Proteins with cardiopreservation activity

Abstract

A Chrdl1l protein or fragment thereof, or a polynucleotide encoding therefor, for use in treating heart failure and / or improving cardiac function in heart failure.

Description

PROTEINS WITH CARDIOPRESERVATION ACTIVITY

[0001] This application claims priority from U.S. Provisional Application No. 63 / 655,568, file on June 03, 2024, the entire contents of which are incorporated herein by reference.

[0002] For countries that permit incorporation by reference, all patents, patent applications and publications cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted being prior art.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights. FIELD OF THE INVENTION

[0004] The present invention relates to the use of Chrdl1 as a medicament, for example in the context of gene therapy or through administration as proteins, such as recombinant or synthetic proteins, for treating heart failure. In particular, the invention relates to treating heart failure by improving cardiac function. Conditions for which the medicaments are effective include, but are not limited to, cardiac ischemia (myocardial infarction and reperfusion injury), cardiac toxic damage and cardiomyopathy of genetic origin. BACKGROUND OF THE INVENTION

[0005] Heart Failure is a clinical syndrome diagnosed from a combination of clinical symptoms and signs plus one or more diagnostic tests / investigation. Heart failure is the world’s leading cause of hospitalization, affecting more than 64 million people worldwide (World Heart Federation). There is no cure for heart failure. SUMMARY OF THE INVENTION

[0006] An aspect of the invention is directed to a method of treating heart failure. In embodiments, the method comprises administering to a subject a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor.

[0007] An aspect of the invention is also drawn towards a method of improving cardiac function in heart failure. In embodiments, the method comprises administering to a subject a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor.

[0008] In embodiments, the heart failure is early heart failure or established heart failure.

[0009] In embodiments, the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0010] In embodiments, the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0011] In embodiments, the Chrdl1 protein consists of an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0012] In embodiments, the polynucleotide consists of a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0013] In embodiments, the heart failure is associated with cardiac ischemia or loss of cardiomyocytes.

[0014] In embodiments, the heart failure is selected from the consequences of myocardial infarction; the reperfusion-injury after percutaneous coronary interventions, thrombolysis, or coronary artery bypass surgery, chronic cardiac ischemia; myocarditis; hypertension; cardiac toxic or metabolic damage; or cardiomyopathy. For example, the percutaneous coronary intervention is coronary angioplasty. For example, the cardiac toxic damage is caused by chemotherapy.

[0015] In embodiments, cardiac remodeling is reduced, LV ejection fraction is improved, left ventricular volume is reduced, and / or myocardial fibrosis is reduced. For example, the LV ejection fraction is increased to at least about 50%. For example, the subject has myocardial fibrosis.

[0016] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to the subject one week or more after cardiomyocyte loss.

[0017] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to the subject one week or more after myocardial infarction.

[0018] In embodiments, the Chrdl1 protein or fragment thereof is comprised in a fusion protein. For example, the fusion protein is an Fc fusion protein.

[0019] In embodiments, the polynucleotide is in the form of a vector. For example, the vector comprises a viral vector. For example, the viral vector comprises an adeno-associated viral (AAV) vector.

[0020] In embodiments, the protein or polynucleotide encoding the protein is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient. For example, the pharmaceutical composition is formulated for injection.

[0021] Aspects of the invention are also drawn towards a method of treating early or established heart failure. In embodiments, the method comprises administering to a subject a vector, wherein the vector comprises a polynucleotide encoding a Chrdl1 protein or a fragment thereof. For example, the vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.

[0022] In embodiments, the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0023] In embodiments, the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0024] In embodiments, the vector is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient. For example, the pharmaceutical composition is formulated for injection.

[0025] Aspects of the invention are also drawn towards the use of a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor, in the manufacture of a medicament for the treatment of heart failure.

[0026] Still further, aspects of the invention are drawn towards the use of a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor, in the manufacture of a medicament for improving cardiac function in heart failure.

[0027] In embodiments, heart failure is early or established heart failure.

[0028] In embodiments, the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0029] In embodiments, the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0030] In embodiments, the Chrdl1 protein consists of an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0031] In embodiments, the polynucleotide consists of a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0032] In embodiments, the heart failure is associated with cardiac ischemia or loss of cardiomyocytes.

[0033] In embodiments, the heart failure is selected from the consequences of myocardial infarction; the reperfusion-injury after percutaneous coronary interventions, thrombolysis, orcoronary artery bypass surgery, chronic cardiac ischemia; myocarditis; hypertension; cardiac toxic or metabolic damage; or cardiomyopathy. For example, the percutaneous coronary intervention is coronary angioplasty. For example, the cardiac toxic damage is caused by chemotherapy.

[0034] In embodiments, the cardiac remodeling is reduced, LV ejection fraction is improved, left ventricular volume is reduced, and / or myocardial fibrosis is reduced. For example, the LV ejection fraction is increased to at least about 50%.

[0035] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject having myocardial fibrosis.

[0036] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject one week or more after cardiomyocyte loss.

[0037] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject one week or more after myocardial infarction.

[0038] In embodiments, the Chrdl1 protein or fragment thereof is comprised in a fusion protein. For example, the fusion protein is an Fc fusion protein.

[0039] In embodiments, the polynucleotide is in the form of a vector. For example, the vector comprises a viral vector. For example, the viral vector comprises an adeno-associated viral (AAV) vector.

[0040] In embodiments, the protein or polynucleotide encoding the protein is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient. For example, the pharmaceutical composition is formulated for injection.

[0041] Still further, aspects of the invention are drawn towards the use of a vector comprising a polynucleotide encoding a Chrdl1 protein or a fragment thereof in the manufacture of a medicament for the treatment of early or established heart failure.

[0042] In embodiments, the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0043] In embodiments, the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0044] In embodiments, the vector is a viral vector, optionally herein the viral vector is an adeno-associated viral (AAV) vector.

[0045] In embodiments, the vector is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient. For example, the pharmaceutical composition is formulated for injection.

[0046] Aspects of the invention are also drawn towards a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor, for use in a method of treating heart failure.

[0047] Still further, aspects of the invention are drawn towards a Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefore, for use in a method of improving cardiac function in heart failure.

[0048] In embodiments, the heart failure is early or established heart failure.

[0049] In embodiments, the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0050] In embodiments, the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0051] In embodiments, the Chrdl1 protein consists of an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0052] In embodiments, the polynucleotide consists of a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0053] In embodiments, the heart failure is associated with cardiac ischemia or loss of cardiomyocytes.

[0054] In embodiments, the heart failure is selected from the consequences of myocardial infarction; the reperfusion-injury after percutaneous coronary interventions, thrombolysis, or coronary artery bypass surgery, chronic cardiac ischemia; myocarditis; hypertension; cardiac toxic or metabolic damage; or cardiomyopathy. For example, percutaneous coronary intervention is coronary angioplasty. For example, the cardiac toxic damage is caused by chemotherapy.

[0055] In embodiments, the cardiac remodeling is reduced, LV ejection fraction is improved, left ventricular volume is reduced, and / or myocardial fibrosis is reduced. For example, the LV ejection fraction is increased to at least about 50%.

[0056] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject having myocardial fibrosis.

[0057] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject one week or more after cardiomyocyte loss.

[0058] In embodiments, the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject one week or more after myocardial infarction.

[0059] In embodiments, the Chrdl1 protein or fragment thereof is comprised in a fusion protein. For example, the fusion protein is an Fc fusion protein.

[0060] In embodiments, the polynucleotide is in the form of a vector. For example, the vector comprises a viral vector. For example, the viral vector comprises an adeno-associated viral (AAV) vector.

[0061] In embodiments, the protein or polynucleotide encoding the protein is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient. For example, pharmaceutical composition is formulated for injection.

[0062] Still further, aspects of the invention are drawn towards a vector comprising a polynucleotide encoding a Chrdl1 protein or a fragment thereof for use in a method of treating heart failure.

[0063] In embodiments, the heart failure is early or established heart failure.

[0064] In embodiments, the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 1, 4, 5 or 6.

[0065] In embodiments, the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

[0066] In embodiments, the vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.

[0067] In embodiments, the vector is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient. For example, pharmaceutical composition is formulated for injection.

[0068] Other objects and advantages of this invention will become readily apparent from the ensuing description. BRIEF DESCRIPTION OF THE FIGURES

[0069] FIG. 1 shows overexpression of Chrdl1 in a mouse model of early heart failure following myocardial infarction improves cardiac function. Panel A: A schematic of the experiment timing indicating the timing of AAV treatment relative to induction of MI. Briefly, MI was induced by permanent ligation of the left descending coronary artery in adult CD-1 mice, with control animal receiving a sham procedure only. Overexpression of Chrdl1, through delivery of 1x1012vg of AAV2 / 9 Chrdl1 or non-expressing control AAV2 / 9 MCS, was initiated 7 days after MI by systemic administration. Mice were monitored for an additional 4 weeks. Panel B: At the termination of the experiment heart size was assessed by measurement of heart weight and tibial length. Animals expressing Chrdl1 showed a non- significantly reduced heart size. Echocardiographic analysis to evaluate heart function at 7-and 35-days post-MI revels that MI had induced a similar reduction in left ventricle ejection fraction (LVEF) in both groups, but after treatment when the animals were assessed again the Chrdl1 expressing animals had significantly improved LVEF. Panel C: When the hearts underwent histological analysis and scar size was quantified after Masson’s trichrome staining (infarct size expressed as % of left ventricle). Treatment with AAV2 / 9-Chrdl1 had no significant change in scar size compared to mice treated with AAV2 / 9 MCS the non- expressing control vector. A trend is present, indicating final scar editing may be ongoing. Data are shown as mean+ / -SEM; *p<0.05; ***p<0.001. LVEF data were analyzed using RM 2-way ANOVA - Bonferroni's multiple comparison test, while scar size and cardiac remodeling were analyzed using unpaired t-test. NB: no differences in body weights between groups across full experiment.

