KLF-induced myocardial regeneration

Abstract

The present technology relates to a method for inducing myocardial regeneration, which comprises administering a therapeutically effective amount of either or both of KLF1 and KLF2b to increase the levels of KLF1 and / or KLF2b in cardiomyocytes, thereby inducing myocardial regeneration.

Description

[Technical field]

[0001] Technical Field The present technology relates to methods for promoting myocardial regeneration (i.e., the formation of new cardiomyocytes as a result of cell division) by administering at least one of KLF1 or KLF2b, or at least one of KLF1 or KLF2b nucleic acids to cardiomyocytes. Additionally, the present technology relates to promoting myocardial regeneration in a subject by administering at least one of KLF1 or KLF2b proteins or nucleic acids to the subject.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Australian Provisional Patent Application No. 2019902703, which is incorporated by reference in its entirety. [Background technology]

[0003] background Cardiomyocytes in the adult mammalian heart are terminally differentiated cells that have exited the cell cycle and have limited proliferative capacity. As a result, the death of mature cardiomyocytes in pathological cardiac disease results in high mortality and morbidity. For example, the high mortality and morbidity associated with myocardial infarction is largely due to the human heart's extremely limited ability to repair itself by regenerating new cardiomyocytes (myocardial regeneration). As a result, the infarcted myocardium is replaced by fibrous scar tissue that cannot be contacted, resulting in reduced cardiac pump activity, heart failure, and / or sudden death due to arrhythmias.

[0004] In contrast to mammals, certain vertebrates, such as the teleost zebrafish, show complete, scar-free regeneration of the heart after myocardial infarction. Fate-mapping studies have shown that cardiomyocytes, rather than stem cells, are the primary source of new myocardium in regenerating zebrafish hearts. Importantly, a similar regenerative capacity was found in neonatal mice. However, the self-renewal capacity of mammalian cardiomyocytes declines soon after birth.

[0005] Cardiovascular diseases can induce a severe and progressive loss of contractile myocardial tissue as a result of the loss of billions of cardiomyocytes (CMs). Because the mammalian heart has a poor regenerative capacity, this ultimately leads to heart failure, and there are currently no therapeutic options to robustly restore lost CMs.

[0006] KLF1 is a Kruppel-like transcription factor known to play an important role in red blood cell development, and mutations in the KLF1 gene cause congenital anemia. The present inventors have discovered a previously unknown role for KLF1 in myocardial regeneration. Furthermore, the present inventors have demonstrated that overexpression of KLF1 in adult cardiomyocytes induces myocardial regeneration in the adult mammalian heart, leading to cardiac regeneration. Summary of the Invention

[0007] summary In a first aspect, a method for inducing myocardial regeneration is provided, comprising administering a therapeutically effective amount of KLF to cardiomyocytes or inducing expression of KLF in cardiomyocytes, e.g., increasing the levels of KLF1 and / or KLF2b in cardiomyocytes, thereby inducing myocardial regeneration.

[0008] In one embodiment, the population of cardiomyocytes are infant, child or adult cardiomyocytes. The population of cardiomyocytes may be isolated from a subject or may be present in a subject.

[0009] In one embodiment, myocardial regeneration promotes cardiac regeneration in a subject. Cardiac regeneration may be characterized by an increase in ejection fraction, fractional shortening, or both.

[0010] Cardiac regeneration can be characterized by an increase in vascular endothelial cells, epicardial cells, or both.

[0011] KLF may induce dedifferentiation of cardiomyocytes to produce proliferative cardiomyocytes, preferably the proliferative cardiomyocytes are mitotic.

[0012] The method may further comprise expanding the proliferative cardiomyocytes in the presence of KLF to produce a population of proliferative cardiomyocytes.

[0013] Proliferating cardiomyocytes preferentially metabolize glucose using the pentose phosphate pathway, the serine synthetic pathway, or both.

[0014] The method may further comprise differentiating the population of proliferative cardiomyocytes to produce a population of cardiomyocytes. Differentiation may occur in the substantial absence of KLF or after induction of KLF is discontinued.

[0015] In some embodiments, KLF induces chromatin remodeling to promote dedifferentiation.

[0016] KLF-induced chromatin remodeling can reduce the accessibility of the binding sites for one or any combination of MEF2C, GATA4, MEF2A and NKX2.5.

[0017] The KLF can be KLF1, KLF2b or both, the nucleic acid of KLF1 and / or KLF2b, or a vector comprising at least one of these nucleic acids. The vector can comprise the nucleic acid operably linked to a promoter. The promoter can be a cardiac specific promoter. In an embodiment in which a population of cardiomyocytes is present in a subject, the KLF is administered to the subject. For example, the KLF can be administered to the heart of the subject.

[0018] The KLF1 protein can be SEQ ID NO:1, 11, or a protein at least 80% identical to SEQ ID NO:1 or 11. The KLF1 nucleic acid can comprise or consist of any one of SEQ ID NO:2, 3, 4, 5, 9, 10, or a nucleic acid at least 80% identical to any one of SEQ ID NO:2, 3, 4, 5, 9, 10.

[0019] The KLF2b protein may be SEQ ID NO: 6, or a protein at least 80% identical to SEQ ID NO: 6. The KLF2b nucleic acid may comprise or consist of SEQ ID NO: 7 or 8, or a nucleic acid at least 80% identical to any one of SEQ ID NO: 7 or 8.

[0020] The promoter may be an alpha-myosin heavy chain (α-MHC) promoter, a myosin light chain 2 (MLC-2) promoter, a cardiac troponin C (cTnC) promoter, an NCX1 promoter, or a TNNT2 promoter. The promoter may be used to provide CM expression of a vector encoding KLF1 or KLF2b.

[0021] The promoter may be an inducible promoter, such as a tetracycline-inducible promoter, a steroid hormone (e.g., progesterone or ecdysone)-inducible promoter, a hypoxia-inducible promoter, a promoter specific to a region near the site of cardiac injury, or a promoter that responds to stress resulting from ischemia and reperfusion.

[0022] The proliferative cardiomyocytes may be cardiomyocyte progenitor cells, immature cardiomyocytes, cardiomyocytes with an embryonic phenotype, or any combination thereof.

[0023] In some embodiments, cardiomyogenesis does not involve reprogramming of the cardiomyocyte lineage.

[0024] In some embodiments, the proliferating cardiomyocytes re-enter the cell cycle.

[0025] In some embodiments, the proliferative cardiomyocytes are characterized by an increased dependency on the pentose phosphate pathway (PPP), the serine synthesis pathway, or both, as compared to cardiomyocytes.

[0026] In one embodiment, myocardial regeneration includes expansion of epicardial and endothelial cells.

[0027] In one embodiment, myocardial regeneration is characterized by an increase in the number of epicardial cells, vascular endothelial cells, or both.

[0028] The subject may have a cardiac disease characterized by loss of cardiac muscle cells, such as myocardial infarction, ischemic cardiomyopathy, dilated cardiomyopathy, or heart failure.

[0029] In a second aspect, there is provided a population of cardiomyocytes or proliferative cardiomyocytes produced by the method of the first aspect.

[0030] In a third aspect, there is provided a composition comprising cardiomyocytes or proliferative cardiomyocytes produced by the method of the first aspect.

[0031] In a fourth aspect, there is provided a method of treating a cardiac disease in a subject comprising administering to the subject a cardiomyocyte or a population of proliferative cardiomyocytes of the second aspect, or a composition of the third aspect.

[0032] In a fifth aspect there is provided the use of a population of cardiomyocytes or proliferative cardiomyocytes of the second aspect, or a composition of the third aspect, in the manufacture of a medicament for the treatment of cardiac disease.

[0033] definition As used herein, unless the context clearly requires otherwise, the term "KLF" refers to either or both of KLF1 and KLF2b.

[0034] Throughout this specification, unless the context clearly requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of other elements, integers or steps, or groups of elements, integers or steps.

[0035] Throughout this specification, the term "consisting of" means consisting only of.

[0036] Any discussion of documents, acts, materials, devices, articles or the like included in this specification is solely for the purpose of providing a context for the present technology and should not be construed as an admission that any or all of such matters formed part of the prior art base or were common general knowledge in the art relevant to the present technology prior to the priority date of each claim herein.

[0037] Unless otherwise required by context or clearly stated to the contrary, integers, steps, or elements of the techniques described herein as singular integers, steps, or elements explicitly encompass both the singular and plural forms of the described integer, step, or element.

[0038] In the context of this specification, the terms "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, a reference to "an element" means one element or more than one element.

[0039] In the context of this specification, the term "about" means that reference to a number or value is not to be taken as an absolute number or value, but includes room for variation above or below the number or value along those that one of ordinary skill in the art would understand in accordance with the art, including within the bounds of typical error or instrument limitations. In other words, use of the term "about" is understood to refer to a range or approximation that one of ordinary skill in the art would consider equivalent to the referenced value in the context of achieving the same function or result.

[0040] As used herein, the terms "treat", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing the disease and / or side effects resulting from the disease. "Treatment", as used herein, is directed to any treatment of disease in a mammal, particularly a human, including: (a) preventing the disease from occurring in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting the onset of the disease; and (c) palliating the disease, i.e., causing regression of the disease.

[0041] The terms "individual," "subject," and "patient," used interchangeably herein, refer to animals such as, but not limited to, fish (e.g., zebrafish), rodents (rats, mice), non-human primates, humans, dogs, cats, and ungulates (e.g., horses, cows, sheep, pigs, quail). In some embodiments, the subject is a human.

[0042] "Therapeutically effective amount" or "efficacious amount" means the amount of a compound sufficient to effectively effect such treatment for a disease when administered to a mammal or other subject. A "therapeutically effective amount" will vary depending on the compound or cells, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0043] Those skilled in the art will understand that the technology described herein is susceptible to variations and modifications other than those specifically described.The technology is understood to include all such variations and modifications.For the avoidance of doubt, the technology also includes all steps, features, and compounds referred to or shown in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features, and compounds. [Brief description of the drawings]

[0044] [Figure 1] Figure 1: Klf1 expression in regenerating zebrafish hearts. (A) qPCR analysis of uninjured (No injury) and injured (7 dpi) ventricles. (B) Semi-qPCR. Cardiomyocytes (CM), endocardial cells (End) and epicardial cells (Epi) were purified by fluorescence-activated cell sorting (FACS) from uninjured and 7-day post-injury (dpi) ventricles of Tg(cmlc2:EGFP), Tg(fli1a:EGFP) and TgBAC(tcf21:DsRed2) fish, respectively. (C) RNAScope analysis. Arrowheads indicate klf1 mRNA in myocardium. Arrows indicate klf1 mRNA in hemocyte progenitor-like cells. ***p < 0.005.

[0045] [Diagram 2] Figure 2: Klf1 function in zebrafish cardiac regeneration. (A) Picromalory staining of ventricles from control (KlfDN-OFF) or transgenic zebrafish expressing dominant-negative Klf1 (KlfDN-ON). Dotted line indicates resection plane. dpi indicates days after injury. Details of the transgenic lines expressing KlfDN are described in Figure 11A. (B) Quantification of proliferating CMs detected by immunofluorescence for myocyte nuclear markers, myocyte enhancer factor 2 (Mef2), and proliferating cell nuclear antigen (PCNA). *P < 0.05.

[0046] [Diagram 3]Figure 3: Expression and function analysis of klf1 in regenerating zebrafish hearts. (A) Quantitative reverse transcription PCR (RT-qPCR) analysis of klf1 in injured ventricles (mean ± SEM, n = 5-6). Gene expression is shown relative to levels in uninjured control groups at 0 dpi (days after injury). (B) RT-qPCR analysis of klf1 in purified cardiac cells (mean ± SEM). Cardiomyocytes (CM), endocardial cells (End) and epicardial cells (Epi) were purified using fluorescence-activated cell sorting (FACS) from uninjured and 7 dpi ventricles of Tg(cmlc2:EGFP), Tg(fli1a:EGFP) and TgBAC(tcf21:DsRed2) fish, respectively. Gene expression is shown relative to levels in uninjured cardiomyocytes. In situ hybridization of klf1 mRNA using RNAscope and immunofluorescence for TnC in regenerating hearts (not shown) demonstrated that klf1 mRNA was detected in hematopoietic cells and myocardium. (C) Cre-dependent conversion of klf1ct from a non-mutagenic to a mutagenic orientation. See Figure 9 for details on the construction and characterization of klf1ct. (D) Pichromallory staining of ventricular sections from control (klf1-CT) and klf1-deficient hearts (klf1-MT) was performed to quantify regeneration (n = 20-22). (E,F) Immunofluorescent staining of myosin heavy chain (MHC) or cardiac troponin C (TnC) using either smooth muscle protein 22α (Sm22) or Alcam, and box plots (n = 5-6) generated to show quantification of Sm22+MHC+ (E) or Alcam+TnC+ (F) regions. (G) Immunofluorescence staining for myocyte nuclear markers, myocyte enhancer factor 2 (Mef2) and proliferating cell nuclear antigen (PCNA). Box plots were generated to show quantification of Mef2+PCNA+ cells (n = 7-9). *p < 0.05, **p < 0.01, ****p < 0.001 by unpaired t-test (E, F, G).

[0047] [Figure 4]Figure 4: Zebrafish Klf1-induced CM dedifferentiation. Klf1 overexpression induces expression of a dedifferentiation marker (runx1; A) and suppresses differentiated muscle markers (vmhc, actc1a, myom2a; B). qPCR analysis of Klf1-ON and OFF ventricles at 7 days after Klf1 overexpression (ON). Details of the transgenic lines used for Klf1 overexpression are described in Figure 12A. *P<0.05; **P<0.01; ***P<0.005.

[0048] [Diagram 5] Figure 5: Zebrafish Klf1 function in CM proliferation. (A) EdU was injected once daily from days 9 to 11 after ON, and S-phase CM was quantified as EdU+ CM at day 12 after ON. (B) Mitotic CM was quantified as phospho-histone H3 (pHH3)+ CM at day 12 after ON. (C) qPCR analysis of cell proliferation markers. Details of the transgenic lines used for Klf1 overexpression are described in Figure 12A. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001. ND, not detectable.

[0049] [Figure 6] Figure 6: Mouse Klf1 function. (A) Semi-qPCR analysis. Neonatal mouse hearts were injured on postnatal day 3 and harvested on day 6. (B) CM proliferation was assessed by co-labeling with cardiac troponin T (TnT) and Ki67 and quantified in non-injured adult mouse hearts injected with control (GFP) or KLF1 adenovirus (KLF1). Co-labeling was confirmed in the xz and yz planes. (C) Analysis of S-phase CMs. EdU+ nuclei were included and quantified by WGA. *P<0.05; **P<0.01. dpt is days post-transfection; LV is left ventricle.

[0050] [Figure 7]Figure 7: Mouse Klf1 function in myocardial repair. (A) CM proliferation was assessed by co-labeling with TnT and Ki67 and quantified at 14 days post-myocardial infarction (MI). (B) CM mitosis was assessed by co-labeling with TnT and pHH3 and quantified at 14 days post-MI. (C) Analysis of hearts treated with control (Ad-GFP) or KLF1 adnovirus (Ad-Klf1) by Gomori's trichrome staining at 28 days post-MI. (D) Echo analysis of hearts treated with Ad-GFP or Ad-Klf1.

