Proteins with cardioprotective activity

Abstract

A protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the protein, for use in treating heart disease or reducing the risk of heart disease.

Description

Field of the Invention

[0001] The present invention relates to the use of Chrdl1, Fam3c and Fam3b as drugs, for example in the context of gene therapy or by administration as a protein (such as a recombinant or synthetic protein), for treating heart disease or reducing the risk of heart disease. In particular, the present invention relates to protecting the heart from developing heart failure (HF) by maintaining the viability of cardiomyocytes. Conditions for which the drug is effective include, but are not limited to, cardiac ischemia (myocardial infarction and reperfusion injury), cardiotoxic damage and cardiomyopathies of genetic origin. Background of the Invention

[0003] Despite recent advances in cardiovascular surgery and therapy, cardiovascular disorders (CVD) still account for approximately 30% of deaths worldwide, of which approximately 50% are caused by ischemic heart disease, according to the World Health Organization (WHO). www.who.int / cardiovascular_ diseases / en / ), with a trend of increasing to more than 23 million by 2030. Coronary artery disease and myocardial infarction are the main causes of heart failure (65-70% of cases). The prognosis of this condition remains poor, with an estimated mortality rate of 40% at 4 years from diagnosis (Owan, TE, et al. Trends in prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med 355, 251-259 (2006)). An essential component of the epidemic burden of HF is the inability of the myocardium to undergo regeneration in adulthood. Cardiac damage caused by ischemia, hypertension, infection, inflammation or toxic insults usually leads to irreversible loss of cardiomyocytes (CMs), resulting in fibrosis and scar formation (Laflamme, MA & Murry, CE Heart regeneration. Nature 473, 326-335 (2011); Xin, M., Olson, EN & Bassel-Duby, R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat Rev Mol Cell Biol 14, 529-541 (2013)).

[0004] Since the mid-1990s, drug development in this field has been marginal in terms of efficacy, and all available drugs are small chemical molecules. If the current ESC guidelines for chronic HF with reduced ejection fraction are considered (Ponikowski, P., et al. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Eur J Heart Fail 18, 891-975 (2016)), the three drugs recommended for all patients (ACE inhibitors, beta-blockers and mineralocorticoid / aldosterone receptor antagonists) have all been introduced in clinical practice in the 1970s or earlier (Gavras, H., Faxon, DP, Berkoben, J., Brunner, HR & Ryan, TJ Angiotensin converting enzyme inhibition in patients with congestive heart failure. Circulation 58, 770-776 (1978); Swedberg, K., Hjalmarson, A., Waagstein, F. & Wallentin, I. Prolongation of survival in congestive cardiomyopathy by beta-receptor blockade. Lancet 1, 1374-1376 (1979); Goldberger, E. Aldosterone and the Edema of Congestive Heart Failure. Am J Cardiol 15, 274 (1965)); Among the drugs recommended only for specific patients, angiotensin II receptor blockers-ARBs can be traced back to the mid-1990s (Gottlieb, SS, et al. Hemodynamic and neurohormonal effects of the angiotensin II antagonist losartan in patients with congestive heart failure.Circulation 88, 1602-1609 (1993)), and the more recent LCZ6969 is based on the combination of an old ARB (valsartan) and sacubitril, which belongs to the neprilysin inhibitor class, which was also developed in the late 1980s (Jhund, PS & McMurray, JJ The neprilysin pathway in heart failure: a review and guide on the use of sacubitril / valsartan. Heart 102, 1342-1347 (2016)). The last drug in the recommendation, ivabradin, is an I. f Channel inhibitors were also developed in the mid-1990s (Thollon, C., et al. Electrophysiological effects of S 16257, a novel sino-atrial node modulator, on rabbit and guinea-pig cardiac preparations: comparison with UL-FS 49. Br J Pharmacol 112, 37-42 (1994)).

[0005] Biochemical studies have shown that candidate biological factors for these pathologies (e.g., relaxin, natriuretic peptides, AVP antagonists) have all failed in phase III clinical trials (Teerlink, JR, et al. Serelaxin in addition to standard therapy in acute heart failure: rationale and design of the RELAX-AHF-2 study. Eur J Heart Fail 19, 800-809 (2017); O'Connor, CM, et al. Effect of nesiritide in patients with acute decompensated heart failure. N Engl J Med 365, 32-43 (2011); Matsuzaki, M., Hori, M., Izumi, T. & Fukunami, M. Efficacy and safety of tolvaptan in heart failure patients with volume overload despite the standard treatment with conventional diuretics: a phase III, randomized, double-blind, placebo-controlled study (QUEST study). Cardiovasc Drugs Ther 25Suppl 1,S33-45(2011);Wang,G.,et al.Efficacy and Safety of 1-Hour Infusion ofRecombinant Human Atrial Natriuretic Peptide in Patients With AcuteDecompensated Heart Failure:A Phase III,Randomized,Double-Blind,Placebo-Controlled,Multicenter Trial. Medicine (Baltimore) 95, e2947 (2016)).

[0006] In particular, there is no drug or treatment of any kind that protects the heart during acute ischemia and after myocardial infarction. When a patient experiences a myocardial infarction, heart cells gradually die due to the sudden lack of oxygen caused by the blockage of the coronary arteries. If the patient undergoes revascularization (percutaneous coronary intervention, angioplasty) within the first few hours after the infarction, most of the myocardium is spared, but a large number of myocardial cells still undergo irreversible death. Angioplasty itself promotes additional damage due to the sudden flow of oxygen that occurs after blood perfusion is restored. Since contractile myocardial cells cannot regenerate significantly in adults, the part of the myocardium lost is irreversibly repaired by scar formation, which is a major determinant of heart failure in the long run.

[0007] Therefore, there remains a strong need to provide a drug, in particular a biological drug that mimics the endogenous survival process, which can rescue cardiomyocytes immediately after an injury that causes cardiomyocyte loss and subsequent cardiac pathological remodeling. In particular, this need is relevant to the treatment of several conditions that lead to HF, including myocardial infarction, reperfusion injury after angioplasty, cardiotoxic damage caused by cancer chemotherapy, myocarditis, and cardiomyopathies of genetic and non-genetic origin.

[0008] Protection of cardiomyocytes from death would be of great relevance as it would preserve the myocardium and allow long-term maintenance of cardiac integrity and function, thereby avoiding the onset of deterioration in cardiac function leading to HF.

[0009] Despite the lack of curative therapies, remarkable progress has been made in understanding the cellular and molecular mechanisms that lead to tissue degeneration.

[0010] Therefore, there is a need for new biological therapies that can specifically interfere with different mechanisms of disease onset and progression, thereby providing therapeutic opportunities.

[0011] Recombinant insulin since 1982 Since its approval, the number of biotech drugs has increased exponentially in the past three decades. If monoclonal antibodies, enzymes, receptor modulators, subunit vaccines and peptides are considered, there are now more than 350 biotech drugs approved for clinical use, and more than 400 have entered clinical trials (Kinch, MS An overview of FDA-approved biologics medicines. Drug Discov Today 20, 393-398 (2015); Rader, RA (Re) defining biopharmaceuticals. Nat Biotechnol 26, 743-751 (2008)).

[0012] In gene research, some breakthrough discoveries are obtained by screening methods. Since the 1980s, genomic DNA libraries were initially selected by hybridization, and then cDNA was identified by antibody screening in phage libraries to promote gene identification. In the late 1980s and 1990s, functional screening of libraries in cultured cells led to the identification of cell receptors for several oncogenes and animal viruses. Most early methods were based on the use of merged libraries (usually cDNA libraries), in which the desired factors were determined by phenotype-based selection. In the 2000s, with the advancement of robotics, based on the use of array libraries, library screening gradually turned to high-throughput screening (HTS) analysis. HTS not only paves the way for the use of cDNA libraries, but also for the use of peptides, nucleic acids (Eulalio, A., et al. Functional screening identifies miRNAs inducing cardiacregeneration. Nature 492, 376-381 (2012)) and small molecule libraries. Today, advances in gene transfer have allowed the field to move one step further, to screen libraries directly in animals, thus moving from in vitro biochemical or phenotypic selection to true in vivo functional screening. SUMMARY OF THE INVENTION

[0014] The inventors utilized a unique protocol based on in vivo functional selection of factors that exert a desired function (FunSel) to identify factors with cardioprotective effects. The inventors' protocol is based on the use of a library of AAV vectors, which are elegant tools for efficient cardiac gene transfer.

[0015] Notably, factor identification by FunSel does not require that the selected factor function in a given tissue during normal physiology, thereby expanding the universe of potential therapeutic proteins to all secreted factors encoded by the genome.

[0016] The inventors surprisingly discovered that the three factors Chrdl1 (tenascin-like protein 1), Fam3c and Fam3b, despite exerting completely different biological activities, generally share a cardioprotective role.

[0017] The three novel cardioprotective proteins, Chrdl1, Fam3c, and Fam3b, have high homology between mouse and human (93%, 94%, and 79%, respectively).

[0018] These three factors protect against cardiac cell death induced by ischemia and other types of insults, including treatment with chemotherapeutic agents.

[0019] The present invention therefore relates to the factors Chrdl1, Fam3c and Fam3b for use as medicaments.

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

[0021] In another aspect, the present invention provides Chrdl1 or a fragment thereof, or a polynucleotide encoding the same, for use in treating heart disease or reducing the risk of heart disease. In another aspect, the present invention provides Fam3c or a fragment thereof, or a polynucleotide encoding the same, for use in treating heart disease or reducing the risk of heart disease. In another aspect, the present invention provides Fam3b or a fragment thereof, or a polynucleotide encoding the same, for use in treating heart disease or reducing the risk of heart disease.

[0022] In another aspect, the present invention provides a method for treating heart disease or reducing the risk of heart disease, wherein the method comprises administering to a subject in need thereof a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the protein.

[0023] In some embodiments, the use reduces the risk of heart failure. In some embodiments, the risk of heart failure is reduced in a subject with heart disease. In some embodiments, the risk of heart failure is reduced in a subject at risk of heart disease.

[0024] In one aspect, the present invention provides a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the protein, for use in reducing the risk of heart failure.