[0070] FIG. 2 shows overexpression of Fam3b or Fam3c in a mouse model of early heart failure following myocardial infarction does not improve cardiac function. Panel A: A schematic of the experiment timing indicating the timing of AAV treatment relative to induction of MI. Briefly, MI was induced by permanent ligation of the left descending coronary artery in adult CD-1 mice, with control animal receiving a sham procedure only. Overexpression of Chrdl1, through delivery of 1x1012vg of AAV2 / 9 Fam3b, AAV2 / 9 Fam3c or non-expressing control AAV2 / 9 MCS, was initiated 7 days after MI. Mice were monitored for an additional 4 weeks. Panel B: At the termination of the experiment heart size was assessed by measurement of heart weight and tibial length. Animals expressing Fam3b or Fam3c showed no alteration heart size compared to control animals. Echocardiographic analysis to evaluate heart function at 7- and 35-days post-MI revels that MI had induced a similar reduction in left ventricle ejection fraction (LVEF) in both groups, after treatment when the animals were assessed again the expression of Fam3b or Fam3c still showed no difference in LVEF compared to control AAV2 / 9 MCS treated animals. Panel C: When the hearts underwent histological analysis and scar size was quantified after Masson’s trichrome staining (infarct size expressed as % of left ventricle). Treatment with AAV2 / 9 Fam3c or AAV2 / 9 Fam3c had no significant effect on scar size compared to mice treated with AAV2 / 9 MCS the non-expressing control vector. Data are shown as mean+ / -SEM. LVEF data were analyzed using RM 2-way ANOVA - Bonferroni's multiple comparison test, while scar size and cardiac remodeling were analyzed using Ordinary one-way ANOVA – Dunnett’s multiple comparison test and no significant differences were found. NB: no difference in body weights between groups across full experiment.

[0071] FIG. 3 shows overexpression of Chrdl1 in a mouse model of late heart failure following myocardial infarction improves cardiac function. Panel A: A schematic of the experiment timing indicating the timing of AAV treatment relative to induction of MI. Briefly, MI was induced by permanent ligation of the left descending coronary artery in adult CD-1 mice, with control animal receiving a sham procedure only. Overexpression of Chrdl1, through delivery of 1x1012vg of AAV2 / 9 Chrdl1 or non-expressing control AAV2 / 9 MCS, was initiated 14 days after MI. Mice were monitored for an additional 4 weeks. Panel B: At the termination of the experiment heart size was assessed by measurement of heart weight and tibial length. Animals expressing Chrdl1 showed a significantly reduced heart size. Echocardiographic (ECG)analysis to evaluate heart function at 14- and 42-days post-MI reveals that MI had induced a similar reduction in left ventricle ejection fraction (LVEF) in both groups, but after treatment when the animals were assessed again the Chrdl1 expressing animals had significantly improved LVEF compared to control AAV2 / 9 MCS transduced animals. Panel C: When the hearts underwent histological analysis and scar size was quantified after Masson’s trichrome staining (infarct size expressed as % of left ventricle). Treatment with AAV2 / 9-Chrdl1 had no significant change in scar size compared to mice treated with AAV2 / 9 MCS the non-expressing control vector. Data indicates an effect of Chrdl1 in established heart failure independent of impact on scar formation / acute MI biology. Data are shown as mean+ / -SEM; *p<0.05; ; ***p<0.001. LVEF data were analyzed using RM 2-way ANOVA - Bonferroni's multiple comparison test, while scar size and cardiac remodeling were analyzed using unpaired t-test. NB: no difference in body weights between groups across full experiment.

[0072] FIG. 4 shows overexpression of Fam3b or Fam3c in a mouse model of late heart failure following myocardial infarction does not improve cardiac function. Panel A: A schematic of the experiment timing indicating the timing of AAV treatment relative to induction of MI. Briefly, MI was induced by permanent ligation of the left descending coronary artery in adult CD-1 mice, with control animal receiving a sham procedure only. Overexpression of Chrdl1, through delivery of 1x1012vg of AAV2 / 9 Fam3b, AAV2 / 9 Fam3c or non-expressing control AAV2 / 9 MCS, was initiated 14 days after MI. Mice were monitored for an additional 4 weeks. Panel B: At the termination of the experiment heart size was assessed by measurement of heart weight and tibial length. Animals expressing Fam3b or Fam3c showed no alteration heart size compared to control animals. Echocardiographic analysis to evaluate heart function at 14-and 42-days post-MI revels that MI had induced asimilar reduction in left ventricle ejection fraction (LVEF) in both groups, after treatment when the animals were assessed again the expression of Fam3b or Fam3c still showed no difference in LVEF compared to control AAV2 / 9 MCS treated animals. Panel C: When the hearts underwent histological analysis and scar size was quantified after Masson’s trichrome staining (infarct size expressed as % of left ventricle). Treatment with AAV2 / 9 Fam3c or AAV2 / 9 Fam3c had no significant effect on scar size compared to mice treated with AAV2 / 9 MCS the non-expressing control vector. Data are shown as mean+ / -SEM. LVEF data were analyzed using RM 2-way ANOVA - Bonferroni's multiple comparison test, while scar size and cardiac remodeling were analyzed using Ordinary one-way ANOVA – Dunnett’s multiple comparison test and no significant differences were found.

[0073] FIG. 5 shows sequence alignment of human Chrdl1-FL Vs human Chrdl1- ΔCTD mutant.

[0074] FIG. 6 shows heart Chrdl1 quantification upon AAV9 systemic administration.

[0075] FIG. 7 shows nucleic acid sequence alignment of Chrdl1-CDS Vs ΔSP-Chrdl- ΔCTD mutant.

[0076] FIG. 8 shows amino acid sequence alignment of Chrdl1-CDS Vs ΔSP-Chrdl- ΔCTD mutant. DETAILED DESCRIPTION OF THE INVENTION

[0077] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0078] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0079] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitlystated otherwise. Similarly “an example,” “exemplary” and the like are understood to be nonlimiting.

[0080] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0081] The terms “comprising”, “including”, “containing” and “having” and “involving” (and similarly “comprises”, “comprised of”, “contains”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of’.

[0082] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0083]

[0084] Proteins

[0085] Chrdl1 is a Bone Morphogenetic Protein (BMP) extracellular inhibitor, mainly expressed in mesenchyme-derived cell types, pericytes in the retina and in neural 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)).

[0086] The Chrdl1 name derives from its sequence similarity with Chordin, another BMP inhibitor identified as a factor dorsalizing Xenopus embryo. Chrdl1 has a spatiotemporalexpression pattern distinct from Chordin, but both genes contain cysteine-rich units designed procollagen repeats (CRs), which are also present in a variety of extracellular matrix proteins. CR1 and CR3 are responsible for Chrdl1-BMPs binding. The protein binds with high affinity to BMP4 and with less affinity to BMP5, BMP6 and BMP7.

[0087] An example amino acid sequence of Chrdl1 is SEQ ID NO: 1 (human) - UniProt ID: Q9BU40-6.

[0088] SEQ ID NO: 1 [human Chrdl1-Full length 456 amino acids] MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYC VNCICSENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQH GELFVAEGLFQNRQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSW EHSDGDIFRQPANREARHSYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQI VINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKY PQKIDGKCCKVCPGKKAKELPGQSFDNKGYFCGEETMPVYESVFMEDGETTRKIALETERPP QVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKLVTRTTLSQWKIFTEGEAQISQMCSSRV CRTELEDLVKVLYLERSEKGHC

[0089] In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In embodiments, the Chrdl1 protein or fragment thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 1. In embodiments, the Chrdl1 protein or fragment thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 1. In embodiments, the Chrdl1 protein or fragment thereof has one or two amino acid insertions compared to SEQ ID NO: 1. In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of SEQ ID NO: 1. In embodiments, the Chrdl1 protein or fragment thereof consists of SEQ ID NO: 1.

[0090] An example nucleotide sequence encoding Chrld1 (human) is SEQ ID NO: 2 - Seq ID: NM_001143981.1 (coding sequence).

[0091] SEQ ID NO: 2 ATGAGAAAAAAGTGGAAAATGGGAGGCATGAAATACATCTTTTCGTTGTTGTTCTTTCTTTT GCTAGAAGGAGGCAAAACAGAGCAAGTAAAACATTCAGAGACATATTGCATGTTTCAAGACA AGAAGTACAGAGTGGGTGAGAGATGGCATCCTTACCTGGAACCTTATGGGTTGGTTTACTGC GTGAACTGCATCTGCTCAGAGAATGGGAATGTGCTTTGCAGCCGAGTCAGATGTCCAAATGT TCATTGCCTTTCTCCTGTGCATATTCCTCATCTGTGCTGCCCTCGCTGCCCAGAAGACTCCT TACCCCCAGTGAACAATAAGGTGACCAGCAAGTCTTGCGAGTACAATGGGACAACTTACCAA CATGGAGAGCTGTTCGTAGCTGAAGGGCTCTTTCAGAATCGGCAACCCAATCAATGCACCCAGTGCAGCTGTTCGGAGGGAAACGTGTATTGTGGTCTCAAGACTTGCCCCAAATTAACCTGTG CCTTCCCAGTCTCTGTTCCAGATTCCTGCTGCCGGGTATGCAGAGGAGATGGAGAACTGTCA TGGGAACATTCTGATGGTGATATCTTCCGGCAACCTGCCAACAGAGAAGCAAGACATTCTTA CCACCGCTCTCACTATGATCCTCCACCAAGCCGACAGGCTGGAGGTCTGTCCCGCTTTCCTG GGGCCAGAAGTCACCGGGGAGCTCTTATGGATTCCCAGCAAGCATCAGGAACCATTGTGCAA ATTGTCATCAATAACAAACACAAGCATGGACAAGTGTGTGTTTCCAATGGAAAGACCTATTC TCATGGCGAGTCCTGGCACCCAAACCTCCGGGCATTTGGCATTGTGGAGTGTGTGCTATGTA CTTGTAATGTCACCAAGCAAGAGTGTAAGAAAATCCACTGCCCCAATCGATACCCCTGCAAG TATCCTCAAAAAATAGACGGAAAATGCTGCAAGGTGTGTCCAGGTAAAAAAGCAAAAGAAGA ACTTCCAGGCCAAAGCTTTGACAATAAAGGCTACTTCTGCGGGGAAGAAACGATGCCTGTGT ATGAGTCTGTATTCATGGAGGATGGGGAGACAACCAGAAAAATAGCACTGGAGACTGAGAGA CCACCTCAGGTAGAGGTCCACGTTTGGACTATTCGAAAGGGCATTCTCCAGCACTTCCATAT TGAGAAGATCTCCAAGAGGATGTTTGAGGAGCTTCCTCACTTCAAGCTGGTGACCAGAACAA CCCTGAGCCAGTGGAAGATCTTCACCGAAGGAGAAGCTCAGATCAGCCAGATGTGTTCAAGT CGTGTATGCAGAACAGAGCTTGAAGATTTAGTCAAGGTTTTGTACCTGGAGAGATCTGAAAA GGGCCACTGTTAG

[0092] In embodiments, the polynucleotide comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2. In embodiments, the polynucleotide comprises or consists of SEQ ID NO: 2. In embodiments, the polynucleotide consists of SEQ ID NO: 2.