[0051] [Figure 8] Figure 8: (A) Zwitch2 gene trap cassette. (B) Schematic diagram of Cre-mediated inactivation of klf1 gene. (C) Myocardial-specific inactivation of klf1 gene expression. (D) Suppression of CM proliferation by myocardial-specific inactivation of klf1 gene expression. (E, F) Reduction of CM dedifferentiation markers by myocardial-specific inactivation of klf1 gene expression.

[0052] [Figure 9]Figure 9: Generation and characterization of zebrafish klf1 conditional alleles. Zwitch2 consists of a removable cassette (LG-tag) flanked by a triplet polyA sequence (3xBGHpA) followed by a splice acceptor site and a flipase recognition target (FRT), in which enhanced green fluorescent protein (EGFP) is expressed under the control of the lens-specific alpha A crystallin (cryaa) promoter and used for screening. The segment containing the splice acceptor site, P2A peptide sequence and 3xBGHpA is flanked by tandem loxP and lox5171 sites in inverted orientations at both ends. Cre-mediated recombination against this Cre target site configuration permanently inverts the cassette, and the splice acceptor disrupts the normal splicing pattern. (A) Schematic diagram of the zebrafish klf1 wild-type (+) allele, the transcription activator-like effector nucleases (TALENs) used to induce a DNA double-strand break in intron 1, and the resulting conditional klf1ct allele. Exons are indicated by numbered filled boxes. Binding sites of the TALEN pair are highlighted in blue, and the TALEN target site is indicated by a dashed arrow. (B) Genomic PCR analysis of the correct insertion of Zwitch2 using the primers indicated in (A). Primers within LA were used as a control. (C) Southern blot analysis of the Zwitch2-modified klf1 allele. The HpaI recognition site is indicated in (A). (D) Representative image of an embryo with a correct insertion of Zwitch2 showing expression of EGFP in the lens (arrow). (E) Genomic PCR analysis of the Zwitch2 inversion in embryos. klf1ct / + fish were crossed with Tg(ubb:Cre-GFP), and genomic DNA from 4-7 dpf embryos of each genotype was analyzed using PCR with the primers indicated in (A). Control primers were the same as those used in (B). Cre-F and Cre-R primers were used to confirm Cre-GFP. (F) RT-qPCR analysis of 7 dpf embryos of each genotype (mean ± SEM, n = 3-4). One embryo per sample was used for RT-qPCR analysis.(G) Genomic PCR analysis of 4-hydrotamoxifen (4-HT)-dependent Zwitch2 inversion in adult hearts. klf1-CT and klf1-MT denote zebrafish carrying the klf1ct / ct and cmlc2:CreER; klf1ct / ct transgenes, respectively. Control primers were the same as in (B). (H) RT-qPCR analysis of 4-HT-treated, non-injured and 7 dpi ventricles of klf1-CT and klf1-MT fish (mean ± SEM, n = 4). BGHp (A) is bovine growth hormone polyadenylation signal; cryaa is alpha A-crystallin; dpf is days postfertilization; dpi is days postinjury; klf1+ / + is clutch-mate control; LA is left arm; NS is not significant; RA is right arm. *p < 0.05, unpaired t test.

[0053] [Figure 10]Figure 10: Klf1 function in zebrafish development. (A) Severe cardiac edema was observed in ubb:Cre-GFP; klf1ct / ct embryos at 7 dpf, but not in ubb:Cre-GFP; klf1+ / + embryos (clutch-mate control). n = 3 (+ / +), 4 (ct / +) or 4 (ct / ct). (B) Severe cardiac edema was observed in ubb:Cre-GFP; actb2-BS-dn-klf1 embryos at 7 dpf (arrowheads), but not in ubb:Cre-GFP embryos (clutch-mate control) (n = 7). Details of actb2-BS-dn-klf1 are described in Figure 11A. (C) Immunofluorescence of myocyte enhancer factor 2 (Mef2) and myosin heavy chain (MHC) using ventricular sections of klf1-CT and klf1-MT embryos. Embryos were treated with 4-hydroxytamoxifen (4-HT) from 1 to 3 dpf and analyzed at 7 dpf. Quantification of Mef2+ nuclei in single optical planes (mean ± SEM, n = 7-9). (D) Immunofluorescence for Mef2 and MHC in ventricular sections of actb2:BS-dn-klf1 (clutch-mate control) and cmlc2:CreER; actb2:BS-dn-klf1 embryos. Embryos were treated with 4-HT from 1 to 3 dpf and analyzed at 7 dpf. Quantification of Mef2+ nuclei in single optical planes (mean ± SEM, n = 6-8). (E) Immunofluorescence for Mef2 and MHC in ventricular sections of klf1ct / ct (clutch-mate control) and ubb:Cre-GFP; klf1ct / ct embryos at 4 dpf. Quantification of Mef2+ nuclei in single optical planes (mean ± SEM, n = 5-7). (F) Immunofluorescence for Mef2 and MHC in ventricular sections of actb2:BS-dn-klf1 (clutch-mate control) and ubb:Cre-GFP;actb2:BS-dn-klf1 embryos at 4 dpf. Quantification of Mef2+ nuclei in single optical planes (mean ± SEM, n = 8-9). Note that cardiac edema is prominent from 5 dpf onwards, but not at 4 dpf. dpf is days post-fertilization. *p < 0.05, ****p < 0.001 by χ2 test (A), Fisher's exact test (B), or unpaired t test (CF).

[0054] [Figure 11] Figure 11: Impairment of cardiac regeneration by expression of dominant-negative Klf1 in zebrafish. (A) Tg(actb2:loxP-TagBFP-STOP-loxP-dn-klf1)vcc22 zebrafish line (hereafter actb2:BS-dn-klf1) was established and crossed with cmlc2:CreER expressing dn-Klf1 specifically in cardiomyocytes upon 4-hydroxytamoxifen (4-HT) treatment. EnR is the engrailed repressor domain. (B) Pichromallory staining was performed to quantify regeneration (n = 5-7). (C) Immunofluorescence for smooth muscle protein 22 alpha (Sm22) and myosin heavy chain (MHC) was performed to generate box plots showing quantification of Sm22+MHC+ regions (n ​​= 4-5). (D) Immunofluorescence for myonuclear markers Mef2 and proliferating cell nuclear antigen (PCNA) was performed and box plots were generated showing quantification of Mef2+PCNA+ cells (n = 9-10). ***p < 0.005, ****p < 0.001 by Fisher's exact test (B) or unpaired t-test (C,D). Scale bar 50 μm.

[0055] [Figure 12]Figure 12: Gain-of-function analysis of zebrafish cardiac Klf1. (A) Tg(actb2:loxP-TagBFP-STOP-loxP-3xHA-klf1)vcc29 fish were crossed with cmlc2:CreER fish, and the double transgenic fish and Cre-negative clutchmates are designated klf1-ON and klf1-OFF, respectively. (B) 4-hydroxytamoxifen (4-HT) treatment regimen used for klf1-OFF and klf1-ON fish. Immunofluorescence was performed for myosin heavy chain (MHC), smooth muscle protein 22 alpha (Sm22), troponin C (TnC) or Alcam. Sarcomere disassembly was detected by immunofluorescence for actinin and TEM from day 7 onwards. (C) Immunofluorescence for myocyte nuclear markers myocyte enhancer factor 2 (Mef2) and proliferating cell nuclear antigen (PCNA) was performed and quantified (mean ± SEM, n = 7). (D) EdU incorporation assay. Images were collected on the xz and yz planes of the enlarged compartment area and quantified (mean ± SEM, n = 8). EdU+ cardiomyocytes were counted only when EdU+ nuclei were in cmlc2:GFP+ myocardium in the z plane. (E) Immunofluorescence for phospho-histone H3 (pHH3) was performed and quantified (mean ± SEM, n = 6). pHH3+ cardiomyocytes were counted only when pHH3+ nuclei were in cmlc2:GFP+ myocardium in the z plane. (F) Tg(cmlc2:3xHA-klf1-ER; cryaa:TagBFP)vcc32 (hereafter klf1-ER), a transgenic line that allows transient nuclear translocation of Klf1 in cardiomyocytes. (G) 4-HT treatment regimen used for klf1-ER fish. klf1-ER fish were treated with vehicle or 4-HT O / N for 7 days, followed by a 30-day recovery period under normal aquarium conditions. (H) Gross morphology of klf1-ER hearts was analyzed 30 days after treatment with vehicle or 4-HT for 7 days. (I) PicroMallory staining of heart sections from (H). (J) Quantification of (I). (K,L) Cell size quantification (K) [n = 332 (vehicle), 326 (4-HT)] and cell number counts (L) of dissociated CMs from (H) (mean ± SEM, n = 3). Representative data from two independent analyses are shown in (K) and (L).at, atrium; ba, bulbus arteriosus; DAPI, 4',6-diamidino-2-phenylindole; dpt, days after treatment; 5dU, 5-ethynyl-2'-deoxyuridine; O / N, overnight; TEM, transmission electron microscope; Veh, vehicle; vt, ventricle. *p < 0.05, **p < 0.01, ****p < 0.001 by Mann-Whitney U test (C, D, E) or unpaired t test (K, L). Scale bars are 500 μm in (H, I).

[0056] [Figure 13] Figure 13: Functional analysis of Klf1-related family members and non-myocardial effects of Klf1 expression. Quantification of vasculature (A) (mean ± SEM, n = 4) and epicardial cell area (B) (mean ± SEM, n = 3-4) was performed using klf1-ON or klf1-OFF cardiac tissue sections. (C) Quantification of pHH3+actinin+cardiomyocytes in sections of hearts expressing Klf1, Klf2a, Klf2b, and Klf4 (mean ± SEM, n = 4). Klf2a, Klf2b, and Klf4 were expressed in cardiomyocytes in an inducible manner as described for klf1-ON (Figure 12A). pHH3+ nuclei within actinin+ myocardium in the z-plane were counted as mitotic cardiomyocytes. pHH3 is phospho-histone H3. *p<0.05, ****p<0.001 by unpaired t-test.

[0057] [Figure 14] Figure 14: Cardiac dysfunction in klf1-ON zebrafish. (A) Heart failure-like phenotypes, such as raised scales (brackets), blood congestion (arrows), and abdominal edema (arrowheads), in klf1-ON fish at 12 dpt. (B) Kaplan-Meier survival curves demonstrating significantly reduced survival of klf1-ON fish (n = 10; p < 0.0001, log-rank test).

[0058] [Figure 15]Figure 15: Epigenetic analysis of Klf1-induced cardiac regrowth in zebrafish. (A,B) Heatmaps of 3xHA-Klf1 ChIP-seq read density were generated from klf1-OFF and ON ventricles at 7 dpt (A) and motifs within ±100 bp from the apex of the Klf1 peak were enriched (B). (C) Functional annotation of Klf1 peaks using GREAT. (D) Sorted heatmaps of 5-methylcytosine (5mC) levels at Klf1 ChIP-seq peaks and normalized ChIP-seq read density for H3K27ac, H3K4me1 or H3K4me3 in klf1-OFF and ON ventricles at 7 dpt. Klf1 ChIP-seq peaks were divided into two categories based on H3K27ac / H3K4me1 (enhancer) and H3K4me3 profiles (promoter) and the heatmaps are displayed accordingly. (E) Heatmap of ATAC-seq reads obtained from klf1-OFF and ON ventricles at 7 dpt. (F) Heatmap of H3K27ac ChIP-seq peaks centered around differentially enriched ATAC-seq peaks obtained in (E). (G) Boxplot showing mRNA expression of all genes and genes closest to the reduced ATAC-seq peaks in klf1-ON hearts in (E). (H) Functional annotation of the reduced ATAC-seq peaks in klf1-ON hearts using GREAT. (I) GSEA plot demonstrating KEGG gene set enrichment scores of myocardial contractile transcripts in RNA-seq data from klf1-OFF and ON hearts at 7 dpt (FDR < 1.0 × 10-6).Bp: base pairs; ChIP: chromatin immunoprecipitation; dpt: days post-treatment; FC: fold-change; FDR: false discovery rate; GREAT: genomic regions enrichment of annotations tool; GSEA: gene set enrichment analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes.

[0059] [Figure 16]Figure 16: Transcriptome and metabolomic analysis of Klf1-induced zebrafish heart regrowth. (A,B) Enrichment analysis of RNA-seq data of klf1-OFF and ON hearts at 7 dpt demonstrating up- and down-regulated gene sets from GO bioprocess (A) and KEGG pathways (B). (C-E) GSEA plots from analysis of RNA-seq data of klf1-OFF and ON hearts at 7 dpt demonstrating enrichment scores of gene signatures such as cell division (C), DNA replication (D) and generation of precursor metabolites and energy (E). (F) Mitochondrial ultrastructure of klf1-OFF and ON ventricular myocardium was analyzed by TEM and clister number was quantified (mean ± SEM, n = 30-32). (G) Quantification of mitochondrial DNA content (mtDNA) in klf1-OFF and ON ventricles at 7 dpt using qPCR (mean ± SEM, n = 3). Expression of mtDNA (mt-co1, mt-nd1) was normalized to nuclear DNA (nDNA, actb2) expression and expressed relative to levels in klf1-OFF controls. (HK) Quantification of NADH (H), NAD+ (I), NADH / NAD+ ratio (J), and ATP (K) in klf1-OFF and ON ventricles at 7 dpt (mean ± SEM, n = 3-4). (L) Expression values ​​(in FPKM) of genes controlling mitochondrial biogenesis and function derived from RNA-seq data in klf1-OFF and ON ventricles at 7 dpt (mean ± SEM, n = 4). (MP) Mass spectrometry quantification of glucose 6-phosphate (M), ribose 5-phosphate (N), sedoheptulose 7-phosphate (O), and serine (P) in klf1-OFF and ON ventricles at 7 dpt (mean ± SEM, n = 5). Quantitative values ​​are expressed relative to the levels in the klf1-OFF control. (QS) Quantification of NADPH (Q), NADP+ (R), and NADPH / NADP+ ratio (S) in klf1-OFF and ON ventricles at 7 dpt (mean ± SEM, n = 3).FDR is false discovery rate; FPKM is fragments per kilobase of exon per million mapped reads; GO is gene ontology; GSEA is gene set enrichment analysis; KEGG is Kyoto Encyclopedia of Genes and Genomes, unpaired t-test, *p < 0.05, **p < 0.01, *** p < 0.005, ****p < 0.001.

[0060] [Figure 17]Figure 17: Comprehensive analysis of Klf1 function in mouse hearts. (A) Time course qRT-PCR analysis of mouse Klf1 (mKlf1) expression in neonatal and adult mouse hearts after myocardial infarction (mean ± SEM, n = 3-4). Gene expression is expressed relative to levels in uninjured controls (0 dpi). MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery in adult and neonatal mice on postnatal day 2. (B) Adenoviral vectors used in this study. (C) Experiments and analyses performed in this study. Echo is echocardiogram. (D) Immunohistochemistry of enhanced green fluorescent protein (EGFP) reporter expression from Ad-mKlf1. Dotted lines outline the area of ​​infarction. (EG) Time course echocardiograms of Ad-GFP (control) and Ad-mKlf1-injected hearts (mean ± SEM, n = 7-11). Ejection fraction (E) and fractional shortening (F) are shown, as well as representative B-mode and M-mode images (G). Baseline cardiac function was measured before MI and designated 0 dpi (E, F). (H) Gomori trichrome staining of tissue sections from Ad-GFP- or Ad-mKlf1-treated hearts. Two independent hearts are shown for each treatment group. (I, J) Quantification of cardiac repair (I) and scar tissue size (J) in (H) (mean ± SEM, n = 8). (K) Immunofluorescence of TnT and WGA in the injured border zone myocardium in cross sections was performed and quantified (mean ± SEM, n = 5). (LN) Quantification of Ki67+TnT+ cardiomyocytes (L; mean ± SEM, n = 5), EdU+TnT+ cardiomyocytes (M; mean ± SEM, n = 3-4), and pHH3+TnT+ cardiomyocytes (N; mean ± SEM, n = 5) in Ad-GFP (control) and Ad-mKlf1-injected hearts. dpi, days post-treatment; EdU, 5-ethynyl-2'-deoxyuridine; pHH3, phospho-histone H3; WGA, wheat germ agglutinin. *p < 0.05, *** p < 0.005 by unpaired t test for all panels except (I), which was analyzed by chi-square test.