[0025] On the other hand, the present invention provides Chrdl1 or a fragment thereof, or a polynucleotide encoding the same, for reducing the risk of heart failure. On the other hand, the present invention provides Fam3c or a fragment thereof, or a polynucleotide encoding the same, for reducing the risk of heart failure. On the other hand, the present invention provides Fam3b or a fragment thereof, or a polynucleotide encoding the same, for reducing the risk of heart failure.

[0026] In another aspect, the present invention provides a method for reducing the risk of heart failure, wherein the method comprises administering to a subject in need thereof a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the protein.

[0027] In some embodiments, the risk of heart failure is reduced in a subject with heart disease. In some embodiments, the risk of heart failure is reduced in a subject at risk for heart disease.

[0028] In another aspect, the present invention provides a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the protein, for use in maintaining the viability of cardiomyocytes.

[0029] In another aspect, the present invention provides Chrdl1 or a fragment thereof, or a polynucleotide encoding the same, for maintaining the viability of cardiomyocytes. In another aspect, the present invention provides Fam3c or a fragment thereof, or a polynucleotide encoding the same, for maintaining the viability of cardiomyocytes. In another aspect, the present invention provides Fam3b or a fragment thereof, or a polynucleotide encoding the same, for maintaining the viability of cardiomyocytes.

[0030] In another aspect, the present invention provides a method for maintaining cardiomyocyte viability, wherein the method comprises administering to a subject in need thereof a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the protein.

[0031] In some embodiments, cardiomyocyte viability is preserved in a subject with heart disease. In some embodiments, cardiomyocyte viability is preserved in a subject at risk for heart disease.

[0032] An exemplary amino acid sequence of Chrdl1 is SEQ ID NO: 1. An exemplary nucleotide sequence encoding Chrdl1 is SEQ ID NO:4.

[0033] An exemplary amino acid sequence of Fam3c is SEQ ID NO: 2. An exemplary nucleotide sequence encoding Fam3c is SEQ ID NO:5.

[0034] An exemplary amino acid sequence of Fam3b is SEQ ID NO: 3. An exemplary nucleotide sequence encoding Fam3b is SEQ ID NO:6.

[0035] In some embodiments, the protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

[0036] In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 3.

[0037] In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6.

[0038] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6.

[0039] For example, cardiac disease can be due to myocardial infarction, reperfusion injury after percutaneous coronary intervention (coronary angioplasty), hypertension, cardiotoxic insult (particularly due to cancer chemotherapy), myocarditis, cardiomyopathy of both genetic and non-genetic origin resulting in loss of cardiomyocytes.

[0040] In some embodiments, the heart disease is associated with cardiac ischemia. In some embodiments, the heart disease is associated with loss of cardiomyocytes.

[0041] In some embodiments, the cardiac disease is selected from myocardial infarction; consequences of myocardial infarction; reperfusion injury after percutaneous coronary intervention (coronary angioplasty); myocarditis; hypertension; cardiotoxic insult (particularly caused by cancer chemotherapy); or cardiomyopathy.

[0042] In some embodiments, the cardiac disease is ventricular dysfunction.

[0043] In some embodiments, the heart is protected from myocardial infarction. In some embodiments, cardiac function is preserved after myocardial infarction or percutaneous coronary intervention (coronary angioplasty). In some embodiments, post-infarction fibrosis is reduced.

[0044] In some embodiments, the protein or polynucleotide results in cardioprotection from myocardial infarction and / or other conditions resulting in cardiomyocyte loss, preferably preserving cardiac function and reducing reparative fibrosis.

[0045] In some embodiments, heart failure is prevented.

[0046] In some embodiments, the protein is administered by direct protein delivery.In some embodiments, the polynucleotides are used in gene therapy.

[0047] In some embodiments, the protein is a recombinant protein.

[0048] In some embodiments, the protein is obtained from bacteria, yeast, or mammalian cell culture. In some embodiments, the protein is obtained from Escherichia coli, Pichia pastoris, or Chinese hamster ovary cells.

[0049] In some embodiments, the protein is glycosylated.

[0050] In some embodiments, the protein is a fusion protein. In some embodiments, the protein is an Fc fusion protein.

[0051] In some embodiments, the polynucleotide is in the form of a vector.

[0052] In some embodiments, the polynucleotide is in the form of a viral vector.

[0053] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector. In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector.

[0054] In some embodiments, the vector is an AAV2 vector.

[0055] In some embodiments, the vector is an AAV9 vector.

[0056] In some embodiments, the vector is an AAV8 vector.

[0057] In another aspect, the present invention provides a vector for treating heart disease or reducing the risk of heart disease, wherein the vector comprises a polynucleotide as disclosed herein. In another aspect, the present invention provides a vector for reducing the risk of heart failure, wherein the vector comprises a polynucleotide as disclosed herein. In another aspect, the present invention provides a vector for maintaining the viability of cardiomyocytes, wherein the vector comprises a polynucleotide as disclosed herein.

[0058] In some embodiments, the vector is a viral vector.

[0059] In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector. In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector.

[0060] In some embodiments, the vector is an AAV2 vector.

[0061] In some embodiments, the vector is an AAV9 vector.

[0062] In some embodiments, the vector is an AAV8 vector.

[0063] In some embodiments, the protein is administered parenterally. In some embodiments, the protein is administered intramyocardially. In some embodiments, the polynucleotide is administered parenterally. In some embodiments, the polynucleotide is administered intramyocardially.

[0064] In another aspect, the present invention provides a pharmaceutical composition comprising a protein as disclosed herein and a pharmaceutically acceptable vehicle and / or excipient.

[0065] In another aspect, the present invention provides a pharmaceutical composition comprising a carrier as disclosed herein and a pharmaceutically acceptable vehicle and / or excipient.

[0066] In some embodiments, the composition is formulated for injection.In preferred embodiments, the composition is formulated for intracardiac or intravenous administration.

[0067] In another aspect, the present invention provides a pharmaceutical composition disclosed herein for treating heart disease or reducing the risk of heart disease. In another aspect, the present invention provides a pharmaceutical composition disclosed herein for reducing the risk of heart failure. In another aspect, the present invention provides a pharmaceutical composition disclosed herein for maintaining the viability of cardiomyocytes.

[0068] In another aspect, the present invention provides a protein selected from the group consisting of Chrdl1, Fam3c, Fam3b and fragments thereof, or a polynucleotide encoding the protein, for use as a medicament.

[0069] BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 .FunSel, an in vivo selection protocol for identifying novel cardiac therapeutics for myocardial infarction (MI)

[0071] A. Schematic representation of the pGi vector plasmid used for secretome library generation. B. Overview of the functional selection (FunSel) protocol for identifying cardiac active factors after myocardial infarction (MI). Briefly, a pool of 50 vectors is used to transduce the left ventricle of the heart; each vector enters a different cardiomyocyte. MI is then induced, which kills most cardiomyocytes but not those expressing cardioprotective factors. Three weeks later, barcoded DNA is recovered from the heart by PCR amplification and the frequency of the vectors is determined by next-generation sequencing (NGS). The number of sequencing reads of the vectors with or without infarction is then compared; the enriched factors are those that exert cardioprotective activity. C. Cumulative results obtained from an in vivo competitive screening of 1198 AAV vectors of the library, organized into 24 AAV9 pools, each consisting of 50 inserts of similar size. The frequency of each factor recovered from the heart after infarction (in the MI group, n = 9 mice per pool) is reported as a ratio to the frequency of the same factor in the absence of selective treatment (in the control group, n = 6 mice per pool), as detected by NGS barcode quantification 3 weeks after AAV pool injection. D. Cumulative results of in vivo screening of the top 200 factors enriched in the first round of selection. From this additional round of selection, 3 factors were selected for further individual study, namely Chrdl1, Fam3c and Fam3b, which have never been associated with cardiac function before. Fold enrichment is expressed as Z score relative to uninjured heart (0 = no selection) (Z>1.96 or Z<-1.96; p<0.05).

[0072] Figure 2 Overexpression of Chrdl1, Fam3c, and Fam3b in a mouse model of myocardial infarction

[0073] AB. Echocardiographic analysis to evaluate the effect of AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, AAV2 / 9-Fam3b, or AAV2 / 9-control (1×10 11vg / animal; n=8 animals per group) transduced with AAV2 / 9 vectors (AAV9 vectors). Cardiac overexpression of the three secreted factors preserved left ventricular ejection fraction (LVEF) (A) and reduced cardiac dilatation (Vd-diastolic left ventricular volume) (B) if compared to AAV2 / 9 control animals. C. Two months after MI, mice were sacrificed for histological analysis and scar size was quantified after Masson's trichrome staining (infarct size expressed as a percentage of the left ventricle). Cardiac treatment with the three factors significantly reduced scar size. D. Measurement of CM cross-sectional area after wheat germ agglutinin (WGA) staining of cardiac sections in mice treated with AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, AAV2 / 9-Fam3b or AAV2 / 9-control at 60 days after MI. Treatment with the three factors counteracted post-MI cardiomyocyte hypertrophy, indicating improved cardiac function. Data are shown as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

[0074] Figure 3 Chrdl1, Fam3c, and Fam3b counteract the pattern of gene expression associated with pathological LV remodeling

[0075] AD. Real-time PCR quantification of cardiac expression levels of α-MHC (A), β-MHC (B), SERCA2a (C), and RYR2 (D) genes in non-infarcted animals and in AAV2 / 9-control or AAV2 / 9-Chrd1, AAV2 / 9-Fam3c, or AAV2 / 9-Fam3b-treated hearts 60 days after MI. Values ​​are normalized to GAPDH and expressed as fold relative to untreated (n=6). Each of the three factors effectively protected the heart from pathological LV remodeling. Untr: untreated control, Ctr: control with MI. Data are shown as mean ± SEM; *P<0.05; **p<0.01.