[0093] Another example nucleotide sequence encoding Chrld1 (human) is SEQ ID NO: 3.

[0094] SEQ ID NO: 3 ATGAGAAAAAAGTGGAAAATGGGAGGCATGAAATACATCTTTTCGTTGTTGTTCTTTCTTTT GCTAGAAGGAGGCAAAACAGAGCAAGTAAAACATTCAGAGACATATTGCATGTTTCAAGACA AGAAGTACAGAGTGGGTGAGAGATGGCATCCTTACCTGGAACCTTATGGGTTGGTTTACTGC GTGAACTGCATCTGCTCAGAGAATGGGAATGTGCTTTGCAGCCGAGTCAGATGTCCAAATGT TCATTGCCTTTCTCCTGTGCATATTCCTCATCTGTGCTGCCCTCGCTGCCCAGACTCCTTAC CCCCAGTGAACAATAAGGTGACCAGCAAGTCTTGCGAGTACAATGGGACAACTTACCAACAT GGAGAGCTGTTCGTAGCTGAAGGGCTCTTTCAGAATCGGCAACCCAATCAATGCACCCAGTG CAGCTGTTCGGAGGGAAACGTGTATTGTGGTCTCAAGACTTGCCCCAAATTAACCTGTGCCT TCCCAGTCTCTGTTCCAGATTCCTGCTGCCGGGTATGCAGAGGAGATGGAGAACTGTCATGG GAACATTCTGATGGTGATATCTTCCGGCAACCTGCCAACAGAGAAGCAAGACATTCTTACCA CCGCTCTCACTATGATCCTCCACCAAGCCGACAGGCTGGAGGTCTGTCCCGCTTTCCTGGGG CCAGAAGTCACCGGGGAGCTCTTATGGATTCCCAGCAAGCATCAGGAACCATTGTGCAAATT GTCATCAATAACAAACACAAGCATGGACAAGTGTGTGTTTCCAATGGAAAGACCTATTCTCA TGGCGAGTCCTGGCACCCAAACCTCCGGGCATTTGGCATTGTGGAGTGTGTGCTATGTACTT GTAATGTCACCAAGCAAGAGTGTAAGAAAATCCACTGCCCCAATCGATACCCCTGCAAGTAT CCTCAAAAAATAGACGGAAAATGCTGCAAGGTGTGTCCAGGTAAAAAAGCAAAAGAACTTCC AGGCCAAAGCTTTGACAATAAAGGCTACTTCTGCGGGGAAGAAACGATGCCTGTGTATGAGT CTGTATTCATGGAGGATGGGGAGACAACCAGAAAAATAGCACTGGAGACTGAGAGACCACCT CAGGTAGAGGTCCACGTTTGGACTATTCGAAAGGGCATTCTCCAGCACTTCCATATTGAGAA GATCTCCAAGAGGATGTTTGAGGAGCTTCCTCACTTCAAGCTGGTGACCAGAACAACCCTGA GCCAGTGGAAGATCTTCACCGAAGGAGAAGCTCAGATCAGCCAGATGTGTTCAAGTCGTGTA TGCAGAACAGAGCTTGAAGATTTAGTCAAGGTTTTGTACCTGGAGAGATCTGAAAAGGGCCA CTGTTAG

[0095] In embodiments, the polynucleotide comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3. In embodiments, the polynucleotide comprises or consists of SEQ ID NO: 3. In embodiments, the polynucleotide consists of SEQ ID NO: 3.

[0096] Any other polynucleotide coding for the above proteins is comprised in the present invention.

[0097] “Fragments” of full length Chrdl1 are also described herein, and the term can refer to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus can refer to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0098] In embodiments, the fragment is a secreted form of Chrdl1. In embodiments, the fragment does not comprise a signal peptide. An example amino acid sequence of a Chrdl1 fragment lacking the signal peptide is SEQ ID NO: 4.

[0099] SEQ ID NO: 4 [human Chrdl1 fragment lacking the signal peptide (429 amino acids)] EQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENGNVLCSRVRCPNVHCLSPV HIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQNRQPNQCTQCSCSEGN VYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARHSYHRSHYDP PPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHP NLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDN KGYFCGEETMPVYESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMF EELPHFKLVTRTTLSQWKIFTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC

[0100] In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4. In embodiments, the Chrdl1 protein or fragment thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 4. In embodiments, the Chrdl1 protein or fragment thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 4. In embodiments, the Chrdl1 protein or fragment thereof has one or two amino acid insertions compared to SEQ ID NO: 4. In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of SEQ ID NO: 4. In embodiments, the Chrdl1 protein or fragment thereof consists of SEQ ID NO: 4.

[0101] Further amino acid sequences could be generated by a person of skill in the art by suitable amino acid substitutions, deletions, and / or insertions. Known amino acid substitutions compared to SEQ ID NO: 1 include M7V, R195Q, R208H, R208L, K317N, R382Q, and Y447H. Known amino acid deletions compared to SEQ ID NO: 1 are described below. Known amino acid insertions compared to SEQ ID NO: 1 include P → PE at position 100 and K → KE at position 328. In embodiments, the Chrdl1 protein or fragment thereof comprises a CR1 domain and a CR3 domain. In embodiments, the Chrdl1 protein or fragment thereof binds to BMP4. The binding to BMP4 may be determined by a co- immunoprecipitation assay (see e.g. Larraín, J., et al., 2000. Development, 127(4), pp.821- 830). In embodiments, the Chrdl1 protein or fragment thereof binds to and reduces the activity of BMP4. The activity of BMP4 may be determined by reduction of SMAD 1 / 5 / 8 phosphorylation upon treatment with recombinant BMP4.

[0102] An example amino acid sequence of a Chrdl1 C-terminal domain deleted protein is SEQ ID NO: 5 - Human Chrdl1-ΔCTD mutant (C-terminal domain deleted) (336 amino acids)

[0103] SEQ ID NO: 5 [Human Chrdl1-ΔCTD mutant (C-terminal domain deleted) (336 amino acids)] MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYC VNCICSENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQH GELFVAEGLFQNRQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSW EHSDGDIFRQPANREARHSYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQI VINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKY PQKIDGKCCKVCPGKKAKELPGQSFD

[0104] In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 5. In embodiments, the Chrdl1 protein or fragment thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 5. In embodiments, the Chrdl1 protein or fragment thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 5. In embodiments, the Chrdl1 protein or fragment thereof has one or two amino acid insertions compared to SEQ ID NO: 5. In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of SEQ ID NO: 5. In embodiments, the Chrdl1 protein or fragment thereof consists of SEQ ID NO: 5.

[0105] An example amino acid sequence of a Chrdl1 C-terminal domain deleted protein lacking the signal peptide is SEQ ID NO: 6.

[0106] SEQ ID NO: 6 [Human Chrdl1-ΔCTD mutant lacking the signal peptide (309 amino acids)] EQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENGNVLCSRVRCPNVHCLSPV HIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQNRQPNQCTQCSCSEGN VYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARHSYHRSHYDP PPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHP NLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFD

[0107] In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6. In embodiments, the Chrdl1 protein or fragment thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 6. In embodiments, the Chrdl1 protein or fragment thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 6. In embodiments, the Chrdl1 protein or fragment thereof has one or two amino acid insertions compared to SEQ ID NO: 6. In embodiments, the Chrdl1 protein or fragment thereof comprises or consists of SEQ ID NO: 6. In embodiments, the Chrdl1 protein or fragment thereof consists of SEQ ID NO: 6.

[0108] An example nucleotide sequence encoding a Chrdl1 C-terminal domain deleted protein lacking the signal peptide is SEQ ID NO: 7.

[0109] SEQ ID NO: 7 GAGCAAGTAAAACATTCAGAGACATATTGCATGTTTCAAGACAAGAAGTACAGAGTGGGTGA GAGATGGCATCCTTACCTGGAACCTTATGGGTTGGTTTACTGCGTGAACTGCATCTGCTCAG AGAATGGGAATGTGCTTTGCAGCCGAGTCAGATGTCCAAATGTTCATTGCCTTTCTCCTGTG CATATTCCTCATCTGTGCTGCCCTCGCTGCCCAGACTCCTTACCCCCAGTGAACAATAAGGT GACCAGCAAGTCTTGCGAGTACAATGGGACAACTTACCAACATGGAGAGCTGTTCGTAGCTG AAGGGCTCTTTCAGAATCGGCAACCCAATCAATGCACCCAGTGCAGCTGTTCGGAGGGAAAC GTGTATTGTGGTCTCAAGACTTGCCCCAAATTAACCTGTGCCTTCCCAGTCTCTGTTCCAGA TTCCTGCTGCCGGGTATGCAGAGGAGATGGAGAACTGTCATGGGAACATTCTGATGGTGATA TCTTCCGGCAACCTGCCAACAGAGAAGCAAGACATTCTTACCACCGCTCTCACTATGATCCT CCACCAAGCCGACAGGCTGGAGGTCTGTCCCGCTTTCCTGGGGCCAGAAGTCACCGGGGAGC TCTTATGGATTCCCAGCAAGCATCAGGAACCATTGTGCAAATTGTCATCAATAACAAACACAAGCATGGACAAGTGTGTGTTTCCAATGGAAAGACCTATTCTCATGGCGAGTCCTGGCACCCA AACCTCCGGGCATTTGGCATTGTGGAGTGTGTGCTATGTACTTGTAATGTCACCAAGCAAGA GTGTAAGAAAATCCACTGCCCCAATCGATACCCCTGCAAGTATCCTCAAAAAATAGACGGAA AATGCTGCAAGGTGTGTCCAGGTAAAAAAGCAAAAGAACTTCCAGGCCAAAGCTTTGAC

[0110] In embodiments, the polynucleotide comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7. In embodiments, the polynucleotide comprises or consists of SEQ ID NO: 7. In embodiments, the polynucleotide consists of SEQ ID NO: 7.

[0111] Any other polynucleotide coding for the above proteins is comprised in the present invention.

[0112] Activity of the protein of the disclosure and fragments thereof can be readily determined by the skilled person. For example, suitable in vitro assays include: (a) protection from hydrogen peroxide- or doxorubicin-induced cell death, for example using TUNEL assays or Caspase activation assays; (b) induction of autophagy, for example the assaying the formation of LC3-positive autophagy vesicles; and / or (c) for Chrdl1: reduction of BMP and TGFbeta activities, for example activation of aSMA expression in cardiac fibroblasts upon treatment with recombinant TGFbeta, or reduction of SMAD 1 / 5 / 8 phosphorylation upon treatment with recombinant BMP4.

