID NO:

6.

25. 25. The use according to any one of claims 19 to 24, wherein the medicament protects the heart from myocardial infarction, maintains cardiac function after myocardial infarction or percutaneous coronary intervention, or reduces fibrosis after infarction.

26. 26. The use according to any one of claims 19 to 25, wherein the medicament prevents heart failure.

27. 27. The use according to any one of claims 19 to 26, wherein the Fam3b is glycosylated.

28. 27. The use according to any one of claims 19 to 26, wherein the Fam3b is a fusion protein.

29. 29. The use according to any one of claims 19 to 28, wherein the Fam3b is an Fc fusion protein.

30. 27. The use according to any one of claims 19 to 26, wherein the polynucleotide is in the form of a vector, optionally a viral vector.

31. 31. The use of claim 30, wherein the polynucleotide is in the form of an adeno-associated virus (AAV) vector.

32. Use of a vector containing a polynucleotide encoding Fam3b in the manufacture of a medicament for treating or reducing the risk of heart disease.

33. 33. The use of claim 32, wherein the vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.

Images