[0076] Figure 4 AAV9-mediated cardiac overexpression of Chrdl1, Fam3c, and Fam3b reduces cell death and promotes beneficial autophagy after MI

[0077] A. Quantification of apoptosis was performed by assessing TUNEL-positive nuclei (percentage of total) in the infarct border zone. Animals were transduced with AAV2 / 9 vectors expressing Chrdl1, Fam3c or Fam3b after MI. TUNEL staining of apoptotic cells was performed on frozen heart sections 2 days later (n=5 per group). All three factors effectively protected cardiomyocytes from apoptotic death. AAV2 / 9 vectors that do not express any protein were used as controls for these experiments. BC. The induction of autophagy was assessed by analyzing LC3 protein lipidation (conversion from LC3-I to LC3-II) in the left ventricle of transduced hearts harvested 2 days after MI. Representative western blots (B) and optical density analysis. Both AAV2 / 9-Chrdl1 and AAV2 / 9-Fam3c are able to induce autophagy (C). D. To analyze autophagic flux in vivo, adult CD1 mice (n=5 per group) were transduced with AAV2 / 9-mRFP-EGFP-LC3 together with AAV2 / 9 expressing Chrdl1, Fam3c, Fam3b or AAV2 / 9-control. Two days after MI, yellow and red LC3 + Points were used to quantify autophagosomes (yellow) and autolysosomes (red). Both AAV2 / 9-Chrdl1 and AAV2 / 9-Fam3c significantly increased autophagic flux in vivo. Data are mean ± SEM; *P < 0.05; **P < 0.01.

[0078] Figure 5 .Therapeutic effects of circulating Chrdl1, Fam3c, and Fam3b after myocardial infarction

[0079] A. Overview of the strategy to evaluate the therapeutic efficacy of factors released from the liver into the circulation to mimic systemic administration of recombinant proteins. AAV2 / 8 vectors expressing Chrdl1, Fam3c, and Fam3b under the control of the hepatocyte-specific human α-1 antitrypsin (hAAT) promoter were injected into the liver via direct intraparenchymal inoculation (n=7; 3 injection sites per liver, 5×10 total 11 vg / animal); 7 days later, animals underwent MI by coronary artery ligation. B. Circulating Chrdl1, Fam3c and Fam3b proteins were detected in animal serum by western blotting 7 days after AAV2 / 8 intraparenchymal delivery. CD. Circulating Chrld1, Fam3c and Fam3b preserved left ventricular ejection fraction (EF) (C) and reduced cardiac dilatation (measured as diastolic volume, μl) (D) 15, 30 and 60 days after MI. E. Scar size was measured as a percentage of LV by Masson's trichrome staining. All three factors reduced infarct size. Data are shown as mean ± SEM; *P<0.05; **P<0.01; ***P<0.001.

[0080] Figure 6 Recombinant Chrdl1, Fam3c, and Fam3b protect cardiomyocytes from doxorubicin-induced cell death

[0081] Rat cardiomyocytes were treated with the indicated doses of doxorubicin and then administered with the indicated factors at 100 ng / ml. After 20 hours, apoptosis was measured by assessing the level of caspase 3 / 7 activation. All three recombinant proteins protected the cells. Data are shown as mean ± SEM; *P < 0.05; **P < 0.01.

[0082] Figure 7 .The role of Chrdl1 in preventing fibroblast activation and cardiac fibrosis after myocardial injury

[0083] A. Overview of experiments evaluating the role of Chrdl1 on Tgfβ1-induced conversion of primary adult fibroblasts into profibrotic myofibroblasts. B. Adult fibroblasts were treated with increasing doses of Tgfβ1 (1-10-50 ng / ml) in the presence or absence of recombinant Chrdl1 (100 ng / ml). Three days after treatment, Col1a1 and α-Sma levels were assessed by immunofluorescence. C. Regulation of MI-induced fibrotic responses by Chrdl1 was confirmed in transgenic Collα1(I)-EGFP mice. Hearts overexpressing AAV2 / 9-Chrdl1 showed reduced levels of fibrotic scars and Col1a1 and α-Sma activation. D. Transcript levels of Col1a1, α-Sma, Tgfβ1, and MMP9 were quantified by qPCR in cardiac tissues of CD1 mice 3 days after MI. Collectively, these results suggest that Chrdl1 exerts an antifibrotic role after MI. Data are shown as mean ± SEM; *P < 0.05; **P < 0.01.

[0084] Figure 8 Expression of Chrdl1, Fam3c, and Fam3b protects mice from doxorubicin-induced cardiotoxicity and death

[0085] CD1 mice (n=8) were injected intramyocardially with AAV9 vectors expressing three proteins (Chrdl1, Fam3c, or Fam3b) and then treated with doxorubicin for 80 days using an infusion pump. A. Kaplan-Meier survival curves showing strong protective activity of Chrdl1, Fam3c, and Fam3b against drug-induced death. B. and C. Left ventricular (LV) ejection fraction and end-systolic diameter, LVEF and LVID, respectively, at different times during treatment. The data showed that treatment with any of the three factors had a significant cardioprotective effect. Data are shown as mean ± SEM; *P < 0.05; **P < 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0087] As used herein, the term "comprising" is synonymous with "including" or "containing", is inclusive or open-ended, and does not exclude additional, unrecited members, elements or steps. The term "comprising" also includes the term "consisting of . . . . "

[0088] protein

[0089] Chrdl1 is an extracellular inhibitor of bone morphogenetic proteins (BMPs) that are primarily expressed in mesenchymal-derived cell types, pericytes, and neurons in the retina (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 bonemorphogenetic 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 bonemorphogenetic proteins in C2C12 cells. Biochem Biophys Res Commun 344, 786-791 (2006); Coffinier, C., Tran, U., Larrain, J. & De Robertis, EM Neuralin-1 is a novel Chordin-related molecule expressed in the mouse neural plate. Mech Dev 100, 119-122 (2001)).

[0090] The name Chrdl1 is derived from its sequence similarity to tenascin, another factor identified as dorsalizing the Xenopus embryo. Chrdl1 has a different spatiotemporal expression pattern than tenascin, but both genes contain cysteine-rich unit-designed procollagen repeats (CRs) that are also present in various extracellular matrix proteins. CR1 and CR3 are responsible for Chrdl1-BMP binding. The protein binds to BMP4 with high affinity and to BMP5, BMP6, and BMP7 with less affinity.

[0091] Fam3b and Fam3c are two members of the FAM3 family with sequence similarity 3 (Zhu, Y., et al. Cloning, expression, and initial characters of a new cytokine-like gene family. Genomics 80, 144-150 (2002)). Fam3b, also known as PANDER, is highly expressed in the pancreas, where it is involved in regulating glucose homeostasis and beta cell function (Robert-Cooperman, CE, Wilson, CG & Burkhardt, BR PANDER KO mice on high-fat diet are glucose intolerant yet resistant to fasting hyperglycemia and hyperinsulinemia. FEBS Lett 585, 1345-1349 (2011); Robert-Cooperman, CE, et al. Targeted disruption of pancreatic-derived factor (PANDER, FAM3B) impairs pancreatic beta-cell function. Diabetes 59, 2209-2218 (2010); Yang, J., et al. Mechanisms of glucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B) in pancreatic beta-cells. Diabetes 54, 3217-3228 (2005)).

[0092] Fam3c, also known as ILEI, is ubiquitously expressed. It induces inner ear cell proliferation (Pilipenko, VV, Reece, A., Choo, DI & Greinwald, JH, Jr. Genomic organization and expression analysis of the murine Fam3c gene. Gene 335, 159-168 (2004)), regulates osteogenic differentiation (Bendre, A., Buki, KG & Maatta, JAFam3c modulates osteogenic differentiation by down-regulating Runx2. Differentiation 93, 50-57 (2017)) and plays a role in epithelial-mesenchymal transition (EMT) during cancer progression (Waerner, T., et al. ILEI: acytokine essential for EMT, tumor formation, and late events in metastasis in epithelial cells. Cancer Cell 10, 227-239 (2006); Lahsnig, C., et al. ILEI requires oncogenic Ras for EMT ofhepatocytes and liver carcinoma progression.Oncogene 28,638-650(2009)). Restoring its level in the liver of obese diabetic mice improves insulin resistance and reduces fatty liver (Chen, Z., et al. Hepatic Activation of the FAM3C-HSF1-CaM Pathway Attenuates Hyperglycemia of Obese Diabetic Mice. Diabetes 66,1185-1197(2017); Chen, Z., et al. FAM3C activates HSF1to suppress hepatic gluconeogenesis and attenuate hyperglycemia of type 1 diabetic mice. Oncotarget 8,106038-106049(2017)).

[0093] An exemplary amino acid sequence of Chrld1 is SEQ ID NO:1 (human) – UniProt ID: Q9BU40-1.

[0094] MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQNRQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARHSYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDNKGYFCGEETMPVYESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKLVTRTTLSQWKIFTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC

[0095] (SEQ ID NO:1)

[0096] An exemplary amino acid sequence of Fam3c is SEQ ID NO:2 (human) - Uniprot ID: Q92520-1.

[0097] MRVAGAAKLVVAVAVFLLTFYVISQVFEIKMDASLGNLFARSALDTAARSTKPPRYKCGISKACPEKHFAFKMASGAANVVGPKICLEDNVLMSGVKNNVGRGINVALANGKTGEVLDTKYFDMWGGDVAPFIEFLKAIQDGTIVLMGTYDDGATKLNDEARRLIADLGSTSITNLGFRDNWVFCGGKGIKTKSPFEQHIKNNKDTNKYEGWPEVVEMEGCIPQKQD

[0098] (SEQ ID NO:2)

[0099] An exemplary amino acid sequence of Fam3b is SEQ ID NO: 3 (human) - Uniprot ID: P58499-1.

[0100] MRPLAGGLLKVVFVVFASLCAWYSGYLLAELIPDAPLSSAAYSIRSIGERPVLKAPVPKRQKCDHWTPCPSDTYAYRLLSGGGGRSKYAKICFEDNLLMGEQLGNVARGINIAIVNYV TGNVTATRCFDMYEGDNSGPMTKFIQSAAPKSLLFMVTYDDGSTRLNNDAKNAIEALGSKEIRNMKFRSSWVFIAAKGLELPSEIQREKINHSDAKNNRYSGWPAEIQIEGCIPKERS

[0101] (SEQ ID NO:3)

[0102] An exemplary nucleotide sequence encoding Chr ld 1 (human) is SEQ ID NO: 4 (Chr ld 1) - Seq ID: NM_001143981.1 (coding sequence).