[0113] The present inventors have surprisingly discovered a previously unknown role of Chrdl1 in preserving and / or improving cardiac function in early or established heart failure.

[0114] According to the present invention, Chrdl1 preserves heart function in early heart failure and established heart failure. For example, Chrdl1 effectively reduces cardiac remodeling, improves LV ejection fraction, and has no significant impact on scar size after intravenous injection of viral vectors expressing these factors.

[0115] While the present inventors do not wish to be bound to theory or mechanism of action, it is currently believed that the Chrdl1 protein exerts it therapeutic effects in the heart by modulating TGFb and BMP-4 signaling to affect autophagy, apoptosis, and fibrosis[ ].

[0116] The Chrdl1 protein and fragments thereof may therefore be used to preserve or improve heart function, thus treating heart failure.

[0117] Protein delivery

[0118] As an alternative to the delivery of polynucleotides, the proteins of the invention or fragments thereof can be delivered by direct protein delivery.

[0119] Proteins or fragments thereof can be administered directly to a subject. In some embodiments, the protein or fragment thereof is a fusion protein, preferably a fusion with a second protein capable of increasing the lifespan of the protein in the subject. For example, the protein may be an immunoglobulin Fc domain-fusion protein.

[0120] Protein delivery can be via vector delivery (Cai, Y. et al. (2014) Elife 3: e01911 ; Maetzig, T. et al. (2012) Curr. Gene Ther. 12: 389-409). Vector delivery involves the engineering of viral particles (e.g., lentiviral particles) to comprise the proteins to be delivered to a cell. Accordingly, when the engineered viral particles enter a cell as part of their natural life cycle, the proteins comprised in the particles are carried into the cell.

[0121] Protein delivery (Gaj, T. et al. (2012) Nat. Methods 9: 805-7) can also be achieved, for example, by utilizing a vehicle (e.g., liposomes).

[0122] Polynucleotide

[0123] Polynucleotides of the invention can comprise DNA or RNA. In embodiments, polynucleotides of the invention can comprise DNA. In embodiments, polynucleotides of the invention can comprise RNA. They can be single-stranded or double-stranded. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons can, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

[0124] The nucleotide sequences of the invention disclosed herein can comprise or lack stop codons at their 3’ end, for example depending on their position in a bicistronic vector. Thus, the present disclosure encompasses the SEQ ID NOs disclosed herein with the stop codons present or absent.

[0125] The polynucleotides can be modified by any method available in the art. Such modifications can be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0126] Polynucleotides such as DNA polynucleotides can be produced recombinantly, synthetically or by any means available to those of skill in the art. They can also be cloned by standard techniques.

[0127] Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0128] Vectors

[0129] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. For example, a vector can allow or facilitate the transfer of DNA that encodes proteins of the invention or fragments thereof.

[0130] In one aspect, the invention provides a vector comprising a polynucleotide of the invention. In embodiments, the vector is a non-viral vector. In embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or adenoviral vector.

[0131] Adeno-associated viral (AAV) vectors

[0132] In one aspect, the invention provides an AAV vector comprising a polynucleotide of the invention.

[0133] For example, the AAV vector can be in the form of an AAV vector particle.

[0134] In some embodiments, the AAV vector particle comprises an AAV2 genome. In some embodiments, the AAV vector particle comprises an AAV9 genome. In some embodiments, the AAV vector particle comprises an AAV8 genome.

[0135] In some embodiments, the AAV vector particle comprises AAV9 capsid proteins. In some embodiments, the AAV vector particle comprises AAV8 capsid proteins.

[0136] In some embodiments, the AAV vector particle comprises an AAV2 genome and AAV9 capsid proteins (AAV2 / 9). In other embodiments, the AAV vector particle comprises an AAV2 genome and AAV8 capsid proteins (AAV2 / 8).

[0137] Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

[0138] The AAV vector may comprise an AAV genome or a fragment or derivative thereof.

[0139] AAV can package genomes up to 5.2 kb in size (Dong, J.-Y. et al. (1996) Human Gene Therapy 7: 2101-2112).

[0140] An AAV genome is a polynucleotide sequence, which can encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the invention is typically replication- deficient.

[0141] The AAV genome can be in single-stranded form, either positive or negative- sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.

[0142] The AAV genome can be from any naturally derived serotype, isolate or clade of AAV. Thus, the AAV genome can be the full genome of a naturally occurring AAV. As is known to the skilled person, AAVs occurring in nature can be classified according to various biological systems.

[0143] Commonly, AAVs can be referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, a virus having a particular AAV serotype does not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype.

[0144] AAV serotypes can include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, and also recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain. Any of these AAV serotypes may be used in the invention.

[0145] In some embodiments, the AAV vector particle can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , Rec2 or Rec3 AAV vector particle.

[0146] In some embodiments, the AAV can be an AAV1, AAV2, AAV5, AAV7, AAV8 or AAV9 serotype.

[0147] In some embodiments, the AAV can be an AAV9 or AAV8 serotype.

[0148] In some embodiments, the AAV can be an AAV9 serotype. In other embodiments, the AAV can be an AAV8 serotype.

[0149] The capsid protein can be a mutant capsid protein such as disclosed in WO 2008 / 124724, which is herein incorporated by reference.

[0150] The capsid protein can be a cardiotropic capsid (e.g., AAV2i8, AAVMyo and variants thereof, MyoAAV and variants thereof, AAVrh.74, Anc80L65). See, for example, Asokan, A.; et al. Reengineering a Receptor Footprint of Adeno-Associated Virus Enables Selective and Systemic Gene Transfer to Muscle. Nat Biotechnol 2010, 28 (1), 79–82; Weinmann, J.; et al. Identification of a Myotropic AAV by Massively Parallel in Vivo Evaluation of Barcoded Capsid Variants. Nat Commun 2020, 11 (1); Tabebordbar, M., et al. Directed Evolution of a Family of AAV Capsid Variants Enabling Potent Muscle-Directed Gene Delivery across Species. Cell 2021, 184 (19), 4919–4938, Katz, M. G., et al. Efficient Cardiac Gene Transfer and Early-Onset Expression of a Synthetic Adeno-Associated Viral Vector, Anc80L65, after Intramyocardial Administration. J Thorac Cardiovasc Surg 2022, 164 (6), e429–e443.

[0151] In some embodiments, the AAV vector can comprise an AAV8 capsid with an Y733F mutation.

[0152] In some embodiments, the AAV can be one of a natural serotype (e.g., AAV1, AAV4, AAV6, AAV8, AAV9 serotype). See, for example, Zhang, H.; Zhan, Q.; Huang, B.; Wang, Y.; Wang, X. AAV-Mediated Gene Therapy: Advancing Cardiovascular Disease Treatment. Front Cardiovasc Med 2022, 9.

[0153] Reviews 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 sequences of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes for use in the invention can be derived from the following accession numbers for AAV whole genome sequences: 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.

[0154] AAV can also be referred to in terms of clades or clones. This can refer to the phylogenetic relationship of naturally derived AAVs, such as to a phylogenetic group of AAVs which can be traced back to a common ancestor and includes all descendants thereof. Additionally, AAVs can 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 can describe a population of AAVswhich has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a recognizably distinct population at a genetic level.

[0155] The skilled person can select an appropriate serotype, clade, clone or isolate of AAV for use in the invention on the basis of their common general knowledge. For instance, the AAV5 capsid has been shown to transduce primate cone photoreceptors efficiently as evidenced by the successful correction of an inherited color vision defect (Mancuso et al. (2009) Nature 461: 784-7).

[0156] The AAV serotype can determine the tissue specificity of infection (or tropism) of an AAV. Accordingly, exemplary AAV serotypes for use in AAVs administered to patients in accordance with the invention can be those which have natural tropism for or a high efficiency of infection of target cells within the heart.

[0157] In embodiments, the AAV genome of a naturally derived serotype, isolate or clade of AAV can comprise at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. In embodiments, one or more ITR sequences flank the nucleotide sequences encoding the protein of the invention. The AAV genome can also comprise packaging genes, such as rep and / or cap genes which encode packaging functions for an 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 make up the capsid of an AAV particle. Capsid variants are discussed below.

[0158] A promoter can be operably linked to each of the packaging genes. Non- limiting 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 can be used to express the rep gene, while the p40 promoter is generally used to express the cap gene.

[0159] As discussed herein, the AAV genome used in the AAV vector of the invention can therefore be the full genome of a naturally occurring AAV. For example, a vector comprising a full AAV genome can be used to prepare an AAV vector or vector particle in vitro. However, while such a vector can be administered to patients, this will rarely be done in practice. In embodiments, the AAV genome can be derivatized for the purpose of administration to patients. Such derivatization is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. Derivatization of the AAVgenome and of the AAV capsid are reviewed in: Coura and Nardi (2007) Virology Journal 4: 99, and in Choi et al. and Wu et al., referenced above.

[0160] Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. In embodiments, the AAV genome can be truncated significantly to include minimal viral sequence yet retain the function described herein. This is desired for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell.

[0161] In embodiments, a derivative can include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs can be derived from AAV genomes having different serotypes or can be a chimeric or mutant ITR. An exemplary mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single- stranded genome which contains both coding and complementary sequences, i.e., a self complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.

[0162] The one or more ITRs can preferably flank the nucleotide sequence encoding the protein of the invention at either end. The inclusion of one or more ITRs can aid concatamer formation of the vector of the invention in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

[0163] In embodiments, ITR elements can be the only sequences retained from the native AAV genome in the derivative. Thus, a derivative does not necessarily include the rep and / or cap genes of the native genome and any other sequences of the native genome. This can be desired for the reasons described herein, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.

[0164] The following portions could therefore be removed in a derivative of the invention: one inverted terminal repeat (ITR) sequence, the replication {rep) and capsid (cap) genes. However, in some embodiments, derivatives can additionally include one or more repand / or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting.

[0165] Where a derivative comprises capsid proteins i.e. VP1, VP2 and / or VP3, the derivative can be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. In embodiments, the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e., a pseudotyped vector).

[0166] Chimeric, shuffled or capsid-modified derivatives can be selected to provide one or more desired functionalities for the AAV vector. Thus, these derivatives can display increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and / or improved targeting of a particular cell type compared to an AAV vector comprising a naturally occurring AAV genome, such as that of AAV2. Increased efficiency of gene delivery can be effected by improved receptor or co-receptor binding at the cell surface, improved internalization, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single- stranded genome to double-stranded form. Increased efficiency can also relate to an altered tropism range or targeting of a specific cell population, such that the vector dose is not diluted by administration to tissues where it is not needed.