[0061] [Figure 18] Figure 18: Effects of mKlf1 overexpression on mouse survival and non-myocardial tissues. (A) Immunofluorescence for Ki67 was performed in liver tissue sections from mice intravenously injected with Ad-GFP or Ad-mKlf1, and immunostaining for EGFP was shown to verify mKlf1 expression. Ki67+ hepatocytes were visualized by autofluorescence, morphologically identified in the images, and quantified (mean ± SEM, n = 10). (B) Immunofluorescence for CD31 was performed in ventricular sections from mice after MI injected with Ad-GFP and Ad-mKlf1, and capillaries were quantified (mean ± SEM, n = 5). Ad-GFP, adenovirus vector containing green fluorescent protein construct (control); Ad-mKlf1, adenovirus vector containing mKlf1 construct; ***p < 0.005 by unpaired t-test (A, B).

[0062] [Figure 19]Figure 19: Role of Hippo and ErbB signaling pathways in Klf1-induced myocardial regeneration. (A, B) GSEA plots demonstrating enrichment scores of KEGG gene sets related to Hippo signaling (A) and ErbB signaling (B) pathways in RNA-seq data of klf1-OFF and ON hearts at 7 dpt. (C, D) Immunofluorescence for myocyte enhancer factor 2 (Mef2) and proliferating cell nuclear antigen (PCNA) in 7 dpt klf1-ON hearts subjected to pharmacological inhibition of YAP or ErbB showed proliferating cardiomyocytes co-labeled with Mef2 and PCNA. Mef2+PCNA+ cells were quantified in hearts with inhibition of YAP (C; mean ± SEM, n = 4) or ErbB (D; mean ± SEM, n = 4). dpt: days post-treatment; FDR: false discovery rate; GSEA: gene set enrichment analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; YAP: yes-associated protein.

[0063] Description of the embodiments Since adult cardiomyocytes are terminally differentiated cells with limited proliferation capacity, many cardiac diseases involve loss of cardiomyocytes that are not replaced. As demonstrated herein, administration of KLF or KLF nucleic acid is useful for promoting myocardial regeneration. As demonstrated herein, each of KLF1 and KLF2b is useful for promoting myocardial regeneration. This process involves dedifferentiation of adult cardiomyocytes, which then proliferate and subsequently differentiate into cardiomyocytes. That is, administration of at least one KLF is useful for promoting or inducing myocardial regeneration in the adult heart.

[0064] In one embodiment, KLF-induced cardiomyocyte regeneration occurs not by reprogramming cell lineages, but by reprogramming the state of adult cardiomyocytes to a proliferative state, which may involve the reprogrammed cardiomyocytes promoting growth in neighboring tissues.

[0065] In some embodiments, myocardial regeneration includes expansion of epicardial and endothelial cells.

[0066] method For example, increasing the level or activity of KLF by administering at least one KLF nucleic acid to cardiac tissue is useful for promoting or inducing myocardial regeneration in vitro or in vivo.Therefore, a method for promoting or inducing myocardial regeneration is provided, comprising administering at least one KLF, or at least one KLF nucleic acid, or at least one expression vector comprising KLF nucleic acid to a subject.In one embodiment, myocardial regeneration is associated with cardiomyocyte dedifferentiation, for example adult cardiomyocyte dedifferentiation.

[0067] Suitable subjects include individuals with heart disease (e.g., mammalian subjects such as humans; non-human primates; experimental non-human mammalian subjects such as mice, rats, etc.). Heart disease can result in ischemic heart tissue, such as individuals with coronary artery disease; and the like. Suitable subjects include those with degenerative heart disease, such as heart failure or familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease that results in ischemic cardiomyopathy.

[0068] The subject may be an infant, a child, or an adult.

[0069] In one embodiment, the method comprises administering KLF, KLF nucleic acid, or an expression vector comprising KLF nucleic acid to a subject's cardiomyocyte or myocardial tissue. Alternatively or additionally, the method may comprise inducing expression of KLF in a subject's cardiomyocyte. Addition or expression of active KLF in cardiomyocytes causes the cardiomyocytes to dedifferentiate. The resulting cells (proliferative cardiomyocytes) can then proliferate to replace at least some of the cardiomyocytes lost, for example, due to an ischemic event. After proliferation, the dedifferentiated cells redifferentiate into functional cardiomyocytes.

[0070] In some embodiments, KLF, KLF nucleic acid, or expression vector containing KLF nucleic acid is administered to cardiomyocytes or cardiac tissue. Administration to cardiomyocytes and / or cardiac tissue can be performed in vitro or in vivo. KLF, KLF nucleic acid, or expression vector containing KLF nucleic acid can be directly contacted with cells, i.e., applied directly to cells, or alternatively, for example, KLF, KLF nucleic acid, or expression vector can be injected into the bloodstream of a subject, thereby carrying the molecule to cells and indirectly binding to the cells. Alternatively or additionally, KLF, KLF nucleic acid, or expression vector containing KLF nucleic acid can be administered directly to the heart, for example, by injection into the cardiac muscle.

[0071] In these methods, a therapeutically effective amount of KLF, a KLF nucleic acid, or an expression vector is administered to the subject. In some embodiments, the administration includes delivering the KLF, a KLF nucleic acid, or an expression vector directly to cardiac tissue or to cardiomyocytes.

[0072] Administration of KLF, KLF nucleic acid or expression vector can be achieved by any method known in the art.In some embodiments, contacting cells with KLF or KLF nucleic acid occurs in vitro or in vivo.KLF or KLF nucleic acid can be directly contacted with cells, i.e., directly applied to cardiomyocytes, or alternatively, can be indirectly combined with cells, for example, by injecting KLF1 or KLF1 nucleic acid into subject's cardiac tissue.

[0073] In some embodiments, administration of KLF, a KLF nucleic acid, or an expression vector increases the level of KLF in cardiomyocytes or cardiac tissue compared to endogenous KLF levels. The term "endogenous" as used in this context refers to the "naturally occurring" levels of KLF expression and / or activity prior to administration of KLF, a KLF nucleic acid, or an expression vector.

[0074] Myocardial regeneration Cardiomyocyte regeneration is a complex process in which cardiomyocytes (CMs), which form the muscle tissue of the heart (myocardium), divide and generate new cells. As disclosed herein, KLF-induced cardiomyocyte regeneration is mediated by reprogramming of CMs and their expansion (proliferation) followed by redifferentiation into mature contractile CMs.

[0075] KLF induces dedifferentiation of cardiomyocytes to produce proliferative cardiomyocytes. That is, cardiomyocytes are terminally differentiated cells that cannot differentiate to form or regenerate other cell types in normal adult individuals. It is generally accepted that cardiomyocytes undergo terminal differentiation after birth, characterized by binucleation and centrosome disassembly, and are unable to regenerate the heart. However, as demonstrated herein, administration of KLF induces CMs to dedifferentiate into proliferative CMs. In some embodiments, the proliferative CMs are mitotic.

[0076] In some embodiments, the method includes expanding proliferative cardiomyocytes in the presence of KLF. This may occur in vitro under appropriate cell or tissue culture conditions, or may occur in vivo. The result of the expansion is that each CM produces a population of proliferative cardiomyocytes.

[0077] Once KLF is removed, or once KLF is metabolized or otherwise degraded, the population of proliferative cardiomyocytes will spontaneously differentiate to produce a population of cardiomyocytes. Thus, KLF can be used to increase the number of cardiomyocytes in vitro or in vivo.

[0078] In some embodiments, KLF induces chromatin remodeling to promote dedifferentiation.

[0079] In one embodiment, CM expressing KLF has a paracrine effect, for example, as exemplified herein, expression of KLF increases the number of epicardial and vascular endothelial cells.

[0080] In one embodiment, expression of KLF in CM under the control of the myosin heavy chain (MHC) promoter does not lead to expression of Raldh2 (also known as retinaldehyde dehydrogenase 2; Aldh1a2), a marker of epicardial and endothelial cells. Thus, expression of KLF in CM does not alter cell lineage. Also, growth for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days does not alter cell lineage. In some embodiments, expression of KLF by the MHC promoter is at reduced levels compared to normal CM.

[0081] Thus, KLF-induced cardiomyogenesis in the adult heart does not involve lineage reprogramming, but rather is characterized by reprogramming the state of adult CMs to a highly proliferative state with the ability to promote the growth of adjacent tissues.

[0082] In some embodiments, KLF-induced myocardial regeneration is characterized by a switch in energy production from oxidative phosphorylation (OXPHOS) to the pentose phosphate pathway (PPP) and / or the serine synthesis pathway (SPP).

[0083] KLF1 As used herein, "Kruppel-like factor" or "KLF" refers to any protein variant of KLF1 or KLF2b from any species (e.g., mouse, human, non-human primate), as well as any mutants and fragments thereof that retain KLF activity. Similarly, "KLF nucleic acid" refers to any nucleic acid sequence encoding KLF, e.g., from any species, e.g., mouse, human, or non-human primate. The amino acid sequence of human KLF1 is shown in SEQ ID NO:1, the nucleotide sequence of human KLF1 is underlined in SEQ ID NO:2, and the coding sequence is shown in SEQ ID NO:3. The nucleotide sequence of mouse KLF1 is shown in SEQ ID NO:4, and the coding sequence is shown in SEQ ID NO:5. KLF1 is also known as erythroid Kruppel-like transcription factor (EKLF), Kruppel-like factor 1, INLU, or HBFQTL6. The amino acid sequence of zebrafish KLF2b is shown in SEQ ID NO:6, the nucleotide sequence of zebrafish KLF2b is shown in SEQ ID NO:7, and the coding sequence is shown in SEQ ID NO:8. In addition, the zebrafish KLF1 amino acid sequence is shown in SEQ ID NO:11, the zebrafish KLF1 nucleotide sequence is shown in SEQ ID NO:9, and the coding sequence is shown in SEQ ID NO:10. [KK1]

[0084] In some embodiments, the KLFs may be from non-human or non-mammalian species, for example, the KLFs may be from a regenerating species such as a zebrafish, or a salamander or snake.

[0085] As described herein, KLF or nucleic acids encoding KLF (KLF nucleic acids) may be used to induce myocardial regeneration in vitro or in vivo. Thus, KLF and KLF nucleic acids may be used to treat conditions in which dedifferentiation, proliferation, or both of cardiomyocytes may be desirable, such as ischemic injury following myocardial infarction (MI); following cardiac injury, such as from cardiotoxic drugs (e.g., anthracycline antibiotics such as doxorubicin, cocaine, methamphetamine, cyclic antidepressants, calcium channel blockers, beta-blockers, and digoxin) or following trauma (whether accidental or intentional as a result of surgery); heart failure; or cardiac decline associated with aging.

[0086] The method disclosed herein can be used for cardiac regeneration. In this context, "cardiac regeneration" refers to the structural and / or functional regeneration or improvement of damaged heart. For example, structural improvement can be an increase in the amount or number of cardiomyocytes in the heart after administration of KLF. Examples of functional improvement include an increase in the contractile force or ejection fraction of the heart after administration of KLF.

[0087] For example, the ejection fraction, left ventricular fractional shortening, or both can be increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or more compared to the ejection fraction or left ventricular fractional shortening before administration of FLK.

[0088] In some embodiments, cardiac regeneration occurs up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days after administration of KLF.

[0089] The methods disclosed herein can utilize KLF mutants or KLF functional fragments. The mutants or functional fragments can bind DNA with the same specificity as wild-type KLF and retain at least one function of wild-type KLF. For example, in one embodiment, the mutants or functional fragments are any mutants of KLF that retain DNA binding activity.

[0090] In some embodiments, the KLF may be a conjugate or fusion protein. For example, a KLF fusion protein. A fusion protein comprises at least a portion of another protein, peptide or polypeptide, such as a nuclear localization sequence or at least a portion of a KLF linked via a peptide bond to an additional KLF or its domain, such as a transactivation domain. In some embodiments, the KLF may be fused to a transactivation domain from another protein, such as the transactivation domain p53 or VP16. The fusion protein may also comprise a marker protein (e.g., a fluorescent protein such as GFP) or a protein that aids in isolation and / or purification (e.g., a FLAG or His tag). The non-KLF sequence may be amino-terminal or carboxy-terminal to the KLF sequence.

[0091] In some embodiments, the KLF is fused to one or more nuclear localization sequences.

[0092] In some embodiments, a KLF nucleic acid encodes a KLF fusion protein.

[0093] In some embodiments, the method requires administering a KLF comprising a mature KLF amino acid sequence. Alternatively, the KLF may be at least 80% identical to the mature KLF amino acid sequence. Generally, the KLF useful in the methods described herein is at least 80% identical to the wild-type KLF amino acid sequence, for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identical to the wild-type amino acid sequence. Useful KFL proteins can be identified by routine experimentation. Generally, the KLF protein functions as a wild-type KLF.

[0094] The method includes administering a KLF nucleic acid that comprises a mature KLF coding sequence. Alternatively, the KLF nucleic acid may be at least 80% identical to the mature KLF coding sequence. Generally, the KLF nucleic acid useful for the methods described herein is at least 80% identical to the wild-type KLF nucleic acid, for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identical to the wild-type nucleic acid. Useful nucleic acids can be identified by routine experimentation. Generally, the nucleic acid must code for a protein that functions as a wild-type KLF.

[0095] In some embodiments, the KLF nucleic acid can be a viral expression vector, such as a ssRNA, dsRNA, dsDNA, or an expression vector, such as a plasmid expression vector, that contains the KLF nucleic acid. The KLF nucleic acid may contain one or more modifications.

[0096] In some embodiments, the KLF nucleic acid includes at least one nucleotide modified at the 2' position of the sugar, such as a 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro modified nucleotide. In other embodiments, the RNA modifications include 2'-fluoro, 2'-amino and 2'-O-methyl modifications on the ribose of pyrimidines, a basic residue at the 3' end of the RNA or an inverted base.

[0097] Many nucleotide and nucleoside modifications render the nucleic acids into which they are incorporated more resistant to nuclease digestion. Specific examples of modified KLF nucleic acids include those containing modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. In some embodiments, the modification of the KLF nucleic acid is a phosphorothioate backbone or a heteroatom backbone, particularly CH2-NH-O-CH2, CH, -N(CH3)-O-CH2 (known as a methylene (methylimino) or MMI backbone), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 or ON(CH3)-CH2-CH2 backbone (where the native phosphodiester backbone is represented as OPO-CH); an amide backbone; a morpholino backbone structure; a peptide nucleic acid (PNA) backbone (where the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone and the nucleotides are attached directly or indirectly to the aza nitrogen atoms of the polyamide backbone). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl hostotriesters, or borane phosphates.