[0103]

[0104] (SEQ ID NO:4)

[0105] An exemplary nucleotide sequence encoding Fam3c is SEQ ID NO:5 (Fam3c)-Seq ID: NM_014888.3 (coding sequence).

[0106] ATGAGGGTAGCAGGTGCTGCAAAGTTGGTGGTAGCTGTGGCAGTGTTTTTACTGACATTTTATGTTATTTCTCAAGTATTTGAAATAAAAATGGATGCAAGTTTAGGAAATCTATTTGCAAGATCAGCATTGGACACAGCTGCACGTTCTACAAAGCCTCCCAGATATAAGTGTGGGATCTCAAAAGCTTGCCCTGAGAAGCATTTTGCTTTTAAAATGGCAAGTGGAGCAGCCAACGTGGTGGGACCCAAAATCTGCCTGGAAGATAATGTTTTAATGAGTGGTGTTAAGAATAATGTTGGAAGAGGGATCAATGTTGCCTTGGCAAATGGAAAAACAGGAGAAGTATTAGACACTAAATATTTTGACATGTGGGGAGGAGATGTGGCACCATTTATTGAGTTTCTGAAGGCCATACAAGATGGAACAATAGTTTTAATGGGAACATACGATGATGGAGCAACCAAACTCAATGATGAGGCACGGCGGCTCATTGCTGATTTGGGGAGCACATCTATTACTAATCTTGGTTTTAGAGACAACTGGGTCTTCTGTGGTGGGAAGGGCATTAAGACAAAAAGCCCTTTTGAACAGCACATAAAGAACAATAAGGATACAAACAAATATGAAGGATGGCCTGAAGTTGTAGAAATGGAAGGATGCATCCCCCAGAAGCAAGACTAA

[0107] (SEQ ID NO:5)

[0108] An exemplary nucleotide sequence encoding Fam3b is SEQ ID NO:6 (Fam3b)-Seq ID: NM_058186.3 (coding sequence).

[0109] ATGCGCCCATTGGCTGGTGGCCTGCTCAAGGTGGTGTTCGTGGTCTTCGCCTCCTTGTGTGCCTGGTATTCGGGGTACCTGCTCGCAGAGCTCATTCCAGATGCACCCCTGTCCAGTGCTGCCTATAGCATCCGCAGCATCGGGGAGAGGCCTGTCCTCAAAGCTCCAGTCCCCAAAAGGCAAAAATGTGACCACTGGACTCCCTGCCCATCTGACACCTATGCCTACAGGTTACTCAGCGGAGGTGGCAGAAGCAAGTACGCCAAAATCTGCTTTGAGGATAACCTACTTATGGGAGAACAGCTGGGAAATGTTGCCAGAGGAATAAACATTGCCATTGTCAACTATGTAACTGGGAATGTGACAGCAACACGATGTTTTGATATGTATGAAGGTGATAACTCTGGACCGATGACAAAGTTTATTCAGAGTGCTGCTCCAAAATCCCTGCTCTTCATGGTGACCTATGACGACGGAAGCACAAGACTGAATAACGATGCCAAGAATGCCATAGAAGCACTTGGAAGTAAAGAAATCAGGAACATGAAATTCAGGTCTAGCTGGGTATTTATTGCAGCAAAAGGCTTGGAACTCCCTTCCGAAATTCAGAGAGAAAAGATCAACCACTCTGATGCTAAGAACAACAGATATTCTGGCTGGCCTGCAGAGATCCAGATAGAAGGCTGCATACCCAAAGAACGAAGCTGA

[0110] (SEQ ID NO:6)

[0111] The present invention encompasses any other polynucleotides encoding the above-mentioned protein.

[0112] The activity of the proteins and fragments thereof disclosed herein can be readily determined by a skilled person. For example, suitable in vitro assays include: (a) protection against hydrogen peroxide or doxorubicin-induced cell death, such as using a TUNEL assay or a caspase activity assay; (b) induction of autophagy, such as assaying the formation of LC3-positive autophagic vesicles; and / or (c) reduction of Chrdl1:BMP and TGFbeta activity, such as activation of aSMA expression in cardiac fibroblasts after treatment with recombinant TGFbeta, or reduction of SMAD 1 / 5 / 8 phosphorylation after treatment with recombinant BMP4.

[0113] The present inventors have surprisingly discovered a previously unknown role for Chrdl1, Fam3c and Fam3b in promoting cardiomyocyte survival, suggesting their therapeutic activity in counteracting ischemia and other forms of cardiac damage and thereby preventing heart failure.

[0114] According to the present invention, Chrdl1, Fam3c and Fam3b effectively increase cardiac function and reduce infarct size following intracardiac injection of viral vectors expressing these factors.

[0115] Although the inventors do not wish to be bound by theory or mechanism of action, it is believed that the proteins Chrdl1, Fam3c and Fam3b exert a therapeutic effect in the heart by preventing cardiomyocyte apoptosis and inducing cardiomyocyte autophagy. Taken together, this leads to the prevention of cardiac function, reduction of fibrosis and pathological remodeling of the left ventricle, and the induction of a beneficial expression pattern of genes associated with pathological cardiac remodeling, such as an increase in Serca2a (sarcoplasmic / endoplasmic reticulum calcium ATPase 2a) and RYR2 (Ranodin receptor 2) and a maintained ratio of α-myosin heavy chain (αMHC) to β-myosin heavy chain (βMHC).

[0116] The mechanism by which Chrdl1, Fam3c, and Fam3b exert cardioprotective effects on the ischemic heart is by maintaining cardiomyocyte viability by preventing apoptotic cell death, for example, following intracardiac injection of viral vectors expressing these factors.

[0117] Chrdl1 and Fam3c promote beneficial autophagy to counteract cardiomyocyte death after myocardial infarction.

[0118] Chrdl1, Fam3c, and Fam3b can preserve cardiomyocyte viability by preventing apoptotic cell death after doxorubicin treatment.

[0119] An additional positive role of Chrdl1 is the prevention of cardiac fibroblast activation and cardiac fibrosis.

[0120] The heart is an organ that cannot undergo significant regeneration in adulthood, so the integrity of cardiomyocytes is maintained by autophagy, a mechanism that allows the renewal of specific intracellular components (including mitochondria). This mechanism is particularly relevant after myocardial infarction, because sudden ischemia or reperfusion after ischemia, such as after percutaneous revascularization, can cause severe damage to mitochondria, which begin to use oxygen to produce destructive chemicals (Yellon, DM., Hausenloy DJ. Myocardial reperfusion injury. N. Engl. J. Med. 357, 1121-1135 (2007); Gustafsson AB., Gottlieb RA. Circ. Res. 104 (2), 150-158 (2009)). Therefore, autophagy and apoptosis are highly interrelated, with the former mechanism being activated after injury to remove damaged organelles as a protective response to avoid apoptotic cell death.

[0121] Chrdl1, Fam3c and Fam3b can therefore be used for cardioprotection, thereby reducing the risk of heart disease or heart failure. Cardioprotection can be achieved by maintaining cardiomyocyte viability.

[0122] Protein delivery

[0123] As an alternative to polynucleotide delivery, the proteins of the invention can be delivered by direct protein delivery.

[0124] The protein can be administered directly to the subject. In some embodiments, the protein is a fusion protein, preferably a fusion with a second protein that can increase the life span of the protein in the subject. For example, the protein can be an immunoglobulin Fc domain fusion protein.

[0125] Protein delivery can be delivered 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 engineering of viral particles (e.g., lentiviral particles) to contain the protein to be delivered to the cell. Thus, when the engineered viral particles enter the cell as part of their natural life cycle, the protein contained in the particles is carried into the cell.

[0126] Protein delivery (Gaj, T. et al. (2012) Nat. Methods 9:805-7) can also be achieved, for example, by utilizing vehicles such as liposomes.

[0127] Polynucleotide

[0128] The polynucleotides of the present invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. The skilled person will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides may encode the same polypeptide. In addition, it should be appreciated that the skilled person may use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention, to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is to be expressed.

[0129] The nucleotide sequences of the invention disclosed herein may contain or lack a stop codon 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 or without a stop codon.

[0130] The polynucleotides may be modified by any method available in the art. Such modifications may be performed to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0131] Polynucleotides such as DNA polynucleotides can be produced recombinantly, synthetically or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0132] Longer polynucleotides are usually produced using recombinant methods, such as using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g., about 15 to 30 nucleotides) flanking the target sequence of the desired clone, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragments (e.g., by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0133] Carrier

[0134] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another.

[0135] On the one hand, the present invention provides a vector comprising a polynucleotide of the present invention. In a preferred embodiment, the vector is a viral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector or an adenoviral vector, preferably an AAV vector.

[0136] Adeno-associated virus (AAV) vector

[0137] In one aspect, the invention provides an AAV vector comprising a polynucleotide of the invention.

[0138] Preferably, the AAV vector is in the form of an AAV vector particle.

[0139] 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.

[0140] In some embodiments, the AAV vector particle comprises an AAV9 capsid protein. In some embodiments, the AAV vector particle comprises an AAV8 capsid protein.

[0141] In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV9 capsid protein (AAV2 / 9). In other embodiments, the AAV vector particles comprise an AAV2 genome and an AAV8 capsid protein (AAV2 / 8).

[0142] Methods for making and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art.

[0143] The AAV vector may comprise the AAV genome or a fragment or derivative thereof.

[0144] AAV is known to be able to package a genome size of up to 5.2 kb (Dong, J.-Y. et al. (1996) Human Gene Therapy 7:2101-2112).

[0145] The AAV genome is a polynucleotide sequence that can encode the functions required to produce AAV particles. These functions include functions that play a role in the replication and packaging cycle of AAV in host cells, including encapsulation of the AAV genome into AAV particles. Naturally occurring AAV is replication-defective and relies on the provision of trans-helper functions to complete the replication and packaging cycle. Therefore, the AAV genome of the AAV vector of the present invention is generally replication-defective.