[0167] Chimeric capsid proteins can include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are co-transfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties. The capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.

[0168] Chimeric capsid proteins can also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.

[0169] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g., those encoding capsid proteins of multiple differentserotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology. A library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality. Similarly, error prone PCR can be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.

[0170] The sequences of the capsid genes can also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence. For example, capsid genes can be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and / or C-terminus of a capsid coding sequence.

[0171] The unrelated protein or peptide can advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population. The unrelated protein can also be one which assists purification of the viral particle as part of the production process, i.e., an epitope or affinity tag. The site of insertion can be selected so as not to interfere with other functions of the viral particle e.g., internalization, trafficking of the viral particle. The skilled person can identify suitable sites for insertion based on their common general knowledge. Particular sites are disclosed in Choi et al., referenced herein.

[0172] Embodiments of the invention can additionally encompass the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also can encompass the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

[0173] The AAV vector of the invention can take the form of a nucleotide sequence comprising an AAV genome or derivative thereof and a sequence encoding the protein of the invention.

[0174] The AAV particles of the invention can include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV particles of the invention can also include mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid. The AAV particle also includes chemically modified forms bearing ligandsadsorbed to the capsid surface. For example, such ligands can include antibodies for targeting a particular cell surface receptor.

[0175] The AAV vector can comprise multiple copies (e.g., 2, 3 etc.) of the nucleotide sequence referred to herein.

[0176] In embodiments, the polynucleotide can further comprise one or more AAV ITRs. For example, the polynucleotide can further comprise two AAV ITRs. In some embodiments, the polynucleotide can comprise an AAV ITR at its 5’ end and an AAV ITR at its 3’ end. In some embodiments, the AAV ITRs are AAV2, AAV9 or AAV8 ITRs.

[0177] Promoters and regulatory sequences

[0178] The polynucleotide or vector of the invention can also include elements allowing for the expression of the nucleotide sequence encoding the protein of the invention in vitro or in vivo. These can be referred to as expression control sequences. Thus, the polynucleotide or vector can comprise expression control sequences (e.g., comprising a promoter sequence) operably linked to the nucleotide sequence encoding the protein of the invention.

[0179] Any suitable promoter can be used, the selection of which can be readily made by the skilled person. The promoter sequence can be constitutively active (i.e., operational in any host cell background), or alternatively can be active only in a specific host cell environment, thus allowing for targeted expression of the transgene in a particular cell type (e.g. a tissue-specific promoter). The promoter can show inducible expression in response to presence of another factor, for example a factor present in a host cell. In any event, where the vector is administered for therapy, embodiments can comprise a promoter that is functional in the target cell background.

[0180] In embodiments, the promoter can be a liver-specific promoter. In embodiments, the promoter can be a liver-specific hAAT promoter.

[0181] In embodiments, the liver-specific hAAT promoter can confer selective specificity for hepatocytes. When vectors are administered through the portal vein, each circulating factor, secreted from the liver, can protect the heart after damage.

[0182] In embodiments, the promoter can be a heart-specific promoter. In embodiments, the promoter can be a cardiac-specific promotor, e.g., Tropinin T (cTnT) promoter or derivatives thereof.

[0183] Suitable ubiquitous promoters can include the chicken beta-actin (CBA) promoter, optionally in combination with a cytomegalovirus (CMV) enhancer element. An example promoter for use in the invention is a CAG promoter.

[0184] In some embodiments, the promoter can be a CMV or CAG promoter.

[0185] The polynucleotide or vector of the invention can also comprise one or more additional regulatory sequences which may act pre- or post-transcriptionally. The regulatory sequence can be part of the native transgene locus or can be a heterologous regulatory sequence. The polynucleotide or vector of the invention can comprise portions of the 5'- UTR or 3'-UTR from the native transgene transcript.

[0186] Regulatory sequences can be any sequences which facilitate expression of the transgene, i.e., act to increase expression of a transcript, improve nuclear export of mRNA or enhance its stability. Such regulatory sequences can include for example enhancer elements, post-transcriptional regulatory elements and polyadenylation sites.

[0187] Suitable enhancers can include the WPRE regulatory element. Suitable poly-A signals can include the Bovine Growth Hormone poly-A signal.

[0188] Additional regulatory sequences can be readily selected by the skilled person.

[0189] Method of administration

[0190] A variety of administration routes and techniques can be utilized, among them parenteral techniques such as intravenous, intracardiac and intra-arterial injections, catheterization and the like. Average quantities of the active agent can vary and in particular should be based upon the recommendations and prescription of a qualified physician.

[0191] The protein, polynucleotide or vector of the invention can be administered systemically (for example by peripheral vein infusion) or can be administered locally or regionally.

[0192] Preferably, the protein or fragment thereof can be administered by parenteral route, in particular intravenous, intraarterial or intramyocardial route.

[0193] The administration of the polynucleotide encoding the protein or fragment thereof disclosed herein can be achieved by gene therapy, see for example WO 2013 / 093870.

[0194] According to the present invention, Chrdl1 or fragment thereof can be active when they reach the damaged or diseased heart through the systemic circulation.

[0195] In embodiments, the protein, polynucleotide or vector of the invention can be administered after cardiomyocyte loss. Cardiomyocyte loss can be indicated by e.g., troponin elevation, replacement fibrosis or post-infarct scaring. For example, the protein, polynucleotide or vector of the invention can be administered at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, or longer than eight weeks after cardiomyocyte loss. In embodiments, the protein, polynucleotide or vector of the invention is administered at least one week after cardiomyocyte loss. In embodiments,the protein, polynucleotide or vector of the invention is administered at least two weeks after cardiomyocyte loss. For example, the protein, polynucleotide or vector of the invention can be administered at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or longer than twelve months after cardiomyocyte loss.

[0196] In embodiments, the protein, polynucleotide or vector of the invention can be administered after myocardial infarction. For example, the protein, polynucleotide or vector of the invention can be administered at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, or longer than eight weeks after myocardial infarction. In embodiments, the protein, polynucleotide or vector of the invention is administered at least one week after myocardial infarction. In embodiments, the protein, polynucleotide or vector of the invention is administered at least two weeks after myocardial infarction. For example, the protein, polynucleotide or vector of the invention can be administered at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or longer than twelve months after myocardial infarction.

[0197] Pharmaceutical compositions and injected solutions

[0198] The medicaments, for example proteins, polynucleotides or vectors, of the invention can be formulated into pharmaceutical compositions. These compositions can comprise, in addition to the medicament, a pharmaceutically acceptable carrier, diluent, excipient, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration.

[0199] According to the administration route chosen, the compositions can be in solid or liquid form, suitable for oral, parenteral, intravenous or intra-arterial administration. The pharmaceutical composition can be in liquid form. Liquid pharmaceutical compositions can include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% can be used.

[0200] For injection at the site of affliction, the active ingredient can be in the form of an aqueous solution which is pyrogen-free, and has suitable pH, isotonicity and stability. The skilled person is well able to prepare suitable solutions using, for example, isotonic vehiclessuch as Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as required.

[0201] For delayed release, the medicament can be included in a pharmaceutical composition which can be formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

[0202] Such compositions are well-known in the art, see for example Remington’s Pharmaceutical Sciences; last edition, Mack Pub.

[0203] Method of treatment

[0204] It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment, although in the context of the invention references to preventing are more commonly associated with prophylactic treatment. Treatment may also include arresting progression in the severity of a disease.

[0205] The treatment of mammals, particularly humans, is preferred. However, both human and veterinary treatments are within the scope of the invention.

[0206] The administration regime, dosage and posology will be determined by the physician according to his experience, the disease to be treated and the patient’s conditions.

[0207] The proteins, fragments thereof, and / or the polynucleotides of the present invention can be administered either singularly or in combination thereof.

[0208] The term “combination”, or terms “in combination”, “used in combination with” or “combined preparation” as used herein can refer to the combined administration of two or more agents simultaneously, sequentially or separately.

[0209] The term “simultaneous” as used herein can refer to agents being administered concurrently, i.e., at the same time.

[0210] The term “sequential” as used herein can refer to agents being administered one after the other.

[0211] The term “separate” as used herein can refer to agents being administered independently of each other but within a time interval that allows the agents to show a combined, preferably synergistic, effect. Thus, administration “separately” can permit one agent to be administered, for example, within 1 minute, 5 minutes or 10 minutes after the other.

[0212] As described herein, we have identified a new treatment for patients diagnosed with heart failure. Heart Failure is a clinical syndrome diagnosed from clinical symptoms andsigns, one or more diagnostic tests / investigation, or a combination thereof. For example, a diagnosis of heart failure can be based on clinical features in combination with a measurement of BNP or NTproBNP via a blood test and an echocardiogram. The major clinical symptoms, signs and investigations used to diagnose heart failure include, but are not limited to:

[0213] Clinical symptoms: breathlessness (aka dyspnoea), dyspnoea on exertion, orthopnoea, paroxysmal nocturnal dyspnoea, reduced exercise capacity, ankle swelling

[0214] Clinical signs: peripheral oedema, fluid overload, pulmonary rales, gallop cardiac rhythm, raised jugular venous pressure (in more advanced HF then also hepatomegaly, anasarca, ascites)

[0215] Investigations: (A) Echocardiogram / cardiac MRI / MUGA: left ventricular ejection fraction, left ventricular end diastolic volume, left ventricular end systolic volume, left ventricular mass, left ventricular strain, left ventricular adverse remodeling, myocardial fibrosis (detected using late gadolinium enhancement). For HFpEF markers of diastolic dysfunction / elevated filling pressures are used, particularly E / e’, left atrial volume index (LAVI), TR Vmax (estimated PASP); (B) Blood tests: NTproBNP, BNP, soluble ST2, troponin-T, troponin-I; (C) Chest x-ray: pulmonary congestion, increased cardiothoracic ratio / cardiomegaly, pleural effusions, pulmonary interstitial oedema, curly B-lines; (D) For HFpEF, sometimes cardiac catheterization is used to measure LV filling pressures and left atrial pressure (often referred to as Pulmonary Capillary Wedge Pressure).

[0216] In embodiments, heart failure can be diagnosed in patients with evidence of LV dysfunction and / or adverse cardiac remodeling even in the absence of / minimal clinical signs and symptoms. In this case, for example, LV dysfunction can be determined by imaging revealing reduced LVEF, abnormal LV strain, increased LVEDV and / or LVESV (adverse remodeling) accompanied by increased BNP or NTproBNP even if they have no / minimal symptoms.