[0098] Modified nucleic acid backbones that do not contain a phosphorus atom therein have ck backbones formed with short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S and CH2 moieties.

[0099] The one or more substituted sugar moieties can also include, for example, at the 2' position, one of the following: OH, SH, SCH3, F, OCN, OCHOCH3, OCHO(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 (n is from 1 to about 10); C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S- or N-alkyl; O-, S- or N-alkenyl; SOCH3; SO2; CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; a group for improving the pharmacokinetic properties of the nucleic acid; or a group for improving the pharmacodynamic properties of the nucleic acid and other substituents having similar properties. As another example, the nucleic acid sequence may include 2'-modified nucleotides such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0100] As another example, a KLF nucleic acid sequence can include at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-O-methyl modification. In another embodiment, the modification is 2'-methoxyethoxy [2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl)]. Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH3) or 2'-fluoro (2'-F), etc. Similar modifications can also be made at other positions on the nucleic acid, particularly the 3' position of the sugar on the 3' terminal nucleotide, and the 5' position of the 5' terminal nucleotide. Nucleic acids can also have sugar mimetics, such as cyclobutyls, in place of pentofuranosyl groups.

[0101] KLF nucleic acids can also additionally or alternatively contain base modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases that occur rarely or only temporarily in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, especially 5-methylcytosine (also called 5-methyl-2'deoxycytosine, often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC or gentobiosyl HMC, as well as synthetic nucleobases, such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolyl alkyl)adenine, 2-(amino alkyl amino)adenine or other hetero-substituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine or 2,6-diaminopurine. Inosine can also be included. Other modifications include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), and other synthetic and natural nucleobases such as 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl or other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl or 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, 3-deazaguanine and adenine.

[0102] It is not necessary for all positions in a given nucleic acid to be uniformly modified, but more than one of the aforementioned modifications may be incorporated within a single oligonucleotide, or even within a single nucleoside within an oligonucleotide.

[0103] In some embodiments, the nucleic acid is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.Such moieties include, but are not limited to, cholesterol moieties, thioethers such as hexyl-S-tritylthiol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylaminocarbonyl-toxycholesterol moieties.

[0104] In some embodiments, the KLF nucleic acid is operably linked to a promoter sequence. The promoter sequence may be constitutively active or constitutively active. In some embodiments, the promoter sequence is cardiac specific, for example, the cardiac specific promoter sequence may be an alpha-myosin heavy chain (α-MHC) promoter, a myosin light chain 2 (MLC-2) promoter, a cardiac troponin C (cTnC) promoter, an NCX1 promoter, or a TNNT2 promoter.

[0105] In some embodiments, the KLF nucleic acid is operably linked to an inducible promoter, such as a tetracycline-inducible promoter, a steroid hormone (eg, progesterone or ecdysone)-inducible promoter, or a hypoxia-responsive promoter.

[0106] In other embodiments, the KLF nucleic acid is operably linked to a promoter that is active or selectively active in regions of the heart adjacent to the site of injury, such as, for example, the GATA-4 promoter.

[0107] In other embodiments, the KLF nucleic acid is operably linked to the promoter of a gene that is overexpressed in cardiac tissue in response to stress resulting from ischemia and reperfusion, such as aminoadipate semialdehyde synthase, apolipoprotein E, flavin-containing monooxygenase 2, NADPH oxidase 4, prostaglandin endoperoxide synthase 2, recombinase activating gene 2, stearoyl-coenzyme A desaturase 1, or solute carrier family 38 (member 1).

[0108] Administration of KLF In the methods described herein, KLF or KLF nucleic acid is administered to a subject. In particular, KLF or KLF nucleic acid is administered to a target cell, tissue or organ. In some embodiments, KLF or KLF nucleic acid is administered to a target cell, tissue or organ, or an expression vector encoding a KLF nucleic acid is administered to a target cell, tissue or organ where the KLF nucleic acid is expressed. In some embodiments, administration is systemic, and the expression vector is incorporated into the target cell, tissue or organ. In some embodiments, the expression vector may be incorporated into non-target cells, tissues or organs, but preferably does not have significant adverse effects on such cells or tissues, or on the subject as a whole.

[0109] Methods for administering or delivering nucleic acids and expression constructs to target cells are known in the art and include those briefly described below. The target cell may be, for example, a cardiomyocyte. In some embodiments, the KLF, KLF nucleic acid, or expression vector is delivered to the target cell, tissue, or organ in vivo. In some embodiments, the KLF, KLF nucleic acid, or expression vector is administered to the target cell ex vivo. In some embodiments, the KLF, KLF nucleic acid, or expression vector is delivered to the target cell in vitro.

[0110] In some embodiments, the target cell is a cardiomyocyte. The cardiomyocyte may be present in a subject or in culture outside the subject. In some embodiments, KLF, KLF nucleic acid or expression vector is administered to the heart or cardiac tissue.

[0111] In other embodiments, the target cells are proliferative cardiomyocytes, i.e., KLF, a KLF nucleic acid, or an expression vector can be administered to cardiomyocytes that have already undergone KLF-induced dedifferentiation.

[0112] In some embodiments, the KLF nucleic acid or expression vector can be transfected or transduced into cells of a subject ex vivo. The transfected or transduced cells capable of expressing KLF can then be expanded and administered to the subject. In some embodiments, the cells are autologous to the subject. Suitable cells include those isolated from blood or bone marrow, such as adult hematopoietic stem / progenitor cells.

[0113] In some embodiments, the KLF, KLF nucleic acid, or expression vector is delivered systemically, such as by intravenous injection. Additional routes of administration may include, for example, oral, topical, intracardiac, and intramuscular. In some embodiments, the KLF, KLF nucleic acid, or expression vector can be delivered ex vivo to cells taken from a subject, and the cells containing the KLF, KLF nucleic acid, or expression vector can then be reintroduced into the subject.

[0114] In some embodiments, the KLF, KLF nucleic acid or expression vector is administered via cardiac catheterization.

[0115] Numerous methods for delivery of nucleic acids (such as KLF nucleic acids) are known in the art. These methods include adeno-associated virus (AAV) or lentivirus mediated delivery, nanoparticle mediated delivery, gelfoam mediated intrapericardial delivery; and direct intramuscular administration of nucleic acids to the heart.

[0116] A preferred method of administering KLF nucleic acid is by using adeno-associated virus (AAV). In some embodiments, KLF nucleic acid can be continuously or conditionally expressed. Furthermore, the use of cardioactive AAV serotypes or variants improves tissue specificity. Thus, for example, the method includes delivering KLF nucleic acid with cardioactive AAV. Suitable AAV includes cardiospecific AAV such as those described in Pacak and Byrne, Mol Ther, 2011, 19(9), pp1582-1590.

[0117] Other viruses, such as retroviruses, lentiviruses, HSV or adenoviruses, can also be used.

[0118] Further specificity can be obtained in addition to the AAV serotype by using cardiac tissue-specific promoters for expression (e.g., NCX1, TNNT2).

[0119] In some embodiments, KLF, KLF nucleic acid or expression vector is administered by transfection using a transfection agent or delivery vehicle. As used herein, the term "delivery vehicle" refers to a compound that enhances the entry of KLF nucleic acid into cells. Examples of delivery vehicles include protein-polymer complexes (polyplexes), polymer-lipid combinations (lipopolyplexes), multi-layered and recharged particles, lipids and liposomes (lipoplexes, e.g., cationic liposomes and lipids), polyamines, calcium phosphate precipitates, polycations, histone proteins, polyethyleneimine, polylysine and polyampholyte complexes, and the like. In some embodiments, the delivery vehicle includes a transfection agent. The transfection agent may be used to condense the nucleic acid. The transfection agent may also be used to associate functional groups with the polynucleotide. Non-limiting examples of functional groups include cellular targeting sites, cellular receptor ligands, nuclear localization signals, compounds that enhance the release of contents from endosomes or other intracellular vesicles (such as membrane active compounds), and other compounds that alter the behavior or interactions of the compounds or complexes to which they are bound (interaction modifiers). For in vivo delivery, complexes made with subneutralizing amounts of cationic transfection agents can be used.

[0120] In some embodiments, the KLF nucleic acid or expression vector can be delivered using exosomes or exosome-like vesicles. For example, the KLF nucleic acid can be introduced into exosome-producing cells and exosomes containing the KLF nucleic acid can be isolated from those cells. Alternatively, exosomes can be isolated or prepared according to any method known in the art and the KLF nucleic acid can be introduced into the exosomes.

[0121] In some embodiments, the KLF nucleic acid or expression vector can be delivered using lipopolymers, liposomes, gelatin complexes, poloxamine nanospheres, or lipoproteins.

[0122] An alternative technique to achieve cardiac-specific delivery of KLF nucleic acid or expression vector is ultrasound targeted microbubble destruction (UTMD). This technique is based on the physical properties of ultrasound contrast agents, which are gas-filled microbubbles that oscillate to destruction when sonicated by ultrasound. Transfection into the heart can be achieved by loading KLF nucleic acid or expression vector into microbubbles, injecting them intravenously, and destroying them in the heart by ultrasound.

[0123] In some embodiments, the KLF nucleic acid or expression vector can be delivered systemically. In some embodiments, the KLF nucleic acid or expression vector can be delivered in combination with one or more pharma- ceutically acceptable carriers. The polymeric reagent for delivering the KLF nucleic acid or expression vector may incorporate compounds that enhance its utility. These groups can be incorporated into the monomer prior to polymer formation or can be attached to the polymer after its formation. A vector translocation-enhancing moiety is a molecule that can modify a nucleic acid complex and direct it to a cellular location (such as a tissue cell) or subcellular location (such as the nucleus) in culture or in a whole organism. By altering the cellular or tissue location of the complex, the desired localization and activity of the KLF nucleic acid or expression vector can be enhanced. The translocation-enhancing moiety can be, for example, a protein, peptide, lipid, steroid, sugar, carbohydrate, nucleic acid, cell receptor ligand, or synthetic compound. The translocation-enhancing moiety can, in some embodiments, enhance cell binding to the receptor, cytoplasmic transport to the nucleus, and nuclear entry or release from endosomes or other intracellular vesicles.

[0124] Nuclear localization signals (NLS) can also be used to enhance targeting of mir-1 nucleic acids or expression vectors to the vicinity of the nucleus and / or entry into the nucleus. Such nuclear transport signals can be proteins or peptides, such as SV40 large tag NLS or nucleoplasmin NLS. These nuclear localization signals interact with various nuclear transport factors, such as the NLS receptor (karyopherin alpha), which in turn interacts with karyopherin beta. Nuclear transport proteins themselves can also function as NLSs in some embodiments, as they are targeted to the nuclear pore and the nucleus.

[0125] One of ordinary skill in the art will be able to select and use an appropriate system for delivering a KLF nucleic acid or expression vector to the heart or cardiac tissue, or to cardiomyocytes or other target cells in vitro, ex vivo or in vivo, without undue experimentation.

[0126] In some embodiments, localized delivery of a KLF nucleic acid or expression vector is desired, particularly delivery of a KLF nucleic acid or expression vector to the heart.

[0127] There are many strategies that allow local delivery of KLF, KLF nucleic acid or expression vector to the heart. For example, osmotic mini-pumps such as Alzet® osmotic pumps can be used for effective local delivery of KLF, KLF nucleic acid or expression vector at sustainable therapeutic concentrations. Pumps with reservoirs are typically implanted in subcutaneous tissue and deliver KLF, KLF nucleic acid or expression vector to target tissues via silicone tubes or cannulas. Osmotic mini-pumps rely on osmotic pressure for constant drug delivery and have already been used in clinical applications.

[0128] Localized delivery of KLF, KLF nucleic acid, or expression vector may be achieved by transplanting cells. In addition to cell replacement therapy, cells containing KLF, KLF nucleic acid, or expression vector may be transplanted into the myocardium.

[0129] KLF Dosage The effective dosage level of KLF, KLF nucleic acid, or expression vector administered will depend on a variety of factors, such as the type of condition being treated and the stage of the condition; the activity and properties of the KLF, KLF nucleic acid, or expression vector employed; the composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration; the route of administration; the duration of the treatment; drugs used in combination or concomitantly with the treatment, and other relevant factors well known in medicine.

[0130] Those skilled in the art will be able to determine, by routine experimentation, the effective, non-toxic dosage required to treat the applicable condition, which will, in most cases, be determined on a case-by-case basis.

[0131] In general, an effective amount is expected to be in the range of about 0.0001 mg / kg to about 1000 mg / kg of body weight per 24 hours; typically, about 0.001 mg / kg to about 750 mg / kg of body weight per 24 hours; about 0.01 mg / kg to about 500 mg / kg of body weight per 24 hours; about 0.1 mg / kg to about 500 mg / kg of body weight per 24 hours; about 0.1 mg / kg to about 250 mg / kg of body weight per 24 hours; or about 1.0 mg / kg to about 250 mg / kg of body weight per 24 hours. More typically, an effective amount range is expected to be in the range of about 10 mg / kg to about 200 mg / kg of body weight per 24 hours.

[0132] Alternatively, an effective dosage may be up to about 5000 mg / m2. In general, effective amounts are expected to range from about 10 to about 5000 mg / m2, typically from about 10 to about 2500 mg / m2, from about 25 to about 2000 mg / m2, from about 50 to about 1500 mg / m2, from about 50 to about 1000 mg / m2, or from about 75 to about 600 mg / m2.

[0133] Moreover, it will be apparent to one skilled in the art that the optimal amount and interval of each individual dose will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the nature of the particular individual being treated, and such optimal conditions can be determined by conventional techniques.

[0134] It will also be apparent to one of skill in the art that an optimal course of treatment, e.g., the number of doses of a composition administered per day for a defined number of days, can be ascertained by one of skill in the art using a conventional course of treatment determination testing.

[0135] The efficacy of treatment may also be assessed by determining the expression level of KLF in a sample from a subject treated with KLF, a KLF nucleic acid, or an expression vector. After a period of time, the expression level of KLF nucleic acid in an additional sample from the subject may be determined, and a change in the expression level of KLF nucleic acid may indicate the efficacy of the treatment regimen. The sample may include plasma or serum.

[0136] Alternatively, or additionally, the efficacy of treatment can be assessed, for example, by determining the level of proliferative cardiomyocytes in a sample from a subject treated with KLF, a KLF nucleic acid, or an expression vector.

[0137] Cardiomyocyte / progenitor cell populations Administration of KLF, KLF nucleic acid or expression vector generates a population of proliferative cardiomyocytes. The proliferative cardiomyocytes can be a population of immature cardiomyocytes, cardiomyocytes with an embryonic phenotype, or a population of cardiac progenitor cells, or any combination thereof. This population of proliferative cardiomyocytes can be incorporated into a pharmaceutical composition for administration to a subject. In some embodiments, the population of proliferative cardiomyocytes can be differentiated into cardiomyocytes prior to incorporation into the pharmaceutical composition.