[0146] The AAV genome can be in single-stranded form, either positive or negative sense, or in double-stranded form. The use of the double-stranded form allows bypassing the DNA replication step in the target cell and can therefore accelerate transgene expression.

[0147] The AAV genome can be from any naturally derived AAV serotype, isolate or clade. Thus, the AAV genome can be the full genome of a naturally occurring AAV. As known to the skilled artisan, AAVs present in nature can be classified according to various biological systems.

[0148] Typically, AAVs are referred to by their serotype. Serotypes correspond to variant subspecies of AAV that have unique reactivity due to the expression profile of their capsid surface antigens, which can be used to distinguish them from other variant subspecies. Typically, viruses with a specific AAV serotype do not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype.

[0149] AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, as well as recombinant serotypes recently identified from primate brains, such as Rec2 and Rec3. Any of these AAV serotypes can be used in the present invention.

[0150] In some embodiments, the AAV vector particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rec2, or Rec3 AAV vector particle.

[0151] In some embodiments, the AAV is an AAV1, AAV2, AAV5, AAV7, AAV8, or AAV9 serotype.

[0152] In some embodiments, the AAV is of AAV9 or AAV8 serotype.

[0153] In some embodiments, the AAV is an AAV9 serotype. In other embodiments, the AAV is an AAV8 serotype.

[0154] The capsid protein may be a mutant capsid protein as disclosed in WO2008 / 124724, which is incorporated herein by reference.

[0155] In some embodiments, the AAV vector comprises an AAV8 capsid having a Y733F mutation.

[0156] A review of AAV serotypes can be found in Choi et al. (2005) Curr. Gene Ther. 5:299-310 and Wu et al. (2006) Molecular Therapy 14:316-27. The sequence of the AAV genome or AAV genome elements including ITR sequences, rep or cap genes used in the present invention can be derived from the accession numbers of the following AAV complete 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 strains DA-1 NC_006263, AY629583; bovine AAV NC_005889, AY388617.

[0157] AAVs may also be referred to in terms of clades or clones. This refers to the phylogenetic relationships of naturally derived AAVs, typically referring to the phylogenetic group of AAVs that can be traced back to a common ancestor and includes all of its descendants. In addition, AAVs may be referred to in terms of specific isolates, i.e., genetic isolates of a particular AAV found in nature. The term genetic isolate describes a population of AAVs that has had limited genetic admixture with other naturally occurring AAVs, thereby defining an identifiable distinct population at the genetic level.

[0158] A skilled person can select a suitable AAV serotype, clade, clone or isolate for use in the present invention based on their common knowledge. For example, AAV5 capsids have been shown to efficiently transduce primate cone photoreceptors, as demonstrated by the successful correction of inherited color vision defects (Mancuso et al. (2009) Nature 461:784-7).

[0159] The AAV serotype determines the tissue specificity of AAV infection (or tropism). Therefore, preferred AAV serotypes for AAV administered to patients according to the present invention are those that have a natural tropism or high infection efficiency for target cells within the heart.

[0160] Typically, the AAV genome of a naturally derived serotype, isolate or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequence acts in cis to provide a functional replication origin and allows integration and excision of the vector from the cell genome. In a preferred embodiment, one or more ITR sequences are located on the flank of the nucleotide sequence encoding the protein of the present invention. The AAV genome also typically contains packaging genes, such as rep and / or cap genes that encode packaging functions for AAV particles. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or their variants. The cap gene encodes one or more capsid proteins, such as VP1, VP2 and VP3 or their variants. These proteins constitute the capsid of the AAV particles. Capsid variants are discussed below.

[0161] A promoter will be operably linked to each packaging gene. Specific examples of such promoters include p5, p19, and p40 promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76:5567-5571). For example, p5 and p19 promoters are generally used to express rep genes, while p40 promoters are generally used to express cap genes.

[0162] As described above, the AAV genome used in the AAV vector of the present invention can therefore be a complete genome of a naturally occurring AAV. For example, a vector comprising a complete AAV genome can be used to prepare an AAV vector or vector particles in vitro. However, although such vectors can be administered to patients in principle, this is rarely done in practice. Preferably, the AAV genome will be derivatized for the purpose of administration to patients. Such derivatization is standard in the art, and the present invention encompasses the use of any known AAV genome derivative, as well as derivatives that can be produced by applying techniques known in the art. A review of the derivatization of the AAV genome and AAV capsid is in: Coura and Nardi (2007) Virology Journal 4:99, and Choi et al. and Wu et al., referenced above.

[0163] Derivatives of the AAV genome include any truncated or modified form of the AAV genome that allows for in vivo expression of transgenes from the AAV vector of the present invention. Generally, it is possible to significantly truncate the AAV genome to include minimal viral sequences, but still retain the above functions. For safety reasons, this is preferred to reduce the risk of vector recombination with wild-type viruses, and also to avoid triggering cellular immune responses due to the presence of viral gene proteins in target cells.

[0164] Typically, the derivative will include at least one inverted terminal repeat (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from an AAV genome with a different serotype, or may be a chimeric or mutant ITR. A preferred mutant ITR is a mutant ITR with a trs (terminal resolution site) deletion. This deletion allows continued replication of the genome to produce a single-stranded genome containing both coding and complementary sequences, i.e., a self-complementary AAV genome. This allows bypassing DNA replication in the target cell, thereby enabling accelerated transgene expression.

[0165] One or more ITRs will preferably be located at the flanks of either end of the nucleotide sequence encoding the protein of the present invention. Preferably, one or more ITRs are included to help the vector of the present invention form concatemers in the nucleus of the host cell, for example after the single-stranded vector DNA is converted to double-stranded DNA under the action of the host cell DNA polymerase. The formation of such additional concatemers protects the vector construct during the life cycle of the host cell, thereby allowing the extended expression of the transgene in vivo.

[0166] In a preferred embodiment, the ITR element will be the only sequence retained from the native AAV genome in the derivative. Therefore, the derivative will preferably not include the rep and / or cap genes of the native genome and any other sequence of the native genome. This is preferred for the reasons mentioned above, and is also to reduce the possibility of vector integration into the host cell genome. In addition, reducing the size of the AAV genome can increase the flexibility of incorporating other sequence elements (such as regulatory elements) in addition to transgenes into the vector.

[0167] The following parts can therefore be removed in the derivatives of the present invention: an inverted terminal repeat (ITR) sequence, replication (rep) and capsid (cap) genes. However, in some embodiments, the derivatives may additionally include one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency at a specific site on human chromosome 19 and shows a negligible frequency of random integration, so retention of integration capacity in the vector can be tolerated in a therapeutic setting.

[0168] Where the derivative comprises a capsid protein, i.e., VP1, VP2 and / or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the present invention encompasses providing capsid protein sequences of different AAV serotypes, clades, clones or isolates from within the same vector (i.e., pseudotyped vector).

[0169] Typically, chimeric, reorganized or capsid-modified derivatives are selected to provide one or more desired functions for AAV vectors. Therefore, compared with AAV vectors comprising naturally occurring AAV genomes (e.g., AAV2), these derivatives can show higher gene delivery efficiency, reduced immunogenicity (body fluid or cell), changed tropism range and / or improved targeting of specific cell types. The improvement in gene delivery efficiency can be affected by cell surface receptor or co-receptor binding improvements, internalization improvements, intracellular transport and improved nuclear transport, improved viral particle uncoating and improved single-stranded genome conversion to double-stranded form. The efficiency improved can also be related to the tropism range of changes or the targeting of specific cell populations, so the carrier dose will not be diluted due to being applied to tissues that do not need it.

[0170] Chimeric capsid proteins include those produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be done, for example, by a marker rescue method, in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and directed selection is used to select for capsid sequences with desired properties. The capsid sequences of different serotypes can be altered by homologous recombination in cells to produce new chimeric capsid proteins.

[0171] Chimeric capsid proteins also include those produced by engineering the capsid protein sequence to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, such as between capsid proteins of two or more different serotypes.

[0172] Shuffled or chimeric capsid proteins can also be produced by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes, such as those encoding capsid proteins of multiple different serotypes, can be created by randomly fragmenting the sequences of related AAV genes, and then reassembling the fragments in a self-priming polymerase reaction, which can also lead to crossovers in regions of sequence homology. Hybrid AAV gene libraries created by shuffling capsid genes of several serotypes can be screened to identify viral clones with desired functions. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to create a diverse library of variants, from which variants with desired properties can then be selected.

[0173] The sequence of the capsid gene can also be genetically modified to introduce specific deletions, substitutions or insertions relative to the natural wild-type sequence. In particular, the capsid gene can be modified by inserting an unrelated protein or peptide sequence within the open reading frame of the capsid coding sequence or at the N and / or C-terminal of the capsid coding sequence.

[0174] The unrelated protein or peptide may advantageously be a protein or peptide that serves as a ligand for a specific cell type, thereby conferring improved binding to a target cell or improving the specificity of the vector for targeting a specific cell population. The unrelated protein may also be a protein or peptide that aids in the purification of viral particles as part of the production process, i.e., an epitope or affinity tag. The insertion site is typically selected so as not to interfere with other functions of the viral particles, e.g., internalization, transport of the viral particles. The skilled person can identify suitable insertion sites based on their common knowledge. Specific sites are disclosed in Choi et al. cited above.

[0175] The invention further encompasses providing sequences of the AAV genome that differ in order and configuration from the native AAV genome. The invention also encompasses replacing 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 consist of sequences of two or more related viral proteins from different viral species.

[0176] The AAV vector of the present invention may take the form of a nucleotide sequence comprising the AAV genome or a derivative thereof and a sequence encoding the protein of the present invention.

[0177] The AAV particles of the present invention include transcapsid forms, in which an AAV genome or derivative having ITRs of one serotype is packaged in a capsid of a different serotype. The AAV particles of the present invention also include mosaic forms, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. AAV particles also include chemically modified forms with ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting specific cell surface receptors.

[0178] An AAV vector may contain multiple copies (eg, 2, 3, etc.) of a nucleotide sequence mentioned herein.