[0217] In embodiments, heart failure can be further subcharacterised by LVEF, in patients with symptoms and / or signs. These are characterized as: HFrEF: LVEF ≤40%, HFmEF: LVEF 41-49%, HFpEF: LVEF ≥ 50%, and evidence of structural or functional abnormalities consistent with the presence of LV diastolic dysfunction or raised LV filing pressures plus raised natriuretic peptides (BNP or NTproBNP). See, for example, (1) McDonagh, T. A.; Metra, M.; Adamo, M.; Gardner, R. S.; Baumbach, A.; Böhm, M.; Burri, H.; Butler, J.; Čelutkienė, J.; Chioncel, O.; Cleland, J. G. F.; Coats, A. J. S.; Crespo-Leiro, M. G.; Farmakis, D.; Gilard, M.; Heymans, S.; Hoes, A. W.; Jaarsma, T.; Jankowska, E. A.; Lainscak, M.;Lam, C. S. P.; Lyon, A. R.; McMurray, J. J. V.; Mebazaa, A.; Mindham, R.; Muneretto, C.; Piepoli, M. F.; Price, S.; Rosano, G. M. C.; Ruschitzka, F.; Skibelund, A. K.; . 2021 ESC Guidelines for the Diagnosis and Treatment of Acute and Chronic Heart Failure. Eur Heart J 2021, 42 (36), 3599–3726. https: / / doi.org / 10.1093 / eurheartj / ehab368.

[0218] The criteria used to diagnose early heart failure include, but are not limited to, the symptoms, clinical signs and / or investigations that are included in the American College of Cardiology / American Heart Association / Heart Failure Society of America (ACC / AHA / HFSA) definition of Stage B heart failure, or Stage C heart failure diagnosed within the previous 12 months and with symptoms at New York Heart Association (NYHA) class I or II.

[0219] The criteria used to diagnose established heart failure include, but are not limited to, the symptoms, clinical signs and investigations that are included in the ACC / AHA / HFSA definition of Stage C heart failure with symptoms at NYHA class II and diagnosed greater than 12 months previously, or Stage C heart failure with symptoms at NYHA class III or IV, or Stage D heart failure. See, for example, Heidenreich PA, Bozkurt B, Aguilar D, Allen LA, Byun JJ, Colvin MM, Deswal A, Drazner MH, Dunlay SM, Evers LR, Fang JC, Fedson SE, Fonarow GC, Hayek SS, Hernandez AF, Khazanie P, Kittleson MM, Lee CS, Link MS, Milano CA, Nnacheta LC, Sandhu AT, Stevenson LW, Vardeny O, Vest AR, Yancy CW. 2022 AHA / ACC / HFSA guideline for the management of heart failure: a report of the American College of Cardiology / American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation. 2022;145:e895-e1032.

[0220] The present inventors have surprisingly discovered a previously unknown role of Chrdl1 in improving cardiac function in heart failure. For example, Chrdl1 effectively improves LV ejection fraction after intravenous injection of viral vectors expressing these factors.

[0221] Cardiac remodeling may be reduced by the method of the invention. Cardiac remodeling may refer to a group of molecular, cellular and interstitial changes that manifest clinically as changes in size, mass, geometry and function of the heart after injury and may be determined by any suitable method, for example by changes in the cavity diameter, mass (hypertrophy and atrophy), geometry (heart wall thickness and shape), areas of scar, fibrosis and inflammatory infiltrate (see e.g. Azevedo, P.S., et al., 2015. Arquivos brasileiros de cardiologia, 106, pp.62-69).

[0222] The subject’s LV ejection fraction may be improved by the method of the invention. Left ventricular ejection fraction (LVEF) may refer to the fraction of chambervolume ejected in systole in relation to the volume of the blood in the ventricle at the end of diastole and may be determined by any suitable method, for example by echocardiography, magnetic resonance imaging (MRI), or computed tomography (CT). LV ejection fraction can be increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared with an untreated subject. In embodiments, the LV ejection fraction is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared with the subject prior to treatment. In embodiments, the subject’s LV ejection fraction prior to treatment is about 40% or less, about 45% or less, or less than about 50%. In embodiments, the LV ejection fraction is increased to at least about 50%, about 55%, or about 60%.

[0223] The subject’s left ventricular volume may be reduced by the method of the invention. LV volume may be determined by any suitable method, for example by echocardiography, magnetic resonance imaging (MRI), or computed tomography (CT). In embodiments, the LV volume is the end-diastolic volume (EDV) or the end-systolic volume (ESV). LV volume can be decreased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared with an untreated subject. In embodiments, the LV volume is decreased by at least about 5%, about 10%, about 15%, or about 20% when compared with the subject prior to treatment.

[0224] In embodiments, the subject has myocardial fibrosis. Myocardial fibrosis may be defined as the expansion of the cardiac interstitium due to net accumulation of extracellular matrix proteins, and may arise as a result of cardiac diseases (see e.g. Frangogiannis, N.G., 2021. Cardiac fibrosis. Cardiovascular research, 117(6), pp.1450- 1488). The presence and extent of myocardial fibrosis may be determined by any suitable method, for example by endomyocardial biopsy (EMB) histopathology or multiparametric cardiac magnetic resonance (CMR) imaging (see e.g. Abecasis, J., et al., 2023. Cardiovascular Pathology, 65, p.107541 and Gräni, C., et al., 2019. Journal of Cardiovascular Magnetic Resonance, 21(1), p.14.). In embodiments, the subject has a myocardial fibrotic content of at least about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In embodiments, the myocardial fibrosis is reduced is reduced by the method of the invention.

[0225] Further, Chrdl1 improves heart function in heart failure while having no significant impact on scar size after intravenous injection of viral vectors expressing thesefactors. Once myocardium dies during a heart attack, it is replaced by scar tissue over the course of several weeks. Scar tissue does not have the elasticity and flexibility of healthy heart muscle, and there can be complications with pumping and transporting blood. Myocardial scaring can be detected in subjects with both recognized myocardial infarction (RMI) and unrecognized myocardial infarction (UMI). Accordingly, aspects of the invention are drawn to methods of treating heart failure in subjects with myocardial scarring but no clinical myocardial infarction, and methods of treating heart failure in subjects with myocardial scarring and clinical myocardial infarction. See, for example, Barbier, Charlotte Ebeling, et al. "Myocardial scars more frequent than expected: magnetic resonance imaging detects potential risk group." Journal of the American College of Cardiology 48.4 (2006): 765-771.

[0226] Variants, derivatives, analogues, homologues and fragment.

[0227] In addition to the specific proteins and nucleotides mentioned herein, the invention also can encompass the use of variants, derivatives, analogues, homologues and fragments thereof.

[0228] In the context of the invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains its function. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring protein.

[0229] The term “derivative” as used herein, in relation to proteins or polypeptides of the invention can include any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its endogenous functions.

[0230] The term “analogue” as used herein, in relation to polypeptides or polynucleotides can include any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.

[0231] In embodiments, amino acid substitutions can be made, for example, from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence substantially retains the required activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogues.

[0232] Proteins used in the invention can also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0233] Conservative substitutions can be made, for example according to the table below. Amino acids in the same block in the second column and in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R H AROMATIC F W Y

[0234] The term “homologue” as used herein can refer to an entity having a certain homology with the wild type amino acid sequence and the wild type nucleotide sequence. The term “homology” can be equated with “identity”.

[0235] A homologous sequence can include an amino acid sequence which may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical, such as at least 90% or 95% or 97% or 99% identical to the subject sequence. In embodiments, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the invention homology can be expressed in terms of sequence identity.

[0236] A homologous sequence can include a nucleotide sequence which can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical, such as at least 90% or 95% or 97% or 99% identical to the subject sequence. Although homology can also beconsidered in terms of similarity, in the context of the invention it is preferred to express homology in terms of sequence identity.

[0237] In embodiments, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein can refer to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0238] Homology comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences.

[0239] Percentage homology can be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence 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 an “ungapped” alignment. In embodiments, such ungapped alignments can be performed only over a relatively short number of residues.

[0240] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence can cause the following codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximize local homology.

[0241] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties can produce optimized alignments with fewer gaps. Most alignment programs can allow the gap penalties to be modified. However, one can use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0242] Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out 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 that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, one can use the GCG Bestfit program. Another tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0243] Although the final percent homology can be measured in terms of identity, the alignment process itself may not be based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix can be used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if 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 in the case of other software, the default matrix, such as BLOSUM62.

[0244] Once the software has produced an optimal alignment, one can calculate percent homology, such as percent sequence identity. The software may not do this as part of the sequence comparison and generates a numerical result.

[0245] “Fragments” of full length Chrdl1, such as those fragments described herein, are also variants and the term can refer to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus can refer to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0246] Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions can be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site can be made. The flanking regions can contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so thatthe sequence can be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0247] The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0248] Features and embodiments of the invention will now be described by way of non-limiting examples.

[0249] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, 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 & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & 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. Each of these general texts is herein incorporated by reference. EXAMPLES

[0250] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

[0251] Example 1: Efficacy of Chrdl1 in improving heart function when given in early heart failure.

[0252] Based on the performance of Chrdl1 as a cardioprotective molecule during myocardial infarction (MI) we decided to characterize the effect of Chrdl1 when given afterMI. An individual AAV9 vector expressing Chrdl1 was used with the specific purpose to assess the capacity of the factor to improve heart function when given after MI and the initial cardiac scar formation.

[0253] Eight to twelve week-old CD1 mice were subjected to MI or a surgical sham procedure and cardiac function was assessed using echocardiography at 7 days after MI. In this work MI was induced by permanent ligation of the left descending coronary artery, sham animals received a thoracotomy which did not occlude a major vessel. Animals subjected to MI were randomized according to the ejection fraction at this time point and were injected with AAV2 / 9 vectors expression Chrdl1, a control empty vector or left untreated (1x1012vg / animal; n=6-7 per group; n=4 sham group). Our previous experience indicates that this procedure results in efficient transgene expression, by using a ubiquitous promoter, CMV, that drives expression in many tissues allowing both local heart expression and systemic expression through transduction of the liver and other tissues. Transduction was confirmed by quantitative PCR in heart and liver. Cardiac function of the animals was further monitored by echocardiography at 40 days post MI.