[0138] KLF, KLF nucleic acid or expression vector can be administered to adult cardiomyocytes taken from a subject (or elsewhere) to induce myocardial regeneration. Whether the obtained cells are a population of cardiomyocytes or a population of proliferative myocardial progenitor cells, the cells can be prepared as a pharmaceutical composition, such as a sterile aqueous or non-aqueous solution, suspension or emulsion, further comprising a physiologically acceptable carrier (i.e., a non-toxic substance that does not interfere with the activity of cardiomyocytes). Any suitable carrier known to those skilled in the art can be used in the pharmaceutical composition. The selection of the carrier will depend in part on the nature of the substance (i.e., cells or chemical compound) to be administered.

[0139] Suitable carriers include saline, gelatin, water, alcohol, natural or synthetic oils, sugar solutions, glycols, injectable organic esters such as ethyl oleate, or combinations of these materials. The pharmaceutical composition may further include preservatives and / or other additives, such as, for example, antimicrobial agents, antioxidants, chelating agents, and / or inert gases, and / or other active ingredients. In some embodiments, the cardiomyocyte or cardiomyocyte progenitor cell populations are encapsulated according to known encapsulation techniques.

[0140] In some embodiments, the cardiomyocyte or cardiomyocyte progenitor cell population is present in a matrix.

[0141] The unit dosage form of the cardiomyocyte or cardiac progenitor cell population is about 10 3 Approximately 10 cells 9 Cells, e.g., about 10 3 Approximately 10 cells 4 cells, about 10 4 Approximately 10 cells 5 cells, about 10 5 Approximately 10 cells 6 cells, about 10 6 Approximately 10 cells 7 cells, about 10 7 Approximately 10 cells 8 cells, or about 10 8 Approximately 10 cells 9 It may contain cells, etc.

[0142] In some embodiments, a method is provided for inducing myocardial regeneration in a population of cardiomyocytes in vitro and transplanting the population of cardiomyocytes into the heart of a subject. The population of cardiomyocytes can be used for allogeneic or autologous transplantation into an individual in need thereof.

[0143] Combination therapy The terms "combination therapy" or "adjunctive therapy" when defining the use of KLF, a KLF nucleic acid or vector with one or more other agents are intended to include the sequential administration of each agent in a regimen that results in the beneficial effect of the combination of agents, and to include co-administration of these agents in a substantially simultaneous manner, such as in a single formulation having a fixed ratio of the active agents, or in multiple, separate formulations of each agent.

[0144] According to various embodiments of the invention, one or more of the KLF, KLF nucleic acid or vector may be formulated or administered in combination with one or more additional therapeutic agents. Thus, according to various embodiments of the invention, at least one of the KLF, KLF nucleic acid or vector may be included in a combination treatment regimen with surgery and / or other known therapeutic or therapeutic agents, and / or adjunctive or prophylactic agents.

[0145] Many drugs are commercially available, under clinical evaluation and preclinical development, which can be selected for the treatment of the above-listed diseases and conditions as part of combination drug therapy.The suitable drugs that can be used in combination therapy will be recognized by those skilled in the art.Suitable drugs are listed, for example, in the Merck Index, An Encyclopaedia of Chemicals, Drugs and Biologicals, 12th Ed., 1996, and later editions, the entire contents of which are incorporated herein by reference.

[0146] The combination regimen may involve administering the active agents together, sequentially, or at intervals, as appropriate in each case. The combination of active agents, such as at least one KLF, KLF nucleic acid, or vector, may be synergistic.

[0147] Co-administration of at least one of the KLF, KLF nucleic acid or vector with an additional agent may be effected by the agent being in the same unit dose as another active agent, or the one or more other active agent(s) may be present in individual separate unit doses administered at the same or similar times or at different times according to a dosing regimen or schedule. Sequential administration may be in any order as needed, and may require that the physiological effect of the first or initial compound is ongoing when the second or subsequent compound is administered, particularly where a cumulative or synergistic effect is desired.

[0148] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0149] In order that the present technology may be more clearly understood, preferred embodiments will now be described with reference to the following drawings and examples. EXAMPLES

[0150] Example 1: Injury induces myocardial expression of klf1 during zebrafish heart regeneration. While investigating the molecular mechanisms of cardiac regeneration in zebrafish, we identified injury-induced, myocardial expression of Krüppel-like factor 1 (klf1) (Figure 1A), a gene encoding the zebrafish orthologue of the zinc-finger transcription factor KLF1 / EKLF. In mammals, KLF1 is expressed in hematopoietic tissues and is essential for red blood cell development, but the role of KLF1 in non-hematopoietic organs was unknown prior to this study.

[0151] Analysis showed that klf1 expression transiently peaked at 7 days post injury (dpi) (Figure 1B), corresponding to the maximal response of cardiomyocyte (CM) regenerative proliferation, using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Using purified cardiomyocytes from 7 dpi ventricles of cell type-specific reporter lines, RT-qPCR with fluorescence-activated cell sorting (FACS) demonstrated that klf1 transcripts were detected in CM, but not in epicardial and endocardial cells (Figure 1B). We also assessed klf1 expression using a genetic CM ablation model in which myocardium can be specifically injured without hemorrhage by 4-hydroxytamoxifen (4-HT) treatment. Using sections from CM-deficient hearts, we performed highly sensitive in situ hybridization and immunofluorescence for the cytoplasmic muscle marker troponin C (TnC) and detected colocalization of klf1 mRNA signal with TnC in the regenerating myocardium (Figure 1C, arrowheads). These data indicate that injury induces myocardial expression of klf1 during zebrafish heart regeneration.

[0152] Example 2: Klf1 is essential for cardiac regeneration in zebrafish. To investigate the function of Klf1 in cardiac regeneration, we induced a dominant-negative form of Klf1 (dn-Klf1) in CM in combination with the myocardial-specific Cre driver line Tg(cmlc2:CreER) (hereafter referred to as cmlc2:CreER), a zebrafish line Tg(actb2:loxP-TagBFP-STOP-loxP-dn-klf1). vcc22We established a 4-HT-dependent phenotype for the cmlc2:CreER; actb2:BS-dn-klf1 (KlfDN-ON) and control actb2:BS-dn-klf1 (KlfDN-OFF) fish and analyzed regeneration at 30 dpi. Strikingly, all KlfDN-ON hearts showed extremely severe scarring at the wound site (7 out of 7, Fig. 2A), whereas no such phenotype was observed in control KlfDN-OFF hearts (0 out of 7, Fig. 2A). Myocardial expression of dn-Klf1 significantly reduced CM proliferation, as detected by reduced co-labeling of myonuclear markers Mef2 and PCNA (Fig. 2B). These data indicate that Klf1 is an essential transcription factor for cardiac regeneration in zebrafish.

[0153] Using quantitative transcriptional analysis, we observed that klf1 expression peaked at 7 days post-injury (dpi), coinciding with the period of maximal regeneration-induced proliferation in cardiomyocytes (Figure 3A). This transient klf1 expression was restricted to purified cardiomyocytes and was not detected in epicardial (Epi) or endocardial (End) cells (Figure 3B). Using a conditional 4-hydroxytamoxifen (4-HT)-induced cardiomyocyte ablation model, which injures the myocardium without cardiac hemorrhage, we observed colocalization of klf1 mRNA with the cytoplasmic muscle marker, troponin C, indicating that regenerating cardiomyocytes express Klf1 during zebrafish cardiac regeneration.

[0154] Example 3: Klf1 expression induces zebrafish cardiac CM dedifferentiation. cmlc2:Tg(actb2:loxP-TagBFP-STOP-loxP-3xHA-klf1), a transgenic zebrafish line that expresses 3xHA-tagged Klf1 in CMs using CreER. vcc29We established double transgenic fish and Cre-negative clutchmates, referred to as klf1-ON and klf1-OFF, respectively. klf1-ON; cmlc2:GFP and control klf1-OFF; cmlc2:GFP fish were treated with 4-HT and the expression of the dedifferentiation marker Sm22α (Sm22) in cmlc2:GFP-labeled CMs was assessed. Strikingly, compared with control hearts (12d klf1-OFF), Klf1-overexpressing hearts had a markedly elevated expression level of Sm22, with expression first detected in the outermost myocardium of the ventricle at 7 days post 4-HT treatment (dpt), and then also observed in the inner cavernous myocardium at 12 dpt. Analysis using another dedifferentiation marker, Tg(gata4:EGFP) (hereafter gata4:GFP), in zebrafish CMs also revealed a spatial and temporal pattern of gata4:GFP similar to that of Sm22. Immunofluorescence for alpha-actinin, a major component of Z-lines, revealed that sarcomere structure was highly disorganized in klf1-ON myocardium compared to klf1-OFF myocardium. Furthermore, consistent with histological evidence of CM dedifferentiation, klf1-ON hearts showed significantly increased expression of runx1, a mouse stem cell and CM dedifferentiation marker (Figure 4A), as well as decreased expression of contractile genes such as myosin heavy chain (vmhc), cardiac actin (actc1a), and myomesin (myom2a) (Figure 4B). These data indicate that expression of Klf1 is sufficient to dedifferentiate CMs to a less mature state in the zebrafish heart.

[0155] Example 4: Klf1 expression induces CM proliferation in zebrafish hearts. Because the state of reduced contractility acquired by CM dedifferentiation has been suggested to promote cell division, we next examined whether Klf1 overexpression promoted CM proliferation using EdU incorporation assays. Surprisingly, background levels of proliferation in klf1-OFF hearts (EdU +Compared to CMs (Fig. 5A), proliferation in klf1-ON hearts was increased by approximately 200-fold (Fig. 5A). We next assessed CM mitosis by immunofluorescence using an antibody against phosphohistone H3 (pHH3). It is extremely difficult to identify mitotic CMs even in regenerating zebrafish hearts, probably due to the short duration of the mitotic phase in the cell cycle. However, we found that mitotic CMs were abundant in Klf1-ON hearts, with approximately six pHH3 CMs per section. + CMs were constantly observed, and no mitotic CMs were detected in control hearts (Figure 5B), strongly indicating the strong effect of Klf1 overexpression on cell cycle reentry in CMs. Also, consistent with the significantly enhanced levels of CM proliferation, Klf1-ON hearts showed significantly increased expression of a broad range of cell cycle regulators, including FoxM1, PCNA, E2F2, Cdc25b, cyclin-dependent kinases (Cdk1 / 2), G1 / S cyclin (CyclinD1), and G2 / M cyclins (CyclinB2 / A2) (Figure 5C). Together, these data indicate that expression of Klf1 is sufficient to induce quiescent CMs to reenter the cell cycle in adult zebrafish hearts.

[0156] Example 5: KLF1 function in the adult mouse heart Similar to zebrafish hearts, mouse Klf1 (mKlf1) mRNA was upregulated from baseline levels in regenerating neonatal hearts after infarction (Neonatal; Fig. 6A). Interestingly, however, mKlf1 expression was not induced in infarcted adult hearts (Adult; Fig. 6A), indicating a correlation between its expression level and the regenerative capacity of mouse hearts. To investigate KLF function in adult mouse hearts, we generated a recombinant adenoviral vector in which mKlf1 cDNA was linked to EGFP cDNA via a P2A peptide sequence to visualize infected cells (Ad-Klf1). We also generated a control vector containing only EGFP cDNA (Ad-GFP). We injected purified Ad-GFP or Ad-Klf1 viruses into the myocardium of uninjured hearts of adult C57BL / 6 mice and assessed the proliferation of CMs by Ki67 immunofluorescence analysis. The results showed that injection of Ad-Klf1 virus significantly increased co-labeling of Ki67 and troponin-T (TnT) (KLF1; Figure 6B) compared with injection of the control virus (GFP; Figure 6B). Immunofluorescence detection of GFP demonstrated the expression of mKlf1 in proliferating CMs.

[0157] To confirm this result, we assessed CM proliferation by delivering EdU using an osmotic minipump and identifying CMs with the membrane marker wheat germ agglutinin (WGA) in conjunction with immunofluorescence for TnT, a more sensitive method to identify proliferating CMs in adult mouse heart slices. Consistent with the results of immunolabeling for Ki67 and TnT, analysis using WGA staining also showed that EdU was significantly increased in hearts injected with Ad-Klf1 virus (KLF1; Figure 6C). + A significant increase in CMs was detected in mouse CMs. Together, these data indicate that mKlf1 has a conserved function in mouse CM proliferation and can induce CM cell cycle re-entry in vivo, even in the absence of injury.

[0158] We also delivered Klf1 to adult mouse hearts after myocardial infarction (MI). We also observed a significant increase in CM proliferation (Figure 7A) and mitosis (Figure 7B) upon Ad-Klf1 transduction, indicating that mKlf1 has a conserved regenerative function in mice. Consistent with the increased CM regeneration, we found that cardiac scarring was significantly reduced in Ad-Klf1-transduced hearts, together with increased cardiomyogenesis in the injured area (Figure 7C) and significant recovery of cardiac function (Figure 7D). Together, these data indicate that mKlf1 has a conserved function in CM proliferation and regeneration in mice.

[0159] Example 6: Conditional gene trap lines To investigate the role of Klf1 in cardiac regeneration, we generated a conditional gene trap line, Tg(klf1:Zwitch2), by inserting a Cre-dependent gene trap cassette, called Zwitch2, into the first intron of the klf1 gene by homologous recombination, as previously described. vcc33Gt (hereinafter referred to as klf1-ct or klf1 ct We generated a mutant Zwitch2 clone (called klf1) (Figure 8A,B). Zwitch2 consists of a splice acceptor (SA) site and a triplet polyA sequence (3xBGHpA) with a removable lens-specific tag expressing enhanced green fluorescent protein (EGFP) for screening (Figure 8A). The segment containing SA and 3xBGHpA is flanked by tandem loxP and lox5171 sites in an inverted orientation and can be permanently inverted via Cre-mediated recombination to inactivate expression of klf1 via aberrant splicing (mutagenicity; Figure 8B).

[0160] In the established lineages, the correct insertion of Zwitch2 was confirmed by genomic PCR, Southern blot and DNA sequencing analysis (Figure 9A-D). We also crossed klf1-ct / + with Tg(ubb:iCre-P2A-EGFP), a strain in which Cre is driven by a strong and ubiquitously expressed ubiquitin B (ubb) promoter, to characterize the established alleles. We obtained embryos carrying WT and / or mutant klf1 alleles through crosses (Figure 9E,F), and analyzed the phenotypes of these embryos, confirming the evolutionarily conserved role of Klf1 in zebrafish erythroid development (Figure 10A).

[0161] We inactivated myocardial klf1 expression by crossing klf1-ct / + fish with Tg(cmlc2:CreER)(cmlc2:CreER), a line in which 4-hydroxytamoxifen (4-HT)-inducible Cre is expressed by regulatory sequences of the contractile gene cardiac myosin light chain 2 (cmlc2 / myl7). We obtained klf1-ct / ct and cmlc2:CreER; klf1-ct / ct fish and treated them overnight with 4-HT for three consecutive days, with excision injury performed two days after the last treatment. The 4-HT treatment regimen successfully reduced myocardial klf1 expression to uninjured control levels in cmlc2:CreER; klf1-ct / ct ventricles at 7 dpi (Figure 8C, Figure 9H). Proliferation of CMs was analyzed by immunofluorescence against myonuclear markers Mef2 and proliferating cell nuclear antigen (PCNA), and demonstrated increased proliferation of Mef2 following inactivation of myocardially expressed klf1. + , PCNA +We identified a significant reduction in the number of CMs (Figure 8D). We performed immunofluorescence for fetal CM markers, such as smooth muscle protein 22 alpha (Sm22a; also known as transgelin) (Figure 8E) and activated leukocyte cell adhesion molecule a (Alcama; also known as DM-GRASP) (Figure 8F). This revealed that the expression of these markers was significantly reduced in hearts lacking klf1 expression (Figure 8F). Together, these data indicate that Klf1 drives a novel mechanism of CM dedifferentiation and proliferation in zebrafish cardiac regeneration, with important roles in erythropoiesis and congenital anemia.