[0179] In some embodiments, the polynucleotide further comprises one or more AAV ITRs. In preferred embodiments, the polynucleotide further comprises two AAV ITRs. In some embodiments, the polynucleotide comprises an AAV ITR at its 5' end and an AAV ITR at its 3' end. In some embodiments, the AAV ITR is an AAV2, AAV9 or AAV8 ITR.

[0180] Promoter and regulatory sequences

[0181] The polynucleotide or vector of the present invention may also include elements that allow the nucleotide sequence encoding the protein of the present invention to be expressed in vitro or in vivo. These may be referred to as expression control sequences. Therefore, the polynucleotide or vector typically comprises an expression control sequence (e.g., comprising a promoter sequence) operably linked to the nucleotide sequence encoding the protein of the present invention.

[0182] Any suitable promoter may be used, and the skilled artisan can readily select. The promoter sequence may be constitutively active (i.e., operable in any host cell background), or may be active only in a specific host cell environment, thereby allowing targeted expression of the transgene in a specific cell type (e.g., a tissue-specific promoter). The promoter may exhibit inducible expression in response to the presence of another factor, such as a factor present in the host cell. In any case, when the vector is administered for therapy, preferably the promoter should function in the target cell background.

[0183] In a preferred embodiment, the promoter is a liver-specific promoter. In a preferred embodiment, the promoter is a liver-specific hAAT promoter.

[0184] The liver-specific hAAT promoter can confer hepatocyte selective specificity. When the vector is administered via the portal vein, the inventors have shown that each circulating factor secreted from the liver can protect the heart after injury.

[0185] Suitable promoters include the chicken beta-actin (CBA) promoter, optionally in combination with a cytomegalovirus (CMV) enhancer element.An exemplary promoter for use in the present invention is the CAG promoter.

[0186] In some embodiments, the promoter is a CMV promoter.

[0187] The polynucleotides or vectors of the present invention may also contain one or more additional regulatory sequences that may function before or after transcription. The regulatory sequences may be part of the native transgenic locus or may be heterologous regulatory sequences. The polynucleotides or vectors of the present invention may contain portions of the 5'-UTR or 3'-UTR from the native transgenic transcript.

[0188] A regulatory sequence is any sequence that promotes transgene expression, i.e., functions to increase expression of a transcript, improve nuclear export of mRNA, or enhance its stability. Such regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

[0189] Suitable enhancers include the WPRE regulatory element. Suitable poly-A signals include the bovine growth hormone poly-A signal.

[0190] Additional regulatory sequences can be readily selected by the skilled artisan.

[0191] Application method

[0192] A variety of administration routes and techniques may be utilized including parenteral techniques such as intravenous, intracardiac and intraarterial injection, catheterization, etc. The average amount of active agent may vary and should particularly be based on the advice and prescription of a qualified physician.

[0193] The proteins, polynucleotides or vectors of the invention may be administered systemically (eg, by peripheral intravenous infusion) or may be administered locally or regionally.

[0194] Preferably, the protein is administered by a parenteral route, in particular an intravenous, intraarterial or intramyocardial route.

[0195] Administration of polynucleotides encoding the proteins disclosed herein can be achieved by gene therapy, see, for example, WO 2013 / 093870.

[0196] According to the present invention, Chrdl1, Fam3c and Fam3b are also active when they reach the infarcted heart via the systemic circulation.

[0197] Pharmaceutical composition and injection

[0198] The drugs of the present invention, such as proteins, polynucleotides or carriers, can be formulated into pharmaceutical compositions. In addition to the drug, these compositions can also contain pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers or other materials known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The technician can determine the exact nature of the carrier or other materials based on the route of administration.

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

[0200] For injection at the site of affliction, the active ingredient can be in the form of an aqueous solution without pyrogen and with suitable pH, isotonicity and stability. The technician can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as needed.

[0201] For delayed release, the drug can be contained in a pharmaceutical composition formulated for slow release, such as in microcapsules formed from biocompatible polymers or in a liposome carrier system, 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] Treatment

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

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

[0206] The administration regimen, dosage and posology will be determined by the physician according to his experience, the disease to be treated and the condition of the patient.

[0207] The proteins and / or polynucleotides of the present invention may be administered alone or in combination.

[0208] As used herein, the term "combination" or the term "combination", "combined use" or "combination preparation" may refer to the simultaneous, sequential or separate combined administration of two or more agents.

[0209] As used herein, the term "simultaneously" means that the agents are administered concurrently, ie, at the same time.

[0210] As used herein, the term "sequentially" refers to the administration of the agents one after the other.

[0211] As used herein, the term "separately" refers to that the medicaments are administered independently of one another but within a time interval that allows the medicaments to exhibit a combined, preferably synergistic effect. Thus, "separately" administration can allow one medicament to be administered within 1 minute, 5 minutes, or 10 minutes after, for example, another medicament.

[0212] Variants, derivatives, analogs, homologs and fragments

[0213] In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologues and fragments thereof.

[0214] In the context of the present invention, a variant of any given sequence is a sequence in which a particular sequence of residues (whether amino acid residues or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question substantially retains its function. Variant sequences may be obtained by adding, deleting, substituting, modifying, replacing and / or mutating at least one residue present in a naturally occurring protein.

[0215] As used herein, the term "derivative" in relation to a protein or polypeptide of the present invention includes any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues to the sequence, provided that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

[0216] As used herein, the term "analog" in relation to a polypeptide or polynucleotide includes any mimetic, ie, a compound having at least one of the endogenous functions of the polypeptide or polynucleotide that it mimics.

[0217] Generally, 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 desired activity or ability.Amino acid substitutions can include the use of non-naturally occurring analogs.

[0218] The proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties 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; amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0219] For example, conservative substitutions can be made according to the following table. Amino acids in the same block in the second column, preferably in the same row in the third column, can be substituted for each other:

[0220]

[0221] As used herein, the term "homologue" refers to an entity having a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be equivalent to "identity".

[0222] Homologous sequences may include amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, preferably at least 95% or 97% or 99% identical. Typically, homologs will contain 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 present invention, it is preferred to express homology in terms of sequence identity.

[0223] Homologous sequences can include nucleotide sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the subject sequence, preferably at least 95% or 97% or 99% identical. Although homology can also be considered in terms of similarity, in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0224] Preferably, reference to a sequence having a percentage identity to any one of the SEQ ID NOs detailed herein refers to the sequence having that percentage identity over the entire length of the referenced SEQ ID NO.

[0225] Homology comparisons can be performed by eye, or more usually with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the homology or identity percentage between two or more sequences.

[0226] The percent homology can be calculated for consecutive sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is compared directly to the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0227] While this is a very simple and consistent approach, it does not take into account that, for example, one insertion or deletion in a nucleotide sequence in an otherwise identical sequence pair may cause subsequent codons to be out of alignment, thus potentially resulting in a substantial reduction in the homology percentage when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes possible insertions and deletions into account without overly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

[0228] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting a higher correlation between the two compared sequences, will receive a higher score than one with many gaps. An "affine gap cost" is often used, which charges a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparison, it is preferred to use the default values. For example, when using the GCG WisconsinBestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0229] Therefore, the calculation of the maximum homology percentage first needs to produce the best comparison, taking into account the gap penalty. The suitable computer program for performing such comparison is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparison include but are not limited to BLAST packages (see Ausubel et al. (1999) ibid–Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al. (1999) ibid, 7-58 to 7-60 pages). However, for some applications, the GCG Bestfit program is preferably used. Another tool called BLAST 2 sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0230] Although the final percentage homology can be measured according to identity, the alignment process itself is usually not based on an all-or-nothing comparison. Instead, a scaled similarity scoring matrix is ​​usually used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the default matrix of the BLOSUM62 matrix-BLAST program suite. The GCG Wisconsin program usually uses public default values ​​or custom symbol comparison tables (if provided) (see user manual for details). For some applications, it is preferred to use the public default values ​​of the GCG package, or in the case of other software, use a default matrix, such as BLOSUM62.

[0231] Once the software has produced an optimal alignment, the percent homology, preferably the percent sequence identity, can be calculated. The software typically does this as part of the sequence comparison and generates a numerical result.

[0232] A "fragment" of a full-length Chrdl1, Fam3c or Fam3b is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0233] Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. In the case of a pending insertion, a synthetic DNA encoding the insertion and 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site can be made. The flanking regions will contain convenient restriction sites corresponding to the sites in the naturally occurring sequence so that the sequence can be cut with a suitable enzyme and the synthetic DNA can be connected to the cut. The DNA is then expressed according to the present invention to prepare the encoded protein. These methods are merely illustrations of numerous standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0234] Those skilled in the art will understand that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[0235] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0236] 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 one of ordinary skill in the art and are explained in the literature. See, for example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FMet 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, JM and McGee, J.O'D. (1990) In SituHybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DMand Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. Example

[0237] Example 1

[0238] FunSel, an in vivo selection protocol for identifying novel cardiac therapeutics for myocardial infarction

[0239] We recently developed FunSel (1,2), a novel protocol for the in vivo functional identification of novel therapeutic factors against degenerative conditions, which we applied here with the goal of identifying factors that ensure cardioprotection after myocardial infarction (MI). This is based on the use of adeno-associated virus (AAV) vectors, which are elegant tools for efficient cardiac gene transfer (3).

[0240] In brief, we generated a gene array library corresponding to the secretome, defined as the subset of proteins secreted into the extracellular milieu of the cell. By computational methods (4), 2033 unique proteins have been identified in the genome that carry a signal peptide and lack any transmembrane domains or intracellular localization signals and are therefore likely secreted from the cell. The size of the AAV genome, which restricted cloning to 4.5 kb, and the availability of cDNA clones, limited the number of cDNAs suitable for cloning to 1198. The coding regions of these genes were individually cloned into the pAAV pGi backbone plasmid under the control of the constitutive CMV IE promoter and confirmed by sequencing. A unique 10-nt barcode that allowed PCR amplification and sequencing unambiguously identified each clone ( Figure 1 A).