[0254] As reported in FIG. 1, AAV2 / 9-mediated overexpression of Chrdl1 successfully improved cardiac function of infarcted mice when compared to control animals 35 days after MI, whereas prior to treatment there was no significant difference between control and Chrdl1 over-expressing animals. Infarcted animals show a significant reduction in EF% if compared with sham animals at 7days post MI (RM 2-way ANOVA - Bonferroni's multiple comparison test; Sham: 57% + / - 1.7, n=4; MCS: 41.4% + / - 5.19, n=6; p<0.05).). At day 7 post MI the animals that subsequently received the control and Chrdl 1 vector showed no significant difference in baseline infarct size. When the same animals were reassessed at 28 days following injection (35 days post-MI) a significantly higher %LVEF was seen in the Chrdl1 vector treated group vs the control vector treated group (RM 2-way ANOVA - Bonferroni's multiple comparison test; MCS: 36.8% + / - 4.41, n=6; Chrdl1: 57.53% + / - 2.8, n=7; p<0.001).

[0255] When myocardial infarction occurs, cardiac fibroblasts proliferate, differentiate into myofibroblasts and produce extracellular matrix to create a scar to replace the gap generated by cardiomyocyte loss. This often results in myocardial stiffness and leads to pathological remodeling of the left ventricle, dilatation and dysfunction. Morphometric analysis on trichromic-stained heart sections at day 35 post injection indicated that AAV2 / 9- Chrdl1-treated mice showed a non-significant reduction of fibrotic area (Infarct size: unpaired t-test; MCS: 38.39% + / - 7.16, n=5; Chrdl1: 29.9% + / - 6, n=6 p>0.05).

[0256] Finally, assessment of heart weight / tibial length gives an assessment of heart size and can be used to detect cardiac remodeling and hypertrophy. Chrdl1 treated animals showed a non-significant lower heart weight / tibial length compared to control vector treated animals (unpaired t-test; MCS: 10.2 + / - 0.79, n=6; Chrdl1: 8.2 + / - 0.65, n=7; p>0.05).

[0257] Taken together, these results indicate that the AAV2 / 9-mediated overexpression of Chrdl1 after acute ischemia is sufficient to improve cardiac function, even without a significant impact on infarct size.

[0258]

[0259] Example 2: Lack of efficacy of cardio protectants Fam3b and Fam3c when given in early heart failure.

[0260] Fam3b and Fam3C were also selected from N in vivo functional screen to identify new therapeutic factors against degenerative conditions, which was applied to identify factors ensuring cardiac protection. Fam3b and Fam3c were identified as having a similar cardioprotective capacity and some shared modes of actions such as on autophagy.11

[0261] AAV2 / 9 Vectors expressing Fam3b and Fam3c or a control empty vector were used in a similar study to assess if these factors also have benefit when given 7 days after MI. Eight to twelve week-old CD1 mice were subjected to MI or a surgical sham procedure and cardiac function was assessed using echocardiography at 7 days after MI. At this timepoint mice subjected to MI were injected with AAV2 / 9 vectors expressing Fam3b or Fam3c, a control empty vector or left untreated (1x1012vg / animal; n=8-9 per treatment group; n=3 sham group). Our previous experience indicates that this procedure results in efficient transgene expression. This data was confirmed by quantitative PCR on transduced tissue showing stable transgene overexpression Cardiac function of the animals was further monitored by echocardiography at 35 days post MI.

[0262] As reported in FIG. 2, AAV2 / 9-mediated overexpression of Fam3b or Fam3b had no significant effect on cardiac function of infarcted mice when compared to control animals 35 days after MI, with baseline cardiac function prior to treatment also showing no significant difference between control and Fam3b of Fam3c over-expressing animals. Infarcted animals show a significant reduction in LVEF% if compared with sham animals at 7days post MI (RM 2-way ANOVA - Bonferroni's multiple comparison test; Sham: 57% + / - 3.7, n=3; MCS: 34% + / - 3.1, n=8; p<0.001). At day 7 post MI the animals that subsequently received the control, Fam3b or Fam3c vector showed no significant difference in baseline infarct size. When the same animals were reassessed at 21 days following injection (35 days post-MI) animals continued to show no differences in %LVEF between the Fam3b- orFam3c-vector treated group vs the control vector treated group.

[0263] Morphometric analysis on trichromic-stained heart sections at day 35 post injection indicated that AAV2 / 9-Fam3b or Fam3c-treated mice had no reduction in fibrotic area (Infarct size: Ordinary one-way ANOVA – Dunnett’s multiple comparison test; MCS: 32.5% + / - 4.8, n=6; Fam3c: 30.3% + / - 2.9, n=6; Fam3b: 31.6% + / - 3.57, n=6; p>0.05).

[0264] In keeping with the lack of impact on MI scar size, the heart weight / tibial length assessment of heart size was also not significantly different from control in the Fam3b- of Fam3c-treated animals (Ordinary one-way ANOVA – Dunnett’s multiple comparison test; MCS: 12.5 + / - 0.42, n=8; Fam3c: 12.59 + / - 0.73, n=8; Fam3b: 12.32 + / - 0.92, n=9; p>0.05) compared to control vector treated animals.

[0265] Taken together, these results indicate that the AAV2 / 9-mediated overexpression of Fam3b or Fam3c after acute ischemia is not sufficient to improve cardiac function or infarct size. This indicates that having cardioprotective biology at the time of infarct does not predict the ability of proteins to treat ongoing heart failure.

[0266]

[0267] Examples 3: Efficacy of Chrdl1 in improving heart function when given in established heart failure.

[0268] The left anterior descending coronary artery ligation model is an irreversible ischemic model which generates large infarct in mice. Mice are resistant to developing terminal HF and thus can maintain cardiac function for a number of week following a large infarct.2This allows an opportunity to investigate long-term post-MI remodeling and heart failure.

[0269] Based on the performance of Chrdl1 when given 7 days after infarct we tested whether the improvements in heart function were still seen when Chrdl1 was expressed 14 days after MI.

[0270] Eight to twelve week-old CD1 mice were subjected to MI or a surgical sham procedure and cardiac function was assessed using echocardiography at 18 days after MI. At this timepoint mice subjected to MI were randomized and injected with AAV2 / 9 vectors expression Chrdl1, a control empty vector or left untreated (1x1012vg / animal; n=6-7 per group). Our previous experience indicates that this procedure results in efficient transgene expression. Cardiac function of the animals was further monitored by echocardiography at 50 days post MI.

[0271] As reported in FIG. 3, AAV2 / 9-mediated overexpression of Chrdl1 successfully improved cardiac function of infarcted mice when compared to control animals 42 days afterMI, whereas prior to treatment there was no significant difference between control and Chrdl1 over-expressing animals. Infarcted animals show a significant reduction in LVEF% if compared with sham animals at 14days post MI (RM 2-way ANOVA - Bonferroni's multiple comparison test; Sham: 57% + / - 1.7, n=4; MCS: 31% + / - 4.46, n=6; p<0.001).). At day 14 post MI the animals that subsequently received the control and Chrdl1 vector showed no significant difference in baseline infarct size. When the same animals were reassessed at 28 days following injection (42 days post-MI) a significantly higher %LVEF was seen in the Chrdl1 vector treated group vs the control vector treated group (RM 2-way ANOVA - Bonferroni's multiple comparison test; MCS: 29.7% + / - 4.12, n=6; Chrdl1: 48.8% + / - 1.7, n=7; p<0.001).

[0272] Strikingly morphometric analysis on trichromic-stained heart sections at day 50 post injection indicated that AAV2 / 9-Chrdl1-treated mice showed no significant difference to control animals in scar size (Infarct size: unpaired t-test; MCS: 28% + / - 3.7, n=6; Chrdl1: 22.8% + / - 2.75, n=7; p>0.05).

[0273] The changes in LVEF indicative of altered heart remodeling after formation of the initial MI scar, which was unchanged by treatment in this study. This conclusion was further supported by the assessment of heart weight / tibial length which indicated that Chrdl1 treated animals showed a significantly lower heart weight / tibial length compared to control vector treated animals (unpaired t-test; MCS: 11.56 + / - 0.72, n=6; Chrdl1: 8.7 + / - 0.64, n=7; p<0.05). This data is supportive of reduced post-MI hypertrophy in the Chrdl1-treated animals.

[0274] Taken together, these results indicate that surprisingly in addition to the protective biology of Chrdl1 at the time of MI it also has a striking ability to improve heart function independent of any effect of MI scar size.

[0275]

[0276] Example 4: Lack of efficacy of cardioprotectants Fam3b and Fam3c when given in established heart failure

[0277] We further confirmed that this property of Chrdl1 is surprising and not a result of the FunSel selection by testing the AAV2 / 9 Vectors expressing Fam3b and Fam3c or a control empty vector in a similar study.

[0278] Eight to twelve week-old CD1 mice were subjected to MI or a surgical sham procedure and cardiac function was assessed using echocardiography at 14 days after MI. At this timepoint mice subjected to MI were injected with AAV2 / 9 vectors expressing Fam3b or Fam3c, a control empty vector or left untreated (1x1012 vg / animal; n=8-9 per treatmentgroup; n=3 sham group). Our previous experience indicates that this procedure results in efficient transgene expression. Cardiac function of the animals was further monitored by echocardiography at 35 days post MI.

[0279] As reported in FIG. 4, AAV2 / 9-mediated overexpression of Fam3b or Fam3b had no significant affect on cardiac function of infarcted mice when compared to control animals 42 days after MI, with baseline cardiac function prior to treatment also showing no significant difference between control and Fam3b of Fam3c over-expressing animals. Infarcted animals show a significant reduction in LVEF% if compared with sham animals at 14days post MI (RM 2-way ANOVA - Bonferroni's multiple comparison test; Sham: 53.4% + / - 0.51, n=3; MCS: 30.8% + / - 2, n=8; p<0.001).). At day 14 post MI the animals that subsequently received the control, Fam3b or Fam3c vector showed no significant difference in baseline infarct size. When the same animals were reassessed at 28 days following injection (42 days post-MI) animals continued to show no differences in %LVEF between the Fam3b- or Fam3c-vector treated group vs the control vector treated group.

[0280] Morphometric analysis on trichromic-stained heart sections at day 42 post injection indicated that AAV2 / 9-Fam3b or Fam3c-treated mice had no reduction in fibrotic area (Infarct size: Ordinary one-way ANOVA – Dunnett’s multiple comparison test; MCS: 28.6% + / - 3.7, n=5; Fam3c: 31.4% + / - 5.9, n=5; Fam3b: 24.2% + / - 0.8, n=5; p>0.05).