[0162] klf1 ct We provide details on the generation and characterization of the ubiquitously expressed, Cre-inducible klf1 gene (Figure 9A-D). ct Not only was global activation of klf1 suppressed (Figure 9E), but expression of klf1 was markedly reduced (Figure 9F) and cardiac edema due to severe anemia occurred (Figure 10A). ct Tissue-specific activation of klf1 was achieved in adult myocardium using the cardiomyocyte-restricted Cre line Tg(cmlc2:CreER). This myocardial trap line and the cmlc2:CreER negative control line, termed klf1-MT and klf1-CT, respectively (Figure 3C), validated by ventricular resection after 4-HT treatment, induced a Cre-dependent, cardiac klf1-CT reversal (Figure 9G) and reduced ventricular klf1 expression at 7 dpi to levels similar to uninjured controls (Figure 9H). Inactivation of myocardial klf1 expression significantly increased the percentage of regenerating hearts exhibiting persistent fibrin and collagenous scars (Figure 3D), indicating that myocardial klf1 is required for zebrafish cardiac regeneration.

[0163] Known cardiomyocyte dedifferentiation markers, such as smooth muscle protein 22α / transgelin (Sm22; Fig. 3E) and activated leukocyte cell adhesion molecule (Alcam; Fig. 3F), were markedly decreased in klf1-MT hearts. This was accompanied by a marked reduction in the number of proliferating cardiomyocytes that were double positive for myocyte enhancer factor 2 (Mef2) and proliferating cell nuclear antigen (PCNA) (Fig. 3G). Similarly, we observed that under conditional expression of a dominant-negative form of Klf1 (dn-Klf1; Fig. 11A-D), cardiomyocyte dedifferentiation and proliferation were significantly reduced and cardiac regeneration was severely impaired, providing evidence for further critical roles of Klf1 in cardiac regeneration. Collectively, these data demonstrate a novel non-hematopoietic function of Klf1 in myocardial regeneration, namely, that Klf1 plays a critical role in successfully inducing cardiomyocyte dedifferentiation and proliferation.

[0164] Klf1 was virtually undetectable during cardiac development (one mRNA punctum per 81 ventricular myocardial sections examined). Whether Klf1 has a functional role in cardiac development was examined. Surprisingly, in contrast to the anemia phenotype observed with global inhibition of Klf1 (Figure ​(Figure10A,B), myocardial Klf1 ct Neither activation (Figure S1C) nor myocardial overexpression of dn-Klf1 (Figure S1D) affected myocardial morphogenesis and cardiomyocyte proliferation during development. We also transfected the Klf1 gene using a ubiquitously expressing Cre driver. ct A similar phenotype was observed in hearts in which activation or constitutive induction of dn-Klf1 expression was performed before the onset of cardiac edema (Fig. S10E, F). Together, these data indicate a regeneration-specific role for Klf1 in the myocardium.

[0165] Example 8: Klf1 triggers an injury-independent regenerative response To examine whether forced expression of klf1 induces a regenerative phenotype in uninjured hearts, we used a conditional strategy to drive 3xHA-tagged Klf1 in the myocardium (klf1-ON; Figure S12A). We treated klf1-ON fish or Cre negative control (klf1-OFF; Figure S12A) with 4-HT overnight (Figure S12B) and detected nuclear localization of 3xHA-Klf1 in the myocardium by immunofluorescence. In klf1-ON hearts, myocardial expression of Sm22 and Alcam was increased at 7 days post treatment (dpt) and more at 12 days post 4-HT treatment (dpt). During myocardial regeneration, cardiomyocyte division is likely promoted by a decrease in contractile force, accompanied by the expression of genes associated with an immature proliferative state. Consistent with partial cardiomyocyte dedifferentiation, impaired α-actin (a major component of Z-lines) sarcomeres were observed in the cortical layer myocardium in klf1-ON hearts at 7 dpt and more extensively in the cavernous myocardium at 12 dpt. These structural abnormalities were visualized as diffuse Z-discs by transmission electron microscopy (TEM) at 7 and 12 dpt.

[0166] To examine whether Klf1-induced cardiomyocyte dedifferentiation was accompanied by increased cardiomyocyte proliferation, we examined the proliferation marker PCNA. + Mef2 + We confirmed that the number of cardiomyocytes was significantly increased (Figure S1C). In klf1-ON cardiomyocytes, 5-ethynyl-2'-deoxyuridine (EdU) incorporation was significantly increased, confirming that many cardiomyocytes were undergoing DNA synthesis (Figure S1D). + (pHH3 + ) visualization of cardiomyocytes is extremely rare even in the robustly regenerating zebrafish heart. However, in the klf1-ON ventricles, many pHH3 + Cardiomyocytes were detected (Figure S12E), clearly indicating that cardiomyocytes had successfully progressed through mitosis to anaphase. Together, these data indicate that overexpression of klf1 is sufficient to induce a robust cardiomyocyte dedifferentiation response and strongly promotes cardiomyocyte cell cycle reentry and proliferation in uninjured zebrafish hearts.

[0167] During the extensive proliferative response observed in klf1-ON hearts, EGFP-labeled cardiomyocytes were restricted to cells of the myocardial lineage and did not contribute to other cardiac cell lineages such as endocardial or epicardial cells. These data indicate that Klf1 induces cardiomyocyte proliferation not by reprogramming mature cardiomyocytes into proliferative multipotent progenitors, but rather by extensively upregulating cardiomyocyte self-renewal without affecting lineage plasticity. Increased numbers of vascular endothelial and epicardial cells were observed in klf1-ON hearts (Figure S13A,B), indicating that myocardial Klf1 expression indirectly stimulates regeneration programs in other cardiomyocytes.

[0168] The KLF1 subfamily members KLF2 and KLF4 control cardiovascular development and function. We inducibly expressed the zebrafish KLF2 and KLF4 orthologues, Klf2a, Klf2b, and Klf4, as described for the klf1-ON to compare the cardiomyocyte regenerative abilities of the KLF1 subfamily (Figure S12A, B). Notably, cardiomyocyte mitosis was most strongly induced during Klf1 overexpression (Figure S13C). During Klf2b overexpression, cardiomyocyte mitosis was moderately induced (Figure S13C), whereas such induction was negligible following overexpression of Klf2a and Klf4 (Figure S13C). The relatively high potency of Klf1 compared with its family members Klf2 and Klf4 attests to the unique role of Klf1 in controlling adult-specific cardiomyocyte regenerative targets.

[0169] Mammalian KLF1 has a similar domain structure to KLF2 and KLF4, which have the remarkable ability to reprogram somatic cells into pluripotent stem cells. KLF1 can also generate induced pluripotent stem cells with other reprogramming factors, albeit with a much lower efficiency than KLF2 and KLF4, and Klf1-induced cardiomyogenesis was shown to be a mechanism via CM reprogramming and expansion of progenitor cells followed by redifferentiation into cardiac muscle. Interestingly, significantly more epicardial and endothelial cells were observed in klf1-ON ventricles, and klf1 + Although the paracrine effect of CM was demonstrated, reprogrammed klf1 + This could also be explained by de novo differentiation from CMs. To investigate the involvement of cellular reprogramming, we performed genetic fate mapping analysis using klf1-ON fish carrying a tamoxifen-dependent indicator transgene, allowing us to permanently label CMs at the onset of klf1 induction. The genetically labeled cells still expressed myosin heavy chain (MHC) at 12 dpt, albeit in a reduced manner, and did not express Raldh2 (also known as retinaldehyde dehydrogenase 2; Aldh1a2), a marker for epicardial and endothelial cells, indicating that klf1 + It has been shown that CMs did not change lineage during the extensive proliferation period.Thus, rather than reprogramming adult CMs into proliferative multipotent cardiac progenitors, overexpression of Klf1 dedifferentiated adult CMs into a proliferative immature state, achieving extensive levels of myocardial regeneration in the adult heart with the ability to promote proliferation of neighboring tissues.

[0170] Example 9: Klf1-induced cardiomyocyte hyperplasia drives myocardial regeneration klf1-ON zebrafish progressively showed signs of heart failure, including swollen scales (Figure S4A), lethargy, and rapid breathing that affected survival by 9 dpt (Figure S4B). To model the transient therapeutic delivery of Klf1, we transformed the Tg(cmlc2:3xHA-klf1-ER; cryaa:TagBFP) vcc32In the klf1-ER model, Klf1 reversibly translocates to the nucleus in response to 4-HT (Fig. S4F).

[0171] Treatment with 4-HT once a day for 7 days (Fig. S12G) induced nuclear klf1-ER localization and a regenerative response in the ventricle. In contrast to klf1-ON, survival of klf1-ER fish after cessation of 4-HT treatment was unaffected, so we analyzed hearts after 30 days, when the Klf1 induction response had ceased (Fig. S12G). Interestingly, macroscopic analysis of experimental hearts confirmed massive hypertrophy of hearts taken from 4-HT-treated klf1-ER fish (Fig. S12H). Sections of hypertrophied hearts showed no pathological dilation or fibrosis (Fig. S12I), and the area of ​​the myocardium was significantly larger (~2-fold; Fig. S12J). Mef2 + The number of nuclei was significantly increased in klf1-ER ventricles, and α-actinin staining revealed well-organized streaks with distinct Z-bands. Furthermore, although individual cardiomyocytes in the hypertrophied ventricles were smaller in size (Fig. S1K) and therefore not hypertrophied, they were significantly increased in number by approximately fivefold compared to those in the control ventricles (Fig. S1L). Thus, after only 7 days of Klf1 activation, cardiomyocytes proliferated approximately fivefold compared to those in uninjured zebrafish hearts, highlighting the extremely pro-proliferative potential of Klf1 to stimulate myocardial regeneration in the adult heart.

[0172] Example 10: Klf1 induces chromatin remodeling to repress cardiac gene programs To gain insight into the myocardial function of Klf1, we performed chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-sequon) in klf1-OFF and klf1-ON ventricles using anti-HA-tag antibody at 7 dpt. 3xHA-Klf1 ChIP peaks were specifically detected in klf1-ON ventricles (Figure 15A). These peaks were most significantly enriched in KLF1 motifs (Figure 15B), validating the specificity of Klf1 purification with anti-HA antibody. Potential target genes under Klf1 peaks were analyzed with Genomic Region Enrichment Annotation Tool (GREAT) to identify genes involved in organogenesis pathways such as cardiomyogenesis (Figure 15C).

[0173] To characterize Klf1 binding sites, we performed ChIP-seq for active promoter marks (histone H3K4me3) and active enhancer marks (histone H3K4me1 and H3K27ac) and plotted the resulting histone peaks against Klf1 peaks (Figure S4D). Contrary to expectations, we found only a small proportion of Klf1 peaks (98 peaks; 8.6%) at active promoters, whereas the majority of the remaining (1,039 peaks; 91.4%) were found at active enhancers, which were constitutively marked with reduced DNA methylation (5-methylcytosine, 5mC; Figure S4D) and H3K27ac and H3K4me1, regardless of Klf1 expression or the cardiomyocyte regenerative response. Furthermore, cross-referencing our data with previously published datasets indicates that Klf1-targeted enhancers acquire an active epigenetic profile as early as 48 hours post-fertilization (hpf), and several Klf1-targeted sites coincide with the genomic locations of functionally validated developmentally active enhancers from the VISTA enhancer browser. These data indicate that Klf1 preferentially targets developmentally and constitutively active enhancers in adult cardiomyocytes.

[0174] We next assessed global changes in chromatin accessibility in klf1-ON hearts by assay of transposase-accessible chromatin using sequencing (ATAC-seq). Strikingly, the majority of differentially enriched ATAC peaks were annotated to regions of reduced chromatin accessibility in klf1-ON hearts (Figure 15E), and were associated with reduced H3K27ac (Figure 15F) and transcription of the nearest genes (Figure 15G). Strikingly, regions of reduced accessibility were enriched in binding sites for MEF2C, GATA4, MEF2A and NKX2.5 (Figure 15H). These were central to cardiac regulatory networks controlling cardiomyocyte fate, structure, morphogenesis and contractile gene expression. Consistent with this result, pathways controlling cardiac tissue development were most significantly associated with regions of reduced accessibility (Figure 15I). Furthermore, RNA-seq analysis of klf1-ON ventricles demonstrated that cardiomyocyte dedifferentiation marker genes were upregulated, while myocardial structural and regulatory genes were largely downregulated (Figure S15I). Taken together, these data demonstrate a model for cardiomyocyte dedifferentiation: Klf1 binds to pre-existing cardiac enhancers in the adult heart and reduces chromatin accessibility at central cardiac transcription factor binding sites, thereby repressing genetic programs that control myocardial development and function.

[0175] Example 11: Klf1 upregulates diverse cell cycle genes and promotes cardiomyocyte proliferation and division Consistent with the histological evidence of increased cardiomyocyte proliferation in klf1-ON hearts (Fig. S12D,E), we found that the majority of gene signatures upregulated upon klf1 overexpression were related to cell cycle machinery (Fig. S16A,B). In klf1-ON hearts, we observed robust increases in the expression of genes encoding essential regulators of DNA replication, cell cycle, and cytokinesis (Fig. S16C,D). In particular, we found that genes encoding many types of cyclins, such as cyclin D (ccnd1, ccnd2a, ccnd2b), cyclin E (ccne1, ccne2), cyclin A (ccna2), and cyclin B (ccnb1, ccnb2), as well as cyclin-dependent kinases (cdk1, cdk2), were significantly upregulated, whereas the expression of a Cdk inhibitor gene (cdkn1ca) was downregulated. Among these cyclin genes, the present inventors detected Klf1-binding regulatory regions in the ccnd1 and ccnd2a genes and demonstrated that Klf1 directly regulates the expression of D-type cyclins.

[0176] We also analyzed the interaction of Klf1 with known gene pathways for adult myocardial regeneration, such as the Hippo-yes associated protein (YAP) pathway and the neuregulin-ErbB2 pathway. Gene set enrichment analysis using RNA-seq data from klf1-ON and klf-OFF hearts detected significant enrichment of gene signatures in the Hippo pathway (Figure 19A), but not the ErbB pathway (Figure 19B), upon Klf1 overexpression. We assessed whether inhibition of these pathways affected cardiomyocyte proliferation in klf1-ON hearts and found that pharmacological inhibition of YAP (Figure 19C), but not ErbB (Figure 19D), significantly reduced cardiomyocyte proliferation in klf1-ON hearts. These findings demonstrate that Klf1 mediates cardiomyocyte proliferation in part through the Hippo-YAP pathway. In klf1-ON hearts, the expression of many YAP target genes was increased, but the expression of genes encoding core components of the Hippo pathway was not significantly altered, and no Klf1 peaks were observed at these core component genes (data not shown), demonstrating that Klf1 activates the Hippo-YAP pathway through an indirect mechanism.