[0241] FunSel is based on the following strategy: Pools of AAV plasmids from a library, each encoding a specific factor and identified by a unique barcode, are used to mass produce AAV serotype 9 (AAV9) vectors (a total of 24 pools consisting of 50 factors of similar size each). Eight-week-old CD1 mice (n=9 animals per AAV9 pool) were subjected to myocardial infarction induced by permanent ligation of the left descending coronary artery, which in this case represents a selective stimulus. Immediately after MI, vectors from each AAV9 pool were injected in vivo into the left ventricular (LV) peri-infarct region at a multiplicity by which each vector, in principle, enters a different cell (10 per animal). ^10 Three weeks later, vector inserts were recovered from surviving LV tissue and the frequency of each vector was determined by next-generation sequencing (NGS) of the barcodes and compared with the vector frequency found in control animals injected with the same AAV9 pool but without MI (n = 6 animals per pool). Most myocytes die after infarction, but cells selectively survive when they express protective factors, so enrichment of a barcode represents positive genetic selection (beneficial effect), while depletion represents negative selection (neutral or deleterious effect) ( Figure 1 Experimental scheme in B).

[0242] Figure 1C reports the cumulative results obtained from the in vivo screen of 1198 factors. In the figure, the frequency of each factor recovered from the heart after MI is reported as a ratio to the frequency of the same factor in the absence of selective treatment. Based on Z score calculations, we selected the top 200 performers and ran 4 additional screens (4 pools of 50 vectors) for these factors. Eleven factors resulted in competitive enrichment of 1.96 Z-scores or higher (P<0.05) ( Figure 1 D). Supporting the robustness of the FunSel approach, among the hits from the second round of screening we found several known cardioprotective factors and unknown factors with some relevance to cardiomyocyte biology. These included Mdk, a pleiotropic molecule that plays a protective role against cardiac injury, and Rln1, a well-described antifibrotic agent that is able to reduce ROS production, apoptosis, and inflammation in infarcted hearts. More notably, however, the top performers included three novel proteins for which no information was currently available and no studies related to cardioprotection had been performed. These were Chordin-like 1 (Chrdl1) and two members of the family with sequence similarity 3, Fam3b and Fam3c. These factors were selected for further individual investigation to assess efficacy and mechanism of action.

[0243] Example 2

[0244] Effects of Chrdl1, Fam3c, and Fam3b on maintaining cardiac integrity and function after myocardial infarction in mice

[0245] Based on the FunSel results, we decided to validate and characterize the effects of selected top secreted factors (Chrdl1, Fam3c, and Fam3b) expressed as individual AAV2 / 9 vectors on myocardial infarction in the mouse heart, with the specific goal of evaluating the ability of each factor to counteract or reduce ischemic damage and promote cardiac function.

[0246] Eight-week-old CD1 mice were subjected to MI and simultaneously injected with AAV2 / 9 vectors expressing Chrdl1, Fam3c, Fam3b or control empty vector (1×10 11 vg / animal; n=8 per group). Our previous experience has shown that this protocol results in efficient myocardial transduction and transgene expression for up to one month (1, 5). Cardiac function of the animals was monitored by echocardiography at 15, 30, and 60 days after MI.

[0247] like Figure 2 As shown in , AAV2 / 9-mediated overexpression of Chrdl1, Fam3c, or Fam3b successfully preserved LV ejection fraction (LVEF) in infarcted mice compared with control-treated animals ( Figure 2A). LVEF values ​​improved significantly starting 15 days post-MI and were maintained (60 days post-MI: AAV9-Chrdl 1 39.96 ± 2.67%, AAV2 / 9-Fam3c 39.02 ± 2.53%, and AAV2 / 9-Fam3b 32.50 ± 2.70% compared to 19.86 ± 0.98% in animals receiving control vector, P < 0.001 for all treatments). As expected, diastolic LV volume (Vd) was significantly greater in control animals compared to treated mice at 30 and 60 days post-MI, when heart failure was onset ( Figure 2 B) (60 days post-MI: AAV2 / 9-Chrdl 101.22±10.31 μl, AAV2 / 9-Fam3c 107.31±8.85 μl, and AAV2 / 9-Fam3b 115.16±11.03 μl, compared with 168.64±7.47 μl in animals receiving control vector, P<0.001 for all treatments).

[0248] When myocardial infarction occurs, cardiac fibroblasts proliferate, differentiate into myofibroblasts, and produce extracellular matrix to form a scar to replace the gaps created by the loss of cardiomyocytes. This usually leads to myocardial stiffness and causes pathological remodeling, dilation, and dysfunction of the left ventricle. Morphometric analysis of trichrome-stained heart sections at day 60 showed that AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, and AAV2 / 9-Fam3b-treated mice showed a significant preservation of LV contractile organization and a reduction in fibrotic areas (infarct size: AAV2 / 9-Chrdl1 6.88±1.60%, AAV2 / 9-Fam3c 10.19±2.05%, AAV2 / 9-Fam3b 13.35±1.79%, vs. 26.26±2.35% of the LV in control animals; Figure 2 C).

[0249] Finally, wheat germ agglutinin (WGA) staining showed that overexpression of Chrdl1, Fam3c, or Fam3b did not induce a hypertrophic response in cardiomyocytes, as there was no increase in fiber cross-sectional area in treated animals compared with controls ( Figure 2 D).

[0250] Taken together, these results suggest that AAV2 / 9-mediated cardiac overexpression of Chrdl1, Fam3c, or Fam3b after acute ischemia promotes cardiomyocyte viability, reduces infarct size, and preserves cardiac function after myocardial infarction.

[0251] Example 3

[0252] AAV2 / 9-mediated expression of Chrdl1, Fam3c, or Fam3b counteracts pathological left ventricular remodeling associated with the onset of heart failure in mice

[0253] Two months after MI, we also investigated the effect of each secreted factor on the expression of a panel of genes previously associated with LV pathological remodeling (overexpression of β-myosin heavy chain (βMHC) and α-myosin heavy chain (αMHC), sarcoplasmic / endoplasmic reticulum Ca 2+ -Decrease in the levels of SERCA2a and RYR2).

[0254] Total RNA was extracted from LV tissue and analyzed by qRT-PCR using TaqMan probes specific for the genes studied. Consistent with echocardiographic and morphometric observations, AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, and AAV2 / 9-Fam3b counteracted the characteristic pattern of gene expression typically associated with pathological LV remodeling observed in AAV2 / 9-control mice, decreasing levels of β-MHC and increasing levels of α-MHC, SERCA2a, and RYR2 ( Figure 3 , Figure AD).

[0255] Example 4

[0256] All three factors maintain tissue viability, thereby protecting cardiomyocytes from cell death, and both Chrdl1 and Fam3c promote beneficial autophagy in the heart after MI

[0257] So far, the available information on the factors Chrdl1, Fam3c and Fam3b is insufficient, certainly not to explain why these factors exert cardioprotective effects after myocardial infarction. Therefore, we set out to explore possible biological mechanisms that could mediate their activity. Since the FunSel method is based on the selection of factors based on cardiomyocyte survival, the first set of experiments was performed by testing the level of cell death due to apoptosis. Compared with the control (1×10 11vg / animal; n = 5 per group), levels of cell death due to apoptosis were normally substantial two days after coronary artery occlusion in hearts of infarcted adult CD1 mice treated with the three AAV vectors (6). Fam3b, Fam3c, and especially Chrdl1 were extremely effective in preventing apoptotic cell death in infarcted hearts as assessed by nuclear TUNEL (TdT-mediated dUTP nick end labeling) staining of snap-frozen heart sections two days after MI (percentage of positive TUNEL nuclei: AAV2 / 9-Chrdl1 4.01 ± 1.21%, AAV2 / 9-Fam3c 10.33 ± 1.43%, AAV2 / 9-Fam3b 19.83 ± 3.01%, vs. 30.67 ± 4.38% in control animals) ( Figure 4 quantified in A).

[0258] The heart is an organ that is incapable of significant regeneration during adulthood, and thus the integrity of cardiomyocytes is maintained by autophagy, a mechanism that allows for the turnover of specific intracellular components, particularly mitochondria. This mechanism is particularly relevant after myocardial infarction, as abrupt ischemia or ischemia followed by reperfusion, such as after percutaneous revascularization, results in significant damage to mitochondria, which begin to use oxygen to produce destructive chemicals (7). Not surprisingly, therefore, autophagy and apoptosis are highly interrelated, with the former mechanism being activated after insult to remove damaged organelles as a protective response to avoid apoptotic cell death (8).

[0259] To evaluate the induction of autophagy after acute cardiac ischemia, we injected another group of adult infarcted CD1 mice with AAV2 / 9 vectors expressing Chrdl1, Fam3c, Fam3b or a control vector (1 × 10 11 vg / animal; n = 5 per treatment). Two days after MI, we found increased conversion of soluble LC3-I protein to lipid-bound LC3-II, particularly in the hearts of Chrdl1- and Fam3c-treated mice, which was associated with the formation of autophagosomes (representative blots and quantification of AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, AAV9-Fam3b, and AAV2 / 9-control hearts, respectively). Figure 4 B and 4C).

[0260] To directly visualize autophagic flux in infarcted hearts, we previously generated an AAV2 / 9 vector expressing monomeric red fluorescent protein (mRFP)-enhanced green fluorescent protein (EGFP) tandemly fluorescently tagged LC3 protein derived from the ptfLC3 plasmid in which green, but not red, fluorescence is sensitive to the pH difference between neutral autophagosomes and acidic autolysosomes (9). This vector was co-administered with AAV2 / 9-Chrdl1, AAV2 / 9-Fam3c, AAV2 / 9-Fam3b, or AAV2 / 9-control (1 × 10 11 vg / animal; n=5 per treatment) were administered together immediately after MI. Two days later, the number of yellow, LC3-positive vesicles, especially those that showed only red fluorescence, increased significantly in the LV peri-infarct region of hearts injected with AAV9-Chrdl1 and Fam3c, indicating that these two factors stimulate autophagic flux in vivo ( Figure 4 Quantification of yellow and red spots for each treatment is reported in D).