[0281] In keeping with the lack of impact on MI scar size, the heart weight / tibial length assessment of heart size was also not significantly different from control in the Fam3b- of Fam3c-treated animals (Ordinary one-way ANOVA – Dunnett’s multiple comparison test; MCS: 14.3 + / - 0.9, n=6; Fam3c: 14.1 + / - 0.72, n=6; Fam3b: 14.2 + / - 0.75, n=6; p>0.05) compared to control vector treated animals.

[0282] Taken together, these results indicate that the AAV2 / 9-mediated overexpression of Fam3b or Fam3c after acute ischemia is not sufficient to improve cardiac function or infarct size. This indicates that having cardioprotective biology at the time of infarct does not predict the ability of proteins to treat ongoing heart failure and indicates the discovery of the ability of Chrdl1 to improve heart function when delivered 7 or 14 days after MI is a surprising and new finding.

[0283] (1) Ruozi, G.; Bortolotti, F.; Mura, A.; Tomczyk, M.; Falcione, A.; Martinelli, V.; Vodret, S.; Braga, L.; Ferro, M. D.; Cannatà, A.; Zentilin, L.; Sinagra, G.; Zacchigna, S.; Giacca, M. Cardioprotective Factors against Myocardial Infarction Selected in Vivo from an AAV Secretome Library. Sci Transl Med 2022, 14 (660), . https: / / doi.org / 10.1126 / scitranslmed.abo0699.

[0284] (2) Martin, T. P.; MacDonald, E. A.; Elbassioni, A. A. M.; O’Toole, D.; Zaeri, A. A. I.; Nicklin, S. A.; Gray, G. A.; Loughrey, C. M. Preclinical Models of Myocardial Infarction: From Mechanism to Translation. Br J Pharmacol 2022, 179 (5), 770–791. https: / / doi.org / 10.1111 / bph.15595. ***** EQUIVALENTS

[0285] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

WE CLAIM:

1. A Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor, for use in a method of treating heart failure.

2. A Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefore, for use in a method of improving cardiac function in heart failure.

3. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 1 or 2, wherein heart failure is early or established heart failure.

4. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 1 or 2, wherein the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

5. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 1 or 2, wherein the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

6. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 1 or 2, wherein the Chrdl1 protein consists of an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

7. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 1 or 2, wherein the polynucleotide consists of a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

8. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 1 or 2, wherein the heart failure is associated with cardiac ischemia or loss of cardiomyocytes.

9. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefore, for use according to claim 1 or 2, wherein the heart failure is selected from the consequences of myocardial infarction; the reperfusion-injury after percutaneous coronary interventions, thrombolysis, or coronary artery bypass surgery, chronic cardiac ischemia; myocarditis; hypertension; cardiac toxic or metabolic damage; or cardiomyopathy.

10. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 9, wherein the percutaneous coronary intervention is coronary angioplasty.

11. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim p, wherein the cardiac toxic damage is caused by chemotherapy.

12. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any one of claims 1-11, wherein cardiac remodeling is reduced, LV ejection fraction is improved, left ventricular volume is reduced, and / or myocardial fibrosis is reduced.

13. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any of claims 1-12, wherein the LV ejection fraction is increased to at least about 50%.

14. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any of claims 1-13, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefore, is administered to a subject having myocardial fibrosis.

15. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any of claims 1-14, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefore, is administered to a subject one week or more after cardiomyocyte loss.

16. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any of claims 1-15, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefore, is administered to a subject one week or more after myocardial infarction.

17. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any one of claims 1-16, wherein the Chrdl1 protein or fragment thereof is comprised in a fusion protein.

18. The Chrdle1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 17, wherein the fusion protein is an Fc fusion protein.

19. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any one of claims 1-17, wherein the polynucleotide is in the form of a vector.

20. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 19, wherein the vector comprises a viral vector.

21. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 20 wherein the viral vector comprises an adeno-associated viral (AAV) vector.

22. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to any one of claims 1-21, wherein the protein or polynucleotide encoding the protein is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient.

23. The Chrdl1 protein or fragment thereof, or a polynucleotide encoding therefor, for use according to claim 22, wherein the pharmaceutical composition is formulated for injection.

24. A vector comprising a polynucleotide encoding a Chrdl1 protein or a fragment thereof for use in a method of treating heart failure.

25. The vector for use according to claim 24, wherein the heart failure is early or established heart failure.

26. The vector for use according to claim 24, wherein the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

27. The vector for use according to claim 24, wherein the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

28. The vector for use according to claim 24, wherein the vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.

29. The vector for use according to claim 24, wherein the vector is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient.

30. The vector for use according to claim 29, wherein the pharmaceutical composition is formulated for injection.

31. A method of treating heart failure, the method comprising administering to a subject a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor.

32. A method of improving cardiac function in heart failure, the method comprising administering to a subject a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor.

33. The method according to claim 31 or 32, wherein the heart failure is early heart failure or established heart failure.

34. The method according to claim 31 or 32, wherein the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

35. The method according to claim 31 or 32, wherein the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

36. The method according to claim 31 or 32, wherein the Chrdl1 protein consists of an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

37. The method according to claim 31 or 32, wherein the polynucleotide consists of a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

38. The method according to claim 31 or 32, wherein the heart failure is associated with cardiac ischemia or loss of cardiomyocytes.

39. The method according to claim 31 or 32, wherein the heart failure is selected from the consequences of myocardial infarction; the reperfusion-injury after percutaneous coronary interventions, thrombolysis, or coronary artery bypass surgery, chronic cardiac ischemia; myocarditis; hypertension; cardiac toxic or metabolic damage; or cardiomyopathy.

40. The method according to claim 39, wherein the percutaneous coronary intervention is coronary angioplasty.

41. The method of claim 39, wherein the cardiac toxic damage is caused by chemotherapy.

42. The method according to any one of claims 31-41, wherein cardiac remodeling is reduced, LV ejection fraction is improved, left ventricular volume is reduced, and / or myocardial fibrosis is reduced.

43. The method according to any one of claims 31-42, wherein the LV ejection fraction is increased to at least about 50%.

44. The method according to any one of claims 31-43, wherein the subject has myocardial fibrosis.

45. The method according to any one of claims 31-44, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to the subject one week or more after cardiomyocyte loss.

46. The method according to any one of claims 31-45, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to the subject one week or more after myocardial infarction.

47. The method according to any one of claims 31-46, wherein the Chrdl1 protein or fragment thereof is comprised in a fusion protein.

48. The method according to claim 47, wherein the fusion protein is an Fc fusion protein.

49. The method according to any one of claims 31-48, wherein the polynucleotide is in the form of a vector.

50. The method of claim 49, wherein the vector comprises a viral vector.

51. The method of claim 50, wherein the viral vector comprises an adeno-associated viral (AAV) vector.

52. The method according to any one of claims 31-51, wherein the protein or polynucleotide encoding the protein is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient.

53. The method according to claim 52, wherein the pharmaceutical composition is formulated for injection.

54. A method of treating early or established heart failure, the method comprising administering to a subject a vector, wherein the vector comprises a polynucleotide encoding a Chrdl1 protein or a fragment thereof.

55. The method according to claim 54, wherein the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

56. The method according to claim 54, wherein the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

57. The method according to claim 54, wherein the vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.

58. The method according to claim 54, wherein the vector is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient.

59. The method according to claim 58, wherein the pharmaceutical composition is formulated for injection.

60. Use of a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor, in the manufacture of a medicament for the treatment of heart failure.

61. Use of a Chrdl1 protein or a fragment thereof, or a polynucleotide encoding therefor, in the manufacture of a medicament for improving cardiac function in heart failure.

62. The use according to claim 60 or 61, wherein heart failure is early or established heart failure.

63. The use according to claim 60 or 61, wherein the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

64. The use according to claim 60 or 61, wherein the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

65. The use according to claim 60 or 61, wherein the Chrdl1 protein consists of an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

66. The use according to claim 60 or 61, wherein the polynucleotide consists of a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

67. The use according to claim 60 or 61, wherein the heart failure is associated with cardiac ischemia or loss of cardiomyocytes.

68. The use according to claim 60 or 61, wherein the heart failure is selected from the consequences of myocardial infarction; the reperfusion-injury after percutaneous coronary interventions, thrombolysis, or coronary artery bypass surgery, chronic cardiac ischemia; myocarditis; hypertension; cardiac toxic or metabolic damage; or cardiomyopathy.

69. The use according to claim 68, wherein the percutaneous coronary intervention is coronary angioplasty.

70. The use according to claim 68, wherein the cardiac toxic damage is caused by chemotherapy.

71. The use according to any one of claims 60-70, wherein cardiac remodeling is reduced, LV ejection fraction is improved, left ventricular volume is reduced, and / or myocardial fibrosis is reduced.

72. The use according to any of claims 60-71, wherein the LV ejection fraction is increased to at least about 50%.

73. The use according to any of claims 60-72, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject having myocardial fibrosis.

74. The use according to any of claims 60-73, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject one week or more after cardiomyocyte loss.

75. The use according to any of claims 60-74, wherein the Chrdl1 protein or fragment thereof, or the polynucleotide encoding therefor, is administered to a subject one week or more after myocardial infarction.

76. The use according to any one of claims 60-75, wherein the Chrdl1 protein or fragment thereof is comprised in a fusion protein.

77. The use according to claim 76, wherein the fusion protein is an Fc fusion protein.

78. The use according to any one of claims 60-77, wherein the polynucleotide is in the form of a vector.

79. The use of claim 78, wherein the vector comprises a viral vector.

80. The use of claim 79, wherein the viral vector comprises an adeno-associated viral (AAV) vector.

81. The use according to any one of claims 60-80, wherein the protein or polynucleotide encoding the protein is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient.

82. The use according to claim 81, wherein the pharmaceutical composition is formulated for injection.

83. Use of a vector comprising a polynucleotide encoding a Chrdl1 protein or a fragment thereof in the manufacture of a medicament for the treatment of early or established heart failure.

84. The use according to claim 83, wherein the Chrdl1 protein comprises an amino acid sequence that has at least 70% identity to any of SEQ ID NOs: 4, 1, 5 or 6.

85. The use according to claim 83, wherein the polynucleotide comprises a nucleotide sequence that has at least 70% identity to any of SEQ ID NOs: 2, 3 or 7.

86. The use according to claim 83, wherein the vector is a viral vector, optionally herein the viral vector is an adeno-associated viral (AAV) vector.

87. The use according to claim 83, wherein the vector is provided as a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and / or excipient.

88. The use according to claim 87, wherein the pharmaceutical composition is formulated for injection.

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