[0177] Example 12: Klf1 induces metabolic reprogramming to support robust cardiomyocyte proliferation. The majority of gene signatures downregulated in klf1-ON hearts were related to mitochondrial metabolism and bioenergetics production (Figure 16A,B,E). TEM analysis of klf1-ON myocardium revealed mitochondria with reduced clisters and enlarged matrix (Figure 16F), a morphological phenotype similar to functionally immature mitochondria in embryonic and neonatal mouse hearts. Mitochondrial DNA (mtDNA) abundance was also significantly reduced in klf1-ON hearts (Figure 16G), accompanied by comprehensive downregulation of genes controlling mitochondrial energy metabolism, such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Key metabolites of these pathways, such as NADH, NAD+, and ATP, were also significantly decreased in klf1-ON hearts (Figure 16H-K), providing further evidence of impaired mitochondrial energy metabolism during klf1 overexpression. These data indicate that the Klf1 pathway, like other cardiogenic pathways, promotes cardiomyocyte proliferation by attenuating OXPHOS, a major mechanism of cell cycle arrest in postnatal cardiomyocytes.

[0178] Klf4 has been shown to control cardiac mitochondrial homeostasis by regulating mitochondrial biogenesis and autophagic clearance. We did not detect significant enrichment of autophagy genes or increased autophagic flux in klf1-ON hearts. Rather, we found that klf1-ON hearts had significantly decreased expression of nuclear genes controlling mitochondrial homeostasis and function (Figure S16L). In particular, we found downregulation of the gene encoding PPARγ coactivator-1α (PGC-1α / PPARGC1a), a master regulatory transcription factor that controls mitochondrial biogenesis and oxidative function (Figure S16L). We also found that klf1 ChIP peaks at the enhancer of the ppargc1a gene and reduced H3K27ac levels in klf1-ON hearts, indicating that this is a mechanism by which Klf1 directly reduces ppargc1a expression to regulate mitochondrial function.

[0179] The shift in energy production from OXPHOS to aerobic glycolysis, known as the Warburg effect, supports highly proliferative cells, including cancer cells, and has recently been shown to support myocardial regeneration. However, expression of glycolytic enzyme genes was downregulated in kfl1-ON hearts, demonstrating that kfl1-ON cardiomyocytes utilize a different metabolic mechanism to support proliferation. To gain further insight into the metabolic role of Klf1, we analyzed the metabolome of klf1-ON and OFF ventricles at 7 dpt. We confirmed the downregulation of glycolysis in klf1-ON hearts, as shown by a significant decrease in glucose 6-phosphate (Figure ​(Figure16M)) and lactate. Meanwhile, genes of key metabolites in the pentose phosphate pathway (PPP; Figure 16N, O, Q, S) and serine synthesis pathway (SSP; Figure 16P), as well as their regulatory enzymes, were upregulated in klf1-ON hearts, demonstrating that klf1 overexpression diverged glucose metabolism from glycolysis to PPP and SSP. In cancer, PPP and SSP play important roles in the synthesis of macromolecules (e.g., nucleic acids, amino acids) and antioxidants (e.g., NADPH) to support cell proliferation and growth. Thus, our data indicate that Klf1 induces metabolic rewiring of the oxidative respiratory pathway to provide the biomass and antioxidant defense required for extensive proliferation of cardiomyocytes.

[0180] Example 13: Conserved regenerative function of Klf1 in mouse heart We investigated the function of Klf1 in mouse hearts. Using RT-qPCR, we detected significant upregulation of mouse Klf1 (mKlf1) gene expression in neonatal mouse hearts after myocardial infarction (MI) (Figure 17A). High-resolution in situ hybridization detected colocalization of mKlf1 mRNA and cardiac troponin T (TnT) staining in the border myocardium of the infarcted area. These findings indicate that, similar to zebrafish hearts, mKlf1 expression is induced upon injury in neonatal mouse hearts. However, mKlf1 gene expression was not significantly upregulated in adult mouse hearts after MI (Figure 17A), contributing to the decline in regenerative capacity with age.

[0181] To address whether expression of mKlf1 unleashes regenerative capacity in adult mouse hearts, we induced MI after measuring baseline cardiac function by echocardiography and injected adenoviral vectors carrying either a control reporter (Ad-GFP; Fig. S17B) or an mKlf1 construct (Ad-mKlf1; Fig. S17B) into the peri-infarct myocardium of MI hearts (Fig. S17C, D). Using echocardiograms, we observed a marked decrease in left ventricular ejection fraction (Fig. S17E) and left ventricular fractional shortening (Fig. S17F) in both groups at 3 dpi, demonstrating the induction of MI in these cohorts. Echocardiographic time course analysis showed that cardiac function in control hearts was further reduced at 28 dpi (Fig. S17E, F), suggesting the development of ischemic heart failure. On the other hand, cardiac function of Ad-mKlf1-treated hearts was significantly improved at 7 dpi and recovered to nearly 50% of baseline levels by 14 dpi (Fig. 17E-G), and the significant improvement over the control group was maintained at 28 dpi (Fig. 17G, H).

[0182] Consistent with these results, histological analysis demonstrated that control hearts developed severe cardiac remodeling, whereas Ad-mKlf1-treated hearts maintained significantly less scarring and remarkably better cardiac morphology (Fig. S4H, I) at 28 dpi (Fig. S4H, J). +We measured the size of cardiomyocytes in the region of the pericardium and found that cardiomyocytes were significantly smaller along the border zone myocardium of Ad-mKlf1-treated hearts (Fig. S4K), indicating that mKlf1 transduction induces cardiomyocyte hyperplasia. Consistent with this observation, we also found that TnT co-labeled with the cell proliferation marker Ki67 (Fig. S4L), the S-phase marker EdU (Fig. S4M), and the mitotic marker pHH3 (Fig. S4N). + We found a significant increase in the number of cardiomyocytes. We evaluated the effect of Ad-mKlf1 transduction in the liver, an organ with high regenerative capacity in adult mice. No significant changes in cell proliferation were observed (Fig. 18A), indicating that the pro-regenerative function of Klf1 is specific to the heart. Together, these data demonstrate that, similar to zebrafish Klf1, mouse Klf1 has a pro-regenerative function in the adult heart and induces repair in the heart after MI.

[0183] These data indicate that pro-survival pathways are unlikely to play a major role in cardiac recovery after MI with Ad-mKlf1 injection. Similar to the observation of increased blood vessels in the klf1-ON zebrafish hearts (Figure S13A), we observed significantly more coronary vessels near the wound site in the Ad-mKlf1-treated mouse hearts (Figure S18B). These data indicate that, as in zebrafish hearts, activation of the Klf1 pathway stimulates a regenerative program that also involves coronary arteries in mouse hearts, and increased blood vessels contribute, at least in part, to the better repair with Klf1 overexpression. Collectively, although the myocardial role of Klf1 is attenuated in adult mammals, exogenous administration of Klf1 resumes myocardial regeneration, indicating that KLF1 is a suitable therapeutic strategy to regenerate myocardium from within the spare myocardium of injured human hearts.

[0184] Example 14: Methods 14.1 Animals Wild-type and genetically modified zebrafish of the exotic Ekwill (EK) background strain, aged from 4 to 12 months, were used in this study. ctAll transgenic lines were analyzed as hemizygotes, except for the line. The publicly available transgenic lines used in this study are as follows: Tg(cmlc2:EGFP) - Burns, CG et al. Nat Chem Biol 1, 263-264 (2005), TgBAC(tcf21:DsRed2) - Kikuchi, K. et al. Development 138, 2895-2902 (2011), Tg(fli1a:EGFP) - Lawson, ND & Weinstein, BM Dev Biol 248, 307-318 (2002), Tg(cmlc2:CreER) - Kikuchi, K., et al. Nature 464, 601-605 (2010), Tg(bactin2:loxP-mCherry-STOP-loxP-DTA176) - Wang, J. et al. Development 138, 3421-3430 (2011), Tg(gata4:EGFP) - Heicklen-Klein & Evans. Dev Biol 267, 490-504 (2004), and Tg(ubb:iCRE-GFP) - Sugimoto, K. et al Elife 6, e24635 (2017).

[0185] Details of the generation of new transgenic lines are described below. Fish were housed at approximately 5 fish per liter and fed three times per day. Water temperature was maintained at 28°C. Excisional injuries were performed in Tricaine-anesthetized zebrafish as previously described (Poss, KD, Wilson, LG & Keating, MT Science 298, 2188-2190 (2002)). Genetic cardiomyocyte depletion was performed as described (Wang, J. et al. Development 138, 3421-3430 (2011)).

[0186] Male C57BL / 6J mice, aged 8 to 12 weeks, were used in this study. Mice were housed up to 5 per cage on racks with a 12:12-h light / dark cycle and given free access to food and water. All animal experiments were performed in accordance with institutional and national animal ethics guidelines and approved by the Garvan Institute of Medical Research / St Vincent's Hospital Animal Ethics Committee.

[0187] 14.2 Myocardial infarction MI was performed on mice anesthetized by intraperitoneal injection of xylazine (13 mg / kg) and ketamine (100 mg / kg) as previously described (Naqvi, N. et al. Cell 157, 795-807 (2014)). Anesthetized mice were ventilated with 1.5%-2% isoflurane in oxygen (0.5 mL stroke volume at 120 strokes per minute) using a Minivent Ventilator (Harvard Apparatus, Hollistion, MA, USA). The fourth intercostal space was opened, the left anterior descending (LAD) artery was permanently ligated using an 8-0 prolene suture on a tapered needle, and the chest was closed with a 6-0 prolene suture.

[0188] 14.3 Generation of actb2:BS-klf1, klf2a, klf2b, and klf4 TagBFP cDNA was PCR amplified from pTagBFP-C (Evrogen, Moscow, Russia) and 3xHA-tag was synthesized using Ultramer Oligo Synthesis (IDT Technologies, Coralville, IA, USA). DsRed and EGFP in actb2:loxP-DsRed-STOP-loxP-EGFP construct (Kikuchi, K. et al., Nature 464, 601-605 (2010)) were replaced by TagBFP and 3xHA-tag, respectively. Klf1, Klf2a, Klf2b and Klf4 cDNA were amplified by PCR using wild-type (Ekkwill) zebrafish cDNA library and cloned downstream and in frame with 3xHA-tag. In each construct, the entire cassette was flanked by I-SceI sites for transgenesis using the meganuclease method (Thermes, V. et al. Mech Dev 118, 91-98 (2002)). The full names of these transgenic lines are as follows: Tg(actb2:loxP-TagBFP-STOP-loxP-3xHA-klf1). vcc29 ; Tg(actb2:loxP-TagBFP-STOP-loxP-3xHA-klf2a) vcc36 ; Tg(actb2:loxP-TagBFP-STOP-loxP-3xHA-klf2b) vcc38 ; and Tg(actb2:loxP-TagBFP-STOP-loxP-3xHA-klf4) vcc35 At least two founder lines for each line were isolated and crossed with cmlc2:CreER to examine the expression of Klf1, Klf2a, Klf2b, and Klf4 proteins in the adult heart by immunofluorescence staining against the 3xHA-tag. Lines expressing 3xHA-Klf2a, Klf2b, or Klf4 at levels equal to or higher than 3xHA-Klf1 were used in this study.

[0189] 14.4 Construction of actb2:BS-dn-klf1 Based on the strategy employed to construct a dominant-negative form of Klf5 (Oishi, Y. et al. Cell Metab 1, 27-39 (2005)), we generated a dominant-negative form of Klf1 using the Engrailed repressor domain (EnR).

[0190] EnR was PCR amplified from pCS2-EnR and fused in frame with the 5' end of Klf1 cDNA. p026 pCS2-EnR was a gift from Dr. Ramesh Shivdasani (Addgene plasmid #11028; http: / / n2t.net / addgene:11028; RRID: Addgene_11028). The resulting EnR-Klf1 chimeric gene was cloned downstream of a loxP-flanked STOP cassette in an actb2:loxP-TagBFP-STOP-loxP backbone construct. Using the meganuclease method, the entire cassette was flanked by I-SceI sites for transgenesis. The full name of this transgenic line is Tg(actb2:loxP-TagBFP-STOP-loxP-dn-klf1). vcc22 It is.

[0191] 14.5 Construction of ubb:loxP-TagBFP-STOP-loxP-EGFP This transgenic construct was generated by inserting a loxP-TagBFP-STOP-loxP-EGFP cassette into the CH211-202A12 BAC after the ubb translation initiation codon using Red / ET recombination (GeneBridges, Heidelberg, Germany). The final construct was purified, linearized with SfiI, and injected into single-cell stage embryos. The full name of this transgenic line is TgBAC(ubb:loxP-TagBFP-STOP-loxP-EGFP). vcc18 It is.

[0192] 14.6 Construction of klf1-ER 3xHA-Klf1 cDNA was PCR amplified from the actb2:BS-klf1 construct and subcloned downstream of the 5.1 kb cmlc2 promoter. Human estrogen receptor (ER) cDNA was PCR amplified from pBabepuro-myc-ER and subcloned downstream and in frame in 3xHA-Klf1. pBabepuro-myc-ER was a kind gift from Wafik El-Deiry (Addgene plasmid #19128; http: / / n2t.net / addgene:19128; RRID:Addgene_19128). A TagBFP cassette controlled by the lens-specific alphaA-crystallin promoter, which allows visual identification of transgenic animals by lens fluorescence, was also inserted in reverse upstream of the cmlc2 promoter. Using the meganuclease method, the entire cassette was flanked by I-SceI sites for transgenics. The full name of this transgenic line is Tg(cmlc2:3xHA-klf1-ER;cryaa:TagBFP). vcc32 It is.

[0193] 14.7 Creating conditional klf1 alleles The published plasmid pZwitch was modified to remove the TagRFP sequence and two repeats of 5x BGHpA to reduce its size. The resulting plasmid was designated pZwitch2. Homologous sequences LA and RA were amplified from genomic DNA isolated from wild-type adult zebrafish (EK) using LA amplification primers klf1-LA-F and klf1-LA-R and RA amplification primers klf1-RA-F and klf1-RA-R. The PCR products of LA and RA were cloned into the corresponding restriction enzyme sites of pZwitch2 by restriction enzyme digestion. The resulting product, pZwitch2-klf1-int1, was co-injected with transcription activator-like effector nucleases (TALENs) into one-cell stage embryos, and the injected embryos were screened to identify founder fish as previously described (Sugimoto, K. et al Elife 6, e24635 (2017)). The full name of this transgenic line is Tg(klf1:Zwitch2). vcc33Gt It is.

[0194] The TALEN kit used to construct Platinum TALENs was a gift from Dr. Takashi Yamamoto (Addgene kit #1000000043). Southern blotting was performed with radiolabeled probes using the Prime-a-Gene Labeling System (Promega, Madison, WI, USA). The detection probe was generated by PCR using wild-type genomic DNA and amplification primers klf1-probe-F and klf1-probe-R (Supplementary Table 1). DNA bands were imaged with an FLA-5100 Bio-Imaging Analyzer (FujiFilm, Tokyo, Japan).

[0195] 14.8 RT-PCR Total RNA was extracted using TRIzol reagent, and then cDNA was synthesized using either the Transcriptor first strand cDNA synthesis kit (Roche, Basel, Switzerland) or the SensiFAST cDNA synthesis kit (Bioline, Eveleigh, Australia). RT-qPCR was performed using the LightCycler 480 System (Roche) or the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The total amount of cDNA was normalized to the amplification of actb2 or rpl13a in the RT-qPCR experiments. All RT-qPCR assays were performed using SYBR Select Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) or TaqMan Universal Master Mix (Thermo Fisher Scientific). For primer details, see Supplementary Table 1.