[0261] Taken together, these results suggest that AAV2 / 9-mediated cardiac overexpression of Chrdl1, Fam3c, and Fam3b preserves cardiomyocyte viability by preventing apoptotic cell death and, in particular, by promoting cardiac beneficial autophagy through Fam3c and Chrdl1.

[0262] Example 5

[0263] Circulating Chrdl1, Fam3c, and Fam3b produced and secreted by the liver upon AAV8-mediated tissue-specific expression counteract pathological left ventricular remodeling after myocardial infarction

[0264] To assess whether circulating Chrdl1, Fam3b, and Fam3c are active after reaching the heart from the circulation, we designed a strategy whereby each of these three factors is expressed by the liver and secreted into the circulation prior to myocardial infarction, as opposed to endogenous expression using viral vectors. Figure 5 A). In more detail, we performed intraparenchymal injections (5 × 10 11 vg / animal; n=6 per group), which selectively transfer genes into hepatocytes; in these vectors, the factors are expressed under the control of the human α-1 antitrypsin (hAAT) promoter, which ensures specific expression only in hepatocytes (10). Seven days after administration, when the liver is actively producing and releasing dosable amounts of each factor into the circulation ( Figure 5 B), Myocardial infarction was induced by ligation of the left descending coronary artery.

[0265] like Figure 5 As shown in C, AAV2 / 8-mediated hepatic production of Chrdl1, Fam3c, or Fam3b successfully preserved LV ejection fraction (LVEF) in infarcted mice compared to control treated animals. LVEF values ​​began to improve significantly 15 days after MI and were sustained (60 days after MI: AAV2 / 8-Chrdl1 28.77±1.66%, AAV2 / 8-Fam3c 28.09±1.61%, and AAV8-Fam3b 31.22±1.40%, compared to 20.05±1.47% in animals receiving control vector). Two months after MI, as expected, diastolic LV volumes were significantly larger in control animals compared to treated mice ( Figure 5 D) (60 days post-MI: AAV2 / 8-Chrdl1 150.2±10.2 μl, AAV2 / 8-Fam3c 137.9±16.8 μl, and AAV2 / 8-Fam3b 134.7±9.2 μl, compared to 193.2±11.6 μl in animals receiving control vector).

[0266] Finally, also in this experiment, morphometric analysis of trichrome-stained cardiac sections at day 60 showed that mice treated with AAV8-Chrdl1, AAV2 / 8-Fam3c, and AAV2 / 8-Fam3b displayed a significant reduction in the area of ​​fibrosis (infarct size: AAV2 / 8-Chrdl1 13.6 ± 3.1%, AAV2 / 8-Fam3c 15.0 ± 3.4%, AAV2 / 8-Fam3b 13.2 ± 2.7%, vs. 28.7 ± 3.3% of the LV in control animals; Figure 5 E).

[0267] Taken together, these results demonstrate that therapeutic expression of each circulating factor from the liver protects cardiomyocytes from ischemic damage and improves cardiac function after MI. This represents an initial efficacy test of injection of Chrdl1, Fam3c, and Fam3b as recombinant proteins.

[0268] Example 6

[0269] Recombinant Chrdl1, Fam3c, and Fam3b protect cardiomyocytes from doxorubicin-induced cell death

[0270] To evaluate the potential role of Chrdl1, Fam3c, and Fam3b in maintaining cell viability after toxic insults, the corresponding recombinant proteins were tested in primary neonatal rat ventricular cardiomyocytes treated with the chemotherapeutic drug doxorubicin ( Figure 6 ).

[0271] Treatment with 100 ng / ml of Chrdl1, Fam3c or Fam3b recombinant protein significantly abrogated caspase 3 / 7 activation (as a measure of apoptotic cell death) 20 hours after doxorubicin treatment (1 and 1.5 μM).

[0272] Example 7

[0273] Chrdl1 prevents fibroblast activation and cardiac fibrosis after myocardial infarction

[0274] When MI occurs, cardiac fibroblasts proliferate, differentiate into myofibroblasts and stimulate collagen deposition to produce scars to replace the gaps created by cardiomyocyte loss. Interestingly, infarcted hearts overexpressing Chrdl1 not only had very small scars, but also did not undergo pathological remodeling and dilation two months after MI. This suggests that in addition to its effects on cardiomyocyte survival, Chrdl1 also has a specific effect on scar formation.

[0275] Transforming growth factor β1 (Tgfβ1) is expressed at high levels in post-MI scars and is a key inducer of collagen deposition and differentiation of fibroblasts into myofibroblasts (11). To evaluate whether Chrdl1 could regulate fibroblast transdifferentiation induced by Tgfβ1, primary adult mouse cardiac fibroblasts were treated with various doses of Tgfβ1 (1-10-50 ng / ml) in the presence or absence of recombinant Chrdl1 (100 ng / ml) for 3 days ( Figure 7 A). Tgfβ1 induced a substantial and dose-dependent increase in the expression of collagen α-1(I) (Col1α1) and α-Sma, whereas Chrdl1 attenuated this effect ( Figure 7 B).

[0276] To further investigate the role of Chrdl1 in the fibrotic response in vivo, hearts from Colla1(I)-EGFP mice (a transgenic mouse model in which EGFP is expressed exclusively in fibroblasts (12)) were transduced with AAV2 / 9-control or AAV2 / 9-Chrdl1 and MI was induced. Hearts from Colla1α1-eGFP mice overexpressing Chrdl1 showed significantly attenuated cardiac fibrosis and reduced expression of collagen 1α1 and α-SMA ( Figure 7 C). These data were also confirmed by quantifying Col1α1, α-SMA, Tgfβ1, and MMP9 transcript levels by q-PCR in CD1 mice 3 days after infarction ( Figure 7 D).

[0277] Example 8

[0278] Expression of Chrdl1, Fam3c, and Fam3b protects mice from doxorubicin-induced cardiotoxicity and death

[0279] Despite their effectiveness as anticancer drugs, anthracyclines (including doxorubicin) can induce acute and chronic cardiotoxicity (Swain, SM et al. (2003) Cancer 97: 2869-2879). In particular, cumulative doses of these drugs can lead to left ventricular systolic dysfunction and heart failure. The reported incidence of ventricular dysfunction due to treatment with these drugs may be as high as about 10% of patients (Cardinale, D. (2015) Circulation 131: 1981-1988), with the vast majority of cases occurring within the first year of treatment. Currently, there is no standard therapy to prevent anthracycline-induced cardiotoxicity (Zamorano, J Let al. (2016) Eur Heart J 37: 2768-2801).

[0280] Six-week-old female C57 / BL6 mice were injected intramyocardially with 30 μl of AAV9 vector preparations expressing Chrdl1, Fam3c, or Fam3b using a 30G needle syringe. One week later, doxorubicin was administered intraperitoneally at a concentration of 4 mg / kg on days 0, 2, 5, 8, 10, and 12 (cumulative dose: 24 mg / kg) according to the established chronic treatment regimen (Li M. et al. (2018) Circulation 138: 696-711). Echocardiography was performed on mice at weeks 0, 6, and 8.

[0281] Each of Chrdl1, Fam3c, and Fam3b showed strong protective activity against drug-induced death ( Figure 8 A). Significant cardioprotection was observed after treatment with any of the three factors, as evidenced by the reduction of left ventricular (LV) ejection fraction deterioration ( Figure 8 B) and adverse effects on LV internal diameter ( Figure 8 C) is shown by the remarkable protection.

[0282] References

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[0295] All publications mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the disclosed reagents, compositions, purposes and methods of the present invention will be apparent to the technician. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. In fact, various modifications of the disclosed mode for implementing the present invention that are apparent to the technician are intended to fall within the scope of the appended claims.

Claims

1. Use of Fam3c or a polynucleotide encoding the same in the preparation of a pharmaceutical composition for treating heart disease or reducing the risk of heart disease, wherein Fam3c consists of the Fam3c amino acid sequence of UniProt ID: Q92520-1, or wherein the Fam3c does not have a signal peptide.

2. Use of Fam3c or a polynucleotide encoding the same in the preparation of a pharmaceutical composition for maintaining the viability of cardiomyocytes, wherein Fam3c consists of the Fam3c amino acid sequence of UniProt ID: Q92520-1, or wherein the Fam3c does not have a signal peptide.

3. The use according to claim 1 or 2, wherein the Fam3c consists of the amino acid sequence of SEQ ID NO:

2.

4. The use according to claim 1 or 2, wherein the polynucleotide comprises a nucleotide sequence having at least 70% identity with SEQ ID NO:

5.

5. The use according to claim 1, wherein the heart disease is associated with cardiac ischemia or myocardial cell loss.

6. The use according to claim 1, wherein the heart disease is selected from myocardial infarction; reperfusion injury after percutaneous coronary intervention; myocarditis; hypertension; cardiotoxic damage; or cardiomyopathy.

7. The use according to claim 1 or 2, wherein the heart is protected from myocardial infarction; cardiac function is preserved after myocardial infarction or percutaneous coronary intervention; or post-infarction fibrosis is reduced.

8. Use according to claim 1 or 2, wherein heart failure is prevented.

9. The use according to claim 1 or 2, wherein the Fam3c is glycosylated.

10. The use according to claim 1 or 2, wherein the Fam3c is an Fc fusion protein.

11. The use according to claim 1 or 2, wherein the polynucleotide is in the form of a vector.

12. The use according to claim 1 or 2, wherein the polynucleotide is in the form of a viral vector.

13. The use according to claim 1 or 2, wherein the polynucleotide is in the form of an adeno-associated virus (AAV) vector.

14. Use of a vector in the preparation of a pharmaceutical composition for treating heart disease or reducing the risk of heart disease, wherein the vector comprises a nucleotide sequence encoding Fam3c, wherein Fam3c consists of the Fam3c amino acid sequence of UniProt ID: Q92520-1, or wherein the Fam3c does not have a signal peptide.

15. Use according to claim 14, wherein the vector is a viral vector.

16. Use according to claim 15, wherein the viral vector is an adeno-associated virus (AAV) vector.

17. The use according to claim 15, wherein the nucleotide sequence encodes Fam3c without a signal peptide.

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