[0196] 14.9 Cell sorting Cardiomyocytes, endocardial cells and epicardial cells were purified from the ventricles of transgenic reporter zebrafish harboring cmlc2:EGFP, tcf21:DsRed2 or fli1a:EGFP, respectively, using fluorescence-activated cell sorting (FACS) as described (Hui, SP et al. Dev Cell 43, 659-672.e5 (2017)).

[0197] 14.10 In situ hybridization Zebrafish klf1 and mouse Klf1 mRNA were detected using RNAscope probes (Advanced Cell Diagnostics, Hayward, CA, USA). Zebrafish klf1 and mouse Klf1 RNAscope probes were designed and synthesized by Advanced Cell Diagnostics. Signals were detected using the manufacturer's protocol of the RNAscope 2.5 HD Detection Kit-Red (Advanced Cell Diagnostics), followed by immunofluorescence with anti-troponin C or anti-troponin T antibodies. Imaging was performed using a Zeiss LSM 710 confocal microscope as described (Hecksher-Sorensen, J. & Sharpe, J. Mech Dev 100, 59-63 (2001)).

[0198] 14.11 Histological Assays PicroMallory staining, 3,3'-diaminobenzidine (DAB) staining, and Gomori trichrome staining were performed using standard protocols. Immunofluorescence was performed on paraformaldehyde-fixed 10 μm frozen sections as previously described (Hui, SP et al. Dev Cell 43, 659-672.e5 (2017)). Supplementary Table 2 details the primary and secondary antibodies used in this study. Hemoglobin staining of embryos was performed with o-dianisidine (Sigma Aldrich, St. Louis, MO, USA) as described in Detrich et al., Proc Natl Acad Sci USA 92, 10713-10717 (1995).

[0199] 14.12 Microscopes PicroMallory, DAB or Gomori trichrome stained sections were imaged with a Leica DM4000 B microscope (Leica Camera AG, Wetzlar, Germany). Immunofluorescent sections were imaged with a Zeiss AXIO imager M1 microscope (Carl Zeiss AG, Oberkochen, Germany) and confocal images were taken with a Zeiss LSM 710 confocal microscope (Carl Zeiss AG). Whole mount images of zebrafish embryos were taken with an MVX10 microscope (Olympus, Tokyo, Japan).

[0200] 14.13 Transmission Electron Microscopy Zebrafish ventricles were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and refixed in fresh fixative in a PELCO BioWave microwave processor (Ted Pella Inc., Redding, CA, USA). Postfixation was with 1% OsO4 in cacodylate buffer. Tissues were embedded in Procure 812 resin (ProSciTech, Kirwan, Australia) and sectioned using a Leica Ultracut EM UC6 (Leica Camera AG). Ultrathin sections were cut onto copper grids and fixed with an FEI Tecnai G 2 The samples were imaged at 200 kV on a 20 transmission electron microscope (FEI, Hillsboro, OR, USA).

[0201] 14.14 4-HT administration Zebrafish were placed overnight for 10-12 hours in a small beaker of aquarium water supplemented with 5 μM 4-HT as described (Kikuchi, K. et al. Development 138, 2895-2902 (2011)). Zebrafish were rinsed with fresh aquarium water and returned to the circulating water system for feeding. This cycle was repeated for multiple treatments. Zebrafish embryos were treated similarly, except that they were not fed.

[0202] 14.15 EdU assay klf1-OFF and ON fish were intraperitoneally injected with 50 μL of 8 mM EdU once a day on days 5, 6, and 7 after 4-HT treatment. For mice, osmotic minipumps (ALZET, Charles River, MA, USA) were subcutaneously implanted 1 week after MI surgery and EdU was infused daily at 10 mg / kg for 7 days.

[0203] 14.16 Quantification of Cardiac Dedifferentiation Zebrafish ventricular tissue co-labeled with myosin heavy chain (MHC) and Sm22 was quantified pixel-by-pixel using ImageJ (NIH, Bethesda, MD, USA). + Area was also quantified in pixels to determine total MHC + Sm22 for the area + MHC + The percentage of area was calculated. For each heart, three selected sections were analyzed. Myocardial expression of Alcam was quantified similarly, except that troponin C was used as the myocardial marker.

[0204] 14.17 Quantifying cardiomyocyte proliferation in the zebrafish heart PCNA + Cardiomyocytes were quantified in injured hearts as previously described (Hui, SP et al. Dev Cell 43, 659-672.e5 (2017)). Briefly, images of the injury border zone were taken using a Zeiss AXIO imager M1 microscope (vertical approx. 185 μm) and Mef2 cells were analyzed using ImageJ software (NIH). + Cells and Mef2 + PCNA + The number of cells was counted manually. Three selected sections were analyzed for each heart. Quantification in the uninjured ventricles was performed similarly, except that images of the mid-ventricular myocardium (490 μm long × 420 μm wide) were used for quantification.

[0205] EdU in the intact zebrafish ventricle +To quantify cardiomyocytes, confocal images of mid-ventricular myocardium (490 μm long × 420 μm wide) were taken with z-stacks spanning the entire thickness of the myocardium. + EdU embedded in myocardium + The number of nuclei was counted manually and total cmlc2:EGFP was quantified using ImageJ software (NIH). + Normalized to area. Three selected sections were analyzed for each heart. pHH3 + Quantification of cardiomyocyte pHH3 in whole ventricles + The same procedure was followed except that nuclei were counted.

[0206] 14.18 Quantification of cardiomyocyte proliferation in mouse hearts Ki67 + For quantification of cardiomyocytes, cross-sectional images of the area adjacent to the scar tissue were taken at the papillary muscle level. Ki67 in the injured border zone myocardium was defined as healthy myocardium in the area within a distance of approximately 700 μm from the scar. + The number of nuclei was counted manually. Ki67 surrounded by WGA staining + Nuclei were defined as non-cardiomyocytes and excluded from the count (Ang, KL et al. Am J Physiol Cell Physiol 298, C1603-9 (2010)). Values ​​are total troponin T quantified using ImageJ software (NIH). + Normalized to area. EdU + Cardiomyocytes or pHH3 + Quantification of cardiomyocytes was performed similarly.

[0207] 14.19 Measurement of Cardiomyocyte Number and Size Cardiomyocyte number in zebrafish embryos was quantified as previously described (Sugimoto, K. et al Elife 6, e24635 (2017)). Cardiomyocyte number in adult hearts was measured as follows. Ventricles were briefly fixed in 3% PFA for 5 min and incubated overnight at 4 °C in PBS containing 1 mg / mL collagenase type 4 (Worthington Biochemical, Lakewood, NJ, USA). Dissociated cells were resuspended in PBS and rod-shaped cells with clear edges and distinct streaks were manually counted as cardiomyocytes using a hemocytometer.

[0208] To measure cardiomyocyte size, zebrafish ventricles were fixed in 3% PFA for 5 min and incubated overnight at 4 °C in PBS supplemented with 1 mg / mL collagenase type 4 (Worthington Biochemical). After cardiomyocytes were dissociated by gentle trituration with a pipette, the cells were resuspended in PBS and deposited on slides using a Cyto-Tek Cytocentrifuge (Sakura Finetek, Tokyo, Japan), after which immunofluorescence was performed using an anti-α-actinin antibody (Supplementary Table 2). α-actinin + Confocal images of the cells were used to measure the size of each cardiomyocyte using ImageJ software (NIH).

[0209] 14.20 Measurement of Ventricular Area Sections of adult zebrafish ventricles were stained with picromallory and ventricular myocardium area was quantified using ImageJ software (NIH). Three selected sections were analyzed for each heart.

[0210] Example 15: Summary This disclosure provides a function of Klf1 in regenerative plasticity of adult cardiomyocytes. Despite its similar domain structure to Klf4, Klf1 controls cardiomyocyte plasticity through a mechanism that is entirely different from the known function of Klf4 in cell reprogramming. Klf4 has the ability to bind methylated DNA and, as a pioneer factor, targets silent chromatin to induce the expression of pluripotency genes. On the other hand, Klf1 associates with hypomethylated constitutively active cardiac enhancers and reduces chromatin accessibility of binding sites for core transcription factors that control cardiac development and function. Recently, it has been shown that a genome-wide reduction in active chromatin occurs during cardiomyocyte regeneration in zebrafish. The data presented herein support this finding and further demonstrate that Klf1 plays a key role in the global repression of the cardiac gene program that triggers cardiomyocyte dedifferentiation.

[0211] The data presented herein show that Klf1 induces cardiomyocyte proliferation in part through YAP (Figure ​(Figure19A,C). Cardiac activation of YAP is controlled by mechanical signals resulting from cytoskeletal and sarcomere changes. Klf1 may activate Hippo signaling as a consequence of sarcomere reduction induced by downregulation of genes controlling cardiac contraction and actin cytoskeleton organization (Figure15H,I). ChIP-seq motif analysis of Klf1 identified enrichment in binding sites for TEA domain transcription factor 4 (TEAD4), a cofactor of YAP (Figure15B), indicating that one of the mechanisms for this work is that Klf1 controls the Hippo pathway via association with the YAP-TEAD4 complex in the nucleus.

[0212] Klf1 induces metabolic reprogramming of adult cardiomyocytes from cellular respiration pathways to the pentose phosphate pathway (PPP) and serine synthesis pathway (SSP). These pathways upregulate the synthesis of macromolecules and antioxidants, helping klf1-ON cardiomyocytes to proliferate extensively, indicating the possibility of targeting these pathways in regenerative therapy for cardiomyocytes. However, because the PPP and SSP do not produce ATP, continued activation of these pathways will likely lead to energy deficiency (Figure S16K) and fatal cardiac dysfunction (Figure S14A,B).

Claims

1. An in vitro method of inducing myocardial regeneration comprising administering a therapeutically effective amount of KLF1 and / or KLF2b to cardiomyocytes or inducing expression of KLF1 and / or KLF2b in cardiomyocytes.

2. The in vitro method of claim 1, wherein the cardiomyocytes are infant, child or adult cardiomyocytes.

3. 3. The in vitro method of claim 1 or 2, wherein the myocardial regeneration promotes cardiac regeneration.

4. 4. The in vitro method of claim 3, wherein cardiac regeneration is characterized by an increase in ejection fraction, fractional shortening or both.

5. 5. The in vitro method according to claim 3 or 4, wherein cardiac regeneration is characterized by an increase in vascular endothelial cells, epicardial cells or both.

6. The method of any one of claims 1 to 5, wherein KLF1 and / or KLF2b induce dedifferentiation of cardiomyocytes to produce proliferative cardiomyocytes.

7. The in vitro method of claim 6, wherein the proliferative cardiomyocytes are mitotic.

8. The in vitro method of claim 6, further comprising expanding the proliferative cardiomyocytes in the presence of KLF1 and / or KLF2b to produce a population of proliferative cardiomyocytes.

9. 9. The in vitro method of claim 8, wherein the proliferative cardiomyocytes preferentially metabolize glucose using the pentose phosphate pathway, the serine synthesis pathway, or both.

10. 10. The in vitro method of claim 8 or 9, further comprising differentiating the population of proliferative cardiomyocytes to produce a population of cardiomyocytes.

11. The in vitro method of claim 10, wherein differentiation occurs in the substantial absence of KLF1 and / or KLF2b or after induction of KLF1 and / or KLF2b has ceased.

12. The in vitro method of any one of claims 6 to 11, wherein KLF1 and / or KLF2b induce chromatin remodeling to promote dedifferentiation.

13. The in vitro method of claim 12, wherein KLF1 and / or KLF2b-induced chromatin remodeling reduces accessibility to binding sites for one or any combination of MEF2C, GATA4, MEF2A and NKX2.

5.

14. The in vitro method according to any one of claims 1 to 13, wherein KLF1 is provided as a KLF1 protein, a KLF1 nucleic acid or a vector comprising a KLF1 nucleic acid.

15. The in vitro method of claim 14, wherein the KLF1 protein is SEQ ID NO: 1 or 11, or a protein at least 80% identical to SEQ ID NO: 1 or 11.

16. 15. The in vitro method of claim 14, wherein the KLF1 nucleic acid comprises or consists of any one of SEQ ID NOs: 2, 3, 4, 5, 9 or 10, or a nucleic acid at least 80% identical to any one of SEQ ID NOs: 2, 3, 4, 5, 9 or 10.

17. The in vitro method according to any one of claims 1 to 13, wherein KLF2b is provided as a KLF2b protein, a KLF2b nucleic acid or a vector comprising a KLF2b nucleic acid.

18. The method of claim 17, wherein the KLF2b protein is SEQ ID NO:6 or a protein at least 80% identical to SEQ ID NO:

6.

19. 18. The in vitro method of claim 17, wherein the KLF2b nucleic acid comprises or consists of SEQ ID NO: 7 or 8, or a nucleic acid at least 80% identical to any one of SEQ ID NO: 7 or 8.

20. 20. The in vitro method of any one of claims 14, 16, 17 or 19, wherein the vector comprises a KLF1 nucleic acid and / or a KLF2b nucleic acid operably linked to a promoter.

21. 21. The in vitro method of claim 20, wherein the promoter is a cardiac-specific promoter.

22. 22. The in vitro method of claim 21, wherein the cardiac-specific promoter is selected from the group consisting of alpha-myosin heavy chain (alpha-MHC) promoter, myosin light chain 2 (MLC-2) promoter, cardiac troponin C (cTnC) promoter, NCX1 promoter and TNNT2 promoter.

23. 21. The in vitro method of claim 20, wherein the promoter is an inducible promoter.

24. The in vitro method of claim 23, wherein the inducible promoter is selected from the group consisting of a tetracycline-inducible promoter, a steroid hormone-inducible promoter, or a hypoxia-inducible promoter, a promoter specific to an area near the site of cardiac damage, or a promoter responsive to stress resulting from ischemia and reperfusion.

25. The in vitro method according to any one of claims 6 to 24, wherein the proliferative cardiomyocytes are cardiomyocyte precursor cells, immature cardiomyocytes or cardiomyocytes with an embryonic phenotype.

26. The in vitro method according to any one of claims 1 to 25, wherein cardiomyogenesis does not involve reprogramming of the cardiomyocyte lineage.

27. 27. The in vitro method according to any one of claims 6 to 26, wherein the proliferating cardiomyocytes re-enter the cell cycle.

28. The in vitro method according to any one of claims 6 to 26, characterized in that the proliferative cardiomyocytes have an increased dependency on the pentose phosphate pathway (PPP), the serine synthesis pathway or both compared to cardiomyocytes.

29. The in vitro method according to any one of claims 1 to 28, wherein myocardial regeneration is characterized by an increase in the number of epicardial cells, vascular endothelial cells or both.

30. Use of KLF1 and / or KLF2b in the manufacture of a medicament for the treatment of cardiac disease in a subject.

31. The use of claim 30, wherein the cardiac disease is associated with loss of cardiac muscle cells.

32. 32. The use according to claim 31 , wherein the cardiac disease is myocardial infarction, ischemic cardiomyopathy, dilated cardiomyopathy or heart failure.

33. The use described in any one of claims 30 to 32, wherein KLF1 and / or KLF2b induce myocardial regeneration in a subject's cardiomyocytes, and the myocardial regeneration is induced in vitro.

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